

**Biotechnology for Sustainability**

_Achievements, Challenges and Perspectives_

**Editors**

**Subhash Bhore,**

**K. Marimuthu & **

**M. Ravichandran**

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**Biotechnology for Sustainability**

**Achievements, Challenges and Perspectives**

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**Editors**

Subhash Bhore, K. Marimuthu & M. Ravichandran

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**Biotechnology for Sustainability**

**Achievements, Challenges and Perspectives**

Subhash Bhore, K. Marimuthu & M. Ravichandran (Editors) ****

Published by AIMST University

2017

ISBN: 978-967-14475-3-6 (Print version)

eISBN: 978-967-14475-2-9 (e-Book version)

2

**Published by**

AIMST University

**Printed by**

AIMST University

**Copyright**

© 2017 by the authors; Licensee, Editors; AIMST University,

Malaysia. This book is an open access book distributed under

the terms and conditions of the Creative Commons Attribution

(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).

CC BY license is applied which allows users to download, copy, reuse and distribute

articles and or data provided the original article and book is fully cited. This open

access aims to maximize the visibility of articles, reviews and or perspectives, much of

which is in the interest of national, regional and global community.

**Disclaimer** : The information provided in this book is designed to highlight the views,

perspectives, achievements and or research findings of respective contributors. While

the best efforts have been used in preparing this book, Editors and or Publisher make

no representations or warranties of any kind and assume no liabilities of any kind with

respect to the accuracy or completeness of the contents and specifically disclaim any

implied warranties. Neither the Editors nor Publisher of this book shall be held liable or

responsible to any person or entity with respect to any loss or incidental or

consequential damages caused, or alleged to have been caused, directly or indirectly,

by the information highlighted herein. Readers should be aware that the information

provided in this book may change.

All articles and or reviews published in this book are deemed to reflect the individual

views of respective authors and not the official points of view, either of the Editors or of

the Publisher.

**Cover image:** A diagram showing the 17 Sustainable Development Goals (Credit:

www.un.org/)

**Edited by**

Dr. Subhash J. Bhore (Senior Associate Professor)1,

Dr. K. Marimuthu (Professor)1, 2, and

M. Ravichandran (Senior Professor)1, 2

_Address for Correspondence:_

_1Department of Biotechnology, Faculty of Applied Sciences, AIMST University,_

_Bedong-Semeling Road, 08100 Bedong, Kedah Darul Aman, Malaysia; Telephone_

_No.: +604 429 8176; e-mail: subhash@aimst.edu.my / subhashbhore@gmail.com_

_2Chancellery, AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah Darul_

_Aman, Malaysia; Tel. No.: +604 429 1054 /8103; e-mail: marimuthu@aimst.edu.my /_

_ravichandran@aimst.edu.my_

**Edition**

First; July 18, 2017

3

_Dedication_

_This book is dedicated to all researchers working in_

_various domains of biotechnology and to all_

_stakeholders those are working for the global_

_sustainable development to improve the health of the_

_people and planet._

4

**Preface**

****

**W** orld Environment Day (WED) is a biggest global annual event celebrated each

year on June 5 to create the positive awareness to preserve the environment and planet

earth. This year, the theme for WED-2017 was "Connecting people to nature". Our

environment should be healthy for our growth, development and to achieve the sustainable

development goals (SDGs) adopted by the international community to transform the world.

Most recently, António Guterres (United Nations Secretary General) precisely

highlighted that "Without a healthy environment we cannot end poverty or build prosperity.

We all have a role to play in protecting our only home: we can use less plastic, drive less,

waste less food and teach each other to care". In fact, to achieve the SDGs by protecting

environment, everyone needs to do their part.

We strongly believe that biotechnology can play an important role directly or

indirectly in achieving various SDGs. Hence, we had decided to publish a book,

"Biotechnology for Sustainability" to commemorate the WED and to highlight the

achievements, challenges and perspectives in various domains of the biotechnology. In

response to our call for articles, we had received 50 manuscripts. The selected articles

published in this book are highlighting various issues, achievements, challenges and

perspectives for the viable development and sustainability. The World Commission on the

Environment and Development defined sustainability as the "development that meets the

needs of the present without compromising the ability of future generations to meet their

own needs". The United Nations recent estimate suggest that the world's food supply needs

to be doubled by the year 2050 to keep up with the growing demand. To achieve this is a

huge challenge; because, the amount of arable land is continuously decreasing as a result of

rising urbanization, saline soils and desertification. Biotechnologists (and plant breeders)

around the world are working persistently to produce crops which will boost the food

production to meet the growing demand. Genetically engineered crop varieties do offer

many promising possibilities to boost nutritive value of the food, sustain farming on

marginal lands, and to minimize the loss by creating pests and disease resistant varieties.

The articles published in this book are going to be useful in creating awareness

about the environmental issues, natural resources, biodiversity conservation, sustainable

development and various biotechnological approaches that could be used to alleviate the

respective challenges.

We would like to express our sincere gratitude and thanks to Dato' Seri Utama Dr.

S. Samy Vellu, Chancellor and Chairman, AIMST University for his support in publishing

this book.

We wish to thank all contributing authors for making a common cause with us. This

book publication project could not have been completed without the courteous cooperation

of the authors to highlight achievements, challenges and or perspectives in using

biotechnological approaches for the sustainability.

We are confident that this book will serve as a reference to various researchers,

scientists, academicians and graduate students involved in biodiversity conservation,

environmental protection and various fields of biology and biotechnology.

It is hoped that a prudent use of biotechnology in the biodiversity conservation,

environmental protection, and production of more and better quality of food, fiber, fuel and

drugs will contribute in accomplishing SDGs and to promote peace in the world.

**Subhash J. Bhore**

**K. Marimuthu**

**M. Ravichandran __**

_ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9_ i

**Contents**

****

**Preface ................................................................................................................................ i**

**Contents ............................................................................................................................ ii**

**Plant Tissue Culture for Sustainability**

C. K. John ....................................................................................................................... 1

**Traditional Medicine of the Tribes in Tamil Nadu and Its Sustainable**

**Use through Biotechnology**

Valli Gurusamy, Kavitha Valampuri John, Usha Raja Nanthini

Ayyakkanu, Ramani Bai Ravichandran ......................................................................... 14

**Vermitechnology – An Eco-Biological Tool for Sustainable**

**Environment**

Mahaly Moorthi, Koilpathu Senthil Kumar Abbiramy, Arumugam Senthil

Kumar and Karupannan Nagarajan................................................................................ 41

**Role of Biotechnology in Food Authentication**

Shobana Manoharan, Raghavan Kuppu and Ramesh Uthandakalaipandian ................... 51

**Management Strategies against Tiny Tigers for Sustainable**

**Development of Agriculture**

Viswa Venkat Gantait ................................................................................................... 58

**Designing Greener Pharmaceuticals and Practicing Green Health Is**

**Required for Sustainability**

Sridevi Chigurupati, Jahidul Islam Mohammad, Kesavanarayanan

Krishnan Selvarajan, Saraswati Simansalam, Shantini Vijayabalan and

Subhash Janardhan Bhore ............................................................................................. 68

**Clonal Propagation of a High Value Multipurpose Timberline Tree**

**Species Quercus semecarpifolia Sm. of West Himalaya, India**

Aseesh Pandey and Sushma Tamta ............................................................................... 79

**Spent Mushroom Substrate of _Hypsizygus ulmarius_** **: A Novel**

**Multifunctional Constituent for Mycorestoration and Mycoremediation**

Padmavathi Tallapragada and Ranjini Ramesh .............................................................. 88

**Biotechnology for Sustainability of Forests**

Kumud Dubey and Kesheo Prasad Dubey ................................................................... 104

**Biotechnological Approaches for Conservation and Sustainable Supply**

**of Medicinal Plants**

Sagar Satish Datir and Subhash Janardhan Bhore ........................................................ 117

_ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9_ ii

**Making Himalayas Sustainable: Opportunities and Challenges in**

**Indian Himalayan Region**

Harsh Kumar Chauhan and Anil Kumar Bisht ............................................................. 129

**Natural Polyphenols and Its Potential in Preventing Diseases Related**

**To Oxidative Stress as an Alternative Green Nutraceutical Approach**

Sreenivasan Sasidharan, Shanmugapriy, Subramanion Lachumy Jothy,

Mei Li Ng, Nowroji Kavitha, Chew Ai Lan, Khoo Boon Yin,

Soundararajan Vijayarathna, Leow Chiuan Herng and Chern Ein Oon ........................ 141

**A Review on Green Synthesis of Nanoparticles and Its Antimicrobial**

**Properties**

Karthika Arumugam and Naresh Kumar Sharma ......................................................... 171

**Production of Secondary Metabolites Using a Biotechnological**

**Approach**

Produtur Chandramati Shankar and Senthilkumar Rajagopal ....................................... 187

**Potential of Marine Algae Derived Extracts as a Natural Biostimulant**

**to Enhance Plant Growth and Crop Productivity**

Lakkakula Satish* and Manikandan Ramesh ............................................................... 200

**Biotransformation of Various Wastes into a Nutrient Rich Organic**

**Biofertilizer - a Sustainable Approach towards Cleaner Environment**

Geetha Karuppasamy, Michael Antony D'Couto, Sangeetha Baskaran and

Anant Achary.............................................................................................................. 212

**Bacterial Endophytes as Biofertilizers and Biocontrol Agents for**

**Sustainable Agriculture**

Amrutha V. Audipudi, Bhaskar V. Chakicherla and Shubhash Janardhan

Bhore .......................................................................................................................... 223

**Microbial Metabolic Engineering: A Key Technology to Deal with**

**Global Climate and Environmental Challenges**

Meerza Abdul Razak, Pathan Shajahan Begum and Senthilkumar

Rajagopal .................................................................................................................... 248

**Biodiesel Production for Sustainability: An Overview**

R. Meena Devi, R. Subadevi and M. Sivakumar .......................................................... 262

_**In vitro**_ **Cell Bioassays in Pollution Assessment**

Narayanan Kannan, Poorani Krishnan and Ahmad Zaharin Aris ................................. 274

**Lipopeptide Biosurfactants from Bioagent, Bacillus as a Weapon for**

**Plant Disease Management**

Sampath Ramyabharathi, Balaraman Meena, Lingan Rajendran and

Thiruvengadam Raguchander ...................................................................................... 287

_ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9_ iii

**Biotechnology as a Tool for Conservation and Sustainable Utilization of**

**Plant and Seaweed Genetic Resources of Tropical Bay Islands, India**

Pooja Bohra, Ajit Arun Waman and Anuraj Anirudhan ............................................... 295

**Plantibodies for Global Health: Challenges and Perspectives**

Prasad Minakshi, Basanti Brar, Manimegalai Jyothi, Ikbal, Koushlesh

Ranjan, Upendra Pradeep Lambe and Gaya Prasad ..................................................... 305

**Renewable Energy from Agro-industrial Processing Wastes: An**

**Overview**

Sudhanshu S. Behera, Ramesh C. Ray and S. Ramachandran ..................................... 322

**Mitigation of Climatic Change by Organic Agriculture**

Mohan Mani, Manohar Murugan, Ganesh Punamalai and Vijayalakshmi

Ganesan Singaravelu ................................................................................................... 336

**Application of Anti-vibrio and Anti-quorum Sensing Technology for**

**Sustainable Development in Shrimp Aquaculture**

Ramesh Kandasamy, Amutha Raju and Manohar Murugan ......................................... 344

**Promiscuous Rhizobia: A Potential Tool to Enhance Agricultural Crops**

**Productivity**

Ikbal, Prasad Minakshi, Basanti Brar, Upendera Praddep Lambe,

Manimegalai Jyothi, Koushlesh Ranjan, Deepika, Virendra Sikka and

Gaya Prasad ................................................................................................................ 358

**Organic Farming and Halalan Toyyiban Foods: An Attempt to Relate**

**Them**

Quamrul Hasan and Zakirah Othman .......................................................................... 376

**Biotechnological Approaches: Sustaining Sugarcane Productivity and**

**Yield**

Ashutosh Kumar Mall and Varucha Misra .................................................................. 386

**Bioremediation: A Biotechnology Tool for Sustainability**

Niharika Chandra, Ankita Srivastava, Swati Srivastava, Shailesh Kumar

Mishra and Sunil Kumar ............................................................................................. 398

**Sea Urchin - A New Potential Marine Bio-resource for Human Health**

M. Aminur Rahman, Fatimah Md. Yusoff, Kasi Marimuthu and Yuji

Arakaki ....................................................................................................................... 417

**Marine Pollution and Its Impacts on Living Organisms**

Thavasimuthu Citarasu and Mariavincent Michael Babu ............................................. 444

**Ecology, Distribution and Diversity of Bioluminescent Bacteria in Palk**

**Strait, Southeast Coast of India**

Srinivasan Rajendran, Ganapathy selvam Govindarasu and Govindasamy

Chinnavenkataraman................................................................................................... 456

_ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9_ iv

**Synthesis of Biocompatible Silver Nanoparticles Using Green Alga**

**( _Ulva reticulata_** **) Extract**

Ganapathy selvam Govindarasu, Srinivasan Rajendran and Sivakumar

Kathiresan................................................................................................................... 475

**Diversity and Ethno-Botanical Potential of Tree Plants of Katarniaghat**

**Wildlife Sanctuary, Bahraich (UP) India: An Overview**

Tej Pratap Mall ........................................................................................................... 486

**Free Radical Scavenging Potential and Anticancer Activity of _Primula_**

_**denticulata**_ **Sm. from North-Western Himalayas**

Bilal Ahmad Wani, Mohammed Latif Khan and Bashir Ahmad Ganai ........................ 512

**Panchakavya: Organic Fertilizer and Its Stimulatory Effect on the Seed**

**Germination of _Abelmoschus_** **_esculentus_** **and _Solanum melongena_** ****

V. Ramya and S. Karpagam ........................................................................................ 525

**Increasing Human Interference in Katarniaghat Wildlife Sanctuary**

Shiv Pratap Singh ....................................................................................................... 534

_ISBN: 978-967-14475-3-6 ; eISBN: 978-967-14475-2-9_ v

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P1-13_

**Plant Tissue Culture for Sustainability**

**C. K. John***

_Plant Tissue Culture Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha_

_Road, Pune 411008, India;*Correspondence: ck.john@ncl.res.in; Tel.: +91-9822531551_

**Abstract:** The United Nations has placed great emphasis on sustainability. Three of the

most important requirements of sustainable development are: eradicating extreme pov-

erty and hunger, protecting the environment, and conserving biodiversity. Because of human activities the stable functioning of earth's life support system – which includes the at-

mosphere, oceans, forests, waterways, biodiversity and biogeochemical cycles, is at risk.

One of the major contributing factors is the large scale destruction of natural forests. Defor-

estation had many adverse effects; most importantly, the effects on climate, environment,

and biodiversity. The three pillars of sustainable development are: sustainable agriculture,

conserving biodiversity, and protecting the environment through reversing the effects of

deforestation by large scale afforestation. Plant Tissue Culture can greatly contribute in all

the three. ****

_**Keywords**_ **:** Afforestation; biodiversity conservation; micropropagation; plant tissue culture;

sustainable agriculture

**1. Introduction**

variation in plant varieties as possible.

Plant tissue culture can contribute to all

The United Nations Summits and

the three. In this paper I will elaborate on

Commission Reports from the 1987

how Plant Tissue Culture, my area of re-

Brundtland Commission (World Com-

search, can contribute to Sustainable Ag-

mission on Environment and Develop-

riculture, Protecting Forests, and Con-

ment) report onwards have placed added

serving Biodiversity.

emphasis on sustainability of all devel-

opment efforts. Three of the most im-

**2. Sustainable development**

portant requirements are: 1. Eradicat-

ing extreme poverty and hunger, 2. Pro-

In 1987 it was the Brundtland

tecting the environment, and 3. Conserv-

Commission (World Commission on En-

ing biodiversity. To eradicate extreme

vironment and Development) report "Our

poverty and hunger two things are essen-

Common Future" which brought the con-

tial: first, sustainable agriculture which

cept of "Sustainable Development" into

makes food available/affordable and se-

common use. The World Commission on

cond, creation of jobs which translates to

Environment and Development was set up

purchasing power. One of the major fac-

by the UN General Assembly in 1983.

tors in protecting the environment is re-

Brundtland Commission Report defined

versing the loss of natural forests. Con-

Sustainable Development as "Develop-

serving biodiversity is of great relevance

ment that meets the needs of the present

now than ever before for the reason that

without compromising the ability of the

our world is fast changing. To have crop

future generations to meet their own

varieties suitable for this changing envi-

needs". According to the Brundtland

ronment is to preserve as much natural

Commission Report, the needs, in particu-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 1

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ lar the essential needs of the world"s poor,

the principles of sustainable develop-

to which overriding priority should be

ment into country policies and programs,

given, and the limitations imposed by the

reversing loss of environmental resources,

State of Technology and Social organiza-

and reducing biodiversity loss.

tion on the Environment"s ability to meet

In 2012, the United Nations

present and future needs should be ad-

Rio+20 summit in Brazil committed gov-

dressed. The Brundtland Commission Re-

ernments to create a set of "Sustainable

port emphasized the need to integrate

Development Goals" (SDGs). On Sep-

economic and ecological factors in deci-

tember 25th 2015, countries adopted a set

sion-making at all levels for sustainable

of goals to end poverty **,** protect the planet **,**

development. These factors include, re-

and **** ensure prosperity for all as part of

viving growth, changing quality of

a 2030 Sustainable Development Agenda.

growth, meeting essential needs for jobs,

Each goal has specific targets to be

food, energy, water and sanitation, ensur-

achieved in 15 years. The 17 Sustainable

ing the resource base, reorienting tech-

Development Goals (SDGs), otherwise

nology and managing risks. In its broadest

known as the Global Goals, are a uni-

sense, the strategy for sustainable devel-

versal call for action to end poverty,

opment aims to promote harmony among

protect the planet and ensure that all

people and between human beings and

people enjoy peace and prosperity al-

environment.

ways. The goals are interconnected.

In 1992, at the Earth Summit (Rio,

The key to success on one will involve

1992) there was consensus that environ-

tackling issues associated with anoth-

ment, and economic and social develop-

er. The SDGs work in the spirit of

ment cannot be considered in isolation,

partnership and pragmatism, to make

and in addition to treaties and agreements

the right choices now to improve life,

on climate change, biological diversity,

in a sustainable way, for future genera-

deforestation, and desertification, the Rio

tions. They provide clear guidelines

Declaration contains fundamental princi-

and targets for all countries to adopt in

ples on which nations can base their fu-

accordance with their own priorities

ture decisions and policies, considering

and the environmental challenges of

the environmental implications of socio-

the world at large. The SDGs are an

economic development.

inclusive agenda. They tackle the root

In 2000 the Millennium Sum-

causes of poverty and unite all nations

mit of the United Nations, following the together to make a positive change for

adoption of the United Nations Millenni-

both people and planet (UNDP).The

um Declaration, established the eight Mil-

15th SDG of UN relates to Life on land,

lennium Development Goals (MDGs) to

and involves protecting, restoring and

be achieved by the year 2015. The MDGs

promoting sustainable use of terrestri-

are: 1. to eradicate extreme poverty and

al ecosystems, sustainably managing for-

hunger, 2. to achieve universal primary

ests, combating desertification, and halt-

education, 3. to promote gender equali-

ing and reversing land degradation and

ty and empower women, 4. to re-

halting biodiversity loss.

duce child

mortality,

5.

to

im-

The stable functioning of Earth's

prove maternal

health,

6.

to

com-

life support system – which includes the

bat HIV/AIDS,  malaria, and other diseas-atmosphere, oceans, forests, waterways,

es, 7. to ensure environmental sustainabil-

biodiversity and biogeochemical cycles, is

ity, and 8. to develop a global partnership

a prerequisite for future human develop-

for development. In the present context

ment. However, as per recent research

Goal 7: Ensuring environmental sustaina-

findings this functioning is at risk (Rock-

bility is very important. Two of the im-

ström _et al_., 2009). Further human pres-

portant targets of MDG 7 are: Integrating

sure may lead to large-scale, abrupt, and

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 2

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ potentially irreversible changes to Earth's

generations depends, stands on three pil-

life support system (Lenton 2011; Bar-

lars:

nosky _et al_., 2012). Likely impacts on

_i._

Sustainable agriculture

humanity include: diminishing food pro-

_ii._

Conserving biodiversity

duction, water shortages, extreme weath-

_iii._

Protecting the environment

er, ocean acidification, deteriorating eco-

systems, and sea-level rise. In this back-

Increasing food production must

drop Griggs _et al_. (2013) suggested that

involve, developing/ introducing better

we redefine sustainable development as

(efficient, high yielding, insect-pest re-

"Development that meets the needs of the

sistant) varieties of crop plants, conserv-

present while safeguarding Earth"s life-

ing biodiversity, and protecting environ-

support system, on which the welfare of

ment. Plant Tissue Culture can greatly

current and future generations depends."

contribute in all these.

Without economic, technological, and

societal transformations, chances of large-

**4. Plant tissue culture**

scale humanitarian crises exist. Such cri-

ses could undermine any gains made by

Plant tissue culture is the _aseptic_

meeting the MDGs. A re-evaluation of the

_growing of whole plants or parts (cells,_

relationship between people and planet is

_tissues/ organs) in/ on defined (synthetic)_

necessary (Griggs _et al_., 2014).

_nutrient media under controlled (envi-_

_ronmental) conditions (temperature, light,_

**3. Three pillars of sustainable devel-**

_humidity)_. Usually in glass vessels (test

**opment**

tubes, conical flasks, jam bottles etc.) -

for a review see John _et al_. (1997).

In the second half of the 20th cen-

Plant tissue culture is based on

tury there was intensification of agricul-

cellular "totipotency", the inherent poten-

ture in most parts of the world. Intensive

tial of a plant cell to regenerate a whole

agriculture involved: (i) expanding farm

plant. Unlike animal cells, most plant

lands, by removing natural forests, (ii)

cells retain the capacity to regenerate the

better irrigation, by constructing big

whole organism even after undergoing the

dams, which again submerged vast forests

final differentiation. In plants, as long as

in their catchment areas (iii) use of chem-

the cells have an intact membrane system

ical fertilizers and pesticides, to produce

and a viable nucleus, even highly mature

high yields. Destruction of natural forests

and differentiated cells retain the ability

had many adverse effects; most im-

to regenerate to a meristematic state.

portantly, the effects on climate, envi-

Though initially, in the first two decades

ronment, and biodiversity. Extensive use

of the 20th Century progress was slow,

of chemical fertilizers and pesticides also

standardization of universal plant tissue

had their own adverse effects. Excessive

culture media - White"s (White, 1933),

use of chemical fertilizers has resulted in

Gamborg"s (Gamborg _et al._ , 1975) and

nitrate accumulation, increased soil salini-

MS (Murashige and Skoog, 1963)

ty, and water eutrophication. High use of

changed the scene. Plant tissue culture

pesticides has resulted in development of

media contain minerals, growth factors

resistance in many pest species. In recent

and a carbon source (usually sucrose).

years there is much concern about envi-

Controlled environmental factors are light

ronmental contamination by fertilizers

(intensity and length – photoperiod), tem-

and pesticides.

perature, relative humidity. On/ in a cus-

Sustainable Development, that

tom standardized medium, and controlled

meets the needs of the present while safe-

environmental conditions, the explant

guarding Earth"s life-support system, on

(starting plant material) - usually young,

which the welfare of current and future

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 3

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ undifferentiated tissue, regenerate into

gregates of few cells. These cells/ cell ag-

whole plants.

gregates grow/ divide/ separate as a result

of agitation, and can be continually main-

_4.1. Types of cultures_

tained in this state. Growth of cells in

Different types of cultures are

suspension culture can be more easily

possible: (i) culture of whole plants, (ii)

manipulated in liquid medium than on

embryo culture (embryo rescue), (iii) or-

semi-solid medium. Slowly agitating the

gan culture (shoot tip culture, root culture,

liquid medium on a rotary shaker is nec-

leaf culture, anther culture etc.), (iv) cal-

essary for the growth of the cultures,

lus culture, (v) cell suspension and single

which can be sub-cultured. Growth in

cell culture, (vi) protoplast culture.

single isolated cells can be induced by

****

culturing them in hanging drops in micro-

_4.2. Callus culture_

chambers. Suspension cultures are useful

Callus is an amorphous mass pro-

in plant production by somatic embryo-

duced by cell proliferation, occurring in

genesis form single cells. In regeneration

an unorganized manner. In nature it is a

of plants from callus established on semi-

wound response, or a plant reaction to the

solid media from small cell aggregates,

presence of micro-organisms, insects, or

and for the production of secondary me-

to some kind of stress. Under _in vitro_

tabolites. Suspension cultures can also be

conditions callusing is a response to en-

initiated from tissue other than callus

dogenous or exogenous growth regula-

(Geile and Wagner, 1980).

tors. The potential for callus formation is

dependent on the tissue (explant) type.

_4.5. Protoplast cultures_

Meristematic tissues are more suitable for

Protoplasts are plant cells without

callus induction than mature tissues. Cal-

cell walls. In 1882, Klercker isolated pro-

lus cultures can be maintained for long by

toplasts mechanically for the first time.

sub-culturing the primary callus (callus

The yield of protoplasts was very low. In

established originally from the explant),

1960, Cocking using enzymes for the first

at periodic intervals.

time could isolate protoplasts in large

numbers. Protoplasts can be isolated from

_4.3. Somaclonal variations_

different plant parts, or from tissues al-

Long term callus cultures can

ready in culture. Enzymatic isolation is

however, suffer from spontaneously aris-

now the most commonly used method. A

ing genetic variations, reflected in the

combination of these two can also be

phenotype of plants regenerated from

used.

such calli. These variations are known as

One of the important applications

_somaclonal variations. Somaclonal varia-_

of protoplasts is in somatic hybridization.

_tions_ are reported in many species. The

Many agents like NaNO3 (Power _et al._ ,

basis of somaclonal variations is not well

1990), a higher pH, and a higher concen-

understood.

Chromosomal

rearrange-

tration of calcium ions in the medium

ments, activation of endogenous trans-

(Melchers and Labib, 1974), polyethylene

posons, and changes in the status of DNA

glycol (Kao and Michayuluk, 1974;

methylation, are considered to be the con-

Wallin _et al._ , 1974), and a high strength

tributing factors.

electric field (Zimmermann and Scheu-

rich, 1981), are used for obtaining fusion

_4.4. Suspension cultures_

between protoplasts. Protoplasts, when

Culture of unorganized plant cells,

placed in appropriate media regenerate

as single cells/ cell aggregates, in liquid

cell walls and form calli, from which

medium. Friable callus when cultured in

plants can be regenerated. Protoplasts are

agitated liquid medium, the cells separate

used for producing somatic hybrids (para-

and form a suspension of single cells/ ag-

sexual hybrids), for genetic manipulation,

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 4

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ and for basic studies on a variety of as-shoot tips of virus infected plants are also

pects. Regenerating plants from proto-

virus-free. Morel and Martin (1950; 1955)

plasts is difficult in some species. Con-

could produce healthy plants from virus-

ventional hybridization depends on affini-

free plants through shoot-tip culture from

ty of gametes. Wide crosses are not pos-

infected mother plants. This is possible

sible because of well-established cross

because the pathogen concentration is not

breeding barriers. Protoplast fusion makes

uniform in the infected plants, and apical

such hybridizations possible.

buds of rapidly growing shoots are often

not invaded by the virus. Morel (1960)

_4.6. Anther (isolated microspore) cultures_

used shoot apices of orchids to obtain

Guha and Maheshwari (1964) ob-

their rapid clonal multiplication. Shoot tip

tained haploid embryos, directly from an-

culture has two important practical appli-

ther cultures of _Datura innoxia_. The

cations: (i) virus eradication and (ii) mi-

origin of these embryos was traced to the

cro-propogation.

These

developments

pollen grains. The potential of anther cul-

were followed by _in vitro_ propagation of

ture for obtaining haploid plants, and

plants from shoot tip culture. Initially

from them by chromosome doubling of

most of the species micropropogated were

homozygous diploid plants was apparent.

herbaceous (Morel, 1964; Murashige,

In 1974, Nitsch had reported regeneration

1974). Now methods are available for the

of haploids and homozygous diploids by

micropropagation of a large number of

chromosome doubling, from isolated mi-

species belonging to a wide range of plant

crospore culture (Nitsch, 1974a; 1974b).

groups.

Culturing the microspores along with an-

ther wall is essential for success. In iso-

_4.8. Embryo culture_

lated microspores, pollen embryogenesis

Very young to mature embryos

is induced only rarely.

can be cultured _in vitro._ Embryo culture is

This technique has great potential

one of the oldest applications of plant tis-

in plant breeding. Normally it takes self-

sue culture in plant breeding. It has many

ing for many generations to obtain homo-

practical applications, and very useful in

zygosity in parental lines required in

obtaining hybrid plants from crosses in

breeding programmes. This time can be

which post-zygotic incompatibility exists.

considerably reduced by haploid culture

In post zygotic incompatibility, fertiliza-

techniques.

tion and zygote formation occur on cross

pollination. The zygote grows, but is not

_4.7. Meristem culture and shoot tip cul-_

accepted by the endosperm. This results

_ture_

in embryo abortion at some stage of de-

When growing points (meristems)

velopment before maturing of the seed. In

of shoots are cultured they continue their

such instances when the ovary/ ovule/

organized growth. The shoots/multiple

embryo with a part of the maternal tissue

shoots produced can be rooted to produce

is excised and cultured on a suitable me-

plantlets. This capacity has practical ap-

dium and under optimum culture condi-

plication and economic significance for

tions, it matures to produce a seedling.

plant propagation.

This procedure is hence called _embryo_

Culture of the meristemic zones

_rescue_. Sharma _et al._ (1980) obtained few

/extreme shoot tip is known as _meristem_

hybrids between _Solanum melongena_ and

_culture_ , and culture of small segments (5-

_S. khasianum_ by this method. Embryo

10 mm in size) from the shoot tip is

culture is useful also in overcoming seed

known as _shoot tip culture_. It was known

dormancy and for obtaining seed germi-

that meristems of virus infected roots are

nation in some vegetatively propagating

free of the pathogen (White, 1933; 1934).

species in which seeds are produced but

Limasset and Cornuet (1949) found that

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_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ normally do not germinate (e.g. some

shoots or leaves. These organs may arise

wild bananas).

out of pre-existing meristems or out of

differentiated cells. Indirect pathway in-

_4.9. Invitro pollination and fertilization_

cludes a callus stage.

Pre-zygotic incompatibility is one

Direct pathway bypasses a callus

of the major limitations in obtaining hy-

stage. The cells in the explant act as direct

bridization between many plant species

precursors of a new primordium, an organ

and varieties. In pre-zygotic incompatibil-

or a part in its most rudimentary form or

ity, the zygote is not formed on cross pol-

stage of development.

lination. The pollen either do not germi-

nate on the stigma of the female parent or

_4.12. Somatic embryogenesis_

the pollen tube gets arrested at some point

In plants, embryo-like structures

of its growth on the stigma/ in the style.

can be generated from non-germ cells

A variety of methods used _in vivo_ to

(somatic cells), by circumventing the

overcome this barrier.

process of normal fertilization. As somat-

Kanta _et al_. (1962) developed an

ic embryos are formed without fertiliza-

_in vitro_ technique for overcoming pre-

tion event, they are genetically identical

zygotic incompatibility. In this method,

to the parent tissue, and are therefore

the mature/nearly mature ovaries/ovules

clones.

are cultured on suitable media and polli-

Somatic embryogenesis may be

nated _in vitro_ with cross pollen to obtain

direct or indirect. Indirect somatic embry-

hybrids

(Zenktler,

1980;

Raghavan,

ogenesis involves a callus phase prior to

1990).

embryo production. Direct somatic em-

bryogenesis involves production of em-

_4.10. Root cultures_

bryos from organized tissue without an

Tip portions from primary and

intervening callus phase. Irrespective of

secondary roots of many plants can be

the mode of production, anatomical and

cultured. In 1922, Kotte and Robbins in-

physiological features of somatic embryos

dependently postulated that true _in vitro_

are highly comparable to zygotic embry-

cultures could be raised from meristemat-

os. The morphological and temporal de-

ic cells from root tips and shoot tips.

velopments of somatic embryos are very

Kotte (1922) could cultivate root tips of

similar to that of zygotic embryos. They

pea and maize in nutrient media for long,

both proceed through a series of distinct

but no sub-culturing were done. Robbins

stages, namely, globular, heart, torpedo

(1922) could subculture his maize root

and cotyledon or plantlet stages for dicot-

cultures. White (1934) obtained unlim-

yledons, and globular, elongated, scutellar

ited growth of tomato roots, using the

and coleoptilar stages for monocotyle-

same medium as Robbins (1922), with

dons. These stages typically span a period

yeast extract. Root cultures are useful in:

of several days. In dicots initially small

(i) secondary metabolite production, and

globular embryos form which undergo

(ii) in basic studies on nematode infec-

isodiametric growth and establish bilat-

tions, mycorrhizal associations, and root

eral symmetry. In monocots, especially in

nodulation by _Rhizobium_ bacteria.

grasses, the transition from globular stage

follows a series of events occurring sim-

_4.11. Organogenesis_

ultaneously; such as the development of

Organogenesis is the process of

scutellum, initiation of the coleoptilar

initiation and development of a structure

notch, tissue differentiation with the de-

that shows natural organ form and/or

velopment of embryogenic vascular sys-

function. It is the ability of non-

tem and accumulation of intracellular

meristematic plant tissues to form various

storage substances. Somatic embryogene-

organs _de novo;_ the production of roots,

sis is used for: large-scale clonal propaga-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 6

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ tion of elite cultivars, as an alternative to

Rapid and large-scale clonal (ge-

conventional Micropropagation, produc-

netically uniform) propagation of plants

ing synthetic (artificial) seeds. Indirect

(micropropagation) may allow faster pro-

somatic embryogenesis (via callus) or

duction of plants that are slow to propa-

secondary embryogenesis is used in gene

gate _in vivo_.

transfer. Somatic embryogenesis also of-

The time required for bulking-up

fers potential model for the study of mo-

of new cultivars before they are commer-

lecular, regulatory and morphogenetic

cially introduced can be drastically de-

events in plant embryogenesis.

creased. Storage of germplasm, e.g. _Cry-_

_opreservation._

_4.13. Micropropagation_

__

Micropropagation is the tissue cul-

_4.13.2. The process of micropropagation_

ture method of clonal propagation of

a. A small piece of the plant to be cloned

plants. Plant tissue culture is rapidly be-

(the explant) is removed from a

coming a commercial method for propa-

healthy, well-maintained stock plant

gating difficult-to-propagate plants, new

and surface sterilized (explant varies

cultivars (selections, hybrids, transgenic),

with species, but shoot tips, leaves,

rare/endangered species. Micropropaga-

stem pieces, lateral buds, and young

tion is usually achieved by the release

flowers or floral parts are used).

(from dormancy), and growth of pre-

b. Surface sterilized explants are rinsed

existing (axillary/ lateral) meristems in

with sterile water, and placed aseptical-

the initial culture. This is followed by re-

ly in/ on specially formulated and steri-

peated enhanced formation of axillary

lized medium in culture vessels.

shoots by sub-culture on medium supple-

c. The explant may proliferate directly by

mented with plant growth regulators. The

enhanced lateral branching, or the tis-

shoots produced are rooted either _in vitro_

sue may undergo a certain period of

or _ex vitro_ (out of culture).

unorganized growth (callus) prior to

There are many advantages of Mi-

shoot differentiation.

cropropagation. Shoot production is relia-

d. The growth of the cultures is principal-

ble and consistent. Multiplication rates

ly determined by the plant growth reg-

can be three-fold to eight-fold a month.

ulator (PGR) content of the culture

Plants produced via shoot culture are usu-

medium (the auxin and cytokinin alone

ally true-to-type and uniform. Allows

or in combination and concentration/s).

propagation of rare/ endangered/ hybrid/

Most cultures are established within 4

induced mutant/ genetically transformed

to 12 weeks depending on the species/

plants. There also are few disadvantages.

cultivar.

PGRs do not release apical dominance in

e. A proliferating shoot culture can be

all species. There may be a difference in

sub-cultured to produce divisions

results between juvenile and mature tissue

which will multiply rapidly.

of perennial species; shoot cultures may

f. Rate of multiplication vary and are af-

require a reversion to juvenility. Rooting

fected by many factors. Production of

of the micro-shoots may be difficult. Get-

thousands, and in some cases millions

ting uniform shoot production _in vitro_ ,

of plants a year from a single explant

which is very important in commercial

has been demonstrated

operations, may not be possible in some

****

instances. The procedure is relatively la-

**5. Role of plant tissue culture in sus-**

bor intensive, with high upfront costs to

**tainable agriculture**

get started.

Sustainable agriculture requires

_4.13. 1. Applications of micropropagation_

efficient, biotic and abiotic stress resistant

crop varieties. Germplasm collection and

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 7

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ storage, simple crop improvement meth-jor driver of loss of biodiversity. This puts

ods such as selection and bulking by rapid

in jeopardy the sustainability of agricul-

and large scale cloning by micropropaga-

ture and ecosystem services and their

tion can be of great use. Plant tissue cul-

ability to adapt to changing conditions.

ture techniques such as: anther culture,

This also poses serious threat to food and

dihaploid production, embryo rescue, and

livelihood security.

_in vitro_ pollination and fertilization can be

very useful in developing crop varieties

_6.4. Plant tissue culture methods for con-_

through hybridization. Callus culture, cell

_serving biodiversity_

suspension culture, organogenesis and

Plant Tissue Culture offers novel

somatic embryogenesis are essential for

options for collection, multiplication and

crop improvement through transgenics

medium/ long-term _ex situ_ conservation

(various genetic engineering techniques).

of plant biodiversity. By plant cell, tissue,

****

and organ culture techniques, rapid and

**6. Role of plant tissue culture in con-**

large scale multiplication and season in-

**serving biodiversity**

dependent production of planting material

****

is possible. This has helped in the conser-

_6.1. Biodiversity_

vation of many endangered species. Me-

"Biological diversity means the

dium-term conservation is achieved by

variability among living organisms from

slow growing cultures. Cryopreservation

all sources including, inter alia, terrestrial,

(at −196 °C, in liquid nitrogen) allows the

marine and other aquatic ecosystems and

safe and cost-effective long-term conser-

the ecological complexes of which they

vation.

are part; this includes diversity within

species, between species and of ecosys-

_6.5. In vitro collection_

tems." - Definition of Biodiversity by

Potential advantages of _in vitro_

CBD.

methods are: (i) Less space requirement,

(ii) Pathogen-fee plants, (iii) No need for

_6.2. Importance of biodiversity_

transfer (under storage conditions), (iv)

Biodiversity is essential to: (i) en-

Stored cultures can be used as stock for

sure the production of food, fibre, fuel,

vegetative preservation, and (v) Interna-

fodder, etc., (ii) maintain other ecosystem

tional exchange of plant material made

services, (iii) allow adaptation to chang-

easy because, no use of soil, and no path-

ing conditions - including climate change,

ogens.

and (iv) sustain rural peoples' livelihoods

Basic goals of an _in vitro_ storage

(Convention of Biological Diversity).

system are: to maintain genetic stability,

to keep in indefinite storage without loss

_6.3. Threats to biodiversity_

of viability, and most importantly, to be

Biodiversity is

under

seri-

economical.

ous threat as a result of human activities.

Three types of Plant Tissue Culture sys-

The main dangers worldwide are: (i) In-

tems are available. They are: (i) Normal

vasion by alien species, (ii) Environmen-

growth, (ii) Slow growth, and (iii) Cryo-

tal degradation, (iii) Climate change and

preservation.

global warming, (iv) Urbanization and

__

habitat conversion, (v) Population growth

_6.5.1. Normal growth_

and ever-increasing demand for resources,

Normal Growth is achieved either

(vi) Unsustainable over-exploitation of

on semi solid media or in liquid media.

natural resources.

Normal growth is similar to multiplication

Agriculture contributes signifi-

stage in micro-propagation, and requires

cantly to conservation and sustainable use

frequent sub-culture. Considered as genet-

of biodiversity. However, it is also a ma-

ically stabile when achieved through di-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 8

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ rect organogenesis from apical buds / ax-Cryopreservation is the storage of

illary buds as explants.

living tissues at ultra-low temperatures

(˗196°C). It is useful in conservation of

_6.5.2. Slow growth storage_

plant germplasm of vegetatively propa-

By deliberate slowing down of

gated species, recalcitrant seed species

growth cultures can be stored at least for

(coconut palm etc.), conservation of tissue

6 months and maximum up to 6 years

with specific characteristics, cell lines

without sub-culturing. There are many

producing secondary metabolites, genet-

ways to achieve slow growth. First, by

ically transformed tissues, tissues compe-

manipulating storage temperature (cold

tent to transformation/ mutagenesis, path-

storage at 1-9°C) and light (low light in-

ogen (virus) eradicated tissue for future

tensity). Second, increasing osmotic po-

multiplication (as is done in Banana).

tential of the media [by using osmotically

Cryopreservation procedures are

active compounds such as sucrose (at

available only for limited number of plant

higher ~6%), mannitol etc.]. Third, by

species. Each species/ variety/ tissue type,

addition of inhibitors or retardants, for

needs standardization for: explant size

e.g. mineral oil overlay (callus), reduced

and type, water content, and natural freez-

oxygen tension etc.

ing resistance. Most studies on cryopres-

Plant Growth Retardants are

ervation of plants involve only one or a

chemicals that slow cell division and

few

genotypes.

Only

few

plant

elongation in shoots. They cause plants to

germplasm collections stored in liquid

be shorter and more compact, interrupt

nitrogen currently exist (with a relatively

cell division, stem elongation, and inflo-

limited number of accessions).

rescence / flower formation. But roots

continue to grow. Plant growth retardants

**7. Role of plant tissue culture in pro-**

may reduce the natural Gibberellic acid,

**tecting the environment**

or may produce more ethylene.

Forests are complex ecosystems,

_6.5.3. Cold storage_

predominantly composed of trees and

Storage at non-freezing temps,

shrubs, and usually have closed canopies.

from 1-9° C dependent on species. Stor-

There is nearly 4 billion hectares of forest

age of shoot cultures (stage I or II) works

in the world (this is about 30% of the total

well for strawberries, grapes, may be for

land cover). Depending on the physical,

many more spp. Transferred to fresh me-

geographical, climatic and ecological fac-

dium every 6 months/ annually/ or longer

tors, there are different types of forests

periods basis. Advantages of cold storage

like evergreen forest (mainly composed of

are: (i) simple, (ii) high rates of survival,

evergreen tree species) and deciduous

and (iii) useful in micropropagation (es-

forest (mainly composed of deciduous

pecially in periods of low demand). The

tree species). India"s recorded forest area

disadvantages are: (i) may not be suitable

is 76.52 million hectares. This is 23.28%

for some tropical, subtropical species be-

of the country"s total geographical area.

cause of susceptibility to cold injury, (ii)

Over 90% of the forest area is under gov-

requires refrigeration, which is more ex-

ernment ownership and is managed by the

pensive than storage at ultra-low tempera-

forest departments of the state govern-

tures (in cryopreservation). An alternative

ments (State of Forests Report, 2009).

to cold storage is the use of a medium

Forests are important both economically

with reduced nutrients and lacking su-

and ecologically, and render many ser-

crose (as reported in coffee).

vices to the life support system of earth.

Forests are the primary source of

_6.5.4. Cryopreservation_

wood. Wood is used to fulfil three basic

needs: (i) energy, (ii) construction materi-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 9

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ al, and (iii) industrial raw material. About

due to conversion to agriculture land and

2 billion people in the developing world

urbanization. Deforestation has severe

are dependent on forests for their basic

consequences for the environment and

energy needs (fuel for cooking food). Un-

climate. More than 2000 times the total

til recently wood has been the chief con-

energy consumption of the world popula-

struction material. High strength to

tion, of solar energy reaches the earth"s

weight ratio and availability in many

surface. Because of deforestation, not on-

kinds, ease of cutting and shaping with

ly this natural source of energy is wasted,

simple tools, and insulation to heat, make

but also has a serious negative impact on

wood an ideal construction material for

the environment, by way of surface heat-

many purposes. Major industrial uses of

ing, and desertification.

wood are: (i) paper and pulp, (ii) rayon,

In the tropical and sub-tropical re-

and (iii) plywood. Besides wood, forests

gions of the world, receiving about 600

are a source of a variety of non-wood for-

mm rainfall and above, plantation forestry

est products (NWFPs). Thus forests con-

of economically important tree species

tribute greatly to the economy. Around

(say teak for timber and eucalypts for

1.6 billion people depend on forests for

pulp) can take away pressure for forestry

their livelihood. This includes some 70

resources from natural forests and can add

million indigenous people.

to the forest cover. For this, large num-

Forests play an important role in

bers (in millions) of plating material of

maintaining ecological balance. Forests

superior varieties (fast growing, better

are atmospheric filters. They are the ma-

adapted, disease and insect-pest resistant

jor suppliers of oxygen. In photosynthesis

etc.) are necessary. But forest tree species

they fix atmospheric carbon dioxide into

are difficult to breed, because of their

carbohydrates, sugars, proteins, and many

long generation cycles, highly heterozy-

forms of biomass, thus playing a signifi-

gous natural populations, openly cross

cant part in the global carbon cycle. For-

pollinated nature, and lack of knowledge

ests contribute large quantities of mois-

about their genetics.

ture to the atmosphere, thus regulating

Clonal propagation of "superior"

climate. Forests also conserve soil and

genotypes (identified for desirable traits)

water resources.

through tissue culture has been used very

The term forest implies "natural

profitably in case of many tree species.

vegetation" of the area, existing from

Eucalypts have been multiplied and used

thousands of years and supporting a varie-

for plantation forestry for a long time

ty of biodiversity. More than half of the

(FAO Report, 1981). Eucalyptus wood

known terrestrial plant and animal species

from plantation forestry has been used as

live in forests (Millennium Ecosystem

timber, industrial raw material and fuel.

Assessment, 2005). The forest ecosystem

Plantation forestry using eucalypts may

has two components - biotic and abiotic.

not be suitable for some places because of

The living component includes plants

high water demand. Teak ( _Tectona gran-_

(trees, shrubs, herbs etc.), animals and

_dis_ Linn. f.) is another success story. In

microorganisms. Forests are home to

early 1980s, scientists from CSIR-

more than 80 per cent of all terrestrial

National Chemical Laboratory, Pune for

species of animals, plants and insects.

the first time regenerated complete plant-

Globally, deforestation is the major cause

lets from an 80 year old "elite" tree (Gupta

of loss of biological diversity, and is a

_et al_., 1983). Poplars ( _Populus_ _spp_.) are

matter of great concern (Laurance, 2007).

another tree species which clonally prop-

Worldwide, the area of natural

agated through plant tissue culture tech-

forests decreases by some 13 million ha

niques, and widely cultivated in planta-

annually (this is about 3% of the total for-

tion forestry in many parts of the world.

est area). This loss of forest area is mostly

In India, poplars ( _Populus_ _spp_.) are the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 10

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ most popular tree species in agro-forestry

**Steffen,** **W. and Shyamsundar, P.**

production systems. Poplars are usually

**(2014).** An integrated framework for

intercropped with agricultural crops like

sustainable development goals. _Ecol-_

wheat, rice and sugar cane. Poplars are

_ogy and Society_ **19, 49.**

well known for their fast growth, out-

**Guha, S. and Maheshwari, P. (1964).** _In_

standing properties and quick and high

_vitro_ production of embryos from an-

financial returns. Timber from poplars

thers of _Datura_. _Nature_ **204, 497-98.**

often forms the backbone of match, paper,

**Gupta, P. K., Mehta, U. and Masca-**

sports goods, plywood, and composite

**renhas, A. F. (1983).** A tissue culture

board industries.

method for rapid multiplication of

_Eucalyptus camaldulensis_. _Plant Cell_

**References**

_Rep._ **2, 296-99.**

****

**Hartley, M. J. (2002).** Rationale and

**Barnosky, A. D., Hadly, E. A., Bas-**

methods for conserving biodiversity

**compte, J., Berlow, E. L., Brown, J.**

in plantation forests. _Forest Ecology_

**H., Fortelius, M., Getz, F. M.,**

_and Management_ **155, 81–95.**

**Harte, J., Hastings, A., Marquet, P.**

**John, C. K., Nadgauda, R. S. and Mas-**

**A., Martinez, M. D., Mooers, A.,**

**carenhas, A. F. (1997).** Tissue Cul-

**Roopnarine, P., Vermeij, G., Wil-**

ture of Economic Plants, Center for

**liams, J. W., Gillespie, R., Kitzes,**

S&T of Non-Aligned and other De-

**J., Marshall, C., Matzke, N., Min-**

veloping Countries, New Delhi, and

**dell, D. P., Revilla, E. and Smith, A.**

Commonwealth Science Council,

**B. (2012).** Approaching a state shift

London. **pp. 3-34.**

in Earth"s biosphere. _Nature_ **486, 52-**

**Kanta, K., Rangaswamy, N.S. and Ma-**

**58.**

**heshwari, P. (1962).** Test tube ferti-

**Cocking E. C. (1960).** A method for the

lization in a flowering plant. _Nature_

isolation of plant protoplasts and

**194, 1214-1217.**

vacuoles. _Nature_ **187, 962-63.**

**Kao, K. N. and Michayluk, M. R.**

**FAO (1999).** State of world"s forests

**(1974).** A method for high frequency

1999, United Nations Food and Agri-

intergeneric fusion of plant proto-

cultural Organization, Rome, **154 pp.**

plasts. _Planta_ **115, 355-67.**

**FAO (2007).** State of world"s forests

**Kotte, W. (1922).** Kulturversuchemitiso-

2007,

ftpfao.org/docrep/fao/009.

liertenWurzelspitzen.

_Beitr._

_Z._

United Nations Food and Agricultur-

_Allgem. Bot_. **2, 413-434.**

al Organization, Rome.

**Klercker, I. A. F. (1892).** Eine method

**Gamborg, O. L., Shyluk, J. and Kar-**

zuIsolierunglebenderProtoplasten.

**tha, K. K. (1975).** Factors affecting

_Oefvers K. Vetensk. Akad.Foerh._ **9,**

the isolation and callus formation in

**463-471.**

protoplasts from shoot apices of _Pi-_

**Laurance, W. F. (2007).** Have we over-

_sum sativum_ L. _Plant Sci. Lett._ **4,**

stated the tropical biodiversity crisis?

**285-92.**

_Trends Eco. Evol_. **22, 65-70.**

**Griggs,**

**D.,**

**Stafford-Smith,**

**M.,**

**Lenton, T. M. (2011).** Beyond 2°C: rede-

**Gaffney, O., Rockström, J., Öh-**

fining dangerous climate change for

**man, M. C., Shyamsundar, P.,**

physical systems. _Wiley Interdiscipli-_

**Steffen, W., Glaser, G., Kanie, N.**

_nary Reviews: Climate Change_ **2(3),**

**and Noble, I. (2013).** Sustainable

**451-461.**

development goals for people and

**Limasset, P. and Cornuet, P. (1949).**

planet. _Nature_ **495, 305-7.**

Recherche du virus de la mosaique

**Griggs,** **D., Stafford Smith,** **M.,Rock-**

du Tabacdans les meristemes des

**ström,** **J., Öhman, ****M. C., Gaffney, **

plantesinfectes. _C. R. Ac. Sc._ **228,**

**O.,Gisbert Glaser, ****G.,Noble, ****I.,**

**1971-72.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 11

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ **Melchchers, G. and Labib, G. (1974).**

lated plant protoplasts. _Nature_ **225,**

Somatic hybridization in plants by

**1016-17.**

fusion of protoplasts - I. Selection of

**Reid, W. V. and Miller, K. R. (1989).** __

light resistant hybrids of a haploid

_Keeping options alive: the scientific_

light sensitive varieties of tobacco.

_basis for conserving biodiversity._

_Mol. Gen. Genet._ **135, 277-94.**

World Resources Institute, Washing-

**Millenium**

**Ecosystem**

**Assessment**

ton, DC. **128 p.**

**(2005).** Ecosystem and human well-

**State of Forests Report (2009).** Ministry

being: current state and trends. Find-

of environment and forests, Govern-

ings of the conditions and trends

ment of India, New Delhi.

working group, In: Hassan, R.,

**Raghavan, V. (1980).** Embryo culture,

Scholes, R. and Ash, N. (eds.) Mille-

In: Vasil, I. K. Ed. _Perspectives in_

nium ecosystem assessment series.

_plant cell and tissue culture_ , _Int. Rev._

Island Press, Washington, U.S.A.

_Cytology_ Suppl. 11B, Academic

**Morel, G. (1950).** Sur la culture des tis-

Press, New York, USA, **pp. 209-240.**

sue de deux Monocotyledones. _C. R._

**Robbins, W. J. (1922).** Effect of auto-

_Ac. Sc._ **230, 1099-1101.**

lyzed yeast and peptone on the

**Morel, G. (1960).** Producing virus free

growth of excised corn root tips in

cymbidium. _Bulletin of American Or-_

the dark. _Bot. Gaz._ **74, 59-79.**

_chid Society_ **29, 495-497.**

**Rockström, J.** _et al_. (2009). A safe oper-

**Morel,**

**G.**

**(1964).**

Action

d

ating space for humanity. _Nature_ **461,**

l"acidepantotheninesur la croissance

**472–475.**

des tissues d"Aubepinecultives _in_

**Sharma, D. R., Chowdhury, J. B., Ahu-**

_vitro_. _C. R. Ac. Sc._ **243, 166-168.**

**ja, U., and Dhankhar, B. S. (1980).**

**Morel, G. and Martin, C. (1950).** Gue-

Interspecific hybridization in genus

rison

de

pommes

de

terreat-

Solanum. A cross between _S._

teintesd"unemaladie a virus. _C. R. Ac._

_melongena_ and _S. khasianum_ through

_Sc._ **235, 1324-25.**

embryo culture. Zeitschrift fur Pflan-

**Morel, G. and Martin, C. (1955).** Gue-

zenzuchtung, **85(3), 248-253.**

rison de pommes de terreatteintes de

**Shindell _,_** **D. Kuylenstierna, J. C. I.,**

maladies a virus. _C. R. Ac. Agri._ **41,**

**Vignati,**

**E.,**

**van**

**Dingenen,**

**470-75.**

**R., Amann, M., Klimont, Z., Anen-**

**Murashige, T. (1974).** Plant propagation

**berg, S. C., Muller, N., Janssens-**

through tissue cultures. _Ann. Rev._

**Maenhout, G.,Raes, F., Schwartz,**

_Plant Physiol._ , **24, 135-65.**

**J., Faluvegi, G., Pozzoli, L., Ku-**

**Murashige, T. And Skoog, F. (1963).** A

**piainen,**

**K.,**

**Höglund-Isaksson,**

revised medium for rapid growth and

**L., Emberson,**

**L.,**

**Streets,**

bioasays with tobacco tissue cultures.

**D., Ramanathan, V., Hicks, K.,**

_Physiol Plant._ **15, 473-497.**

**Kim**

**Oanh,**

**N.**

**T., Milly,**

**Nitsch, C. (1974a).** La culture de pollen

**G., Williams, M., Demkine, V. and**

isolesurmihensynthetique. _C. R. Ac._

**Fowler, D. (2012).** Simultaneously

_Sc._ **278, 1031-34.**

mitigating near-term climate change

**Nitsch, C. (1974b).** Pollen culture – a

and improving human health and

new technique for mass production of

food security. _Science_ **335, 183–189.**

haplid and homozygous plants. In:

**UNEP (1994).** Convention on biological

Kasha, K. J. Ed. _Haploids in Higher_

diversity, UNEP/CBD, Switzerland,

_Plants – Advances and Potential_.

November 1994, **34 p.**

University of Guelph, Guelph, Swe-

**United Nations (2005).** _2005 world_

den. **pp. 123-35.**

_summit outcome [adoption resolu-_

**Power, J. B., Cummins, S. E., and**

_tion, 60th session]_.United Nations,

**Cocking, E. C. (1990).** Fusion of iso-

New York, New York, USA. [online]

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 12

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability John_ URL: http://www.who.int/hiv/univers

**SDSN) (2013).** _An action agenda for_

alaccess2010/worldsummit.pdf

_sustainable development_. Report for

**United Nations (2012).** _The future we_

the UN Secretary-General. Sustaina-

_want: outcome document adopted at_

ble Development Solutions Network,

_Rio+20_. United Nations, New York,

New York, New York, USA. [online]

New

York,

USA.

[online]

URL: http://unsdsn.org/resources/pub

URL: http://www.un.org/futurewewa

lications/an-action-agenda-for-

nt

sustainable-development/

**United Nations (2012 _a_** **).** _The millennium_

**United Nations Environment Pro-**

_development goals report 2012_. Unit-

**gramme (UNEP)** **(2013).** _Embedding_

ed Nations, New York, New York,

_the environment in sustainable devel-_

USA.

[online]

_opment goals._ UNEP Post-2015 Dis-

URL: http://www.un.org/millennium

cussion Paper 1.UNEP, Nairobi,

goals/pdf/MDG%20Report%202012.

Kenya.

[online]

pdf

URL: http://www.unep.org/pdf/embe

**United Nations (2013).** _A new global_

dding-environments-in-SDGs-v2.pdf

_partnership: eradicate poverty and_

**Wallin, A., Glimelius, K. G. and Eriks-**

_transform economies through sus-_

**son, T. (1974).** The induction of ag-

_tainable development_. The report of

gregation and fusion of _Daucus caro-_

the High-Level Panel of eminent per-

_ta_ protoplasts by polyethylene glycol.

sons on the post-2015 development

_Zeitschrift fur Planzenphysiol._ **74,**

agenda.United Nations, New York,

**64-80**

New

York,

**White, P. R. (1933).** Plant tissue culture:

USA.  http://www.post2015hlp.org/w

Results of preliminary experiments

p-content/uploads/2013/05/UN-

on the culturing of isolated stem tips

Report.pdf

of _Stellaria media_. _Protoplasma_ **19,**

**United Nations Open Working Group**

**97-116.**

**(UN OWG) (2013).** _Interim progress_

**White, P. R. (1934).** Multiplication of the

_report to UN General Assembly_.

viruses of tobacco and ancuba mosaic

United Nations, New York, New

in growing excised tomato roots.

York,

USA.

[online]

_Phytopathol_. **24, 1003-11.**

URL: http://sustainabledevelopment.

**White, P. R. (1933).** Potentially unlim-

un.org/content/documents/1927interi

ited growth of excised tomato root tip

mreport.pdf

in a liquid medium. _Plant Physiol_ , **9,**

**United Nations Open Working Group**

**585-600.**

**(UN OWG) (2014).** _Outcome docu-_

**Zenktler, O. M. (1980).** Intra-ovarian

_ment - Open Working Group on Sus-_

and _in vitro_ pollination. _In_ : Vasil, I.

_tainable Development Goals (19th_

K. Ed. _Perspectives in plant cell and_

_July 2014)_.United Nations, New

_tissue culture_ , _Int. Rev. Cytology_

York, New York, USA. [online]

Suppl. 11B, Academic Press, New

URL: http://sustainabledevelopment.

York, USA, **pp. 137-156.**

un.org/content/documents/1579SDGs

**Zimmerman, U. and Scheurich, P.**

%20Proposal.pdf

**(1981).** High frequency fusion of

**United Nations Sustainable Develop-**

plant protoplasts by electric fields.

**ment**

**Solutions**

**Network**

**(UN**

_Planta_ **151, 26-32.**

© 2017 by the author. Licensee, Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 13

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P14-40_

**Traditional Medicine of the Tribes in Tamil Nadu and Its**

**Sustainable Use through Biotechnology**

****

**Valli Gurusamy1, Kavitha Valampuri John2, Usha Raja Nanthini Ayyakkanu2,**

**Ramani Bai Ravichandran3,***

****

_**1** Vice Chancellor, Mother Teresa Women's University; **2** Department of Biotechnology, _

_Mother Teresa Women's University; **3** Department of Zoology, University of Madras, India; _

_*Correspondence: rramani8@hotmail.com; Tel:_ _+91 9444020828_

****

****

**Abstract:** India is a land of mega biodiversity representing about 7% of the world"s flora

and 6.5 per cent of world"s fauna. Tamil Nadu, one of the southern most states of India, is

rich in forest cover and cultural diversity. Genomic evidence supports the peopling of Tamil

Nadu from the 1st wave of migration of humans from the "Out of Africa" exodus and points

out that the tribes of state were among the earliest settlers in the region. The tribal popula-

tion of Tamil Nadu represents 1.02% of the total population of the state. Living in close as-

sociation with the forest, they have accumulated a treasure trove of ethno botanical

knowledge in the form of traditional medicine. The future of sustainable use of renewable

forest product lies with the molecular tools of Biotechnology. We present here an analysis

of the documented literature of the medicinal plants used by the tribes of Tamil Nadu for

treatment of common disorders. We also present the challenges and prospects within the

scope of Biotechnology to ensure sustainable use of traditional medicine for the betterment

of mankind and environment. ****

_**Keywords**_ **:** Biotechnology; sustainable; tribes; traditional medicine; Tamil Nadu.

**1. Introduction**

along with their 1st wave of migration out

of Africa. Traditional Medicine is the sum

India is a land of enormous cultur-

total of long-standing information on the

al, linguistic and religious diversity pre-

knowledge, skills, and health practices

sumably because of Man"s long stay, for

based on the theories, beliefs, and experi-

the past 50-70,000 years in this continent.

ences indigenous to different cultures or

This is an outcome of various migrations

local communities. Traditional medicine

that took place into India, serving as a

incorporates plant, animal and mineral

major corridor for the dispersal of modern

based medicines and encompasses spir-

humans out of Africa (Cann, 2001). Ar-

itual therapies, manual techniques and

chaeological evidences indicated that the

exercises which can be applied singularly

Indian subcontinent was peopled by vari-

or in combination for the maintenance of

ous migrations since Palaeolithic (300-

health through the prevention, diagnosis,

400,000 BCE), starting with the Late

improvement or treatment of physical and

Pleistocene (Misra, 2001). "The Castes

mental illness. Traditional knowledge has

and Tribes of Southern India" was an at-

been well preserved and orally passed

tempt to catalogue these populations

from one generation to the next in the

(Thurston, 1909).The knowledge of the

form of stories, legends, folklore, rituals,

medicinal value of plants, animals and

songs, art, and even laws. Since there is

other substances and their uses goes back

no written script the exchange of know-

to the time of the earliest settlers probably

how between diverse communities is a

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

process of evolution through trial and er-

cient lineage from the 1st wave of the "out

ror which makes documentation and rec-

of Africa" exodus and thus in vast ethno-

ord-keeping almost impossible. This pro-

botanical knowledge. According to the

cess of exchange and assimilation is con-

2011 census, 104 million tribal people

tinuous, and today there is a growing

speaking over 227 linguistic groups in-

awareness among the medical community

habit varied geographic and climatic

about the intrinsic value of traditional

zones of the Indian subcontinent. Ethno-

medicine, and as a result in India Ayurve-

medicine includes plants, animal products

da, Unani and Siddha have entered the

and minerals used by tribal communities

mainstream to compliment biomedicine.

of a particular region or country for me-

Contemporary Indian society faces the

dicinal purposes other than those men-

challenge of integrating the best of the

tioned in classical streams of the respec-

different healing traditions to provide a

tive cultures. Tribal people have been us-

holistic health care.

ing a large number of wild plants as doc-

umented by ethnobotanical investigations.

**2. Traditional knowledge**

The application of most of the plants rec-

orded are either lesser known or hitherto

Even before classical medical

unknown to the outside world. India has

knowledge of ancient India was codified

been the country most concerned about

into the canonical texts of Ayurveda in

the conservation of its medicinal plants.

the 6th century BC, there were abundant

There are over 45,000 species of vascular

sources of medical knowhow in the sub-

plants reported from India of which as

continent from prehistoric times. Tradi-

many as 15,000 may be used medicinally.

tional healers can be either folk or tribal

The folk medicine system of India use

healers and have worked in intimate rela-

about 5,000 plant species with about

tion with their environment. Traditional

25,000 formulation, whereas the tribal

healing ranges from simple home reme-

medicine involves the use of over 8,000

dies related to nutrition and treatment for

plant species with about 1,75,000 specific

minor illnesses, to more sophisticated

preparations (Pushpangadan and George,

procedures such as midwifery, bone set-

2010). More than 90% of the raw material

ting, blood-letting (therapeutic phleboto-

for traditional medicine comes from wild

my) and treatment of snake bites and

harvesting as this the common method

mental disorders. Some healing practices

used for collecting them (Tandon, 1996;

were considered to be sacred and were

Gupta 1998; Ved _et al_., 1998). About 71

associated with rituals that helped safe-

medicinal plant species are classified as

guard them for there is a substantial over-

"rare", and of this 92% are in active trade,

lap between healing plants and sacred

and 74% are traded nationally. It has been

plants. Categories of traditional healers

estimated that between 4,000 and 10,000

are traditionally trained healers, old indi-

medicinal plant species in India face ex-

viduals of the community, educated indi-

tinction in the local, regional and national

viduals acquiring certain knowledge from

levels (Hamilton, 2004). In an effort to

their predecessors, ancient inscriptions in

create leadership in affordable and holis-

the form of copper plate/palm leaf writ-

tic health care, India is committed to

ings, old and recent publications in re-

promoting traditional medicines like

gional language.

Ayurveda which remained untapped due

to inadequate scientific scrutiny. Steps

**3. Indian ethnobotany**

are being taken to bring in regulatory

amendments in research and effective en-

India is one of the richest coun-

forcement for integration of quality prod-

tries in the world not only in biodiversity

ucts, practices and practitioners into the

but also in different ethnic groups of an-

AYUSH (Ayurveda, Yoga and Naturopa-

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

thy, Unani, Siddha and Homoeopathy)

in moderate altitude of the district name-

system at central and state level.

ly: New Kotagiri (Aggal), Kil-Kotagiri,

Kundah, Kallimalai, Gudalur, Trichigadi

**4. Traditional knowledge of the tribes**

and Sholur Kokal.Their settlement is

**of Tamil Nadu**

called the "Kokkal" with linear row of

****

houses in streets, "Keri". Each village has

According to the 2001 census,

three Keri known as Kizhkeri, Nadukeri

tribal population in Tamil Nadu is 6,

and Melkeri. Keri, clan, exogamy is note-

51,321 which constitute 1.02% of the to-

worthy among Kotas (Kavitha V.J.,

tal population. There are 36 tribes and sub

2008). Their chief diety is Kambattrayan.

tribes in Tamil Nadu. Out of the 36

Their population was 1,894 in 2001 cen-

Scheduled Tribe communities in the

sus.

state, about six tribal populations Todas,

_Irulas:_ They are also called as Iu-

Kotas, Krumbas, Paniyas, Irulas, Kat-

van,Villiar. The Irulas are distributed in

tunayakas have been classified as primi-

the lower altitudes of the Nilgiri hills (dis-

tive tribes with incredibly high anthropo-

trict). They are negrotoid in appearance

logical significance. The primitive tribes

whose chief occupation is as plantation

occupy the length and breadth of the Nil-

labourers in the estates. Their settlements

giri district in the Western Ghats of Tamil

are called "Aral". Their dialect is Tamil

Nadu. One sixth of the land mass of Tam-

mixed with Malayalam. Their community

il Nadu is covered by forests. The tribes

is divided into seven exogamous clans

of Tamil Nadu live in and around the re-

(Kuems): Kupper, Sambe, Kalkatti, Ku-

served forests and have gained immense

runagar, Devanan, Peradar and Punger

knowledge on the use of forest produce to

(Rajan and Sethuraman, 1991). They are

treat common disorders.

basically hunter gatherers. Their popula-

_Todas:_ They are called by other

tion was 6,700 in 2001 census.

names like Tudas, Thuduvans, and Todar

_Kurumbas:_ The Kurumbas prac-

(Kavitha V.J., 2008). They are profes-

tice hunting food gathering economy,

sional pastoralists and dairy men, a purely

well-versed in honey collection tech-

pastoral economy in India today, living in

niques. They are plain dwelling people

the higher altitudes in the traditional

living in the interior forests of the district.

houses called "Munds" that are half barrel

Their staple foods are wild tubers (Di-

shaped and are vegetarians. Their dialect

oscorea bulbosa), wild fruits and other

is independent form of Dravidian Tamil-

minor forests produces. Their settlements

Malayalam. They are fair skinned and

are called "Mottam". They are dark

wear ornaments and their dress is akin to

skinned and speak the Kurumba dialect.

the Roman "toga". They have two exoga-

Kurumbas are a heterogenous population

mous divisions called Tarthar and Teivali.

having divisions such as Halu Kurumbas,

There are five socially distinguishable

Betta Kurumbas, Mullu Kurumbas, Jess

sects (clans) such as Pelki, Pekkan,

kurumbas and Urali Kurumbas. Their

Kuttan, Kenna and Jodi (Rajan and Sethu-

population was 6,872 in the 2001 census.

raman, 1993). Their population was 1,600

_Paniyas:_ The Paniyas are negro-

in the 2001 census.

toid people living in bamboo huts at the

_Kotas:_ Their other names are Ko-

junction of Kerala and Tamil Nadu bor-

ter, Kothewars, and Kohatur. The Kotas

der. They work as labourers with Waya-

are musicians and excellent craftsmen

nad Chettis though they were basically

having mastery over ironworking. They

hunter gatherers. Their settlements are

are light skinned, with copper hair. They

called "Paddi". They possess excellent

speak the "Kota" a Dravidain language.

skills in the art of fishing by employing

Their distribution in the Nilgiri district is

certain plant parts like bark of Eugenia sp.

confined only to seven villages inhabiting

and leaves of Aibizzia sp. as stupefying

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

agents. They speak the Paniya dialect,

dicinal knowledge of the tribes of Tamil

practice Illam, patilineage, and their in-

Nadu.

heritance is by "Marumakkathayam".

They numbered 6,700 in 2001 census.

**5. Ethnomedical wealth of tribes of**

_Kattunayakas:_ They are also

**Tamil Nadu**

called as Shola Nayakans, Jenu or Teen

Kurumbas. They are another group of for-

A total of 229 medicinal plants

est dwellers who are nomadic in nature,

used by the tribes of Tamil Nadu belong-

their staple foods are honey, wild fruits

ing to 79 families for the treatment of

and tubers. Their settlements are called

more than 40 disorders were documented.

"Paadi". They are short and black with

The percent representation of the families

protruding forehead. They have curly hair

of plants used as medicine by the tribes of

and speak Kannada language. Eating bi-

Tamil Nadu is represented in Figure 1.

son flesh is a cultural taboo with them.

Euphorbiaceae is the largest family repre-

The social customs and religious practices

sented by 18 species at 12% followed by

of Kattunayakas are akin to Kurumbas in

Fabaceae by 14 species (9%), Lamaceae

many respects. They population was

by 13 species (8%), Asteraceae by 11

1,425 in the 2001 census.

species (7%), Solaneaceae and Rutaceae

_Paliyars:_ They are found in the

by 10 species each (6%) and Ascelpiada-

hilly regions of Madurai, Dindigul, Theni,

cea by 8 species(5%). This data marks a

Tirunelveli and Virudhunagar districts. It

direction for scientific researchers as to

is believed that Paliyars are indigenous

which of the families to search for to

people of Palani hills of Kodaikanal and

identify bio active compounds.

speak Tamil. Physically they are similar

Of the primary parts of the plant

to the Semong of Malaya and other Indian

used the leaves formed 40% of usage in

tribal communities. They can be grouped

traditional medicine followed by the root

into three categories based on their life

and bark at 11%, whole plant and fruit at

styles, namely, nomadic, semi nomadic

8%, stem at 7% and seed at 5% (Figure

and settled. Nomadic Paliyars don't build

2). It is of utmost importance to see this

houses; they live temporarily in rock

data in the light of the major families rep-

caves called 'Pudai'.

resented for medicinal use by the tribes of

Ethnobotanical

traditional

Tamil Nadu. It is also important to test

knowledge for the tribes of Tamil Nadu

these bioactive compounds from different

was retrieved using Pubmed and Google

parts of the plant as a combination and to

using the keywords ethnomedicne, tribes,

use bioactive compounds different fami-

Tamil Nadu. The ethnobotanical data pre-

lies in conjunction for any particular dis-

sented here included knowledge form

order.

seven tribal groups of Irula, Pani-

The data when analysed for major

ya,Kurumba, Kota, Thoda, Kattunayak-

remedies against diseases it revealed that

kans and Paliyars (Table 1). These pub-

the tribes of Tamil Nadu had traditional

lished research articles were then ana-

remedies for wounds (31) and skin prob-

lysed manually to ascertain the traditional

lems (29), followed by stomach aches

knowledge of these communities related

(17), diahorrea and headache (13), Cold,

to the study area and the usage pattern of

cough, fever (12) and rheumatic diseases,

the medicinal plant species. The data was

gastric disorders and toothache (9). It is

further analysed using graphical represen-

interesting to note that the ethnomedicine

tations for summarizing and interpreting

of the tribals has 9 remedies for women to

for the major families of plant species

ease labour pain and 3 to induce lactation

represented, part of the plant used and

(Figure 3). Tribal ethnomedicine also has

cure for disorder from the traditional me-

remedies for diabetes and jaundice (5).

****

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

Anacardiaceae Aplaceae

Boraginaceae

Menispermaceae Myrataceae

2%

2%

2% Meliaceae

2%

2%

2% Mimosaceae

2% Pandanaceae

Euphorbiaceae

2%

12%

Piperaceae

2%

Combretaceae

Fabaceae

3%

9%

Convolvulaceae

3%

Malvaceae

Lamaceae

3%

8%

Zingiberaceae

3%

Sapindaceae

Asteraceae

3%

7%

Verbenaceae

3%

Amaranthaceae

4%

Solanaceae

Caesalpiniacea

6%

Rutaceae

4%

Asclepiadaceae

Acanthaceae

6%

5%

4%

****

****

**Figure 1:** Percent representation of major plant families used by Tamil Nadu tribes as medi-

cine.

****

Branches

Heartwood

Nuts Res in

Fruits Flowers

Rhizome

0%

0%

0%

0% 1%

1%

2%

Tuber

2% Latex

3%

Seed

5%

Stem

Leaves

7%

40%

Fruit

8%

Whole Plant

8%

Bark

Root

11%

11%

****

****

**Figure 2:** Parts of the Plant used in traditional healing among the tribes of Tamil Nadu. ****

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_Biotech Sustainability (2017)_

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**Table 1:** Ethnomedical traditional knowledge of the tribes in Tamil Nadu ****

**No.**

**Botanical Name**

**Local name**

## Part Used

### **Medicinal uses**

**Tribe Reference**

**Acanthaceae**

****

****

****

****

****

1

_Adhatoda zeylanica_ Medicus __

Adathodai

Leaves

Asthma, Cold, fever

PL

e

_Adhatoda zeylanica_ Medicus __

Auduthoda

Root, bark,

Cough, asthma ,eye pain

IR

g

flowers

2

_Andrographis lineata_ Wallich ex

Siriyanangai

Leaves

Cough, diabetes, scorpion and

PL

e

Nees __

snake bite

3

_Andrographis paniculata_ (Burm.f.)

Periyanangai or

Leaves

Scorpion sting

PL

e

Wall. ex Nees __

Nilavembu

and snakebites, menorrhagia

4

_Asystasia gangetica_

Valukai keerai

Leaves

Appetite

PL

e

5

_Blepharis maderaspatensis_ (L.) Roth. __

Vettukaaya pachilai Leaves

Fractured bones, Cuts

PL

e

6

_Phlebophyllus kunthianum_ Nees __

Kurinji chedi

Leaves

Nervous disorder

PL

e

**Alangiaceae**

****

****

****

****

****

7

_Alangium salvifolium_ (L.f.) Wangerin __

Alinji

Fruit

Eye infections

PL

e

**Amaranthaceae**

****

****

****

****

****

8

_Achyaranthes aspera_ L. __

Uthrunk

Leaves

Cuts, wounds and sores

KO

b

_Achyranthes aspea_ L. __

Cherukadalai

Whole Plant Sprain ached in the Joints

KT

b

_Achyranthes aspera_ L __

Nayuruvi

Leaves

Diverticulosis & Diverticulitis

KU

i

_Achyranthes aspera_ L. __

Nayurvi Geeda

Whole Plant Ease child birth and labour

KU

b

pain

_Achyranthes aspera_ L. __

leaves

New born babies, lactation

IR

g

9

_Achyranthes bidentata_

Blume, Kithoop

Leaves

Rapid healing of wounds

KU

i

10

_Achyranthus bidentata_ Blume __

Naiyur

Leaves

Skin disorders including sca-

KO

a

bies

11

_Aerva lanata_ (L.) Juss.ex Schult __

Kannupila & Pan-

leaves

New born babies, lactation

IR

g

_Celosia argentea_ L. __

naipoo

12

_Alternanthera sessilis_ (L.) __

Nilakirai

Leaves

Diarrhoea

KU

i

_Alternanthera sessilis_ (L.) __

Nilakirai

Leaves

Roughage

KU

i

13

_Amaranthus gangeticus_ L. __

Mulai keerai

Whole plant Good digestion,Constipation,

KU

i

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_Table 1: Continued..._

****

****

****

****

****

**Anacardiaceae**

****

****

****

****

****

14

_Anacardium occidentale_

Bark, Fruit

Fever, warts

IR,

c

latex

PA

15

_Mangifera indica_

Maavae pattae

Bark

Stomach pain

IR,

c

PA

16

_Spondias pinnata_

Kattu Maa

Bark

Acute diarrhoea

IR,

c

PA

**Annonaceae**

****

****

****

****

****

17

_Annona squanmosa_ L _._

Seetha mara

Seeds

Vermifuge

PA

b

**Apiaceae**

****

****

****

****

****

18

_Centella asiatica_ (L) Urban __

Vallarai

Whole Plant Refrigerant

TH

b

_Centella asiatica_ (L) Urban __

Gottala

Whole Plant Toothache

KT

b

_Centella asiatica_ (Linn.) Urban. __

Kidth Kot

Leaves

Stomach problems Cools the

KO

a

body

19

_Coriandrum sativum_ Linn. __

Kothumull

Leaves

Refrigerant and diuretic

KO

a

**Aplaceae**

****

****

****

****

****

20

_Buplerum wightii_ P.K. Mukherjee, __

Malai seragam

Root

Easy delivery

IR-S

d

21

_Centella asiatica_ (L) Urban, __

Kutheraikokku

Leaves

Digestive agent, blood circula-

IR-S

d

tion

22

_Heracleum ceylanicum_ Gardner ex C.B.

Poonaikal sedi

Leaves

Insect allergy

IR-S

d

Clarke __

**Apocymeeae**

****

****

****

****

****

23

_Rauvolfia serpentine_ (L.) Benth. ex Kurz __ Chivanamelpodi

Root

Stomachache

KT

f

24

_Alstonia scholaris_ (L.) R.Br. __

Paalooram pattai

Stem

Lactation

PL

e

**Araceae**

****

****

****

****

****

25

_Acorus calamus_ L. __

Vasambu

Rhizome

Speech

PL

e

26

_Colocasia esculenta_ (L.) Schott __

Kattu shembu

Tuber

Worms

KT

f

_Colocasia esculenta_ (L.) Schott. __

Chembu

Leaves &

Small red colour boils appear-

KU

h

Rhizome

ing on the skin

**Arecaceae**

****

****

****

****

****

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_Table 1: Continued..._

27

_Phoenix sylvestris_ (L.) Roxb. __

Injai

Stem

Easy Delivery

IR

g

**Aristolochiaceae**

****

****

****

****

****

28

_Aristolochia indica_ L. __

Perumanthikodi

Root

Fever

IR

g

29

_Aristolochia tagala_ Cham. __

Modhalaikodi.

Leaves

Diarrhea and vomiting

IR-S

d

**Asclepiadaceae**

****

****

****

****

****

30

_Ceropegia candelabrum_ L. __

Perun kodi

Leaves

Headache

PL

e

31

_Cryptolepis buchananii_ Roem &

Paalkodi/Karunkodi Latex

Wound

PL

e

Schul __

32

_Gymnema hirsutum_ W&A __

Sakarasedi

Leaves

Diabetes

KU

i

33

_Gymnema sylvestre_ (Retz.) R. Br. Ex __

Sirukurinjan

Leaves

Diabetes and nervous disorder

PL

e

_Gymnema sylvestre_ R.Br. __

Sirukurinjan & ha-

Leaves

Diabetes

IR

g

karikolli

34

_Hemidesmus indicus_ H.f. __

Nannari

Whole

Fever, menorrhagia, stomach-

PL

e

Plant, Root, ache

leaves

35

_Holostemma ada-kodien_ Schult. __

Ada kizhangu

Tuber

Fever

KT

f

36

_Pergularia daemia_ (Fors.) Chio __

Veli parutthi

Leaves

Headache

PL

e

37

_Tylophora indica_ (Burm. f.) Merr. __

Nangilai

Leaves,

Snakebite

PL

e

Root

**Asparagaceae**

****

****

****

****

****

38

_Asparagus racemosus_ Willd. __

Ammaikodi

Tuber

Stomachache

KT

f

**Asteraceae**

****

****

****

****

****

39

_Adenostemma lavenia_ (L.) Kuntze __

Kasirukai

Leaves

Skin diseases

IR-S

d

40

_Ageratum conyzoides_ L. __

Nasar soppu

Leaves

Cough and cold

KU

b

_Ageratum conyzoides_ Linn. __

Pugudu thalai

Leaves

Wound

KO

a

41

_Artemisia nilagarica_ (C. B Clarke)

Manikoland

Leaves &

Removing worms from

KU

i

Pamp. __

stem

wounds both in humans and

animals

42

_Artemisia parviflora_ Buch – ham. Ex

Railpundu

Leaves

Headache

IR-S

d

Roxb. __

43

_Bidens pilosa_ L. __

Katu kunni

Leaves

White patches on the legs

KU

h

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_Table 1: Continued..._

44

_Blumea lacera_ (Burm.f.) DC. __

Navakkarandai

Leaves

Improving vision

KT

f

45

_Chromolaena odorata_ (L.) King & Rob-

Vettukkaya pacha

Leaves

Cuts and wounds

KT

f

ins. __

46

_Elephantopus scaber_ L. __

Anashovadi

Root

Stomach ache

KT

f

47

_Siegesbeckia orientalis_ Linn _._

Potaz

Leaves

Skin rashes, insect bites and

KO

a

allergies

_Sigesbeckia orientalis_ L. __

Nadukadachi

Leaves

Wounds and parasitic skin

KU

h

problems

48

_Sonchus oleraceus_ L. __

Kaalaadi pachilai

Leaves

Wound

PL

e

49

_Tridax procumbens_ L. __

Vettukayapoondu

leaves

Wounds to stop bleeding

IR

g

_Tridax procumbens_ L. __

Vettukkaya thalai

Leaves

Sores

KT

f

**Balanophoraceae**

****

****

****

****

****

50

_Balanophora fungosa_ Fors and Fors. __

Vaer chedi

Whole Plant Skin disease

PL

e

**Berberidaceae**

****

****

****

****

****

51

_Mahonia leschenaultii_ (Wight &

Mullu kadambu

Bark

Skin disease

PL

e

Arn.) Tak. ex Gamble __

52

_Berberis tinctoria_ Lesch __

Jakkala

Leaves &

Dysentery, Bloating of stom-

KU

i

stem

ach

**Bignoniaceae**

****

****

****

****

****

53

_Radermackera xylocarpa_ (Roxb.) K.

Vadencarni

Stem

Fever

KT

f

Schum. __

**Bischofiaceae**

****

****

****

****

****

54

_Bischofia javanica_ Blume _._

Romaviruksha patta Bark

Nervous disorder, hair growth

PL

e

**Boraginaceae**

****

****

****

****

****

55

_Carmona retusa_ (Vahl) Masam. __

Kurangu vetthilai

Leaves

Fertility

PL

e

56

_Nasturtium indicum_ (L.) DC. __

Kadge

Root Por-

Ear diseases

KU

i

tion

57

_Trichodesma zeylanica_ R.Br __

Jalke maram

Root

Round patches appearing on

KU

h

the skin

**Burseraceae**

****

****

****

****

****

58

_Boswellia serrata_ Roxb. Ex Colebr. __

Kungiliyam

Resin

Cold

PL

e

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_Table 1: Continued..._

**Caesalpiniacea**

****

****

****

****

****

59

_Caesalipinia bonduc_ (L) Roxb. __

Porumaielai

Root, seeds

Gastric disorders Increase

IR-S

d

body weight

60

_Bauhinia racemosa_

Mara avara

Bark

Boils

IR,

c

PA

61

_Cassia auriculata_ L. __

Avarai

Fruit,

Skin and scalp

IR

g

leaves,

flowers

62

_Cassia fistula_

Gaggai pattai,

Bark

Sudden "sicknesses" , diar-

IR,

c

Konnai mara

rhoea

PA

and stomach pain

_Cassia fistula_ L. __

Konnei

Stem bark

Stomach ache

KT

f

63

_Pterolobium hexapetalum_

Kari indu

Leaves

Ease delivery pain

PL

e

64

_Tamarindus indica_

Puli

Fruit

Nursing mothers, eczema

IR,

c

PA

**Caparidaceae**

****

****

****

****

****

65

_Capparis sepiaria_ L. __

Thoratti

Root

Wounds and scratches

IR

g

_Capparis zeylanica_ L. __

Adandai

leaves

Increase appetite

IR

g

66

_Celome monophylla_ L. __

Kadugu sedi

Leaves

Earache

IR-S

d

**Caricaceae**

67

_Carica papaya_ L. Poppilli __

Poppilli mara

Fruit

Indigestion and Constipation

KU

i

**Caryophyllaceae**

68

_Drymaria cordata_ (L.) Roem. &

Kodi charai

Leaves

Heel cracks

PL

e

Schult. __

**Celastraceae**

69

_Celastrus paniculatus_ Willd _._

Valulurai

Root

Body pain

KT

f

**Chenopodiaceae**

70

_Chenopodium ambrosioides_ L. __

Jaregida

Whole plant Intestinal cramps

KU

h

**Colchicaceae**

71

_Gloriosa superba_ L. __

Kodanki kizhangu.

Tuber

Sleeping tablet

IR-S

d

**Combretaceae**

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_Table 1: Continued..._

72

_Pterocarpus marsupium_ Roxb. __

Vengai

Stem

Rheumatic pain

KT

f

73

_Terminalia bellirica_ (Gaertn.) Roxb. __

Tani

Bark

Body pain

KT

f

74

_Terminalia chebula_ Retz _._

Kadukai

Fruit

Muscular dislocation

KT

f

_Terminalia chebula_ Retz _._

Kadukkai maram

Leaves

Cold, Cough, Fever, stomach-

PL

e

ache

75

_Terminalia crenulata_ Heyne ex Roth __

Karimathi

Bark

Internal bleeding

KT

f

**Compositae**

76

_Bidens pilosa_ L., Katu __

Katu kunni

Leaves

White patches on the legs

KU

i

**Convolvulaceae**

77

_Aroyreia hirsute_ Wight & Arn. __

Meenidal

Leaves

Male Child

KO

a

78

_Ipomoea alba_ L. __

Velutha

Leaves

Skin diseases

KU

h

79

_Melothria maderaspatana_ Cogn. __

Solapushni kai

Stem

Prolonged cough

KU

i

80

_Trichosanthes cucumerina_ L. __

Peyppadal

Fruit

Headaches

IR

g

**Dioscoreaceae**

81

_Dioscorea oppositifolia_ L. var.

Nurulai/Valli ki-

Rhizome

Stomacache

PL

e

tomentosa. __

langu

**Ebenaceae**

82

_Diospyros ferrea_ (Wild.) Bahk. Var. bux-

Veeraii

Fruit

Bood circulation

IR

g

ifolia __

**Ericaceae**

83

_Gaultheria fragrantissima_ Wall _._

Ameerpan

Leaves

Headache Relieve body

KO

a

sprains and pains

**Erthoroxylaceae**

84

_Erythroxyium monogynum_ Roxb _._

Jeevadalli maram

Stem Bark

Acute skin disease

KU

b

_Erythroxylum monogynum_

Jeevathalimara

Bark

Scabies

IR,

c

PA

**Euphorbiaceae**

85

_Acalypha fruticosa_ Forsskal _._

Chinni chedi

Leaves

Dysentery, skin disease

PL

e

86

_Acalypha indica_ L. __

Kuppaimeni

leaves

Ear pain, snake bite and sca-

IR

g

bies

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_Table 1: Continued..._

87

_Acalypha paniculata_ Miq _._

Paruva thazhai

Leaves

Pimples, stomachache

PL

e

88

_Breynia rhamnoides,_ Muell __

Poolan

Root &

White patches on the skin all

KU

h

Leaves

over the body

89

_Euphorbia antiquorum_ L. __

Sathura kalli

Latex

Body pain

PL

e

90

_Euphorbia hirta_ L. __

Ammanpatcharisi

Leaves &

Pimples

KU

h

Latex

91

_Euphorbia rothiana_

Spreng, Kopot

Latex

Sores, grow hairs, insect repel-

KU

i

lant

92

_Excoecaria agallocha_ L. __

Thillai

Latex

Antiseptic

IR

g

93

_Excoecaria crenulata_ L. __

Vellai thillai

Stem

Skin disease

PL

e

94

_Jatropha curcas_ L. __

Kaatu amanku

Leaves

Headache

KT

f

95

_Jatropha tanjorensis_ Ellis & Saroja __

Katamanukku

Latex

Antiseptic

IR

g

96

_Mallotus philippensis_

Chaneri mara

Bark

Stomach pain and diarrhoea

IR,

c

PA

97

_Phyllanthus amarus_ Schum. & Thonn _._

Kila nelli

Whole plant Jaundice

KT

f

_Phyllanthus amarus_ Schum. & Thonn _._

Kizhanelli

leaves

Jaundice

IR

g

98

_Phyllanthus emblica_ L. __

Nelli

Fruit

Stomachache

KT

f

99

_Ricinus communis_

Kottamuthu

Bark

Quick delivery, sprains,

IR,

c

breathing

PA

problems

100

_Ricinus communis_ Linn _._

Amanaku

Leaves,

Headache

KO

a

Seeds

101

_Securinega virosa_ (Willd.) Baill. __

Pula

Root

Joint pain

KT

f

102

_Euphorbia rothiana_ Spreng. __

Kapsi

Leaves

Sudden sickness and giddiness KO

a

**Fabaceae**

103

_Acacia nilotica_ (L.) Willd. ex. Del. __

Karuvelam

leaves

Dysentery, burns or scalds

IR

g

104

_Albizia lebbeck_ (L.) Benth. __

Vagai

seeds

Lesions of lepers

IR

g

105

_Caesalpinia bonduc_ (L.) Roxb. __

Kazhchikai

seeds

Hydrocele

IR

g

106

_Canavalia lineata_ (Thunb.) DC. __

Kozhiavarai

seeds

Several Disorders, General

IR

g

health

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_Table 1: Continued..._

107

_Clitoria ternatea_ L. __

Sangu pushpam

Infection of eyes, headache,

IR

g

snake bites

108

_Dalbergia sissoides_

Veetimara

Stem

Acute diarrhoea

IR,

c

PA

109

_Flemingia strobilifera_ (L.) R. Br. ex Ait. __ Kaduthuvarai

Whole plant Mental disorders

KT

f

110

_Macrotyloma uniflorum_ (Lam.) verdc __

Kollu

Seeds

Abortifacient

PA

b

111

_Millettia splendens_

Manalikodi

Stem

Burns and scalds , acute diar-

IR,

c

rhoea

PA

112

_Mucuna pruriens_ (L.) DC. __

Poonikali

seeds

Several Disorders, General

IR

g

health

113

_Pongamia pinnata_ (L.) Pierre __

Pongan

seeds

Rheumatic disease

IR

g

114

_Pterocarpus marsupium_

Pennae pattae

Bark

Abortifacient

IR,

c

PA

115

_Shuteria vestita_ W&A __

Kadu belaga

Leaves

Boils appearing on the skin

KU

h

116

_Tephrosia purpurea_ (L.) Pers. __

Averi

Headaches

IR

g

**Gentianaceae**

117

_Enicostema axillare_ (Lam.) Raynal __

Vellarugu

Root

Toothaches

IR

g

**Hypoxidaceae**

118

_Curculigo orchioides_ Gaertn. __

Nelapanai

Rhizome

Snake bite

IR-S

d

**Labiatae**

119

_Coleus malabaricus_

Periya tulasi

Leaves

Asthma

KU

i

120

_Plectranthus nilghericus_ Benth. __

Sone gida

Whole Plant Minor wounds

KU

i

**Lamaceae**

121

_Geniosporum tenuiflorum_ (L.) Merr. __

Nilathulasi

Whole plant Catfish bites

IR

g

122

_Leucas aspera_ (Willd.) Link __

Thumbai

Root

Tooth brush, resistant to snake IR

g

poison

123

_Anisochilus carnosus_ (L.f.) Wallich. __

Saetthupun thazhai

Leaves

Skin disease

PL

e

124

_Anisomeles malabarica_ (L.) R. Br. Ex.

Paei miratti

Stem

Wound

PL

e

Sims __

125

_Coleus parviflorus_ Benth __

Nila

Tuber

Itching, boils on the skin

KU

h

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_Table 1: Continued..._

126

_Leucas biflora_ (Vahl.)R.Br. __

Kaduthumbae

Whole plant Skin irritations

KU

h

127

_Leucas indica_ (L.) R. Br. ex Vatke __

Mosappullu

Leaves

Cough

KT

f

128

_Ocimum basilicum_ Var purpurasens __

Katu thulasi

Whole plant Skin inflammations after the

KU

h

insect bites

129

_Plectranthus coleoides_ Benth _._

Mudupattan or

Leaves

Cold, delivery pain, hair

PL

e

Omavalli chedi

growth, wounds

130

_Plectranthus malabaricus_ (Benth.) R.H.

Ellamabai

Leaves

Heart attack

IR-S

d

Willemse __

131

_Prunella vulgaris_ Linn _._

Kadthur

Root

Refrigerant Haematanic

KO

a

132

_Aloe vera_ (L.) Burm.f __

Sotru Kattrazhai

Leaves

Hair and skin Diseases

KU

h

133

_Asparagus racemosus_ Willd _._

Thanneer vittan

Leaves

Heel cracks

PL

e

kilangu

**Lobeliaceae**

134

_Lobelia heyneana_ Roem. & Schult. __

Upperi chedi

Leaves,

Skin disease

PL

e

Flowers

135

_Lobelia leschenaultiana_ (Presl) Skottsb. __

Bombari thalai.

Whole Plant Sickness in cattle

KO

a

**Loganiaceae**

136

_Strychnos nux-vomica_

Yetti

Bark

Acute stomach pain

IR,

c

PA

**Lythraceae**

137

_Lagestroemia microcarpa_ Wight __

Tindiyam

Bark

Burns

KT

f

**Malvaceae**

138

_Hibiscus rosa sinensis_ L. __

Chembarathi

Flowers

Strenthening hair

KU

h

139

_Malvatrum coromandelianum_ (L) Garke __

Kalakenikai

Root

Stomach pain

IR-S

d

140

_Sida acuta Burm_ f. __

Pilla valatthi chedi.

Leaves

Dandruffs , strengthening hair

PL

e

141

_Sida rhombifolia_ L. __

Kal gadale

Leaves

Wounds

KU

i

_Sida rhombifolia_ L. __

Chitra mutti

Root

Rheumatic pain

KT

f

142

_Side cordifolia_ L. __

Arathae

Leaves

Snakebite

PA

b

**Meliaceae**

143

_Azadirachta indica_

Veppamaram

Stem

Toothache, post-natal compli-

IR,

c

cations

PA

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

_Table 1: Continued..._

144

_Azadirachta indica_ A. Juss. __

Veppam

leaves

Stomach worms

IR

g

145

_Cipadessa baccifera_ (Roth.) Miq. __

Pulipancheddi

Leaves

Unconsciousness and anaemia

KT

f

_Cipadessa baccifera_ (Roth.) Miq. __

Pulippan chedi

Leaves

Diarrhoea

PL

e

_Cipadessa baccifera_ (Roth.) Miq. __

Marundha soppu

Leaves

Rheumatism

IR-S

d

****

**Menispermaceae**

146

_Cissampelos pareira_ L. var. hirsuta

Urikkakodi

Tuber

Snakebite

KT

f

(Ham. ex DC.) Forman __

147

_Cissampelos pureira,_ L. __

Koodibatale

Leaves

Headache, fever, burning sen-

KU

i

sation in chest

148

_Cyclea peltata_ (Lam.) Hook. f. & Thom-

Para

Tuber

Body pain

KT

f

son __

_Cyclea peltata_ (Lam.) Hook.f. __

Sethari Kodi

Leaves

Cough, cold and body pain

IR-S

d

149

_Tinospora cordifolia_ (Wild) Miers ex

Amrithavalli

Leaves

White rashes appearing on the

KU

h

Hook.F. & Thoms __

body

**Mimosaceae**

150

_Acacia caesia_ (L.) Willd. __

Nanjupattai

Bark

Wound

PL

e

_Acacia caesia_ (L.) Willd. __

Kari Indu

Stem bark

Body pain

KT

f

151

_Acacia leucophloea_ (Roxb.) Willd. __

Sarayapattai maram Bark

Cuts

PL

e

152

_Mimosa pudica_ L. (Mimosaceae) __

Thotalvadi

Whole plant Body pain

KT

f

**Moraceae**

153

_Ficus infectoria_ Roxb __

Selakai

Fruit

Food

KU

i

154

_Ficus racemosa_ L __

Athikai

Fruit

Eye sight

KU

i

**Moringaceae**

155

_Moringa concanensis_ Nimmo __

Kattu murukka

Bark

Abortifacient

IR,

c

PA

156

_Moringa oleifera_

Murunga

Bark

Dog or scorpion bite

IR,

c

PA

**Musaceae**

157

_Musa paradisiaca_

Vazhai

Stem

Acute diarrhoea

IR,

c

PA

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_Table 1: Continued..._

**Myrataceae**

158

_Eucalyptus polybractea_ R. T Baker __

Karpura mara

Leaves &

Round patches between fin-

KU

h

bark

gers

159

_Psidium guajava_ L. __

Koyyapazham

Fruit

Gastric troubles and ulcers in

PA

b

stomach

_Psidium guajava_ L. __

Koyyapazham

Fruit &

Anti-dysenteric and Antidiar-

KU

i

Leaves

rhoeal

160

_Syzygium cumini_ (L.) __

Skeels Naval Pa-

Stem Bark,

Sore throat, dysentery, ulcers,

KU

i

zham,

Fruit &

purifying blood, antidiarrhoeal

Seeds

and anti diabetic

_Syzygium cumini_ (L.) Skeels __

Naval

bark

Diarrhoea

IR

g

**Nyctaginaceae**

161

_Mirabilis jalapa_ L. __

Thottanembi

Root ,

Cuts and wounds

KO

b

Leaves

**Orchidaceae**

162

_Cymbidium aloifolium_ (L.) Sw. __

Ottai

Root

Ear pain

IR

g

163

_Malaxis densiflora_ (A. Rich.) Kuntze __

Kuntze, Nelnethch

Leaves

Wounds

KU

i

**Oxalidaceae**

164

_Oxalis corniculata_ L. __

Pulichen segae

Whole Plant Febrifuge

PA

b

_Oxalis corniculata_ L. __

Puliyankeerai

Leaves

Vomiting and headache

IR-S

d

_Oxalis corniculata_ Linn _._

Pulch

Leaves

Anti-emetic Restorative tonic

KO

a

after child birth

**Pandanaceae**

165

_Pandanus odoratissimus_

Kaithae

Stem

Fracture

IR,

c

PA

166

_Passiflora calcarata_ Mast __

Potul

Leaves

Skin diseases

KU

h

167

_Passiflora foetida L._

Narati chedi

Whole Plant Arthritic problems

KU

b

**Periplocaceae**

168

_Hemidesmus indicus_ (L.) R. Br. __

Nannari

Root

Mouth ulcers

KT

f

**Piperaceae**

169

_Piper betle_ L. __

Thabulam

Whole plant Cuts and wounds

KT

f

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_Table 1: Continued..._

170

_Piper brachystachyum_ Wall _.,_

Kadu kurumulaku

Fruit

Cough

KU

i

171

_Piper nigrum_ L. __

Milagu

Seeds

Throat infection

PL

e

**Plantaginaceae**

172

_Plantago erosa_ Wall _._

Kalthal

Leaves

Wounds as a antiseptic

KO

a

173

_Plantago lanceolata_ L. __

Neela kare

Leaves

Boils on the legs

KU

h

**Plumbaginaceae**

174

_Plumbago zeylanica_ L. __

Chitthira moolam

Root

Stomachache

PL

e

_Plumbago zeylanica_ L. __

Cithiramalliver.

Root

Insect bite

IR-S

d

**Poaceae**

175

_Cymbopogon citratus_ L _._

Karppura pul

Root

Pimples

KU

h

176

_Cynodon dactylon_ (L.) Pers. __

Arugampul

Branches

Body coolant

IR

g

**Polygonaceae**

177

_Rumex nepalensis_ Spreng _._

Gundott or Sukkutu Root

Refrigerant and laxative

KO

a

Keerai

_Rumex nepalensis_ spreng _._

Kekal Ott, Gund

Root

Jaundice

KO

b

Ott

**Proaceae**

178

_Cynodon dactylon_ (Linn.) Pers. __

Nagirki

Leaves

Relief from sudden sickness

KO

a

**Ranunculaceae**

179

_Clematis gauriana_ Roxb _._

Meenae

Leaves &

Wounds & skin Diseases

KU

h

stem

_Clematis gouriana_ Roxb. Ex. DC. __

Attumeesai chedi

Leaves

Skin disease

PL

e

**Rhamnaceae**

180

_Ziziphus mauritiana_

Yelluchi maram

Bark

Gastric disturbance

IR,

c

PA

_Ziziphus mauritiana_ Lam _._

Elanthai

Bark

Old wounds

IR

g

_Ziziphus mauritiana_ Lam _._

Ilantha

Whole Plant Mouth freshener

KT

f

**Rubiaceae**

181

_Catunaregam spinosa_ (Thunb.)

Madukarei

Root

Ulcers

KT

f

Tirvengadum __

182

_Rubia cordifolia_ L. __

Sappli Koth

Stem

Restorative, jaundice

KO

b

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_Table 1: Continued..._

_Rubia cordifolia_ L. __

Kalutharupan chedi Leaves

Heel cracks

PL

e

_Rubia cordifolia_ L. __

Periya nangai.

Leaves

Cough, cold and nervous dis-

IR-S

d

orders

_Rubia cordifolia_ Linn _._

Maral

Leaves

Injuries caused by fire

KU

i

_Rubia cordifolla_ L. __

Muthang

Root

Dysmenorrhoea

KT

b

**Rutaceae**

183

_Citrus aurantium_ L. __

Eravae kai

Fruit

Digestion, Hemorrhoids

KU

i

184

_Clausena dentata_ (Willd.) Roem. __

Anai thazhai

Leaves

Wound

PL

e

185

_Glycosmis mauritiana_ (Lam.) Yaich. __

Panasedi

Leaves

Headache

IR-S

d

186

_Glycosmis pentaphylla_ (retz.) DC. __

Eruputtal

Whole Plant Stomach ache and abdominal

KT

b

discomfort

187

_Murraya koenigii_ L. __

Karivepilla

Leaves

Skin inflammations

KU

h

188

_Murraya paniculata_

Chedichi

Bark

Toothache

IR,

c

PA

189

_Naringi crenulata_ (Roxb) Nicolson __

Naivalampattai

Bark

General health

IR-S

d

190

_Ruta chalepensi_ L. __

Aruvatha Geeda

Leaves

Infant convulsions

KO

b

191

_Ruta graveolens_ L. __

Aruvadam

Leaves

Skin diseases

KU

h

_Ruta graveolens_ L. __

Arubathansedi

Leaves

Diarrhea, stomach pain and

IR-S

d

vomiting

192

_Toddalia asiatica_ (L.) Lam. __

Surai

leaves, Fuit

Fever, headache

IR

g

_Toddalia asiatica_ (L.) Lam. __

Kindu mullu

Leaves,

Stomachache, toothache

PL

e

stem, Root

bark

_Toddalia asiatica_ (Linn). Lam. __

Vaseri

Leaves ,

Vermifuge

KO

a

Seeds

**Salvadoraceae**

193

_Azima tetracantha_ Lam _._

Sankan

leaves

Fever

IR

g

194

_Salvadora persica_ L. __

Vagai

Fruit, Root

Rheumatic pains, tooth brush

IR

g

**Santalaceae**

195

_Santalum album_

Santhana mara

Heartwood

Refrigerant, skin diseases

IR,

c

PA

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_Table 1: Continued..._

_Santalum album_ L. __

Sanddanamara

Seed

Skin troubles, refrigerant

KU

i

196

_Thesium wightianum_ Wall. ex Wight __

Anaikchi

Whole Plant Cheek to prevent bulging

IR-S

d

**Sapindaceae**

197

_Cardiospermum halicacabum_ L. __

Mudakkathan

whole plant

Rheumatoid arthritis

IR

g

_Cardiospermum halicacabum_ L. __

Poovanthi

Nuts

Body wash

IR

g

198

_Dodonaea angustifolia_ L.f. __

Marundha soppu

Leaves

Rheumatism

IR-S

d

199

_Dodonaea viscose_ (Linn.) Jacq. __

Vilari thalai

Leaves

Wounds and injuries, joint

KO

a

sprains and bone fracture

200

_Dodonea viscosa_ Linn. __

Manantha

Leaves

Fracture

KU

i

201

_Schleichera oleosa_

Jagada mara

Bark

Abortifacient

IR,

c

PA

**Sapotaceae**

202

_Manilkara hexandra_ (Roxb.) Dubard __

Pala maram

Latex

Toothaches

IR

g

203

_Tinospora cordifolia_ (Willd.) Miers __

Seenthil

stem

Many Disorders

IR

g

**Simaroubaceae**

204

_Ailanthus excelsa_ Roxb _._

Pekalathi

Bark, leaves After child birth

IR

g

**Solanaceae**

205

_Datura stramnium_ L _._

Umbathi

Leaves

Inflamed wound and sores

KO

b

_Datura stramonium_ L _._

Yemmuth

Leaves &

Piles

KU

i

Fruit

206

_Physalis peruviana_ L _._

Urechithuvar

Leaves

Wound

KU

i

207

_Solanum anguivi_ Lam _._

Kandan kathiri

Fruit, leaves Colds, coughs

IR

g

and fever, intestinal worms

208

_Solanum denticulatum_

Periya midinje

Whole Plant Migraine

KU

i

209

_Solanum erianthum_ D.Don __

Malai sundai

Fruit

Toothache

PL

e

210

_Solanum indicum_ Linn _._

Sunda maram

Root &

Toothache and snakebite

KU

i

Leaves

211

_Solanum nigrum_ L _._

Mana thakkali

Leaves

Ulcer, wound

PL

e

_Solanum nigrum_ Linn _._

Ikki sop

Leaves

Stomach disorders Skin rashes KO

a

212

_Solanum sisymbrifolium_ Limk _._

Vadadana

Seeds

Vermifuge

KO

a

213

_Solanum surattrense_ Burm. F __

Kandankathiri

Fruit

Toothache

PL

e

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_Table 1: Continued..._

214

_Solanum trilobatum_ L _._

Thoodhuvalai

Leaves

Asthma, Cold

PL

e

**Tamaricaceae**

215

_Tamarix indica_ willd __

Kattuchaukku

leaves

Laxative

IR

g

**Thunberigiacea**

216

_Thunbergia fragrans_ Roxb _._

Kakka Valli

Root

Snake-bite

KT

b

**Tiliaceae**

217

_Grewia aspera_ Roxb __

Dadchi maram

Bark

Diarrhoea

KU

i

218

_Grewia tiliifolia_ Vahl. __

Unu

Root bark

Swellings

KT

f

**Ulmaceae**

219

_Holoptelea integrifolia_

Vellaya

Bark

Swellings

IR,

c

PA

**Umbelliferae**

220

_Centella asiatica_ (L.) Urban _._

Vallarai

Leaves

Jaundice

PL

e

**Verbenaceae**

221

_Gmelina arborea_ Roxb __

Perungilai/

Root bark

Piles

PL

e

Kumilamaram

222

_Gmelina asiatica_ L _._

Kumalai Nochi

Fruit

Bathing

IR

g

223

_Lantana camara_ L _._

Unni chedi

Whole plant Cuts and wounds

KT

f

_Lantana camara_ L _._

Thusik

Leaves

Gum bleeding and tooth-ache

KO

b

_Lantana camera_ L _._

Parale gida

Flowers

Skin inflammations

KU

h

224

_Tectona grandis_

Thekku

Bark

Constipation

IR,

c

PA

_Tectona grandis_ F. __

Thekku

Bark

Ease child birth and labour

KU

b

pain

_Tectona grandis_ L _._

Thekku

Bark

Ease child birth and labour

PA

b

pain

_Tectona grandis_ L.f. __

Thekku

Stem

Stomach ache and dysentery

KT

f

225

_Vitex negundo_ L _._

Nochhi

Leaves

Rejunvating skin

KU

h

_Vitex negundo_ L _._

Kumalai Nochi

leaves

Repel mosquitoes, body pain

IR

g

_Vitex negundo_ L _._

Notchi

Fruit

Cold, Cough, Fever, headache

PL

e

__

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_Table 1: Continued..._ ****

**Zingiberaceae**

226

_Alpinia calcarata_ Rosc _._

Arathi poo

Rhizome

Immunity

PL

e

227

_Costus speciosus_ (J. Koen.) Smith. __

Koshtam

Leaves

Diabetes

PL

e

228

_Curcuma longa_ L. __

Manjal

Rhizome

Itching, glowing skin

KU

h

_Curcuma longa_ L. __

Manjal

Rhizome

Scorpion bite

KT

f

229

_Elatteria cardamomum_ (L.) Maton. __

Yelakkai

Fruit

Stomachache

PL

e

KU = Kurumba, IR = Irula, KT = Kota, TH = Thoda, PA = Paniya, PL = Paliyar, KT = Kattunayakas. a, (Rajan and Sethuraman, 1991); b, (Ra-

jan _et al_., 1997); c, (Rajan _et al_., 2001); d, (Murugesan _et al_., 2005); e,

(Ignacimuthu _et al_., 2006); f, (Udayan _et al_., 2007); g, (Ragupathy

and Newmaster, 2009); h, (Deepak _et al_., 2014a); I, (Deepak _et al_., 2014b)

Thus, the medicinal uses of the tribal traditional medicines depict

The tropical countries are gifted with vast resources of medicinal

their medical history through the ages. They have tried and found

plants and the recent global renaissance in traditional medicines has

cure to their major health problems such as wounds and skin prob-

created a large market for herbal products that can be exploited by

lems. This data also throws light on a cultural aspect of their life

these countries they meet up to quality and safety specifications.

style, mainly that they held their women in high esteem for they

Population explosion, incidence of side effects of synthetic medi-

have painstakingly developed remedies form medicinal plants to al-

cines and our inability to provide modern medicines to a vast section

leviate labour pains and post-partum medical care.

of the population living in rural and remote areas of the country due

****

to non-availability, in accessibility and unaffordability have been the

**6. Towards sustainable local production of traditional medicines**

prime reasons for the growing popularity of alternative medicines

****

amongst rural and remote population, and neo-rich people in the de-

Sustainable development denotes a development that meets the

veloped countries. Access to quality health care is an enormous pub-

needs of the present without compromising the ability of future gen-

lic health global issue at the scientific, clinical, economic, political

erations to meet their own needs. It encompasses two key concepts,

and policy levels. It is one aspect of the "great divide" that exists

the concept of needs, in particular meeting the essential needs of the

between and within every country in the world; it is the difference in

poor and the idea of limitations that is the environment"s ability to

access to health care between the rich and the poor. The very basis

meet these needs. Thus sustainable development seeks to relieve

for promoting the local production of herbal remedies is to provide

poverty, create equitable standards of living, satisfy the basic needs

cost effective medicines to populations who cannot afford costly

of all peoples, and establish sustainable political practices, while en-

medicines. The World Health Organization (WHO) has been repeat-

suring that there are no irreversible damages to natural resources and

edly stressing that the goal of "health for all" cannot be accom-

nature.

plished without herbal medicines.

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

Nervous disorder

Hair

Burns

Antiseptic

Lactation

Heel cracks

Fracture

Eye infections

Boils

Asthma

Vermifuge

Ear pain

Abortifacient

Snake bite

Jaundice

Diabetes

Body pain

Refrigerant

Fever

Toothache

Gastric disorders

Labour pain

Rheumatic disease

Cold, Cough, Fever

Headache

Diahorrea

Stomacache

Skin Problems

Wound

0

5

10

15

20

25

30

35

****

****

**Figure 3:** Traditional Medicine of the Tribes of Tamil Nadu in the treatment of common ail-

ments.

**7. Strategies for promoting sustainable**

ered and collected from the wild and rela-

**production of traditional medicines**

tively few are cultivated in farmlands.

World Wildlife fund report (2004) report-

The first step in promoting sustainable

ed that 20% of the medical plants world-

production of traditional medicine is de-

wide are in the treat of disappearing (Pan

veloping a standardized mode of produc-

_et al_., 2013). A paradigm shift from wild

tion which will meet the standards of qual-

collection to structured cultivation of me-

ity, efficacy and safety as defined in the

dicinally important plants will further en-

WHO guidelines. Thus bringing these

sure the purity, authenticity and sustaina-

plants into large scale cultivation ensures

ble supply of raw drugs. Suitable infra-

that endangered plant are protected by cul-

structure like production equipment, pota-

tivation and by involving the indigenous

ble water, storage facilities and post-

people over-exploitation will be avoided

harvest quality monitoring and marketing

while adding to income for the indigenous

are very critical. Trained man power with

tribes. Majority of the plants are still gath-

an appropriate background in pharmaceu-

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

tical sciences in bioprocess technology is

accessible globally. Biotechnological as-

an integral part of the quality of the fin-

pect of herbal remedies involves extraction

ished product (Cordell and Colvard, 2012).

of plant"s active ingredients and formula-

The concept of sustainability directs us to

tion into final product. First, it must be

shift form non-renewable chemical drug

established unequivocally that the source

discovery programs to natural renewable

of the plant material is authentic. A plant

sources (Cordell, 2011). Sustainable pro-

extract usually contains hundreds of active

duction of traditional medicine will require

ingredients and in some cases the bioactive

an integrated approach encompassing the

compound is usually not known. Bioactivi-

different disciples of science like ethno-

ty guided processes are then used to sepa-

batany, chemistry, biomedicine, mathemat-

rate the active compound. This is further

ics and physics. Traditional medicine has

tested on in-vitro or in-vivo models for

still not come to the forefront because of

their functional efficacy. There are many

the following challenges faced by the

disease conditions for which such biologi-

pharmaceutical companies in providing the

cal models are not easily available, or if

capital investment in developing and mar-

even available, would be beyond the

keting them namely, collection of plants is

means of many researchers. Therefore a

a time consuming process and requiring

more practical and cost effective way is to

extensive negotiations related to access,

use total extracts of traditional medicinal

insufficient documentation and lack of

plants for which abundant ethnomedical

ethno medical knowledge, lack of institu-

evidence exits. The mode of formulation

tional and financial support, limited avail-

can then be based on the traditional meth-

ability of trained man power, low yield,

ods used to prepare that particular remedy,

long discovery process and expensive syn-

taking steps to establish safety through in

thesis, lack of scientific validation of the

vivo and in vitro studies followed by ap-

quality, safety and efficacy of traditional

propriate pilot clinical trials for cytotoxic,

formulations, lack of appropriate technol-

mutagenic and therapeutic perspectives.

ogy for post-harvest and pre-processing

The formulation should be free of poten-

purposes, low market value for traditional

tially toxic insecticides, pesticides and

medicinal plants, lack of methodologies

heavy metals. The formulation must be

for the preservation of medicinal extracts

evaluated for microbial contamination both

for extended shelf life.

fungal and bacterial, and radiation contam-

ination during the stages of processing of

**8. Biotechnology: challenges and pro-**

the material (Tan _et al._ , 2004). Quality

**spects for the sustainable use of tra-**

control of the product is then done by con-

**ditional medicine**

ducting accelerated stability tests as well

as on-shelf stability tests.

Biotechnology with its robust tools

The pharmacological approach in

and state of art technology will play a cru-

developing a drug includes bringing as

cial role in the sustainable use of tradition-

much chemical diversity as possible to the

al medicinal drugs in future. Although the

biological screening interface but with no

use of transgenic plants is a debatable for

consideration given to the origin of the

the preservation of biodiversity, genetic

plant derived materials, chemo-diversity,

engineering will play an important role in

functional diversity of the constituents,

saving medicinal plants, which are rare or

ethno-medical association of the plant or

endangered (Cordell, 2011). Ethnomedical

known or novel active constituent of the

information about biological evaluation of

plant extracts (Tan _et al_., 2004). The other

plant extracts and their constituents, the

challenge is that the active constituent is

chemistry of natural sources, and the clini-

often extracted and analyzed only at a sin-

cal evaluation of plant extracts are still not

gle point in time, ignoring daily metabolic

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 36

_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

flux, seasonal variation in enzyme activi-

There are three main types of

ties, and the biosynthetic genes which are

search strategies: biorational, chemo-

present, but not fully functional. Method-

rational and random approaches. An anti-

ologies that are able to characterise the

HIV bioactive compound Conocurvone

majority of the constituents without indi-

was discovered as a result of random ap-

vidual isolation of active constituents need

proach of screening strategies. Drugs dis-

to be developed to help validation and

covered using bio-rational approaches

standardization. India has a vast and di-

were artemisinin, morphine, quinine, and

verse wealth of traditional medicinal

ephedrine. Bio-rational approach is mostly

knowledge that can be used for bio-

guided by the ethnomedical information

prospecting to benefit both the country and

generated from the traditional medicines

the indigenous people.

and the most effective approach to date.

These medicinal plants contain reservoir of

**9. Bioprospecting of traditional medi-**

etho-medical and ethno-botanical tradi-

**cine to combat disease**

tional knowledge, which is an important

guide to discovery of many new drug lead

The search for new drugs is the

molecules (Table 2). As there are many

vast opportunity in the ambit of Biotech-

existing and emerging diseases that cannot

nology given the vast majority of diseases

be treated by the current plethora of drugs

encountered today and improved health

and the additional burden of increasing

care services.

****

**Table 2:** Bioactive compounds from medicinal plants and their clinical uses ****

SN

Bioactive compound

Species

Clinical Uses

Cholesterol

1

Mevastatin & lovastatin

_Penicillin_ _spp_.

lowering

Anthelmintic and antipar-

2

Ivermectins

_Streptomycetes_ _spp._

asitic

3

Reserpine

_Rauwolfia serpentine_

Antihypertensive

4

Ephedrine

_Ephedra sinca_

Antiasthma

5

Atropine

_Belladonna_

Anticholinergic

6

Teprotide

_Bothrops jaracaca_

Cardiovascular diseases

Vincristine and

7

_Catharanthus roseus_

Anti-cancer drug

vinblastine

8

Paclitaxel

_Taxus brevifolia_

Anti-cancer drug

9

Camptothecian

_Camtotheca acuminate_

Anti-cancer drug

10

Podophylotoxin

_Podophylum peltatum_

Skin Cancer

Ovarian

11

Bryostatin-1

_Bugula neritina_

carcinoma and non-

Hodgkin"s" lymphoma

Antimicrobial and anti-

12

Cyclosporins and rapamycin

_Penicillium notatum_

plasmodial

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_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

drug resistant pathogens, the need for the

drugs, the costs escalate and these drugs

hour is the development of new arsenal of

will not be available to the local people

drugs to combat them based on the tradi-

because of the costs which is also a critical

tional knowledge.

an ethical issue (Pan _et al_., 2013).

**10. Biotechnological tools to be exploited**

_11.1 The Kani model of benefit sharing of_

**for sustainable production of tradi-**

_traditional medicine_

**tional medicines**

The Kanis inhabit the forests of the

Thiruvananthapuram district of Kerala and

Biotechnology produces a vast array of

Thirunelveli district of Tamil Nadu in

tools for the successful discovery and vali-

southwestern India. In 1987, scientists

dation of traditional medical drugs. These

from Tropical Botanic Garden and Re-

tools have been developed in the 80"s and

search Institute (TBGRI) while collecting

90"s and today there is vast improvement

ethnomedical data form Kani tribal people

in their technological aspects after subse-

discovered that the tender fruits of Arog-

quent modification of the initial technolo-

yappacha ( _Trichopus zeylanicus_ subsp.

gy. Each technique has its own pros and

_travancoricus_ ) have anti-ageing, anti-

cons and care needs to exercise in the em-

depressant and anti-fatigue property. This

ployment of a suitable technology to pro-

paved a way for the scientists at TBGRI to

vide an appropriate traditional medicine

develop a scientifically validated and

based drug. Techniques like in vitro regen-

standardised herbal drug called Jeevani, a

eration through mciropropagation, callus

formulation consisting of four ingredients

mediated organogenesis, somatic embryo-

and Arogyappacha was one of the constit-

genesis, cryopreservation, production of

uents. Jeevani has been found to be thera-

secondary metabolites and genetic trans-

peutically effective having anti-fatigue and

formation holds a tremendous potential for

immuno-enhancing properties and it has

the production of high quality plant based

also shown good hepato-protective and

medicines mainly because of the multipli-

anti-stress

properties.

Subsequently,

cation rate, pathogen free material, plant

TBGRI decided to share 50% of the li-

preservation and regeneration success to

cence fee and royalty with the Kani people

yield bioactive compound that ensures re-

to encourage an equitable sharing of the

duced costs compared to the natural syn-

benefits arising from the utilisation of such

thesis by the plants.

knowledge, innovations and practices as

stated in the mandate of Article 8(j) of the

**11. Intellectual property rights (IPR) for**

Convention

on

Biological

Diversity

**sustainable use of traditional medi-**

(CBD). This is considered to be one of the

**cine**

first models for benefit sharing in the

world, which is popularly known as the

It is very vital to consider intellec-

TBGRI Model for Benefit Sharing.

tual property rights of the local people

when the ethnomedical documentation is

**12. Conclusion**

done for traditional medical knowledge.

The economic implication of the eventual

Sustainable local production of tra-

commercial production of standardized

ditional medicines requires an enabling

traditional medicinal drug should include

environment and effective partnerships

the welfare of the people from whom the

between traditional health practitioners,

traditional knowledge was documented.

researchers, public and the private sector.

Biopiracy becomes an ever looming issue

There is a strong need for indexing eco and

when large pharmaceutical companies con-

ethno information of medicinal plants, sus-

fiscate medicinal plants to make new

tainable cultivation of the traditional medi-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 38

_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

cal plants, protecting the intellectual prop-

nopharmacology. _Journal of Eth-_

erty rights of the indigenous people and

_nopharmacology_ , **1-2, 5-14.**

using the various tools of Biotechnology in

**Cordell, G.A. (2011).** Sustainable medi-

formulating a superior product that is cost

cines and global health care. _Planta_

effect to the very same indigenous people.

_Medica.,_ **11 (77), 1129-1138.**

Recent innovations in Biotechnology have

**Deepak, P. and Gopal, G.V. (2014).** Nil-

impacted the use of molecular tools for

giris: A Medicinal Reservoir. _The_

sustainable use of a renewable natural re-

_Pharma Innovation Journal,_ **3(8),**

source for the betterment of India and its

**73-79.**

indigenous communities. Modern tools of

**Deepak, P and Gopal, G.V. (2014).** Eth-

Genomics can be applied to traditional

nomedicinal practices of Kurumba

medicine without the need for transgenics.

tribes of Nilgiri District, Tamil Nadu,

Protection of intellectual property rights of

India, in treating skin diseases.

the traditional medicinal knowledge of in-

_Global J Res. Med. Plants & Indi-_

digenous people in the form of benefit

_gen. Med_., **3 (1), 8-16.**

sharing and bioprospecting the vast biodi-

**Fabricant, D.S. and Farnsworth, N.R.**

versity available in the country is the need

**(2001).** The value of plants used in

of the hour to march India in the field of

traditional medicine for drug discov-

bio pharmaceuticals as a global leader. It is

ery. _Environmental Health Perspec-_

now is the hands of young researchers to

_tives_ , **1 (109), 69-75.**

work under well placed regulatory frame-

**Gupta, A. Vats, S. K. Lal, B. (1998).**

work in converting traditional medicinal

_Curr Sci_. **75, 565**.

knowledge into a safe and novel bio drug

**Hamilton, A. C. (2004).** _Biodivers Con-_

for the treatment of existing and emerging

_serv.,_ **13, 1477.**

disorders.

**Ignacimuthu, S. Ayyanar, M. Sivara-**

**man, S.K. (2006).** Ethnobotanical 

## Acknowledgements

### investigations among tribes in Madu-

****

rai District of Tamil Nadu (In-

The

authors

gratefully

dia). _Journal of Ethnobiology and_

acknowledge the DST NRDMS (2016 to

_Ethnomedicine_ , **2, 25**.

2018) project to Ramani Bai, R. We are

**Kavitha, V.J. (2008).** Studies on the ge-

also thankful for the UGC MRP (2015-

nomic diversity of Southern Indian

2018) project to Kavitha, V.J. We are

breeding

isolates.Ph.D

The-

grateful to all the tribes of Tamil Nadu for

sis.Madurai

Kamaraj

University,

their vast knowledge in Traditional Medi-

Tamil Nadu, India.

cine.

**Misra, V.N. (2001).** Prehistoric colonisa-

tion of India. _J Biosci_ , **26,491-531.**

**References**

**Murugesan, M. Balasubramaniam, V.**

****

**and Arthi, H. (2005).** Ethno Medical

**Cann, R.L. (2001)**. Genetic clues to dis-

Knowledge of Plants Used By Irula

persal of human populations: Retrac-

Tribes, Chengal Combai, The Nilgi-

ing the past from the present. _Sci-_

ris, Tamilnadu. _Ancient Science of_

_ence,_ **291, 1742-1748**.

_Life_ , **24(4),179-182**.

**Cordell, G. A. and Colvard, M. D.**

**Pan, S.Y. Zhou, S.F. Gao, S.H. _et al_** **.,**

**(2012)**. Natural products and tradi-

**(2013).** New Perspectives on How to

tional medicine: turning on a para-

Discover Drugs from Herbal Medi-

digm. _Journal of Natural Products,_ **3**

cines: CAM's Outstanding Contribu-

**(75), 514-525.**

tion to Modern Therapeutics. Evi-

**Cordell, G.A. and Colvard, M.D. (2005).**

dence-Based Complementary and Al-

Some thoughts on the future of eth-

ternative

Medicine.

URL:

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 39

_Biotech Sustainability (2017)_

_Traditional Medicine of the Tribes in Tamil Nadu... Gurusamy et al._

http://dx.doi.org/10.1155/2013/6273

Tamil Nadu. _Journal of Natural_

75.

_Remedies,_ **1, (49-54).**

**Pushpangadan, P. and George, V.**

**Sethuraman, M. and Suresh, D.B.**

**(2010).** Ethnomedical practices of ru-

**(1997).** Plants from the Traditional

ral and tribal populations of India

Medical System of the Nilgiri

with special reference to mother and

Tribes. _Ancient Science of Life,_ **26(4),**

child care. _Indian journal of Tradi-_

**1742-1748**.

_tional Knowledge_ , **9 (1), 9-17**.

**Tan, B.H. Bay, B.H. Zhu, Y.Z. (2004).**

**Ragupathy, S. and Newmaster, S.G.**

Plants in Drug Discovery – Creating

**(2009).** Valorizing the 'Irulas'tradi-

a New Vision". In: Novel Com-

tional knowledge of medicinal plants

pounds from Natural Products in the

in the Kodiakkarai Reserve Forest,

New Millennium. Cordell G. A.

India. _Journal of Ethnobiology and_

(eds). World Scientific Publishing,

_Ethnomedicine,_ **5,10**.

Singapore, **pp. 1-19.**

**Rajan, S. and Sethuraman, M. (1991).**

**Tandon, V. (1996).** Med Plant Con-

Plants Used In Folk Medicine by the

serv.Newslett, **pp 2, 12.**

_**Kotas**_ of Nilgiri District, Tamil Na-

**Thurston, E. (1909).** The Castes and

du. _Ancient Science of Life,_ **4, 223-**

Tribes of Southern India.7 volumes.

**230.**

Madras.

**Rajan, S. and Sethuraman, M. (1993).**

**Udayan, P.S. Tushar, K. V.George. S.**

In-digenous folk practices Among

**Balachandran, I.** ( **2007** ). Ethtnome-

Nilgiri Irulas. _International J. Indig-_

dicinal information from Kattuna-

_enous knowledge and development_

yakas tribes of Mudumalai wildlife

_monitor (Netherland)_ , **1(3), 19-20**.

sanctury, Nilgiris district,Tamilnadu.

**Rajan, S. Baburaj, D.S. Sethuraman, M.**

_Indian J traditional knowledge,_ **6 (4),**

**Parimala, S. (2001).** Stem and stem

**574-78.**

bark used medicinally by the Tribals

**Ved, D. K. Anjna, M. Shankar, D.**

Irulas and Paniyas of Nilgiri District,

**(1998).** _Curr Sci_., **75, 341.**

****

© 2017 by the authors. Licensee, Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 40

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P41-50_

**Vermitechnology – An Eco-Biological Tool for**

**Sustainable Environment**

****

**Mahaly Moorthi1, Koilpathu Senthil Kumar Abbiramy1,*, Arumugam Senthil Kumar2**

**and Karupannan Nagarajan3**

****

_1PG and Research Department of Zoology & Wildlife Biology, A.V.C. College (Autono-_

_mous), Mannampandal - 609305, Mayiladuthurai, Nagapattinam District, Tamilnadu, In-_

_dia; 2PG and Research Department of Zoology, Chikkaiah Naicker College, Erode – 638_

_004, Tamilnadu, India; 3PG and Research Department of Zoology, Sri Vasavi College,_

_Erode – 638 316, Tamilnadu, India; ***** Correspondance: moorthideksha@gmail.com / ksab-_

 _biramy@gmail.com; Tel;_ _+91-8526385977_

__

****

**Abstract:** The word vermi, typically indicates earthworms. Vermitechnology is a simple

process, which uses earthworms to produce earthworms, good quality compost (vermicom-

post) through organic waste recycling and other products involving earthworms. This tech-

nology is inevitable in managing biodegradable wastes, biomass or organic material that

can be degraded or composted thus contributing to the environment indirectly. Solid waste

management through Vermitechnology contributes more for the sustainability of the envi-

ronment. The major components of Vermitechnology can be considered as Vermiculture

(mass production of earthworms), Vermicomposting (production of vermicompost) and

Vermiwash (the extract of vermicompost). In 1996, "Vermitech", the vermicomposting

technique was developed by Mr. A. Thimmaiah at the Indian Agricultural Research Insti-

tute (IARI), New Delhi, India. As IARI is also known as "Pusa Institute", this innovative

technology was dedicated to the institute and named "Pusa Vermitech". "Pusa Vermitech"

was developed to provide a simple solution to poor farmers. This method has now become

popular in Bhutan, Costa Rica, India, Italy, Nepal and Sri Lanka. It appears that Ver-

mitechnology is going to play an important role for the sustainability of agriculture and en-

vironment. This chapter is highlighting the Vermitechnology, as an eco-biological tool for

the sustainable environment.

****

_**Keywords:**_ Solid waste management; vermicomposting techniques; vermiculture; ver-

mitechnology; vermiwash

**1. Introduction**

manure comes in handy especially for

small-holders who do not have the money

The advent of organic farming has

to buy expensive, chemical fertilizers.

made farmers innovative and nature

The manure is used rampantly for all sorts

friendly. Vermitechnology, an effective

of crops. The biggest beneficiaries are

replacement for chemical input is the

women who have formed themselves into

most sought after due to its cost-

NGO-trained Self-Help Groups (SHGs).

effectiveness and quality of enriching the

These women can easily prepare the

soil. Vermicompost is becoming the prin-

Vermitech products in their backyard and

cipal manure for crops in the field of or-

sell the excess one left after their own use

ganic farming. The market crisis for agri-

to neighbouring estates or farmers. The

cultural products has also contributed to

NGO-run institutes like these are assisted

the popularity of vermicomposting. The

by the local bodies like grama pancha-

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_Biotech Sustainability (2017)_

_Vermitechnology – An Eco-Biological Tool for Sustainable... Moorthi et al._

yats, zilla panchayats, which organize

cessed potato; wastes from supermarkets

seminars and teach farmers the methods

and restaurants; wastes from poultry,

of Vermitechnology. Government agricul-

pigs, cattles, sheeps, goats, horses (Ed-

tural departments also buy earthworms in

wards and Bater, 1992) as well as horti-

bulk from these SHGs to conduct their

cultural residues from dead plants and

own projects on vermiculture. In fact, the

spent wastes from mushroom industry

Supreme Court"s ruling criteria is that to

(Edwards,1988).

decide on metro status would be the par-

The degradable organic matter

ticular district"s level of participation in

from these wastes when dumped in open

organic farming.

undergoes either aerobic or anaerobic

But the low sense of awareness

degradation.

These

un-engineered

still remains a problem even within the

dumpsites permit fine organic matter to

municipalities that have undergone semi-

become mixed with percolating water to

nars on Vermitechnology. Neither the

form leachate. The potential for this

Boards nor the local bodies have ideas on

leachate to pollute adjoining water and

the perspectives of Vermitechnology.

soil is high. India where a lot of solid or-

Technical expertise alone does not make

ganic waste is available in different sec-

organic farming. Unless there is a uni-

tors with no dearth of manpower, the en-

formity of procedure, the advantages of

vironmentally acceptable Vermitechnolo-

organic farming – low cost, more soil fer-

gy using earthworms can very well be

tility and eco-friendliness - will not come

adopted for converting waste into wealth.

through.

Considerable work has been carried out

After all, by preparing Vermitech

on vermicomposting of various organic

products, the farmer is making the soil

materials and it has been established that

healthy. In turn, he"s supplying healthy

epigeic forms of earth-worms can hasten

crops into the market. The content of or-

the composting process to a significant

ganic carbon, the index for the presence

extent, with production of a better quality

of humus in the soil, is in high Ranges. So

of composts as compared with those pre-

the farmers must contribute considerably

pared through traditional methods. The

more towards ecology and food produc-

viability of using earthworms as a treat-

tion. He deserves all the support he can

ment or management technique for nu-

get. With the right financial support from

merous organic waste streams has been

the Government and a more organized

investigated by a number of workers

network of cultural units, Vermitech

(Logsdon, 1994; Madan, 1988; Singh,

products, as a form of enrichment can

2002). Similarly a number of industrial

generate a steady source of income for the

wastes have been vermicomposted and

impoverished folk of agricultural areas.

turned

into

nutrient

rich

manure

There are numerous sources of

(Sundaravadivel, 1995). Hand _et al._

waste produced in India where degradable

(1988) defined vermicomposting as a low

organic matter is either partially or fully

cost technology system for the processing

generated. Solid waste consists of the dis-

or treatment of organic wastes.

carded portion of the households, dead

A growing awareness of some of

animals, trade, commercial, agricultural

the adverse economic and environmental

and industrial waste and other large waste

impacts of agrochemicals in crop produc-

like debris from construction site, furni-

tion has stimulated greater interest in the

ture etc. Solid wastes are generally cate-

utilization of organic amendments such as

gorized as domestic, industrial and haz-

compost or vermicompost for crop pro-

ardous or biomedical waste. Studies were

duction (Follet, 1981). Therefore, the sus-

made on some solid wastes like sewage

tainability has to be restored by some

sledges and solids from waste water

means of regular food security. Utiliza-

(Mitchell _et al_., 1980); wastes from pro-

tion of earthworms may be an answer as

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_Biotech Sustainability (2017)_

_Vermitechnology – An Eco-Biological Tool for Sustainable... Moorthi et al._

an ecologically sound, economically via-

id wastes (John Paul _et al_., 2011), as a

ble and socially acceptable technology.

source of live feed in poultry and aqua-

The present chapter reviews on various

culture industries. Worms have a number

aspects involved in Vermitechnology and

of other possible uses on farms, including

thus managing the organic waste leading

value as a high quality animal feed. The

to a sustainable environment.

earthworms are also used as bait in

freshwater sport fishing. They are also

**2. Vermiculture**

sold to small scale business people who

maintain garden at home and to nurseries

The process of culturing of earth-

that do organic gardening and composting

worms using scientific methods is known

and sell saplings. Also there are demands

as Vermiculture. Earthworms are known

for the pure form of vermicastings in var-

as biological indicators of soil health. In

ious places.

soil earthworms" activity and their casting

support a lot amount of microbial popula-

_2.1._ **** _Methods for Vermiculture_

tions. Microbial populations such as bac-

Vermiculture is the culture of

teria, fungi, Actinomycetes and protozo-

earthworms. The goal is to continually

ans grow well, also insects like spiders,

increase the number of worms in order to

millipedes, and other nematodes that are

obtain a sustainable harvest. The worms

essential for sustaining the soil fertility

are either used to expand a vermicom-

grow well. Presence of soil biome enrich-

posting operation or sold to customers

es the soil fertility. Thus earthworms

who use them for the same or other pur-

which form the base for the survival of

poses. If the goal is to produce ver-

other organisms can be cultured artificial-

micompost then we want to have maxi-

ly and used for many purposes. The ulti-

mum worm population density all of the

mate goal of this technology is the bet-

time. If the goal is to produce worms then

terment of soil fertility and health of hu-

we keep the population density low

man beings.

enough that reproductive rates are opti-

In recent years considerable atten-

mized. Vermiculture as a business must

tion has been focused upon the potential

be started in a small scale units. After

role of intensive earthworm culture, or

learning the technique of culturing, one

vermiculture. It is now accepted that the

can start his business at large scale. Cul-

economic value of vermiculture lies in (i)

turing of earthworms needs minimum re-

reduction of noxious qualities associated

quirements and care on a regular sched-

with organic wastes, e.g. elimination of

ule. Culturing can be done by two meth-

smell; (ii) generation of a useful compost;

ods.

and (iii) production of earthworm bio-

i. Container method.

mass. Various Vermiculture systems,

ii. Tank method.

which have been designed primarily for

The first method consists of cul-

biological waste control, are producing

turing earthworms in containers made of

earthworms in large quantities. This chap-

plastic and the medium or their bedding

ter covers the production of the earth-

(commonly called as vermibeds) can be

worms which can be used as a source of

done in it. Containers in the shape of rec-

food, primary proteins, and as drugs.

tangular boxes or tubes of one foot width,

Now-a-days, people are very

3 feet length and 2 feet height can be uti-

much eager in culturing earthworms as a

lized. The shape of the containers can

part time business, as a source of income.

change according to the availability in the

The culturing of worms becomes a prom-

area of culturing. These containers with

ising business because of its need in

vermibeds must be placed in a proper en-

enormous amount in organic farming, in

vironment. Either it can be placed in sepa-

big municipalities for the treatment of sol-

rate thatched roof shed if economically

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_Biotech Sustainability (2017)_

_Vermitechnology – An Eco-Biological Tool for Sustainable... Moorthi et al._

viable or can be placed in spaces availa-

organic matter for partial decomposition,

ble inside the house itself. Thus depend-

care must be taken that, both the organic

ing on the economical status, the place

matter and the cow dung are shad dried.

can be selected. The conditions in main-

This is because; wet organic matter and

taining the vermibeds are, direct sunlight

cow dung may contain the cyst and larvae

must be avoided and it must be safe from

of other microorganisms and insects. So if

insects and rodents.

implemented wet, they may grow along

The second method of culturing

with earthworms and do hindrance.

earthworms is by tank method. By this

The alternate layers of organic

way multiplication of earthworms can be

matter and cow dung are arranged in the

done in large scale. For this method, ce-

form of heap and 50% moisture must be

ment tanks of dimensions one meter

maintained. Cover the heap with thatched

width × one meter height × 3-5 meter

coconut leaves. This set up must be kept

length can be constructed. These tanks

for atleast 30 days. The organic matter,

must be constructed inside a thatched roof

along with cow dung mixture must be

shed so that direct sunlight and rain can

turned over with a spade once in a week.

be avoided. The shed can be of any di-

The microorganisms which are present in

mensions according to the land available.

the cow dung slowly degrade the organic

Thus the length of the tank and the no of

matter. After 40 days the process of par-

tanks to be constructed can also be done

tial decomposition will be finished and

accordingly. The shed must be construct-

now it can be used as a medium for ver-

ed in East-West direction length wise to

mibed.

avoid direct sunlight and preferably open

In the containers, the vermibed

from all sides with unpaved floor with

must be prepared by mixing the partially

raised ground (atleast 6 inches) to protect

decomposed organic waste and cow dung

the area from flooding during the rains.

(shade dried and powdered) in 1: 1 ratio.

This ratio is recommended by many sci-

_2.1.1. Vermibed preparation_

entists and has been proved in many liter-

The vermibed for both the meth-

atures also. Nearly 50% of moisture must

ods can be prepared using any kind of de-

be maintained in the bed. Earthworms can

gradable, non-toxic organic waste like

be introduced after two days. Leave the

leaf litters, agricultural wastes (Suthar,

setup for 45 days. The worms eat the de-

2010), shredded newspaper (Updegraff,

graded organic matter and convert them

1971), cardboard, etc... can be utilized as

into vermicastings. At the same time they

medium. Wastes from fruit and vegetable

also reproduce and increase in number.

market **** such as potato peels **,** onion peels,

Vermibed for tank method can al-

cabbage leaves, carrot and radish leaves,

so be prepared in the same way but the

lettuce leaves, moldy bread can also be

partially degraded waste and the cow

used (Suthar, 2009). Though organic

dung must be added in large quantities

wastes serve as food for earthworms, they

into the tank. Earthworms can also be re-

can"t be directly implemented for ver-

leased in large quantities. Water must be

miculture. In other words, the worms

sprinkled on the vermibed once or twice

can"t eat the organic matter directly. Thus

in a week according to the humidity pre-

it must be subjected to partial decomposi-

vailing in the atmosphere. Temperature of

tion and then used as a medium for ver-

about 37 degree Celsius must be main-

mibed.

tained. If the climate is hot, wet jute bags

The organic matters must be

can be screened on the sides of the tank.

shredded and alternate layers of organic

By the tank method, earthworms can the

matter and mixture of cow dung is spread

reproduced 300 times greater within 45

on the floor of shed constructed for ver-

days or 60 days (depending upon the

miculture. Before the implementation of

worms cultured). The reproducing capaci-

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ty of exotic worms is greater than the in-

small worms (juveniles) present segregate

digenous worms. It is said that eight red

in the centre of the heap and can be col-

worms multiply into 1,500 redworms ( _Ei-_

lected.

_senia fetida_ ) in six months. These earth-

worms can be used to prepare vermicom-

_2.2._ **** _Application of earthworms_

post or for other purposes or can be sold.

Adult earthworms can be sold as

The vermibeds must be covered

fish bait and the young ones can be used

with a Jute mat to protect earthworms

as inoculums for fresh bedding. Many

from birds and insects. Water is sprinkled

industries like aquaculture and Poultry

on the vermibeds daily according to re-

will buy earthworms for using it as live

quirement and season to keep them moist.

feed. Business people who are interested

The waste is turned upside down once in

in constructing vermicomposting units

a week. The appearance of black granular

(house hold, small scale or large scale

crumbly powder on top of vermibeds in-

unit) will be purchasing the earthworms.

dicates the casts of earthworm. Addition

Earthworms can be sold to new business

of more amount of cow dung ensures the

people who are about to start their ver-

fecundity rate of earthworms.

miculture or composting unit. Worms are

also bought by academic institutions for

_2.1.2. Harvesting of earthworms_

research purposes. Earthworms can be

The vermibed after 45 days is

sold to wholesalers who then resell the

converted into vermicastings. This indi-

worms to bait shops, home and organic

cates that all the organic matter along

nurseries, and other users.

with cow dung is eaten up by the worms

and converted into vermicastings. Now

**3. Vermicomposting**

there is no food available for the worms.

The content in the container or in the tank

Earthworms facilitate the stabili-

must be removed and new beds must be

zation of organic wastes because their ac-

replaced. Before the replacement, the

tivity maintains aerobic conditions and

worms must be harvested. The very re-

ingested solids are converted into discrete

cent and new technique followed to har-

odourless casts (Edwards, 1988). Thus the

vest earthworms is using "fresh cow dung

end product of vermicomposting is re-

balls". Fresh cow dung attracts the earth-

ferred to as "vermicasting" or vermicom-

worms very much. Thus balls of fresh

post. This is a nutrient rich organic sub-

cow dung, of 15 to 20 cm in diameter are

stance that can be added to soil to in-

placed inside the vermibeds. For contain-

crease its organic matter content and

er method, one ball is enough while for

available nutrients. Vermicomposting is

tank method, four to six balls can be used

getting enormous importance in the ame-

to harvest earthworms.

lioration of severe problems associated

As soon as the cow dung balls are

with the disposal of large quantities of

placed, the earthworms start migrating

organic wastes (John Paul, 2005). Earth-

into the fresh cow dung balls. The cow

worms feed on organic matter, in which

dung balls were kept for 6 to 8 hours, af-

5-10% is taken as food intake for their

ter which are removed. Thus the earth-

growth and the rest is excreted. Exotic

worms can be harvested and used as in-

earthworms like Red worms ( _Eisenia feti-_

oculums for the new vermibed. The ver-

_da_ ) and African worm ( _Eudrilus eugeni-_

micastings in the container or the tank is

_ae_ ) and Indigenous species like _Lampito_

removed and made into heap. This heap is

_mauritti_ and _Perianyx excavatus_ are

left undisturbed for a day. Next day, the

proved to be effectively in vermicompost-

vermicastings are removed slowly from

ing (Karmegam and Daniel, 2009, Kaur,

the outer side with the help of spade,

_et al_., 2010). Vermicomposting can be

sieved, and packed as biofertilizer. The

done in three ways.

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i.

Container method

at the bottom, about 15 to 20 cm thick

ii.

Tank method

above a thin layer (5 cm) of broken bricks

iii.

Pit method

and coarse sand. Earthworms are intro-

For all the three methods, the pre-

duced into the loamy soil, which the

processing of organic matter, i.e. the par-

worms will inhabit as their home. 150

tial decomposition of organic matter is

earthworms may be introduced into a

essential and it can be done as said above

compost pit of about 2m x 1m x 0.75m,

for vermiculture.

with a vermibed of about 15 to 20 cm

thick. Handful lumps of fresh cattle dung

_3.1. Container method_

are then placed at random over the ver-

This method of composting is

mibed. The compost pit is then layered to

normally followed in small scale ver-

about 5 cm of dry leaves or preferably

micomposting units or in houses where

chopped hay/straw or agricultural waste

vermicomposting is done. The containers

biomass or any non-toxic organic waste.

and the vermibed preparations can be fol-

This layer of organic waste and cow dung

lowed as said for vermiculture. The only

must be repeated till the top of the sur-

difference is the ratio of organic matter

face. For the next 30 days the pit is kept

and cow dung. For vermiculture, the ratio

moist by watering it whenever necessary.

of 1: 1 must be maintained in order to in-

The bed should neither be dry or soggy.

crease the fecundity rate. Because, the

The pit may then be covered with coconut

main aim of vermiculture is production of

or Palmyra leaves or an old jute (gunny)

earthworms. While during vermicompost-

bag to discourage birds. Plastic sheets on

ing, the ratio can be altered as 2: 1 or

the bed are not recommended as they trap

even to 3: 1 according to the availability

heat. All these organic wastes can be

of cow dung. An advantage of container

turned over or mixed periodically with a

method over traditional composting, is

pick axe or a spade. If the weather is very

that they can be kept inside the compost-

dry it should be dampened periodically.

ing shed during the winter, thus allowing

Red worms and African worms consume

this process to be done all over the year.

large amounts of organic matter and

hence they are recommended for com-

_3.2. Tank method_

posting. Though exotic earthworm spe-

Same as for vermiculture, the tank

cies are used in pit method, the indige-

can be constructed and the process of

nous worms as invade the pit and do their

vermicomposting can be done. Thus this

role.

method counts for large scale of ver-

micomposting. The only difference in

_3.4. Precautions_

both is, production of large amount of

Vermicomposting unit or pit should

earthworms is the aim in vermiculture

be protected from direct sun light. To

while production of large amount of ver-

maintain moisture level, spray water on

micompost is the aim in vermicompost-

the composting unit as and when re-

ing. The same type of shed can be con-

quired. Large amounts of waste can cause

structed for this purpose. The size can ac-

odours and attract dogs or rodents, ant, rat

cording to the availability of organic

and bird. Thus, preventive measures

waste (the shed can be constructed in the

should be taken like net or anti-insect

same way as said for vermiculture).

propellants to avoid them.

_3.3. Pit method_

_3.5. Harvesting Vermicompost_

A

pit

of

approximately

Vermicompost can be harvested fol-

4m×6m×4m (breath× length× depth) must

lowing the same method as that of ver-

be constructed. Vermibed is actually con-

miculture, using fresh cow dung balls and

structed with a layer of loamy soil placed

heap method. Some consumers are picky

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in getting the pure form of vermicompost,

 It neutralizes the soil acidity or alka-

i.e. the vermicasts, which are collected on

linity.

the top of vermibed. At that case, ver-

 Vermicompost is free from patho-

micasts can be collected with a spatula

gens, toxic elements, weed seeds

once in a week and can be packed and

etc.

sold. The rate of pure form of vermi-

 Vermicompost minimizes the inci-

castings is also higher when compared to

dence of pest and diseases.

the vermicompost. When large amount of

 It contains valuable vitamins, en-

compost are to be harvested, for example

zymes and hormones like auxins,

the compost obtained from tank method

gibberellins etc.

or from pit method, first the worms are

 The nutrients available in ver-

harvested with the help of fresh cow dung

micompost (in general) are Organic

balls, then the whole material is moved to

carbon (9.5 – 17.98%), Nitrogen

a plain area or on a plastic sheet and made

(0.5 – 1.50%), Phosphorous (0.1 –

into a single heap and is exposed to light,

0.30%), Potassium (0.15 – 0.56%),

scooped, sieved and packed for sales.

Sodium (0.06 – 0.30%), Calcium

Earthworms or undecomposed materials

and Magnesium (22.67 to 47.60

if seen any, are collected and returned to

meq/100g), Copper (2 – 9.50 mg

the compost pile.

kg-1), Iron (2 – 9.30 mg kg-1), Zinc

For faster rate of harvesting ver-

(5.70 – 11.50 mg kg-1) and Sulphur

micompost, the original heap is better di-

(128 – 548 mg kg-1).

vided into several smaller pyramids. To

enhance the earthworm movement to-

_3.7. Storing and packing of vermicompost_

wards the centre of the heap, ball of raw

Watering is stopped for atleast 5

or fresh cow dung can be placed at the

days at this stage. The first lot of Ver-

centre. Always earthworms have high af-

micompost is ready for harvesting after 2-

finity towards fresh cow dung. Thus after

2 ½ months and the subsequent lots can

few hours the worms can be easily sepa-

be harvested after every 6 weeks of load-

rated and the compost can be harvested

ing. The vermibed is loaded for the next

and packed.

treatment cycle. The harvested ver-

micompost should be stored in dark, cool

_3.6. Advantages of vermicompost_

place. It should have minimum 40%

 Vermicompost is rich in all essential

moisture. Sunlight should not fall over the

plant nutrients, hence provide excel-

composted material. It will lead to loss of

lent effect on overall plant growth,

moisture and nutrient content. It is advo-

encourages the growth of new

cated that the harvested composted mate-

Shoots / leaves

rial is openly stored rather than packed in

 Vermicompost is free flowing, easy

over sac. Packing can be done at the time

to apply, handle and store and does

of selling. If it is stored in open place, pe-

not have bad odour.

riodical sprinkling of water may be done

 It improves soil structure, texture,

to maintain moisture level and also to

aeration, and water holding capaci-

maintain beneficial microbial population.

ty.

If the necessity comes to store the materi-

 Vermicompost is rich in beneficial

al, laminated over sac is used for packing.

microorganisms which fix the Ni-

This will minimize the moisture evapora-

trogen and Phosphorous in the soil.

tion loss. Vermicompost can be stored for

 Vermicompost may contain earth-

one year without loss of its quality, if the

worm cocoons from which juveniles

moisture is maintained at 40% level.

may come and increases in popula-

tion in the soil where applied.

**4. Vermiwash**

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Vermiwash is the liquid bio-

rel setup while 100-200 earthworms is

fertilizer collected after the passage of

enough for a bucket. Keep a pot at the

water through a column of worms. It is

bottom of the stop cork of the bucket so

very useful as a foliar spray. It is a collec-

that waterfalls drop by drop. Every day

tion of excretory products and excess se-

30-40 litres in barrel and about 3-4 liters

cretions of earthworms along with micro-

of vermiwash in the bucket setup can be

nutrients from soil organic molecules.

collected.

Vermiwash units can be set up in a plastic

As the tap is closed and water is

or iron barrel of 200 litre capacity for a

sprinkled on top of the unit, the water

large scale production. While for produc-

slowly percolates through the compost

tion in small scale, a plastic bucket of 15

carrying with it nutrients through the filter

litre capacity is enough.

unit. When the tap is opened after two

days, vermiwash is collected, which is

_4.1._ **** _Method of preparation_

sprayed on plants as a foliar spray. The

The holding unit, i.e. the plastic

vermicasts formed on the surface of the

barrel must be taken and fixed in a stand

unit may also be collected periodically.

or on a high platform. A hole is drilled on

one side at the bottom and a vertical limb

_4.2._ **** _Application_

of a T joint tube is attached in a way that

The vermiwash may be diluted

half to one inch of the tube projects inside

with water in 1:1 ratio or it may be dilut-

the barrel. A tap is attached to the end of

ed with 10 per cent cow"s urine, which is

the horizontal limb and the other end is

an effective growth tonic and pesticide.

closed with a dummy nut. The whole set

Mix 1 liter of vermiwash with 7-10 liters

up is mounted on a suitable pedestal.

of water and spray the solution on the leaf

Keeping the tap open, a layer of broken

(upper lower side) in the evening at the

bricks or pebbles is filled up to 25-30cm

growing crop. Mix 1 liter of vermiwash

inside the barrel. Water is made to flow

with 1 litre of cow urine and then add 10

through this layer, followed by 20-30 cm

liters of water and mix thoroughly and

layer of coarse sand. This forms the basic

keep it over night before spraying. 50-60

filtering unit. Over this a 60-75 cm layer

litres of such solution can be sprayed in

of good loamy soil along with organic

one hectare of land to control various

waste and cow dung is kept moistened. In

crop diseases.

this layer earthworms like, _E. fetida, E._

_eugeniae and lumbricus terrestris_ may be

**5. Perspectives**

introduced. Cattle dung pats and hay are

placed on top of this layer of soil for

As a processing system, the ver-

mulching purpose. The unit is moistened

micomposting of organic waste is very

every day. For two days after the intro-

simple. Worms ingest the waste material -

duction of earthworms the tap must be

break it up in their rudimentary gizzards,

kept closed. After two days, open the tap,

consume the digestible, putrefiable por-

about 30 – 40 litres of vermiwash drains

tion and then excrete a stable, humus-like

out.

material that can be immediately market-

Similar setup can also be made in

ed and has a variety of documented bene-

bucket for production in small scale. The

fits to the consumer. Vermitechnology is

layer of organic matter here last for 30-45

a promising technique that has shown its

cm thickness and the mulching of 2-3 cm

potential in certain challenging areas like

thicknesses must be spread to prevent

augmentation of food production, waste

evaporation. Spray water regularly for 7-8

recycling, management of solid wastes

days for both of the setup so that 60% of

etc. In most of the countries, soil pollu-

moisture is maintained. Introduce 1000-

tion is increasing due to accumulation of

2000 numbers of earthworms for the bar-

organic wastes and on the other side there

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_Biotech Sustainability (2017)_

_Vermitechnology – An Eco-Biological Tool for Sustainable... Moorthi et al._

is shortage of organic manure. Organic

vermicomposting of

different

waste could be converted into vermicom-

types of organic substrates. _Envi-_

post using Vermitechnology to increase

_ronmentalist_ **29, 287–300.**

the fertility and productivity of the agri-

**Kaur, A., Singh, J., Vig, A.P., Dhaliwal,**

cultural land and to produce nutritive and

**S.S., and Rup, P.J. (2010).** Co-

safe food. Hence we need to use and

composting with and without _Ei-_

promote this ecofriendly technology for

_senia fetida_ for conversion of

the sustainability of agriculture and envi-

toxic paper mill sludge to a soil

ronment.

conditioner. _Bioresource Tech-_

_nology_ **101, 8192–8198.**

**References**

**Logsdon, G. (1994).** Worldwide progress

****

in vermicomposting. _Biocycle_

**Edwards, C. A. (1988).** Breakdown of

**35(10),63-5.**

animal, industrial and organic

**Madan, M., Sharma, S., Bisaria, R. and**

wastes

by

earthworms.

In:

**Bhamidimarri, R. (1988).** Recy-

_Earthworms in Waste and Envi-_

cling of organic wastes through

_ronment Management._ Edwards

vermicomposting and mushroom

CA, Newhauser EF (Eds). __ SPB

cultivation.

_Alternative_

_waste_

Academic

Publishing,

The

_treatment systems_ **132-141.**

Hague. **pp. 21 – 31.** __

**Mitchell, M. J., Hornor, S. G. and**

**Edwards, C. A. and Bater, J. E. (1992).**

**Abrams, B. I. (1980).** Decompo-

The use of earthworms in envi-

sition of sewage sludge in drying

ronmental Management, soil biol.

beds and the potential role of the

_Biochemical Journal_ **24 (12),**

earthworm, _Eisenia fetida._ _Jour-_

**pp.1683-1689.**

_nal of Environmental Qualilty_ **9,**

**Hand, P., Hayes, W. A., Satchell, J. E.,**

**373-378.**

**Frankland, J. C., Edwards, C. A.**

**Singh, A. and Sharma, S. (2002).** Com-

**and Neuhauser, E. F. (1988).** The

posting of a crop residue through

vermicomposting of cow slurry.

treatment with microorganisms

_Pedobiologia_ **31,** __**49-63.**

and subsequent vermicompost-

**Follet, R., Donahue, R. and Murphy, L.**

ing.

_Bioresource_

_Technology_

**(1981).** _Soil and Soil Amendments._

**85:107-11.**

Prentice- hall. Inc., New Jersey.

**Sundaravadivel, S. and Ismail, S. A.**

**John Paul, J.A. (2005).** Municipal solid

**(1995).** Efficacy of a biological

waste generation, characteriza-

filter unit in the treatment of dis-

tion, microbial activity, ver-

tillery effluents. _Journal of Eco-_

micomposting and management

_toxicology and Environmental_

in Dindigul Town. Ph.D. Thesis.

_Monitoring_ **5(2), 125-9.**

The Gandhigram Rural Institute –

**Suthar, S. (2009).** Vermicomposting of

Deemed University, Gandhigram,

vegetable-market solid waste us-

Tamil Nadu, India.

ing _Eisenia fetida_ : Impact of

**John Paul., Karmegam, N. and Daniel,**

bulking material on earthworm

**T. (2011).** Municipal solid waste

growth and decomposition rate.

(MSW) vermicomposting with an

_Ecological Engineering_ **35, 914–**

epigeic

earthworm,

_Perionyx_

**920.**

_ceylanensis_ Mich. _Bioresource_

**Suthar, S. (2010).** Recycling of agro-

_Technology_ **102, 6769–6773.**

industrial sludge through ver-

**Karmegam, N. and Daniel, T. (2009).**

mitechnology. _Ecological Engi-_

Investigating efficiency of _Lam-_

_neering_ **36, 1028–1036.**

_pito mauritii_ (Kinberg) and _Peri-_

**Updegraff, D.M. (1971).** Utilization of

_onyx ceylanensis_ Michaelsen for

cellulose from waste paper by

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 49

_Biotech Sustainability (2017)_

_Vermitechnology – An Eco-Biological Tool for Sustainable... Moorthi et al._

Myrothecium verrucaria. _Bio-_

**13, 77–97.**

_technology and Bioengineering_

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 50

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P51-57_

**Role of Biotechnology in Food Authentication**

****

**Shobana Manoharan1, Raghavan Kuppu1 and Ramesh Uthandakalaipandian2,***

****

_1Department of Molecular Biology, School of Biological Sciences, Madurai Kamaraj Uni-_

_versity, Madurai - 625021, Tamil Nadu, India; 2Assistant Professor, Department of Mo-_

_lecular Biology, School of Biological Sciences, Madurai Kamaraj University, Madurai -_

_625021, Tamil Nadu, India; *Correspondence: ramesh.biological@mkuniversity.org; Tel:_ __

_+91-9489014892_

****

****

**Abstract:** Food is the most essential component of life for survival. The quality of food

products is essentially to be authenticated as they are closely related to the health of human

beings. "Foodomics", is a concept to utilize technology for improvement of food and nutri-

tion. Food products are authenticated in benefit of both consumers as well as commercial

traders. Food authentication is done by analytical techniques like chromatography, Fourier

Transform Infrared Spectroscopy (FTIR), Nuclear Magnetic Resonance Spectroscopy

(NMR), Gas Chromatography - Mass Spectroscopy (GC-MS) and Liquid Chromatography

\- Mass Spectroscopy (LC-MS). Plant or animal based food products are widely authenticat-

ed for their origin, species nomenclature using DNA barcodes - a genomics based approach.

The protein molecules of food products serve as key molecules for authentication using

proteomics approach such as 2-dimensional gel electrophoresis and 2-dimensional differ-

ence gel electrophoresis (DIGE). Thus, food authentication is mandatory to substantiate the

geographical origin, species nomenclature, food composition, genetic modification of a

food product that reaches to the hands of consumers in the food markets. Thus, the devel-

opment of rapid, novel food authentication methods to validate sea food products, Genet-

ically Modified (GM) food products based on biotechnological approaches helps to provide

quality assured food products to the human community. This review briefs about the vari-

ous techniques employed in food authentication, their advantages and applications in food

biology.

****

_**Keywords**_ **:** Analytical techniques; food authentication; genomics; proteomics

****

****

**1. Introduction**

objective, the optimization of human

****

****

health and well-being" (Capozzi and Bor-

Nowadays wide range of food

doni, 2013). According to Hippocrates

products are available from various coun-

"Let food be thy medicine and medicine

tries to the consumers but the quality of

be thy food"; hence, the ratification of

the food consumed is of great concern as

food components becomes vital to shield

they are directly linked to human health.

human health. Development of quality

"Foodomics" is a new term coined at the

assurance methods to authenticate the

International Conference, Cesena (2009),

food products based on their geographical

Italy (foodomics.eu) which deals with the

origin, composition, originality, certifica-

application of the recent "omics" technol-

tion of their species in case of animal or

ogy to promote the field of food and nu-

plant derived products are of prodigious

trition. Foodomics is thus defined as, "a

attention from both consumer and com-

new approach to food and nutrition that

mercial trader"s opinion. The need for

studies the food domain as a whole with

food authentication and its application

the nutrition domain to reach the main

differs from country to country. For ex-

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_Biotech Sustainability (2017)_

_Role of Biotechnology in Food Authentication Shobana et al._

ample, geographic origin of the food is a

Food labelling is a common practice

principal authenticating criterion to be

to be followed in order to approve the

mandatorily confirmed in Europe, numer-

originality of food products bought in

ous schemes for food quality analysis like

hand to the consumers. Food authentica-

PDO (Protected Designation of Origin),

tion is the process of confirmation a food

PGI (Protected Geographical Indication)

product for its originality as described in

and TSG (Traditional Specialities Guar-

their labels (Figure 1). This process has

anteed) are also followed by them in order

grabbed wide attention because of the up-

to look after the conventional production

surging perception among the people

methods (Drivelos and Georgiou, 2012).

about food quality and safety (Danezis _et_

There are a huge number of valid reports

_al.,_ 2016). Food authentication is mainly

till date stating about the adulterants, sub-

done at the following circumstances,

stitutes and confused species in herbal

_i._ Validation of imported traditional

formulations as like the recounted in-

foods,

stances of Chinese herbs that caused se-

_ii._ Identification of Genetically Mod-

vere intoxications and even deaths due to

ified Products (GMPs) from unau-

adulterants or substitutes (But, 1994).

thorised GMP"s,

Thus, food authentication is indispensa-

_iii._ Identification on adulterants or

ble, in earlier days it was carried out

substitutes,

based on analytical chemistry techniques.

_iv._ Corroboration of medicinally im-

Conversely, in recent days it is accom-

portant plant species that may con-

plished by various other methods like ge-

tain confused species,

nomics, proteomics and metabolomics for

_v._ Verification

of

Geographical

the reason that food authentication being

origin of plant or animal derived

a multi-disciplinary research that utilizes

food products (Figure 2).

data from biology, chemistry, chemomet-

rics and bioinformatics (Ibanez _et al.,_

**3. Chemometric methods**

2013; Moore _et al.,_ 2012).

****

****

Chemometric methods comprises

**2. Food authentication**

of chromatographic and spectroscopic

methods. Chromatographic analysis sepa-

Food Labelling

Food Authentication

Food Composition Database

****

**Figure 1:** Important steps involved in developing a database for food industry.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 52

_Biotech Sustainability (2017)_

_Role of Biotechnology in Food Authentication Shobana et al._

Food

Food Product

Authentication

Chromatographic

techniques

Adulteration

DNA Barcodes

Species

Proteome

Confusion

analysis

****

**Figure 2:** Importance of food authentication.

****

rates analogous chemical compounds in

ly facilitate the identification of individual

food products. Food is composed of pro-

molecular components from which unique

teins, lipids, carbohydrates, phytochemi-

marker compounds for a food may be

cals and many other small molecules such

identified. Nuclear Magnetic Resonance

as food additives, colourants and preserv-

Spectroscopy (NMR) analysis is a fast

atives. In general, these compounds are

and more reliable technique to profile the

chemically distinct based on their molecu-

complete metabolome of a food. The

lar weight, polarity, charge etc., Devel-

peaks pertaining to the functional groups

opment of fingerprint pattern using chro-

are analysed and unique peaks are used

matography (High Performance Liquid

for differentiating the food in terms of

Chromatography - HPLC) to differentiate

quality (Cozzolino, 2012; Longobardi _et_

food free from adulterants or constituents

_al.,_ 2013; Hohmann _et al.,_ 2015). Proton

succours in food labelling (Cserhati _et al.,_

Transfer Reaction Mass Spectroscopy are

2005; Reinholds _et al.,_ 2015; Georgiou

used for analysis of volatile organic com-

and Danezis, 2015).

pounds, reports based on these techniques

Fourier Transform Infrared Spec-

distinguish organically grown tomatoes

troscopy (FTIR) analysis can be done for

from conventional ones (Hohmann _et al.,_

the functional assessment of nominal fre-

2015). In few instances, highly complex

quency wave number obtained from the

food products are difficult to be differen-

metabolites studied. They identify the

tiated using HPLC, FTIR or NMR at such

presence of fatty acids and other metabo-

rationale Gas Chromatography (GC) or

lites of the food and help in development

Liquid Chromatography (LC) coupled to

of the FTIR fingerprint region which can

Mass Spectrometry (MS), have flourished

serve as a unique, preliminary tool to in-

as better food authentication tools (Figure

dicate the nutritional index of the food.

3). Few applications of chemometric

Further analysis of metal concentrations

methods are sated below,

can be done using Atomic Absorption

i.

Authentication of wild, farmed

Spectra (AAS) or Inductively Coupled

fish food species (Capuano _et_

Plasma Mass Spectroscopy (ICP - MS).

_al.,_ 2012),

Fatty acids and Protein analysis using LC

ii.

Fatty acid content confirma-

\- MS and GC - MS techniques respective-

tion in animal and plant ex

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 53

_Biotech Sustainability (2017)_

_Role of Biotechnology in Food Authentication Shobana et al._

**HPLC**

**Barcode**

**FTIR**

**Food**

**Authentication**

**DGGE**

**NMR**

**GC-MS**

****

**Figure 3:** Techniques involved in food authentication. ****

tracted oils (Yang _et al.,_

formulations are mostly in processed or

2013),

powdered form, in those cases mini DNA

iii.

Validation of mineral content

barcodes are employed for authentication.

in eggs (Giannenas _et al.,_

And most of the food products of animal

2009).

and plant origin are in processed forms

where the food source cannot be verified

**4. Genomic methods**

by other chemometric methods and in

****

****

such circumstances DNA barcodes play a

This approach involves the use of

vital role in validation of plant or animal

genetic material, Deoxyribonucleic Acids

species used in manufacturing of a food

(DNA) either as complete genome for

product. DNA barcodes are short, con-

evaluation or amplification of signature

served sequences that are employed in

DNA sequences to authenticate a food

molecular taxonomy classification of

product. Molecular techniques such as

plants and animal"s species. Cytochrome

Random Amplified Polymorphic DNA

Oxidase I [CO I] gene of mitochondrial

(RAPD), Restriction Fragment Length

(mt) DNA is a universally acceded mo-

Polymorphism (RFLP), Inter Simple Se-

lecular marker used for DNA barcoding

quence Repeats (ISSR), Simple Sequence

in animals (Hebert _et al.,_ 2003). In plants,

Repeats (SSR), Sequence Characterized

maturase K (mat K) and RubisCO L

Amplified Region (SCAR) use the ge-

(Ribulose 1, 5 bisphosphate carboxylase /

nome variation in certification of food

oxygenase) gene abet in barcoding (Jan-

products (Ali _et al.,_ 2014). Meta DNA

zen _et al.,_ 2009).

barcoding, _de novo_ sequencing and Next

Generation Sequencing are some of the

**5. Proteomic methods**

techniques that lead to the high through-

****

put sequencing of entire genome. Dena-

****

Food proteomics research helps in

turing Gradient Gel Electrophoresis

analysis of individual marker protein

(DGGE) is used in microbial food author-

components from mixture of proteins us-

ization such as cheese, curd, probiotic yo-

ing Polyacrylamide gel electrophoresis

ghurts and other fermented dairy products

(1D or 2D) along with LC - MS for pro-

(Arcuri _et al.,_ 2013). Commercial herbal

tein characterisation. Characterisation of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 54

_Biotech Sustainability (2017)_

_Role of Biotechnology in Food Authentication Shobana et al._

**Table 1:** Applications of food authentication ****

**No Food Product**

**Authentication Method**

**Source**

1.

Olive Oil

GC - MS

Yang _et al.,_ 2013

2.

Eggs

ICP - MS

Giannenas _et al.,_ 2009

3.

Salmon

1H NMR

Capuano _et al.,_ 2012

4.

Tomatoes

Proton Mass Spectroscopy

Hohmann _et al.,_ 2015

5.

Italian Sweet Cherry

NMR

Longobardi _et al.,_ 2013

6.

_Nigella sativa_ seed oil

FTIR

Nurrulhidayah _et al.,_ 2011

7.

Cheese

16s rDNA PCR and DGGE

Arcuri _et al.,_ 2013

genetically modified products, soy pro-

databases will be fruitful for high

teins in foods and dairy products are done

throughput validation of food products.

based on proteomic analysis (Gallardo _et_

_al.,_ 2013). This proteomics based ap-

**Acknowledgement**

proach is of wide importance and applica-

tions in food industry as they accurately

Authors are thankful to the Educa-

pin point the difference between original

tional

fellowship,

UGC-NON-NET

and adulterated food products.

Scheme. Authors are also thankful to the

State-of-art infrastructure facility provid-

**6. Immunological methods**

ed by CEGS, School of Biological Sci-

****

ences, Madurai Kamaraj University, In-

****

Immunological methods rely on

dia.

the binding specificity of the antibodies

designed to an atypical antigen _i.e.,_ aller-

**References**

gens, toxins etc. in food by use of En-

****

zyme Linked Immuno Sorbent Assay

**Ali, M. E., Razzak, M. A., and Hamid,**

(ELISA) (Asensio _et al.,_ 2008). For in-

**S. B. A. (2014).** Multiplex PCR in

stance, they are used to spot the presence

species authentication: probability

of potential fish allergen parvalbumin

and prospects - a review. _Food Ana-_

(Gajewski and Hsieh _,_ 2009) in seafood

_lytical Methods_ , **7(10), 1933-1949.**

industry. These techniques rely on speci-

**Arcuri, E. F., El Sheikha, A. F., Rych-**

ficity and also have an added advantage

**lik, T., Piro Metayer, I., and Mon-**

of validating a large number of samples

**tet, D. (2013).** Determination of

with high precision in a short span of

cheese origin by using 16S rDNA

time.

fingerprinting of bacteria communi-

ties by PCR–DGGE: Preliminary

**7. Perspectives**

application to traditional Minas

****

cheese. _Food Control_ , **30(1), 1-6**.

****

Food authentication not only en-

**Asensio, L., Gonzalez, I., Garcia, T.,**

sures food quality but also assures human

**and Martin, R. (2008).** Determina-

health. **** Emergence of new research fields

tion of food authenticity by enzyme-

such as transgenics, foodomics, nutri-

linked immunosorbent assay (ELI-

genomics has resulted in wide range of

SA). _Food Control_ , **19(1), 1-8.**

neoteric products that need to be validated

**But P. P. H. (1994).** Herbal poisoning

for substitutes prior to their distribution in

caused by adulterants or erroneous

commercial markets for edible purpose.

substitutes. _Journal of Tropical_

Some of the practical applications of food

_Medicine and Hygiene_ , **97,371–374.**

authentication based on the above-

**Capozzi, F., and Bordoni, A. (2013).**

mentioned techniques are tabulated in Ta-

Foodomics: a new comprehensive

ble 1. Construction of food authentication

approach

to

food

and

nutri-

tion. _Genes and nutrition_ , **8(1), 1-4.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 55

_Biotech Sustainability (2017)_

_Role of Biotechnology in Food Authentication Shobana et al._

**Capuano, E., Lommen, A., Heenan, S.,**

trometry (ICP-MS). _Food Chemis-_

**de la Dura, A., Rozijn, M., and**

_try_ , **114(2), 706-711.**

**van Ruth, S. (2012)**. Wild salmon

**Hebert, P. D., Cywinska, A., and Ball,**

authenticity can be predicted by 1H‐

**S. L. (2003).** Biological identifica-

NMR spectroscopy. _Lipid Technol-_

tions

through

DNA

bar-

_ogy_ , **24(11), 251-253.**

codes. _Proceedings of the Royal So-_

**Cozzolino, D. (2012).** Recent trends on

_ciety of London B: Biological Sci-_

the use of infrared spectroscopy to

_ences_ , **270(1512), 313-321.**

trace and authenticate natural and

**Hohmann, M., Monakhova, Y., Erich,**

agricultural food products. _Applied_

**S., Christoph, N., Wachter, H.,**

_Spectroscopy Reviews_ , **47(7), 518-**

**and Holzgrabe, U. (2015).** Differ-

**530.**

entiation of organically and conven-

**Cserhati, T., Forgacs, E., Deyl, Z., and**

tionally grown tomatoes by chemo-

**Miksik, I. (2005).** Chromatography

metric analysis of combined data

in authenticity and traceability tests

from proton nuclear magnetic reso-

of vegetable oils and dairy products:

nance and mid-infrared spectrosco-

a review. _Biomedical Chromatog-_

py and stable isotope analy-

_raphy_ , **19(3), 183-190.**

sis. _Journal of Agricultural and_

**Danezis, G. P., Tsagkaris, A. S., Camin,**

_Food Chemistry_ , **63(43), 9666-9675.**

**F., Brusic, V., and Georgiou, C. A.**

**Ibanez, C., Garcia Canas, V., Valdes,**

**(2016).** Food authentication: tech-

**A., and Simo, C. (2013).** Novel

niques, trends and emerging ap-

MS-based approaches and applica-

proaches. _TrAC Trends in Analytical_

tions in food metabolomics. _Trends_

_Chemistry_ , **85, 123-132.**

_in Analytical Chemistry_ , **52, 100-**

**Drivelos, S. A., and Georgiou, C. A.**

**111.**

**(2012).** Multi-element and multi-

**Janzen, D. H., Hallwachs, W., Blandin,**

isotope-ratio analysis to determine

**P., Burns, J. M., Cadiou, J., Cha-**

the geographical origin of foods in

**con, I., and Franclemont, J. G.**

the European Union. _Trends in Ana-_

**(2009).** Integration of DNA barcod-

_lytical Chemistry_ , **40, 38-51.**

ing into an ongoing inventory of

**Gajewski, K. G., and Hsieh, Y. H. P.**

complex

tropical

biodiversi-

**(2009).** Monoclonal antibody specif-

ty. _Molecular_

_Ecology_

_Re-_

ic to a major fish allergen: parval-

_sources_ , **9(s1), 1-26**.

bumin. _Journal of Food Protec-_

**Longobardi, F., Ventrella, A., Bianco,**

_tion_ , **72(4), 818-825.**

**A., Catucci, L., Cafagna, I., Gallo,**

**Gallardo, J. M., Ortea, I., and Carrera,**

**V., and Agostiano, A. (2013).** Non-

**M. (2013).** Proteomics and its appli-

targeted 1 H NMR fingerprinting

cations for food authentication and

and multivariate statistical analyses

food-technology research. _Trends in_

for the characterisation of the geo-

_Analytical Chemistry_ , **52, 135-141.**

graphical origin of Italian sweet

**Georgiou, C. A., and Danezis, G. P.**

cherries. _Food_

_Chemistry_ , **141(3),**

**(2015).** Elemental and isotopic mass

**3028-3033.**

spectrometry. _Comprehensive Ana-_

**Moore, J. C., Spink, J., and Lipp, M.**

_lytical Chemistry_ , **131-143**.

**(2012).** Development and applica-

**Giannenas, I., Nisianakis, P., Gavriil,**

tion of a database of food ingredient

**A., Kontopidis, G., and Kyria-**

fraud and economically motivated

**zakis, I. (2009).** Trace mineral con-

adulteration

from

1980

to

tent of conventional, organic and

2010. _Journal_

_of_

_Food_

_Sci-_

courtyard eggs analysed by induc-

_ence_ , **77(4), R118-R126.**

tively coupled plasma mass spec-

**Nurrulhidayah, A. F., Man, Y. B., Al-**

**Kahtani, H. A., and Rohman, A.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 56

_Biotech Sustainability (2017)_

_Role of Biotechnology in Food Authentication Shobana et al._

**(2011).** Application of FTIR spec-

contaminants in condiments: A re-

troscopy coupled with chemomet-

view. _Journal of Food Composition_

rics for authentication of _Nigella sa-_

_and Analysis_ , **44, 56-72.**

_tiva_ seed oil. _Journal of Spectrosco-_

**Yang, Y., Ferro, M. D., Cavaco, I., and**

_py_ , **25(5), 243-250.**

**Liang, Y. (2013).** Detection and

**Reinholds, I., Bartkevics, V., Silvis, I.**

identification of extra virgin olive

**C., van Ruth, S. M., and Essling-**

oil adulteration by GC-MS com-

**er, S. (2015).** Analytical techniques

bined with chemometrics. _Journal_

combined with chemometrics for

_of Agricultural and Food Chemis-_

authentication and determination of

_try_ , **61(15), 3693-3702.**

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 57

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P58-67_

**Management Strategies against Tiny Tigers for**

**Sustainable Development of Agriculture**

****

**Viswa Venkat Gantait***

****

_Zoological Survey of India, M-Block, New Alipore, Kolkata-700053, West Bengal, India;_

_*Correspondence: v.gantait@rediffmail.com; Tel:_ _09433463555_

****

****

**Abstract:** Amongst different pests of agriculture, the plant parasitic nematodes are consid-

ered the worst one because of devastations they cause to the crops. Due to their microscopic

nature, they are the hidden enemies of soil and crop damages caused by them have not been

fully formulated. For sustainable development of agriculture and to stop the yield loses

causes by these tiny pests of crops, potential control measures should be explored and

adopted against them. By following various physical, chemical, biological and botanical

methods, some of cultural practices and also by regulatory methods nematode infestation in

agricultural fields must be stopped or checked at certain level. This will help to enhance

crop production, which will ultimately be helpful to improve the gross national product

(GNP) of country and agricultural sustainability. This article provides an overview of man-

agement strategies which could be used against plant parasitic nematodes to boost the sus-

tainable development of agriculture. ****

****

_**Keywords**_ **:** Agriculture; control measure; plant parasitic nematode; sustainable development

****

**1. Introduction**

the phylum Platyhelminthes and Annelida

****

in the animal kingdom. They generally

_1.1. What are tiny tigers?_

have a cylindrical body while a few may

Tiny tigers! How funny the term

be fusiform, saccate or kidney-shaped.

is? Tiger is one of the most ferocious car-

Those are characterized by having a body

nivores of the world. Not a joke, the nem-

cavity, complete digestive tract, well de-

atodes are now treated as tiny tigers in

veloped reproductive system, excretory

crop fields. Even, these are more harmful

and nervous system; but lacking circula-

than tigers, as far as agriculture is con-

tory and respiratory system. Most of them

cerned. They represent one of the most

are microscopic in size, but may be seen

abundant groups and probably the second

with naked eyes.

largest one in the animal kingdom, imme-

diately behind the arthropods (Hugot _et_

_1.2. Where we can find them?_

_al._ , 2001).

Nematodes are highly diverse in

Nematodes represent sharply dif-

their habitats ranging from Himalayan

ferentiated primitive group of inverte-

peak to the sea floor, from Arctic to Ant-

brates, commonly known as round

arctic (Lal, 1998). They can withstand

worms, thread worms or eelworms, dis-

extreme adverse environmental condi-

tinctly different from segmented worms

tions and may be found in Polar Regions

like earthworms and flatworms. Simply,

to tropics. They occur almost everywhere

these are also called 'nemas'. These are

on the earth like in ocean, river, lake,

multicellular, vermiform, triploblastic,

pond, estuary, island, soil, hill and rocks,

pseudocoelomate, bilaterally symmetrical,

desert hot springs etc. Interestingly, their

unsegmented animal, possessed between

habitat is unsurpassed by any other meta-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 58

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ zoan invertebrate group, because they are

where fine plant roots are concentrated;

found in all types of habitats (Bohra and

adequate soil moisture and oxygen also

Baqri, 1997). They are so numerous that

favours dense populations of nematodes.

if everything on the earth were to disap-

pear except the nematodes, the outlines of

_1.3. What they feed?_

habitats would still be dimly visible; the

Nematodes

are

microphagous,

mountains, lakes and oceans, the plants

mycetophagous, saprophytic, phytopara-

and the animals would all be outlined by

sitic, predatory, carnivorous and even

the nematodes present (Cobb, 1914).

cannibalistic in nature. Most of them feed

Based on the habitat, the total

on bacteria, fungi, other microorganisms

nematode population can be categorized

and decaying matter. They can parasitize

into three groups: marine, animal-

plants, different animals including man

parasitic, soil and freshwater nematodes

and even other nematodes also. Depend-

(Ayoub, 1980). The marine nematodes

ing on the diverse diets Yeates _et al._

constitute about 50% of the total nema

(1993) categorized nematodes into eight

population, where as animal-parasitic

feeding groups: plant feeders, algal feed-

nematodes make up only 15% of the

ers, hyphal feeders, substrate ingesters,

known nematode species. The soil and

unicellular eukaryote feeders, bacterial

freshwater nematodes can be spitted into

feeders, predators and omnivorous.

two finer divisions: free-living and plant-

****

parasitic. Free living nematodes compris-

**2. Important roles in agricultural as-**

ing about 25% of the total nematode pop-

**pects**

ulation. The plant-parasitic nematodes

****

constitute only 10% of the total nema-

Plant parasitic and soil-inhabiting

todes (Figure 1).

nematodes have been known for their vir-

According to Crofton (1966) most

ulence causing significant loss to agricul-

of the soil holds about 90% nematodes at

tural and horticultural crops (Bohra and

top six inches. (Nicholas, 1984) opined

Baqri, 2004). Several species have come

that the soil nematodes are usually most

to be recognized as useful predators in the

abundant near the soil surface, with the

control of different insects and nematodes

majority within the top 10 cm, though

also. The possibility of using nematodes

some may be found much deeper. He also

for control of different plant parasitic

stated that, populations are much denser

nematodes was first suggested by Cobb

in the zones, rich in organic matter or

(1917). The role of predatory nematodes

****

__

**Figure 1** : Pie diagram

showing the different

groups of nematodes.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 59

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ in biological control of plant parasitic

 They can also act as modifiers of

nematodes was described by Jairajpuri _et_

host substrates and render them more

_al._ (1990). They regulate microbial bio-

susceptible to other plant pathogens.

mass and nitrogen mineralization by kill-

ing and feeding on nematodes and other

**3. Important symptoms of their attack**

microorganisms that pass from bottom to

****

top tropic levels in an ecosystem (Wardle

****

Being protected under soil and

and Yeates, 1993). Some species are

having microscopic size, nematodes are

known to play a vector role in transmit-

practically the hidden enemies of crops.

ting so many soil-borne bacterial, fungal

Symptoms of their attacking are not strik-

as well as viral pathogens to their hosts

ing in most cases and therefore over-

(Jairajpuri and Ahmad, 1992). The innoc-

looked. The important symptoms may be

uous nematodes, those have no concern to

tabulated as follows (Table 1).

the farmer or gardener; those feed on bac-

teria, fungi, algae and even other nema-

**4. Common names of some important**

todes, play an important role in control-

**nematodes**

ling soil nutrient cycling (Tahseen, 2006).

****

Each species possibly plays a significant

The plant parasitic nematodes be-

role in the ecosystem inhabits and un-

longing to the order Dorylaimida are mi-

doubtedly has a major role in maintaining

gratory root ectoparasitic in nature. The

the natural ecological balance. Nematodes

genera, _Trichodorus_ and _Paratrichodorus_

are intimately involved in many parts of

under the family Trichodoridae browse

the soil ecosystem, so they can be used as

along the root surface of plant. On the

bioindicators of sustainability for soils.

other hand, genera like _Longidorus,_ _Pa-_

Because of their ubiquity and diversity,

_ralongidorus_ and _Xiphinema_ feed for

nematodes are used in measuring the im-

longer period at specific sites of deeper

pact of various perturbations on ecosys-

root tissues. Apart from causing direct

tems, such as pollution, organic enrich-

minor or major root damages, these

ment and physical disturbance (Tahseen,

nematodes are of great economic im-

2006).

portance as vectors of nearly 22 soil-

borne plant viruses. All the Tylenchids

_2.1. How they depress crop yields_

i.e. the nematodes belonging to the order

Plant parasitic nematodes depress

Tylenchida are totally plant parasites.

crop yields by the following important

They are ectoparasitic, semi-endoparasitic

ways.

or endoparasitic in nature. Most of the

 They feed on plant parts and deprive

endoparasitic nematodes form root-galls

the host of its nutrients.

and leaf-galls of plants. The common

 During feeding they cause mechani-

names of some important plant parasitic

cal injury to different parts of plants

nematodes are as follows.

and their feeding sites serve as entry-

points of other pathogenic fungi and

**5. Management strategies**

bacteria.

****

 Act as vectors of different fungal,

For sustainable development of

bacterial and viral pathogens of

agriculture, the adverse effects of nema-

plants.

todes have to be eradicated or minimized.

 They can secret various enzymes in

For

this

purpose

different

control

the plant tissues during feeding and

measures should be adopted against them.

the effected plants show abnormal

The first and foremost effort is to reduce

growth responses to these secre-

increasing populations of these hidden

tions/excretions like hypertrophy.

enemies of crops, under the soil. The di-

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 60

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ **Table 1:** Symptoms of nematode infestation

**Symptoms**

**Caused by the species**

**Produced by above-ground feeders**

 Crinkled and distorted stems and foliages

_Anguina_ _spp_.

 Dead and devitalized buds

_Aphelenchoides spp_.

 Leaf lesions

_Aphelenchoides besseyi_

 Leaf and seed galls

_Anguina_ _spp_.

 Necrosis and discoloration

_Rhadinaphelenchus spp_.

**Produced by below-ground feeders**

 Poor growth, stunting patchiness, discol-

Most of the species

ored foliage, wilting etc.

****

 Root galls

_Meloidogyne spp_., _Nacobbus spp_., _Ditylen-_

_chus spp_., _Xiphinema spp_. etc.

 Root lesions

_Pratylenchus_ spp., _Radopholus spp_.

 Reduced root system

_Trichodorus_ spp., _Belonolaimus spp_.

 Rots of fleshy plants

_Ditylenchus_ _spp_.

rect and indirect benefits to control these

Fallowing is a very common prac-

tiny tigers of agricultural fields improved

tice and cheaper way to minimize the

plant health, thereby reducing their

nematode problems by keeping the lands

changes of suffering from nematode dis-

from all vegetations for a certain period.

eases and also increased the plants ability

Complete fallow without allowing any

to withstand adverse growing conditions,

plant or weed to grow invariably ensures

resulted improvement of crop production.

the parasitic nematodes will have no host

Principal strategies for nematode man-

to feed. Thus, those are deprived of food

agement are cultural practices, physical,

and killed by the solar heat and soil desic-

biological, botanical, chemical and regu-

cation.

latory methods.

Deep summer ploughing involves

exposure of soils to solar heat and desic-

**6. Cultural Practices**

cation, helps to kill the nematode pests

****

along with other pathogens. For en-

To control nematode pests, cultur-

hancement of the efficacy of solar heat-

al practices are the most effective and

ing, polyethylene mulching of moist soil

economical means which can be achieved

during hottest period is being advocated.

by crop rotation, fallowing, deep summer

It considered as a very effective and good

plaughing, water flooding, growing an-

control measure against nematode pests

tagonistic or trap crops, removal of in-

and can be used with other cultural prac-

fected plant debris, selection of healthy

tices like crop rotation, green manuring,

planting materials, application of organic

inter cropping as well as with nematicides

manures and fertilizers etc.

etc. (Mathur _et al._ , 1987).

Some species of nematodes are

In the field where there is enor-

able to feed and multiply on certain crops,

mous availability of water and nematode

but not on others. The crop plants on

infested area is uniformly leveled, flood-

which they cannot feed and abundantly

ing can be adopted as a routine practice

reproduce are called non-host. By crop

and very effective measure. Under sub-

rotation system, the crops to be grown in

merged condition, chemicals lethal like

between the susceptible host crops should

hydrogen sulphide and ammonia to these

be immune or resistant to nematodes or at

noxious pests are released. Asphyxiation

least non-host plants which help to eradi-

and microbial decomposition products

cate or minimize the nematodes problems.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 61

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ due to anaerobic condition help to kill the

terials like banana corns, onion bulbs, tu-

nematodes.

ber seeds and roots of seeding can be

The crops like mustard, marigold,

dipped in 5-55°C hot water for about 10

neem etc. are treated as antagonistic

minutes.

crops, those have chemicals or alkaloids

The most primitive way of heating

as root exudates which repeal or suppress

the soil on large scale is by putting a fire

the plant parasitic nematodes. These

over it. Burning a layer of dry leaves on

plants can be grown along with main crop

soil killed nematode pests. Rabbing of

or may be included in crop rotation.

bajara husk, paddy husk, wheat straw etc.

The basic concept with trap crop-

become most effective against nematodes.

ping system is that two crops are grown in

Burning materials outside and incorporat-

the field, out of which one crop is highly

ing ash into the soil surface satisfaction

susceptible to nematode. When nema-

nematode kill.

todes attack such crop, very carefully it

Soil solarization is the most recent

should be remove and destroyed or burnt

method for control of plant parasitic nem-

totally. Thus, the main crop escapes from

atodes (Sharma & Trivedi, 1991). The

nematode infestation. Cowpea is a very

technique consists of covering the moist

good example of such susceptible crop for

soil with a good quantity, clear and trans-

destroying root-knot nematodes.

parent plastic film during the period of

Early detection of infected plants,

intense sunshine and increasing substan-

immediate removal and destruction of

tial soil temperature over the non-

those helps to reduce the spreading of

solarized soil. Increased soil temperature

nematodes in the field. Selection of nema-

coupled with soil moisture invariably re-

tode-free healthy planting materials or

sult the significant reduction in popula-

plantation of nematode-resistant varieties

tion densities of phytoparasitic nema-

is also a very effective method to avoid

todes. The nematode pests of crops are

nematode infestation in field.

effectively controlled by this method but

Soil treatments with organic ma-

it is restricted to summer season and only

nures, green crop residues or green leaf

in the tropical and subtropical region of

manures, farm yard or poultry manures,

the world.

oil cakes, and oils of neem, karanja, cas-

Careful washing of tubers, bulb

tor etc. significantly checked nematode

and other planting materials prevent this

population. The use of such materials also

nematode infestation in new planting

encourages the development of nematode-

fields. Modern mechanical seed cleaning

antagonistic microbes and predacious

methods have been developed to remove

nematodes also those help to control 
the seed galls to form normal healthy

nematode infestation in

agricultural

seeds. Sanitation, the use of clean tools

fields.

and equipments in field also prevent nem-

atodes infestation. Soil amendments and

_6.1. Physical methods_

frequent irrigation can also help to reduce

The hot water treatment, hot water

nematode-damage of crops.

drenching, rabbing with slow burning ma-

terials, soil solarization, electrical soil

_6.2. Biological methods_

heating, washing and cleaning of seed etc.

Biological control method of

are the most effective physical methods to

nematodes include the use of predaceous

control nematodes infestations to crops.

and parasitic organisms such as fungi,

For denamatization, rhizomes,

bacteria, protozoans, viruses, nematodes,

bulbs, corns, tubers and fleshy roots of

tardigrades, collembolans, mites etc, even

plantations and also other planting mate-

antagonistic higher plants also. This

rials are submerged into hot water for cer-

method, in fact, should be considered a

tain periods. Prior to plating the seed ma-

skillful manipulation of the biosphere

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 62

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ against nematodes pest of agricultural

safety to non-target organisms and the

fields for achieving maximum benefits.

environment as a whole and renewable

There are three types of components of

nature, the botanical pesticides offer al-

biological control of nematodes.

ternate strategy to the prevalence use of

Natural: Where the agent are al-

synthetic nematicides (Mishra, 1998). In-

ready present at levels to be sufficient for

discriminate use of chemical pesticides to

suppression of nematode development.

control nematode pest in agriculture give

Induced: The agents are already

rise to serious problems like food contam-

present in the soil and only their activities

ination, adverse effects on non-target or-

are stimulated by modifying the environ-

ganisms and environment, as well as de-

ment or by applying inciters.

velopment of pesticidal resistance in

Introduced: The agents are applied

many nematode pests. For this reason, the

by man from outside.

use of bio-pesticides of botanical origin

There are more than 50 species of

for the management of plant parasitic

predaceous fungi which have the capacity

nematodes has been increased presently.

to kill nematodes in agricultural field

Different parts of botanicals directly, the

(Jain, 2003). These fungi capture nema-

extracts of botanical parts or the product

todes by traps, mechanical traps and con-

of botanicals are used for nematode man-

stricting rings.

agement.

There are several reports of bacte-

Parts of different plants having

ria, present inside the nematode body.

nematicidal value are used directly

_Pasteuria penetrans_ has been described

against phytonematodes, infesting various

as potential biological agent against nem-

crops (Table 2). Chopped leaves of pine-

atodes. They prevent reproduction and

apple, karanja and neem leaves etc. could

eventually kill the root-knot nematodes

be significantly reduced the root-knot,

and many other species. Some rhizospher-

reniform and other nematodes also. Vari-

ic bacteria like _Azotobactor chroococcum_ ,

ous parts of _Crotolaria_ , marigold, Ken-

_Azospirilum lipoferum_ , and some _Pseu-_

tucky blue grass etc. in powdered form

_domonas_ spp. have found to be promising

also

reduced

nematode

population.

in reducing nematode population. Root-

Chopped castor leaves, Subabool leaves

knot nematode larvae infected with virus-

prevent gall nematodes. Chopped shots of

es were observed to exhibit sluggishness.

latex-bearing plants significantly sup-

pressed the population build up of reni-

_6.3. Botanical methods_

form and root-knot nematodes.

Due to their facile biodegradabil-

ity, selective toxicity only to target pests,

****

**Table 2:** Common names of some important phytonematodes

No.

Genera/Species

Common names

1.

_A. fragariae_

Spring dwarf nematode

2.

_Anguina_ spp. __

Seed gall, Leaf gall nematodes

3.

_Anguina tritici_

Ear-cockle nematode, Wheat gall nematode.

4.

_Aphelenchoides besseyi_

Rice white tip nematode, White tip nematode

5.

_Aphelenchoides ritzemabosi_

Chrysanthemum foliar nematode

6.

_Aphelenchoides_ spp. __

Bud and leaf nematodes, foliar nematodes

7.

_Belonolaimus_ spp. __

Sting nematodes

8.

_Belonololaimus gracilis_

Pine sting nematode

9.

_Cacopaurus_ spp. __

Sessile nematodes

10.

_Criconema_ spp. __

Spine nematodes

11.

_Criconemoides citri_

Citrus ring nematode

12.

_Criconemoides_ spp. __

Ring nematodes

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 63

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ **Table 2** _: Continued..._

13.

_Ditylenchus angustus_

Rice nematode

14.

_Ditylenchus destructor_

Potato root nematode, Potato tuber nematode,

Iris nematode

15.

_Ditylenchus dipsaci_

Stem nematode, Tulip root nematode, Bulb

nematode

16.

_Ditylenchus myceliophagus_

Mushroom spawn nematode

17.

_Dolichodorus_ spp. __

Awl nematodes

18.

_Dorylaimus_ spp. __

Spear nematodes

19.

_Globodera rostochiensis_

Golden nematode of potato

20.

_Globodera_ spp. __

Cyst nematode

21.

_Helicotylenchus_ spp. __

Spiral nematodes

22.

_Hemicriconemoides_ spp. __

Sheathoid nematodes

23.

_Hemicycliophora_ spp. __

Sheath nematodes

24.

_Heterodera avenae_

Great root nematode, Cereal nematodes

25.

_Heterodera cruciferae_

Cabbage cyst nematode

26.

_Heterodera glycines_

Soybean cyst nematode

27.

_Heterodera goettingiana_

Pea cyst nematode, Pea root nematode, Alfalfa

root nematode

28.

_Heterodera schachtii_

Sugar beet nematode

29.

_Heterodera_ spp. __

Cyst-forming nematodes

30.

_Hirschmanniella oryzae_

Rice root nematode

31.

_Hoplolaimus_ spp. __

Lance nematode, Spear nematode

32.

_Longidorus_ spp. __

Needle nematode

33.

_Meloidodera_ spp. __

Cystoid nematode

34.

_Meloidogyne arenaria_

Peanut root knot nematode

35.

_Meloidogyne brevicauda_

Indian root knot nematode

36.

_Meloidogyne exigua_

Coffee root knot nematode, Brazilian root knot

nematode

37.

_Meloidogyne incognita_

Southern root knot nematode

38.

_Meloidogyne javanica_

Javanese root knot nematode

39.

_Meloidogyne_ spp. __

Root knot nematodes, Root-gall nematodes

40.

_Nacobbus_ spp. __

False root knot nematodes

41.

_Paratylenchus_ spp. __

Pin nematodes

42.

_Pratylenchus_ spp. __

Root lesions nematodes, Meadow nematodes

43.

_Radopholus similis_

Burrowing nematode

44.

_Rhadinaphelenchus cocophilus_ Coconut palm nematode, Red ring nematode

45.

_Rotylenchulus reniformis_

Reniform nematode

46.

_Rotylenchus_ spp. __

Spiral nematodes

47.

_Trichodorus_ spp. __

Stubby root nematodes

48.

_Tylenchorhynchus claytoni_

Stunt nematode, Teaselate stylet nematode

49.

_Tylenchorhynchus martini_

Sugarcane stylet nematode

50.

_Tylenchorhynchus_ spp. __

Stunt nematode, Stylet nematode.

51.

_Tylenchulus semipenetrans_

Citrus root nematode

52.

_Xiphenema_ spp. __

Dagger nematodes

Certain botanicals in the form of aqueous

_charantia_ L.; leaves of _Ageratum coni-_

extracts of various parts have great poten-

_zoides_ L., _Anacardium occidentale_ L.,

tial against nematodes, the aqueous ex-

_Argemone mexicana_ L., _Datura stramo-_

tracts of fresh neem leaves; fruit skin of

_nium_ L. etc.; aqueous root extract of _Oci-_

_Citrus reticulata_ Blanco and _Momordica_

_mum sanctum_ L.; seed extracts of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 64

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ _Vernonia anthelmintica_ Wild, _Holarrhea_

****

Quarantine principles are tradi-

_antidysenterica_ Wall; bulb extracts of _Al-_

tionally employed to restrict the move-

_lium sativum_ L. and many other plant ex-

ment of infected plant materials and con-

tracts have potentiality to prevent nema-

taminated soil into a state or country.

tode infestation in agricultural fields.

Many serious plant parasitic nematodes

Different plant products like oil

spread from one country to another and

seed cakes, oils, seeds, and various other

from one state to other. The potato cyst

formulations are extensively used for the

nematode,

_Globodera_

_rostochiensis_

management of plant parasitic nematodes.

spread from Peru to almost whole of Eu-

rope and UK through seed potatoes and

_6.4. Chemical methods_

gunny bags. The stem and bulb nematode,

The chemicals those are used for

_Ditylenchus dipsaci_ got introduced in

controlling nematodes are the nemati-

southern parts of Sweden also through

cides. These are the soil fumigants, ap-

seeds. For this reason, plant quarantine

plied to the soil and diffuse through the

has been introduced at state, national and

soil as gas and acted against nematodes.

international levels as a legal restriction to

The use of nematicides for the manage-

check the spreading of nematode pest.

ment of plant parasitic nematodes in agri-

Regulatory control of pests and diseases

culture becomes essential when other

is the legal enforcement of measures to

methods are unable to protect the crops

prevent them from spreading. Strict regu-

from these pests, or spreading of nema-

lations have been made against _G. rosto-_

todes is so high in the field. Before plant-

_chiensis_ and _Rhadinaphelenchus cocophi-_

ing, the nematicidal application in the

_lus_ , the red ring nematode of coconut.

field in proper doses resulted in nema-

Domestic quarantine regulations have al-

tode-free rhizosphere, healthy root sys-

so been imposed to restrict the movement

tem, efficient use of minerals, moisture

of potato to prevent the spread of potato

and also reduces the chances of invasion

cyst nematode from Tamil Nadu to other

of other harmful soil microorganism.

states in India.

Kuhn (1881) first used chemical (CS2)

****

against _Heterodera schachtii_ in Germany.

**7. Conclusion**

The discovery of DD-mixture in 1943,

****

EDB in 1945 and DBCP in 1954 played

The plant parasitic nematodes are

remarkable role in demonstrating the

undoubtedly the most widespread and in-

nematode damage and crop loses. The use

sidious pests of crops. The management

of methylisothiocyanate, precursor com-

practices against these hidden enemies of

pounds like daromet, methamsodium, me-

agriculture to be adopted depend upon the

thylisothiocyanate mixture like vorlex etc.

degree of infection, relative value of the

also help in controlling nematodes. The

crop, filed size, level of capital invest-

non-volatile

nematicides

like

fen-

ment, practicability and feasibility of the

sulphothion, aldicarb, carbofuran, etho-

control strategy. The cultural practices are

prop etc. are also very promisible nemati-

simple and effective methods of nema-

cides. But the use of nematicides is a

tode pest control, adopted by the farmers.

costly proposition and creates toxic haz-

Physical methods are also simple and

ards and environmental pollution. Few of

popular for management of nematode in-

them like DBCP, MBr, aldicarb etc. have

festation. The biological and botanical

been banned already. The use of nemati-

methods are eco-friendly rather than oth-

cides is not so popular in agriculture ex-

ers. Though chemical methods may create

cept in few cases where drastic spreading

health hazards and causes environmental

of nematodes occurs in the field.

pollution but for urgent need and to check

severe attack by serious nematode pests,

_6.5. Regulatory methods_

and when other control measures are not

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 65

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ so fruitful, the chemical methods may be

**Jairajpuri, M. S. and Ahmad, W.**

adopted against these noxious pests of

**(1992).** Dorylaimida: Free-living,

various crops. For sustainable develop-

Predaceous and Plant __ parasitic

ment of agriculture, a combination of dif-

Nematodes _._ Oxford and IBH Pub-

ferent management systems integrated in

lishing Company Private Limited,

the correct manner can help to manage

New Delhi. **pp. 458.**

the nematode problems.

**Jairajpuri, M. S., Alam, M. M. and**

****

**Ahmad,**

**I.**

**(1990) _._** __

Nematode

**Acknowledgement**

biocontrol: Aspects and Prospects. __

****

CBS Publishers and Distributors,

I am thankful to Dr. Kailash

Delhi, India. **pp. 155.**

Chandra, Director, Zoological Survey of

**Kuhn, I. (1881).** Die Ergebnisse der

India, Kolkata for providing facilities and

versuche zur Ermittelung der ursach

encouragement to prepare this article.

der Ruben mudigkeit und zur

Erforschung

der

Natur

der

**References**

nematode _. Ber physiol. Lab. Univ._

****

_Halle_ **3, 1-53.**

**Ayoub, S. M. (1980).** Plant Nematology:

**Lal, A. (1998).** Application of computers

An Agricultural Training Aid. Nema

in nematode identification. _In:_ Re-

Aid Publication. USA. **pp. 195.**

cent Advances in Plant Nematology.

**Bohra, P. and Baqri, Q. H. (1997).** Plant

Trivedi, P. C. (ed.). CBS Publishers

and soil nematodes. _State fauna_

and Distributors, Daryaganj, New

_Series_

_6;_

_Fauna_

_of_

_Delhi_ ,

Delhi, **pp. 107-114.**

Zoological Survey of India **75-108.**

**Mathur, B. N., Handa, D. K. and**

**Bohra, P. and Baqri, Q. H. (2004).** _State_

**Swarup, G. (1987).** Effect of deep

_Fauna Series 8_ , _Fauna of Gujarat_

summer ploughings on the cereal cyst

_(Part-2)_. Zoological Survey of India

nematodes, _Heterodera avenae_ and

**355-400.**

yield of wheat in Rajasthan, India. _In-_

**Cobb, N. A. (1914).** North American

_dian Journal of Nematology_ **17, 292-**

free-living fresh water nematodes.

**295.**

_Transactions of the American Mi-_

**Mishra, S. D. (1998).** Botanicals in the

_croscopical Society_ **33,** **35-100.**

management of plant parasitic nem-

**Cobb, N. A. (1917).** The mononchs

atodes. In: Recent advances in plant

(Mononchus, Bastian): a genus of

nematology. Trivedi, P. C. (ed.).

free-living nematodes. _Soil Science_

CBS Publishers and Distributors,

**3,** **431-486.**

New Delhi. **pp. 226-246.**

**Crofton, H. D. (1966).** _Nematodes._

**Nicholas, W. L. (1984).** The biology of

Hutchinson

University

Library,

free-living nematodes _._ Clarendon

London. **pp. 160.**

Press, Oxford, Second Edition. **pp.**

**Hugot, J. P., Baujard, P. and Morand,**

**251.**

**S. (2001).** Biodiversity in hel-

**Sharma, R. and Trivedi, P. C. (1991).**

minthes and nematodes as a field of

Nematicidal properties of some leaf

study: an overview. _Nematology_ **3**

extracts against _Meloidogyne incogni-_

**(3), 199-208.**

_ta. **** Journal of Phytopathology Re-_

**Jain, R. K. (2003).** Integrated pest

_search_ **4 (2), 131-137.**

mengement

for

plant

parasitic

**Tahseen, Q. (2006).** The non parasitic

nematodes. _In:_ Nematode manage-

nematodes. _In:_ _Plant Nematology in_

ment in plants. Trivedi, P. C. (ed.).

_India_. Mohilal, N. and Gambhir, R.

Scientific

Publishers

(INDIA),

K. (eds.). Parasitology Laboratory,

Jodhpur. **pp. 293-304.**

Dept. of Life sciences, Manipur

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 66

_Biotech Sustainability (2017)_

_Management Strategies against Tiny Tigers... Gantait_ University, Manipur, India. **pp. 159-Yeates, G. W., Bongers, T., De Goede,**

**177.**

**R. G. M., Freckman, D. W. and**

**Wardle, D. A. and Yeates, G. W.**

**Georgieva, S. S. (1993).** Feeding

**(1993).** The dual importance of

habits in soil nematode families and

competition and predation as regula-

genera: An outline for soil ecol-

tory forces in terrestrial ecosystem:

ogists. _Journal of Nematology_ **25** ,

evidence from decomposer food

**315-331.**

webs. _Oecologia_ **93** , **303-306.**

****

© 2017 by the author. Licensee, Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 67

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P68-78_

**Designing Greener Pharmaceuticals and Practicing**

**Green Health Is Required for Sustainability**

****

**Sridevi Chigurupati1,*, Jahidul Islam Mohammad2, Kesavanarayanan Krishnan Sel-**

**varajan3, Saraswati Simansalam4, Shantini Vijayabalan1, Subhash Janardhan Bhore5**

_1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, AIMST University,_

_Semeling, 08100, Bedong, Kedah, Malaysia; 2Department of Pharmacology, Faculty of_

_Medicine, CUCMS, Cyberjaya, Selangor, 63000, Malaysia; 3Department of Pharma-_

_cology and Toxicology, College of Pharmacy, University of Hail, Hail, Kingdom of_

_Saudi Arabia; 4Department of Clinical Pharmacy, Faculty of Pharmacy, AIMST Uni-_

_versity, Semeling, 08100, Bedong, Kedah, Malaysia; 5Department of Biotechnology,_

_Faculty of Applied Sciences, AIMST University, Semeling, 08100, Bedong, Kedah, Ma-_

_laysia; *Correspondence: sridevi_ch@aimst.edu.my; _ _Tel: +6-04-429-8000 extn., 1284_

****

**Abstract:** The global demand for drugs is increasing day-to-day and in the same fashion

pharmaceutical effluents released from industry adversely affect the environment and hu-

man health. Discharge of pharmaceutical effluents either directly or indirectly into the envi-

ronment results in substantial pollution. Disposal of such toxic effluents into the environ-

ment also affects the ecosystems either by direct or indirect pathway. Several researchers

from environmental biotechnology domain have reported the presence of pharmaceuticals

in water and soil. To minimize the environmental pollution and to develop the sustainable

solutions, various methods to convert pharmaceutical effluents into non-toxic and biode-

gradable organic matter has been proposed and discussed by the scientific community. This

chapter discusses the fate of pharmaceutical waste in the environment, its impact on human

health, methods adopted to treat effluents before its disposal into the environment, QSAR

studies in the design of biodegradable agents and future research directions to protect the

environment for sustainability.

_**Keywords**_ **:** Bioaccumulation; biodegradation; environment; green pharmaceuticals; phar-

maceutical effluents

**1. Introduction**

ates, drug metabolites and pharmaceutical

****

waste should be performed by the manu-

In recent years, improper disposal

facturer in order to know their fate in the

of chemical waste has greatly affected

environment. Toxic concentrations of the-

both environment and human health. Dis-

se chemical wastes get accumulated in

posal of chemical waste products as such

seawater, wastewater, sewage sludge's,

into the environment can accumulate and

water bodies and in the living organisms

affect both the biotic and abiotic factors

that are part of affected ecosystems. En-

of ecosystems. Hence, the fate of chemi-

vironmental pollution caused by humans

cals or pharmaceuticals after its disposal

can adversely affect the wellbeing of both

into the environment should be consid-

living and non-living things of ecosystem.

ered by the health care industry and regu-

The health of both animals and humans

latory agencies. Ecotoxicological studies

are affected when food materials contam-

of chemicals, drugs, chemical intermedi-

inated with the toxic substances are con-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 68

_Biotech Sustainability (2017)_

_Designing Greener Pharmaceuticals and Practicing Green... Chigurupati et al._

sumed. Constant pollution of the envi-

downs, warehouses or tanks used

ronment has disturbed several species as

in fuel and chemical industries.

the toxic effluents can either destroy them

 Carriage i.e. air, road, rail, pipe-

or affect their reproductive life cycle

lines, and water.

permanently (Goudie, 2006; Chigurupati

 Impurities emission such as Nitro-

_et.al_., 2016).

gen dioxide, Carbon monoxide,

Recently, the concerns about envi-

particulate matter (≤10μm),

ronmental pollution on endocrine- related

total suspended particulate matter,

disease and disorders have been identi-

Sulphur dioxide and volatile or-

fied. Some environmental pollutants (nat-

ganic compounds such as acetoni-

ural and anthropogenic) interfere with

trile, dichloromethane, ethylene

endocrine system and their hormones,

glycol, methanol, N,N dimethyl

which can affect both animal and human

formamide and toluene.

health. These chemicals are called as en-

 Various types of effluents (may be

docrine disruptors. Proper disposal meth-

lethal in nature) those are not ef-

ods of chemical wastes should be consid-

fectively biodegraded. The efflu-

ered to minimize their adverse effects on

ents those are able to go straight

environment. To minimize the risk of

towards oceans, lakes, rivers,

environmental pollution, biodegradation

streams or other bodies of water.

is the method adopted by several chemi-

The discharge because of over-

cals and pharmaceutical industries before

flow, including storm water run-

a chemical or pharmaceutical waste has

offs, could likewise be a potential

been released into the environment. Bio-

risk.

degradation is a process whereby the

complex organic compounds are deterio-

Figure 1 depicts that how drug

rated aerobically by microbes into simple

metabolites or drugs reaches water bodies

organic matter, for instance CO2, H2O,

and results in contamination of environ-

NH3, and CH4 etc. (OECD Statistics

ment. In addition to health benefits,

Directorate, 2002).

pharmaceuticals (drugs, chemicals, pills,

This chapter highlights the fate

medicine, sedatives, stimulants, narcotics

and impact of pharmaceutical waste in the

etc.) are also known to cause various

environment and on human health, re-

types of damage and or pollution. The

spectively. Various methods used to treat

risks from the pharmaceuticals could be

effluents, the role of QSAR studies in bi-

categorized as (Calleja _et al_., 1994):

odegradation and the possible research

 Ecotoxicological damage elicited to

directions to protect the environment

the environment.

from toxic effluents are also discussed.

 Malignant- contribute to the causality

of cancer.

**2. Pharmaceutical biowaste and its**

 Persistent-remain hazardous for a long

**environmental impact**

time.

****

 Bio-accumulative–gathers as it make

In general, man-made biowaste

its way up the food chain.

causes the environmental pollution and

 Disastrous due to a catastrophe, mis-

can pose a threat to the public health.

hap, calamity or grave manifestation

Biowaste originates from various sources

in any area

as stated below (Rand-Weaver _et al._ ,

2013):

Seepage of the chemicals or

 Formulation and industrial set up.

pharmaceuticals to the environment is

 Management and packing of haz-

common from the usage of diagnostics,

ardous chemicals along with go-

antiseptics and individual care products.

The pharmaceuticals, their metabolites

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**Figure 1:** A diagrammatic sketch showing imaginable sources and paths for the pharma-

ceutical deposits to reach into the water bodies including our drinking water. ****

and transformation products if not eradi-

teams in few countries. However, the

cated during sewage handling then they

presence of approximately 160 various

may come in the aquatic environment and

drugs and or various pharmaceuticals

in the long run can reach the drinking wa-

chemical associates is reported in STPs

ter bodies and may come to our homes

effluent and in ground and surface water

through water supply (see Figure 1). The

bodies (WHO, 2011). Even in drinking

active amalgams pharmaceuticals or its

water samples, some Active Pharmaceuti-

derivatives could enter into environment

cal Ingredients (APIs) were found (WHO,

by various routes or non-point sources;

2011). They are additionally distin-

for instance waste, sewage treatment

guished in the freezing environment. Con-

plants (STPs) and landfill effluent or from

trasted with the free water, phase analysis

animals treatment facilities (Calamari _et_

of APIs is difficult in bio solids and sew-

_al._ , 2003).

age sludge for proper comprehension

Pharmaceuticals,

particularly

(Daughton and Ternes, 1999).

hormones were the primary focus of re-

Medications are useful in aquacul-

search and public consciousness in the

ture, livestock farming, and veterinary

1970s. The hormones do not (bio) de-

and in other sectors of agriculture. How-

grade completely. However, the concept

ever, improper practices results in con-

produced enthusiasm in 1980s. Other

tamination of environment; for instance,

constituents like heavy metals, aromatic

seepage of growth promoters are found in

polycyclic hydrocarbons, pesticides and

contaminated soil. Various types of chem-

detergents were also the issue of wide-

icals used in agriculture and those become

spread examination during 1980s (Jones

source of environmental contamination or

_et al._ , 2008).

biowaste can have an adverse impact on

Deliberate research on contamina-

human health (Table 1). Veterinary anti-

tion of environment by various pharma-

biotics could end up in the groundwater

ceuticals is undertaken by some research

or in the soil. By runoff, it may be cleared

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**Table 1:** Different types of wastes and their impact on human health (Ferrari _et al._ , 2003;

Tauxe-Wuersch _et al._ , 2005; Isidori _et al._ , 2006; Khanal _et al._ , 2006) ****

**No**

**Type of waste**

**Effect on humans**

1.

Nuclear waste

**Hair:** The losing of hair occurs with radiation exposure at 200

rems.

**Brain:** Brain cells do not replicate, they damaged directly at

the exposure is 5,000 rems.

**Thyroid:** The thyroid gland is vulnerable to radioactive iodine.

In adequate amounts, radioactive iodine can destroy all or part

of the thyroid.

**Blood System:** The blood's lymphocyte cell count will be re-

duced once a person is exposed to around 100 rems, leaving

the victim more susceptible to infection. This is often referred

to as mild radiation sickness.

**Heart:** Immediate damage to small blood vessels and perhaps

cause heart failure and death directly by intense exposure to

radioactive material at 1,000 to 5,000 rems.

**Gastrointestinal Tract:** Exposure to 200 rems or more radia-

tion harm to the intestinal tract lining will cause nausea,

bloody vomiting and diarrhea. The radiation will begin to de-

stroy the cells in the body that goes division rapidly. These in-

cluding blood, GI tract, reproductive and hair cells, and harms

their DNA and RNA of living cells.

**Reproductive Tract:** Since reproductive tract cells multiply

rapidly, these areas of the body can be damaged at rem levels

as low as 200. Long-term, some radiation sickness victims will

become sterile.

2.

Environmental

Interaction of humans to agrichemicals is common and results

waste

in acute and chronic health threats, together with acute and

chronic neurotoxicity **(fumigants, insecticides and fungi-**

**cides,** ), lung chemical burns (anhydrous ammonia), newborn

methemoglobinemia and also impairment (paraquat).

3.

Agrichemicals

Soil pollution effects causes leukemia and it is danger for

young children as it can cause developmental damage to the

brain furthermore it illustrated that mercury in soil increases

the risk of neuromuscular blockage, causes headaches, kidney

failure, depression of the central nervous system, eye irritation

and skin rash, nausea and fatigue. Soil contamination closely

related to air and water pollution, so numerous things come out

as similar as caused by water and air pollution.

into surface water bodies. Wide variety of

namics in soils. The profusion of these

indications of various active ingredients

natural antibiotics was less and appeared

in liquid state and in the soil has been es-

as restricted to the adjacent surroundings

tablished. The primary reviews that have

(Kümmerer, 2004). The composition of

researched the transfer and the related

the soil-lodging species is found to be in-

risks have been published only recently

fluenced by antimicrobial agents. Antibi-

(Corcoran _et al._ , 2015).

otics in the soil appear to support fungal

Antibiotics transpire naturally into

development. The circumstances in the

the soils and the resistance against these

areas beneath fish farms are critical due to

antibiotics influences the inhabitant's dy-

more concentrations of antimicrobial

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agents as a result of seepage. Some anti-

well as affects the flora and fauna at vari-

microbial agents are responsible to dimin-

ous extents. Hence, the dumping of phar-

ish the number of microscopic organisms

maceuticals into the environment is now

around aquaculture facilities or where the

recognized as a serious issue (Boxall,

seepage of antimicrobial agents was no-

2004).

ticeable (Oliveira _et al_., 1995). The de-

There are many techniques and or

gree of the impact of ciprofloxacin on mi-

processes utilized to overcome this type

crobial salt marsh groups was contrarily

of pollution problem. Few of them are -

related to the level of sorption to the sed-

(i) Thermal process; (ii) Chemical pro-

iments. Even though ciprofloxacin is a

cess; (iii) Irradiation process; (iv) Biolog-

wide-spectrum antibiotic, its influence on

ical process (biodegradation); and (v)

sediment microbial organisms was selec-

Mechanical process.

tive and seemed to favor Gram-negative

Among the five above stated pro-

and sulfate-reducing bacteria. (Sengupta __

cesses used to minimize the waste or pol-

_et al._ , 2013). This type of the environ-

lution, biodegradation techniques (biolog-

mental contamination can cause the im-

ical process) are considered as greener

balance in ecosystems.

technique/technology that involves the

The impacts of oxytetracycline in

use of various biological processes to

environmentally applicable concentra-

overcome the adverse effects of pharma-

tions on enchytraeids, springtails and

ceutical effluents into the environment.

earthworms have been analyzed. Neither

Biodegradation is a complex and natural

one of the antibiotics demonstrated any

decomposition process of organic sub-

harmfulness against the organisms under

stances with the help of biochemical reac-

scrutiny. However, the capability of a pol-

tions of microbes (OECD Statistics

lutant to accumulate in organisms must be

Directorate, 2002). Although, pharmaceu-

considered seriously. Antibiotics that are

ticals signify a minor fraction of all chem-

inadequately water soluble, particularly if

icals that are dumped into the environ-

the bio-concentration factor is between

ment, exceptional care must be taken

500 and 1000 or if the octanol/water dis-

about their presence in the environment.

tribution coefficient crosses the value of

Because, they are ubiquitous and dissem-

1000, have a tendency to get accumulated

inate easily; they act on biological sys-

in organisms. The substance enrichment

tems; they show a lot of side effects in

in organisms has been proved for a few

non-targeted bodies; and they are known

antibiotics, e.g., sulphadimethoxine (Call

to cause chronic toxicity even at low con-

_et al._ , 2013).

centrations (Enick and Moore, 2007).

****

As stated by Jones _et al_. (2005),

**3. Biodegradation of pharmaceutical**

the occurrence of pharmaceutical contam-

**products**

inants in the environment was reported

first time in late 1970's (Jones _et al_ ,

Globally, the production of phar-

2005). The major source of pharmaceuti-

maceuticals continues to grow year by

cals in the environment is due to their im-

year, and with its environmental concerns

proper disposal and leaching from landfill

pertaining to not just production, but also

sites to natural water (Jones _et al._ , 2004)

consumer waste and disposal (Wu _et al._ ,

and most often the drugs enter into sew-

2010). The drugs (pharmaceuticals) con-

ages and culminate in water sources.

sumed are eventually either excreted by

There is no any guarantee that the phar-

humans and animals and or disposed

maceutical drugs and their effluents are

which causes environment pollution.

removed by merely dumping in sewage

Most of the pharmaceuticals produced,

(Boxall, 2004). Moreover, many times, it

finally make their way to the ecosystems

is proven that the water waste treatment

and causes environmental pollution as

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plants are inefficient in managing the

_Example_ :

Compounds

like

pharmaceutical effluents (Ternes, 1998).

mefenamic acid and ibuprofen, the greater

The main biodegradation tech-

part of the expulsion is because of bio-

niques are - (i) aerobic biodegradation;

degradation (Jones _et al_., 2007). The sim-

(ii) anaerobic biodegradation, and (iii)

pler a molecule, the higher chance for bi-

biodegradation in activated sludge and

odegradation to occur (Jones _et al._ , 2004).

pure culture.

During biodegradation, the xenobiotic

****

compounds can be changed in three di-

_3.1. Biodegradation by Aerobes_

verse ways namely, hydrophobic, hydro-

Aerobic biodegradation is the

philic and or mineralization transfor-

breakdown of organic substances by mi-

mation. Carbamazepine (CBZ) is one of

croorganisms in the presences of oxygen.

the pharmaceuticals with the lowest re-

The microorganisms used in the process

moval efficiency when treated with acti-

require oxygen for growth; hence these

vated sludge treatment (Ternes, 1998).

microbes are called as aerobes. Aerobic

Subsequently, CBZ is not influenced ei-

degradation of hydrocarbons is an exam-

ther by sorption or by the microbes. Most

ple of this biological process. Aromatic

positive outcomes with respect to the bio-

hydrocarbons could be converted into

degradation of CBZ are by a pure culture.

natural intermediates; for instance, proto-

catechuate and catechol (Figure 2)

**4. QSAR studies in biodegradation**

(Boxall, 2004). Gram negative bacteria

****

that possess plasmids produce enzymes

Persistent, bioaccumulative, and

needed for the aromatic compounds deg-

toxic chemicals show high lipid solubility

radation. This is possible because of hy-

and low water solubility which lead to

droxylation reaction which introduces a

high potential for bioaccumulation. The

hydroxyl group (-OH) into substrate, in

European REACH regulation is the first

this case hydrocarbons (Chatterji, 2003).

to work on quantitative structure–activity

****

relationship (QSAR) models for biodeg-

_3.2. Biodegradation by anaerobes_

radation. REACH inspires the use of sub-

Anaerobic biodegradation is the

stitutes to animal testing which includes

breakdown of organic substances by us-

predictions from QSAR models. QSAR

ing microbes in the absences of oxygen.

Model mainly involves three types of data

When the anaerobes are predominant over

generation - Persistence data generation;

the aerobic microbes then anaerobic bio-

biodegradation data generation; and tox-

degradation is preferred. It is broadly

icity data generation. For estimating bio-

used to treat biodegradable waste and

degradation, there are few important data

wastewater sludge since it gives mass and

bases such as Syracuse BIODEG, BIO-

volume reduction. There are four key bio-

DEG database, BIOLOG Database, MITI

logical and chemical reactions involved in

Database, and ESIS Database.

anaerobic biodegradation namely, hydrol-

The partially observed persistence; bioac-

ysis, acidogenesis, acetogenesis, and

cumulation and toxicity data, the expen-

methanogenesis (Figure 3).

sive testing composed with the regulatory

limitations and the international encour-

_3.3. Biodegradation in activated sludge_

agement to minimize animal models mo-

_and pure culture_

tivates a greater dependence on QSAR

Activated sludge treatment is the

models in the biodegradation assessment.

most

well-known

process

for

the

Several QSAR biodegradation models

wastewater treatment. Two disposal

have been established for selected groups

forms act simultaneously in the activated

of structurally similar compounds like:

sludge, either the biodegradation by the

specific number of alcohols, chlorophe-

microbes or sorption to solids.

nols, n-alkyl phthalates, chloroanisoles,

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**Figure 2:** A diagrammatic sketch showing degradation of natural aromatic and some xeno-

biotic compounds.

****

**Figure 3:** Schematic diagram showing anaerobic biodegradation of xenobiotics.

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meta-substituted

anilines

and

para-

stance in the environment. The infor-

substituted phenols etc. Most of the

mation obtained from the degradability

Quantitative structure- biodegradation

test is mainly required for its hazard or

relationships (QSBRs) depend on the oc-

risk assessment in aquatic environment.

tanol/water partition coefficients, alkaline

The waste water get in contact with the

hydrolysis rate constants, van der Waals

sewage treatment microbes and chemical

radii and also on molecular connectivity

substances enter into the oceanic envi-

indices. Generally, the association be-

ronment. The process whereby the chem-

tween physicochemical properties or mo-

ical substance gets bioaccumulated in an

lecular descriptors and biodegradation

(aquatic) organism is known as bioaccu-

rates were satisfactory; but, generally the-

mulation

se models have not been used much. Be-

The bioaccumulation degree of a

cause, use of these models is limited to

chemical substance at a given period is

the specific classes of chemicals for

the collective data of the competing pro-

which these models were created. Hence,

cesses of uptake, distribution, transfor-

these models are unsuitable to predict bi-

mation and excretion. The information on

odegradation rates for chemicals outside

degradation and accumulation could be

of those classes (Howard _et al._ , 1992;

obtained by performing

appropriate

Dimitrov _et al._ , 2005).

standardized tests recommended by the

Therefore, in recent years a very

OECD (Table 2).

rigorous development of new and better

****

qualitative and quantitative biodegrada-

**6. Malaysian government regulations**

bility models by the usage of new and

**on pharmaceutical pollution**

well developed statistical and computa-

****

tional methods are developed. Weighted

In 2005, the Ministry of Natural

molecular fragments are used as model

Resources and Environment (MNRE) has

descriptors with an idea that molecular

published guidelines on the disposal of

fragments may have an attractive or hin-

clinical and pharmaceutical wastes. Any

dering effect on biodegradability. Numer-

waste listed in the First Schedule of the

ous statistical techniques have been used

Environmental

Quality

(Scheduled

in determining weights: linear and non-

Waste) Regulations known as Scheduled

linear regression modelling partial least

Waste, this type of waste can only be dis-

square (PLS) and neural networks. The

posed in the predetermined premises and

results of QSBR studies are strongly in-

waste must be treated before its disposal.

fluenced by the way the molecule is

These wastes may possess either inorgan-

fragmented. To overcome this, the Multi-

ic or organic components, e.g. discarded

CASE approach has been established to

drugs comprising psychotropic or danger-

generate all possible fragments of the

ous substances such as carcinogens, mu-

molecules and to subsequently select the

tagens or teratogens (SW 403) (Ministry

statistically most significant ones to suit

of Natural Resources and Environment,

proper results. These fragments are then

2009).

used to develop regression models be-

In 2010, the Pharmaceutical Ser-

tween screened fragments and the final

vices Division initiated 'Return Your

results (Banerjee _et al._ , 1984; Niemi _et_

Medicines Programme' in which patients

_al._ , 1987).

were encouraged to return the expired and

unused drugs to the pharmacies in the

**5. Test guidelines for degradation and**

government hospitals. The aim of this

**bioaccumulation studies**

programme was to promote safe disposal

****

of medications and mitigate the undesired

Biodegradability test is performed

adverse effects of APIs on the environ-

to evaluate the fate of a chemical sub-

ment as well as living beings. At the end

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_Designing Greener Pharmaceuticals and Practicing Green... Chigurupati et al._

**Table 2:** OECD test guidelines to assess the environmental fate of chemicals

(OECD Guidelines for the testing of chemicals - OECD, no date) ****

**Test No Test Title**

301 Ready Biodegradability

302A Inherent Biodegradability: Modified SCAS Test

302B Inherent Biodegradability: Zahn-Wellens/ EVPA Test

302C Inherent Biodegradability: Modified MITI Test (II)

303 Simulation Test - Aerobic Sewage Treatment - A: Activated Sludge

Units; B: Biofilms

304A Inherent Biodegradability in Soil

305 Bioaccumulation in Fish: Aqueous and Dietary Exposure

Bioconcentration: Flow-through Fish Test ****

306 Biodegradability in Seawater

307 Aerobic and Anaerobic Transformation in Soil

308 Aerobic and Anaerobic Transformation in Aquatic Sediment Systems

309 Aerobic Mineralization in Surface Water – Simulation Biodegradation

Test

310 Ready Biodegradability - CO2 in sealed vessels (Headspace Test)

311 Anaerobic Biodegradability of Organic Compounds in Digested Sludge:

by Measurement of Gas Production

312 Leaching in Soil Columns

313 Estimation of Emissions from Preservative - Treated Wood to the Envi-

ronment

314 Simulation Tests to Assess the Biodegradability of Chemicals Dis-

charged in Wastewater

315 Bioaccumulation in Sediment-dwelling Benthic Oligochaetes

316 Phototransformation of Chemicals in Water – Direct Photolysis

317 Bioaccumulation in Terrestrial Oligochaetes

of year 2016, the Ministry of Health

long time in the environment. The organic

(MoH) had disposed nearly RM 2 million

carcinogenic solvents should be replaced

worth of medicines, most of which were

with green solvents wherever it is possi-

obtained through the 'Return Your Medi-

ble. Industries should more focus on the

cines Programme'. The most common

minimum utilization of atoms for the syn-

medicines returned under this programme

thesis of pharmaceutical products.

were the ones used for treatment of diabe-

Biological based products are naturally

tes, hypertension, hyperlipidemia and gas-

less poisonous and promote the principles

tritis. The common reason for the patients

of green chemistry by expanding pro-

to incomplete the course of medicines

spects to develop expected processes ex-

was a change or discontinuation of a

ploiting renewable resources. Ultimately,

treatment ( _Ministry destroys RM 2 million_

renewable resources do have a potential

_worth of expired meds - Nation | The Star_

to produce a significant amount of phar-

_Online_ , 2016).

maceutical materials which are currently

produced using either hazardous and or

**7. Concluding remarks**

nonrenewable materials. Green chemical

****

synthetic procedures should replace the

The pharmaceuticals should be

conventional procedures of producing

designed by using green chemistry tech-

pharmaceuticals. It is not only essential to

niques; so that at the end, products can be

promote the health of people but also for

easily broken down into innocuous deg-

the sustainability of the industry and

radation products and do not persist for

planet. ****

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**References**

**Daughton, C. G., and Ternes, T. A.**

****

**(1999).** Pharmaceuticals and personal

**Banerjee, S., Howard, P. H., Rosen-**

care products in the environment:

**berg, A. M., Dombrowski, A. E.,**

agents

of

subtle

**Sikka, H., and Tullis, D. L. (1984).**

change? _Environmental health per-_

Development of a general kinetic

_spectives_ , **107(Suppl 6), 907-938.**

model for biodegradation and its ap-

**Dimitrov, S., Dimitrova, G., Pavlov, T.,**

plication to chlorophenols and related

**Dimitrova, N., Patlewicz, G., Nie-**

compounds. _Environmental Science_

**mela, J., and Mekenyan, O. (2005).**

_and Technology_ , **18(6), 416–422.**

A stepwise approach for defining the

**Boxall, A. B. A. (2004).** The environmen-

applicability domain of SAR and

tal side effects of medication. _EMBO_

QSAR models. _Journal of Chemical_

_Reports_ , **5(12), 1110–1116.**

_Information and Modeling,_ **45(4),**

**Calamari, D., Zuccato, E., Castiglioni,**

**839–849.**

**S., Bagnati, R., and Fanelli, R.**

**Enick, O. V, and Moore, M. M. (2007).**

**(2003).** Strategic survey of therapeu-

Assessing the assessments: pharma-

tic drugs in the rivers Po and Lambro

ceuticals in the environment. _Envi-_

in northern Italy. _Environmental Sci-_

_ronmental Impact Assessment Re-_

_ence and Technology,_ **37(7), 1241–**

_view_ , **27(8), 707–729.**

**1248.**

**Ferrari, B., Paxeus, N., Giudice, R. Lo,**

**Call, D. R., Matthews, L., Subbiah, M.,**

**Pollio, A., and Garric, J. (2003).**

**and Liu, J. (2013).** Do antibiotic res-

Ecotoxicological impact of pharma-

idues in soils play a role in amplifica-

ceuticals

found

in

treated

tion and transmission of antibiotic re-

wastewaters: study of carbamazepine,

sistant bacteria in cattle populations?

clofibric acid, and diclofenac. _Eco-_

_Frontiers in Microbiology_ , **4, 193-**

_toxicology and Environmental Safety,_

**196.**

**55(3), 359–370.**

**Calleja, M. C., Persoone, G., and Gela-**

**Goudie, A. (2006).** The Human Impact

**di, P. (1994).** Comparative acute tox-

on the Natural Environment: Past,

icity of the first 50 multicentre evalu-

Present, and Future. _John Wiley and_

ation of in vitro cytotoxicity chemi-

_Sons._

cals to aquatic non-vertebrates. _Ar-_

**Jones, O. A., Voulvoulis, N., and**

_chives of Environmental Contamina-_

**Lester, J. N. (2005).** Human phar-

_tion and Toxicology,_ **26(1), 69–78.**

maceuticals in wastewater treatment

**Chatterji, A. K. (2003).** Introduction to

processes. _Critical Reviews in Envi-_

environmental biotechnology. _Pren-_

_ronmental Science and Technology_ ,

_tice-Hall of India._

**35(4), 401–427.**

**Chigurupati, S., Mohammad, J. I., and**

**Howard, P. H., Stiteler, W. M., Meylan,**

**Krishnan, K. (2016).** Focus on Envi-

**W. M., Hueber, A. E., Beauman, J.**

ronment. _Focus on Environment,_ **1,**

**A., Larosche, M. E., and Boethling,**

**74-88.**

**R. S. (1992).** Predictive model for

**Corcoran, J., Winter, M. J., Lange, A.,**

aerobic biodegradability developed

**Cumming, R., Owen, S. F., and Ty-**

from a file of evaluated biodegrada-

**ler, C. R. (2015).** Effects of the lipid

tion data. _Environmental Toxicology_

regulating drug clofibric acid on

_and Chemistry,_ **11(5), 593–603.**

PPARα-regulated gene transcript lev-

**Isidori, M., Nardelli, A., Parrella, A.,**

els in common carp (Cyprinus car-

**Pascarella, L., and Previtera, L.**

pio) at pharmacological and envi-

**(2006).** A multispecies study to as-

ronmental exposure levels. _Aquatic_

sess the toxic and genotoxic effect of

_Toxicology_ , **161, 127–137.**

pharmaceuticals: furosemide and its

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 77

_Biotech Sustainability (2017)_

_Designing Greener Pharmaceuticals and Practicing Green... Chigurupati et al._

photoproduct. _Chemosphere,_ **63(5),**

**OECD Statistics Directorate. (2002).**

**785–793.**

OECD glossary of statistical terms -

**Jones, O. A. H., Dondero, F., Viarengo,**

household definition.

**A., and Griffin, J. L. (2008).** Meta-

**Oliveira, R. da G. B., Wolters, A. C.,**

bolic profiling of Mytilus gallopro-

**and Elsas, J. D. van. (1995).** Effects

vincialis and its potential applications

of antibiotics in soil on the popula-

for pollution assessment. _Marine_

tion dynamics of transposon Tn5 car-

_Ecology Progress Series,_ **369, 169–**

rying

Pseudomonas

fluorescens.

**179.**

_Plant and Soil,_ **175(2), 323–333.**

**Jones, O. A. H., Voulvoulis, N., and**

**Rand-Weaver, M., Margiotta-Casaluci,**

**Lester, J. N. (2004).** Potential eco-

**L., Patel, A., Panter, G. H., Owen,**

logical and human health risks asso-

**S. F., and Sumpter, J. P. (2013).**

ciated with the presence of pharma-

The read-across hypothesis and envi-

ceutically active compounds in the

ronmental risk assessment of phar-

aquatic environment. _Critical Re-_

maceuticals. _Environmental Science_

_views in Toxicology,_ **34(4), 335–350.**

_and_

_Technology_ , **47(20),**

**11384-**

**Jones, O. A. H., Voulvoulis, N., and**

**11395.**

**Lester, J. N. (2007).** The occurrence

**Sengupta, S., Chattopadhyay, M. K.,**

and removal of selected pharmaceuti-

**and Grossart, H. P. (2013).** The

cal compounds in a sewage treatment

multifaceted roles of antibiotics and

works utilising activated sludge

antibiotic

resistance

in

na-

treatment. _Environmental Pollution,_

ture. _Frontiers in microbiology_ , **4,1-**

**145(3), 738–744.**

**13.**

**Khanal, S. K., Xie, B., Thompson, M.**

**Tauxe-Wuersch, A., De Alencastro, L.**

**L., Sung, S., Ong, S.-K., and Van**

**F., Grandjean, D., and Tarradellas,**

**Leeuwen, J. (2006).** Fate, transport,

**J. (2005).** Occurrence of several

and biodegradation of natural estro-

acidic drugs in sewage treatment

gens in the environment and engi-

plants in Switzerland and risk as-

neered systems. _Environmental Sci-_

sessment. _Water Research,_ **39(9),**

_ence and Technology,_ **40(21), 6537–**

**1761–1772.**

**6546.**

**Ternes, T. A. (1998).** Occurrence of

**Kümmerer, K. (2004).** Resistance in the

drugs in German sewage treatment

environment. _Journal of Antimicro-_

plants and rivers. _Water Research_ ,

_bial Chemotherapy,_ **54(2), 311–320.**

**32(11), 3245–3260.**

**The Star Online.** **(2016).** Ministry de-

**W. H. O. (2011).** Pharmaceuticals in

stroys RM 2 million worth of expired

Drinking-water Public Health and

meds – Nation.

Environment Water, Sanitation, _Hy-_

**Ministry of Natural Resources and En-**

_giene_

_and_

_Health,_

**vironment. (2009).** Guidelines on

(WHO/HSE/WSH/11.05).

The Handling and Management of

**Wu, M., Atchley, D., Greer, L.,**

Clinical Wastes In Malaysia, 1–29.

**Janssen, S., Rosenberg, D., and**

**Niemi, G. J., Veith, G. D., Regal, R. R.,**

**Sass, J. (2010).** Dosed Without Pre-

**and Vaishnav, D. D. (1987).** Struc-

scription: Preventing Pharmaceutical

tural features associated with de-

Contamination

of

Our

Nation's

gradable and persistent chemicals.

Drinking Water (Natural Resources

_Environmental_

_Toxicology_

_and_

Defense

Council).

URL:

_Chemistry_ , **6(7), 515–527.**

http://docs.nrdc.org/health.

© 2017 by the authors. Licensee, Editors and AIMST University, Malaysia. This arti-

cle is an open access article distributed under the terms and conditions of the Creative

Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 78

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_BiotechSustainability (2017), P79-87_

**Clonal Propagation of a High Value Multipurpose**

**Timberline Tree Species _Quercus semecarpifolia_** **Sm. of**

**West Himalaya, India**

****

**Aseesh Pandey** 1, 2 **and Sushma Tamta** 2, * ****

_1Govind Ballabh Pant National Institute of Himalayan Environment and Sustainable De-_

_velopment, Sikkim Unit Pangthang 737101, East Sikkim, Gangtok, India; 2Plant Tissue Cul-_

_ture Laboratory, Department of Botany, DSB Campus, Kumaun University, Nainital_

_263001, Uttarakhand, India; *Correspondence: sushmatamta@gmail.com; _ _Tel:_

_+918126966284_

**Abstract:** Tree species across the alpine timberline are most vulnerable to climate change

and requires immediate attention for their conservation. _Quercus semecarpifolia_ Sm. forms

the extensive forests in alpine timberline region and it is among the highly exploited tree

species of western Himalaya. Attempts were made to develop _in vitro_ clonal propagation

procedure for _Q. semecarpifolia._ Nodal explants, derived from a single mature tree growing

in natural stands, were cultured on different nutrient media for the optimization of nutrient

medium. Woody plant (WP) medium supplemented with 8.88 µM 6-benzylaminopurine

(BA) and 0.72 µM Gibberellic acid (GA3) has yield significant shoot multiplication re-

sponse. For root induction a two- step method was applied. Microshoots treated with 100

µM indole-3-butyric acid (IBA) for 24h, showed the significantly higher rooting response.

Present study leads a way forward to conservation and sustainable utilization of this high

value tree species. Moreover, further strengthening and optimization efforts are required to

develop a low cost procedure for the development of the nursery of clonally propagated _Q._

_semecarpifolia_ plants. Thus, the threat-mitigation strategies can be developed through the

introduction of these _in vitro_ raised plants into the natural forest. ****

_**Keywords**_ **:** Clonal propagation; nodal explant; _Quercus semecarpifolia_ ; timberline

****

****

**1. Introduction**

sphere has increased by approximately 5-

10% over mid and high latitudes (IPCC,

Alpine timberline is the most sen-

2013). Due to diverse climatic conditions,

sitive ecotone to changing climate (Smith

topography, and precipitation regimes the

_et al.,_ 2009). The regeneration of tree

composition of timberline varied across

species in this region is expected to affect

the globe and dominated by different tree

adversely in coming years. Ample evi-

species. The Himalaya harbors a unique

dences of a typical regeneration of tim-

mountain system and represents the high-

berline tree species are already document-

est alpine timberline of the world. The

ed by various researchers across timber-

timberline of Indian Himalayan region

lines of different mountains located in

(IHR) is dominated by different tree spe-

diverse geographical provinces (Harsch _et_

cies from east to west. The IHR consists

_al_., 2009; Walck _et al_., 2011; Kirdyanov

of more than 35 species of genus _Quercus_

_et al_., 2012). The global mean surface

(Singh and Singh 1992). The genus _Quer-_

temperature has risen by 0.6ºC over the

_cus_ is represented by five evergreen tree

last 100 years (Wang _et al_., 2016), and the

species ( _Q. leucotrichophora, Q. flori-_

level of precipitation in Northern Hemi-

_bunda, Q. glauca, Q. lanuginosa_ and _Q._

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 79

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ _semecarpifolia_ ) and one deciduous exotic

is comprises of various propagation

tree species ( _Q. serrata_ ) in Uttarakhand,

methods. However, the clonal propaga-

Western Himalaya, India (Pandey and

tion through nodal explants derived from

Tamta,

2012).

Among

these,

_Q._

mature trees growing in natural forest is

_semecarpifolia_ (Kharsu) possess highest

tedious, still one of the best method to

elevational range (2500-3300m asl) and

develop true-to-type plants of desired

forms timberline in the region (Singh and

quality. In present study attempts were

Singh, 1992).

made to develop clonal propagation pro-

The world's highest timberline is

cedure for _Q. semecarpifolia u_ sing nodal

also utilized by local peoples for their dai-

explant derived from mature elite tree

ly needs (agricultural tools, fuel, food,

growing in _Q. semecarpifolia_ dominated

fodder, spiritual rituals etc.) and liveli-

forest, west Himalaya India.

hoods (agriculture, silk worm rearing,

livestock rearing, etc.). This dependency

**2. Material and methods**

creates enormous pressure on multipur-

_****_

pose evergreen tree species like _Q._

_2.1. Plant material_

_semecarpifolia._ This species is considered

Nodal explants were collected

as the oldest and overexploited plant of

from the mature tree growing in China

sub-alpine zones (Singh _et al.,_ 2010). The

peak forest area (2619m asl; 29 27N and

green leaves of _Q. semecarpifolia_ are

79 29E) of district Nainital, Uttarak-

used in tasar silk-worm rearing and live-

hand, India. Twigs (10-15 cm) were col-

stock fodder; bark and galls for tannin;

lected from different parts of mature trees

dried branches and boles are used in the

(from stump sprouts, or from juvenile

preparation of agricultural implements

parts) and brought to the Plant Tissue

and used as fuel wood (Tamta _et al.,_

Culture laboratory, Department of Bota-

2008; Pandey, 2013). This high magni-

ny, D.S.B. campus Kumaun University,

tude of human pressure along with low

Nainital, India in a mini-chillier (-10C;

regeneration (Bisht _et al.,_ 2012), short

Genei, Banglore). In laboratory, the twigs

viability of seeds (Pandey and Tamta,

were dipped in water and kept in room

2013), and unavailability of a decent seed

temperature for 1h. Within same day, the

crop every year (Pandey 2013) could be

twigs were defoliated and cut in to small

precariousfor thenatural regeneration of

segments (1.0-1.5cm). These nodal seg-

this species in changing climate. Consid-

ments having at least one dormant bud

ering the role of _Q. semecarpifolia_ in the

were used as explant. ****

economy as well as ecology of Himalaya,

****

immediate attention is required for the

_2.2_. _Chemicals, glassware and culture_

conservation of this species through sus-

_conditions_

tainable utilization of its genetic re-

Each chemical used during the ex-

sources. The _ex situ_ conservation through

periment was of analytical grade and pur-

plant tissue culture technique is extensive-

chased from Himedia, Pvt. Ltd. Mumbai,

ly used method for the conservation and

India except labolene (Qualizen, India)

sustainable harnessing of use values of

and Bavistin (purchased from local mar-

threatened and high value woody species

ket). The plant growth regulators were

such as _Citrus sinensis_ (Pandey and Tam-

procured from Duchefa Biochemie, The

ta, 2016); _Berberis aristata_ (Brijwal _et al.,_

Netherlands. All the glasswares used dur-

2015); _Quercus serrata_ (Pandey and

ing the experiment were purchased from

Tamta, 2014); _Berberis chitria_ (Pandey _et_

Borosil India. Cultures were maintained

_al.,_ 2013); _Quercus semecarpifolia_ (Tam-

at 25±1°C under a 16h/8h light/dark pho-

ta _et al.,_ 2008) and other _Quercus_ species

toperiod with an irradiance of 42 μmol

(Wilhelm, 2000). Based on the source of

−

−

m 2 s 1 inside and 60 μmol m−2

explant, the plant tissue culture technique

−

s 1outsidethe culture vessels/ flasks pro-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 80

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ vided by cool fluorescent tubes (40 W;

(w/v) and the media was solidified with

Philips from Saveer Biotech Limited,

0.8% agar (w/v) and/or 0.24% clarigel

New Delhi, India).

(w/v). Besides, casein hydrolysate (CH,

w/v, 500mg l-1) and activated charcoal

_2.3. Explant preparation_

(AC, w/v, 20mg l-1) were also incorpo-

rated in to each shoot multiplication me-

Nodal

explant

were

initially

dium. Each experiment consisted of 12

washed in running tap water for 10 min

explants and repeated twice. Subculturing

and then immersed in detergent solution

was carried out at 6 week interval and da-

(labolene, 0.1%, v/v; 20 min), and rinsed

ta on shoot number and shoot length were

thoroughly with distilled water. Washed

recorded during subculture.

explants were treated with fungicide (ba-

Explant selection experiment was

vistin, 0.5%, w/v; 30 min) with gentle

carried out in culture vessels (50 ml vol-

shaking. Those treated explants were in-

ume, 20 ml medium per tube). The con-

troduced to laminar air flow cabinet,

tamination-free explants were transferred

where after 5 rinses of autoclaved double

to conical flasks (250 ml volume, 100 ml

distilled water, nodal segments were sur-

medium per flask) for further growth and

face disinfected with mercuric chloride

development. WP basal media devoid of

(HgCl2, 0.1%, w/v; 10 min). This treat-

growth regulators served as control in all

ment was followed by subsequent 5 rinses

experiments. All culture vessels and

of autoclaved double distilled water (2

flasks were plugged with non-absorbent

min each). The exposed ends of nodal

cotton plugs and sterilized at 121°C for

segments were excised out prior to inocu-

15 min. The pH was adjusted to 5.7- 5.8

lation. To check the contamination nodal

by 0.1M NaOH or HCl before autoclaving

explants were cultured in WA: water agar

at 1.06 kg cm-2 (121°C) for 20 min.

medium for one week (Figure 1a).

_2.6. Rooting of microshoots_

_2.4. Shoot induction and nutrient medium_

Microshoots (2.0-3.0 cm, with

_selection_

well-developed leaves) were introduced

To select the nutrient medium for

to a two step-rooting procedure; as de-

shoot induction and multiplication, con-

scribed by Pandey and Tamta et al (2012).

tamination-free nodal segments were in-

Briefly, during the first step excised mi-

oculated either in full strength MS: Mu-

croshoots were cultured in full strength

rashige and Skoog (1962) or WP: Lloyd

WP media supplemented with different

and McCown (1980) nutrient medium.

concentrations of indole-3-butyric acid

Each medium was supplemented with 6-

(IBA 50.0 or 100.0 M) for 24 or 48 h

benzyleamenopurene (BA, 4.44 µM) and

and cultures were placed in a dark during

activated charcoal (AC, w/v, 20mg l-1) to

this step (Table 2). In the second step the-

monitor the shoot induction responses.

se treated shoots were transferred to PGR-

free half-strength WP medium, solidified

_2.5_. _Shoot multiplication_

with clarigel (0.25%) and exposed to 16 h

After 30 day on shoot induction

photoperiod. The percentage of root for-

media, explants with positive shoot induc-

mation, lengths of the formed roots and

tion response were transferred to shoot

length of longest root were recorded after

multiplication media (Figure 1). Different

4 weeks of incubation in PGR-free half-

concentrations of cytokinin (BA, 4.44-

strength WP medium.

22.20 μM) alone or in combination with

IAA (1.43 μM) or GA3 (0.73-1.44 μM)

_2.7_. _Acclimatization_

were tested for shoot multiplication re-

After 4 week of transfer to PGR-

sponses in WP full strength nutrient me-

free medium, these shoots with roots were

dium. The sucrose concentration was 3.0%

taken out from the culture flasks and wa-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 81

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ **Figure 1:** _In vitro_ propagation of _Quercus semecarpifolia_. **a** ) Explant inoculation in WA

medium to check contamination; **b)** contamination free explants having positive response;

**c-d)** Shoot proliferation in WP supplemented with (BA 4.44 M); **e)** shoot elongation in

BA+GA3 (4.44 M +1.45 M); **f)** shoot multiplication in BA+GA3 (8.88 M +1.45 M); **g)**

root induction in IBA 100 M for 24 h. __

-shed gently with distilled water to re-

col pots (8 cm width and 10 cm height)

move traces of clarigel. After recording of

containing soil and farmyard manure (3:1,

data, plantlets were transferred to therma-

w/w). These plants were placed inside

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 82

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ growth chamber under16 h photoperiod

was recorded in MS medium supplement-

−

(60μmol m−2s 1) at 25±2°C temperatures

ed with similar concentration of BA

and 60% relative humidity. Plants were

(4.44M) with higher response time of

watered on alternate days with 1/4 basal

17.44±2.12 days. The activated charcoal

WP medium devoid of sucrose and accli-

particles (gas absorber) were found more

matized over a period of 4 week. ****

suitable then activated charcoal powder.

****

The incorporation of activated charcoal in

_2.8_. _Statistical analysis_

to shoot-induction media has shown

Experiments were performed in a

promising results (data not shown) in

completely randomized design to deter-

shoot induction and incorporated in to the

mine the effect of treatments and concen-

shoot multiplication media.

trations on plant vigour. Data presented as

mean values ± standard error (SE) and

collected from three independent experi-

MS

Response time (days)

WP

ments. The statistical analyses were per-

formed using SPSS (Statistical Package

for Social Science, version 20). Level of

Shoot induction (%)

significance was determined by analysis

of variance (ANOVA) and statistical sig-

nificance mean values were grouped by

0

20

40

60

80 100 120

using Duncan's multiple range post hoc

Numbers

test ( _p <0.05_).

**Figure 2:** Effect of nutrient mediums on

**3. Results**

_in vitro_ propagation of _Q. semecarpifolia._

****

_MS_ , Murashige and Skoog (1962) medi-

_3.1. In vitro culture establishment_

um; _WP_ , woody plant medium (Lloyd and

Nodal explants cultured on WA

McCown, 1980). __

medium had shown high contamination

and poor survival (10-15%) rate. The sur-

_3.3. Shoot multiplication_

vival rate could not be maximized even

Explants with positive responses

after altering the concentrations and expo-

(induced shoots; Figure 1b-c) were sub-

sure time of disinfectants (data not

cultured in the shoot multiplication medi-

shown). Further, the high phenolic secre-

um, supplemented with different concen-

tion from basal end of nodal segment was

trations of BA alone or in combination

also observed. Nonetheless, with the in-

with GA3 or IAA (Table-1). After six

crease in concentration and exposure time

week of culture, the shoot multiplication

of disinfectant, explants started to die.

rate differed significantly (p<0.05) among

The contamination-free explants were

the treatments. In alone BA supplemented

used for shoot induction.

media, the shoot number was significantly

(p<0.05) increased with the increasing

_3.2. Shoot induction_

BA concentrations from 4.44 M to 22.22

Both tested media (MS or WP)

M. The significantly (p<0.05) highest

supplemented with BA (4.44M) were

number of shoots were (7.44±0.44) ob-

able to induce shoots (Figure2), neverthe-

served in WP medium supplemented with

less, the shoot induction rate and response

BA+IAA (17.76+1.43 M) (Plate-1d,f).

time was different. WP basal medium for-

Overall, the significantly (p<0.05) best

tified with BA (4.44M) gave better re-

shoot multiplication response (5.89±0.59

sults. Average shoot induction (88.89%)

shoots/explant) with the average shoot

was recorded within (14.00±2.03 days)

length of 3.37±0.29 cm and 4.02±0.40 cm

response time. While comparatively less

average length of longest shoot was ob-

percentage of shoot induction (77.78%)

served in WP medium supplemented with

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 83

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ BA+GA3 (8.88+0.72M) (Figure 1e).

because of the ability of HgCl2 to pene-

Though the number of shoots were signif-

trate the cutinized cell wall, which leads

icantly (p<0.05) less in BA+GA3 supple-

to precipitation of cell protein (Sharma

mented media but the other parameters

and Sharma, 1980).

were recorded significantly (p<0.05) bet-

Charcoal is a form of carbon hav-

ter in comparison to other used treat-

ing a high adsorptive capacity of gases,

ments. The explants cultured in control

vapours and colloidal solids (Pan and

treatment had not shown any shoot multi-

Staden, 1998). The activated charcoal

plication response.

used in WP nutrient media has an adsorp-

tion preference for moderately polar ra-

_3.4. Rooting of microshoots_

ther than a polar or highly polar organic

Root induction visually observed

chemicals and they show greater adsorp-

after 10 days of transfer in ½ strength WP

tion for aromatic than olefinic unsatura-

media. Shoots having root length >1mm

tion products (Yam _et al.,_ 1990). There-

considered as rooted. All IBA treatments

fore, aromatic compounds such as the

were able to induce roots in microshoots;

phenolic compounds secreted from nodal

however,

the

maximum

rooting

explants and their oxidates, could have

(88.89±1.11) was observed in the medium

great adsorption affinity for activated

supplemented with 100.0 M IBA **(** Figure

charcoals. Nevertheless, the highly polar

1g). A 3.63±0.23 number of roots were

and readily water-soluble sugars (glucose,

observed within 4 weeks with an average

sorbitol, mannitol and inositol) might not

root length of 0.86±0.01cm (Table-2).

be removed from the medium and/or solu-

Root formation was not observed in mi-

tion (Pan and Staden, 1998). This proper-

croshoots that lack IBA in the first step of

ty of activated charcoal might have en-

root induction method. Although root in-

hanced the shoot induction rate in present

duction takes place by applying two step

study without altering the basic composi-

methods but these rooted plantlets were

tion of WP nutrient medium. Activated

not able to survive in transplanted soil

charcoal also controlled browning and

conditions and died after 20 days.

stimulated shoot growth of _Strelitzia re-_

_ginae_ and _Anemone oronaria_ (Mensuali-

**4. Discussion**

Sodi, 1993), and found more effective

****

than ascorbic acid or PVP in reducing

Sterilization of _explants_ is the

browning in _Dipterocarpus intricatus_

most important step of plant tissue cul-

(Linington, 1991).

ture. For present study two well-studied

WP basal medium supplemented

sterilizing agents (Mercuric chloride and

with BA+GA3 (8.88+0.72 µM) was found

Bavistin) were used. Bavistin is systemic

to be the best media for shoot multiplica-

fungicide generally used against the

tion. WP medium is reported to be the

members of ascomycetes, deuteromycetes

best and effective in several earlier stud-

and various basidiomycetes (Vyas, 1984).

ied in _Quercus_ species, _viz_. _Q. leucotri-_

Mercuric chloride (HgCl

_chophora_ and _Q. glauca_ (Purohit _et al.,_

2) used to control

various microbial infestations (Bonga and

2002b), _Q. semecarpifolia_ (Tamta _et al.,_

Aderkas, 1992), and it is effectively used

2008), _Q. rubra_ (Vieitez _et al.,_ 1993;

for the surface sterilization of several hard

Vengadesan and Pijut, 2009; Pandey and

wood species (Chalupa, 1987). In present

Tamta, 2015). The used cytokinin combi-

study both used sterilents were not able to

nation BA+GA3, was also found suitable

control contamination rate and only 10-

for _Q. rubra_ (Vengadesan and Pijut,

20% contamination-free explants were

2009), _Q. leucotrichophora_ and _Q. glauca_

achieved. On increasing the concentration

(Purohit _et al.,_ 2002b) and in _Quercus flo-_

or treatment time of HgCl

_ribunda_ (Purohit _et al.,_ 2002a). During

2 the percent

survival was further decreased; this may

the study the alone cytokinin BA was

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 84

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ used; BA reported to be the best plant

yan Environment and Sustainable Devel-

growth regulator in _Quercus shumardii_

opment, Almora, Uttarakhand, India for

for shoot multiplication (Bennett and Da-

providing financial assistance through

vies, 1986). 20 μM BA was found to be

integrated eco-development research pro-

best for adventitious shoot induction and

gramme (IERP) for Indian Himalayan re-

multiplication of individual shoots of _Q._

gion (IHR) during 2009-2011. The head

_semecarpifolia_ (Tamta _et al.,_ 2008). In

department of botany, D.S.B. Campus and

contrast to multiplication from petiolar

department of biotechnology, Bhimtal

tube of _Q. semecarpifolia_ the higher con-

Campus of Kumaun University, Nainital,

centrations of BA were not found to be

Uttarakhand India are highly acknowl-

the best concentration although it induced

edged for providing necessary facilities

highest number of shoots/bud but the

for experimentation.

length of shoots was reduced. Shoot mul-

****

tiplication was followed by rooting. Earli-

**References**

er study in _Q. semecarpifolia_ suggests

that IBA is the better responded auxin

**Bennet, L.K. and Davies, F.T. (1986).** _In_

(root inducer) than NAA and a two step-

_vitro_ propagation of _Quercus shu-_

rooting procedure resulted 100.0% rooting

_mardii_ seedlings. _Horticulture Sci-_

response, without the formation of basal

_ence_ **21(4), 1045-1047**.

callus (Tamta _et al.,_ 2008). In present

**Bisht, H., Prakash, V. and Nautiyal,**

study two step method was used and

**A.R. (2012).** Factors Affecting Re-

about 88.89percent rooting was recorded

generation Potential of _Quercus_

within 20 days of culture. Similar method

_semecarpifolia_ , Smith: A Poor Re-

of rooting has also been reported earlier in

generated Oak of Himalayan Tim-

other _Quercus_ species such as _Q. suber_

berline. _Research Journal of Seed_

(Manzanera and Pardos, 1990) and _Q._

_Science_

**5,**

**63-70.**

**doi:**

_leucotrichophora_ and _Q. glauca_ (Purohit

**10.3923/rjss.2012.63.70.**

_et al.,_ 2002b). During acclimatization the

**Bonga, J. and Aderkas, P. (1992).** _In_

semi-rooted microshoots were not able to

_vitro_ culture in trees, Kluwer Aca-

survive. The survival rate of microshoots

demic

Publ,

Dordrecht-Boston-

was poor due to undeveloped rooting sys-

London **. pp. 207.**

tem to survive _ex vitro_ conditions.

**Brijwal, L., Pandey, A. and Tamta, S.**

**(2015).** _In vitro_ propagation of the

**5. Conclusion**

endangered species _Berberis aris-_

_tata_ DC. via leaf-derived callus. _In_

Results of present study are en-

_Vitro Cellular and Developmental_

couraging and could be useful in develop-

_Biology-Plant_ **51(6), 637-647.**

ing a rapid clonal propagation method for

**Chalupa, V. (1987).** Effect of benzyla-

this important multipurpose tree species.

minopurine and thidiazuron on _in_

However, the low rate of multiplication

_vitro_ shoot proliferation of Tilia

and poor survival rate of plantlets in the

cordata Mill., _Sorbus aucuparia_ L.

soil are the main challenges at the present.

and _Robinia pseudoacacia_ L. _Biolo-_

Therefore, to address the low regeneration

_gia Plantarum_ **29, 425-429.**

and to improve survival rate of _Q._

**Harsch, M.A.,**

**Hulme,**

**P.**

_semecarpifolia_ further research is re-

**E., McGlone, M. S. and Duncan,**

quired.

**R. P. (2009).** Are tree lines advanc-

ing? A global meta-analysis of tree-

## Acknowledgements

### line response to climate warm-

ing. _Ecology Letters_ **12,1040–1049.**

The authors express their gratitude

**IPCC, (2013).** Climate Change 2013: The

to G.B. Pant National Institute of Himala-

Physical Science Basis. Contribu-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 85

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ tion of Working Group I to the Fifth

ods. Ph.D. thesis submitted to

Assessment Report of the Intergov-

Kumaun University Nainital, Utta-

ernmental Panel on Climate Change,

rakhand, India.

Cambridge, UK. **pp. 1535.**

**Pandey, A. and Tamta, S. (2013).** Effect

**Kirdyanov, A. V., Hagedorn, F.,**

of pre-sowing treatments on seed

**Knorre, A. A., Fedotova, E.**

germination in _Quercus serrata_

**V.,Vaganov, E. A., Naurzbaev, M.**

Thunb. and _Quercus semecarpifolia_

**M., Moiseev, P. A. and Rigling, A.**

Sm. _International Journal of Biodi-_

**(2012).** 20th century treeline ad-

_versity and Conservation_ **5(12),**

vance and vegetation changes along

**791-795.**

an altitudinal transect in the Putora-

**Pandey, A. and Tamta, S. (2014).** _In_

na Mountains, northern Siberia. Bo-

_vitro_ propagation of the important

reas **41, 56–67.**

tasar oak ( _Quercus serrata_ Thunb.)

**Linington, I.M. (1991).** _In vitro_ propaga-

by casein hydrolysate promoted

tion

of

_Dipterocarpusintricatus_.

high frequency shoot proliferation.

_Plant Cell Tissue Organ Culture_ **27,**

_Journal of sustainable forestry_

**81–88.**

**33(6), 590-603.**

**Lloyd, G. and McCown, L.B. (1980).**

**Pandey, A. and Tamta, S. (2016).** Effi-

Commercially feasible micropropa-

cient micropropagation of _Citrus_

gation of mountain laurel, Kalmia

_sinensis_ (L.) Osbeck from cotyledo-

latifolia, by use of shoot-tip culture.

nary explants suitable for the devel-

Comb. Proc. International Plant

opment of commercial variety. _Afri-_

Propagation Society **30, 421-427.**

_can_

_Journal_

_of_

_Biotechnology_

**Manzanera, J.A. and Pardos, J.A.**

**15(34), 1806-1812.**

**(1990).** Micropropagation of juve-

**Pandey, A. Brijwal, L. and Tamta, S.**

nile and adult _Quercus suber_

**(2013).** _In vitro_ propagation and phy-

L. _Plant Cell Tissue Organ Culture_

tochemical assessment of _Berberis_

**21, 1-8.**

_chitria_ : An important medicinal

**Mensuali-Sodi, A., Panizza, M., Serra,**

shrub of Kumaun Himalaya, India.

**G. and Tognoni, F. (1993).** In-

_Journal of Medicinal Plants Re-_

volvement of activated charcoal in

_search_ **7(15), 930-937.**

the modulation of abiotic and biotic

**Purohit, V.K., Palni, L.M.S., Nandi,**

ethylene levels in tissue-cultures.

**S.K. and Rikhari, H.C. (2002a).** _In_

_Scientia Horticulture_ **54, 49–57.**

_vitro_ plant regeneration through cot-

**Murashige, T. and Skoog, F. (1962).** A

yledonary nodes of _Quercus flori-_

revised medium for rapid growth

_bunda_ Lindl. ex A. Camus (Tilonj

and bioassays with tobacco tissue

oak), a high value tree species of

cultures. _Physiologia Plantarum_ **15,**

central Himalaya. _Current Science_

**473-497.**

**833, 101-104.**

**Pan, M. J. and Staden, J. V. (1998).** The

**Purohit, V.K., Palni, L.M.S., Nandi,**

use of charcoal in _in vitro_ culture–A

**S.K., Vyas, P. and Tamta, S.**

review. _Plant growth regulation_

**(2002b).** Somatic embryogenesis in

**26(3), 155-163.**

_Quercus floribunda_ , an important

**Pandey, A. and Tamta, S. (2012).** Influ-

Central Himalayan oak. In: Role of

ence of kinetin on _in vitro_ rooting

plant tissue culture in biodiversity

and survival of banj oak ( _Quercus_

conservation and economic devel-

_leucotrichophora_ L.). _African Jour-_

opment. Nandi SK, Palni LMS,

_nal of Biotechnology_ **62 (11),**

Kumar A (Ed) Gyanodaya Prakash-

**12538-12545.**

an, Nainital, **pp. 41-52.**

**Pandey, A. (2013).** Propagation of two

**Singh, G. and Rawat, G.S. (2010).** Is the

species of oaks using _in vitro_ meth-

future of oak ( _Quercus_ spp.) forest

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 86

_Biotech Sustainability (2017)_

_Clonal Propagation of Quercus semecarpifolia ... Pandeyand Tamta_ safe in the Western Himalaya? _Cur-Vyas, S.C. (1984). Systemic fungicides,_

 _

_rent Science_ **98, 1420-1421.**

Tata McGraw hill Publishing Co.

**Singh, J.S. and Singh, S.P. (1992).** For-

Ltd. New Delhi.

ests of Himalaya: Structure, func-

**Walck, J. L., Hidayati, S. N., Dixon, K.**

tioning

and

impact

of

man.

**W.,**

**Thompson,**

**K.E.N.**

**and**

Gyanodaya

Prakashan,

Nainital,

**Poschlod,**

**P.**

**(2011).**

Climate

**pp.294**.

change and plant regeneration from

**Smith, W. K., Germino, M. J., Johnson,**

seed. _Global Change Biology_ **17,**

**D. M. and Reinhardt, K. (2009).**

**2145–2161.**

The altitude of alpine treeline: a

**Wang, B., Chen, T., Xu, G., Liu, X.,**

bellwether of climate change ef-

**Wang, W., Wu, G. and Zhang, Y.**

fects. _The Botanical Review_ **75(2),**

**(2016).** Alpine timberline popula-

**163-190.**

tion dynamics under climate change:

**Tamta, S., Palni, L.M.S., Purohit, V.K.**

a comparison between Qilian juni-

**and Nandi, S.K. (2008).** _In vitro_

per and Qinghai spruce tree species

propagation of brown oak ( _Quercus_

in the middle Qilian Mountains of

_semecarpifolia_ Sm.) from seedling

northeast Tibetan Plateau. Boreas.

explants. _In Vitro Cellular and De-_

**10.1111/bor.12161. ISSN 0300-**

_velopment Biology-Plant_ **44(2),136-**

**9483.**

**141.**

**Wilhelm, E. (2000).** Somatic embryogen-

**Vengadesan, G. and Pijut, P.M.**

esis in oak ( _Quercus_ spp.). _In Vitro_

**(2009).** _In_

_vitro_

propagation

of

_Cellular and Developmental Biolo-_

northern red oak ( _Quercus rubra_

_gy-Plant_ **36(5), 349-357.**

L.). _In Vitro Cellular and Develop-_

**Yam, T.Y., Ernst, R., Arditti, J., Nair,**

_ment Biology-Plant_ **45, 474-482.**

**H.**

**and**

**Weatherhead,**

**M.A.**

**Vieitez, A. M., Pintos, F., San-Jose, M.**

**(1990).** Charcoal in orchid seed

**and Ballester, A. (1993).** _In vitro-_

germination and tissue culture me-

shoot proliferation determined by

dia: a review. _Lindleyana_ **5, 256–**

explant orientation of juvenile and

**265.**

mature _Quercus rubra_ L. _Tree Phys-_

_iology_ **12, 107-117.**

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 87

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P88-103_

**Spent Mushroom Substrate of _Hypsizygus ulmarius_** **: A**

**Novel Multifunctional Constituent for Mycorestoration**

**and Mycoremediation**

**Padmavathi Tallapragada1, * and Ranjini Ramesh2**

_1Department of Microbiology, Centre for Post Graduate Studies, Jain University, 18/3,9th_

_Main, Jayanagar 3rd Block, Bangalore, India; 2Department of Environmental Science,_

_Mount Carmel College, Autonomous, 58, Palace Road, Vasanthnagar, Bangalore, India;_

_*Correspondence: vam2010tpraviju@gmail.com _ _/ t.padmavathi@jainuniversity.ac.in; Tel_ _:_

_+91 9448533337_

__

**Abstract:** _'Spent Mushroom_ _Substrate'_ (SMS) is a composted growing medium that results

from the mushroom growing process. The spent substrate remains after harvesting the

mushrooms, which is entangled with innumerable mushroom threads (collectively referred

as _'mycelia'_ ), would have been biochemically modified by the mushroom enzymes into a

simpler and more readily digestible form, which could then be used in _'mycorestoration'_

and _'mycoremediation'_. Mushroom mycelia can produce a group of complex extracellular

enzymes that can degrade and utilize the lignocellulosic wastes found in nature, which also

reduces their potential for pollution. It has been revealed recently that mushroom mycelia

can play a significant role in the restoration of damaged environments. Saprotrophic, endo-

phytic, mycorrhizal and even parasitic fungi or mushrooms can be used in _'mycorestora-_

_tion'_ , which can be performed in four different ways: _'mycofiltration'_ (using mycelia to fil-

ter contaminated water), _'mycoforestry'_ (using mycelia to restore degraded forests), _'my-_

_coremediation'_ (using mycelia to eliminate toxic wastes from soil and water) and _'my-_

_copesticides'_ (using mycelia to control insect pests). These methods represent the potential

to create a clean ecosystem, where no damage will be left after fungal implementation. _'Ap-_

_plied Mushroom Biology'_ can not only convert this huge amount of lignocellulosic wastes

into human food but also can produce notable nutraceutical products, which have several

health benefits and it is discussed in this chapter.

****

_**Keywords**_ **:** Applied mushroom biology; _Hypsizygus ulmarius;_ mycoremediation; my-

corestoration; spent mushroom substrate

**1. Introduction**

and high-yielding varieties have been fol-

lowed to overcome the constraints (Dal-

Soils in the tropical regions of the

gaard _et al.,_ 2003). With the help of these

world are fragile, contain very less organ-

technologies, there has been a world-wide

ic matter and are prone to severe degrada-

doubling of food crop production, but at

tion, especially with increased deforesta-

the cost of environmental degradation of

tion and loss of topsoil. These attributes

soil and water quality, reduction in biodi-

of tropical soils put constraints on food-

versity and suppression of ecosystem

crop production in these regions of high

functions (Vance, 2001). Today, more

and dense human populations. In the last

than one billion people lack in food secu-

few decades, _Green Revolution_ practices

rity and many village communities in the-

like using pesticides, synthetic fertilizers

se areas are continuously affected by a

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 88

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ steady reduction of food grains. In addi-being utilized extensively in industry, ag-

tion, the increase in industrialization has

riculture, medicine, food and textile in-

polluted our environment with chemicals

dustries (Prabhakaran _et al.,_ 2011).

and toxins of various kinds (Singh _et al.,_

_Hypsizygus ulmarius_ , the _'Elm_

2011) Most contaminated sites usually

_Oyster Mushroom'_ (Figure 1), is a new

contain a mixture of non biodegradable

variety of edible mushroom, developed by

persistent compounds, which increase the

the Indian Institute of Horticultural Re-

difficulties of remediation. This is due to

search

(IIHR),

Bangalore

\-

the intensification of agriculture, range of

www.iihr.res.in. It is a type of basidiomy-

crops grown and the diversity of manu-

cete, also known as _'white rot fungi',_ of

facturing industries. The excess usage of

which there are about 1,400 known spe-

chemical fertilizers has also contributed

cies. It can be commercially cultivated by

to the deterioration of the environment,

solid-state fermentation method, using

with soil degradation, loss of soil fertility

agricultural wastes such as paddy straw,

and agricultural productivity being the

coconut husk, tea and saw dust, among

main consequences (Khan and Ishaq,

others. Mushroom cultivation is environ-

2011).

ment-friendly, in addition to providing a

For improving the long-term sus-

cost-effective source of food protein for

tainability of industry and agriculture,

vegetarians and a source of income for

emphasis should be on the holistic man-

rural women (Ahmed _et al_., 2009). Mush-

agement of natural resources. Microor-

rooms are a good source of vitamins and

ganisms can control pollution and pests,

minerals, while having low content of

maintain the fertility of soil and enhance

fats, carbohydrates and dietary fiber. With

plant growth, with no major adverse ef-

their nutritional value, mushrooms can

fects on the environment or other non-

reduce malnutrition in the rural poor to a

target organisms (Gomathi and Ambika-

large extent, and are also effective in re-

pathy, 2011). These types of mechanisms

ducing the occurrence of life-style diseas-

rely on stimulating the growth of specific

es like hypercholesterolemia, hyperten-

species of micro-organisms or mixtures of

sion, diabetes and cancer (Alam _et al.,_

microflora native to the contaminated

2007).

sites and are thus, able to remediate the

area more easily and efficiently (Kumar _et_

_al._ , 2010).

Recent research has favored the

techniques of _'bioremediation'_ for clean-

ing up the above types of sites, as it is

both environment-friendly and of relative-

ly low-cost (Sasek, 2003). Bioremediation

is the addition of biological agents, main-

ly microbes like yeast cells, fungi or bac-

teria to detoxify the contaminated soil and

water. When fungi are specifically used, it

is known as _'mycoremediation'_. Lignino-

__

**Figure 1:** _Hypsizygus ulmarius_ : The Elm

lytic basidiomycete fungi such as _Phan-_

Oytser Mushroom, growing naturally on

_erochaete chrysosporium, Pleurotus os-_

the

bark

of

Elm

trees

_treatus, Lentinula edodes_ , etc. are well

(http://www.mushroomexpert.com/hypsiz

known mycoremediation agents. These

ygus_ulmarius.html) ****

microbes use the xenobiotics to be de-

__

graded as nutrients or as sources of ener-

_'Spent_

_mushroom_

_substrate'_

gy (Tang _et al.,_ 2007). Fungi play an im-

(SMS) is the by-product of mushroom

portant role in bioremediation, besides

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_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ cultivation, and contains the fungal myce-lic acid, 2,6-dimethoxyphenol, etc (Ike-

lium, fermented substrate, residues of in-

hata _et al.,_ 2004).

organic nutrients and secreted enzymes

Phenol, also known as carbolic ac-

such as ligno-cellulases, proteases and

id, is a highly toxic element that is added

peroxidases (Medina _et al.,_ 2009). SMS

in the manufacture of resins, herbicides

is also a rich source of carbon, nitrogen

and various other industrial processes

and other nutrients, and can be added to

(Amara and Salem, 2010). It is one of the

enhance crop growth and maintain soil

most persistent chemicals, with high tox-

fertility. It contains a consortium of bacte-

icity even at low concentrations, and is

ria and fungi which can mediate the for-

considered a _'priority pollutant'_ under the

mation and weathering of soil, nutrient

Environment

Protection

Act,

1986

and water mobilization, nitrogen fixation

(Chandrakant _et al.,_ 2006). Phenols can

and denitrification processes. The fungal

be degraded by various white rot fungi

mycelium on the spent substrate is similar

like _P.florida, L.edodes and H.ulmarius_

to _'Arbuscular mycorrhizal fungi'_ – AMF

(Ranjini and Padmavathi, 2013; 2012).

(Jonathan _et al.,_ 2013). AM fungi espe-

_Pleurotus florida_ , _Pleurotus_ _os-_

cially function to mobilize water and

_treatus, Pleurotus flabellatus_ and _Pleuro-_

phosphorus for plants (Manimegalai _et_

_tus_ _sajor-caju_ have also been studied for

_al_., 2011). ). _Pleurotus florida_ is a known

their potential in dye decolourization

_'mycorestoration agent'_ , as it improves

(Faraco _et al.,_ 2009). Azo dyes are added

soil fertility by phosphate solubilization,

in textile, pharmaceutical, cosmetic and

increases aeration and water movements

food industries. After processing, almost

through soil and enhances plant growth

forty percent of the dye is released into

(Kumar _et al.,_ 2010).

wastewater. This affects the aesthetics,

_White rot fungi_ are some of na-

transparency and oxygen levels of the re-

ture's most efficient lignin degraders from

ceiving water, making it toxic (Ali _et al.,_

the microbial world, due to their ability to

2008). Even at very low concentrations

produce several kinds of lignin and phe-

(<1 mg/L) in the effluent, they are visible

nol-degrading enzymes such as laccases

and cause turbidity, especially red colour

and peroxidases. Laccases are copper-

(Forgacs and Oros, 2004). Reactive dyes

containing, glycosylated polyphenol oxi-

are significant because of their bright col-

dases. Their broad substrate specificity

our and low energy usage during applica-

increases their significance in industrial

tion (Aksu, 2005). In exhausted dye baths

and biotechnological applications such as

and rinsing water, they are not recyclable

biomechanical pulping of cellulosic mat-

or biodegradable.

ter, bleaching of pulp and degradation of

Dyes can cause allergies, skin irri-

dyes, chloro-phenols and a variety of xe-

tation and skin cancers, in addition to ge-

nobiotic and aromatic compounds, mainly

netic mutations (Inbaraj _et al.,_ 2002).

by reduction of oxygen to water (Patel _et_

Bacterial degradation of azo and reactive

_al.,_ 2008). Extracellular laccase is usually

dyes usually occurs anaerobically, trans-

secreted into the medium in small quanti-

forming them into carcinogenic interme-

ties. Its production is affected by typical

diates; thus it is not considered suitable

fermentation factors such as media com-

on a large scale (Manikandan _et al.,_

position, carbon-nitrogen ratio of growth

2012). Fungi, in aerobic conditions, can

media, pH, temperature and diffusion of

uptake and remove dyes without creating

oxygen into the media (Revankar and

the above carcinogens. Physical adsorp-

Lele, 2006). Their production is stimulat-

tion onto the surface of spent mycelium,

ed by the addition of inducers such as

followed by enzymatic breakdown is the

phenolic and aromatic compounds like

usual mechanism of fungal remediation of

catechol, guaiacol, veratryl alcohol, ferru-

dyes (Zumriye and Karabayir, 2008). The

effectiveness of fungi in bioremediation

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 90

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ of dye-polluted areas is because of their

mainly produce cellulases (Singh _et al_.,

ability to penetrate and break the chemi-

2002).

cal structure of the dye molecules, as they

Carbon sources also have a regu-

have long hyphae and also their capability

lating effect on enzyme secretion by _white_

to degrade a wide range of dyes, especial-

_rot fungi_. In _Phanerochaete chrysospori-_

ly anthraquinone and triphenylmethane

_um_ , ligninolytic genes are triggered by the

dyes (Palmieri _et al.,_ 2005). According to

depletion of carbon in the media (Wang _et_

studies by Kodam _et al._ , (2005), azo dyes

_al.,_ 2008). In _Trametes pubescens_ , signif-

can be decolorized by _white-rot fungi_.

icant laccase secretion occurs when glu-

Microbial decolourization uses both oxi-

cose is reduced to a critical level. Glucose

dative and reductive steps, oxidation

and cellobiose are good inducers of lac-

brought about by peroxidases (lignin pe-

case, while fructose and cellulose inhibit

roxidase, manganese peroxidase, versatile

it (Bettin _et al.,_ 2008). The above obser-

peroxidase, etc.) and laccases, usually

vations indicate that carbohydrates can

found in these fungi for breaking down

regulate laccase secretion in _white rots_. __

lignin, the main constituent of woody

The source of carbon used is fungus-

substrates (Gomaere and Govindwar,

specific.

2009).

Other factors like pH, tempera-

The production of ligninolytic en-

ture, the presence of inducers and inhibi-

zymes by _white rot fungi_ is regulated to a

tors have their own effects on enzyme

great extent by the concentration and car-

secretion. The effect of extremes of pH

bon-nitrogen sources added. Mikiashvili

may be due to the fact that it alters the

_et al._ (2005) proved this during his re-

three-dimensional structure of the en-

search on _Trametes versicolor_. It was ob-

zymes. Higher temperatures can reduce

served that both the nature (i.e.) organic

enzyme production by drying of the sub-

or inorganic source of nitrogen and con-

strate (Patel _et al.,_ 2008). Surfactants like

centration of nitrogen are important fac-

Tween 20 and Tween 80 are inhibitors,

tors that regulate their secretion. Media

while veratryl alcohol, vanillic acid, feru-

with high nitrogen content produced

lic acid, guaiacol and copper sulphate are

higher quantity of laccase activity in _Len-_

inducers (Ikehata _et al.,_ 2004).

_tinus edodes, Rigidoporus lignosus_ and

The science of _'Applied Mushroom_

_Trametes pubescens_ , while nitrogen-

_Biology_ can provide solutions to the

limited conditions enhance enzyme pro-

above environmental problems in the fol-

duction in _Pycnoporus cinnabarinus, P._

lowing ways:

_sanguineus_ and _Phlebia radiata_. In some

i. _Mushroom cultivation_ \- Production

cases, high nitrogen content of the sub-

of inexpensive food protein (mush-

strate suppresses enzyme activity. This

rooms)

using

agricultural

by-

occurs in substrates with high lignin con-

products like paddy straw, coconut

tent, correlated with a reduction in activi-

husk, tea, and saw dust, which also

ty of peroxidase or phenol oxidase. How-

generates the _spent mushroom sub-_

ever, substrates with low lignin content

_strate_ (SMS).

degrade well, which is correlated with

ii. _Mycorestoration –_ Addition of the

increase of cellulase activity. This shows

above generated spent mushroom

that _white-rot fungi_ are more important in

substrate to improve the fertility of

high-lignin substrates, where they are the

marginal and contaminated soils by

primary organisms responsible for the

increasing soil aeration and the

secretion of phenol oxidase. This enzyme

availability of phosphorus and po-

is suppressed by the addition of excess

tassium to plants.

nitrogen. In low-lignin substrates, excess

iii. _Mycoremediation –_ Uptake and/ or

nitrogen stimulates a wider group of cel-

degradation of environmental pollu-

lulose-degrading fungi and bacteria that

tants like phenol and dyes using

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 91

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ fungal enzymes present in the above

_bacteria_ (PGPR). The nitrogen content of

generated spent substrate (Figure 2).

SMS varies from 0.4-13.7% with a C: N

****

ratio of 9 to 15: 1. It contains cations like

**2. Mycorestoration of soil using spent**

K+, Na+, Ca2+, Mg2+; and anions like Cl-,

**mushroom substrate from mush-**

NO -

2-

3 , SO4 ; all essential for optimal plant

**room cultivation**

growth. It improves physical soil proper-

__

ties by decreasing its density, surface

_Spent mushroom substrate_ (SMS)

crust formation and diurnal temperature

also known as _'spent mushroom compost'_

changes; in addition to increasing infiltra-

(SMC), is generated as a by-product after

tion, aeration __ and water-retaining capaci-

the harvest of mushroom crop. Recently,

ties. It maintains a high organic content of

it has been proposed to re-name it as _'post_

soil. It can be added singly or as a sup-

_mushroom substrate'_ because it is not re-

plement to conventional bio-fertilizers,

ally _'spent'_ and has many uses remaining.

though it functions better in combination

The composition of spent mushroom sub-

with other bio-fertilizers (Rinker and Kan

strate varies depending on the type of

Zeri, 2004).

mushroom cultivated and the agricultural

Restoring a degraded or stressed

waste material used as substrate. It is an

soil using mycorrhizae and _myco-bio-_

excellent source of humus, although much

_fertilizers_ is known as ' _mycorestoration_ '.

of its nitrogen content is used up by the

Humans have the ability to synergize my-

growing mushrooms. Overall, it is a good

corrhizae and use them for healing forest

source of the macro nutrients _viz_., nitro-

habitats that have suffered from stress,

gen, phosphorus and potassium, in addi-

toxic waste or poor nutrition, making

tion to having trace elements. This makes

them critical to our mutual evolutionary

it suitable for supporting plant growth

survival (Stamets, 2006). Most plants also

(Kulshreshtha and Sharma, 2014 **)**. This is

have associated with them diverse groups

also because it behaves similar to ' _arbus-_

of _plant-growth-promoting-fungi_ (PGPF)

_cular mycorrhizal fungi'_ (AMF), associ-

and AMF.

ated with _plant-growth-promoting rhizo-_

**Research Process Flow**

**Applied Mushroom Biology**

**Effective**

**Can remove**

**SWM**

**malnutrition**

**Mushroom Cultivation**

**Food protein**

**Generation of SMS**

**Mycoremediation**

**Mycorestoration**

**Nutraceuticals**

**Food**

**Uptake of phenol and dyes**

**Release of Phosphorus**

**Industry**

**Pharmaceuticals**

**Soil fertility improved**

**Bioremediation of soil pollutants**

**Medicines**

**Reduced fertilizer dependence**

7

****

**Figure 2:** The science of applied mushroom biology.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 92

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ _2.1._ **** _The mushroom industry and mush-For obtaining the spent mushroom_

 _

_room cultivation_ ****

substrate (SMS) of _H.ulmarius_ , it is culti-

The ever-increasing demand for

vated on agricultural wastes such as pad-

protein-rich vegetarian food and the inef-

dy straw and coconut husk _****_ (Khan _et al.,_

ficiency of conventional methods have

2008), by solid-state fermentation meth-

resulted in the need to explore alternatives

od, as prescribed by Chang (1999).

for low cost production of protein-rich

food like mushrooms (Mukherjee and

_2.2.1. Substrate preparation and sterili-_

Nandi, 2004). The mushroom industry

_zation_

has a world production greater than 25

Crushed rice straw is used for cul-

million tonnes. The largest producer is

tivation. Straw is cut to 2-6 cm pieces,

China, which cultivates more than 20 mil-

soaked overnight and autoclaved, at

lion tonnes and account for over 80% of

121oC and 15 psi, while coconut husk is

the world's mushroom production (Li,

separated and soaked for 5-6 hours, then

2012). Research has proved that produc-

autoclaved as above.

tion of 1 kg mushrooms will generate 5

kg of spent residual material called _spent_

_2.2.2. Spawn rate_

_mushroom substrate_ or SMS. An average

The quantity of spawn used for

farm discards about 24 tonnes of SMS per

inoculation is 5% of its total weight (50

month (Singh _et al.,_ 2011). In Ireland,

gm spawn for 1 kg substrate).

approximately 2,54,000 tonnes of SMS is

generated each year (Barry _et al.,_ 2012)

_2.2.3. Spawning of substrate bag_

and in The Netherlands, more than

The pasteurized substrate is filled

8,00,000 tonnes (Oei and Albert, 2012).

into transparent perforated polyethylene

In some countries, waste management of

bags; incubated at 23-25oC for 12 to 14

SMS is a major problem faced by farmers

days. Mushrooms form around the edges

and the government. The obvious solution

of bag perforations and are harvested ap-

is to increase the demand for SMS

proximately 3 to 4 weeks later.

through exploration of new applications,

being recycled and reused.

_2.2.4. Spawn broadcasting_

The spent substrate is a composted

After spawning, the bags are

organic medium, made from renewable

moved to a room where temperature is

agricultural residues such as paddy straw,

around 18–20oC and relative humidity is

wheat straw, sawdust, sugarcane bagasse,

close to 95-98%. The first 12-21 days are

hay, poultry manure, ground corncobs,

completed without artificial lighting. At

cottonseed meal, cocoa shells, gypsum

the end of this period, 4 hours light is

and other substances (Jordan _et al.,_ 2008).

provided daily using fluorescent bulbs. At

Generally, each cultivation cycle lasts for

the time of formation of mushrooms,

5 to 6 months, after which the spent sub-

fresh air is let in to lower CO2 levels.

strate would be disposed. In Malaysia, an

Studies have shown that paddy

average farm producing 100 tonnes of

straw is the preferred substrate for culti-

fresh mushrooms per annum generates

vation of _H.ulmarius_. However, coconut

approximately 438 tonnes of SMS. The

husk could be used as a supplement to

current disposal strategy of SMS in Ma-

enhance stipe length. Increasing stipe

laysia is by burning, spreading on land,

length can make picking the fruit during

burying, composting with animal manure

harvest much easier and thereby, increase

or land-filling.

its competitiveness in the commercial

market. Rice straw, cotton waste, coir,

_2.2._ **** _Cultivation process for hypsizygus_

baggase and banana leaves are all also

_ulmarius_ ****

considered suitable substrates for growing

oyster mushrooms (Belewu and Belewu,

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 93

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ 2005). The yield and quality depends on

__

_There is a reduction or complete ab-_

the C: N ratio, composition of vitamins,

_sence in the growth of weeds_ \- when SMS

phytohormones, macro, and micro ele-

is added to soil.

ments present in the substrate (Ad-

__

_SMS is already supplemented with_

enipekun and Gbolagade, 2006). The use

_nitrogen_ \- along with its innate ability to

of these agricultural wastes in mushroom

release phosphorus and potassium, it

cultivation provides a solution for their

makes the soil enriched in all the three

disposal, and effective solid waste man-

major plant nutrients – nitrogen, phospho-

agement.

rus and potassium.

The cultivation of edible mush-

__

_There is absence of heavy metals and_

rooms offers one of the most feasible and

_toxins_ \- in soil supplemented with SMS **.**

economic methods for environment-

****

friendly bio-conversion and disposal of

_2.4. Use of spent mushroom substrate_

agro-lignocellulose waste (Cohen _et al.,_

_(SMS) of Hypsizygus ulmarius as a_

2002). The spent substrate after two or

_mycorestoration agent_

three harvests of mushrooms can be used

The potential of the SMS of _H._

for mycorestoration and mycoremediation

_ulmarius_ has been studied for improving

studies.

soil fertility and plant growth by the re-

lease of soil phosphorous, improving aer-

_2.3. Applications of spent mushroom sub-_

ation and disease resistance. Phosphorous

_strate_

is one of the limiting factors for plant

The most significant applications of

growth in most soils. SMS added singly

spent mushroom substrate (SMS) is its

increased soil phosphorus; with conven-

ability to increase and retain the organic

tional bacterial biofertilizers such as _Azo-_

content of soil or the potting medium (by

_tobacter sp._ and fungal biofertilizers like

increasing the release of major plant nu-

_G.intraradices_ , it enhances soil porosity,

trients such as phosphorus and potassi-

production of leaves, auxiliary buds and

um); it also increases the porosity of soil

flowers; also root biomass, soil carbon

by creating air spaces (due to the thread-

and nitrogen. Hence, it is more beneficial

like nature of fungal mycelia) –

when the SMS of _H.ulmarius_ was used as

www.mushroom-sms.com.

a supplement to conventional fertilizers,

Some of the other applications of

rather than as a stand-alone bio-fertilizer.

SMS are as follows:

A lot of literature has indicated that there

__

_Consistency of quantity and quality_ –

is a stimulatory effect of _'plant growth-_

A consistent amount of SMS can be pro-

_promoting Rhizobacteria'_ (PGPR) on the

duced annually as mushrooms can be cul-

growth of plants, due to the presence of

tivated throughout the year with regulated

beneficial micro-organisms. However the

temperature and humidity conditions.

mechanisms of this stimulation have not

Consistent high quality can also be pro-

been discussed in detail in most of the

duced by standardizing the process and

literature. The modes of action that have

materials used for cultivation.

been studied, are, however, as follows:

__

_High water and nutrient retention_

Increased supply of nitrogen to the host

_capacity -_ Water and nutrient retention

by microbial nitrogen-fixation; increase in

capacity of the soil increases by addition

supply of phosphorus, sulphur and iron;

of SMS due to the filamentous nature of

increase in surface area of roots due to

the mycelia that creates a network, similar

production of phytohormones and stimu-

to _'mycorrhizal fungi'_ , which traps the

lation of mutualistic relationships be-

water molecules and also the released nu-

tween the host plant and other algae and

trients.

fungi (Banerjee, 2006).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 94

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ **3. Mycoremediation of soil using white**

macro-fungi grown in submerged fermen-

**rot fungi**

tation. Enzymes can also be extracted

****

from _'solid substrate fermentation tech-_

_Bioremediation_ mainly depends

_nology'_. Laccase is the most common en-

on the ability of microorganisms like bac-

zyme isolated from the spent mycelium

teria and fungi to produce enzymes that

substrate (SMS) of _Agaricus bisporus_

break down the pollutants to non-

(Mayolo-Deloisa _et al.,_ 2009), _Pleurotus_

hazardous products. As bioremediation is

_sajor-caju_ , _P.ostreatus_ , _Lentinus edodes,_

effective only where environmental con-

_Flammulina velutipes_ , _Hericium erina-_

ditions permit microbial growth and ac-

_ceum_ and _Hypsizygus ulmarius_. However,

tivity, its usage often involves the manip-

the productivity of lignin peroxidase (per

ulation of environmental parameters to

microgram of SMS) was found to be the

allow faster microbial growth and degra-

highest in the SMS of _P.sajor-caju_ ; it was

dation (Karigar and Rao, 2011). The limi-

twice, 22, 30 and 86-fold higher than that

tations of bacterial growth are due to var-

of β-glucosidase, laccase, xylanase and

iations in pH, temperature, oxygen, soil

cellulase, respectively. Certain methods

organic matter, moisture and optimum

for extraction and purification of enzymes

level of nutrients, poor bioavailability of

like laccase are dialysis, ultra-filtration,

contaminants and the presence of toxins

anion-exchange chromatography and gel

(Vidali, 2001). In this respect, fungi are

filtration (Quaratino _et al.,_ 2007). How-

far better adapted, as they exhibit high

ever, most of these experiments have

tolerance toward low pH and drought

been carried out using the fruiting bodies

conditions, characteristic of contaminated

or mycelia of mushroom, not the spent

and marginal lands. Most bioremediation

substrate. The important parameters in-

systems operate under aerobic conditions,

fluencing enzyme yield are pH, tempera-

which is also a condition well suited for

ture, extraction medium, incubation time,

fungi (Leung, 2004).

inoculum density and nitrogen source.

Processes such as formation of in-

soluble metal oxalates, biosorption or

_3.2. Mycoremediation of phenol using_

chelation on the surface of polymers are

_spent mushroom substrate_

used by fungi for removing pollutants

Phenol, if ingested, inhaled or ab-

from soil and water (Sasek, 2003). Some

sorbed through the skin, quickly pene-

of the white rot fungi produce all the lig-

trates the surface and causes severe irrita-

ninolytic enzymes, while others produce

tion to the eyes and respiratory tract. It is

only one or two of them. _Lentinus edodes_

potentially carcinogenic to humans (Muf-

and _Pleurotus_ spp. are fungi with im-

tah _et al.,_ 2009). The _Hazardous Waste_

portant medicinal, biotechnological and

_Management Rules 1989_ permits only 5

environmental applications (Elisashvili _et_

kg of phenol per year for disposal. The

_al.,_ 2008). They are capable of producing

actual quantities disposed are much great-

hydrolytic and oxidative enzymes like

er. In addition, the present treatment

laccases and peroxidases, which are es-

methods are chemical intensive and fur-

sential in breaking down the lignocellulo-

ther contaminate the environment. This

sic biomass into low molecular weight

has made it imperative that new non-

compounds that support mycelial growth

chemical methods like _mycoremediation_

and fruiting (Reddy _et al.,_ 2003).

are devised (Nuhoglu and Yalcin, 2005).

There is a heightened concern over public

_3.1._ **** _Recovery of ligninolytic enzymes_

health and environmental hazards due to

_from spent mushroom substrate of_

the presence of organic toxins like phe-

_white rot fungi_

nols and dyes in waste water (Eriksson _et_

Enzymes reported in the literature

_al.,_ 2007). Phenols and azo dyes are well

are derived mainly from the mycelia of

known for their bio-recalcitrant nature

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 95

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ and acute toxicity. They are being contin-the formation of dihydroxy benzene. Fur-

uously introduced into ponds, lakes and

ther degradation proceeds through the

rivers through from chemical and textile

cleavage of dihydroxy benzene to give

industries of different scale and category.

pent 2-enedioic acid and formaldehyde, in

The major sources of phenol pol-

addition to benzoquinone and maleic acid.

lution are waste water coming from paint

The decarboxylation of maleic acid and

industries, pesticides, resin production

ring opening of hydroquinone result in the

and petrochemical industries. Phenols are

formation of oxalic acid. Decarboxylation

considered as primary pollutants under

of oxalic acid leads to the formation of

directive 80/778/EC, since they are harm-

carbon dioxide and water.

ful to organisms even at very low concen-

****

trations (Calace _et al.,_ 2002). The maxi-

_3.2.1. Application of spent mushroom_

mum concentration has been set at 0.5

_substrate of white rot fungi in my-_

mg/l for total phenols in drinking water,

_coremediation of phenolic com-_

and individual concentration should be

_pounds_

under 0.1 mg/l.

The enzyme systems of _'white rot_

The main routes of exposure to

_fungi'_ contain laccase, lignin peroxidase

phenol include breathing contaminated

and manganese-dependent peroxidases

air; inhaling cigarette smoke, drinking

which catalyses metabolism of many lig-

water from contaminated surface or

nin-like structures, for example, PAHs

groundwater supplies, swallowing or in-

and phenols (Eggen and Sasek, 2002).

haling products containing phenol or

Phenol oxidation using SMS from

coming into contact with contaminated

_A.bisporus_ was reported and laccase was

water and products containing phenol

identified as the main enzyme responsible

through bathing (Ahmaruzzaman, 2008).

(Trejo-Hernandez _et al.,_ 2001). Studies

The common method to detect in-

have also supported the idea of de-

termediate products of phenol breakdown

contaminating phenolic compounds using

is extraction by organic solvent after es-

SMS from cultivation of edible mush-

terification or acetylation, followed by

room.

gas chromatography and mass spectros-

copy (GC-MS). However, this method

_3.2.2. Phenol tolerance and degradation_

has two disadvantages: (1) as the inter-

_by spent mushroom substrate of_

mediates are mostly polar compounds,

_Hypsizygus ulmarius_

they dissolve easily in water and the non-

_H.ulmarius_ tolerated and degraded

or low-polar solvents don't get extracted

phenol better under carbon and nitrogen

completely (2) Esterification is suitable

limiting conditions of the growth medi-

only for derivatizing acids, while acetyla-

um. The optimum carbon sources for its

tion only for hydroxylated compounds. In

growth were glucose, mannitol and cellu-

addition, it is not possible to detect these

lose, while ammonium nitrate, ammoni-

intermediates by _high-performance liquid_

um chloride and sodium nitrate were the

_chromatography_ (HPLC). This method

optimum nitrogen sources. Peroxidase

needs not only many calibration standards

and manganese peroxidase were the en-

but takes a lot of time as there are several

zymes secreted in maximum quantity dur-

unknown compounds in the intermedi-

ing phenol degradation, followed by lac-

ates, and some compounds would have

case (Ranjini and Padmavathi, 2012;

the same retention time (Guo _et al.,_

2013).

2006).

****

A possible degradation mecha-

_3.3 Mycoremediation of dyes using spent_

nism for phenol was given by Devi and

_mushroom substrate_

Rajashekhar (2011). The hydroxyl radi-

The majority of natural dyes are

cals attack the phenol molecule leading to

produced from plant sources like roots,

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_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ berries, bark, leaves, wood and also fungi

Chen (2002 a, b) also reported the use of

and lichens. Azo compounds have aryl or

biomass for the removal of basic dyes.

alkyl functional groups, with vivid colors

Decolourization by living and

like reds, oranges and yellows. About

dead microbial cells involves mechanisms

50% of dyes produced in the world are

such as surface adsorption, ion-exchange,

azo dyes (Perumal _et al.,_ 2007). They are

complexation (coordination), complexa-

extremely recalcitrant and when released

tion–chelation and micro-precipitation.

with the wastewater, remains in the water

Cell walls consist of polysaccharides, pro-

or soil and adversely impacts the photo-

teins and lipids and offer many functional

synthetic ability of phytoplanktons in wa-

groups for the above reactions. Dyes can

ter. They interfere with the functioning of

interact with these active groups on the

chlorophyll in the plankton, as they color

surface of the cell. The accumulation of

the water and prevent the proper absorp-

dyes by biomass may involve a combina-

tion of light (Duran and Esposito, 2000).

tion of active, metabolism-dependent and

Many methods have been tried for achiev-

passive transport mechanisms. It starts

ing decolorization of azo dyes in

with diffusion of the adsorbed solute to

wastewater, but most of them like nano-

the surface of the microbial cell

filtration, specific coagulation, use of ac-

(O'Mahony _et al.,_ 2002).

tivated carbon and multiple effect evapo-

Laccase oxidizes the phenolic

rators are very expensive. Bio-treatment

group of the azo dye with the participa-

is a cheaper and environmentally better

tion of one electron, generating a phenoxy

alternative (Olukanni _et al.,_ 2006). Deg-

radical and then oxidizes it to a carboni-

radation by micro-organisms utilizes their

um ion. A nucleophilic attack on the phe-

enzymes, mainly laccases and peroxidas-

nolic ring carbon bearing the azo linkage

es produced by few bacteria and white

to produce 3-diazenyl-benzenesulfonic

rot, brown rot and soft fungi (Duran and

acid (III) and 1, 2-naphthoquinone then

Esposito, 2000). Only these enzymes are

takes place (Camarero _et al_., 2005). Phe-

found to be effective in breaking down

nolic radicals get oxidized further to yield

the high structural integrity and variety of

oligomers. Under certain conditions, the

azo dyes. Decolourization and/or _'bio-_

C-C-formed dimers take part in coupling

_adsorption'_ of dye-containing wastewater

reactions to form extended quinines (Zille

by dead or living biological matter (bio-

_et al_., 2005).

mass), white-rot fungi and other microbial

In reactive dyes _,_ the chromophore

cultures are the subject of many studies

contains a substitute that is activated and

reviewed in several recent papers (Aksu,

allows the dye to directly react to the sub-

2005). In particular, these studies demon-

strate surface. They are added for dyeing

strate that _'bio-sorbents'_ from suitable

of cotton or flax.

microbial biomass can be used for dye

The spent mushroom substrate

decolourization; this is because certain

(SMS) of _P.sajor-caju_ offer an economi-

dyes have an affinity for binding with

cal source of industrially important en-

some microbial species. The use of bio-

zymes decolourize dyes (Singh _et al.,_

mass is becoming popular due to its

2002). Singh _et al._ (2011) has shown the

availability in large quantities and cost-

decoloriza **t** ion of eight dyes (viz.) trypan

effectiveness. It is produced in fermenta-

blue, amido black, remazol brilliant blue

tion processes to synthesize antibiotics

R, bromophenol blue, crystal violet, me-

and enzymes. Here, a large amount of by-

thyl green, congo red and methylene blue

products are generated, which can be used

using lignin peroxidase extracted from 5-

in bio-sorption of pollutants. Aksu and

month-aged SMS of _P. sajor-caju_ cou-

Tezer (2005) have shown the uptake of

pled with veratryl alcohol as a redox me-

588.2 mg of reactive black 5 per g of bi-

diator. Further, three azo group dyes, re-

omass of _Rhizopus arrhizus_. Chu and

active black 5, reactive orange 16 and

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 97

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ disperse blue 79; two anthraquinone

_corestoration_. Using the spent mushroom

group dyes, disperse red 60 and disperse

substrate (SMS), a by-product of its culti-

blue 56 and textile wastewater from all

vation, as a _bio-fertilizer_ , either singly or

the five reactive and disperse dyes were

in combination with conventional bio-

also successfully decolourized by crude

fertilizers improves soil fertility, especial-

enzymes from SMS of _P.sajor-caju_. The

ly phosphorus, which is mostly insoluble

mechanisms of enzymatic dye decolouri-

and unavailable to plants. Phosphorus is

zation were most probably due to laccase

one of the three macro nutrients of soil

and manganese peroxidase, as a recent

that can determine the yield of crops and

study showed that under stimulation by

agricultural income to farmers, and re-

malachite green, a triphenylmethane dye,

duce their dependence on chemical ferti-

the laccase and manganese peroxidase

lizers.

levels in the enzyme extracts of

Soil contamination by phenolic

_P.ostreatus_ were increased by 1.4 and

compounds and dyes can be remedied us-

2.1-fold, respectively (Papinutti and For-

ing the spent mushroom substrate (SMS)

chiassin, 2010). Thus, dyes can be re-

of _H.ulmarius_. Phenolic waste is an inte-

moved, degraded and detoxified by en-

gral part of biomedical and industrial

zymatic biological processes and also

waste. Present treatment methods are

physical adsorption using SMS (Gao _et_

chemical-intensive and further pollute the

_al.,_ 2011). Use of SMS in bioremediation

environment.

of dyes is both time saving and cost-

The spent mushroom substrate

effective.

(SMS) of _H. ulmarius_ can also be used to

decolorize dyes. Dyes color and pollute

_3.3.1. Dye degradation using spent mush-_

water bodies, kill aquatic organisms by

_room substrate (sms) of hypsizygus_

increasing toxicity, and known toreduce

_ulmarius_

of light and dissolved oxygen in water.

The SMS of _H.ulmarius_ was ef-

The present treatment methods are mostly

fective in degrading three categories of

physico-chemical in nature.

dyes (viz.) azo (Congo red), heterocyclic

(Methylene blue) and reactive (Solo-

**References**

chrome black), with Methylene blue be-

****

ing most effectively decolourized, fol-

**Adenipekun, C.O. and Gbolagade, J.S.**

lowed by Solochrome black and Congo

**(2006).** Nutritional requirements of

red. Here, laccase was the enzyme secret-

_Pleurotus florida_ (Mont.) Singer, a

ed in greater quantity, followed by man-

Nigerian mushroom. _Pakistan Jour-_

ganese peroxidase. Maximum secretion of

_nal of Nutrition_ **6, 597-600.**

both enzymes was observed during de-

**Ahmaruzzaman, Md. (2008).** Adsorp-

colourization of Congo red (Ranjini and

tion of phenolic compounds on low-

Padmavathi, 2015).

cost adsorbents: a review. _Advances_

_in Colloid and Interface Science_

**4. Conclusion**

**143, 48-67.**

**Ahmed, S.A., Kadam, J.A., Mane, V.P.,**

To conclude the effective man-

**Patil, S.S. and Baig, M.M.V.**

agement of agricultural wastes as a part of

**(2009).** Biological efficiency and

solid waste management is mushroom

nutritional contents of _Pleurotus_

cultivation which utilizes agricultural

_florida_ (Mont.) Singer cultivated on

wastes in an environment-friendly manner

different agro-wastes. _Nature and_

and solves their disposal problem, as they

_Science_ **7, 44-48.**

are generated in large amounts each year.

**Aksu, Z.** **(2005).** Application of biosorp-

The fertility of marginal soils increases in

tion for the removal of organic pol-

an environment-friendly manner - _my-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 98

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ lutants, a review. _Process Biochem-lation to carbon and organic nitro-_

 _

_istry_ **40, 997-1026.**

gen sources, antifoams and Tween

**Aksu, Z. and Tezer, S.** **(2005)**. Biosorp-

80. _Journal of Industrial Microbiol-_

tion of reactive dyes on the green

_ogy and Biotechnology_ **36, 1–9.**

alga _Chlorella vulgaris_. _Process Bi-_

**Calace, N., Nardi, E., Petronio, B.M.**

_ochemistry_ **40, 1347–1361.**

**and Pietroletti, M. (2002).** Adsorp-

**Alam, N., Hossain, M.S., Khair, A.,**

tion of phenols by paper mill sludg-

**Amin, S.M.R. and Khan, A.**

es. _Environmental Pollution_ **118,**

**(2007).** Comparative study of oyster

**315-319.**

mushrooms on plasma lipid profile

**Camarero, S., Ibarra, D., Martinez,**

of hypercholesterolemic rats. _Bang-_

**M.J. and Angel, T.M.** **(2005).** Lig-

_ladesh Journal of Mushrooms_ **1, 15-**

nin-derived compounds as efficient

**22.**

laccase mediators for decolouriza-

**Ali, N., Ikramullah, G., Lutfullah, A.,**

tion of different types of recalcitrant

**Hameed and Ahmed, S. (2008).**

dyes. _Applied Environmental Mi-_

Decolourization of Acid red 151 by

_crobiology_ **1775–1784.**

_Aspergillus niger_ _SA1_ under differ-

**Chandrakant,**

**K.,**

**Aravind,**

**M.,**

ent physico-chemical conditions.

**Manjunath, N. and Dae Jin Yun.**

_World Journal of Microbiology and_

**(2006).** Phenol degradation by im-

_Biotechnology_ **24, 1099-1105.**

mobilized cells of _Arthrobacter_

**Amara, A.A. and Salem, S.R.** **(2010).**

 _citreus_. _Bi_ _odegradation_ **17, 47-55.**

Logical and experimental design for

**Chang, S. T.** **1999.** Global impact of edi-

phenol degradation using immobi-

ble and medicinal mushrooms on

lized _Acinetobacter spp._ culture.

human welfare in the 21st century:

_IIUM Engineering Journal_ **11,** **89-**

non-green revolution. _International_

**104.**

_Journal of Medicinal Mushrooms_ , **1,**

**Banerjee, M.R., Yesmin, L. and Vessey,**

**1-7.**

**J.K.**

**(2006).**

Plant

Growth-

**Chu, K.H. and Chen, K.M.** **(2002) a**.

promoting Rhizobacteria as Biofer-

Reuse of activated sludge biomass:

tilizers and Bio-pesticides. _In:_ Rai,

I. Removal of basic dyes from

M. K. (Ed.), _Handbook of Microbial_

wastewater by biomass. _Process Bi-_

_Biofertilizers_. Food Products Press,

_ochemistry_ **37, 595** – **600.**

New York, p. **137-215.**

**Chu, K.H. and Chen, K.M.** **(2002) b**.

**Barry, J., Doyle, O., Grant, J. and**

Reuse of activated sludge biomass:

**Grogan, H. (2012).** Supplementary

II. The rate processes for the adsorp-

of spent mushroom substrate (SMS)

tion of basic dyes on biomass. _Pro-_

to improve the structure and produc-

_cess Biochemistry_ **37, 1129–1134.**

tivity as a casing material. _In:_ _18th_

**Cohen, R., Persky, L. and Hadar, Y.**

_Congress of the International Socie-_

**(2002).** Biotechnological applica-

_ty for Mushroom Science_. Beijing,

tions and potential of wood degrad-

China, pp. **735–742.**

ing mushrooms of the genus _Pleuro-_

**Belewu, M.A. and Belewu, K.Y.** **(2005).**

_tus_. _Applied Microbiology and Bio-_

Cultivation of mushroom ( _Volva-_

_technology_ **58, 582–594.**

_riella volvacea_ ) on banana leaves.

**Dalgaard, T., Hutchings, N.J. and Por-**

_African Journal of Biotechnology_ **4,**

**ter, J.R. (2003).** Agroecology, scal-

**1401-1403.**

ing and interdisciplinarity. _Agricul-_

**Bettin, F., Montanari, Q., Calloni, R.,**

_tural Ecosystems and Environment_

**Gaio, T.A., Silveira, M.M. and**

**100, 39-51.**

**Dillon, A.J.P.** **(2008).** Production of

**Devi, L.G. and Rajashekhar, K.E.**

laccases in submerged process by

**(2011).** A kinetic model based on

_Pleurotus sajor-caju_ PS-2001 in re-

nonlinear regression analysis is pro-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 99

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ posed for the degradation of phenol

sorbent for the removal of Reactive

under UV/solar light using nitrogen

Red 15 from aqueous solutions.

doped TiO2. _J. Molecular Catalysis_

_Green Environment Bulletin_ **20, 51–**

_A: Chemical_ **334, 65-76.**

**62.**

**Duran, N. and Esposito, E.** **(2000).** Po-

**Gomaere, S.S. and Govindwar, S.P**.

tential applications of oxidative en-

**(2009).** _Brevibacillus laterosporus_

zymes and enzymatic activity in re-

_MTCC_ _2298_ : a potential azo dye

lation to litter composition, N depo-

degrader. _Journal of Applied Micro-_

sition and mass loss. _Biogeochemis-_

_biology_ **106, 993-1004.**

_try_ **60, 1-24.**

**Gomathi, S. and Ambikapathy, V.**

**Eggen, T. and Sasek, V.** **(2002).** Use of

**(2011).** Antagonistic activity of fun-

edible and medicinal oyster mush-

gi against Pythium debaryanum

room

[ _Pleurotus_

_ostreatus_

(Hesse) isolated from Chilli field

(Jacq.:Fr.) Kumm.] spent compost

soil. _Advances in Applied Sciences_

in remediation of chemically pollut-

_Research_ **2, 291-297.**

ed soils. _International Journal of_

**Guo, Z., Ma, R. and Li, G.** **(2006).** Deg-

_Medicinal Mushrooms_ **4, 255–261.**

radation of phenol by nano materials

**Elisashvili, V., Penninckx, M., Ka-**

TiO2 in wastewater. _Chemical Engi-_

**chlishvili, E., Tsiklauri, N., Me-**

_neering Journal_ **119, 55-59.**

**treveli, E., Kharziani, T.** **and**

**http://www.mushroomexpert.com/hyps**

**Kvesitadze, G.** **(2008).** _Lentinus_

**izygus_ulmarius.html**

_edodes_ and _Pleurotus spp._ lignocel-

**Ikehata, K., Buchanan, D.I. and Smith,**

lulolytic enzymes activity in sub-

**D.W.** **(2004).** Recent developments

merged and solid-state fermentation

in the production of extracellular

of lignocellulosic wastes of different

fungal peroxidises and laccases for

composition. _Bioresources Technol-_

waste treatment. _Journal of Envi-_

_ogy_ **99, 457-462.**

_ronmental Engineering and Science_

**Eriksson, E., Baun, A., Mikkelsen, P.S.**

**3, 1-19.**

**and Ledin, A.** **(2007).** Risk assess-

**Inbaraj, B., Selvarani and Sulochana,**

ment of xenobiotics in stormwater

**N.** **(2002).** Evaluation of a carbona-

discharged to Harrestup Ao, Den-

ceous sorbent prepared from pearl

mark. _Desalination_ **215, 187-197.**

millet husk for its removal of basic

**Faraco, V., Pezzella, C., Miele, A.,**

dyes. _Journal of Scientific and In-_

**Giardina, P. and Sannia, G.**

_dustrial Research_ **61, 971-978.**

**(2009).** Bioremediation of coloured

**Jonathan,**

**S.G.,**

**Oyetunji,**

**O.J.,**

industrial wastewaters by the white-

**Olawuyi, O.J. and Uwukhor, P.O.**

rot fungi _Phanerochaete chryso-_

**(2013).** Application of _Pleurotus os-_

_sporium_ and _Pleurotus ostreatus_

_treatus_ SMC as soil conditioner for

and their enzymes. _Biodegradation_

the growth of soybean ( _Glycine_

**20, 209-220.**

_max_ ). _Academia Arena_ **5, 55-61.**

**Forgacs, T.C. and Oros, G.** **(2004).** Re-

**Jordan, S.N., Mullen, G.J. and Mur-**

moval of synthetic dyes from waste

**phy, M.C.** **(2008).** Composition

waters: a review. _Environment In-_

variability of spent mushroom com-

_ternational_ **30, 953-971.**

post in Ireland. _Bioresource Tech-_

**Gadd, G.M.** **(2001).** Fungi in bioreme-

_nology_ **99, 411–418.**

diation.

_Cambridge_

_University_

**Karigar, C.S. and Rao, S.S.** **(2011).**

_Press_ , Cambridge.

Role of microbial enzymes in the

**Gao, J.F., Wang, J.H., Wu, X.L.,**

bioremediation of pollutants: A re-

**Zhang, Q.A. and Peng, Y.Z.**

view. _Enzyme Research_ Article ID

**(2011).** Protonated spent mushroom

805187, doi **:10.4061/2011/805187.**

substrate as a potential low-cost bio-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 100

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ **Khan, F. and Ishaq**. **(2011).** Chemical

**Manimegalai, V., Ambikapathy, V. and**

nutrient analysis of different com-

**Panneerselvam, A.** **(2011).** Popula-

posts (Vermicompost and Pitcom-

tion dynamics of soil mycoflora in

post) and their effect on the growth

the paddy fields of Thanjavur Dis-

of a vegetative crop _Pisum sativum_.

trict, Tamilnadu. _European Journal_

_Asian Journal of Plant Science and_

_of Experimental Biology_ **1, 14-19.**

_Research_ **1,116-130.**

**Mayolo-Deloisa, K., Trejo-Hernández,**

**Khan, M.A., Tania, M., Amin, S.M.R.,**

**M.D.R. and Rito-Palomares, M.**

**Alam, N. and Udin, M.N.** **(2008).**

**(2009).** Recovery of laccase from

An investigation on the nutritional

the residual compost of _Agaricus_

composition of mushrooms ( _Pleuro-_

_bisporus_ in aqueous two-phase sys-

_tus florida_ ) cultivated on different

tems. _Process Biochemistry_ **44,**

substrates. _Bangladesh Journal of_

**435–439.**

_Mushrooms_ **2, 17-23.**

**Medina, E., Paredes, C., Perez-Murcia,**

**Kodam, K.M., Soojhavon, I., Lo-**

**M.D., Bustamante, M.A. and**

**khande, P.D. and Gawai, K.R.**

**Moral, R. (2009).** Spent mushroom

**(2005).** Microbial degradation of re-

substrate as component of growing

active azo dyes under aerobic condi-

media for germination and growth

tions. _World Journal of Microbiolo-_

of horticultural plants. _Bioresource_

_gy and Biotechnoogy_ , **21, 367-370.**

_Technology_ **100, 4227-4232.**

**Kulshreshtha, S. and Sharma, K**.

**Mikiashvili, N., Elisashvili, V., Wasser,**

**(2014).** Perspectives of bioremedia-

**S. and Nevo, E. (2005).** Carbon and

tion through mushroom cultivation.

nitrogen sources influence the lig-

_Journal of Bioremediation and Bio-_

ninolytic

enzyme

activity

of

_degradation_

5,

5.

_Trametes versicolor_. _Biotechnology_

**http://dx.doi.org/10.4172/2155-**

_Letters_ **27,** **955-959.**

**6199.1000e154.**

**Muftah, H., El-Naas, Shaheen, A., Al-**

**Kumar, A., Bisht, B.S., Joshi, V.D. and**

**Muhtaseb**

**and**

**Makhlouf,**

**S.**

**Dhewa, T.** **(2010).** Review on Bio-

**(2009).** Biodegradation of phenol by

remediation of Polluted Environ-

_Pseudomonas putida_ immobilized in

ment: A Management Tool. _Interna-_

polyvinyl alcohol (PVA) gel. _Jour-_

_tional Journal of Environmental_

_nal of Hazardous Materials_ **164,**

_Science_ **1, 1079-1093.**

**720-725.**

**Leung, M.** **(2004).** Bioremediation: tech-

**Mukherjee, R. and Nandi, B. (2004).**

niques for cleaning up a mess. _Jour-_

Improvement of in vitro digestibility

_nal of Biotechnology_ , **2, 18–22.**

through biological treatment of wa-

**Li, Y. (2012).** Present development situa-

ter hyacinth biomass by two _Pleuro-_

tion and tendency of edible mush-

_tus spp. International Journal of Bi-_

room industry in China. _In: 18th_

_odeterioration and Biodegradation_

_Congress of the International Socie-_

**53, 7–12.**

_ty for Mushroom Science_. Beijing,

**Nuhoglu, A. and Yalcin, B. (2005).**

China, pp. **3–9.**

Modeling of phenol removal in a

**Manikandan, N., Kuzhali, S.S. and**

batch reactor. _Process Biochemistry_

**Kumuthakalavalli, R.** **(2012).** De-

**40, 1233–1239.**

colourization of textile dye effluent

**O' Mahony, T., Guibal, E. and Tobin,**

using fungal mycoflora isolated

**J.M. (2002).** Reactive dye biosorp-

from spent mushroom substrate

tion by _Rhizopus arrhizus_ biomass.

(SMS). _Journal of Microbiology_

_Enzyme and Microbial Technology_

_and Biotechnology Research_ **2, 57-**

**31, 456–463.**

**62.**

**Oei, P. and Albert, G.** **(2012).** Recycling

casing soil. _In: 18th Congress of the_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 101

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ _International Society for Mushroom_

and reactive dyes using spent myce-

_Science_. Beijing, China, pp. **757–**

lium substrate (SMS) of _Hypsizygus_

**765.**

_ulmarius_. _Journal of Environmental_

**Olukanni O.D., Osuntoki, A.A. and**

_Biology, ****_**36, 1083-1088.**

**Gbenle, G.O. (2006).** Textile efflu-

**Ranjini R. and Padmavathi T.** **(2012).**

ent biodegradation potentials of tex-

Phenol tolerance of _Pleurotus flori-_

tile

effluent-adapted

and

non-

_da_ under varying conditions of ni-

adapted bacteria, _African Journal of_

trogen sufficiency. _European Jour-_

_Biotechnology_ **5, 1980-1984.**

_nal of Experimental Biology ****_**2, 75-**

**Palmieri, G., Cennamo, G. and Sannia,**

**82.**

**G.** **(2005).** Remazol brilliant blue R

**Ranjini, R. and Padmavathi, T. (2013).**

decolourization by the fungus _Pleu-_

A preliminary assessment of phenol

_rotus ostreatus_ and its oxidative en-

tolerance and degradation by spent

zymes. _Enzyme and Microbial_

mycelium substrate (SMS) of novel

_Technology_ **36, 17-24.**

edible mushroom _Hypsizygus ul-_

**Papinutti, L. and Forchiassin, F.**

_marius_. _Journal of Scientific and_

**(2010).** Adsorption and decolouriza-

_Industrial Research_ **72, 767-771.**

tion of dyes using solid residues

**Reddy, G.V., Babu, P.R., Komaraiah,**

from _Pleurotus ostreatus_ mushroom

**P., Roy, K.R.R.M. and Kothari,**

production. _Biotechnology and Bio-_

**I.L. (2003).** Utilization of banana

_process Engineering_ **15, 1102–**

waste for the production of lignolyt-

**1109.**

ic and cellulolytic enzymes by solid

**Patel, H., Gupte, A. and Gupte, S.**

substrate fermentation using two

**(2008).** Biodegradation of fluoran-

_Pleurotus_ species ( _P ostreatus and_

thene by basidiomycetes fungal iso-

_P. sajor-caju_ ). _Process Biochemis-_

late _Pleurotus ostreatus_ HP-1. _Ap-_

_try_ **38, 1457–1462.**

_plied Biochemistry and Biotechnol-_

**Revankar, M.S. and Lele, S.S.** **(2006).**

_ogy_ **157, 367-376.**

Enhanced production of laccase us-

**Perumal, S. M., Munuswamy, M.,**

ing a new isolate of white rot fungus

**Sellamuthu, P.S., Kandasamy, M.**

WR-1. _Process Biochemistry_ **41,**

**and Thangavelu, K. P. (2007).** Bi-

**581-588.**

osorption of textile dyes and efflu-

**Rinker, D.L. and Kang, S.W. Zeri.**

ents by _P.florida_ and _T. hirsuta_ with

**(2004).** Recycling of oyster mush-

evaluation of their laccase activity.

room substrate. _In:_ Mushroom

_Iraninan Journal of Biotech_ n _ology_

Growers' Handbook - **1.9, 187-191.**

**5, 114-118.**

**Sasek, V.** **(2003).** _In:_ Sasek, V., Glaser,

**Prabhakaran, M., Merinal, S. and**

J.A. and Baveye, P. (ed.) The utili-

**Paneerselvam, A.** **(2011).** Investi-

zation of bioremediation to reduce

gation of phylloplane mycoflora

soil contamination: problems and

from some medicinal plants. _Euro-_

solution, Kluwer Ac. Pub., Amster-

_pean Jourmal of Experimental Biol-_

dam **.**

_ogy_ **1,** **219-225.**

**Singh, A.D., Vikineswary, S. and Noor-**

**Quaratino, D., Federici, F., Petruccioli,**

**lidah, A.** **(2002).** Extraction of en-

**M., Fenice, M. and D'Annibale, A.**

zymes from spent compost of _Pleu-_

**(2007).** Production, purification and

_rotus sajor-caju_ and its potential use

partial characterization of a novel

for decolourization of synthetic dye.

laccase from the white-rot fungus

_Malaysian Journal of Science_ **21, 9–**

_Panus tigrinus_ _CBS 577.79_. _Antonie_

**16.**

_Van Leeuwenhoek_ **91, 57–69.**

**Singh, A.D., Vikineswary, S., Abdullah,**

**R. Ranjini and T. Padmavathi. (2015).**

**N. and Sekaran, M.** **(2011).** En-

Decolourization of azo, heterocyclic

zymes from spent mushroom sub-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 102

_Biotech Sustainability (2017)_

_Spent Mushroom Substrate of Hypsizygus ulmarius Tallapragada and Ramesh_ strate of _Pleurotus sajor-caju_ for the

Plant nutrition in a world of declin-

decolourization and detoxification

ing renewable sources. _Plant Physi-_

of textile dyes. _World Journal of_

_ology_ **127, 390-397.**

_Microbiology and Biotechnol_ ogy

**Vidali, M. (2001).** Bioremediation. An

**27, 535–545.**

overview. _Pure and Applied Chem-_

**Stamets, P. (2006).** Mycelium Running:

_istry_ **73, 1163** – **1172.**

How mushrooms can help save the

**Wang, P., Hu, X., Cook, S., Begonia,**

world. _Ten Speed Press_ , New York,

**M., Lee, K.S. and Hwang, H, M.**

p. **19-29, 69-105,111-113, 151, 196-**

**(2008).** Effect of culture conditions

**199.**

on the production of ligninolytic en-

**Tang, C.Y., Criddle, Q.S., Fu, C.S. and**

zymes by white rot fungi _Phanero-_

**Leckie, J.O.** **(2007).** Effect of flux

_chaete_

_chrysosporium_

(ATCC

(trans membrane pressure) and

20696) and separation of its lignin

membranes properties on fouling

peroxidase. _World Journal of Mi-_

and rejection of reverse osmosis and

_crobiology and Biotechnology_ **24,**

nanofiltration membranes treating

**2205–2212.**

perfluorooctane sulfonate contain-

**Zille, A., Gornacka, B., Rehorek, A.**

ing waste water. _Journal of Envi-_

**and Paulo, A.C.** **(2005).** Degrada-

_ronmental Science and Technology_

tion of azo dyes by _Trametes villosa_

**41, 2008-2014.**

laccase over long periods of oxida-

**Trejo-Hernandez,**

**M.R.,**

**Lopez-**

tive conditions. _Applied Environ-_

**Munguia, A. and Ramirez, Q.R.**

_mental Microbiology_ **6711–6718.**

**(2001).** Residual compost of _Agari-_

**Zumriye, G. and Karabayir, G. (2008).**

_cus bisporus_ as a source of crude

Comparison of biosorption proper-

laccase for enzymatic oxidation of

ties of different kinds of fungi for

phenolic compounds. _Process Bio-_

removal of Gryfalan Black RL met-

_chemistry_ **36, 635–639.**

al-complex dye. _Bioresource Tech-_

**Vance, C.P.** **(2001).** Symbiotic nitrogen

_nology_ **99, 16, 7730 - 7741.**

fixation and phosphorus acquisition.

****

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 103

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P104-116_

**Biotechnology for Sustainability of Forests**

**Kumud Dubey1 and Kesheo Prasad Dubey _2,_** *****

_1Centre for Social Forestry and Eco-Rehabilitation, 3/1, Lajpat Rai Road, Allahabad,_

_Uttar Pradesh, India; 2GM (East) Forest Corporation, Allahabad 24 B Agnipath, Forest_

_Corporation, Allahabad, Uttar Pradesh, India; *Correspondence: dkumud@yahoo.com_

_/ dkesheo@yahoo.co.in; Tel.: +91 9415536077_

****

****

**Abstract:** Biotechnology has various applications which could be utilized to enhance the

sustainability in various sectors. Forest Biotechnology was started only during 1990s in

the earnest sense. It encompasses structural and functional studies of genes and genomes

(including development and application of genetic molecular markers); various methods

of vegetative reproduction, and asexual insertion of genes into forest plant species. Tra-

ditionally, productivity of forests has been improved by introduction of new germplasm

developed through tree genetics and breeding, as trees are strategically and efficiently

able to utilize both horizontal as well as vertical space in an optimum way. The applica-

tion of modern biotechnological tools and techniques that span the diverse fields of plant

biology, genetic transformation and discovery of genes associated with complex multi-

genic traits. Modern innovations have added an exclusively new dimension to forest tree

improvement programs. However, forest biotechnology is still lagging behind because of

longer time periods required for planning, investigation and field research, poor juvenile-

mature correlations and multiplicity of selection criteria. But in the present scenario,

with growing population, economic development, environmental degradation and Cli-

mate Change, the demand for more biomass production from trees for production of

miscellaneous forest products and services, renewable energy alternatives to fossil fuels

and carbon sequestration etc. is ever increasing. Currently, the major challenge is to

maintain the original characteristics of pristine virgin forests for _in situ_ biodiversity con-

servation for a sound natural genetic foundation. Genetic trees for pest and disease re-

sistance will help in enhancing plantation survival, productivity and yield that would

lead to eco-restoration of native tree species. This chapter highlights various application

of biotechnology in forestry which could help in boosting sustainability. ****

_**Keywords**_ **:** Bioreclamation; biotechnology; carbon sequestration; markers; micropropaga-

tion; phytoremediation ****

****

****

**1. Introduction**

processes

for

specific

use"

(www.biodiv.org;  FAO, 2001). It pro-

Over the past few years, tech-

vides important tools for the sustainable

niques of cell biology, genetic screening,

development of agriculture, fisheries and

and gene manipulation are being used to

forestry and can be of significant help in

develop improved plant varieties. The

meeting the growing needs of population.

term "biotechnology" is coined for the

Rapid advancements have been

collective use of these techniques. Bio-

and are being made in the scientific en-

technology may be defined as "any tech-

quiry and investigation with regards to

nological application that uses biological

plant biotechnology throughout the uni-

systems, living organisms, or derivatives

verse today. Considerable progress has

thereof, to make or modify products or

already been achieved in the field of med-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 104

_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ ical biotechnology, animal biotechnology

diseases. Improvements through tree bio-

and agricultural biotechnology. Research

technology may also improve weed con-

and applications of biotechnology in the

trol enabling young trees to compete with

field of forestry are also advancing rapid-

weeds in natural ecosystem. Trees genet-

ly. The term forest tree biotechnology

ically engineered for pest resistance may

started during 1980s. It involves a broad

promote plantation survival and yield, and

collection of tools for breeding, propaga-

also lead to restoration of native tree spe-

tion and modern innovations that focus on

cies. Other potential benefits include en-

a portion of a biological system (Yan-

hancing the ability of trees to tolerate abi-

chuk, 2001). As commonly used, forest

otic stress; restoring contaminated sites

tree biotechnology encompasses structur-

through phytoremediation; facilitating

al and functional studies of genes and ge-

weed control using more environmentally

nomes (including development and appli-

benign treatments; producing new indus-

cation of genetic markers); various meth-

trial products; modifying biomass chemis-

ods of vegetative reproduction such as

try to improve pulp and biofuels produc-

micropropagation, tissue culture, and so-

tion; and improving carbon sequestration

matic embryogenesis and genetic engi-

to mitigate greenhouse gas emissions. In

neering, which is the physical manipula-

addition, biotechnology, especially Ge-

tion and asexual insertion of genes into

netic Engineering methods, offers unique

organisms (FAO, 2004). However, forest

and important tools to conduct research to

biotechnology is still aged behind because

identify the biological mechanisms for

of inherent problems related with forestry,

control of many ecologically and eco-

such as longer time periods for planning

nomically significant traits. The applica-

and investigation, poor juvenile- mature

tion of biotechnology offers a great poten-

correlations (i.e. the characteristics fea-

tial to hasten the pace of tree improve-

tures of young trees are not necessarily

ment for desired need and can improve

accurate indicators of those found in ma-

the productivity of forest and environ-

ture individuals), the multiplicity of selec-

ment.

tion criteria (e.g. timber quality, quantity,

Most of the biotechnologies used

fuel wood, medicinal values, fodder etc.).

in forestry today involve vegetative re-

Moreover, biotechnology in forestry re-

production through tissue culture and mo-

quires collaborative and integrated appli-

lecular marker applications. However,

cation of knowledge and techniques

Genetically Engineered Plants are also

drawn from several diverse disciplines

likely to play a major role in forestry.

like agriculture, silviculture, genetics, mi-

crobiology, molecular biology, plant

**2. Application of biotechnology in for-**

physiology etc. Biotechnology applica-

**estry**

tions in forestry are a growing area of in-

terest. Initial applications of forest tree

In the present scenario, with grow-

biotechnology targeted to improved

ing population and economic develop-

productivity and quality of plantation for-

ment, the demand of wood products is

ests. Such use, with appropriate social

increasing which will increase the de-

controls, can help to reduce impacts on

mand for more biomass production from

natural forest ecosystems from timber

trees in the future for carbon sequestra-

harvest-related

perturbations

(Sedjo,

tion and to meet the demand for forestry

2001). Through biotechnology faster

products and renewable energy alterna-

growing trees can be produced thereby

tives to fossil fuels (Scholes and Noble,

decreasing the growth as well as harvest-

2001). Forestry products are the third

ing period. Biotechnology can also reduce

most valuable commodity after oil and

threats to tree health by introducing the

gas. Trees supply the bulk of fiber for

traits that confer resistance to different

pulp, paper, packaging and building

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ needs. Three billion people depend on

such newly developed planting stocks as

wood for fuel. So we must harvest wood.

new varieties generated through clonal

But forests also are an essential compo-

propagation and advanced breeding pro-

nent of our ecology. It is essential to en-

grams or as transgenic trees with high-

hance the productivity of plantation for-

value traits, is expected in the near future,

ests in order to meet the ever increasing

and these trees will enhance the quality

future world demand for wood and wood-

and productivity of our plantation forests.

based products in a sustainable manner

The application of plant biotechnology

that preserves natural forests and biodi-

techniques promises potentially signifi-

versity (Scholes and Noble, 2001). The

cant genetic improvement of forestry spe-

major challenge of foresters today is to

cies and may become very attractive in

maintain the natural characteristics of for-

view of several constraints traditionally

ests while meeting society"s need for

imposed by the long life cycles and phys-

products produced from trees. Implemen-

ical size of trees (Schuch,1991). The po-

tation of improved silvicultural tech-

tential applications of biotechnology in

niques and forest management practices

forestry may be classified in three broad

are being used to manage the forest in

categories: Plant Culture, Plant Protec-

sustainable manner. Productivity of the

tion, and Plant Utilization.

forest has also been improved by the in-

troduction of new germplasm developed

_2.1. Plant tissue culture_

through genetics and breeding efforts for

Genetic improvement in planta-

tree species. The application of new bio-

tion forestry relies significantly on con-

technological tools and techniques that

ventional breeding techniques which have

span the fields of plant biology, genetic

been extensively used to improve various

transformation and discovery of genes

characteristics in forest trees such as

associated with complex multigenic traits

growth and form, volume yield, resistance

along with genetic engineering have add-

to pathogens and quality of the end prod-

ed a new dimension to forest tree im-

uct (Walter _et al.,_ 1998). Traditional

provement programs. Significant progress

breeding techniques involve identification

has been made during the past few years

of superior trees with desired traits and

in the area of plant regeneration via or-

selection of the offspring having desired

ganogenesis and somatic embryogenesis

traits. It was the major technique used for

(SE) for economically important tree spe-

the planting stock improvement in 1970s.

cies. These advances have not only

In the 1990s, biotechnology was intro-

helped the development of efficient gene

duced in forestry in earnest (Sedjo, 2001).

transfer techniques but also have opened

Forest biotechnology offers new perspec-

up avenues for using new high growth

tives in the genetic improvements of for-

performance clonally replicated planting

est trees through tissue culture and genet-

stocks in forest plantations. Advance-

ic engineering. Plant tissue culture broad-

ments in gene cloning and genomics

ly refers to the techniques of growing

technology in forest trees have enabled

plant tissues or parts on a nutrient medi-

the discovery and introduction of value-

um containing minerals, sugars, vitamins

added traits for wood quality and re-

and plant hormones, all under sterilized

sistance to biotic and abiotic stresses into

conditions. It is a basic technique to be

improved genotypes. One of the greatest

used

for

multiplying

elite

clonal

challenges today is the ability to extend

germplasm or genetically engineered

this technology to the most elite

plants of forestry species. Plant tissue cul-

germplasm, such that it becomes an eco-

ture of forestry species are aimed to fulfill

nomically feasible means for large-scale

following objectives:

production and delivery of improved

_i._ Micro-Propagation: To develop pro-

planting stock. Commercialization of

tocol for micro propagation of im-

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ portant forestry species and improve

Micropropagation by microcut-

vegetative propagation procedures

tings consists of mass producing vegeta-

including tissue culture procedures.

tive copies of desired genotypes by either

_ii._ Genetically engineering: To identify,

axillary or adventitious budding. Micro-

characterize and isolate genes of in-

propagation by microcuttings is carried

terest and genetically engineer tree

out on more than twenty species including __

seedlings to acquire desirable traits

_Populus alba, P. deltoides, P. tremula_ and

and qualities.

_Populus_ hybrids in Germany and India

_iii._ To develop molecular markers to

(Cornu, 1994), Spain (Bueno _et al.,_ 2003)

help aid in progeny selection.

and Lithuania (Kuusiene, 2002); _Euca-_

_lyptus_

_camaldulensis,_

_E. globulus,_

_2.1.1. Micro-propagation of forestry spe-_

_E. grandis, E. nitens, E. tereticornis_ and

_cies_

_E. urophylla_ in South Africa, Spain and

In India, forestry biotechnologies

Portugal (Watt _et al.,_ 2003), India (Watt

involve vegetative propagation mostly

_et al.,_ 2003; Nadgauda in press), Vietnam

through tissue culture. The method offers

and Thailand (O. Monteuuis personal ob-

substantial advantages over plants ob-

servation) and Australia; _Acacia mangi-_

tained through seed origin. It controls ge-

_um,_

_A. melanoxylon,_

_A. mangium_

×

netic diversity, ensures greater uniformity

_A. auriculiformis_ in Malaysia and South

and high proportion of non-additive ge-

Africa (Galiana _et al.,_ 2003; Monteuuis _et_

netic variance, eliminates inbreds, pro-

_al.,_ 2003; Quoirin, 2003); _Tectona gran-_

vides clones of desired traits and helps in

_dis_ in India (Bonga and Von Aderkas,

predicting the yield of plantations. Vege-

1992; Nicodemus _et al.,_ 2001; Nadgauda,

tative propagation allows cloning of supe-

in press), Vietnam, Brazil and Indonesia

rior lines and prevents the loss of desira-

(O. Monteuuis personal observation),

ble traits and the uncertainties associated

Thailand (Kjaer _et al.,_ 2000), Costa Rica

with sexual reproduction. In addition, the

(Schmincke, 2000), Malaysia (Goh &

ability to propagate transformed cells

Monteuuis, 2001); _Pinus sp. in_ United

(cells to which DNA has been introduced

States (Rahman _et al.,_ 2003); _Anogeissus_

via the techniques of biotechnology _viz_.

_latifolia_ and _A. pendula_ in India (Saxena

Genetic Transformation) and to regener-

and Dhawan, 2001); _Gmelina arborea,_

ate plants from cultured cells, microprop-

_Artocarpus chaplasha, A. heterophyllus,_

agation is prerequisite to genetic engi-

_Azadirachta indica_ and _Elaeocarpus ro-_

neering. Micro-propagation is a term used

_bustus_ in Bangladesh (Sarker _et al.,_ 1997;

here to describe methods of _in vitro_ vege-

Roy _et al.,_ 1998).

tative multiplication including rooted mi-

Somatic embryogenesis, or pro-

cro-cuttings, organogenesis and somatic

duction of embryos from somatic cells, is

embryogenesis.

Micro-propagation

is

in fact a cloning technique, as opposed to

aimed at cloning superior individuals or at

zygotic embryogenesis, in which germi-

"bulk" (in mixture) propagating new

nal cells give rise to seedlings that are all

genotypes with high genetic potential but

genetically different. Somatic embryo-

available in limited quantities (such as

genesis is a type of plant tissue culture

materials obtained by controlled pollina-

that starts with a piece of donor plant and

tion). Vegetative propagation bypasses

forms new embryos. In the right culture

the genetic mixing associated with sexual

conditions, embryos could be developed

reproduction. Researches are mainly fo-

from the somatic cells. The process of

cused on the improvement of rooting pro-

somatic embryogenesis derives usually

cedures

for

cuttings

and

micro-

from callus formation induced by apply-

propagation through organogenesis and

ing cytokinic or auxinic exogenous

somatic embryogenesis.

growth regulators to very juvenile plant

tissues. In the most favourable situations,

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ some undifferentiated cells of these calli

it more reliable, especially when using

can evolve into somatic embryos charac-

mature selected genotypes.

terized similarly to zygotic embryos, by a

Organogenesis or the creation of

shoot–root bipolar structure. This basical-

plantlets from tissues such as cotyledons

ly distinguishes somatic embryogenesis

is not common and rarely used in forestry.

from microcuttings consisting first of a

Organogenesis and somatic embryogene-

shoot from which an adventitious root

sis have been achieved in a large number

must subsequently develop. Somatic em-

of woody plant species. However, in a

bryogenesis offers the advantage of rapid

majority

of

tree

species,

micro-

embryo multiplication in a small space.

propagation has been carried out by em-

Somatic embryogenesis is of specific in-

ploying juvenile explants, for example

terest in the long term because it has the

embryos, cotyledons and shoots tips from

potential for producing, inexpensively,

seedlings. Clonal propagation from ma-

large numbers of clones that can be prop-

ture trees, in particular conifers, is still

agated as artificial seeds. In the case of

very difficult by tissue culture and re-

conifers, somatic embryogenesis, espe-

mains a challenging biotechnological

cially when derived from a single cell,

problem. Micro propagation of commer-

seems the most suitable regeneration and

cial forest tree species is a major research

propagation technique. In broad-leaved

and development goal.

species, vegetative propagation of genet-

ically engineered materials is likely to use

_2.1.2. Genetic engineering_

a combination of micropropagation and

Future prospects for genetic engi-

rooted stem cuttings, at least in the begin-

neering of forest trees are high. Through

ning. The advantages of somatic embryo-

the use of genetic engineering techniques,

genesis in comparison with micropropa-

individual genes of interest may be isolat-

gation through microcuttings are especial-

ed from the donor organism and trans-

ly with regard to multiplication rate and

ferred to a target microbe, animal, or

genetic modification applications. Somat-

plant cell. Forest trees have generally

ic embryogenesis is preferred to micro

been more difficult to work with mainly

propagate conifers (Sutton, 2002; Lelu-

due to their long generation times and life

Walter and Harvengt, 2004). However,

cycles. Through genetic engineering, for-

there are still serious obstacles to large-

eign genes can be transferred to a forest

scale operational application of somatic

tree resulting in faster tree improvement

embryogenesis to forest trees, for exam-

and unique gene combinations which

ple:

cannot be achieved by traditional tree

 Only few species and within these

breeding. The successful genetic engi-

species, only few genotypes can pro-

neering of a forest tree requires four fac-

duce somatic embryos.

tors:

 Success has been obtained with few

_i._

A desirable gene must be identi-

exceptions, mainly with juvenile tis-

fied and isolated from a donor or-

sues coming for instance from imma-

ganism.

ture zygotic embryos.

_ii._

A plant regeneration protocol

 There are risks that somaclonal varia-

from single cells or a small group

tion may decrease the value of the

of cells.

genotypes produced by somatic em-

_iii._

A mechanism for inserting or

bryogenesis, resulting in a considera-

transferring foreign DNA into a

ble waste of time, material and mon-

target cell

is required.

ey.

True-to-typeness,

particularly,

_iv._

Additional DNA sequences for

may remain a problem for certain

regulating the target gene, e.g. a

genotypes and efforts are still needed

promoter is necessary to cause the

for optimizing this technique to make

target gene to function in the

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ proper tissue when and where de-troporation and polyethylene glycol. Most

sired. ****

successful work on genetic transformation

****

of forest tree species genomes so far has

Considerable research is focused

been obtained by using juvenile material,

on identifying, characterizing and isolat-

for example from an explants produced

ing genes in trees that are responsible for

from juvenile tissues which have much

traits of particular interest, including

higher regeneration capacities than older

stress responses viz. drought, frost and

material. Successful genome modification

water-logging etc., disease tolerance and

reports of adult selected plant material are

or resistance growth characteristics, wood

very rare, except in poplars precisely be-

quality and flowering. The examples of

cause of its greater capacity to regenerate.

traits for which genes are being sought in

Transformation of a number of

commercial forest trees include lignin

tree species by genetically engineering

composition, disease and insect re-

trees has been reported. Populus spp.

sistance, dormancy, cold hardiness and

serves as the model for much of the re-

growth rate etc. Genes of interest can be

search because techniques for genetic en-

divided into two categories. First, genes

gineering and micropropagating are rela-

those are responsible in governing agro-

tively advanced. However, research in

nomic traits or growth of trees. These

this area is still modest because several

genes are expected to lower the cost of

technical problems have to be solved.

wood. This category includes genes for

Limitations to the broad use of

disease and pest resistance or tolerance of

genetic engineering to improve forest

environmental stresses. Genes that help a

trees include primarily the following

crop to grow more efficiently, such as

facts:

herbicide tolerance, also fall into this

_i._

Only a few genes for specific

group. A second category includes genes

traits of commercial interest have

for value-added traits that improve pro-

been identified,

duction efficiency, product quality, or

_ii._

Culturing cells of many tree spe-

product value and are expected to in-

cies is difficult and

crease the value of wood or quantity and

_iii._

Regenerating whole plants from

quality of active bio-chemicals. For in-

cultured cells of commercial tree

stance, genes for reduced lignin content

species has met with only limited

or lignin type which are more easily re-

success.

moved during pulping fall into this cate-

Those biochemical activi-

gory. Genes responsible for improved fi-

ties that are specific to woody plants or

ber characteristics would also be included

even to single species or varieties of trees,

here. Several value-added traits are being

such as wood fiber formation and re-

actively studied in order to isolate valua-

sistance (or susceptibility) to specific in-

ble genes.

sects or diseases often must be studied in

Several gene transfer systems are

the tree species of interest. It is important

available for movement of foreign DNA

to note, however, that much of the re-

into target plants. The most common

search on non-forest plants is relevant to

method uses _Agrobacterium tumefaciens_.

commercial forest trees. For example, the

This soil-borne bacterium is able to natu-

research underway on cellulose biosyn-

rally genetically engineer plants to create

thesis in cotton plants is focused on un-

an environment in which the bacterium

covering enzymes and genes for which

can thrive. Molecular biologists have al-

similar counterparts can be anticipated in

tered this organism to insert target genes

trees. The same is true for photosynthesis

into plants without causing plant disease.

research on spinach. Major research ef-

Other methods for gene transfer in forest

forts are underway in many laboratories

trees include particle bombardment, elec-

on the small plant _Arabidopsis_ _thaliana_

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ because it completes its life cycle in 6

like drought and water logging add to it,

weeks. Funds for much basic biochemical

quite substantially. Shoot borer in _Shorea_

research are more effectively spent on

_robusta_ and _Fusarium_ wilt of _Dalbergia_

plants other than forest trees.

_sissoo_ greatly damage these important

****

species of timber. Through the application

_2.1.3. Molecular markers_

of Biotechnology threats to tree health

A serious problem today is in

can be reduced. Research is showing

identifying desired progeny before they

promise in the introduction of traits that

get too old to propagate vegetatively, by

confer resistance to pests and pathogens

root cuttings, for example. Marker-

that weaken or cause heavy mortality of

assisted selection (MAS) has given fur-

trees. Improvements through tree bio-

ther impetus to tree breeding and selec-

technology may also improve weed con-

tion. Molecular markers allow rapid iden-

trol enabling young trees to get a head

tification of the gene or genes of interest

start over nutrient-robbing competitors.

in minute samples. Molecular markers are

The research falls into two categories: (a)

genetically linked to a given allele on a

direct protection through control of in-

given locus and can therefore, be used to

sects and diseases and (b) indirect protec-

predict the presence of the allele with

tion through development of resistant tree

great accuracy (FAO, 2004). Biochemical

varieties.

and molecular markers play a significant

role in many forest biotechnology activi-

_3.1. Direct protection_

ties for the selection of desired proge-

The biotechnology product ""Bt

ny.Research aimed at developing molecu-

toxin" is already being used commercially

lar markers for screening is accelerating.

in agriculture and forestry. Produced by

Marker applications for fingerprinting and

the bacterium _Bacillus thuringiensis_ , this

paternity analysis been used for character-

toxin is effective against lepidopterous

ization of genetic diversity. Isozymes,

insects. One biological approach is to use

randomly amplified polymorphic DNAs

pheromones to attract the insects to a cen-

(RAPDs) and restriction fragment length

tral point for control. Techniques of mo-

polymorphisms (RFLPs) have been wide-

lecular biology apparently have not yet

ly used for genetic diversity and mapping

been applied to these pests. Direct bio-

studies, though the current trend favours

control of rusts and other fungal, bacterial

microsatellites (nuclear and cytoplasmic)

and viral diseases of trees is labour inten-

and AFLPs (amplified fragment length

sive and costly at best.

polymorphisms). Currently, ESTs (ex-

pressed sequence tags) and SNPs (single

_3.2. Indirect protection_

nucleotide polymorphisms) represent the

Trees have a wide variety of natu-

most active area of marker development

ral defenses against insects and diseases,

(FAO, 2004).

which is why most microbes, insects and

viruses do not cause tree damage. Con-

**3. Plant protection**

siderable research is underway to identify

****

genes that confer resistance to fusiform

****

Forest trees are affected by a

rust and white pine blister rust in resistant

large number of different insect pests and

varieties and species of pines. When these

diseases, the latter caused by fungi, bacte-

genes have been identified, they can theo-

ria and viruses. Biotechnology offers a

retically be inserted into susceptible trees

powerful approach to mitigating the dam-

to provide resistance. This is fairly long-

age caused by insects and diseases as well

range research. In the near term, the iden-

as by environmental stressors. Insects and

tification of markers for these resistance

diseases probably cause great damage to

genes will allow marker-assisted tree

Indian forests; environmental stressors

breeding and asexual progeny selection to

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ hasten the development of resistant lines

qualitatively modified through genetic

of trees. Stress tolerance genes are also

engineering for expected financial gains

being sought. Another biotechnological

from pulp processing improvements. Lig-

approach to indirectly controlling insects

nins, which enhance cell wall mechanical

and diseases in trees is to insert foreign

properties and hardness, are difficult to

genes that confer resistance. Thus, several

process and are a significant limitation in

tree species have been transformed with

processing wood into paper pulp by

insect-controlling genes such that for Bt

chemical treatment. Genetic transfor-

toxin. ****

mation to modify lignin characteristics is

The production of insect resistant

a key research feature on species used in

plants via genetic engineering has gener-

the paper industry. The aim is to regulate

ally taken one of two approaches. The

the activity of key enzymes involved in

first approach makes use the Bt toxin de-

the lignin biosynthesis pathway (Jouanin

rived from _Bacillus thuringiensis_. This

_et al.,_ 2000; Le _et al.,_ 2003).

toxin damages the digestive mechanisms

****

of the larvae that feed upon it. The toxin

**5. Benefits of biotechnology in forestry**

specifically affects insects belonging to

the lepidopteran, dipteran and coleopteran

Forestry is in the take-of stage today as

orders of insects, which include a number

biotechnology is introduced into its sev-

of major herbivores of forest tree species.

eral operations bringing promising devel-

The Bt toxin gene was first used to trans-

opmental changes having tremendous po-

form hybrid poplars by McCown _et al.,_

tential. The use of biotechnology for tree

(1991) via direct (biolistics) gene transfer.

improvement can bring economic, social

The introduction of the Bt toxin gene re-

and environmental benefits. Biotechnol-

sulted in a significant reduction in forest

ogy has many useful applications in for-

tent caterpillar ( _Malacosoma disstria_ )

estry viz. lignin reduction, fiber modifica-

survival and growth rates of the gypsy

tion, pest and disease resistance, bio con-

moth larvae ( _Lymantria dispar_ ). Herbi-

trol methods against weeds, pests and dis-

cide resistant crops have been one of the

eases, cellulose content enhancement, de-

major products of the first generation of

velopment of cold and hot tolerant species

agricultural biotechnology. They are in-

of a desired species, modification of bi-

tended to reduce weed control costs, in-

omass chemistry to improve biofuels and

crease control flexibility, facilitate the use

pulp production, phyto-remediation of

of low-tillage (and thus reduced erosion)

problem contaminated sites viz. arid, ra-

cropping systems and enable broad-

vine, saline and usar sites, improving

spectrum and environmentally benign

Carbon sequestration potential to mitigate

herbicides to be more readily employed.

greenhouse gas emissions and sterility,

The first successful transformation of a

which is an important factor to prevent

woody species was reported in _Populus_

modified genes from "leaking" into the

_alba × P. grandidentata_ using _Agrobac-_

natural environment. The short term eco-

_terium tumefaciens_ (Fillatti _et al_., 1987).

nomic gains from the introduction of bio-

Transgenic hybrid poplars, with a reduced

technology to forestry will be lower costs

sensitivity to glyphosate, an extensively

and increased availability of wood and

used broad-spectrum herbicide, were pro-

wood products at shorter rotation than

duced.

usual. Innovations in forest biotechnology

have the potential to address important

**4. Plant utilization**

environmental issues, including the reha-

Genetic transformation of tree for

bilitation of habitats altered by diseases

its commercial end-use can be achieved

like the Sal Borer Attack in Sal Forests,

through biotechnology. For example lig-

Drying of Sheesham, or invasive exotics.

nin composition can be quantitatively and

Moreover, the increased productivity of

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ biotechnology driven tree plantations may

desirable traits would be the easy break-

free huge chunks of natural or primary

down of wood fibers and the removal of

forest areas from ever increasing pres-

lignin

during

chemical

processing.

sures to supply industrial wood and thus

Through Biotechnology, the raw material

improve their ability to maintain and pre-

for paper production can be customized to

serve biodiversity. And as trees are genet-

meet the requirements of producers there-

ically modified to be able to grow in pre-

by wood values increase to that extent.

viously unsuitable areas such as arid or

Usar lands and saline soils the newly cre-

_5.1.2. Environmental benefits_

ated forests could not only produce more

Forestry Biotechnology may also

wood but also enhance watershed protec-

be used to create a number of desirable

tion and sequester carbon for climate

environmental outputs (Table 1).

change mitigation.

****

**Table 1:** Utilization of biotechnology for

_5.1. Benefits of biotechnology_

environmental benefits

_5.1.1. Economic benefits_

**No.**

**Biotechnological Environmental**

__

__ Introduction of any technology

**innovation**

**output**

for the consumer simply means that rela-

1

Cheaper planta-

Pressure to log

tive prices of the desired goods fall com-

tion wood substi-

primary forests

pared to that which would have been in

tutes for wood

can be reduced ****

the absence of the particular technological

from natural for-

innovation. In other words, technology

ests ****

features increased productivity, that is,

2

Trees are genet-

Ecological forests

enhanced output per unit of input. Alter-

ically modified to can be estab-

natively, technology can be either cost

grow in arid, usar lished on de-

(input) reducing or yield (output) enhanc-

or saline condi-

graded lands ****

ing. For society, more output for the

tions ****

equivalent expenditure of inputs means

3

Trees are genet-

Carbon-

societal increase in efficiency. Incidental-

ically modified to sequestrating for-

ly, Plantation Forestry has received some

adapt to tradi-

ests can be estab-

success in recent decades because of its

tionally unsuita-

lished on sites

associated cost-reducing technology that

ble sites ****

previously not

has given wood from planted forests a

suitable for for-

competitive price advantage over that

estry ****

harvested from natural forests. The poten-

4

Cold-tolerant

The altitudinal

tial applications of immediate interest of

species of a de-

and geographical

cost-reducing biotechnology to forestry

sired genus are

range of desirable

are increased wood production, improved

developed

tree species can

tree form and wood quality, enhanced

be extended

survival, quicker growth rates and en-

hanced resistance to insects, diseases and

Trees genetically engineered for

herbicides. In addition, production and

pest and disease resistance may promote

processing costs of wood or chips could

plantation survival and yield and also lead

be reduced as well as financial and envi-

to eco-restoration of native tree species

ronmental costs for pulping.

like Sal. Biotechnology has the potential

Paper production, for instance,

to enhance the ability of trees to tolerate

requires fiber with adequate strength to

abiotic stresses; restoration of contami-

allow sheets to be produced on high-

nated sites through phytoremediation; fa-

speed machines, an attribute determined

cilitation of weed control using more en-

by the wood fiber characteristics. There-

vironmentally safe treatments; modifica-

fore, in pulp making for paper production,

tion of biomass chemistry to improve

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_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ pulp and biofuels production; and im-ment. Thus, the presence of that modified

proving carbon sequestration to mitigate

gene in the natural environment appears

greenhouse gas emissions. Biotechnol-

unlikely to constitute any serious envi-

ogy also offers the potential to assist in

ronmental problem, either short- or long-

ecosystem restoration and repair. Similar-

term.

ly, biotechnology may help deal with in-

For qualitative genes that affect

vasive exotics, which have in many plac-

tree form or wood fibre characteristics,

es threatened locally and easily available

release into the natural environment is

indigenous species. Modified tree species

unlikely to provide a competitive ad-

also prove useful in providing ecological

vantage in survival and therefore, unlikely

and environmental services in areas

to have significant or adverse conse-

where trees now have difficulty in surviv-

quences on the ecology. However, the

ing, such as arid or drought-prone areas

consequences could be different if a sur-

areas and areas with saline soil or frost

vival gene is involved. For example, the

zones and industrial waste effluents viz.

introduction of _Bacillus thuringiensis_ (Bt)

Dairy waste disposal sites, Alumunium

gene makes a plant toxic to certain pests.

waste effluents-Red Mud, Fly Ash Ponds

The release into the wild of such a gene

in Thermal Power Plants etc.. Another

could constitute a major problem if it al-

contemporary and very important applica-

tered the comparative competitive posi-

tion of biotechnology from the present

tion of wild vegetation vis-a-vis those

Climate Change scenario involves crea-

pests. However, the seriousness of this

tion of biological sinks, a potential tool to

problem depends on the probability of the

mitigate the build-up of greenhouse gases

transfer of a survival gene into the wild,

associated with global warming. Land

the scale of the transfer and the compara-

Areas and Wetlands not currently forested

tive change in the competitive ecological

could grow carbon-sequestering planta-

balance within the natural habitat. Since

tions of Genetically Modified transgenic

pests adapt via natural selection to modi-

trees (GM Trees). __

fied genes, the long-term impact of the

The most threatening cost of Bio-

release of the modified gene into the natu-

technology is the after-effect of transgen-

ral environment will be mitigated. Subse-

ic plants on the natural ecosystem, when

quently, wild populations would gradual-

there would be genetic exchange(s) be-

ly become resistant to the _Bacillus thurin-_

tween domestic and wild populations. In

_giensis_ (Bt) gene, thereby undermining its

cases where plantation tree species are

long-term effectiveness against those

exotic, genetic "outcrossing" to the natu-

pests.

ral environment would not be a factor.

Transgenic biotechnology has be-

Where genetic exchange could be a prob-

come controversial in agriculture. Some

lem, planting sterile trees or varieties with

of those controversies appear to be spill-

reduced or delayed flowering would less-

ing over to forestry too. The controversy

en the likelihood of their "escape" to the

revolves around a number of issues. One

natural environment. In the case of the

such issue involves the effects of biotech-

herbicide-tolerant gene, the consequences

nology, particularly the introduction of

of release into the wild are probably

transgenic plants on human health. The

small. Herbicides are unlikely to be ap-

food safety issue is not generally raised

plied to most of the natural environment,

for plants such as forest trees, which are

and where necessary, other types could be

not usually a food source. However, cel-

used to which the escaped genes do not

lulose is increasingly being used as filler

confer tolerance. In the long term, the

in food products, and the food safety issue

herbicide in question will almost surely

could become a concern, subsequently to

be replaced periodically in the normal

be encountered by the forest biotechnolo-

course of product change and develop-

gists. ****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 113

_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ Therefore, the most visible costs

of genetic exchange between genetically

of forest biotechnology are those associ-

modified trees and wild populations. ****

ated with Modified Genetically Engi-

Genetic engineering, by identify-

neered (GE) trees which include among

ing and isolating specific genes to serve a

others, effects of the newly acquired traits

particular purpose, can allow a novel trait

on forest ecosystem structure and func-

to be transferred to any genotype in a sin-

tion; unintended consequences of insert-

gle generation with little or no alteration

ed genes on tree and forest biology; relia-

to its other genetic properties. Thus, if

bility of the newly encoded traits to pro-

there is to be a fertile crescent in forestry,

duce the desired outcomes; and persis-

it is to be found in the Biotechnology La-

tence and potential impacts of the intro-

boratories and Field Experiments that op-

duced genes in native populations through

erate at the interface of basic plant mo-

the dispersal of pollen, seeds or vegeta-

lecular genetics and forestry. ****

tive propagules (Frankenhuyzen and

Genetic engineering can provide

Beardmore, 2004). Other apparent risks

exceptional particular genotypes that can

from biotechnology are associated with

be characterised by corresponding specif-

loss of genetic diversity from vegetatively

ic molecular markers and subsequently

propagating a small number of highly se-

integrated in the clonal forestry and

lected varieties. Another serious disad-

breeding programs. Further progress in

vantage of vegetative propagation of

this area will depend on reliable regenera-

highly selected varieties may ultimately

tion and automation for mass production

result in vulnerability to insect and mi-

of selected particular genotypes. It is ex-

crobial pests and also to stressful climatic

pected that future research in biotechnol-

events.

ogy will provide insight into the control

of maturation and rejuvenation, directed

**6. Concluding remarks**

gene transfer, genome structure, gene se-

****

****

quence and basic mechanisms involved in

Before taking any new biotechno-

growth and differentiation of trees. Bio-

logical strides in the forestry sector, ow-

technology is going to play an important

ing to the complexity of forest ecosys-

role in the 21st century in boosting sus-

tems, several ecological benefits, costs

tainability of Forests.

and risks and the effectiveness of risk

management practices should be com-

**References**

pletely evaluated through careful review

****

of scientific literature and well-designed

**Bueno, A., Gomez, A. and Manzarena,**

field experiments. ****

**J.A. (2003).** Propagation and DNA

****

The benefits of biotechnology in

markers characterization of _Populus_

forestry go beyond economic advantages

_tremula_ L. and _Populus alba_ L. _In_

including increased production, lower

S.M. Jain & K. Ishii, eds. _Micro-_

costs to consumers and trees modified for

_propagation of woody trees and_

easy wood processing or specific produc-

_fruits, forestry sciences_ , pp. 37–74.

tion objectives etc. to environmental sur-

Dordrecht, Netherlands, Kluwer.

pluses viz. preservation of biodiversity,

**Cornu, D. (1994).** Forêt, de la gélose à la

eco rehabilitation of degraded lands, eco

terre. _Biofutur_ , Février **1994, 25–31.**

restoration of contaminated sites and mit-

**FAO. (2001).** Glossary of biotechnology

igation of global warming etc.. But bio-

for food and agriculture: a revised

technological innovations raise immediate

and augmented edition of the glos-

serious concerns about biosafety and the

sary of biotechnology and genetic

effects of transgenic plants on the re-

engineering. FAO Research and

sistance of pathogens and on the natural

Technology Paper No. 9. Rome.

ecosystem too, particularly the question

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 114

_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ **FAO. (2004).** Preliminary review of bio-Teaknet Publication No. 3, pp. 161-

technology in forestry, including

189.

genetic modification. Rome.

**Le Dantec, L., Chagné, D., Pot, D., Be-**

**Fillatti, J.J., Sellmer, J., McCown, B.,**

**don, F., Géré-Garnier, P., De Da-**

**Haissig, B. and Comai, L. (1987).**

**ruvar, A. & Plomion, C. (2003). **

Agrobacterium mediated transfor-

_Data mining in pine ESTs: II. De-_

mation and regeneration of populus.

_velopment of SNP markers_. _In_ Tree-

_Mol. Gen. Genet_. 206, 192-199.

biotechnologies, Umea, Sweden, 7–

**Frankenhuyzen,V. K.; Beardmore, T.**

12 June 2003, s6.22.

**(2004).** Current status and environ-

**LeluWalter, M.A. and Harvengt, L.**

mental impact of transgenic trees.

**(2004).** L"emeryogenèse somatique

Canadian Journal of Forest Re-

des conifers, état et perspectives.

search **34, 1163-1180.**

_Afocel Inf.-For._ , **694, 6.**

**Galiana, A., Goh, D., Chevallier, M.H.,**

**McCown, B.H., McCabe, D.E., Russell,**

**Gidiman, J., Moo, H., Hattah, M.**

**D.R., Robinson, D.J., Barton,**

**& Japarudin, Y. (2003). ** Micro-

**K.A. and Raffa, K.F. (1991)**. Sta-

propagation of _Acacia mangium ×_

ble transformation of _Populus_ and

_A. auriculiformis_ hybrids in Sabah.

incorporation of pest resistance by

_Bois For. Trop.,_ **275, 77–82.**

electric discharge particle accelera-

**Goh, D. and Monteuuis, O. (2001).**

tion. _Plant Cell Rep._ **9, 590-594.**

_Production of tissue-cultured teak:_

**Monteuuis, O., Alloysius, D., Garcia,**

_the plant biotechnology laboratory_

**C., Goh, D. and Bacilieri, R.**

_experience._ _In_ Proceedings, 3rd Re-

**(2003).** Field behavior of an _in_

gional Seminar on Teak: "Potential

_vitro_ -issued _Acacia mangium_ ma-

and opportunities in marketing and

ture selected clone compared to its

trade of plantation teak: challenge

seed-derived progeny. _Aust. For._ ,

for the new millennium", pp. 237-

**66, 87–89.**

247. Yogyakarta, Indonesia, 31 July

**Nehra, N. S. , Becwar, M. R., Rott-**

– 4 August, 2000.

**mann, W. H. , Pearson, L. ,**

**Jouanin, L., Goujon, T., de Nadaï,V.,**

**Chowdhury, K. , Chang, S., Day-**

**Martin, M.T., Mila, I., Vallet, C.,**

**ton Wilde, H., Kodrzycki, R. J. ,**

**Pollet, B., Yoshinaga, A., Chab-**

**Zhang, C., Gause, K. C. , Parks,**

**bert, B., Petit-Conil, M. and**

**D. W. and Hinchee, M. A. (2005).**

**Lapierre, C. (2000).** Lignification

Forest biotechnology: innovative

in transgenic poplars with extremely

methods, emerging opportunities. In

reduced

caffeic

acid- _O_ -

___
___

Vitro Cellular and Developmental

methyltransferase activity. _Plant_

Biology–Plant **41(6),701-717.**

_Physiol._ , **123, 1363–1373.**

**Nicodemus, A., Nagarajan, B., Mandal,**

**Kuusiene, S. (2002).** __ Sexual and non-

**A.K. and Suberbramanian, K.**

sexual plant reproduction in the la-

**(2001).** _Genetic improvement of teak_

boratory

(available

at

_in India_. _In_ Proceedings, 3rd Re-

www.genfys.slu.se/staff/dagl/nova0

gional Seminar on Teak: "Potential

2/Abstracts_Nova02.doc).

and opportunities in marketing and

**Kjaer, E.D., Kaosa-Ard, A. and**

trade of plantation teak: challenge

**Suangtho, V. (2000).** Domestica-

for the new millennium", pp. 277-

tion of teak through tree improve-

294. Yogyakarta, Indonesia, 31 July

ment: Options, potential gains and

– 4 August, 2000.

critical factors. _In_ _Site, technology_

**Quoirin, M. (2003).** Micropropagation of

_and productivity of teak plantations_ ,

_Acacia_ species. _In_ S.M. Jain & K.

FORSPA Publication No. 24/2000,

Ishii, eds. _Micropropagation of_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 115

_Biotech Sustainability (2017)_

_Biotechnology for Sustainability of Forests Dubey and Dubey_ _woody trees and fruits,_ pp. 245-268.

for forestry research. In Vitro

Dordrecht, Netherlands, Kluwer.

Cellular & Developmental Biology -

**Rahman M.S., Messinamg, M.G. & **

Plant ******27,** **99-103.**

**Newton, R.J. (2003).** Performance

**Scholes, R.J. and Noble, I.R. (2001).**

of loblolly pine ( _Pinus taeda_ L.)

Storing carbon on land. _Science_ ,

seedlings

and

micropropagated

**294, 1012.**

plantlets on an east Texas site. I.

**Sutton, B. (2002).** Commercial delivery

Above- and belowground growth.

of genetic improvement to conifer

_For. Ecol. Manage.,_ **178, 245–255.**

plantations using somatic embryo-

**Roy, S., Islam, M. & Hadiuzzaman, S. **

genesis. _Ann. For. Sci._ , **59, 657–**

**(1998).** Micropropagation of _Elaeo-_

**661.**

_carpus robustus_ Roxb. _Plant Cell_

**Walter, C., Carson, Menzies, M.I. ,**

_Rep_., **17(10), 810–813.**

**Richardson**

**T. and Carson,**

**Sarker, R.H., Rafiqul Islam, M. & **

**M..(1998).** Review: Application of

**Hoque, M.I. (1997).** _In vitro_ propa-

biotechnology to forestry – molecu-

gation of neem ( _Azadirachta indica_

lar biology of conifers. World Jour-

A. Juss) plants from seedlings ex-

nal of Microbiology and Biotech-

plants. _Plant Tissue Cult_., **7(2),**

nology **14,** **321-330.**

**125–133.**

**Watt, M.P., Blakeway, F.C., Mokotedi,**

**Saxena, S. & Dhawan, V. (2001). ** Large-

**Meo, Jain, S.M. (2003).** Micro-

scale production of _Anogeissus pen-_

propagation of _Eucalyptus. In_ S.M.

_dula_ and _A.latifolia_ by micropropa-

Jain & K. Ishii, eds. _Micropropaga-_

gation. _In Vitro Cell. Dev. Biol.-_

_tion of woody trees and fruits,_ pp.

_Plant_ **37, 586–591.**

217–244. Dordrecht, Netherlands,

**Sedjo, R.A. (2001).** Biotechnology in

Kluwer.

forestry: Considering the costs and

**Yanchuk, A.D. (2001).** The role and im-

benefits. Resources for the Future

plications of biotechnological tools

**145, 10-12.**

in forestry. _Unasylva_ **204, 53–61.**

__ **Schuch. W. (1991).** Advances in plant

biotechnology and their implication

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 116

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P117-128_

**Biotechnological Approaches for Conservation and Sus-**

**tainable Supply of Medicinal Plants**

**Sagar Satish Datir1, * and Subhash Janardhan Bhore2**

_1Department of Biotechnology, Savitribai Phule Pune University, Pune – 411007, MS, In-_

_dia; 2Department of Biotechnology, Faculty of Applied Sciences, AIMST University,_

_Bedong-Semeling Road, 08100, Bedong, Kedah Darul Aman, Malaysia;_

_subhashbhore@gmail.com / subhash@aimst.edu.my (SJB);_

_*Correspondence: datirsagar2007@gmail.com; Tel.: +91 8412013810_

****

****

**Abstract:** Food and medicines are integral part of human life. Continuously increasing

global population and food demand has created an alarm about sustainable use of natural

resources. Due to adverse environmental conditions such as drought, salinity, temperature

and pathogens, it is very challenging to achieve high yield with current agricultural practic-

es. Due to deterioration of food quality and unpredictable environmental conditions, there is

a major public health concern about various diseases. Plant-derived compounds are playing

significant role in combating various human diseases since prehistoric times and therefore,

there is an increasing demand for production of plant-derived secondary metabolites. How-

ever, due to mismanagement of natural resources and faulty agricultural practices, several

medicinal plant species have become rare, vulnerable and endangered. Hence, alternative

strategies are needed to protect medicinally important plant species. Biotechnology has be-

come a center of attraction due to its innumerable advantages in agriculture, pharmaceuti-

cals, forestry and food sectors. In recent years, plant-derived compounds (also called as

natural compounds) are widely studied and biotechnological tools such as, _in vitro_ propaga-

tion, transgenic for secondary metabolite production and cryopreservation not only provid-

ed alternative but also offer sustainable approaches towards conservation of medicinally

important plant species. This brief review highlights various biotechnological approaches

for conservation and sustainable supply of medicinal plants. Achievements, challenges and

perspectives on _in vitro_ propagation for the conservation of medicinal plants are also high-

lighted. ****

_**Keywords**_ **:** Biotechnology; conservation; medicinal plants; plant tissue culture; secondary

metabolites; sustainable development

**1. Introduction**

problems have magnified the aforemen-

****

tioned threats and necessitated the sus-

Climate change, biotic and abiotic

tainable use of natural resources. Food

stress, depletion of natural resources, de-

and medicines are integral part of human

forestation and loss of biodiversity are

life and to fulfil the growing demand,

major challenges in the process of sus-

continuous global efforts are underway

tainable global development. In order to

for increasing agricultural productivity.

fulfill the basic requirements such as

The United Nations Food and Agricultur-

food, fuel, medicines and shelter, humans

al Organization (FAO) assuming that

are completely dependent on natural re-

global population will be about 9.1 billion

sources. However, continuous increase in

in 2050 (Godfray _et al_., 2010). It is re-

global population and associated food

ported that 83% medicinal plants have

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_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ become endangered mainly due to the

though they are facing the threat of be-

human activity (European Commission,

coming endangered and or extinct (Ma-

2008; Ibrahim _et al.,_ 2013). Whereas,

nohar, 2012).

over-utilization of natural resources, pol-

One third of the global plant spe-

lution of the soil, water and the atmos-

cies are threatened at different level ac-

phere, and introduction of invasive spe-

cording to International Union of Conser-

cies have resulted into reduced biodiversi-

vation of Nature (IUCN, 2013). Further-

ty (Hunde, 2007).

more, the habitat destruction and loss also

Medicinal plants are important for

leads to the fragmentation of the remain-

the wellbeing of human population and

ing habitat which eventually results in

there is an increasing demand for the pro-

further isolation of the respective plant

duction of plant-derived secondary me-

species population. The destructive har-

tabolites/ novel drug leads (Atanasov _et_

vest of underground parts of slow repro-

_al.,_ 2015). Currently, there is constant

ducing, slow growing and habitat-specific

demand for plants and plant parts in

plant species are the crucial factors in

pharmaceutical industries as well as from

making

them

vulnerable

and

rare

Ayurveda professionals. Furthermore, due

(Ghimire _et al.,_ 2005; Kala, 2005).

to major public concerns about dreadful

Providing high quality planting material

diseases such as cancer, HIV etc., phar-

for sustainable use and thereby saving the

maceutical industries are actively engaged

genetic diversity of plants in the wild is

in production of plant-derived drugs. Due

important (Krishnan _et al.,_ 2011). How-

to the toxicity and side effects of synthet-

ever, due to the human intervention there

ic drugs, the plant-derived drugs are be-

is rapid dwindling of plant resources for

coming more popular and as a result there

medicines; hence, alternative strategies

is increase in the number of herbal drug

and or innovative approaches are needed

manufacturers (Verma and Singh, 2008;

for their conservation. Bukuluki _et al._

Agrawal, 2005; Lahlou, 2013).

(2014) had scrutinized the harvesting

The projected escalating demand

practices of medicinal plants in Uganda

for medicinal plants is increasing which

and identified harvesting methods for sus-

leads to unscrupulous collection from the

tainable supply of medicinal plants. The

wild and adulteration of raw material

good harvesting practices suggested in-

supplied to the manufacturers. Ultimately,

clude, careful harvesting of roots without

this practice has resulted into the over-

affecting tap root, careful removal of stem

harvesting of many plants from wild and

bark to avoid damaging the innermost

disturbed the population of various me-

layer that contributes to drying of the

dicinal plant species and several species

plant, plucking of leaves without breaking

even became endangered (Kala _et al.,_

the shoots, picking flowers those are fall-

2006; Rao _et al.,_ 2004). It has been re-

en down or selecting only a few in order

vealed that more than 50,000 plant spe-

to allow the plant to bear fruits and repro-

cies are used in phytotherapy and medi-

duce. Recently, Hishe _et al._ (2016) re-

cine of which 2/3 are harvested from na-

viewed the value chain of medicinal

ture leading to local extinction of many

plants and the associated challenges. They

species or degradation of their habitats

have conducted detailed studies of modes

(Tasheva and Kosturkova, 2012). Due to

of harvesting, storage, packaging, supply

the constant expansion of herbs trade, the

and distribution of medicinal plants. They

insufficient cultivation fields, and the

highlighted that the medicinal plants sup-

weak management of harvesting and

ply chains have varying requirements for

overharvesting of medicinal plants have

their cultivation, resource management in

led to exhaustion of the natural resources

the wild, harvesting, processing and mar-

and reduction in the biodiversity. Medici-

keting. Considering these facts, they had

nal plants are always in demand even

concluded that in order to become com-

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_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ petitive in the medicinal plants global

for food, pharmaceutical and cosmetic

market place, value chain must become

industries (Nalawade _et al.,_ 2003). A sys-

more flexible, innovative and efficient, so

tematic concept of sustainability was pro-

it can bring to market new products in a

posed by Prescott-Allen and Prescott-

timely fashion.

Allen (1996). According to them, both

As medicinal plants represent con-

humans and ecosystem are interrelated

sistent part of biodiversity, their utiliza-

and dependent on each other. Hence, in

tion and conservation strategies needs

conceptual terms, the essence of sustaina-

planned management for sustainability.

ble development is expressed by the rela-

Therefore, systematic efforts should not

tionship between people and the ecosys-

only be directed towards preservation of

tem around them. They further stated that

the plant populations but also elevating

the society is thought to be sustainable

the level of knowledge for sustainable

when both the human condition and the

utilization of these plants in medicine

condition of the ecosystem are satisfacto-

(WHO 2010). Developing strategies for

ry or improving. They concluded that the

long-term sustainable supply of medicinal

system improves only when both the con-

plants is challenging; therefore, it has

dition of the ecosystem and the human

been suggested that to meet future public

condition improve (Prescott-Allen and

food and healthcare demand, integration

Prescott-Allen, 1996).

of conventional methods and biotechnol-

In order to supply medicinal

ogy are essential. Biotechnological meth-

plants or medicinal plant-based raw mate-

ods not only offer faster cloning and con-

rial in a sustainable manner, various _in_

servation of the genotype of the plants;

_situ_ and _ex situ_ strategies (which includes

but also enable genetic modification, gene

_in vitro_ techniques, botanical gardens,

regulation and expression for an efficient

plant banks, GenBank, gene sanctuaries

production of valuable natural substances

and seed banks) have been suggested for

in higher amounts or with better proper-

the conservation of critically endangered

ties (Tasheva and Kosturkova, 2012). Be-

plant species (Khan _et al.,_ 2012). Genetic

cause of innumerable advantages of bio-

diversity preservation is of prime im-

technology in agriculture, pharmaceuti-

portance while conserving plant genetic

cals, forestry, food industry and other sec-

resources. For the conservation of plant

tors, the field of biotechnology has be-

and or their germplasm, _ex situ_ and _in situ_

come a center of attraction for conserva-

strategies are used. The _in situ_ approach

tion and sustainable supply of medicinal

includes the maintenance of plant species

plants.

and or their populations in their habitats,

where they can naturally occur, grow and

**2. Biotechnological approaches for con-**

reproduce. Whereas, _ex situ_ approach of

**servation of medicinal plants**

conservation focuses on the maintenance

of plant species germplasm under con-

It appears that biotechnology is

trolled conditions (Pathak and Abido,

emerging dramatically as a key enabling

2014; Rai _et al.,_ 2010). The multiplication

technology for environmental protection

of plants by classical methods such as

and stewardship in a sustainable manner

cuttings, budding, layering, and or graft-

(Cantor, 2000; Gavrilescu, 2004; Arai,

ing in nurseries produces enormous num-

2006). Biotechnological advances have

ber of plants. However, biotechnological

encompassed almost every aspect of hu-

methods such as micropropagation, meta-

man life including food, fuel, cosmetics,

bolic engineering and genetic manipula-

medicines and beverages. Most im-

tions are especially appropriate for spe-

portantly,, biotechnology based-methods

cies which are difficult to propagate _in_

are reliable and provides continuous sup-

_vivo_ (Tasheva and Kosturkova, 2012).

ply of raw material and natural products

Hence, _in situ_ approach of conservation

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_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ alone would not be efficient and effective

(Sreekumar and Renuka, 2006). Biotech-

strategy for conservation and multiplica-

nological approaches for sustainable sup-

tion of medicinal plants. Krishnan _et al._

ply and conservation of medicinal plants

(2011) suggested that prudent application

include micropropagation, mycorrhiza-

of propagating biotechnology tools in

tion, genetic transformation and devel-

plant conservation program is a prerequi-

opment of the DNA banks (Sheikhpour _et_

site to succeed (in sustainable use of me-

_al.,_ 2014; Rai _et al.,_ 2010). Figure 1 de-

dicinal plants) and to complement the ex-

picts the biotechnological approaches use-

isting _ex situ_ measures. The systematic

ful in sustainable supply of medicinal

study on genetic diversity of rare and en-

plants. Biotechnological approaches can

dangered plant species is very important

be used to conserve plants from any

mainly because, it will be helpful in for-

group. Table 1 shows some examples of

mulating plans for management and pre-

successfully conserved plant species us-

serving their genetic diversity as well as

ing biotechnology approaches.

ensuring

their

long

term

survival

**Figure 1:** Biotechnological approaches for the sustainable supply of medicinally important

plants.

****

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_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_

****

**Table 1:** Names of some endangered medicinal plants those are propagated using plant tis-

sue culture techniques

**Name**

**Family**

**Common**

**Explant used**

**Reference**

**name**

_Aerva lanata_ (L.)

Amaranthaceae

Chaya

Node

Shekhawat and

Juss.

Revathi, 2017

ex Schult.

_Bacopa monnieri_ L.

Scrophulariaceae

Bramhi

Leaves, node

Rathore and

&shoot apex

Singh, 2013

_Commiphora wightii_

Burseraceae

Guggul

node, shoot tip,

Tejovathi _et al.,_

(Ar.) Bhandari

& axillary bud

2011

_Gentiana kurroo_ L.

Gentianaceae

Indian

Node

Verma _et al.,_

gentian

2012

_Paris polyphylla_ sm.

Trilliaceae

Bulb

Verma _et al.,_

2012

_Picrorhiza kurroa_

Scrophulairaceae

Kaur

Nodal sector

Jan _et al.,_ 2010

Royle ex. Benth

_Picrorrhiza kurroa_

Scrophulariaceae

Satuva

Leaf/ node

Verma _et al.,_

Royle ex Benth.

2012

_Psoralea corylifolia_

Fabaceae

Indian

Apical meristem

Pandey _et al.,_

Linn

bread root

2013

_Psoralea corylifolia_

Fabaceae

Kutki

cotyledons, hypo-

Sehrawat _et al.,_

Linn

cotyls

2013

_Rheum emodii_

L.

Polygonaceae

Himalayan

Basal disc

Verma _et al.,_

rhubarb

2012

_Salvia sclarea_ L.

Labiateae

Clary sage

Node

Verma _et al.,_

2012

_Saussurea esthonica_

Asteraceae

\--

Seeds

Gailīte _et al.,_

Baer ex Rupr.

2010

_Stevia rebaudiana_

Asteraceae

Candy leaf

Leaf/Node

Verma _et al.,_

(Bertoni) Bertoni

2012

**Table 2:** Some examples of successfully conserved plant species using biotechnology ap-

proaches ****

**Plant species**

**Approach**

**Explant used**

**Reference**

_Calophyllum apetalum_

Micropropagation

Lakshmi and Seeni,

2003

_Cineraria maritima_ L.

Cryopreservation- Shoot tips and

Srivastava _et al.,_ 2009

Encapsulation

nodal seg-

ments

_Chlorophytum borivilianum_

Micropropagation

Floral buds

Sharma and Mohan,

Sant. Et Fernand

2006

_Decalepis arayalpathra_

Micropropagation

Gangaprasad _et al.,_

2005

_Dioscorea floribunda_

Cryopreservation

Shoot tip

Ahuja _et al.,_ 2002

_Gomortega keule_ (Mol.)

Micropropagation

Zygotic em-

Muñoz-Concha and

Baillon

bryos

Dave, 2011

_Psoralea corylifolia_ L.

Micropropagation

Apical meri-

Pandey _et al.,_ 2013

stem

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_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ _2.1. In vitro culture and micropropaga-successfully hardened under glasshouse_

 _

_tion_

conditions.

Plant tissue culture is the _in vitro_

_S_ eed encapsulation techniques are

culture of plant cells, tissues, and or or-

considered as very promising for conser-

gans in sterile nutritionally and environ-

vation purposes as the protection provid-

mentally controlled conditions. In micro-

ed to the plant material by encapsulation

propagation, _in vitro_ multiplication of

could increase its resistance to dehydra-

large number of plants from explants such

tion and low temperature, thus opening

as leaves, seeds, nodes, anthers, ovary and

new possibilities for medium-term storage

tubers etc. is carried out. The plantlets

(Ray and Bhattacharya, 2010). These

thus propagated can be used for produc-

studies clearly suggest that the efficient _in_

tion and multiplication of plantlets which

_vitro_ propagation system developed for

are genetically similar. In addition to the

rare, endangered and or economically im-

conservation of rare and endangered me-

portant medicinal plants are very useful in

dicinal plants, micropropagation is con-

their conservation and supply for a sus-

sidered as the oldest commercial biotech-

tainable growth and development of the

nology based clonal plant propagation

industry.

method (Rai _et al.,_ 2010). Development

of reliable _in vitro_ culture protocols is of

_2.2. Metabolite Engineering and Genetic_

great importance for conservation of rare

_Manipulations_

and endangered medicinal plants. So far

Plant-derived secondary metabo-

number of _in vitro_ protocols has been de-

lites are in great demand and researchers

veloped for rare/endangered medicinal

are actively engaged in secondary metab-

plants to propagate them at large scale

olite production using various _in vitro_

(Shahzad and Saeed, 2013). Table 2

techniques. For instance, transgenic for

shows some examples of successfully

overexpression of gene/s in secondary

propagated rare/endangered medicinally

metabolite pathway have not only provid-

important plants using micropropagation

ed alternative but also offer sustainable

technique. An efficient micropropagation

approaches towards conservation of me-

system for endangered Chinese medicinal

dicinally important plants. Genetic trans-

plant, _Saussurea involucrata_ has been de-

formation or transgenic technology (also

veloped from leaf explants. Similarly, _In_

referred as GM technology) have been

_vitro_ culture of _Saussurea esthonica_ , an

successfully resulted in adjunct to classi-

endangered wild plant species in Latvia

cal plant breeding, in that it allows the

was performed using seeds (Gailīte _et al.,_

targeted manipulation of specific charac-

2010). Tropical Botanic Garden and Re-

ters using genes from a range of sources

search Institute, India has developed _in_

(Shewry _et al.,_ 2008). _Agrobacterium_

_vitro_ protocol for rapid regeneration and

mediated plant transformation approach

establishment of about 40 medicinally

was successful in a number of non-food

important rare and threatened plants of

and food crops mainly due to its simplici-

Western Ghats. Simila attempts of medic-

ty and efficiency; but, it is still not used

inal plants conservation were made by

widely to improve the quality of medici-

Verma _et al._ (2012). Their studies includ-

nal plants (Tashdeva and Kosturkova,

ed _in vitro_ conservation of 23 over-

2012). One of the most appropriate meth-

exploited medicinal plants that belonging

ods for medicinal plants engineering is

to the Indian Sub-Continent (Verma _et al.,_

genetic transformation using _Agrobacte-_

2012). It is important to note that Synthet-

_rium rhizogenes_ leading to increased syn-

ic seeds were also produced from highly

thesis of secondary metabolites in root

proliferating shoot cultures of some

cultures or in regenerated plantlets. For

plants. Out of 23 plants, 18 plants were

instance, studies on hairy root cultures

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 122

_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ using _Agrobacterium rhizogenes_ sug-plant-based medicine (Sheikhpour _et al.,_

gested that the intact plant synthesizes a

2014; Tripathi and Tripathi, 2003). As

large quantity of biologically active sub-

plant-derived drugs have lesser side ef-

stances and therefore, their production

fects in comparison to allopathic medi-

could be increased significantly by trans-

cine, medicinal plant species have made

formed roots cultures as well as by trans-

an outstanding contribution in many tradi-

formed plants (Wang _et al.,_ 2015;

tional herbal therapies practiced in vari-

Tashdeva

and

Kosturkova,

2012;

ous parts of the world. Considering the

Georgiev _et al.,_ 2007; Guillon _et al.,_

importance of medicinal plants, efforts

2006). For example, _Psammosilene tuni-_

should be made at different levels for

_coides_ is a medicinal herb endemic to

their sustainable supply (Kala _et al.,_

China and due to excessive destructive

2006). Due to accelerated local, national

exploration; natural resources of this plant

and international interest, the demand for

species have dwindled and species be-

medicinal and aromatic plants has grown

come threatened (Wang _et al.,_ 2015).

rapidly and therefore, public–private col-

Considering its medicinal importance,

laboration is essential (Van De Kp _et al.,_

successful hairy root culture has been es-

2006).

tablished using genetic transformation of

plant tissues by _Agrobacterium rhi-_

_2.3. Cryopreservation_

_zogenes_ aiming to enhance the secondary

One of the important biotechno-

metabolites production __ (Wang _et al.,_

logical tools in conservation of plant spe-

2015). As the continuous harvesting of

cies is cryopreservation which includes

medicinal plants lead to the exhaustion,

freeze- preservation or cryogenic storage

use of _Agrobacterium rhizogenes_ to trans-

of biological material at a very low tem-

form medicinal plants for secondary me-

perature (Jain _et al.,_ 2012). Cryopreserva-

tabolite production under laboratory con-

tion approach is used to conserve plant

ditions would not only provide the protec-

germplasm when other traditional ap-

tion and conservation of rare or endan-

proaches such as seed banking and vege-

gered medicinal plant species but also of-

tative propagation do not work efficiently

fers a sustainable approach.

for the respective plant species. Hence,

The sustainable plant production

for long-term conservation of _in vitro_ -

is also possible by using microbial inocu-

derived plant germplasm is stored in liq-

lants as substitution for chemical fertiliz-

uid nitrogen (-196°C) (Engelmann, 2011).

ers and pesticides (O'Gara, 1996). Inocu-

It has been reported that, as threatened

lation of mycorrhizal fungi into the roots

and endangered species produce little or

of plants is referred as mycorrhization

no viable seeds or are dormant; hence, the

(Williams _et al.,_ 1994). This can be

preservation of remaining individuals is

achieved by delivering microbial inocu-

considered of paramount importance

lants via micropropagation (Dolcet-

(Bunn _et al.,_ 2007) and such problematic

Sanjuan _et al.,_ 1996). Several reports

species needs to be maintained through

suggest that inoculation of arbuscular

cryo-collections (Kaczmarczyk _et al.,_

mycorrhizal fungi (AMF) into the roots of

2012). Cryopreservation ensures safe and

micropropagated plantlets plays an advan-

cost-efficient long-term conservation of

tageous role (Sylvia _et al.,_ 2003; Voets _et_

species without the loss of viability, so

_al.,_ 2005; Chandra _et al.,_ 2010).

when required, material can be readily

Tools such as _in-vitro_ propaga-

retrieved and reinitiated, reestablishing

tion, mycorrhization and genetic engi-

desirable clonal lines (Shibli _et al.,_ 2004).

neering not only hold tremendous poten-

Plant materials such as cells, tissues,

tial to select, multiply and conserve the

gametes, oocytes, organs, DNA samples

critical genotypes of medicinal plants but

etc. are stored so that they can be used in

also offer the production of high-quality

future (Sharma and Sharma, 2013). Cryo-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 123

_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ preservation is considered as one of the

traditional applications and for the devel-

most important conservation techniques

opment of novel drugs and or supplemen-

and has been a successfully approach for

tary products. However, plant-derived

the conservation of number of medicinal

drugs as medicines have been based on

plants. Conservation of _Atropa belladon-_

the assumption that the plants will be

_na, Digitalis lanata, Hyoscyamus sp_. and

available on a continuing basis. As much

_Rauvolfia serpentine_ are some examples

more attention is given for discovery of

to state (Jain _et al.,_ 2012). Initiatives for

new drugs from medicinal plants, the de-

conservation of medicinally important

mand for raw material is steadly growing.

endangered plants have already been tak-

However, there are no enough meticulous

en by several institutions. For instance,

efforts to ensure the availability of target-

more than 110 accessions of rare or

ed medicinal plants for harvesting to fulfil

threatened plant species are stored using

the industry's demand. Moreover, there

cryopreservation approach of conserva-

are no sustainable approaches developed

tion at the Kings Park and Botanic Gar-

for harvesting as well as for conservation

den in Perth, Australia (Touchell and

of in-demand medicinal plants. Due to

Dixon, 1994). Likewise, The National

this awful situation, many medicinal plant

Bureau for Plant Genetic Resources

species are becoming rare, vulnerable,

(NBPGR, New Delhi, India) has stored

endangered and or extinct. Unfortunately,

more than 1,200 accessions from 50 dif-

very meager efforts have been undertaken

ferent plant species (Mandal, 2000).

for their conservation and sustainable

Shoot tips of endangered and medicinally

supply. Therefore, there is a need to in-

important plant, _Picrorhiza kurroa_ have

tensify the efforts for not only for the

been preserved using same approach

conservation but also to ensure sustaina-

(Sharma and Sharma, __ 2003). Cryo-

ble supply of medicinal plants. Biotech-

preserved embryogenic cultures of _Di-_

nological tools such as micropropagation,

_oscorea bulbifera_ was performed using an

cryopreservation and transgenic approach

encapsulation-dehydration procedure. Af-

should be used efficiently for the conser-

ter cryopreservation, the sub-culturing

vation of medicinal plants for the sustain-

showed 53.3% recovery of growth of em-

able growth and development of the in-

bryogenic culture (Mandal _et al.,_ 2009).

dustry, people and the planet.

These studies clearly provide the insights

about the usefulness of cryo-techniques in

**References**

conservation of valuable medicinal plants

****

(Tashdeva and Kosturkova, 2012). How-

**Agrawal, A. (2005).** Current issues in

ever, research on cryopreservation tech-

quality control of natural products.

niques is relatively limited; hence, further

_Pharma Times_ **37, 9-11.**

research and development is essential in

**Ahuja, S. Mandal, B.B. Dixit, S. and**

this area to utilize this technique/approach

**Srivastava, P. S. (2002).** Molecular

efficiently for the conservation of medici-

phenotypic and biosynthetic stabil-

nal plants.

ity of plants recovered from cryo-

preserved shoot-tips of _Dioscorea_

**3. Perspectives**

_floribunda_. _Plant Science_ **3, 971-**

****

**977.**

Understanding of the challenges

**Arai, K. (2006).** Toward biotechnology:

and opportunities in conservation of me-

The mission of IUBMB in the 21st

dicinal plants and to develop the biotech-

century. _IUBMB Life_ **58, 267-268.**

nological approaches to deal with it is

**Atanasov, A. G. Waltenberger, B.**

important for the sustainable supply of

**Pferschy-Wenzig, E. Linder, T.**

medicinal plants. Medicinally important

**Wawrosch, C. Uhrin, P. Temml,**

plants are continuously harvested for their

**V. Wang, L. Schwaiger, S. Heiss,**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 124

_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ **E. Rollinger, J. M. Schuster, D.**

demic and endangered ethano me-

**Breuss, J. M. Bochkov, V. Mi-**

dicinal plant of Western Ghats. _In-_

**hovilovic, M. D. Kopp, B. Bauer,**

_dian Journal of Biotechnology_ **4,**

**R. Dirsch, V. M. and Stuppner,**

**265-270.**

**H. (2015).** Discovery and resupply

**Gavrilescu, M. (2004).** Removal of

of pharmacologically active plant-

heavy metals from the environment

derived natural products: A review.

by bio-sorption. _Engineering in_

_Biotechnology Advances_ **33, 1582-**

_Life Sciences_ **4, 219-232.**

**1614.**

**Ghimire, S. K. McKey, D. and Aumee-**

**Bunn, E. Turner S. Panaia M. and Dix-**

**ruddy-Thomas, Y. (2005).** Hetero-

**on K. W. (2007).** The contribution

geneity

in

ethno

ecological

of invitro technology and cryogenic

knowledge and management of me-

storage to conservation ofindige-

dicinal plants in the Himalayas of

nous plants. _Australian Journal of_

Nepal: Implication for conservation.

_Botany_ **55, 345–355.**

_Ecology and Society_ **9, 6.**

**Bukuluki, P. Luwangula, R. and Wala-**

**Georgiev, M.I. Pavlov, A.I. and Bley, T.**

**kira E. J. (2014).** Harvesting of

**(2007).** Hairy root type plant _in_

Medicinal Plants in Uganda: Prac-

_vitro_ systems as sources of bioac-

tices, Conservation and Implica-

tive substances. _Applied Microbiol-_

tions for Sustainability of Supplies.

_ogy and Biotechnology_ **74, 1175-**

_Online International Journal of_

**1185.**

_Medicinal Plant Research_ **3, 1-10.**

**Guillon, S. Tremouillaux-Guiller, J.**

**Cantor, C. R. (2000).** Biotechnology in

**Pati, P.K. Rideau, M. and Gantet,**

the 21st century. _Trends in Biotech-_

**P. (2006).** Harnessing the potential

_nology_ **18, 6-7.**

of hairy roots: dawn of a new era.

**Chandra, S. Bandopadhyay, R. Kumar,**

_Trends in Biotechnology_ **24, 403-**

**V.and Chandra, R. O. (2010).** Ac-

**409.**

climatization of tissue cultured

**Godfray, H. C. J. Beddington, J. R.**

plantlets: from laboratory to land.

**Crute, I. R. Haddad, L. Law-**

_Biotechnology Letters_ **32, 1199-**

**rence, D. Muir, J. F. Pretty, J.**

**1205.**

**Robinson, S. Thomas. S. M. and**

**Dolcet-Sanjuan,**

**R.**

**Claveria,**

**E.**

**Toulmin, C. (2010).** The Challenge

**Camprubi, A. Estaun, V. and**

of Feeding 9 Billion People. _Sci-_

**Calvet, C. (1996).** Micropropaga-

_ence_ **327, 812-818.**

tion of walnut trees ( _Juglans regia_

**Hishe, M. Asfaw, Z. and Giday, M.**

L.) and response to arbuscular my-

**(2016).** Review on value chain

corrhizal inoculation. _Agronomie_

analysis of medicinal plants and the

**16, 639-645.**

associated challenges. _Journal of_

**Engelmann, F. (2011).** Use of biotech-

_Medicinal Plants Studies_ **4, 45-55.**

nologies for the conservation of

**Hunde, D. (2007).** Human influence and

plant Biodiversity. _In Vitro Cellular_

threat to biodiversity and sustaina-

_& Developmental Biology _**47, 5-16.**

ble living. _Ethiopian Journal of Ed-_

**Gailīte, A. Kļaviņa, D. and Ievinsh, G.**

_ucation and Science_ **1, 85-94.**

**(2010).** _In vitro_ propagation of an

**Ibrahima, M. A. Nad, M. Oha J. Schi-**

endangered plant _Saussurea es-_

**nazie, R. F. McBrayerg, T. R.**

_thonica. Environmental and Exper-_

**Whitakerg , T. Doerksenb, R. J.**

_imental Biology_ **8, 43-48.**

**Newmani, D. J. Zachosj, L. J. and**

**Gangaprasad, A. Decruse, S. W. Seeni,**

**Hamanna, M. T. (2013).** Signifi-

**S. and Nair, G. M. (2005).** Micro-

cance of endangered and threatened

propagation and restoration of __ (Jo-

plant natural products in the control

seph and Chandra) Venter- an en-

of human disease. _Proceeding of_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 125

_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ _National Academy of Science USA_

**Lakshmi, G. N. and Seeni, S. (2003).** _In_

**110** , **16832–16837.**

_vitro_ multiplication of _Calophyllum_

**Jan, A. Thomas, J. Shawl, A. S. Jabeen,**

_apetalum_ (Clusiaceae), an endemic

**N. and Kozgar, M. I. (2010).** Im-

medicinal tree of the Western

proved Micropropagation Protocol

Ghats. _Plant Cell Tissue and Organ_

of an Endangered Medicinal Plant-

_Cult_ ure **75, 169-174.**

_Picrorhiza Kurroa_ Royleex Benth.

**Lahlou, M. (2013).** The success of natu-

Promptly Through Auxin Treat-

ral products in drug discovery.

ments. _Chiang Mai Journal of Sci-_

_Pharmacology & Pharmacy_ **4, 17-**

_ence_ **37, 304-313.**

**31.**

**Jain, M. Johnson, T. S. and Krishnan,**

**Manohar,** **P. R. (2012).** Sustainable harP. (2012). Biotechnological ap-

vesting of medicinal plants: Some

proaches to conserve the wealth of

thoughts in search for solutions.

nature: endangered and rare medic-

_Ancient Science of Life_ **32, 1-2.**

inal plant species, a review. _Journal_

**Mandal, B. B. (2000).** Cryopreservation

_of Natural Remedies_ **12, 93-102.**

research in India: current status and

**Kaczmarczyk, A. Funnekotter, B.**

future perspectives. _In_ : Cryopreser-

**Menon, A. Phang, P. Y. Al-**

vation of tropical plant germplasm-

**Hanbali,**

**A.**

**Bunn,**

**E.**

**and**

current research progress and appli-

**Mancera R. L. (2012).** Current is-

cations. Engelmann F.; Takagi H.

sues in plant cryopreservation. _In_ :

(eds). JIRCAS, Tsukuba, **pp 282-**

Current Frontiers in Cryobiology.

**286.**

Katkov I. (ed.). In Tech, Croatia,

**Mandal, B. B.** **Sharma, D. S.** **Srivasta-**

**pp. 417-438.**

**va, P. S.** **(2009).** Cryopreservation **Kala, C. P. (2005).** Ethnomedicinal bota-of embryogenic cultures of _Di-_

ny of the Apatani in the Eastern

_oscorea bulbifera_ L. by encapsula-

Himalayan region of India. _Journal_

tion- dehydration _. Cryo Letters_ **30,**

_of Ethnobiology and Ethnomedicine_

**440-448.**

**1, 111-118.**

**Muñoz-Concha, D. and Davey, M. R.**

**Kala, C. P. Dhyani, P. P. and Sajwan,**

**(2011).** Micropropagation of the

**B. S. (2006).** Developing the medic-

endangered Chilean tree, Gomorte-

inal plants sector in northern India:

ga keule. _In Vitro Cellular & De-_

challenges and opportunities. _Jour-_

_velopmental Biology_ **47, 170-175.**

_nal of Ethnobiology and Ethnomed-_

**Nalawade, S. M. Sagare, A. P. Lee, C.**

_icine_ **2, 1-15.**

**Y. Kao, C. L. and Tsay, H. S.**

**Khan, S. Al-Qurainy, F. and Nadeem,**

**(2003).** Studies on tissue culture of

**M. (2012).** Biotechnological ap-

Chinese medicinal plant resources

proaches for conservation and im-

in Taiwan and their sustainable uti-

provement of rare and endangered

lization. _Botanical Bulletin of Aca-_

plants of Saudi Arabia. _Saudi Jour-_

_demia Sinica_ **44, 79-98.**

_nal of Biological Sciences_ **19, 1-11.**

**O'Gara, F. (1996).** The biotechnology

**Krishnan, P. N. Decruse, S. W. Radha,**

and ecology of rhizosphere micro-

**R. K. (2011).** Conservation of me-

organisms. _In_ : Novel biotechnolog-

dicinal plants of Western Ghats, In-

ical approaches to plant production:

dia and its sustainable utilization

from sterile root to mycorrhizo-

through _in vitro_ technology. _In_

sphere. Proceeding of the Joined

_Vitro Cellular and Developmental_

Meeting between 8.21 and 8.22, Pi-

_Biology_ **47, 110-122.**

sa, Italy, 14-15 July, 1996.

**IUCN (2013).** www.iucn.organization

**Pandey, P. Mehta, R. and Upadhyay,**

access on May 25, 2014. ****

**R. (2013).** _In vitro_ propagation of

an endangered medicinal plant _Pso-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 126

_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ _ralea corylifolia_ L. _Asian Journal of_

_In_ : Recent Trends in Biotechnology

_Pharmaceutical and Clinical Re-_

and Therapeutic Applications of

_search,_ **6, 115-118.**

Medicinal Plants Mohd. S. A.

**Pathak, M. R. and Abido, M. S. (2014).** __

Abida, M. and Aastha, S. (eds). **pp**

The role of biotechnology in con-

**263-292.**

servation of biodiversity. _Journal of_

**Sharma, N. B. and Sharma B (2003).**

_Experimental Biology and Agricul-_

Cryopreservation of shoot tips of

_tural Sciences_ **2, 352-363.**

_Picrorhiza kurroa_ Royle ex Benth,

**Prescott-Allen, R. and Prescott-Allen,**

an indigenous endangered medici-

**C. (1996).** Assessing the sustaina-

nal plant through vitrification. _Cryo_

bility of uses of wild species. Case

_Letters_ **24, 181-90.**

studies and initial assessment pro-

**Sharma, U. and Mohan, J. S. S. (2006).**

cedure. – Gland & Cambridge,

_In vitro_ clonal propagation of _Chlo-_

IUCN (Occasional Paper of the

_rophytum borivilianum_ Sant et Fer-

IUCN Species Survival Commis-

nand., a rare medicinal herb from

sion 12). ****

immature floral buds along with in-

**Rai, M. K. (2010).** Review: Biotechno-

florescence axis. _Indian Journal of_

logical strategies for conservation

_Experimental Biology_ **44, 77-82.**

of rare and endangered medicinal

**Sheikhpour, S. Yadollahi, P. Fakheri,**

plants. _Biodiversitas_ **11, 157-166.**

**B. A. and Amiri, A. (2014).** Bio-

**Rao, M. R. Palada, M. C. and Becker,**

technological strategies for conser-

**B. N. (2004).** Medicinal and aro-

vation of rare medicinal plants. In-

matic plants in agro-forestry sys-

ternational Journal of Agriculture

tems. _Agroforestry Systems_ **61, 107-**

and Crop Sciences. _International_

**122.**

_Journal of Agriculture and Crop_

**Rathore, S. and Singh, N. (2013).** _In_

_Sciences_ **7, 79-82.**

_vitro_ conservation of bacopa mon-

**Shekhawat, M. S. Manokari, M. and**

neri – An endangered medicinal

**Revathi, J. (2017).** _In vitro_ propa-

plant. _Global Journal of Bio Sci-_

gation and ex vitro rooting of _Aerva_

_ences and Biotechnology_ **2, 187-**

_lanata_ (L.) Juss. ex Schult.: a rare

**192.**

medicinal plant. _Indian Journal of_

**Ray, A. Bhattacharya, S. (2010).** Stor-

_Plant Physiology_ **22, 40-47.**

age and conversion of _Eclipta alba_

**Shewry, P.R. Jones, H.D. and Halford,**

synseeds and RAPD analysis of

**N.G. (2008).** Plant biotechnology:

converted

plantlets.

_Biologia_

transgenic crops. _Advances in Bio-_

_Plantarum_ **54, 547-550.**

_chemcial Engineering/ Biotechnol-_

**Report of the European Commission,**

_ogy_ **111, 149-86.**

**(2008)**.

Available

online

at

**Sylvia, D. M. Alagely, A. K. Kane, M.**

http://ec.europa.eu/; accessed on

**E. and Philman, N. L. (2003)**.

April 18, 2017. ****

Compatible host/mycorrhizal fun-

**Sehrawat, N. Yadav, M. and Jaiwal, P.**

gus combinations for micropropa-

**K. (2013).** Development of an effi-

gated sea oats. I. Field sampling and

cient _in vitro_ regeneration protocol

greenhouse evaluations. _Mycorrhiza_

for rapid multiplication and genetic

**13, 177-183.**

improvement of an important en-

**Shibli, R. A. Al-Ababneh, S. S. and**

dangered medicinal plant _Psoralea_

**Smith M. A. L. (2004).** Cryopres-

_corylifolia. Asian Journal of Plant_

ervation of plant germplasm: a re-

_Science and Research_ **3, 88-94.**

view (scientific review). _Dirasat_

**Shahzad. A. and Saeed, T. (2013).** _In_

_Agricultural Sciences_ **31, 60-73.**

_Vitro_ Conservation Protocols for

**Sreekumar, V. B. and Renuka, C.**

Some Rare Medicinal Plant Species.

**(2006).** Assessment of genetic di-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 127

_Biotech Sustainability (2017)_

_Biotechnological Approaches for Plants Conservation Datir and Bhore_ versity in _Calamus_ _thwaitesii_ BECC

and Nijhoff, H. (eds.). Springer,

(Arecaceae) using RAPD markers.

Netherlands **pp 191-202.**

_Biochemical Systematics and Ecol-_

**Verma, P. Mathur, A. K. Jain, S. P.**

_ogy_ **34, 397-405.**

**and Mathur, A. (2012).** _In Vitro_

**Srivastava, V. Khan, S. A. and**

Conservation

of

Twenty-Three

**Banerjee, S. (2009).** An evaluation

Overexploited Medicinal Plants Be-

of genetic fidelity of encapsulated

longing to the Indian Sub Conti-

microshoots of the medicinal plant:

nent. _The Scientific World Journal_

_Cineraria_ _maritima_ following six

**2012, 1-10.**

months of storage. _Plant Cell Tissue_

**Verma, S. and Singh, S.P. (2008).** Cur-

_and Organ Culture_ **99, 193-198.**

rent and future status of herbal med-

**Tasheva, K. and Kosturkova, G.**

icines. _Veterinary World_ **11, 347-**

**(2012).** Towards Agrobacterium-

**350.**

mediated transformation of the en-

**Voets, L. Dupré de Boulois, H. Renard,**

dangered medicinal plant golden

**L. Strullu, D. G. and Declerck, S.**

root. _AgroLife Scientific Journal_ **1,**

**(2005).** Development of an auto-

**132-138.**

trophic culture system for the in

**Tejovathi,**

**G.**

**Goswami,**

**H.**

**and**

vitro mycorrhization of potato

**Bhadauria, R. (2011).** _In vitro_

plantlets. _FEMS Microbiology Let-_

propagation of endangered medici-

_ters_ **248, 111-118.**

nal plant– _Commiphora_ _wightii_. _In-_

**Wang, H. Gao, S. da Silva, J. A. T. and**

_dian Journal of Science and Tech-_

**Guohua, M. (2015).** _Agrobacte-_

_nology_ **4, 1537-1540.**

_rium rhizogenes_ -mediated genetic

**Touchell, D. H. and Dixon, K. W.**

transformation of _Psammosilene tu-_

**(1994).** Cryopreservation for seed

_nicoides_ and identification of high

banking of Australian species. _An-_

saponin-yielding clones. _Environ-_

_nals of Botany_ **40, 541-546.**

_mental and Experimental Biology_

**Tripathi, L F. and Tripathi, J. N.**

**13: 19-23**.

**(2003).** Role of biotechnology in

**Williams, P. G. Roser, D. J. and Sep-**

medicinal plants. _Tropical Journal_

**pelt, R. D. (1994).** Mycorrhizas of

_of Pharmaceutical Research_ **2, 243-**

hepatics in continental Antarctica.

**253.**

_Mycological Research_ **98, 34-36**. ****

**Van De Kop, P. Alam, G. and De**

**WHO,**

**(2010).**

**URL,**

**Steenhuijsen, B. P. (2006).** Devel-

http://www.who.int/mediacentre/fac

oping a sustainable medicinal-plant

tsheets/fs-134/en/.

Accessed

on

chain in India - Linking people,

April 3, 2017. ****

markets and values. _In:_ Agro-food

chains and networks for develop-

ment. Ruben, R. Slingerland, M.

© 2017 by the authors. Licensee Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 128

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P129-140_

**Making Himalayas Sustainable: Opportunities and**

**Challenges in Indian Himalayan Region**

**Harsh Kumar Chauhan and Anil Kumar Bisht***

_Department of Botany, D.S.B. Campus, Kumaun University, Nainital, 263001, Uttarak-_

_hand, India; *Correspondence: bishtakb@rediffmail.com; _ _Tel: +91 9412044500_

****

****

**Abstract:** Himalayas are among the most vulnerable ecosystems of the world. They harbor

unique geology/geography, ecosystem, biodiversity, and several other life sustaining biotic

and abiotic resources. Sustainability of Himalayas is being challenged by increased tourism

activities, deforestation, pollution, unmanaged exploitation of bio-resources, climate change

and unplanned developmental activities. Besides, the region has several resources of aes-

thetic and economic interest that can be harnessed for generation of income and employ-

ment to millions of the people residing in the region. Understanding the specific problems

of Himalaya and carving out the prospects for its sustainable development is a difficult task.

Looking at the present scenario, the chapter provides an overview of the opportunities and

challenges for achieving sustainability in Indian Himalayan Region. The present trends

suggest that the existing interventions in the region are unsustainable. Further, the unscien-

tific exploitation of natural resources is increasing the environmental degradation in the re-

gion. Proper policies and their implementation for harnessing the potential of the natural

resources are urgently needed for the sustainable development of the region.

****

_**Keywords**_ **:** Himalayas; **** hotspot; sustainability; unscientific exploitation; vulnerable

****

****

**1. Introduction**

cal and economic security of the people

****

living downstream. They are also consid-

The Himalayas are among the

ered as the repository of geological and

most vulnerable mountain ecosystem

agriculture assets and harvested wild

stretching between the Indus and Brah-

goods (Badola _et al_., 2015).

maputra river valleys (Bawa _et al_., 2010).

Unfortunately, the entire region is

They spread across the eight countries;

prone to several disasters due to fragile

Afghanistan, Bangladesh, Bhutan, China,

geophysical structures, high peaks, and

India, Myanmar, Nepal and Pakistan. The

high angle of slope and variable climatic

Himalayas harbor unique biodiversity,

conditions (Chhetri, 2001). This vulnera-

ecosystem composition and several other

bility increases several folds with the in-

life sustaining resources. They are the

creasing human population, exploitation

source of 10 of the largest rivers in Asia

of the natural resources and the effects of

which provides water to about 1.3 billion

the climate change (Liu and Chen, 2000;

people (Xu _et al_., 2007; Bates _et al_.,

Dyurgerov and Meier, 2005). Poverty in

2008). In addition to provide water, the

the Himalayan region is high and persis-

Himalayas provide huge inputs to agricul-

tent (Hunzai _et al_., 2011). Some of the

ture through regulating micro-climates as

areas in the region are under territory dis-

well as wind and monsoon circulation

putes between the nations which are asso-

(Rasul, 2010) supporting life of about 40

ciated with the military presence along

million people (Zurik and Pacheco, 2006).

the international border; for instant the

They are known to facilitate vital ecologi-

degradation of the Hind Kush, Karako-

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_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ rum, Western Himalaya and the Kashmir.

degradation (Singh, 2002; Blaikie and

This situation is particularly damaging to

Muldavin, 2004). Several scientific and

fragile ecosystem whose recovery is par-

political forums have emphasized the

ticularly slow (Bawa _et al_., 2010). The

uniqueness, environmental challenges and

political situation thus prevail prevent the

political legacies of the Himalayan region

proper policy implementation for devel-

so that sustainable planning and manage-

opmental programs causing the unfair

ment in the region can be worked out.

treatment of the people residing in these

Global change and World's Mountains

areas. These synergistic effects seem to

conference held at Perth, Scotland in 2010

make Himalayas the hotspot for the phys-

also identified several research gaps in

ical, economic and social vulnerability.

sustainable mountain development. Look-

The studies carried out on the

ing at the current scenario, the present

mountain ecosystems throughout the

chapter provides the overview of the op-

world concluded that mountains are in

portunities and challenges of sustainabil-

dire need of relief from anthropogenic

ity in the Himalayas with special refer-

activities (Jodha, 2005). Sustainability of

ence to Indian Himalayan Region.

Himalayas is being challenged by in-

creased tourism activities, deforestation,

**2. Indian Himalayan region (IHR)**

pollution, climate change and unplanned

****

development. Besides all this, the region

IHR occupies a special place in

has several resources of symbolic and

the mountain ecosystem of the world

economic values whose harnessing can

(Singh, 2006). The region extends be-

provide income and employment to mil-

tween latitude 26o20' and 35o40' North,

lions of the people residing in the region.

and between longitudes 74o50' and 95o40'

Understanding the specific Himalayan

East covering 530, 795 sq. km of geo-

problems and prospects of the sustainable

graphic area. It spreads across the states

development is not an easy task. Interna-

of Jammu and Kashmir, Himachal Pra-

tional failure to recognize the economic

desh, Uttarakhand, Sikkim, Arunachal

value of the issues of sustainability using

Pradesh, Meghalaya, Nagaland, Manipur,

policy tools in the Himalayas has been

Mizoram, Tripura and the hill regions of

cited as the major cause of this continued

Assam and West Bengal (Figure 1). It co-

**Figure 1:** A picture of the Himalayan landscape.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 130

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ nstitutes about 16.2% of India's adminis-veloped and 9.09% is under construction

tered geographical area. Most of the area

(CEA, 2009). Accordingly the Prime

in the region is covered with the snow

Minister of India launched a 50,000 MW

clad mountain peaks, glaciers in the high-

hydroelectric initiative program, formu-

er Himalayas and dense forest in the mid-

lated by CEA for preparation of Prelimi-

Himalayas thus harbouring a rich variety

nary Feasibility reports of 162 new hy-

of flora, fauna and cultural diversity.

dropower schemes (47,930MW) and out

of these 133 are in IHR (Agarwal _et al_.,

**3. Opportunities and challenges for**

2010).

**sustainability in the IHR __**

The development of hydropower

_****_

offers several advantages to the economy

_3.1. Water potential_

of the nation. It is the source of clean re-

Himalayas

has

been

rightly

newable source of energy offering the

acknowledged as the 'Water Tower of

mitigation of climate change issues and

Asia'. Approximately 10-20 % of the area

achieving the sustainability goals. It pro-

is covered by glaciers while 30-40% re-

vides the inexpensive power, especially

mains under seasonal snow cover (Baha-

when the project achieves financial

dur, 2004). Being the source of Asia's 10

breakeven. The development of the hy-

largest rivers (Amu Darya, Indus, Ganges,

dropower project is expected to improve

Brahmaputra, Irrawaddy, Salween, Me-

the infrastructure of the remote areas as

kong, Yangtze, Yellow and Tarim) (Xu _et_

well as it helps in flood moderation, irri-

_al_., 2007) they provide drinking water,

gation, navigation and providing drinking

irrigation, fisheries, hydropower, and

water all the year around.

supports several terrestrial and aquatic

Unfortunately several challenges

ecosystems. The Ganges River system is

are associated with the development of

the main source of fresh water to more

the hydropower in IHR. It has been real-

than half the population of India and

ized that the development of the hydro-

Bangladesh and nearly entire population

power projects has significant environ-

of Nepal (Rasul, 2014). About 60% of the

mental and social impact (Goldsmith and

India's irrigated area of 546,820 Km2 is in

Hildyard, 1984). These projects alter the

the Ganges basin (National Ganga River

vital ecological process such as flow of

Basin Authority, 2011). Indus irrigation

water, sediments, nutrients, energy and

system irrigates about 14.3 million hec-

biota (Franklin _et al_., 1995). The IHR is

tares of farmland constituting the world's

among the most seismically active zone

largest contiguous irrigation system; ena-

of the world; the construction of the hy-

bling the production of more than 80%

dropower project increases the chances of

food grains of Pakistan (GoP, 2010). Amu

the earthquakes which may shatter the

Darya irrigates 385,000 ha of farmland in

lives of millions of people. Proper Envi-

Afghanistan contributing a reliable source

ronment Impact Assessment appears to be

of Afghanistan's food and water security

the biggest challenge for the implementa-

(NAS, 2012). The ground water flow

tion of the hydropower project in IHR. In

through bedrocks is approximately six

a nutshell it may cause multi-dimensional

times the annual contribution from glacial

unpredictable ecological disturbances and

ice melt and snow melt to central Himala-

loss of biodiversity, productive land, so-

yan Rivers (Andermann _et al_., 2012).

cial and cultural heritage. Hence, the de-

The government of India had rec-

velopment interventions related to hydro-

ognized the hydropower potential of the

power in the IHR should have different

IHR. The country's hydropower potential

approach. As per the reports of Chopra

is 148,701 MW out of which more than

committee, formulated by the order of

75% (117,139 MW) resides in IHR; how-

Supreme Court of India in the year 2014

ever only 22.37 % potential has been de-

to assess the role of hydroelectric projects

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_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ in Uttarakhand, the negative impacts of

and biodiversity in the Himalayas

the small hydropower projects can be less

(Shrestha _et al_., 2012). The complex

intense and therefore mitigated more easi-

orogeny of the Himalayas, coupled with

ly. On the other hand, large projects often

climatic and edaphic changes facilitate the

lead to massive impacts that are hard to

colonization of floral and faunal diversity

mitigate and may result in permanent

in the region (Pandit _et al_., 2000). Hima-

scarring of nature and society. Further,

layas are the repository of the extremely

the apex court also warned of negative

rich and endemic biodiversity (Chatterjee,

impacts on geological environment, river

1939; Nayar, 1996; Pandit _et al_., 2000).

ecosystem & forests and terrestrial biodi-

The region hosts the parts of four global

versity (Table 1).

biodiversity hotspots (viz. the Himalayas

hotspot, the Indo-Burma hotspot, South-

_3.2. Biodiversity_

West China Hotspot and the Mountains of

Climatic, topographic, geological

Central Asia hotspot) (Mittermeier _et al_.,

and altitudinal variations have generated

2004). Himalayas are very important in

unique landscape (Figure 2), ecosystems

terms of sustaining high levels of the

****

**Table 1:** Negative impact of hydropower projects* ****

**Activity**

**Impact**

**I. Pre-project construction**

1. Construction of ap-

 Land acquisition (displacement, loss of lands,

proach roads

homes and livelihoods)

 Deforestation (loss of tree cover, access to CPRs,

soil erosion and landslides, loss of flora and fauna,

changes in micro-climate)

 Disposal of debris and earth (loss of trees, river wa-

ter pollution)

2. Construction of housing

 Deforestation

for staff and labour

 Pollution due to sewage release

3.Quarrying

 Noise pollution, slop destabilization, disruption of

underground seepage and damage to house.

**II. Project construction**

4.Tunneling

Air and soil pollution, destabilization of slopes, damage to

houses, disturbing wildlife, drying of springs, disposal of

muck into the river, psychological trauma to people and

animals due to repeated blasts

5.Dam construction

Disruption of river flows (biotic changes, disruption of nat-

ural functions, e.g. sediment disposal, land shaping, nutri-

ent cycling), river pollution, loss of aesthetic, cultural, eco-

nomic and recreational values.

**III. Project operation**

6. Testing of tunnels

Slope destabilization (loss of tree cover, land, livelihoods,

water sources and access to CPRs)

7.Water storage and re-

 Sedimentation (effect on river water quality)

lease

 Disruption of river flow

 Secondary effects (release of greenhouse gases,

warming of valleys, increased earthquake risks,

floods, downstream urban and industrial develop-

ment

8.Laying of power lines

Deforestation (loss of wild life habitat), soil erosion

*Information source: http://iced.cag.gov.in

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 132

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ **Figure**

**2:**

Indian

Himalayan

Region

(IHR)

(Source:

www.http://gbpihedenvis.nic.in/indian_him_reg.htm)

biodiversity. However, biodiversity loss

for the biodiversity loss in IHR. At the

from the region had become a matter of

current rate of deforestation in IHR, the

great concern. There are several factors

total forest cover (84.9 % in 2000) and

such as anthropogenic pressure and the

coverage of dense forest (75.4% in 2000)

climate change which contributes to the

is expected to be reduced to 52.8% and

loss of biodiversity in the region.

34% respectively by the year 2100 (Pan-

Due to the collation of several ge-

dit _et al._ 2007). This could have serious

ographic and climatic features IHR pro-

implications on the diversity of the flora

vides very suitable environment for flour-

and fauna of the region. Looking at the

ishing the huge biodiversity. IHR sup-

ever-increasing threats to the biological

ports nearly 50% of the total flowering

diversity in the region; there is an urgent

plants in India of which 30% are endemic

need of the proper actions or otherwise it

to the region (Singh _et al_., 2006). It har-

may bring huge setback to the economic

bours 816 tree species, 675 edibles and

benefits of the local populations.

1748 species of medicinal value (Samant

_et al_., 1998). The forests of the region

_3.3. Medicinal plants_

have phenomenal diversity that meets the

Among the biodiversity elements,

diverse needs of the people (Singh and

the roles of medicinal plants is remarka-

Singh, 1992). The forest of the region acts

ble in the health care of the Himalayan

as the sink of the carbon dioxide and pro-

people as most of the them resides in the

vides timber, wild edibles, gums, resins

remote locations where the allopathic sys-

and other numerous products of immense

tem of medicines is not practiced to such

value.

an extent. Besides the health care, medic-

****

Massive deforestation, extensive

inal plants have high socio-cultural, sym-

shifting cultivation and the demands for

bolic and economic value, providing in-

agricultural land are the primary drivers

come and employment to millions of peo-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 133

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ ple living in the region (Ghimire, 2008).

(2001) estimated 3200 medicinal plants

Himalayan medicinal plants meet most of

from Indian Himalayas including 700

the

international

demand.

Over-

from trans-Himalaya (Table 2). India is

exploitation seems the biggest challenge

ranked 2nd after China in terms of medici-

for the survival of these plants though

nal plants export and exports about

they also have slow growth rates, low

32,600 tons of medicinal raw material

population densities and narrow geo-

worth about US $46 million annually

graphical ranges (Kala _et al_., 2006).

(Lange, 1997). High nativity and ende-

IHR host 1748 plants species of

mism of medicinal plants is associated

medicinal value including 1685 angio-

with the IHR. Out of 1748 medicinal

sperms, 12 gymnosperms and 51 pterido-

plants, about 548 species are identified in

phytes (Samant _et al_., 1998). Ved _et al_.

HP, 707 in Sikkim and Darjeeling and

****

**Table 2:** Medicinal plants, species diversity and representative species of different biogeo-

graphic zones of India# ****

Biogeographic

re- Estimated no. of Examples of some typical medicinal species

gion

medicinal plants

Trans-Himalayas

700

_Ephedra_

_geradiana_ Wall., _Hippophae_

_rham-_

_noides_ L., _Arnebia euchroma_ (Royle) John

Himalayan

2500

_Aconitum heterophyllum_ Wall. ex Royle,. _Ferula_

_jaeshkeana_ Vatke and _Saussurea costus_ (Balc).

Lipsch., _Nardostachys_

_grandiflora_ D.C. _Taxus_

_wallichiana_ Zucc,. _Rhododendron_

_anthopo-_

_gon_ D.Dun and _Ponax pseudoginseng_ Wall.

Desert

500

_Convolvulus microphyllus_ Seib ex Spreng.,

_Tecomella undulata_ (Sm.) Seem., _Citrulus colo-_

_cynthis_ (L.), __ Schraderand _Cressa crertica_ L.

Semi-Arid

1000

_Commiphora_

_wightii_ (Arn.)

Bhandari, _Caesalpinia_

_bonduc_ (L.)

Roxb, _Balanites_

_aegyptiaca_ (L.),

Delilie

and _Tribulus rajasthanensis_ Bhandari & Sharma.

Western Ghats

2000

_Myristica_

_malabarica_ Lam., _Garcinia_

_indi-_

_ca_ (Thou.)

Choisy, _Utleria_

_salicifolia_ Bedd

and _Vateria indica_ L.

Deccan Peninsula

3000

_Pterocarpus santalinus_ L.f., _Decalepis hamilto-_

_nii_ Wigh & Arn, _Terminalia pallida_ Brandis

and _Shorea tumbuggaia_ Roxb

Gangetic Plain

1000

_Holarrhenaq pubescens_ (Buch-Ham.) Wall. ex

DC., _Mallotus philippensis_ (Lam.) Muell –

Arg., _Pluchea_

_lanceolata_ C.B.

Clarke

and _Peganum harmala_ L.

North-East India

2000

_Aquilaria_

_malaccensis_ Lam., _Smilax_

_gla-_

_bra_ Roxb., _Ambroma_

_augusts_ (L.)

L.f.

and _Hydnocarpus hurzii_ (King) Warb.

Islands

1000

_Claophyllum inophyllum_ L. _Adnanthera pavoni-_

_na_ L., _Barringtonia_

_asiatica_ (L.),

Kurz

and _Aisandra butyracea_ (Roxb.), Baehni.

Coasts

500

_Rhizophora mucronata_ Lam., _Acanthus ilicifoli-_

_us_ L., _Avicennia marina_ Vierth and _Sonneratia_

_caseolaris_ (L.) engl.

#Source: Ved _et al_., 2001; http://www.fao.org/docrep/007/ad871e/ad871e09.htm

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 134

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ 701 in the Uttarakhand (Badola and Ait-medicines (Fuentes _et al_., 1993). Using _in_

ken, 2003). The economic potential of the

_vitro_ propagation methods several rare

medicinal plants has been recognized in

and endangered plant species can be

the IHR. It possesses various forest types

quickly and successfully propagated with

with diverse habitats, slopes, aspects and

low impact on wild population (Cuenca _et_

altitudinal ranges which offers congenial

_al_., 1999). Plant cell cultures represent a

environment for the natural and artificial

potential source of valuable secondary

propagation of the diverse medicinal

metabolites which can be used as food

plants. Such diversity of the medicinal

additives, nutraceuticals, and pharmaceu-

plants would be helpful for further scien-

ticals. The major advantage of the synthe-

tific research on exploring their medical

sis of phytochemicals by the cell cultures

efficacy (Kala _et al_., 2006). The aim set

is that they are independent of environ-

by India for establishing golden triangle

mental conditions and quality fluctua-

between traditional medicine, modern

tions. Therefore, this sector undoubtedly

medicine and modern science will be a

offers several opportunities for the sus-

boon for development of traditional herb-

tainable development of the region how-

al medicine and medicinal plant sector

ever there are lots of challenges associat-

(Mashelkar _et al_., 2005).

ed with the sound development of the sec-

However, there is a paucity of re-

tor in the region.

search on the biology, habitat and adapta-

tion mechanism of the majority of the

_3.4. Agriculture assets and food security_

threatened trade taxa of the medicinal

Agriculture assets and food securi-

plants (Dhar _et al_., 2000). Further, proper

ty is a critical issue of Himalayas due to

agro techniques for the cultivation of the

complete dependency on rain besides

medicinal plant are lacking in the Hima-

general characteristics of remoteness, low

layas. It is noteworthy to mention here

market integration and underdeveloped

that < 90% of the plant raw material for

agrarian resources. However, agriculture

herbal and industries in India and for ex-

and allied sector forms the pivotal part of

port is drawn from natural habitats (Tan-

the people living in the Himalayas. The

don, 1996, Ved _et al_., 1998; Dhar _et al_.,

huge diversity in the Himalayas has been

2000). Several challenges are associated

maintained through a variety of crop

with the sustainable development of the

composition, indigenous methods of

medicinal plant sector in IHR. Most im-

maintaining soil fertility, socio-cultural

portant among these are low population

and religious rituals (Negi and Maikhuri,

size, habitat specificity, narrow distribu-

2013).

tion ranges, unscientific collection for

IHR is the storehouse for the di-

commercial purposes, land use disturb-

verse genetic stocks. For instance, farmers

ances, introduction of non-native species,

of the Central Himalaya grow about 100

habitat loss and alteration, climate chang-

varieties of paddy, 170 varieties of kidney

es, heavy livestock grazing, unregulated

beans, 8 varieties of wheat,4 varieties of

tourism, construction of dams and roads,

barley and about a dozen varieties of

explosion of human population, popula-

pulses and oil seeds each year and farmers

tion bottlenecks and genetic drift (Kala,

of Uttarakhand Himalaya are known for

2005).

cultivating 34 crop species comprising of

Recent advancement in the field of

6 types of cereals, 5 types of pseudo cere-

Plant Biotechnology especially Plant tis-

als, 6 types of millets, 16 types of pulses,

sue culture has emerged as the promising

4 types of oilseeds, 5 types of condiments

technique for the conservation of these

and 8 types of vegetables (Negi and

medicinal plants. _In-vitro_ propagation of

Maikhuri, 2013). The wild fruits of IHR

plants holds tremendous potential for the

have significantly attracted the attention

production of high-quality plant-based

of the entire world from the Nutraceutical

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 135

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ point of view. However, at present the

tion and social equality. In order to utilize

decline in the interest of farming has been

the tourism industry market, uncontrolled

observed as a result of climatic uncertain-

number of tourist there should be proper

ty and cultural transformation. The con-

infrastructural facilities. The ecological

sequences of this may be disastrous be-

pressures are threatening land, water and

cause of genetic erosion of some unique

wildlife resources through direct and indi-

and diverse gene bank.

rect environmental impacts together with

generation of solid and liquid wastes

_3.5. Tourism /aesthetic/ ecological ser-_

(Singh, 2002). So, ecotourism or green

_vices_

tourism should be promoted for the sus-

Tourism industry typically de-

tainable development of the region.

pends on the quality of the natural re-

sources. Himalayas are blessed with natu-

_3.6. Other challenges (disasters, climate_

ral beauty and pilgrim centers which at-

_change, population increase, pov-_

tracts the millions of International tourists

_erty and waste management)_

throughout the year. They have abundant

People living in IHR, are facing the prob-

of natural resources like spectacular land-

lems and damages due to some sudden

scapes, mountains, glaciers, rivers, lakes,

events such as forest fire, avalanches,

fountains, snow clad peaks, forests having

cloud bursting, land and mudslides, earth-

high floral and faunal diversity, which

quakes, debris flows, flash floods, paraly-

offers strong resonance with tourism.

ses the life and property of the people

These bioresources make Himalayas suit-

(Nyaupane and Chhetri, 2009). There are

able for establishing Sanatoria and reju-

several evidences of Climate change and

venating centers. Thus, they have huge

its impact in Himalayas (Beniston, 2003;

potential for promoting tourism in the

Cruz _et al_., 2007; Xu _et al_., 2009).

form of natural and cultural heritage.

Among these impacts, the most widely

Economic potential of tourism that

reported is the receding of glaciers which

could promote to sustainability in IHR is

could have disastrous impact on the sur-

well recognized.IHR host several tourist

vival of the millions of people. The ongo-

destinations which provides livelihood to

ing climate change over succeeding dec-

millions of people residing in the region.

ades will likely to have additional nega-

With the arrival of Britishers in 19th cen-

tive impacts across these mountains, in-

tury, several hill stations like Nainital,

cluding significant cascading effects on

Darjelling, Mussoorie, Shimla etc. were

the river flows, ground water recharge,

established. They are the tourist hotspots

natural hazards, and biodiversity; ecosys-

for thousands of national and international

tem composition, structure and human

tourist. In India, both the state and central

livelihoods (Xu _et al_., 2009). The popula-

government have declared tourism to be

tion in Himalayas is rapidly increasing;

an industry and provides same conces-

however there are finite resources to en-

sions and incentivizes of the industrial

hance the production ultimately promot-

sector (Cole and Sinclair, 2002).

ing the poverty. Recently proper waste

However, several challenges are

management appears as the critical prob-

associated with the sustainable develop-

lem in the Himalayas. Reducing forest

ment of the tourism industry in IHR. Hol-

cover, accelerated soil erosion, drying

len,(2010) had identified lack of confi-

springs, biodiversity loss etc. seems the

dence in the economic certainty of tour-

ever increasing challenges for the sustain-

ism as the major challenge in the sustain-

ability in the Himalayas.

able development of the tourism in Hima-

****

layas. He also advocates the philosophy

**4. Concluding remarks**

of sustainable development constructed

****

upon conservation, community participa-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 136

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ Himalayas have undoubtedly huge

**Bawa, K. S., Koh, L. P., Lee, T. M.,**

ecological, life sustaining, recreational,

**Liu, J., Ramakrishnan, P., Yu,**

educational and scientific values. Several

**D. W., Zhang, Y.P. and Raven,**

challenges are associated for the sustaina-

**P. H. (2010).** China, India and the

bility in the region. The present trends

Environment. _Science_ **327, 1457-**

suggest that the existing interventions in

**1459.**

the region are unsustainable; further un-

**Beniston, M. (2003).** Climatic change in

scientific exploitation of natural resources

mountain regions: a review of

is increasing the environmental degrada-

possible impacts. _Climatic Change_

tion in the region (Singh, 2006). Proper

**59, 5–31.**

policies and their implementation for har-

**Blaikie, P. M. and Muldavin, J. S.**

nessing the potential of the natural re-

**(2004).** Upstream, downstream,

sources is the need of the hour for the sus-

China, India: the politics of envi-

tainable development in the region.

ronment in the Himalayan re-

****

gion. _Annals of the Association of_

**References**

_American_

_Geographers_

**94(3),**

****

**520-548.**

**Agrawal, D. K., Lodhi, M. S. and**

**Central Electricity Authority (CEA).**

**Panwar, S. (2010).** Are EIA stud-

**(2009).** Status of hydroelectric po-

ies sufficient for projected hydro-

tential development, 31 August

power development in the Indian

2009; http://www.cea.nic.in/

Himalayan region? _Current Sci-_

**Chatterjee, D. (1939)**. Studies on the en-

_ence_ **98(2), 154-161.**

demic flora of India and Burma. _J_

**Andermann, C., Longuevergne, L.,**

_R Asiat Soc Bengal (Sci)_ **5, 19–67.**

**Bonnet, S., Crave, A., Davy, P.,**

**Chhetri, M. B. P. (2001).** A Practitioner's

**and Gloaguen, R. (2012).** Impact

view of disaster management in

of transient groundwater storage

Nepal: organisation, system, prob-

on the discharge of Himalayan

lems and prospects. _Risk Man-_

rivers. _Nature_

_Geoscience_ **5(2),**

_agement_ **3(4), 63-72.**

**127-132.**

**Cole, V. and Sinclair, A. J. (2002).**

**Badola, R., Hussain, S. A., Dobriyal, P.**

Measuring the ecological footprint

**and Barthwal, S. (2015).** As-

of a Himalayan tourist cen-

sessing the effectiveness of poli-

ter. _Mountain Research and de-_

cies in sustaining and promoting

_velopment_ **22(2), 132-141.**

ecosystem services in the Indian

**Cruz, R. V., Harasawa, H., Lal, M.,**

Himalayas. _International Journal_

**Wu, S., Anokhin, Y., Pun-**

_of Biodiversity Science, Ecosystem_

**salmaa, B., Honda, Y., Jafari,**

_Services & Management_ **11(3),**

**M., Li, C. and Huu Ninh, N.**

**216-224.**

**(2007).** Asia. _In:_ Parry, M. L.,

**Bahadur, J. (2004).** Himalayan Snow

Canziani, O.F., Palutikof J.P., van

and Glaciers – Associated Envi-

der Linden, P. J. and Hanson, C.E.

ronmental Problems, Progress and

(eds). _Climate change 2007: im-_

Prospects, Concept Publishing Co,

_pacts, adaptation and vulnerabil-_

New Delhi.

_ity._ Contribution

of

Working

**Bates, B.C., Kundzewicz, Z. W., Wu,**

Group II to the Fourth Assessment

**S.,**

**and**

**Palutik**

**J.**

**P.**

Report of the Intergovernmental

**(2008).** Climate change and water.

Panel on Climate Change. Cam-

Technical paper of intergovern-

bridge University Press, Cam-

mental panel on climate change,

bridge, UK. **pp. 469-506.**

IPCC Secretarist, Geneva.

**Cuenca, S., Amo-Marco J. B. and Par-**

**ra, R.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 137

_Biotech Sustainability (2017)_

_

__Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ **(1999).** Micropropagation from in-pitality Planning & Develop-_

florescence stems of Spanish en-

_ment_ **7(4), 337-351.**

demic plant _Centaurea pauri_ Los-

**Hunzai, K., Gerlitz, J. Y. and Ho-**

cos ex. Willk (Compositae). _Plant_

**ermann, B.(2011).** Understanding

_Cell Reproduction_ **18, 674-679.**

mountain poverty in the Hindu

**Dhar, U., Rawal, R. S. and Upreti, J.**

Kush-Himalayas: regional report

**(2000).** Setting priorities for con-

for Afghanistan, Bangladesh, Bhu-

servation of medicinal plants––a

tan, China, India, Myanmar, Ne-

case study in the Indian Hima-

pal, and Pakistan. International

laya. _Biological_

_conserva-_

Centre for Integrated Mountain

_tion_ **95(1): 57-65.**

Development (ICIMOD).

**Dyurgerov, M. B. and Meier, M. F.**

**Jodha, N. S. (2005).** Economic globalisa-

**(2005).** Glaciers and the changing

tion and its repercussions for frag-

Earth system: a 2004 snap-

ile mountains and communities in

shot (Vol. 58). Boulder: Institute

the Himalayas. In Global change

of Arctic and Alpine Research,

and mountain regions, Springer

University of Colorado.

Netherlands, 583-591.

**Franklin, J. (1995).** Predictive vegetation

**Kala, C. P. (2005).** Indigenous uses, pop-

mapping: geographic modelling of

ulation density, and conservation

biospatial patterns in relation to

of threatened medicinal plants in

environmental gradients. _Progress_

protected areas of the Indian Him-

_in physical geography_ **19(4), 474-**

alayas. _Conservation_

_Biolo-_

**499.**

_gy_ , _19_ (2), 368-378.

**Fuentes, S. I., Suarez, R., Villegas, T.,**

**Kala, C. P., Dhyani, P. P. and Sajwan,**

**Acero, L. C. and Hernandez, G.**

**B. S. (2006).** Developing the me-

**(1993).** Embryogenic response of

dicinal plants sector in northern

Mexican alfalfa (Medicago sativa)

India: challenges and opportuni-

varieties. _Plant Cell Tissue Organ_

ties. _Journal of Ethnobiology and_

_Culture_ **34, 299- 302.**

_Ethnomedicine_ **2(1), 32.**

**Ghimire, S.K. (2008).** Sustainable har-

**Lange, D. (1997).** Trade figures for bo-

vesting and management of me-

tanical drugs worldwide. _Medici-_

dicinal plants in the Nepal Hima-

_nal Plant Conservation Newsletter_

laya: current issues, knowledge

**3, 16-17.**

gaps

and

research

priori-

**Liu, X. and Chen, B. (2000).** Climatic

ties. _Medicinal Plants in Nepal:_

warming in the Tibetan Plateau

_an Anthology of Contemporary_

during

recent

dec-

_Research_ **25-44.**

ades. _International journal of cli-_

**Goldsmith, E. and Hildyard, N.**

_matology_ **20(14), 1729-1742.**

**(1984).** The social and environ-

**Mashelkar, R. A. (2005).** India's R&D:

mental effects of large dams. Vol-

Reaching

for

the

ume 1: overview. Wadebridge

Top. _Science_ **307(5714),**

**1415-**

Ecological Centre.

**1417.**

**GoP (Government of Pakistan). (2010).**

**Mittermeier, R. A. (2004).** Hotspots re-

Final Report of the Task Force on

visited. Cemex.

Climate Change. Islamabad: Plan-

**NAS (National Academy of Sciences).**

ning Commission.

**(2012).** Himalayan Glaciers: Cli-

**Holden, A. (2010).** Exploring stakehold-

mate Change, Water Resources,

ers' perceptions of sustainable

and Water Security. Washington,

tourism development in the Anna-

DC. National Academies Press.

purna Conservation Area: Issues

**National Ganga River Basin Authority.**

and challenge. _Tourism and Hos-_

**(2011)** **.** National Ganga River Ba-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 138

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ sin Authority. Environmental and

change in the Himalayas and asso-

Social Analysis, Vol. 1, Govern-

ciated changes in local ecosys-

ment of India, New Delhi, India.

tems. _PLoS One_ **7(5), e36741.**

**Nayar, M. P. (1996).** Hot-spot of endem-

**Singh, J. S. (2002).** The biodiversity cri-

ic plants of India, Nepal and Bhu-

sis: a multifaceted review. _Current_

tan. Tropical Botanic Garden and

_Science_ **82(6), 638-647.**

Research Institute, Thiruvanan-

**Singh, J. S. (2006).** Sustainable devel-

thapuram, Kerala, India.

opment of the Indian Himalayan

**Negi, V. S. and Maikhuri, R. K. (2013).**

region: Linking ecological and

Socio-ecological

and religious

economic concerns. _Current Sci-_

perspective of agrobiodiversity

_ence_ **90(6), 784.**

conservation: issues, concern and

**Singh J. S. and Singh S. P. (1992).** For-

priority for sustainable agriculture,

ests of Himalaya: Structure, Func-

Central Himalaya. _Journal of ag-_

tioning and Impact of Man,

_ricultural and environmental eth-_

Gyanodaya Prakashan, Nainital.

_ics_ **26(2), 491-512.**

**Singh, R. B. (2002).** Tourism develop-

**Nyaupane, G. P. and Chhetri, N.**

ment and environ- mental implica-

**(2009).** Vulnerability to climate

tions for the Indian Frontier Re-

change of nature-based tourism in

gion: A Study of Himachal Hima-

the Nepalese Himalayas. _Tourism_

laya. _In:_ S. Krakover and Y. Gra-

_Geographies_ **11(1), 95-119.**

dus. Tourism in Frontier Areas.

**Pandit, M. K., Bhakar, A. and Kumar,**

Lanham: Lexington Books.

**V. (2000).** Floral diversity of

**Tandon, V. (1996).** CAMP workshop-

Goriganga Valley in the Central

plants under threat new list forged.

Himalayan highlands. _J Bombay_

_Medicinal_

_Plant_

_Conservation_

_Nat Hist Soc_ **97,184–192.**

_Newsletter_ **2, 12-13.**

**Pandit, M. K., Sodhi, N. S., Koh, L. P.,**

**Ved, D. K., Mudappa, A. and Shankar,**

**Bhaskar, A. and Brook, B. W.**

**D. (1998).** Regulating export of

**(2007).** Unreported yet massive

endangered medicinal plant spe-

deforestation driving loss of en-

cies: Need for scientific rig-

demic biodiversity in Indian

our. _Current Science_ **75(4), 341-**

Himalaya. _Biodiversity and Con-_

**344.**

_servation_ **16(1) 153-163.**

**Ved P. (2001).** Indian Medicinal Plants;

**Rasul, G. (2010).** The role of the Hima-

Current Status. _In:_ Samant SS,

layan mountain systems in food

Dhar U, Palni, LMS (eds), Hima-

security and agricultural sustaina-

layan Medicinal Plants: Potential

bility in South Asia. _International_

and

Prospects.

Nainital:

_Journal_

_of_

_Rural_

_Manage-_

Gyanodaya Prakashan. **pp. 45–65.**

_ment_ **6(1), 95-116.**

**Xu, J. C., Shrestha, A. B., Vaidya, R.,**

**Rasul, G. (2014).** Food, water, and ener-

**Eriksson, M. and Hewitt, K.**

gy security in South Asia: a nexus

**(2007).** The melting Himalayas:

perspective from the Hindu Kush

regional challenges and local im-

Himalayan region. _Environmental_

pacts of climate change on moun-

_Science & Policy_ _**39**_ **, 35-48.**

tain ecosystems and livelihoods.

**Samant, S. S., Dhar, U. and Palni, L.**

Technical

paper.

International

**M. S. (1998).** Medicinal Plants of

Center for Integrated Mountain

Indian Himalaya: Diversity, Dis-

Development, Kathmandu, Nepal.

tribution,

Potential

Values,

**Xu, J., Grumbine, R. E., Shrestha, A.,**

Gyanodaya Prakashan, Nainital.

**Eriksson, M., Yang, X., Wang,**

**Shrestha, U. B., Gautam, S. and Bawa,**

**Y. U. N. and Wilkes, A. (2009).**

**K. S. (2012).** Widespread climate

The melting Himalayas: cascading

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 139

_Biotech Sustainability (2017)_

_Opportunities and Challenges in Indian Himalayan Region Chauhan and Bisht_ effects of climate change on wa-Zurick, D., Pacheco, J., Shrestha, B. R.,

ter,

biodiversity,

and

liveli-

**and**

**Bajracharya,**

**B.**

hoods. _Conservation_

_Biolo-_

**(2006).** Illustrated atlas of the

_gy_ **23(3), 520-530.**

Himalaya. India Research Press.

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 140

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P141-170_

**Natural Polyphenols and its Potential in Preventing Dis-**

**eases Related to Oxidative Stress as an Alternative Green**

**Nutraceutical Approach**

****

**Sreenivasan Sasidharan1,*, Shanmugapriy1, Subramanion Lachumy Jothy1, Mei Li**

**Ng2, Nowroji Kavitha1, Chew Ai Lan1,** **Khoo Boon Yin1, Soundararajan Vijayara-**

**thna1, Leow Chiuan Herng1 and Chern Ein Oon1**

_1Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 USM, Pe-_

_nang, Malaysia; 2Integrative Medicine Cluster, Advanced Medical and Dental Institute_

_(AMDI), Universiti Sains Malaysia, 13200, Kepala Batas Penang, Malaysia;_

_*Correspondence: srisasidharan@yahoo.com; Tel: +60 46534820_

__

****

**Abstract:** Green bioactive polyphenol from Mother Nature especially from medicinal

plants are a rich source of novel therapeutics. Therefore, the search for bioactive molecules

from nature continues to play an important role in the invention of new medicinal agents.

Most plants do provide an array of phytochemicals that may contribute to reduction of dis-

ease and slowing of aging. Oxidative stress which is continuously produced _in vivo_ by oxy-

gen-centred free radicals and other reactive oxygen species may leads to various diseases.

Since the oxidative stress has a great impact on the human health, it is appropriate to exam-

ine the role of natural antioxidant as a defence system. In this line, medicinal plant based

natural antioxidants such as polyphenols with free radical scavenging activity are emerging

as the primary components of holistic approaches in impeding adverse effect of oxidative

stress. This chapter focuses on biological effects of natural polyphenols on oxidative stress

and related diseases as an aalternative green nutraceutical approach. ****

_**Keywords:**_ Free radicals; medicinal plant; natural antioxidants; oxidative stress; polyphenol

_****_

**1. Introduction**

all eukaryotes. They vary in number and

location according to cell type. Converse-

Oxidative damage caused by free rad-

ly, numerous mitochondria are found in

icals to macromolecules outlines the

human liver cells, with about 1000–2000

foundation of what is arguably the most

mitochondria per cell, making up 1/5 of

popular current explanation of ageing re-

the cell volume (Alberts _et al._ , 1994).

lated diseases (Lawrence _et al._ , 2005).

Given the role of mitochondria as the

Recent years have seen a surge of interest

cell's powerhouse, there may be some

in the role of mitochondrial dysfunction,

leakage of the high-energy electrons in

reactive oxygen species production and

the respiratory chain to form reactive ox-

mitochondrial DNA mutation as driving

ygen species. This was thought to result

factors in the ageing and various diseases

in significant oxidative stress in the mito-

(Balaban _et al._ , 2005; Trifunovic _et al._ ,

chondria with high mutation rates of mi-

2005; Bender _et al_., 2006; Passos _et al._ ,

tochondrial DNA (mtDNA) (Richter _et_

2007). Hypothesized links between aging

_al._ , 1988). A vicious cycle was thought to

and oxidative stress are not new and were

occur, as oxidative stress leads to mito-

proposed over 50 years ago (Harman,

chondrial DNA mutations, which can lead

1956). Mitochondria are found in nearly

to enzymatic abnormalities and further

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 141

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

oxidative stress. The accumulation of the-

Reactive nitrogen species (RNS)

se damaged macromolecules is proposed

and reactive oxygen species (ROS) are

to contribute significantly to aging and

free radicals that arise from normal cellu-

various diseases (Ames _et al._ , 1993).

lar metabolism or as a consequence to

Hence, plants polyphenol with free radi-

pathological exposures. Nitric oxide, per-

cal scavenging activities are likely to

oxynitrite and nitrogen dioxide are nitro-

make a considerable contribution for the

gen-containing oxidants, often referred to

protection against the free radical oxida-

as reactive nitrogen species. ROS are r _e-_

tion. Bioactive products from Mother Na-

_active_ molecules derived from oxygen

ture are a rich source of novel therapeu-

molecules __ such as superoxide, hydroxyl,

tics. Therefore, the search for bioactive

peroxyl, alkoxyl and singlet oxygen. They

molecules from nature continues to play

are usually generated as by-products in

an important role in the invention of new

the mitochondria, peroxisomes, cyto-

medicinal agents. Most plants do provide

chrome P450 and other organelles. Free

an array of phytochemicals that may con-

radicals include reactive oxygen and ni-

tribute to reduction of diseases and slow-

trogen species which are also collectively

ing of aging. This chapter focuses on bio-

termed as reactive oxygen nitrogen spe-

logical effects of natural polyphenols on

cies (RONS). RONS are known for being

oxidative stress and related diseases as an

both beneficial and harmful. On the good

alternative green nutraceutical approach.

side, they have been reported to have a

crucial role in cell signalling (Adams _et_

**2. What are free radicals?**

_al._ , 2015; Reczek and Chandel, 2014),

homeostasis (Kuster _et al._ , 2010; Shadel

The human body is, composed of dif-

and Horvath, 2015) and immune defence

ferent types of living cells which are or-

in response to inflammatory stimuli (Miz-

ganized into tissues, organs, and systems.

gerd and Brain, 1995; Reshi _et al._ , 2014).

Cells are made of different types of mole-

However, these radicals are over-

cules which consist of atoms. Atoms

generated in response to unfavourable en-

comprise of a nucleus, neutrons, protons

vironmental conditions such as poor nutri-

and electrons. Protons are positively

tion, stress, smoking, alcohol, exercise,

charged particles in the nucleus that de-

radiation, inflammation, drugs or expo-

termine the number of negatively charged

sure to air pollutants and chemicals (Fig-

particles known as electrons in the atomic

ure 1). At high concentrations, free radi-

orbital.

cals have the ability to alter proteins, car-

An atom"s chemical behaviour is

bohydrates, lipids and nucleic acids thus

largely dictated by the number of elec-

impairing cellular functions and leading

trons in its outermost shell. An atom is

to pathogenesis of cancer, aging, diabetes,

stable when its outermost shell is full. A

atherosclerosis and other inflammatory

free radical is an atom or group of atoms

diseases (Pham-Huy _et al._ , 2008).

that has an unpaired electron due to split-

Antioxidants are electron donors that

ting of weak bonds between electrons on

can neutralize free radicals, thus prevent-

the outermost shell and is therefore unsta-

ing them from causing cellular damage

ble and highly reactive. It will attempt to

(Figure 1). A balance between free radi-

stabilize itself by reacting with another

cals generated and antioxidant protective

atom or molecule to donate its electron or

defence system is required for optimal

to aggressively capture an electron to fill

physiological function (Bouayed and

its outermost shell, thus stimulating a cas-

Bohn, 2010). Oxidative stress or nitrosa-

cade of free radicals when the attacked

tive stress occurs when there is an imbal-

atom or molecule receives an extra elec-

ance between the production of free radi-

tron or loses its electron (Halliwell,

cals and the ability of the body neutralize

1993).

the deleterious effects through the role of

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_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**Figure 1:** Free radicals are generated due to factors such as stress, alcohol, tobacco, harm-

ful chemicals including pesticides and drugs, fried food and radiation. Found abundantly in

natural food, antioxidants are free radical scavengers that react with free radicals to neutral-

ize them. Antioxidants function by donating an electron to the free radical before the latter

oxidizes other components within the cell. The free radical is stabilized and becomes non-

damaging to cells once it receives a free electron from the antioxidant.

antioxidants. The human body manufac-

was first introduced by Selye (1955). A

tures endogenous antioxidant enzymes in

continuously activity of human body re-

order to control these destructive free rad-

acts with oxygen when breathing resulted

ical chain reactions (Valko _et al._ , 2007).

in energy production by cells and gener-

However, the body also relies on dietary

ate highly reactive molecules within the

antioxidants to fulfil the needs of other

cells known as free radicals. The effect

antioxidants it cannot produce (Diplock _et_

exerted by free radicals in the body is

_al._ , 1998), such as those found abundantly

called "oxidative stress" (Finaud _et al.,_

in bioactive food components including

2006).

fruits, vegetables, grains and mushrooms.

Since overwhelming research has

Examples of dietary antioxidants include

been developed, the contemporary con-

lycopene, beta-carotene, lutein, zeaxan-

cept of "oxidative stress" was updated and

thin, alpha-tocopherol, vitamin A and vit-

briefly redefined as disturbance in the

amin C.

balance between oxidants production and

antioxidants defences (Sies and Jones,

**3. Oxidative stress**

2007; Sies, 2015) which are associated

with electron transfer influencing the re-

The origin of term "oxidative stress"

dox state of cells and organism. Subse-

from its very nature defined as the combi-

quently, the imbalance redox mechanism

nation of electron transfer, free radicals,

leads to the production of reactive oxygen

oxygen metabolites such as the superox-

species (ROS) that includes free radicals

ide anion radical, hydrogen peroxide, hy-

(superoxide, hydroxyl radical, peroxyl,

droxyl radical and singlet molecular oxy-

alkoxyl, and hydroperoxyl) and non-free

gen with a biological concept of stress

radicals

(hydrogen

peroxide,

hypo-

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chlorous acid, ozone, singlet oxygen) are

**4. Oxidative stress and immune re-**

mainly involved in growth, differentia-

**sponse**

tion, progression and death of the cells.

(Rahman _et al.,_ 2012; Rajendran _et al.,_

Immune system is important for our

2014).

body to protect from the invasive patho-

Free radicals known as small mol-

gens. The human defence system has been

ecules and unpaired electron amid highly

well developed and can be divided into

reactive proliferation characteristic which

two immunity reactions, namely innate

differ from most biological molecules that

immunity and adaptive immunity. As its

tend to involve in initiate chain reactions

name suggests, the innate immunity is a

from single free radical until propagate to

non-specific first barrier of defence which

damage multiple molecules. Oxidative

is able to act fast towards microbial inva-

stress occurs when excessive free radical

sion. The adaptive immunity, on the other

formation within a cell or organism as a

hand, is a highly specific barrier of de-

form of ROS due to aerobic metabolism

fence due to possess immunological

and

immune

activation

(Delmastro-

memory function that can rapidly and ef-

Greenwood and Piganelli, 2013), UV ra-

ficiently remove the pathogens that in-

diation (Halliwell and Gutteridge, 2015),

vaded into body (Poland _et al._ , 2013).

heme-oxygenase accumulation (Vanella __

Components of the adaptive immune sys-

_et al.,_ 2013), and hypoxia (Yang _et al._ ,

tem are normally silent; however, when

2011). Failure of the cell"s defence mech-

activated, these components "adapt" to the

anisms to neutralize or balance the accu-

presence of infectious agents by activat-

mulation of free radicals may leads to mi-

ing, proliferating, and creating potent

tochondrial dysfunction, DNA damage

mechanisms for neutralizing or eliminat-

and lipid peroxidation can trigger pro-

ing the microbes. In general, there are two

grammed cell death pathways (Dixon and

types of adaptive immune responses

Stockwell, 2014). The findings could ex-

where humoral immunity, mediated by

plain that low concentration of ROS pro-

antibodies produced by B lymphocytes

ductions play role in intracellular signal-

(B-cells), and cell-mediated immunity,

ling and defence against pathogens, while

mediated by T lymphocytes (T-cells). Un-

the higher concentration of ROS has been

like B-cells, T-cells recognize circulating

linked to clinically relevant diseases in-

antigens of many chemical structures, the

cluding cancer, cardiovascular disease,

vast majority of T cells (> 95%) are only

asthma, ischemia, diabetes and neuro-

able to recognize peptide fragments that

degenerative diseases (Reynolds _et al.,_

are displayed by specialized molecules,

2007; Halliwell and Gutteridge, 2015;

MHC molecules, on the surfaces of anti-

Phaniendra _et al.,_ 2015). Mitochondria is

gen presenting cells. Therefore, this sys-

a major part of cellular sources of ROS

tem ensures that T-cells are able to recog-

which consume oxygen with the process

nize antigens that might be floating in the

of oxidative phosphorylation. Excessive

cytosol or contained within ingested vesi-

formation of free radicals is known to

cles of various cells (Thomas _et al._ ,

weaken defence mechanisms against oxi-

2013).

dation and it results in more cell damages

An imbalance between reactive

(Zorov, 2014). Consequently, the increas-

oxygen species (ROS) and reducing

ing level of oxidative stress involved in

agents (antioxidants) towards a pro-

pathological redox reaction, process of

oxidant state can always result to oxida-

ageing which can initiate tissue damage

tive stress (Sies, 1997). The damaging of

via apoptosis and necrosis (Cui _et al.,_

macromolecular in the form of protein

2012).

carbonylation, lipid peroxidation and

DNA oxidation has historically has been

proven as harmful to particular functional

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cells and being part of the main factors to

are greatly debated in healthcare man-

hypertension and aging problems (Annor __

agement.

_et al._ , 2015; Zhao _et al._ , 2017). Up to

date, ROS is widely recognised as signal-

**5. Oxidative stress and cancer**

ling molecular substances (Suzuki and

Sevanian, 1997) that enable to trigger a

Oxidative stress is closely related to

wide range of biological effects. With re-

all aspects of cancer, from carcinogenesis

gard to the immune system, high levels of

to the tumor-bearing state, from treatment

ROS can also be beneficial. For instances,

to prevention. Cancer cells generally dis-

neutrophils generate ROS and release

play elevated ROS level compared to

them intracellularly and extracellularly in

normal cells that give them a proliferative

the form of an "oxidative burst" to defend

advantage and promote malignant pro-

against and destroy pathogens, thus

gression. The excess level of ROS typi-

providing

antimicrobial

protection

cally observed in cancer cells are the re-

(Dahlgren and Karlsson, 1999). However,

sult of accumulation of intrinsic and envi-

excessive ROS are generated in the pres-

ronmental factors. In cancer cells, high

ence of immune complexes with auto-

levels of ROS can result from hypoxia,

antigens where further macromolecular

mitochondrial dysfunction, peroxisome

damage is induced. Prolonged exposure to

activity, enhanced cellular metabolic ac-

high ROS concentrations can inhibit T-

tivity, increased cellular receptor signal-

cell proliferation and lead to apoptosis

ing, oncogene activity, increased activity

(Thoren _et al._ , 2007), and incubation of

of oxidases, cyclooxygenases, lipoxigen-

T-cells with the reactive nitrogen species

ases and thymidine phosphorylase, and

(RNS) peroxynitrite can also inhibit their

the crosstalk between cancer cells and

proliferation (Kasic _et al._ , 2011). Previous

immune cells recruited to the tumor site

study has revealed that different T-cell

(Holmström and Finkel, 2014). Environ-

responses to ROS production may be due

mental sources of ROS that can signifi-

to the extent of change to the cellular re-

cantly contribute to tumorigenesis include

dox environment (Griffiths, 2005). In

ionizing radiation, xenobiotics, tobacco

some cases such as absence of antigen

components, chlorinated compounds, bar-

presenting cell, reactive carbonyls includ-

biturates, metal ions and phorbol esters

ing 4-hydroxy-2-nonenal and malondial-

etc. Figure 2 illustrates the potential out-

dehyde (MDA), which are generated on

comes when such ROS level exceeds the

proteins and lipids randomly in the pres-

capacity of the oxidation-reduction sys-

ence of ROS, will promote differentiation

tem of the cell, may cause DNA, protein

towards a Th2 phenotype (Moghaddam _et_

and lipid damage, leading to cellular re-

_al._ , 2011). These data emphasize that the

sponses such as chromosomal instability,

importance of ROS homeostasis and flux

genetic mutation, alterations in cellular

in governing cell maturation and that the

metabolism and modulation of cell

balance between oxidising and reducing

growth that may lead to malignant trans-

agents is a delicate process which must be

formation by affecting crucial hallmarks

tightly regulated and well managed, de-

of cancer.

pending on whether the requirement is for

When present at high and sus-

protecting against bacteria, in an immune

tained levels, ROS can cause severe dele-

response, or requirements for T-cell sig-

terious modifications to DNA, protein,

nalling, activation and regulation of func-

and lipids. During the initiation stage,

tion. Owing to their pivotal role, the effect

ROS may produce DNA damage by in-

of the oxidative stress on T-cell biochem-

troducing gene mutations and structural

istry and its implications in autoimmune

alterations into the DNA. ROS-induced

disease such as rheumatoid arthritis (RA)

DNA damage can result in single- or dou-

ble-strand breakage, base modifications,

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**Inciting factors**

**Intrinsic source of ROS**

**Extrinsic source of ROS**

Increased metabolic activity

Ultraviolet rays

Mitochondrial dysfunction

Environmental agents

Peroxisome activity

Pharmaceuticals

Oncogene activity

Industrial chemicals

Increased cellular receptor signalling

Increased activity of enzymes

**High oxidative stress**

**in cancer cells**

**ROS / Oxidative stress**

**Cellular**

Oxidative dam-

Oxidative dam-

Increase

**responses**

age to DNA,

age to proteins

lipid peroxi-

RNA

dation

Altered gene

Loss of DNA repair

Chromo-

Altered sig-

expression

activity (decreased

somal

nal trans-

efficiency of DNA pol-

instability,

duction

ymerase and DNA re-

gene mu-

pair enzymes)

tations

Altered cell growth, differentiation and apoptosis

**Carcinogenesis**

****

Initiation

Promotion

Progression

Invasion

Metastasis

****

**Figure 2:** ROS and their role in the development of human cancer.

deoxyribose modification and DNA

mutations and may contribute to increased

cross-linking. Cell death, DNA mutation,

risk of carcinoma (Caramori _et al._ , 2011).

replication errors and genomic instability

Proteins and lipids are also significant

can occur if the oxidative DNA damage is

targets for oxidative attack and modifica-

not repaired prior to DNA replication

tion of these molecules can increase the

(Valko _et al._ 2006). Oxidative DNA dam-

risk of mutagenesis. Lipid peroxidation

age/repair imbalance due to loss of DNA

results in production of reactive metabo-

repair activity can lead to disease-causing

lites which demonstrate high reactivity

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with protein and DNA and have been im-

Kamendulis, 2007). Most initiating muta-

plicated in the pathogenesis of cancer

tions affect proto-oncogenes or tumor

(Tuma, 2002). ROS also contribute to

suppressor genes. Proto-oncogenes code

chromosomal instability and mutation

for a variety of growth factors, growth

through its role in increasing the rates of

factor receptors, enzymes or transcription

mutation, enhancing sensitivity to muta-

factors that promote cell growth and cell

genic agents and compromising the sur-

division while oncogenes are mutated

veillance systems. In addition to inducing

versions of proto-oncogenes that promote

DNA, lipid and protein damage, oxidative

abnormal cell proliferation. Activation of

damage to protein-coding or –non coding

oncogenes and loss of tumor suppressors

RNA may potentially cause errors in pro-

cause alterations to multiple intracellular

tein synthesis or dysregulation of gene

signaling pathways that promote metabol-

expression. ROS-induced alteration of

ic reprogramming in cancer, resulting in

gene expression can occur through modu-

enhanced nutrient uptake to supply ener-

lation of a host of signaling pathways in-

getic and biosynthetic pathways for en-

cluding cAMP-mediated cascades, calci-

hanced growth and survival (Zhang _et al._ ,

um-calmodulin pathways and intracellular

2013).

signal transducers such as nitric oxide

Oxidative stress may participate in

(Bertin and Averbeck, 2006). ROS stimu-

the progression stage of the cancer pro-

lation on signal transduction pathways

cess by adding further DNA alterations to

can lead to activation of key transcription

the initiated cell population (Qanungo _et_

factors such as Nrf2 and NF-κB.

_al._ , 2005). It can also promote many as-

ROS interact with the multistage

pects of tumor development and progres-

processes in carcinogenesis including ini-

sion through various biological processes.

tiation, promotion, proliferation, invasion,

It acts on cellular proliferation through

angiogenesis and metastasis. ROS act as

extracellular-regulated

kinase

1/2

essential signalling molecules and modu-

(ERK1/2) activation and ligand independ-

late a number of redox-sensitive signal-

ent receptor tyrosine kinases (RTK) acti-

ling pathways in initiation of carcinogen-

vation. ROS were shown as positive regu-

esis. Well-characterized targets include

lators of tumor cell proliferation by

ROS-mediated regulation of the mitogen-

modulating key proteins in cell cycle pro-

activated protein (MAP) kinase/Erk cas-

gression such as cyclin, ATM (ataxia tel-

cade,

phosphoinositide-3-kinase

angiectasia mutated) and antioxidant en-

(PI3K)/Akt-regulated signaling cascades,

zymes like MnSOD, catalase and gluta-

as well as the IκB kinase (IKK)/nuclear

thione peroxidase (Browne _et al._ , 2004;

factor κ-B (NF-κB)-activating pathways

Lewis _et al._ , 2005). ROS are involved in

(Ray _et al._ , 2012). Oxidative stress-

Anoikis resistance, PTEN inactivation,

mediated signaling pathways are persis-

activation of Src, NF-κB, CREB and

tently elevated in many types of cancers

phosphatidylinositol-3 kinase (PI3K)/Akt,

and affect all characters of cancer cell be-

enabling the tumor cells to escape from

havior, where they participate in cell

apoptosis (Giannoni _et al._ , 2009; Zhu _et_

growth/proliferation, differentiation, pro-

_al._ , 2011). In invasion and metastasis,

tein synthesis, glucose metabolism, cell

ROS play a role in Met over-expression,

survival and inflammation (Storz, 2005).

matrix metalloproteinase secretion into

In promotion stage, ROS can con-

the extracellular matrix (ECM), invado-

tribute to abnormal gene expression, inhi-

podia formation, Rho-Rac interaction,

bition of intercellular communication and

plasticity in cell motility and epithelial–

modification of second-messenger sys-

mesenchymal transition (EMT). These

tems, thus resulting in an increase in cell

help cancer cells to escape the primary

proliferation or a decrease in apoptosis of

tumor, invade matrix of different organs,

the initiated cell population (Klaunig and

find a suitable metastatic niche and then

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___
___

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

grow in the secondary site. In addition,

tions (atherosclerotic diseases affecting

oxidative stress is involved in continuous

arteries that supply heart, brain and lower

angiogenesis through its role in endotheli-

extremities) (Greene _et al._ , 1992; Rosen __

al progenitor activation, release of VEGF

_et al._ , 2001) and onset of diabetes (Kaya-

and angiopoietin and recruitment of peri-

ma _et al._ , 2015). Vincent and colleagues

vascular cells.

has demonstrated that ROS production

and neuron injury are activated within 1-2

**6. oxidative stress (os) in diabetes pa-**

hours of hyperglycaemic insult. Majority

**thology**

of the patients with impaired glucose tol-

erance have significant peripheral neu-

A growing body of evidence suggest

ropathy, suggesting that ROS induced by

that increased oxidative stress and deficit

hyperglycaemia is critical to cause major

in antioxidant defense mechanism are

diabetes complications (Vincent _et al._ ,

central players in pathogenesis of diabetes

2002).

complications, in particular β-cell dys-

Metabolic abnormalities such as

function and failure (Folli _et al._ , 2011).

hyperglycaemia,

hyperlipidaemia,

in-

Under physiological condition, reactive

creased free fatty acids, insulin resistance

oxygen species (ROS) serve as second

and hyperinsulinaemia, each of which

messenger regulates signal transduction

was noted to induce oxidative stress in

and gene expression. Oxidative stress de-

endothelial cells of the blood vessels and

velops from imbalance in redox homeo-

myocardium. In addition, genetic suscep-

stasis (overproduction of mitochondrial

tibility of an individual and presence of

reactive oxygen species (ROS) that ex-

accelerating factors (e. g. hypertension

ceeds the level of antioxidants) leads to

and dyslipidaemia) also contribute to de-

aberrant β-cell function and apoptosis.

velopment of diabetes complications

ROS are heterogenous molecules com-

(general features of chronic hyperglycae-

prises of free radicals, such as nitric oxide

mia-induced tissue damage are depicted

(NO **.** ), superoxide (O **.** -

2 ), hydroxyl radical

in Figure 3). Several large scale perspec-

(OH **.** ), non-radicals such as hydrogen per-

tive studies, such as the (Diabetes Control

oxide (H2O2), anions such as superoxide

and Complication Trial DCCT/EDIC

(O -

2 ) and peroxynitrite (ONOOK) (Chang __

(The Diabetes Control and Complications

_et al._ , 1993; Pieper _et al._ , 1997; Lenzen,

Trial Research Group, 1993), UK pro-

2008; Newsholme _et al._ , 2012; Cao and

spective Diabetes Study (UKPDS) (UK

Kaufman, 2014; Keane _et al._ , 2015).

Prospective Diabetes Study (UKPDS)

Sources of free radicals production in-

Group, 1998), and Steno 2 Study have

clude the mitochondrial electron transport

concluded that chronic hyperglycaemia as

system, NADPH oxidases, xanthine oxi-

a key risk factor underlying diabetes pa-

dase (primary source in cardiomyocytes),

thology (Gaede _et al._ , 2008). Hypergly-

uncoupled nitric oxide synthase (NOS)

caemia is known to trigger oxidative

and arachidonic acid (primary source in

stress through FIVE major molecular

vascular cells) pathway. Mitochondria are

mechanisms (Figure 4 depicts the mecha-

major source of free radicals production

nism underlying oxidative stress and dia-

in cells. ROS was noted as a key upstream

betes pathology): (1) Activation of Polyol

signaling event mediates downstream

pathway (2) Increased intracellular ad-

metabolic pathways, leading to loss of

vanced glycation end products (AGEs)

cellular biological function and ultimately

pathway activity and receptor expression

cell death (Brownlee, 2005). Ample evi-

for AGEs (RAGE) (3) Activation of Pro-

dence indicate that ROS damage plays a

tein Kinase C isoforms (PKC) (4) In-

major role in pathogenesis of micro- (dia-

creased Hexosamine pathway flux (5)

betic retinopathy, nephropathy, and neu-

Decreased antioxidant defenses (Sima _et_

ropathy) and cardiovascular complica-

_al._ , 1990; Engerman _et al._ , 1994; Brown-

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_****_

**Figure 3:** General features of chronic hyperglycaemia-induced diabetic tissue damage

(Giacco and Brownlee, 2010).

****

****

**Figure 4:** Mechanisms underlying hyperglycaemia-induced pathophysiology of diabetes

via the generation of ROS and activation of stress-sensitive signaling pathways. Each

mechanism is discussed in the text (Vincent _et al._ , 2004).

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lee, 1995; Lee _et al._ , 1995; Ganz and

1988; Li _et al._ , 1996). These results in

Seftel, 2000; Brownlee, 2005). The effect

auto-oxidation of glucose to glyoxals, de-

of oxidative stress damage is aggravated

composition of the Amadori product (glu-

by inactivation of anti-atherosclerotic en-

cose-derived 1-amino 1-deoxyfructose

zymes (endothelial nitric oxide synthase

lysine adducts, to 3-deoxyglucosone, and

(eNOS) and prostacyclin synthase. In ad-

fragmentation

of

glyceraldehyde-3-

dition, oxidative stress also activates

phosphate and dihydroxyacetone phos-

stress-sensitive signaling pathways, such

phate to methylglyoxal (Brownlee, 2001).

as nuclear redox sensitive transcription

In addition, increased AGEs production

factor (NF-κB), p38 MAPK, and NH2-

promotes the binding of AGEs to its re-

terminal Jun kinases/stress-activated pro-

ceptors (RAGE). Binding of AGEs to

tein kinases (JNK/SAPK) leads to both

RAGE induces overproduction of ROS

insulin resistance and impaired insulin

and activation of NF-kB signaling and

secretion (Folli _et al._ , 2011; Brownlee,

upregulation of intracellular adhesion

2001).

molecule-1(ICAM-1), vascular adhesion

cell molecule-1 (VCAM-1), monocyte

**7. Molecular mechanisms of hypergly-**

chemotactic protein-1 (MCP-1), PAI-1,

**caemia-induced oxidative stress in**

tissue factor, and VEGF (Yamagishi _et_

**diabetes**

_al._ , 1997; Bierhaus _et al._ , 2001).

Previous studies demonstrated that

Hyperglycaemia-induced activation of

PCK activity was increased in the retina,

polyol pathway was the first mechanism

kidney and microvasculature of diabetic

discovered (Gabbay _et al._ , 1966). This

rats (Craven, P.A. and F.R. DeRubertis,

pathway has been therapeutic target for

1989; Lee _et al._ , 1989), suggested that the

diabetes neuropathy (Oates and Mylari,

lipolytic pathway and production of di-

1999). Recent human genetic study has

acylglycerol induces PKC activation

implicated polymorphisms of the aldose

(Ishii _et al._ , 1998). Hyperglycaemia in-

reductase gene associated with increased

creases diacylglycerol synthesis, which is

risk for diabetes complications (Oates and

a critical activating co-factor for PKC

Mylari, 1999). Excess glucose activates

isoforms (Derubertis and Craven, 1994;

polyol pathway. Aldose reductase (de-

Xia _et al._ , 1994; Koya _et al._ , 1997; Koya

pendent upon NADPH as co-factor) in-

and King, 1998). PKC activation has been

creases conversion of glucose to polyal-

shown to have diverse effects on gene ex-

cohol sorbitol. Excessive activation of

pression in different cell types. PKC acti-

polyol pathway results in depletion of in-

vation inhibits insulin-stimulated endothe-

tracellular NADPH and GSH which is an

lial Nitric Oxide Synthase (eNOS) ex-

important intracellular antioxidant (Lee

pression in the endothelial cells and de-

and Chung, 1999). Accumulation of sor-

creases nitric oxide production in the

bitol forms cellular osmotic stress (Ste-

smooth muscle cells (Vlassara _et al._ ,

vens _et al._ , 1993).

1995). In vascular smooth muscle cells,

Excess glucose induces auto-

PKC activation induces over-expression

oxidation through activation of the AGEs

of fibrinolytic inhibitor, plasminogen ac-

pathway.

tivator inhibitor (PAI-1) and activation of

The AGE precursor damages cells by

NF-kB (Abordo and Thornalley, 1997).

three mechanisms: modification of pro-

PKC enhances accumulation of microvas-

teins involve in gene transcription

cular matrix protein by up-regulation of

(Giardino _et al._ , 1994; Shinohara _et al._ ,

transforming growth factor (TGF-β), fi-

1998), modification of extracellular ma-

bronectin and type 4 collagen in both cul-

trix molecules (McLellan _et al._ , 1994),

ture mesangial cells and glomeruli of dia-

and modification of circulating protein in

betic rats (Doi _et al._ , 1992). PKC also en-

the blood (e. g. albumin) (Vlassara _et al._ ,

hances vascular permeability by increas-

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ing the expression of vascular endothelial

creatic β-cells triggers oxidative stress

growth factor (VEGF) (Skolnik _et al._ ,

and ER stress, exacerbated by high circu-

1991).

lating glucose and lipids (non-esterified

Lastly, hyperglycaemia causes

fatty acid). Oxidative stress and ER stress

damage to the blood vessel through acti-

induce chemokine production and acti-

vation of hexosamine pathway. The end

vates inflammatory cells in the pancreatic

product of this pathway, uridine diphos-

islet. In turn, the activated inflammatory

phate (UDP)-N-acetyl glucosamine regu-

cells produce cytokines that further exac-

lates gene expression implicated in vascu-

erbate oxidative and ER stress and disrupt

lar complications (such as PAI -, TGF-α,

β-cell secretory pathway function. In ad-

TGF-β1). In addition, activation of hex-

dition, oxidative stress induces unfolded

osamine pathway impairs Insulin Recep-

protein response (UPR) and NF-κB acti-

tor Substrate (IRS)/phosphatidylinositol

vation. In early diabetes (manifested by

3-kinase (PI3-K)/Akt pathway, resulting

chronic ER stress and inflammation), in-

in deregulation of eNOS activity (Bucala __

creased proinsulin:insulin ratio impairs

_et al._ , 1991; Kolm-Litty _et al._ , 1998).

insulin signaling further aggravates hy-

perglycaemia. Overall, this vicious cycle

**8. Oxidative stress and β-cell dysfunc-**

leads to β-cell apoptosis and progression

**tion in diabetes**

to diabetes (summarized schematically in

Figure 5). Several mechanisms have been

Diabetes mellitus (DM) is character-

implicated in β-cell apoptosis (Nakagawa __

ized by failure of the pancreatic β-cells to

_et al._ , 2000; Oyadomari _et al._ , 2002;

maintain glucose homeostasis. Physiolog-

Puthalakath _et al._ , 2007; Song _et al._ ,

ically, the pancreatic β-cells secrete hor-

2008; Mahdi _et al._ , 2012; Supale _et al._ ,

mone insulin and regulate glucose home-

2012;

Uruno __

_et_

_al._ ,

2015).

The

ostasis. Insulin drives glucose uptake in

PERK/ATF4-mediated

activation

of

the liver (reducing hepatic gluconeogene-

CHOP

and

IRE1a/TRAF2/ASK1-

sis both directly and in conjunction with

mediated activation of JNK are important

suppression of glucagon secretion), mus-

molecular mechanisms (reviewed in Papa

cle and fat (Könner, 2007). Because of

FR 2012) (Papa, 2012). A growing body

their high biosynthetic load and require-

of evidence suggests that ER stress induc-

ment for oxygen, pancreatic β-cells are

es autophagy (an important mechanism

very vulnerable to oxidative stress (Len-

for removal of terminally misfolded pro-

zen, 2008; Newsholme _et al._ , 2012; Cao

tein from the endoplasmic reticulum (ER)

and Kaufman, 2014; Kaneto and Mat-

leads to induction of apoptosis (Wang _et_

suoka, 2015). ****

_al._ , 2014; Li _et al._ , 2012; Quan _et al._ ,

Oxidative stress and endoplasmic

2012). Another mechanism involves NF-

reticulum stress (ER) are key pathological

kB and interleukin 1 beta (IL1b) activa-

features in particular type 2 diabetes

tion (reviewed in Hasnain SZ _et al._ , 2012;

mellitus (T2DM), contribute to pancreatic

Hasnain _et al._ , 2014).

β-cell dysfunction, inducing inflammation

(immune activation) and β-cell apoptosis.

**9. Current therapeutics in diabetes**

Previous studies have suggested the oxi-

dative stress is able to suppress insulin

Good glycaemic control is the most effec-

transcription and associated with accumu-

tive mean of mitigating diabetes compli-

lation of β-amyloid in the human pancre-

cations in particular type 1 diabetes

atic islet (Kaneto and Matsuoka, 2015). In

(Greene _et al._ , 1992; Molitch _et al._ ,

obesity and early stage of diabetes, nutri-

1993). In general, drug available for dia-

ent overload leads to development of mild

betes work by reducing stress on β-cells

insulin resistance and hyperglycaemia.

biosynthesis pathway. In diabetes pa-

Increased insulin production by the pan-

tients, the use of drug that promotes insul-

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****

**Figure 5:** Schematic representation of the cycle of oxidative and ER stress and its effects

on glucose homeostasis in diabetes (Hasnain _et al._ , 2015).

-in secretion (such as sulfonylureas) is

**10. Biological effects of natural poly-**

known to causes loss of β-cell function.

**phenols on oxidative stress**

Another class of GLP-1 receptor agonist,

which promotes insulin secretion in a glu-

Oxygen is an essential element of life

cose dependent-manner, may also have

used by cells to generate energy in the

long-term damaging effect on β-cell

form of ATP whereby this process occurs

(Hasnain _et al._ , 2014). Drug that sup-

within the mitochondria (Turrens, 2003).

presses gluconeogenesis (metformin), in-

The process, however, causes the produc-

crease glucose excretion (SGLT-2 inhibi-

tion of free radicals, such as reactive oxy-

tor) or reduces peripheral insulin re-

gen species (ROS) and reactive nitrogen

sistance (thiazolidinediones) or exoge-

species (RNS) due to the cellular redox

nous insulin.

process in the cells (Pham-Huy _et al._ ,

Given that pronounced oxidative

2008). At low or moderate concentration,

stress mediates major diabetes complica-

these species exert beneficial effects on

tions, antioxidant therapy remains a novel

cellular responses and immune function,

therapeutic approach for diabetes patients.

but when the species exist at higher lev-

Antioxidant drugs target NADPH oxidas-

els, oxidative stress is generated (Young

es are unable to combat high level of oxi-

and Woodside, 2001; Halliwell, 2007;

dative stress (Li _et al_., 2012). In view that

Pham-Huy _et al._ , 2008). Oxidative stress

the IL22 receptor is the most highly ex-

refers to the balance between the produc-

pressed in human pancreatic islet cells,

tion of free radicals and antioxidant de-

studies have been identifying IL22 as

fences in a cell (Betteridge, 2000). The

novel antioxidant target in diabetes (Co-

mechanism arises when there is an unfa-

bleigh and Robek, 2013; Kumar _et al._ ,

vourable balance between the free radical

2013; Rutz _et al._ , 2013; Hasnain _et al._ ,

production and antioxidant defences,

2014; Sabat _et al._ , 2014).

which result in the damage of a broad

range of molecular species, including li-

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pids, proteins and nucleic acid in the cells

Polyphenols are a large group of

(Rock _et al._ , 1996; McCord, 2000).

natural antioxidants found mostly in

The concept of oxidative stress

fruits, vegetables, cereals and beverages

was first hypothesised in the 1950s by

(Arts and Hollman, 2005; Pandey and

researchers that investigated the toxic ef-

Rizvi, 2009). There are more than 8,000

fects of ionizing radiation, free radicals,

polyphenolic compounds that have been

and the similar toxic effects of molecular

identified in various plant species, which

oxygen (Gerschman _et al._ , 1954), as well

arise from a common intermediate, phe-

as its possible contribution to the aging

nylalanine or an immediate precursor, and

process (Harman, 1956). Interest in this

shikimic acid. Polyphenols contain phenol

field of research grew (Hybertson _et al._ ,

rings in the basic structure. Based on the

2011) when studies reported that the bio-

number of phenol rings and the basis of

logical systems are capable of producing

the structural elements that binds to these

substantial amounts of superoxide free

rings, polyphenols can be classified as

radical, O -

2 through the natural metabolic

phenolic acids, flavonoids, stilbenes and

pathways (McCord & Fridovich, 1968)

lignans (Spencer _et al._ , 2008; Pandey and

and the activity of antioxidant enzymes,

Rizvi, 2009). Epidemiological studies

superoxide dismutases (SODs) is neces-

have shown that high consumption of

sary for the survival of aerobic organisms

polyphenols lowered the risk of chronic

(McCord and Fridovich, 1969; McCord _et_

human diseases (Scalbert _et al._ , 2005;

_al._ , 1971). Besides the aging process, oxi-

Arts and Hollman, 2005). Besides that,

dative stress has been postulated to play a

the consumption of polyphenols have also

role in many other conditions, as well.

been linked with cardio-protective effect,

These conditions includes inflammatory

anti-cancer effect, anti-diabetic effect,

diseases (arthritis, vasculitis, glomerulo-

anti-aging effect and neuroprotective ef-

nephritis, lupus erythematous, adult res-

fect (Pandey and Rizvi, 2009). Several

piratory diseases syndrome), ischemic

types of research have also concluded that

diseases (heart diseases, stroke, intestinal

high phenolic content correlates with a

ischemia), hemochromatosis, acquired

good antioxidant capability and lower ox-

immunodeficiency syndrome, emphyse-

idative stress-related chronic diseases

ma, carcinogenesis, gastric ulcers, hyper-

(Aruoma, 1998; Kohen and Nyska, 2002).

tension and preeclampsia, neurological

_Clinacanthus nutans_ is an example of a

disorders (Alzheimer's disease, Parkin-

traditional herb that is rich in polyphenols

son's disease, muscular dystrophy), alco-

and has potent antioxidant capabilities

holism and smoking-related diseases

(Aromdee _et al._ , 2007; Yong _et al._ , 2013).

(Pham-Huy _et al._ , 2008; Lobo _et al._ ,

It is traditionally used to treat oxidative

2010).

stress-related diseases, such as diabetes

To protect cellular components

and various kinds of cancers (Ching _et al._ ,

from the free radical-induced damage, the

2013; P"ng _et al._ , 2013).

body possess several mechanisms to

The antioxidants protect cellular

counteract oxidative stress by producing

components by acting as radical scaven-

an extensive range of antioxidant, both

gers, hydrogen and electron donors, per-

endogenous (generated in situ) and exog-

oxide

decomposer,

singlet

oxygen

enous (supplied through food, e.g. poly-

quencher, enzyme inhibitors, synergist,

phenols) in the cells. The antioxidants can

and metal-chelating agents or by gene ex-

be divided into three main categories

pression regulation (Frei _et al._ , 1988; Lo-

which are antioxidant enzymes, chain

bo _et al._ , 2010). The antioxidant process

breaking antioxidants and transition metal

has been proposed to have two principle

binding proteins (Young and Woodside,

mechanisms of action, a chain-breaking

2001; Pham-Huy _et al._ , 2008).

mechanism and a prevention mechanism.

The first mechanism donates an electron

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from the primary antioxidant to the free

by Yim _et al._ (1990), CuZnSOD catalyzes

radical. The free radical is unstable and

hydroxyl radical (OH●) from H2O2. Dele-

highly reactive. Therefore, it either

tion of _sod1_ gene in knock-out mice was

releases or steals an electron from another

reported to show high level of oxidative

molecule in order to stabilise itself. This

damage to proteins, lipids, and DNA in

activity causes the molecule to become

skeletal muscle tissue due to lack of

unstable, thus forming a second radical.

CiZnSOD (Muller _et al._ , 2006). Compre-

The second radical then releases or steals

hensive oxidative damage was also ob-

an electron from another molecule, thus

served in liver tissues of _sod1-/-_ mice

forming

the

following

radical.This

(Elchuri _et al._ , 2005). Hence, CuZnSOD

process is continued until a chain-

is a potential antioxidant enzyme.

breaking antioxidant helps to stabilise the

Another enzymatic antioxidant de-

free radical. On the other hand, the second

fence mechanism is glutathione perox-

mechanism involves the removal of

idise (GSHPx) activity where free radicals

ROS/RNS initiators by inhibiting chain-

are reduced by glutathione into water and

initiation catalyst in the cells (Pham-Huy __

its analogous alcohols (Wendel, 1980).

_et al._ , 2008; Lobo _et al._ , 2010). Both

The selenieum-containing GSHPx is

mechanisms play a significant role in pre-

mainly localized in cytoplasm and mito-

venting oxidative stress-related diseases.

chondria (Zarowski and Tappel, 1978;

Epp _et al._ , 1983; Tappel, 1984; Timcen-

**11. The defences against free radical**

ko-Youssef, 1985). The free radical de-

**attack and oxidative stress**

fence mechanism through GSHPx is evi-

dent through the study conducted by

Human body is naturally adapted with

Hawker _et al._ (1993) who demonstrated

several defence mechanisms to counter-

an excessive ROS generation in selenium-

balance the excessive free radicals pro-

deficient mice. Although GSHPx-1 was

duced through the oxidative phosphoryla-

reported to be insignificantly contribute

tion. Enzymatic antioxidant mechanism is

against oxidative damage by evaluating

the most important defence system

carbonyl content, lipid peroxidation activ-

against free radical. One such major free

ity and rate of extracellular H2O2 con-

radical scavenging enzyme is the superox-

sumption in several tissues of _GPSH-1-/-_

ide dismutase (SOD). Manganese SOD

knock-out mice samples (Ho _et al._ , 1997),

(MnSOD) was reported to be directly in-

studies conducted by Zhang _et al._ (2009)

volved in the suspension of superoxide

revealed an elevation of free radicals in

_−_

anions which was evident by the death of

_Sod2_ +/ _−Gpx1_ / _−_ mice through evaluation

_sod2_ gene-knocked out mice within 10

of DNA and protein oxidation in several

days after birth due to dilated cardiomyo-

tissues of the transgenic mice. In addition,

+/−

pathy, lipid amassing in liver and skeletal

_Gpx4_

mice was reported to be highly

muscle and metabolic acidosis caused by

sensitive to oxidative stress (Yant _et al._ ,

the deficiency of MnSOD (Li _et al._ ,

2003; Ran _et al._ , 2003).

1995). Furthermore, tissues of those mice

Furthermore, catalase is also a po-

lacking MnSOD exhibited an elevated

tential free radical defence enzyme which

degree of oxidative damage DNA with

eliminates hydrogen peroxide by catalys-

higher rate cancer prevalence (Remmen _et_

ing it into water and oxygen in which one

_al._ , 2003). Another type of SOD is the

molecule of catalase decomposes approx-

Copper, Zinc SOD (CuZnSOD) which is

imately six million of H2O2 molecules per

mainly localized in cytoplasm, nucleus,

minute (Kellin and Hartree, 1938; Mates __

lysosome and intermembrane space of

_et al._ , 1999). Several studies have demon-

mitochondria (Chang _et al._ , 1988; Keller __

strated the defence mechanism of free

_et al._ , 1991; Crapo _et al._ , 1992; Sturtz _et_

radicals by catalyse. Transgenic mice

_al._ , 2001). Based on the study conducted

over expressing catalase ( _hCat_ Tg+/0) was

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shown to decrease the generation of H2O2

antioxidants that quench free radicals, a

which eventually resulted in reduced

physiological response usually not present

blood pressure in contrast to the wild-type

in lower elevation plants (Alonso-Amelot,

mice (Yang _et al._ , 2002). In addition,

2008).

knockout mice with under expression of

catalase by disruption of _cat_ or _cas1_ gene

_12.1._

_Verbascum_

_sinaiticum_

Benth.

was reported to show decreased rate of

(Scrophulariaceae)

decomposition of hydrogen peroxide with

The plant, _Verbascum sinaiticum_

an increased tendency to oxidative stress

is a biennial rosette plant (a taproot with a

(Ho _et al._ , 2004).

cluster of leaves on the soil surface), upon

On the other hand, there are sever-

where this species is listed as rare in

al other non-enzymatic defence mecha-

Egypt and wide-reaching to Sinai Penin-

nisms against free radicals such as vita-

sula (Hegazy, 2000). The leaves and

min E, vitamin C and Thiol antioxidants.

flowers from _Verbascum_ species __ had been

Vitamin E is a fat soluble vitamin which

utilized in the treatment of bronchitis, dry

was reported to act against lipid peroxida-

coughs, tuberculosis and asthma while

tion (Pryor, 2000). Vitamin C is a water

studies have confirmed expectorant, mu-

soluble vitamin which acts in correspond-

colytic and demulcent properties (Kozan __

ence with vitamin E. α-tocopherol is an

_et al._ , 2011). Extraordinary results were

active form of vitamin E which eliminates

obtained from _V_. _sinaiticum_ when it was

lipid peroxyl radical by donating a hydro-

observed

for

selectivity

in

anti-

gen atom and itself becoming an α-

proliferation response against carcinoma

tocopherol radical while vitamin C reduc-

and normal cells. The effect of anti-

es α-tocopherol radical into its original

proliferate was evaluated in hepatocellu-

form (Carr and Frei, 1999; Kojo, 2004).

lar carcinoma (Hep-G2) and normal

Thiol antioxidants include the tripeptide

(MRC-5) cells after exposing the cells to

glutathione (GSH), thioredoxin (TRX),

the extract for 48 hours __ (Tauchen _et al._ ,

and α-lipoic acid (ALA) also acts against

2015). __ The extract comprises composi-

free radical through redox reactions

tions of luteolin, chrysoeriol, hydrocarpin

(Rahman, 2007).

and sinaiticin which were earlier tested

for cytotoxicity reaction towards leukae-

**12. Medicinal plants as green approach**

mia P-388 cells, providing a positive

**defences against free radical attack**

feedback (Afifi _et al._ , 1993). These com-

pounds falls under the classification of

In recent times, substantial evidence

flavonoids and flavonolignans (Afifi _et_

emerged indicating a mutual relationship

_al._ , 1993 and Mahmoud _et al._ , 2007) to

between the consumption of antioxidant-

which they will not cause possible haz-

rich foods and the episodes of human

ards to consumer and consequently an

health deterioration (Sies, 1997). Con-

attribution

to

the

selective

anti-

versely, when synthetic antioxidants

proliferative activity of _V_. _sinaiticum_

namely butylated hydroxytoluene (BHT)

against Hep-G2 and MRC-5 cell line

and butylated hydroxyanisole (BHA)

(Tauchen _et al._ , 2015).

were extensively employed as additives in

food manufacturing business, a multitude

_12.2. Ugni myricoides_ (Kunth) O.Berg

of cases were reported on liver injuries

_Ugni myricoides_ are inhabitants of

and carcinogenesis (Grice, 1988; Wichi,

western Latin America (WCSP, 2016),

1986). Considering this fact, attentions

possessing the properties for anti-

were shifted into utilizing natural antioxi-

hypernociceptive effect (Quintão _et al._ ,

dants. Plants, given the fact that can grow

2010) and anti-nociceptive effects in mice

successfully adapting to the exposure of

(Campêlo, 2011). Brovo _et al._ , had car-

ultraviolet, are also reservoir for potent

ried out an investigation between _U. my-_

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_ricoides_ fruits antioxidant properties and

indigenous medicine for the administra-

their inhibitory interaction against skin

tion of many ailments. It also has measur-

aging-related enzymes. _U. myricoides_ has

ably multitude biological activities asso-

been measured for high values in mitigat-

ciated to antioxidant, anti-inflammatory

ing free radical damages of UV light in

and cancer preventive properties (Teiten __

skin, by both the ORAC (Oxygen radical

_et al._ , 2010; Goel _et al._ , 2008; Hatcher _et_

absorbance capacity) and TEAC assays

_al._ , 2008).

(The Trolox Equivalent Antioxidant Ca-

In a paper reported by Dall'Acqua __

pacity). As a result, _U. myricoides_ were

_et al._ , a pattern of significant changes

determine to retain high total phenolic

were observed in the urinary metabolome

content, with high correlation coefficient

of healthy rats, orally treated with curcu-

above 0.80 and its aptitude in __ hindering

min, compared to controls in data sets ob-

collagenase, elastase, hyaluronidase and

tained both by NMR and HPLC–MS

tyrosinase enzymes propose that the pres-

(Dall'Acqua _et al._ , 2016). The effect of

ence of high values of polyphenols are

Curcuma extract on urinary composition

potential conducive for these responses

in healthy rats disclosed evidence for _in_

(Bravo _et al._ , 2016).

_vivo_ reduction of the amount of urinary

biomarkers of oxidative stress namely al-

12.3. _Black highland barley (BHLPE)_

lantoin, 3-nitrotyrosine, m-tyrosine, 8-

The black highland barley known

OHdG. The m-Tyrosine is examined as a

in Tibet for a crop of high importance is

biomarker for oxidative damage to pro-

referred as Qing Ke in Chinese. The po-

teins while urinary 8-OHdG (Orhan _et al._ ,

tential of BHLPE was measured and the

2004) is measured as a biomarker of

findings exhibited potent superoxide radi-

comprehensive cellular oxidative stress

cal, hydroxyl radical and 2,2-diphenyl-1-

due to its prevalent generation of oxidized

picrylhydrazyl radical-scavenging activi-

DNA repair (Tsikas _et al._ , 2005).

ty, ferric reducing antioxidant power and

moderate metal ion-chelating activity

_12.5. Beta vulgaris_ L.

(Shen _et al._ , 2016). Two groups of mice

Beetroot ( _Beta vulgaris_ ) is well

were prepared; a high fat diet (HFD)

known for its high total phenolic content

group and a group that has been adminis-

(50–60 μmol/g dry weight) (Kähkönen _et_

trated with 600 mg BHLPE/kg body

_al._ , 1999; Vinson _et al._ , 1998), and the

weight. The treated group of mice dis-

presence of considerable amount of phe-

played remarkable reduction in total cho-

nolic acids: ferulic, protocatechuic, vanil-

lesterol, low-density lipoprotein choles-

lic, _p_ -coumaric, _p_ -hydroxybenzoic and

terol and the atherosclerosis index. The _in_

syringic acids (Kujala, _et al._ , 2000). It al-

_vivo_ test further provides information on

so a source of betalains, water-soluble

antioxidant defence system and antioxi-

nitrogenous pigments that feed on reac-

dant gene expression where significant

tive oxygen species (ROS) to protect its

amelioration had occurred within BHLPE

plant from wound injuries and pathogenic

treated group as compared to HFD mice

penetration as studied in red beet

group. Conclusively, the study highlight-

(Sepúlveda-Jiménez _et al._ , 2004). Five

ed the tendency of natural antioxidant of

beetroot pomace extracts from different

BHLPE in generating good health by re-

cultivar were studied upon where Detroit

ducing the incident of disease (Shen _et al._ ,

beetroot pomace extract (DBPE) exhibit-

2016).

ed the highest antiradical properties _in_

_vitro_ , by effectively scavenging DPPH

_12.4. Curcuma longa_ L.

radicals (EC50=2.06±0.10 μg/ml) and high

_Curcuma longa_ L is a spice that

reducing

power

(EC50=123.39±06.05

has considerable usage in the practise of

μg/ml). The effect of _in vivo_ antioxidant

Ayurveda, Unani, Siddha, and Chinese

and hepatoprotective activities indicated

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_Biotech Sustainability (2017)_

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reduction in the measurement levels of

**Adams, L., Franco, M. C. and Estevez,**

(xanthine oxidase, catalase-CAT, peroxi-

**A. G. (2015).** Reactive nitrogen spe-

dase, glutathione peroxidase-GSHPx, glu-

cies in cellular signaling. _Experi-_

tathione reductase, glutathione-GSH and

_mental Biology and Medicine (May-_

thiobarbituric acid reactive substance with

_wood)_ **240, 711-7.**

the administration of DBPE in doses of 2

**Adefegha, S.A. and Oboh, G. (2012).** In

and 3 ml/kg body weight (Vulic _et al._ ,

vitro inhibition activity of polyphenol-

2012; Vulic _et al._ , 2014).

rich extracts from Syzygium aromati-

cum (L.) Merr. & Perry (Clove) buds

_12.6. Syzygium aromaticum_ L.

against carbohydrate hydrolyzing en-

Clove ( _Syzygium aromaticum_ ) is a

zymes linked to type 2 diabetes and

spice regularly used in Asia and many

Fe2+-induced lipid peroxidation in rat

parts of the world for cooking. The com-

pancreas. _Asian Pacific Journal of_

position of _S. aromaticum_ widens its

_Tropical Biomedicine_ **2, 774–781.**

properties ranging from __ antioxidant, anti-

**Afifi, M. S. A., Ahmed, M. M., Pez-**

fungal, anti-viral, anti-microbial to anti-

**zuto, J. M. and Kinghornt, D. A.**

diabetic, anti-inflammatory, antithrombot-

**(1993).** Cytotoxic flavonolignans and

ic, anaesthetic, pain reliving and insect

flavones from verbascum sinaiticum

repellent properties (Parle and Khanna,

leaves. _Phytochemistry_ **34, 839–841.**

2011). Clove buds creates insulin-like in-

**Aguilar-Santamaría, L., Ramírez, G.,**

teraction with hepatocytes and hepatoma

**Nicasio, P., Alegría-Reyes, C. and**

cells by reducing phosphoenolpyruvate

**Herrera-Arellano, A. (2009).** Antidi-

carboxykinase and glucose 6-phosphatase

abetic activities of Tecoma stans (L.)

gene expression (Prasad _et al._ , 2005). It is

Juss. ex Kunth _. Journal of Eth-_

a vital step to reduce postprandial hyper-

_nopharmacology_ **124, 284–8.**

glycemia peak for the treatment of diabe-

**Alberts, B., Alexander, J., Julian, L.,**

tes (Aguilar-Santamaría _et al._ , 2009).The

**Martin, R., Keith, R. and Peter, W.**

presence of phenolic (free and bound) ap-

**(1994).** Molecular Biology of the Cell,

pears to hinder alpha-amylase and alpha-

Garland Publishing Inc., New York,

glucosidase in a dose-dependent manner.

USA.

The extracts displayed high antioxidant

**Alonso-Amelot, M. E. (2008).** High alti-

activities as exemplified by their high

tude plants, chemistry of acclimation

reducing power, 1,1 diphenyl-2- pic-

and adaptation. _Studies in Natural_

rylhydrazyl (DPPH) and 2, 2-azinobis-3-

_Products Chemistry_ **34, 883–982.**

ethylbenzo-thiazoline-6-sulfonate (ABTS)

**Ames, B.N., Shigenaga, M.K. and Ha-**

radical scavenging abilities, as well as

**gen, T.M. (1993).** Oxidants, Antioxi-

inhibition of Fe2+-induced lipid peroxida-

dants, and the Degenerative Diseases

tion in rat pancreas _in vitro_ (Adefegha and

of Aging. _Proceedings of the National_

Oboh, 2012).

_Academy of Sciences USA_ **90, 7915-**

**7922.**

**References:**

**Annor, F. B., Goodman, M., Okosun, I.**

**S., Wilmot, D. W., Il'yasova, D.,**

**Abordo, E. A. and Thornalley, P. J.**

**Ndirangu, M. and Lakkur, S.**

**(1997).** Synthesis and secretion of tu-

**(2015).** Oxidative stress, oxidative

mour necrosis factor-α by human

balance score,

and hypertension

monocytic THP-1 cells and chemotax-

among a racially diverse population.

is induced by human serum albumin

_Journal of the American Sociecty of_

derivatives modified with methylgly-

_Hypertension_ **9, 592-599.**

oxal and glucose-derived advanced

**Aromdee, C., Kongyingyoes, B., Lau-**

glycation endproducts. _Immunology_

**pattarakasem, P., Kukongviriya-**

_Letters_ **58, 139-147.**

**pan, U., Kukongviriyapan, V. and**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 157

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**Pannangpetch, P. (2007).** Antioxi-

swords in cellular redox state: Health

dant activity and protective effect

beneficial effects at physiologic doses

against oxidative hemolysis of Clina-

versus deleterious effects at high dos-

canthus nutans (Burm.f) Lindau.

es. _Oxidative Medicine and Cellular_

_Songklanakarin Journal of Science_

_Longevity_ **3, 228-237.**

_and Technology_ **29, 1–9.**

**Bravo,K., Alzate, F. and Osorio, E.**

**Arts, I.C.W. and Hollman, P.C.H.**

**(2016).** Fruits of selected wild and

**(2005).** Polyphenols and disease risk

cultivated Andean plants as sources of

in epidemiologic studies. _The Ameri-_

potential compounds with antioxidant

_can Journal of Clinical Nutrition_ **81,**

and anti-aging activity. Industrial

**317S–325S.**

Crops and Products, In Press, Correct-

**Aruoma, D.O.I. (1998).** Free radicals,

ed Proof.

oxidative stress, and antioxidants in

**Browne, S.E., Roberts, L. J., Dennery,**

human health and disease. _Journal of_

**P. A., Doctrow, S. R., Beal, M. F.,**

_the American Oil Chemists' Society_

**Barlow, C. and Levine, R. L. (2004).**

**75, 199–212.**

Treatment with a catalytic antioxidant

**Balaban, R.S., Nemoto, S. and Finkel,**

corrects the neurobehavioral defect in

**(2005).** Mitochondria, oxidants, and

ataxia-telangiectasiamice. _Free Radi-_

aging. _Cell_ **120, 483–495**.

_cal Biology and Medicine_ **36, 938–**

**Bertin G., Averbeck D. (2006).** Cadmi-

**942.**

um: cellular effects, modifications of

**Brownlee, M. (1995).** Advanced protein

biomolecules, modulation of DNA re-

glycosylation in diabetes and aging.

pair and genotoxic consequences. _Bi-_

_Annual Review of Medicine_ **46, 223-**

_ochimie_ **88, 1549–59.**

**234.**

**Betteridge, D. J. (2000).** What is oxida-

**Brownlee, M. (2001).** Biochemistry and

tive stress? _Metabolism, Clinical and_

molecular cell biology of diabetic

_Experimental_ **49, 3–8.**

complications. _Nature_ **414, 813-820.**

**Bender, A., Krishnan, K.J., Morris,**

**Brownlee, M. (2005).** The pathobiology

**C.M., Taylor, G.A., Reeve, A.K.,**

of diabetic complications: a unifying

**Perry, R.H., Jaros, E., Hersheson,**

mechanism. _Diabetes_ **54, 1615-1625.**

**J.S., Betts, J., Klopstock, T., Taylor,**

**Bucala, R., Tracey, K. J. and Cerami,**

**R.W. and Turnbull, D.M. (2006).**

**A. (1991).** Advanced glycosylation

High levels of mitochondrial DNA de-

products quench nitric oxide and me-

letions in substantia nigra neurons in

diate

defective

endothelium-

aging and Parkinson disease. _Nature_

dependent vasodilatation in experi-

_Genetics_ **38, 515–517.**

mental diabetes. _Journal of Clinical_

****

_Investigation_ **87, 432-438.**

**Bierhaus, A., Schiekofer, S., Schwan-**

**Campêlo, L. M. L., Almeida, A. A. C.,**

**inger, M., Andrassy, M., Humpert,**

**Freitas, R. L. M., Cerqueira, G. S.,**

**P. M., Chen, J., Hong, M., Luther,**

**Sousa, G. F., Saldanha, G. B.,**

**T., Henle, T., Klöting, I., Morcos,**

**Feitosa, C. M. and Freitas, R. M.**

**M., Hofmann, M., Tritschler, H.,**

**(2011).** Antioxidant and Antinocicep-

**Weigle, B., Kasper, M., Smith, M.,**

tive Effects of Citrus limon Essential

**Perry, G., Schmidt, A. M., Stern, D.**

Oil in Mice. _Journal of Biomedicine_

**M., Häring, H. U., Schleicher, E.**

_and Biotechnology_ **2011, 678673.**

**and Nawroth, P. P. (2001).** Diabetes-

**Cao, S. S. and Kaufman, R. J. (2014).**

associated sustained activation of the

Endoplasmic reticulum stress and ox-

transcription factor nuclear factor-

idative stress in cell fate decision and

kappaB. _Diabetes_ **50, 2792-808.**

human disease. _Antioxidants and Re-_

**Bouayed, J. and Bohn, T. (2010).** Exog-

_dox Signaling_ **21, 396-413.**

enous

antioxidants-Double-edged

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 158

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**Caramori, G., Adcock, I. M., Casolari,**

betic rats. Possible mediation by glu-

**P., Ito, K., Jazrawi, E., Tsaprouni,**

cose. _Journal of Clinical Investiga-_

**L., Villetti, G., Civelli, M., Carnini,**

_tion,_ **83, 1667-1675.**

**C., Chung, K. F., Barnes, P. J. and**

**Cui, H., Kong, Y. and Zhang, H.**

**Papi A. (2011).** Unbalanced oxidant-

**(2012).** Oxidative stress, mitochondri-

induced DNA damage and repair in

al dysfunction, and aging. _Journal of_

COPD: a link towards lung cancer.

_signal transduction_ **2012, 646354**.

_Thorax_ **66, 521-527.**

**Dahlgren, C. and Karlsson, A. (1999).**

**Carr, A., and Frei, B. (1999).** Does vit-

Respiratory burst in human neutro-

amin C act as a pro-oxidant under

phils. _Journal of Immunological_

physiological

conditions?

_FASEB_

_Methods_ **232, 3-14.**

_Journal_ **13, 1007-1024.**

**Dall'Acqua,**

**S.,**

****

**Stocchero,**

**M.,**

**Chang, K.C., Chung, S. Y., Chong, W.**

**Boschiero, I., Schiavon, M., Gol-**

**S., Suh, J. S., Kim, S. H., Noh, H.**

**ob,S., Uddin,J., Voinovich, D.,**

**K., Seong, B. W., Ko, H. J. and**

**Mammi,S. and Schievano E. (2016).**

**Chun, K. W. (1993).** Possible super-

New findings on the in vivo antioxi-

oxide radical-induced alteration of

dant activity of Curcuma longa extract

vascular reactivity in aortas from

by an integrated 1H NMR and HPLC–

streptozotocin-treated rats. _Journal of_

MS metabolomic approach. _Fitotera-_

_Pharmacology_

_and_

_Experimental_

_pia_ **109, 125-131.**

_Therapeutics_ **266, 992-1000.**

**Delmastro-Greenwood, M. M. and Pi-**

**Chang, L. Y., Slot, J. W., Geuze, H. J.,**

**ganelli, J. D. (2013).** Changing the

**and Crapo, J. D. (1988).** Molecular

energy of an immune response. _Amer-_

immunocytochemistry of the CuZn

_ican Journal of Clinical and Experi-_

superoxide dismutase in rat hepato-

_mental Immunology_ **2, 30-54.**

cytes. _The Journal of Cell Biology_

**Derubertis, F. R. and Craven, P. A.**

**107, 2169-2179.**

**(1994).** Activation of protein kinase C

**Ching, S. M., Zakaria, Z. A., Paimin, F.**

in glomerular cells in diabetes. Mech-

**and Jalalian, M. (2013).** Comple-

anisms and potential links to the path-

mentary alternative medicine use

ogenesis of diabetic glomerulopathy.

among patients with type 2 diabetes

_Diabetes_ **43, 1-8.**

mellitus in the primary care setting: a

**Diplock, A. T., Charleux, J. L., Cro-**

cross-sectional study in Malaysia.

**zier-Willi, G., Kok, F. J., Rice-**

_BMC Complementary and Alternative_

**Evans, C., Roberfroid, M., Stahl,**

_Medicine_ **13, 148**.

**W. and Viña-Ribes, J. (1998).** Func-

**Cobleigh, M. A. and Robek, M. D.**

tional food science and defence

**(2013).** Protective and pathological

against reactive oxidative species. _The_

properties of IL-22 in liver disease:

_British Journal of Nutrition_ **80, S77-**

implications for viral hepatitis. _Ameri-_

**112.**

_can Journal of Pathology_ **182, 21-28.**

**Dixon, S. J. and Stockwell, B. R. (2014).**

**Crapo, J. D., Oury, T., Rabouille, C.,**

The role of iron and reactive oxygen

**Slot, J. W. and Chang, L. Y. (1992).**

species in cell death. _Nature chemical_

Copper,zinc superoxide dismutase is

_biology_ **10, 9-17.**

primarily a cytosolic protein in human

**Doi, T., Vlassara, H., Kirstein, M.,**

cells _. Proceedings of The National_

**Yamada, Y., Striker, G. E. and**

_Academy of Sciences of the United_

**Striker, L. J. (1992).** Receptor-

_States of America_ **89(21), 10405-**

specific increase in extracellular ma-

**10409**.

trix production in mouse mesangial

**Craven, P. A. and DeRubertis, F. R.**

cells by advanced glycosylation end

**(1989).** Protein kinase C is activated

products is mediated via platelet-

in glomeruli from streptozotocin dia-

derived growth factor. _Proceedings of_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 159

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

_the National Academy of Sciences of_

**Ganz, M. B. and Seftel, A. (2000).** Glu-

_the United States of America_ **89,**

cose-induced changes in protein ki-

**2873-2877.**

nase C and nitric oxide are prevented

**Elchuri, S., Oberley, T. D., Qi, W., Ei-**

by vitamin E. _American Journal of_

**senstein, R. S., Roberts, L. J.,**

_Physiology. Endocrinology and Me-_

**Remmen, V. H., Epstein, C. J., and**

_tabolism_ **278, E146-152.**

**Huang, T. (2005).** CuZnSOD defi-

**Gerschman, R., Gilbert, D. L., Nye, S.**

ciency leads to persistent and wide-

**W., Dwyer, P., Fenn, W. O. (1954).**

spread oxidative damage and hepato-

Oxygen Poisoning and X-irradiation:

carcinogenesis later in life. _Oncogene_

A Mechanism in Common. _Science_

**24 367–380.**

**119,**

**623–626.**

**Engerman, R. L., Kern, T. S. and Lar-**

DOI:10.1126/science.119.3097.623.

**son, M. E. (1994).** Nerve conduction

**Giacco, F. and Brownlee, M. (2010).**

and aldose reductase inhibition during

Oxidative stress and diabetic compli-

5 years of diabetes or galactosaemia

cations. _Circulation Research_ **107,**

in dogs. _Diabetologia_ **37, 141-144.**

**1058-1070.**

**Epp, D., Landenstein, R., and Wendel,**

**Giannoni, E., Fiaschi, T., Ramponi, G.,**

**A. (1983).** The refined structure of the

**Chiarugi, P. (2009).** Redox regula-

selenoenzyme glutathione peroxidase

tion of anoikis resistance of metastatic

at 0.2-nm resolution. _European Jour-_

prostate cancer cells: key role for Src

_nal of Biochemostry_ 133(1): 51–69

and EGFR-mediated pro-survival sig-

**Finaud, J., Lac, G., and Filaire, E.**

nals. _Oncogene_ **28, 2074–2086.**

**(2006).** Oxidative stress. _Sports medi-_

**Giardino, I., Edelstein, D. and Brown-**

_cine_ **36, 327-358.**

**lee, M. (1994).** Nonenzymatic glyco-

**Folli, F., Corradi, D., Fanti, P., Davalli,**

sylation in vitro and in bovine endo-

**A., Paez, A., Giaccari, A., Perego,**

thelial cells alters basic fibroblast

**C. and Muscogiuri, G. (2011).** The

growth factor activity. A model for in-

role of oxidative stress in the patho-

tracellular glycosylation in diabetes.

genesis of type 2 diabetes mellitus

_Journal of Clinical Investigation_ **94,**

micro- and macrovascular complica-

**110-117.**

tions: avenues for a mechanistic-based

**Goel, A., Kunnumakkara, A. B. and**

therapeutic approach. _Current Diabe-_

**Aggarwal, B. B. (2008).** Curcumin as

_tes Reviews_ **7, 313-24.**

"Curecumin": from kitchen to clinic.

**Frei, B., Stocker, R., Ames, B.N. (1988).**

_Biochemical Pharmacology_ **75, 787–**

Antioxidant defenses and lipid perox-

**809.**

idation in human blood plasma. _Pro-_

**Greene, D. A., Sima, A. A. F., Stevens,**

_ceedings of the National Academy of_

**M. J., Feldman, E. L. and Lattimer,**

_Sciences of the United States of Amer-_

**S. A. (1992).** Complications: neuropa-

_ica_ **85, 9748–9752.**

thy, pathogenetic considerations. _Dia-_

**Gabbay, K. H., Merola, L. O. and**

_betes Care_ **15, 1902-1925.**

**Field, R. A. (1966).** Sorbitol Path-

**Grice, H. P. (1988).** Enhanced tumour

way: Presence in Nerve and Cord with

development by butylated hydroxyan-

Substrate Accumulation in Diabetes.

isole (BHA) from the prospective of

_Science_ **151, 209-210.**

effect on fore-stomach and oesopha-

**Gaede,**

**P.,**

**Lund-Andersen,**

**H.,**

geal squamous epithelium. _Food_

**Parving, H. and Pedersen, O.**

_Chemical Toxicology_ **26, 717-723.**

**(2008).** Effect of a multifactorial in-

**Griffiths, H. R. (2005).** ROS as signal-

tervention on mortality in type 2 dia-

ling molecules in T cells--evidence for

betes. _The New England Journal of_

abnormal redox signalling in the auto-

_Medicine_ **358, 580-591.**

immune disease, rheumatoid arthritis.

_Redox Report_ **10, 273-280.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 160

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**Halliwell, B. (1993).** The chemistry of

**Hegazy, A. K. (2000).** Intra-population

free radicals. _Toxicology and Indus-_

variation in reproductive ecology and

_trial Health_ **9, 1-21.**

resource allocation of the rare biennial

**Halliwell, B. (2007).** Biochemistry of ox-

species Verbascum sinaiticum Benth.,

idative stress. Biochemical Society

in Egypt. _Journal of Arid Environ-_

Transactions.

**35,**

**1147–1150.**

_ments_ **44, 185–196.**

DOI:10.1042/BST0351147

**Ho, Y. S., Xiong, Y., Ma, W., Spector,**

**Halliwell, B. and Gutteridge, J. M.**

**A., and Ho, D. S. (2004).** Mice lack-

**(2015).** Free radicals in biology and

ing catalase develop normally but

medicine. Oxford University Press,

show differential sensitivity to oxidant

USA.

tissue injury. _The Journal of Biologi-_

**Harman, D. (1956).** Aging: a theory

_cal Chemistry_ **279(31), 32804-32812**.

based on free radical and radiation

**Ho, Y., Magnenat, J., Bronsoni, R. T.,**

chemistry. _The Journals of Gerontol-_

**Cao, J., Gargano, M., Sugawara,**

_ogy_ **11, 298–300.**

**M., and Funk, C. D. (1997).** Mice

**Hasnain, S. Z., Lourie, R., Das, I.,**

Deficient in Cellular Glutathione Pe-

**Chen, A. C. and McGuckin, M. A.**

roxidase Develop Normally and Show

**(2012).** The interplay between endo-

No Increased Sensitivity to Hyperox-

plasmic reticulum stress and inflam-

ia. _The Journal of Biological Chemis-_

mation. _Immunology and Cell Biology_

_try_ **272(26), 16644–16651**.

**90, 260-270.**

**Holmström, K. M. and Finkel T.**

**Hasnain, S. Z., Prins, J. B. and**

**(2014).** Cellular mechanisms and

**McGuckin, M. (2015).** Oxidative and

physiological consequences of redox-

endoplasmic reticulum stress in β-cell

dependent signalling. _Nature Reviews_

dysfunction in diabetes. _Journal of_

_Molecular Cell Biology_ **15, 411–421.**

_Molecular_

_Endocrinology_.

DOI:

**Hybertson, B. M., Gao, B., Bose, S. K.,**

10.1530/JME-15-0232.

**McCord, J. M. (2011).** Oxidative

**Hasnain, S.Z., Borg, D. J., Harcourt,**

stress in health and disease: The ther-

**B. E.,**

**Tong, H.,**

**Sheng, Y.**

apeutic potential of Nrf2 activation.

**H., Ng, C. P, Das, I., Wang, R.,**

_Molecular Aspects of Medicine, Oxi-_

****

**Chen, A. C., Loudovaris,**

**T.,**

_dative Damage and Disease_ **32, 234–**

**Kay, T. W., Thomas, H. E., White-**

**246.**

**head, J. P.,**

**Forbes,**

**J.**

**M.,**

**Ishii, H., Koya, D. and King, G. L.**

****

**Prins, J. B. and McGuckin, M.**

**(1998).** Protein kinase C activation

**A. (2014).** Glycemic control in diabe-

and its role in the development of vas-

tes is restored by therapeutic manipu-

cular complications in diabetes melli-

lation of cytokines that regulate beta

tus. _Journal of Molecular Medicine_

cell stress. _Nature Medicine_ **20, 1417-**

_(Berlin, Germany)_ **76, 21-31.**

**1426**.

**Kähkönen, M. P., Hopia, A. I., Vuorela,**

**Hatcher, H., Planalp, R., Cho, J., Torti,**

**H. J., Rauha, J. P., Pihlaja, K.,**

**F. M. and Torti, S. V. (2008).** Cur-

**Kujala, T. S. and Heinonen, M.**

cumin: from ancient medicine to cur-

**(1999).** Antioxidant activity of plant

rent clinical trials. _Cellular and Mo-_

extracts containing phenolic com-

_lecular Life Science_ **65, 1631–1652.**

pounds. _Journal of Agricultural and_

**Hawker, F. H., Ward, H. E., Stewart, P.**

_Food Chemistry_ **47, 3954–3962.**

**M., Wynne, L. A., and Snitch, P. J.**

**Kaneto, H. and Matsuoka, T. A. (2015).**

**(1993).** Selenium deficiency augments

Role of Pancreatic Transcription Fac-

the pulmonary toxic effects of oxygen

tors in Maintenance of Mature β-Cell

exposure in the rat. _European Respir-_

Function. _International Journal of_

_atory Journal_ 6: 1317–1323.

_Molecular Sciences ****_**16, 6281.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 161

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**Kasic, T., Colombo, P., Soldani, C.,**

**Kojo, S. (2004).** Vitamin C: basic metab-

**Wang, C. M., Miranda, E., Roncal-**

olism and its function as an index of

**li, M., Bronte, V. and Viola, A.**

oxidative stress. _Current Medical_

**(2011).** Modulation of human T-cell

_Chemistry_ **11, 1041-1064.**

functions by reactive nitrogen species.

**Kolm-Litty, V., Sauer, U., Nerlich, A.,**

_European Journal of Immunology_ **41,**

**Lehmann, R. and Schleicher, E. D.**

**1843-1849.**

**(1998).** High glucose-induced trans-

**Kayama,**

**Y.,**

**Raaz,**

**U.,** **Jagger,**

forming growth factor beta1 produc-

**A.,** **Adam,**

**M.,** **Schellinger,**

**I.**

tion is mediated by the hexosamine

**N.,** **Sakamoto,**

**M.,** **Suzuki,**

pathway

in

porcine

glomerular

**H.,** **Toyama,**

**K.,** **Spin,**

**J.**

**M.**

mesangial cells. _Journal of Clinical_

_

_**and Tsao, P. S. ****(2015).** Diabetic Car-Investigation_ **101, 160-169.**

diovascular Disease Induced by Oxi-

**Könner, A.C., Janoschek, R., Plum, L.,**

dative Stress. _International Journal of_

**Jordan, S. D., Rother, E., Ma, X.,**

_Molecular Sciences_ **16, 25234-25263.**

**Xu, C., Enriori, P., Hampel, B.,**

**Keane, K.N.,Cruzat, ****V. F., Carlessi, **

**Barsh, G. S., Kahn, C. R., Cowley,**

**R., **

**Bittencourt,**

**P.**

**I.**

**M. A., Ashcroft, F. M. and Brüning,**

**H.** **and Newsholme, ****P. (2015).** Mo-J. C. (2007). Insulin Action in AgRP-

lecular Events Linking Oxidative

Expressing Neurons Is Required for

Stress and Inflammation to Insulin

Suppression of Hepatic Glucose Pro-

Resistance and beta-Cell Dysfunction.

duction. _Cell Metabolism_ **5, 438-449.**

_Oxidative Medicine and Cellular_

**Koya, D. and King, G. L. (1998).** Pro-

_Longevity_ **2015, 181643.**

tein kinase C activation and the de-

**Keller, G. A., Warner, T. G., Steimer,**

velopment of diabetic complications.

**K. S., and Hallewell, R. A. (1991).**

_Diabetes_ **47, 859-66.**

Cu,Zn superoxide dismutase is a pe-

**Koya, D., Jirousek, M. R., Lin, Y. W.,**

roxisomal enzyme in human fibro-

**Ishii, H., Kuboki, K. and King, G.**

blasts and hepatoma cells. _Proceed-_

**L. (1997).** Characterization of protein

_ings of The National Academy of Sci-_

kinase C beta isoform activation on

_ences of the United States of America_

the gene expression of transforming

**88(16), 7381-7385.**

growth factor-beta, extracellular ma-

**Kellin, D., and Hartree, E. F. (1938).**

trix components, and prostanoids in

On the Mechanism of the Decomposi-

the glomeruli of diabetic rats. _Journal_

tion of Hydrogen Peroxide by Cata-

_of Clinical Investigation_ **100, 115-**

lase. _Proceedings of the Royal Society_

**126.**

_of London. Series B, Biological Sci-_

**Kozan, E., Çankaya, I. T., Kahraman,**

_ences ****_**124, 397-405.**

**C., Akkol, E. K. and Akdemir, Z.**

**Klaunig, J. E. and Kamendulis, L. M.**

**(2011).** The in vivo anthelmintic effi-

**(2007).** Chapter 8: chemical carcino-

cacy of some Verbascum species

genesis. In Casarett & Doull"s Toxi-

growing in Turkey. _Experimental_

cology: The Basic Science of Poisons

_Parasitology_ **129, 211-214.**

(Klaassen C. D., ed.), 7th ed.

**Kujala, T. S., Loponen, J. M., Klika, K.**

McGraw-Hill, New York. **pp. 329–**

**D. and Pihlaja, K. (2000).** Phenolics

**380.**

and betacyanins in red beetroot (Beta

**Kohen, R. and Nyska, A. (2002).** Invited

vulgaris) root: distribution and effect

Review: Oxidation of Biological Sys-

of cold storage on the content of total

tems: Oxidative Stress Phenomena,

phenolics and three individual com-

Antioxidants, Redox Reactions, and

pounds. _Journal of Agricultural and_

Methods for Their Quantification.

_Food Chemistry_ **48, 5338–5342.**

_Toxicologic Pathology_ **30, 620–650.**

**Kumar, P., Rajasekaran, K., Palmer, J.**

**M., Thakar, M. S. and Malarkan-**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 162

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**nan, S. (2013).** IL-22: An Evolution-

**Li, N., Li, B., Brun, T., Deffert-**

ary Missing-Link Authenticating the

**Delbouille, C., Mahiout, Z., Daali,**

Role of the Immune System in Tissue

**Y., Ma, X. J., Krause, K. H. and**

Regeneration. _Journal of Cancer_ **4,**

**Maechler, P. (2012).** NADPH Oxi-

**57-65.**

dase NOX2 Defines a New Antago-

**Kuster, G. M., Hauselmann, S. P.,**

nistic Role for Reactive Oxygen Spe-

**Rosc-Schluter, B. I., Lorenz, V. and**

cies and cAMP/PKA in the Regula-

**Pfister, O. (2010).** Reactive oxy-

tion of Insulin Secretion. _Diabetes_ **61,**

gen/nitrogen species and the myocar-

**2842-2850.**

dial cell homeostasis: an ambiguous

**Li, Y. M., Mitsuhashi, T., Wojciechow-**

relationship. _Antioxidants and Redox_

**icz, D., Shimizu, N., Li, J., Stitt, A.,**

_Signaling_ **13: 1899-910.**

**He, C., Banerjee, D. and Vlassara,**

**Lawrence, A.L., Douglas, C.W. and**

**H. (1996).** Molecular identity and cel-

**George, M.M.** **(2005).** The mito-

lular distribution of advanced gly-

chondrial theory of aging and its rela-

cation endproduct receptors: relation-

tionship to reactive oxygen species

ship of p60 to OST-48 and p90 to

damage and somatic mtDNA muta-

80K-H membrane proteins. _Proceed-_

tions", _Proceedings of the National_

_ings of the National Academy of Sci-_

_Academy of Sciences_ _USA_ **102(52),**

_ences USA._ **93, 11047-11052.**

**18769–18770**.

**Li, Y., Huang, T. T., Carlson, E. J., Me-**

**Lee, A. Y. and Chung, S. S. (1999).**

**lov, S., Ursell, P. C., Olson, J. L.,**

Contributions of polyol pathway to

**Noble, L. J., Yoshimura, M. P.,**

oxidative stress in diabetic cataract.

**Berger, C., Chan, P. H., Wallace, D.**

_The FASEB Journal_ **13, 23-30.**

**C., and Epstein, C. J. (1995).** Dilated

**Lee, A. Y., Chung, S. K., & Chung, S. **

cardiomyopathy and neonatal lethality

**S. (1995).** Demonstration that polyol

in mutant mice lacking manganese

accumulation is responsible for dia-

superoxide dismutase. _Nature Genet-_

betic cataract by the use of transgenic

_ics_ **11, 376-381.**

mice expressing the aldose reductase

**Lobo, V., Patil, A., Phatak, A. and**

gene in the lens. Proceedings of the

**Chandra, N. (2010).** Free radicals,

National Academy of Sciences of the

antioxidants and functional foods:

United States of America, 92(7), **pp.**

Impact on human health. _Pharmacog-_

**2780–2784.**

_nosy_

_Reviews_

**4,**

**118–126.**

**Lee, T. S., Saltsman, K. A., Ohashi, H.**

DOI:10.4103/0973-7847.70902

**and King, G. L. (1989).** Activation of

**Mahdi, T., Hänzelmann, S., Salehi, A.,**

protein kinase C by elevation of glu-

**Muhammed, S. J., Reinbothe, T.**

cose concentration: proposal for a

**M., Tang, Y., Axelsson, A. S., Zhou,**

mechanism in the development of di-

**Y., Jing, X., Almgren, P., Krus, U.,**

abetic vascular complications. Pro-

**Taneera, J., Blom, A. M., Lyssenko,**

ceedings of the National Academy of

**V., Esguerra, J. L., Hansson, O.,**

Sciences U S A. 86, **pp. 5141-5145.**

**Eliasson, L., Derry, J., Zhang, E.,**

**Lenzen, S. (2008).** Oxidative stress: the

**Wollheim,**

**C.**

**B.,**

**Groop,**

**L.,**

vulnerable β-cell. _Biochemical Society_

**Renström, E. and Rosengren, A. H.**

_Transactions_ **36, 343-347.**

**(2012).**

Secreted

Frizzled-Related

**Lewis, A., Du, J., Liu, J., Ritchie, J. M.,**

Protein 4 Reduces Insulin Secretion

**Oberley, L. W. and Cullen, J. J.**

and Is Overexpressed in Type 2 Dia-

**(2005).** Metastatic progression of pan-

betes. _Cell Metabolism._ **16, 625-633.**

creatic cancer: changes in antioxidant

**Mahmoud, S. M., Abdel-Azim, N. S.,**

enzymes and cell growth. _Clinical and_

**Shahat, A. A., Ismail, S. I. and**

_Experimental Metastasis_ **22, 523–532.**

**Hammouda, F. M. (2007).** Phyto-

chemical and biological studies on

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 163

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

verbascum sinaiticum growing in

tions Trial. _Kidney International_ **43,**

Egypt. _Natural Product Sciences_ **13,**

**668-674.**

**186–189.**

**Muller, F. L., Song, W., Liu, Y.,**

**Mates, J. M., Perez-Gomez, C., and**

**Chaudhuri, A., Pieke-Dahl, S.,**

**Nunez de Castro, I. (1999).** Antioxi-

**Strong, R., Huang, T., Epstein, C.**

dant enzymes and human diseases.

**J., Roberts II, L. J, Csete, M.,**

_Clinical Biochemistry_ **32, 595-603.**

**Faulkner, J. A., and Remmen, V. H.**

**McCord, J. M. (2000).** The evolution of

**(2006).** Absence of CuZn superoxide

free radicals and oxidative stress.

dismutase leads to elevated oxidative

American Journal of Medicine **108,**

stress and acceleration of age-

**652–659.**

dependent skeletal muscle atrophy.

**McCord, J. M. and Fridovich, I. (1969).**

_Free Radical Biology and Medicine_

Superoxide dismutase. An enzymic

**40, 1993–2004.**

function for erythrocuprein (hemocu-

**Nakagawa, T., Zhu, H., Morishima, N.,**

prein). _The Journal of Biological_

**Li, E., Xu, J., Yankner, B. A. and**

_Chemistry_ **244, 6049–6055.**

**Yuan, J. (2000).** Caspase-12 mediates

**McCord, J. M., Fridovich, I. (1968).**

endoplasmic-reticulum-specific apop-

The Reduction of Cytochrome c by

tosis and cytotoxicity by amyloid-

Milk Xanthine Oxidase. _The Journal_

[beta]. _Nature_ **403, 98-103.**

_of Biological Chemistry_ **243, 5753–**

**Newsholme, P., Rebelato, E., Abdulka-**

**5760.**

**der, F., Krause, M., Carpinelli, A.**

**McCord, J. M., Keele, B. B. and Fri-**

**and Curi, R. (2012).** Reactive oxygen

**dovich, I. (1971).** An Enzyme-Based

and nitrogen species generation, anti-

Theory of Obligate Anaerobiosis: The

oxidant defenses, and β-cell function:

Physiological Function of Superoxide

a critical role for amino acids. _Journal_

Dismutase. _Proceedings of the Na-_

_of Endocrinology_ **214, 11-20.**

_tional Academy of Sciences of the_

**The Diabetes Control and Complica-**

_United States of America_ **68, 1024–**

**tions Trial Research Group. (1993).**

**1027.**

The effect of intensive treatment of

**McLellan, A. C., Thornalley, P. J.,**

diabetes on the development and pro-

**Benn, J. and Sonksen, P. H. (1994).**

gression of long-term complications

Glyoxalase system in clinical diabetes

in insulin-dependent diabetes mellitus.

mellitus and correlation with diabetic

The Diabetes Control and Complica-

complications.

_Clinical_

_Science_

tions Trial Research Group. _The New_

_(Lond)_ **87, 21-29.**

_England Journal of Medicine_ **329,**

**Mizgerd, J. P. and Brain, J. D. (1995).**

**977-986.**

Reactive oxygen species in the killing

**UK**

**Prospective**

**Diabetes**

**Study**

of Pseudomonas aeruginosa by human

**(UKPDS) Group. (1998).** Intensive

leukocytes. _Current Microbiology_ **31,**

blood-glucose control with sulpho-

**124-128.**

nylureas or insulin compared with

**Moghaddam, A. E., Gartlan, K. H.,**

conventional treatment and risk of

**Kong, L. and Sattentau, Q. J.**

complications in patients with type 2

**(2011).** Reactive carbonyls are a ma-

diabetes (UKPDS 33). UK Prospec-

jor Th2-inducing damage-associated

tive Diabetes Study (UKPDS) Group.

molecular pattern generated by oxida-

_Lancet_ **352, 837-853.**

tive stress. _The Journal of Immunolo-_

**Oates, P. J. and Mylari, B. L. (1999).**

_gy_ **187, 1626-1633.**

Aldose reductase inhibitors: therapeu-

**Molitch, M. E., Steffes, M. W., Cleary,**

tic implications for diabetic complica-

**P. A. and Nathan, D. M. (1993).**

tions. _Expert Opinion on Investiga-_

Baseline analysis of renal function in

_tional Drugs_ **8, 2095-2119.**

the Diabetes Control and Complica-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 164

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

**Orhan, H., Vermeulen, N. P. E., Tump,**

oxidants in Disease and Health. _Inter-_

**C., Zappey, H. and Meerman J. H.**

_national Journal of Biomedical Sci-_

**N. (2004).** Simultaneous determina-

_ence_ **4, 89–96.**

tion of tyrosine, phenylalanine and

**Phaniendra, A., Jestadi, D. B. and Per-**

deoxyguanosine oxidation products by

**iyasamy, L. (2015).** Free radicals:

liquid chromatography-tandem mass

properties, sources, targets, and their

spectrometry as non-invasive bi-

implication in various diseases. _Indian_

omarkers for oxidative damage. _Jour-_

_Journal of Clinical Biochemistry_ **30,**

_nal of Chromatography, B: Analytical_

**11-26.**

_Technologies in the Biomedical and_

**Pieper, G. M., Langenstroer, P. and**

_Life Sciences_ **799, 245–254.**

**Siebeneich, W. (1997).** Diabetic-

**Oyadomari, S., Koizumi, A., Takeda,**

induced endothelial dysfunction in rat

**K., Gotoh, T., Akira, S., Araki, E.**

aorta: role of hydroxyl radicals. _Car-_

**and Mori, M. (2002).** Targeted dis-

_diovascular Research_ **34, 145-156.**

ruption of the Chop gene delays en-

**Poland, G. A., Quill, H. and Togias, A.**

doplasmic reticulum stress-mediated

**(2013).** Understanding the human

diabetes. _Journal of Clinical Investi-_

immune system in the 21st century:

_gation_ **109, 525-532.**

the Human Immunology Project Con-

**P'ng, X. W., Akowuah, G. A. and Chin,**

sortium. _Vaccine_ **31, 2911-2912.**

**J. H. (2013).** Evaluation of the Sub–

**Prasad, R. C., Herzog, B., Boone, B.,**

acute Oral Toxic Effect of Methanol

**Sims, L. and Waltner-Law, M.**

Extract of Clinacanthus nutans Leaves

**(2005).** An extract of Syzygium aro-

in Rats. _Journal of Acute Disease_ **2,**

maticum represses genes encoding

**29–32.**

hepatic

gluconeogenic

enzymes.

**Pandey, K. B. and Rizvi, S. I. (2009).**

_Journal of Ethnopharmacology_ **96,**

Plant polyphenols as dietary antioxi-

**295–301.**

dants in human health and disease.

**Pryor, W. A. (2000).** Vitamin E and heart

_Oxidative Medicine and Cellular_

disease: basic science to clinical inter-

_Longevity_ **2, 270–278.**

vention trials. _Free Radical Biology_

**Papa, F. R. (2012).** Endoplasmic Reticu-

_and Medicine_ **28, 141-164.**

lum Stress, Pancreatic β-Cell Degen-

**Puthalakath, H., O'Reilly, L. A., Gunn,**

eration, and Diabetes. _Cold Spring_

**P., Lee, L., Kelly, P. N., Huntington,**

_Harbor Perspectives in Medicine_ **2(9),**

**N. D., Hughes, P. D., Michalak, E.**

**a007666.**

**M., McKimm-Breschkin, J., Moto-**

**Parle, M. and Khanna D. (2011).** Clove:

**yama, N., Gotoh, T., Akira, S.,**

a champion spice. _International Jour-_

**Bouillet, P. and Strasser, A. (2007).**

_nal of Research in Ayurveda and_

ER Stress Triggers Apoptosis by Ac-

_Pharmacy_ **2, 47–54.**

tivating BH3-Only Protein Bim. _Cell_

**Passos, J.F. Saretzki, G. and Von**

**129, 1337-1349.**

**Zglinicki, T. (2007).** DNA damage in

**Qanungo, S., Das, M., Haldar, S. and**

telomeres and mitochondria during

**Basu, A. (2005).** Epigallocatechin-3-

cellular senescence: is there a connec-

gallate induces mitochondrial mem-

tion? _Nucleic Acids Research_ **35,**

brane depolarization and caspase-

**7505–7513**.

dependent apoptosis in pancreatic

**Pham-Huy, L. A., He, H. and Pham-**

cancer cells. _Carcinogenesis_ **26, 958–**

**Huy, C. (2008).** Free radicals, antiox-

**67.**

idants in disease and health. _Interna-_

**Quan, W., Hur, K. Y., Lim, Y., Oh, S.**

_tional Journal of Biomedical Sciences_

**H., Lee, J. C., Kim, K. H., Kim, G.**

**4, 89-96.**

**H., Kim, S. W., Kim, H. L., Lee, M.**

**Pham-Huy, L. A., He, H. and Pham-**

**K., Kim, K. W., Kim, J., Komatsu,**

**Huy, C. (2008).** Free Radicals, Anti-

**M. and Lee, M. S. (2012).** Autophagy

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 165

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

deficiency in beta cells leads to com-

**Huang, T. T., Nelson, J., Strong, R.,**

promised unfolded protein response

**and Richardson, A. (2003).** Life-

and progression from obesity to diabe-

long reduction in MnSOD activity re-

tes in mice. _Diabetologia_ **55, 392-403.**

sults in increased DNA damage and

**Quintão, N. L., da Silva, G. F., Antoni-**

higher incidence of cancer but does

**alli, C. S., Rocha, L. W., Cechinel,**

not accelerate aging. _Physiological_

**A., Filho, V. and Cicció, J. F.**

_Genomic_ **16, 29-37.**

**(2010).** Chemical composition and

**Reshi, M. L., Su, Y. C. and Hong, J. R.**

evaluation

of

the

anti-

**(2014).** RNA viruses: ROS-mediated

hypernociceptive effect of the essen-

cell death. _International Journal of_

tial oil extracted from the leaves of

_Cell Biology_ **2014, 467452.**

Ugni myricoides on inflammatory and

**Reynolds, A., Laurie, C., Mosley, R. L.**

neuropathic models of pain in mice.

**and Gendelman, H. E. (2007).** Oxi-

_Planta medica_ **76, 1411-1418.**

dative stress and the pathogenesis of

**Rahman, K. (2007).** Studies on free radi-

neurodegenerative disorders. _Interna-_

cals, antioxidants, and co-factors.

_tional review of neurobiology_ **82, 297-**

_Clinical Interventions in Aging_ **2,**

**325.**

**219-236.**

**Richter, C., Park, J.W. and Ames, B.N.**

**Rahman, T., Hosen, I., Islam, M. T.**

**(1988).** Normal oxidative damage to

**and Shekhar, H. U. (2012).** Oxida-

mitochondrial and nuclear DNA is ex-

tive stress and human health. _Advanc-_

tensive. _Proceedings of the National_

_es in Bioscience and Biotechnology_ **3,**

_Academy of Sciences USA_ **85, 6465–**

**997.**

**6467.**

**Rajendran,**

**P.,**

**Nandakumar,**

**N.,**

**Rock, C. L., Jacob, R. A. and Bowen, P.**

**Rengarajan, T., Palaniswami, R.,**

**E. (1996).** Update on the biological

**Gnanadhas, E. N., Lakshminara-**

characteristics of the antioxidant mi-

**saiah, U., Gopas, J. and Nishigaki,**

cronutrients: vitamin C, vitamin E,

**I. (2014).** Antioxidants and human

and the carotenoids. _Journal of the_

diseases. _Clinica Chimica Acta_ **436,**

_American Dietetic Association_ **96,**

**332-347.**

**693–702.**

**Ran, Q., Remmen, H. V., Gu, M., Qi,**

**Rosen, P., Nawroth, P. P., King, G.,**

**W., Roberts II, L. J., Prolla, T., and**

**Möller, W., Tritschler, H. J. and**

**Richardson, A. (2003).** Embryonic

**Packer, L. (2001).** The role of oxida-

fibroblasts from Gpx4+/− mice: a

tive stress in the onset and progression

novel model for studying the role of

of diabetes and its complications: a

membrane peroxidation in biological

summary of a Congress Series spon-

processes. _Free Radical Biology and_

sored

by

UNESCO-MCBN,

the

_Medicine_ **35, 1101-1109.**

American Diabetes Association and

**Ray P.D., Huang B.W., Tsuji Y. (2012).**

the German Diabetes Society. _Diabe-_

Reactive oxygen species (ROS) ho-

_tes/Metabolism Research and Reviews_

meostasis and redox regulation in cel-

**17, 189-212.**

lular signaling. _Cell Signal_ **24, 981–**

**Rutz, S., Eidenschenk, C. and Ouyang,**

**90.**

**W. (2013).** IL -22, not simply a Th17

**Reczek, C. R. and Chandel, N. S.**

cytokine.

_Immunological_

_Reviews_

**(2014).** ROS-dependent signal trans-

**252, 116-132.**

duction. _Current Opinion in Cell Bi-_

**Sabat, R., Ouyang, W. and Wolk, K.**

_ology_ **33, 8-13.**

**(2014).** Therapeutic opportunities of

**Remmen, V. H., Ikeno, Y., Hamilton,**

the IL-22-IL-22R1 system. _Nature_

**M., Pahlavani, M., Wolf, N.,**

_Reviews Drug Discovery_ **13, 21-38.**

**Thorpe, S. R., Alderson, N. L.,**

**Scalbert, A., Manach, C., Morand, C.,**

**Baynes, J. W., Epstein, C. J.,**

**Rémésy, C. and Jiménez, L. (2005).**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 166

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

Dietary polyphenols and the preven-

ously diabetic Bio-Breeding rat. _The_

tion of diseases. _Critical Reviews in_

_Journal of Clinical Investigation_ **85,**

_Food Science and Nutrition_ **45, 287–**

**1410-1420.**

**306.** doi:10.1080/1040869059096

**Skolnik, E.Y., Yang, Z., Makita, Z.,**

**Selye, H. (1955).** Stress and disease. _The_

**Radoff, S., Kirstein, M. and Vlassa-**

_Laryngoscope_ **65, 500-514.**

**ra, H. (1991).** Human and rat mesan-

**Sepúlveda-Jiménez, G., Rueda-Benítez,**

gial cell receptors for glucose-

**P., Porta, H. and Rocha-Sosa, M.**

modified proteins: potential role in

**(2004).** Betacyanin synthesis in red

kidney tissue remodelling and diabetic

beet (Beta vulgaris) leaves induced by

nephropathy. _The Journal of Experi-_

wounding and bacterial infiltration is

_mental Medicine_ **174, 931-939.**

preceded by an oxidative burst. _Phys-_

**Song, B., Scheuner, D., Ron, D., Pen-**

_iological and Molecular Plant Pa-_

**nathur, S. and Kaufman, R. J.**

_thology_ **64, 125–133.**

**(2008).** Chop deletion reduces oxida-

**Shadel, G. S. and Horvath, T. L. (2015).**

tive stress, improves beta cell func-

Mitochondrial ROS signaling in or-

tion, and promotes cell survival in

ganismal homeostasis. _Cell_ **163, 560-**

multiple mouse models of diabetes.

**9.**

_Journal of Clinical Investigation_ **118,**

**Shen, Y., Zhang, H., Cheng, L., Wang,**

**3378-3389.**

**L., Qian, H. and Qi, X. (2016).** In

**Spencer, J. P. E., Abd El Mohsen, M.**

vitro and in vivo antioxidant activity

**M., Minihane, A.-M. and Mathers,**

of polyphenols extracted from black

**J. C. (2008).** Biomarkers of the intake

highland barley. _Food Chemistry_ **194,**

of dietary polyphenols: strengths, lim-

**1003-1012.**

itations and application in nutrition re-

**Shinohara, M., Thornalley, P. J.,**

search. _British Journal of Nutrition_

**Giardino,**

**I.,**

**Beisswenger,**

**P.,**

**99, 12–22.**

**Thorpe, S. R., Onorato, J. and**

**Stevens, M. J., Lattimer, S. A., Kamijo,**

**Brownlee, M. (1998).** Overexpression

**M., Van Huysen, C., Sima, A. A.**

of glyoxalase-I in bovine endothelial

**and Greene, D. A. (1993).** Osmoti-

cells inhibits intracellular advanced

cally-induced nerve taurine depletion

glycation endproduct formation and

and the compatible osmolyte hypothe-

prevents hyperglycemia-induced in-

sis in experimental diabetic neuropa-

creases in macromolecular endocyto-

thy in the rat. _Diabetologia_ **36, 608-**

sis. _Journal of Clinical Investigation_

**614.**

**101, 1142-1147.**

**Storz, P. (2005).** Reactive oxygen species

**Sies, H. (1997).** Oxidative stress: oxidants

in tumor progression. _Frontiers in Bi-_

and antioxidants. _Experimental Physi-_

_oscience_ **10, 1881–1896.**

_ology_ **82, 291-295.**

**Sturtz, L. A., Diekert, K., Jensen, L. T.,**

**Sies, H. (2015).** Oxidative stress: a con-

**Lill, R., and Culotta, V. C. (2001).** A

cept in redox biology and medicine.

fraction of yeast Cu,Zn-superoxide

_Redox biology_ **4, 180-183.**

dismutase and its metallochaperone,

**Sies, H. and Jones, D. (2007).** Oxidative

CCS, localize to the intermembrane

stress, 2nd ed.,in: G. Fink (Ed.), En-

space of mitochondria. A physiologi-

cyclopedia of Stress, 3, Elsevier, Am-

cal role for SOD1 in guarding against

sterdam. **pp. 45–48.**

mitochondrial oxidative damage. _The_

**Sima, A. A.,Prashar, A., ****Zhang, W.**

_Journal of Biological Chemistry_ **276,**

**X.,** **Chakrabarti, S.** **and Greene, D. **

**38084-38089.**

**A.** **(1990).** Preventive effect of long-Supale, S., Li, N., Brun, T. and Maech-

term aldose reductase inhibition

**ler, P. (2012).** Mitochondrial dysfunc-

(ponalrestat) on nerve conduction and

tion in pancreatic β cells. _Trends in_

sural nerve structure in the spontane-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 167

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

_Endocrinology & Metabolism_ **23,**

**N.G.** (2005). Somatic mtDNA muta-

**477-487.**

tions cause aging phenotypes without

**Suzuki, Y. J., Forman, H. J. and**

affecting reactive oxygen species pro-

**Sevanian A. (1997).** Oxidants as

duction. _Proceedings of the National_

stimulators of signal transduction.

_Academy of Sciences USA_ , **102,**

_Free Radical Biology and Medicine_

**17993–17998**.

**22, 269-285.**

**Tsikas, D., Mitschke, A., Suchy, M.,**

**Tappel, A. L. (1984).** Selenium--

**Gutzki, F. and Stichtenoth, D. O.**

glutathione peroxidase: properties and

**(2005).**

Determination

of

3-

synthesis. _Current Topics in Cellular_

nitrotyrosine in human urine at the ba-

_Regulations_ **24, 87–97**

sal state by gas chromatography-

**Tauchen, J., Doskocil, I., Caffi, C.,**

tandem mass spectrometry and evalu-

**Lulekal, E., Marsik, P., Havlik, J.,**

ation of the excretion after oral intake.

**Van Damme, P. and Kokoska L.**

_Journal of Chromatography, B: Ana-_

**(2015).** In vitro antioxidant and anti-

_lytical Technologies in the Biomedical_

proliferative activity of Ethiopian me-

_and Life Sciences_ **827, 146–156.**

dicinal plant extracts. _Industrial Crops_

**Tuma, D. J. (2002).** Role of malondial-

_and Products_ **74, 671-679.**

dehyde-acetaldehyde adducts in liver

**Teiten, M., Eifes, S., Dicato, M. and.**

injury. _Free Radical Biology and_

**Diederich M. (2010).** Curcumin-the

_Medicine_ **32, 303–308.**

paradigm of a multi-target natural

**Turrens, J. F. (2003).** Mitochondrial

compound with applications in cancer

formation of reactive oxygen species.

prevention and treatment. _Toxins_ **2,**

_The Journal of Physiology_ **552, 335–**

**128–162.**

**344.**

**Thomas, N., Heather, J., Pollara, G.,**

DOI:10.1113/jphysiol.2003.049478

**Simpson, N., Matjeka, T., Shawe-**

**Uruno, A., Yagishita, Y. and Yamamo-**

**Taylor, J., Noursadeghi, M. and**

**to, M. (2015).** The Keap1–Nrf2 sys-

**Chain B. (2013).** The immune system

tem and diabetes mellitus. _Archives of_

as a biomonitor: explorations in innate

_Biochemistry and Biophysics_ **566, 76-**

and adaptive immunity. _Interface Fo-_

**84.**

_cus_ **3(2), 20120099.**

**Valko, M., Leibfritz, D., Moncol, J.,**

**Thoren, F. B., Betten, A., Romero, A. I.**

**Cronin, M. T., Mazur, M. and**

**and Hellstrand, K. (2007).** Cutting

**Telser, J. (2007).** Free radicals and

edge: Antioxidative properties of my-

antioxidants in normal physiological

eloid dendritic cells: protection of T

functions and human disease. _The In-_

cells and NK cells from oxygen radi-

_ternational Journal of Biochemistry & _

cal-induced inactivation and apopto-

_Cell Biology_ **39, 44-84.**

sis. _The Journal of Immunology_ **179,**

**Valko, M., Rhodes, C. J., Moncol, J.,**

**21-25.**

**Izakovic, M. and Mazur M. (2006).**

**Timcenko-Youssef, L., Yamazaki, R.**

Free radicals, metals and antioxidants

**K., and Kimura, T. (1985).** Subcellu-

in oxidative stress-induced cancer.

lar localization of adrenal cortical glu-

_Chemico-Biological Interactions_ **160,**

tathione peroxidase and protective

**1–40.**

role of the mitochondrial enzyme

**Vanella, L., Sodhi, K., Kim, D. H., Puri,**

against lipid peroxidative damage.

**N., Maheshwari, M., Hinds, T. D.**

_The Journal of Biological Chemistry_

**and Abraham, N. G. (2013).** In-

**260, 13355–13359.**

creased heme-oxygenase 1 expression

**Trifunovic, A., Hansson, A., Wreden-**

in mesenchymal stem cell-derived ad-

**berg, A., Rovio, A.T., Dufour, E.,**

ipocytes decreases differentiation and

**Khvorostov, I., Spelbrink, J.N., Wi-**

lipid accumulation via upregulation of

**bom, R., Jacobs, H.T. and Larsson,**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 168

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

the canonical Wnt signaling cascade.

alleviates metabolic disorders and re-

_Stem Cell Research & Therapy_ **4, 28.**

stores mucosal immunity in diabetes.

**Vincent, A. M., Brownlee, M. and Rus-**

_Nature_ **514, 237-241.**

**sell, J. W. (2002).** Oxidative stress

**WCSP. (2016).** 'World Checklist of Se-

and programmed cell death in diabetic

lected Plant Families. Facilitated by

neuropathy. _Annals of the New York_

the Royal Botanic Gardens, Kew.

_Academy of Sciences_ **959, 368-383.**

Published

on

the

Internet;

**Vincent, A. M., Russell, J. W., Low, P.**

http://apps.kew.org/wcsp/. Retrieved

**and Feldman, E. L. (2004).** Oxida-

on 1 Feb 2016.

tive stress in the pathogenesis of dia-

**Wendel, A. (1980)**. in Enzymatic Basis of

betic neuropathy. _Endocrine Reviews_

Detoxification (Jakoby, W., ed) Vol.

**25, 612-628.**

1. Academic Press, New York. **pp.**

**Vinson, J. A., Hao, Y., Su, X. and Zu-**

**333–353.**

**bik, L. (1998).** Phenol antioxidant

**Wichi, H. C. (1986).** Safety evaluation of

quantity and quality in foods: vegeta-

butylated hydroxytoluene (BHT) in

bles. _Journal of Agricultural and_

the liver, lung and gastrointestinal

_Food Chemistry_ **46, 3630–3634**.

tract. _Food Chemical Toxicology_ **24,**

**Vlassara, H., Brownlee, M., Manogue,**

**1127-1130.**

**K. R., Dinarello, C. A. and Pasagian**

**Xia, P., Inoguchi, T., Kern, T. S.,**

**A. (1988).** Cachectin/TNF and IL-1

**Engerman, R. L., Oates, P. J. and**

induced by glucose-modified proteins:

**King, G. L. (1994).** Characterization

role in normal tissue remodeling. _Sci-_

of the mechanism for the chronic acti-

_ence_ **240, 1546-1548.**

vation of diacylglycerol-protein ki-

**Vlassara, H., Li, Y. M., Imani, F.,**

nase C pathway in diabetes and hy-

**Wojciechowicz, D., Yang, Z., Liu, F.**

pergalactosemia. _Diabetes_ **43, 1122-**

**T. and Cerami, A. (1995).** Identifi-

**1129.**

cation of galectin-3 as a high-affinity

**Yamagishi, S., Yonekura, H., Yamamo-**

binding protein for advanced gly-

**to, Y., Katsuno, K., Sato, F., Mita,**

cation end products (AGE): a new

**I., Ooka, H., Satozawa, N., Kawa-**

member of the AGE-receptor com-

**kami, T., Nomura, M. and Yama-**

plex. _Molecular Medicine_ **1, 634-646.**

**moto, H. (1997).** Advanced glycation

**Vulić, J., Ĉanadanović-Brunet, J.,**

end products-driven angiogenesis in

**Ćetković, G., Tumbas, V., Djilas, S.,**

vitro. Induction of the growth and

**Ĉetojević-Simin, D. and Ĉana-**

tube formation of human microvascu-

**danović, V. (2012).** Antioxidant and

lar endothelial cells through autocrine

cell growth activities of beet root

vascular endothelial growth factor.

pomace extract. _Journal of Functional_

_The Journal of Biological Chemistry_

_Foods_ **4, 670-678.**

**272, 8723-8730.**

**Vulić, J., Ćebović, X., Ĉanadanović-**

**Yang, C., Ling, H., Zhang, M., Yang,**

**Brunet, T.N., Ćetković, J.M., Ĉana-**

**Z., Wang, X., Zeng, F. and Feng, J.**

**danović, G.S., Djilas, V.M. and**

**(2011).** Oxidative stress mediates

**Tumbas Šaponjac, S.M. (2014).** In

chemical hypoxia-induced injury and

vivo and in vitro antioxidant effects of

inflammation by activating NF-κB-

beetroot pomace extracts. _Journal of_

COX-2 pathway in HaCaT cells. _Mol-_

_Functional Foods_ **6, 168-175.**

_ecules and cells_ **31, 531-538.**

**Wang, X., Ota, N., Manzanillo, P.,**

**Yang, H., Shi, M., VanRemmen, H.,**

**Kates, L., Zavala-Solorio, J., Ei-**

**Chen, X, L., Vijg, J., Richardson,**

**denschenk, C., Zhang, J., Lesch, J.,**

**A., and Guo, Z. M. (2002).** Reduc-

**Lee, W. P., Ross, J., Diehl, L., van**

tion of pressor response to vasocon-

**Bruggen, N., Kolumam, G. and**

strictor agents by overexpression of

**Ouyang, W. (2014).** Interleukin-22

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 169

_Biotech Sustainability (2017)_

_Natural Polyphenols and Its Potential in ... Sasidharan et al._

catalase in mice. _American Journal of_

stress causes epigenetic alteration of

_Hypertension_ **16, 1-5.**

CDX1 expression in colorectal cancer

**Yant, L. J., Ran, Q., Rao, L., Remmen,**

cells. _Gene_ **524, 214–219.**

**H. V., Shibatani, T., Belter, J. G.,**

**Zhang,**

**Y.,**

**Ikeno,**

**Y.,Qi,**

**W.,**

**Motta, L.,Richardson, A., and**

**Chaudhuri, A., Li, Y., Bokov, A.,**

**Prolla, T. A. (2003).** The selenopro-

**Thorpe, S. R., Baynes, J. W., Ep-**

tein GPX4 is essential for mouse de-

**stein, C., Richardson, A., and**

velopment and protects from radiation

**Remmen, H. V. (2009).** Mice Defi-

and oxidative damage insults. _Free_

cient in Both Mn Superoxide Dis-

_Radical Biology and Medicine_ **34,**

mutase and Glutathione Peroxidase-1

**496-502.**

Have Increased Oxidative Damage

**Yim, M. B., Chock, P. B., Stadtman, E.**

and a Greater Incidence of Pathology

**R. (1990).** Copper, zinc superoxide

but No Reduction in Longevity. _The_

dismutase catalyzes hydroxyl radical

_Journals of Gerontology Series A: Bi-_

production from hydrogen peroxide.

_ological sciences and Medical Scienc-_

_Proceedings of the National Academy_

_es_ **64, 1212-1220.**

_of Sciences USA_ **87, 5006-5010.**

**Zhao, J., Yu, S., Zheng, Y., Yang, H.**

**Yong, Y. K., Tan, J. J., Teh, S. S., Mah,**

**and Zhang J. (2017).** Oxidative

**S. H., Ee, G. C. L., Chiong, H. S.**

Modification and Its Implications for

**and Ahmad, Z. (2013).** Clinacanthus

the Neurodegeneration of Parkinson's

nutans Extracts Are Antioxidant with

disease. _Molecular Neurobiology_ **54,**

Antiproliferative Effect on Cultured

**1404-1418.**

Human Cancer Cell Lines. _Evidence-_

**Zhu, P., Tan, M.J., Huang, R.L., Tan,**

_based Complementary and Alternative_

**C. K., Chong, H. C., Pal, M., Lam,**

_Medicine_ , **2013, 462751.**

**C. R., Boukamp, P., Pan, J. Y., Tan,**

**Young, I. and Woodside, J. (2001).** An-

**S. H., Kersten, S., Li, H. Y., Ding, J.**

tioxidants in health and disease. _Jour-_

**L. and Tan, N. S. (2011).** Angiopoi-

_nal of Clinical Pathology_ **54, 176–**

etin-like 4 protein elevates the prosur-

**186.**

vival intracellular O2-:H2O2 ratio and

**Zarowski, J., and Tappel, A. L. (1978).**

confers anoikis resistance to tumors.

Purification and properties of rat liver

_Cancer Cell_ **19, 401–415.**

mitochondrial glutathione peroxidise.

**Zorov, D. B., Juhaszova, M. and Sol-**

_Biochimica et Biophysica Acta_ **526,**

**lott, S. J. (2014).** Mitochondrial reac-

**65–76.**

tive oxygen species (ROS) and ROS-

**Zhang, R., Kang, K. A., Kim, K.C., Na,**

induced ROS release. _Physiological_

**S-Y, Chang, W.Y., Kim, G.Y., Kim,**

_reviews_ **94, 909-950.**

**H.S., Hyun, J.W. (2013).** Oxidative

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 170

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P171-186_

**A Review on Green Synthesis of Nanoparticles and its**

**Antimicrobial Properties**

****

**Karthika Arumugam1,* and Naresh Kumar Sharma2 __**

__

_1Department of Biotechnology, Kalasalingam University, Krishnankoil, Srivilliputtur,_

_Tamil Nadu, India; 2Department of Biotechnology, Kalasalingam University, Krishnankoil,_

_Srivilliputtur, Tamil Nadu, India; *Correspondence: karthika1386@gmail.com; Tel: +91-_

_9489142440_

**Abstract** : The most important aspect of nanotechnology is the synthesising and characteriz-

ing the nanoparticles (NPs). Nano particles are manufactured in large quantities because of

their wide range of applications. All Physical, Chemical and Biological methods have been

used in the metal nanoparticles production. The major problems in the production are the

stability, aggregation, morphology, crystal growth, size and size distribution. In recent

years, the green synthesis of metal NPs has become more attractive because of cost effec-

tiveness and its various applications in developing new technologies. It is considered as an

eco-friendly technology for the production of well characterized NPs. The metal nanoparti-

cles produced by the plant are more stable and the rate of synthesis is faster as compared to

other methods. Currently, researchers are mainly focused on searching new antimicrobial

agents against various pathogenic bacteria which cause infectious diseases. Moreover, the

NPs have effective antimicrobial properties against infectious pathogens. In this review, we

have discussed different kind of plants which are used in synthesising NPs and highlighted

their antimicrobial applications. We also discussed the basic mechanism by which nanopar-

ticles interact with microbes.

_**Keywords**_ : Antimicrobial activities; bacteria; green synthesis; nanoparticles; plants

**1. Introduction**

electrochemicals (Colvin _et al_., 1994;

****

Wang and Herron _et al_., 1991).

In modern science, nanotechnolo-

Additionally, tremendous applica-

gy is one of the most interesting areas of

tion are increasing in the field of antimi-

research. The synthesis of nano particle

crobials catalysis, microelectronics, bimo-

and nano materials is an important area of

lecular detection, diagnostics and thera-

the nanotechnology. The size of synthe-

peutics (Veera _et al_., 2013) This is be-

sized nano particle could be in the range

cause of newly enhance physical, chemi-

of 1 to 100 nm. Nowdays it has gaining a

cal and biological properties based on

great importance in area such as cosmet-

their size and morphology distribution. It

ics, health care, food and feed, biomedi-

was found that metallic nano particles are

cal, environment, mechanics, drug-gene

considered to have high antibacterial

delivery, health, optics, electronics, ener-

properties because of their large surface

gy science, space industries, chemical in-

area. These nanoparticles find their appli-

dustries, catalysis, light emitters, single

cation in the field of nanocomposites,

electron transistors, nonlinear optical de-

medical imaging, filters, hyperthermia of

vices (Hoffman _et al_., 1992) and photo-

tumors and drug delivery (Tan _et al_.,

2006; Panigrahi _et al_., 2004). The antibac-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 171

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ terial efficiency of nanoparticles made

Green synthesis is a green biologi-

researcher to find the resistant strains

cally based method of synthesising nano

against metal ions, antibiotics (Khalil _et_

particles using microorganisms and plants

_al_., 2013). Since there are the many ap-

in a cost effective, and an environment-

proaches are available for the synthesis of

friendly manner (Gowramma _et al_., 2015;

nano particle like photochemical, thermal

Makarov _et al_., 2014). In Green synthesis

decomposition, microwave decomposition

method nano particle can be synthesized

(Akl _et al_., 2012).

by utilizing (a) microorganisms like fun-

Comparing all this, the best one is

gi, yeasts (eukaryotes), bacteria and acti-

the eco-friendly way of green synthesis

nomycetes (prokaryotes), (b) plants and

approach for the production of nanoparti-

plant extracts (c) templates like mem-

cles. This can be done by using plant ex-

branes, viruses DNA and diatoms. Micro-

tract, fungi, bacteria and enzyme. As there

organism and plants are very stable to ab-

is the lack of chemicals this green

sorb and accumulate inorganic metallic

synthezied nanoparticles have numerous

ions and heavy metals from their sur-

benefits in the field of pharmaceutical ge-

rounding environment (Beveridge and

nomics, immune response enhancement,

Murray, 1980; Singh _et al_., 2011).

biosensors, clinical chemistry and other

During synthesising nano particles

biomedical applications (Diva _et al_.,

using microorganism, optimisation of cul-

2012). These biosynthesized nanoparti-

turing medium, light, pH and temperature

cles were found to be highly toxic against

are very important. Thereby significantly

human pathogen which varies from sim-

increase enzyme activity (Iravani, 2011;

ple prokaryotes to complex eukaryotes.

Mukherjee _et al_., 2001). Accoring to Mit-

The development of natural nano factories

tal _et al_. (2013) biosynthesis of nanoparti-

will depend on the ability of organism in

cles using plants or plant based extracts

the production of metal nano particles

are biologically safe and cost effective.

(Korbekandi _et al_., 2009). The main as-

Moreover nano particle synthesis using

pects of producing highly stable and well

plant extract perform both reducing and

characterized nano particle is achieved by

stabilizing (capping) agents (Singh _et al_.,

selecting best organism and providing

2010; Sathishkumar _et al_., 2009a).

best optimal condition for the growth and

enzyme activity.

**3. Nano particle synthesized using**

Instead of using organism for syn-

**plant**

thesis of nanoparticles, plant extracts is

****

consider to be cost effective and therefore

_3.1. Gold nanoparticle_

can be used as an economic and best al-

Gold nano particle act as a good

ternative for the large-scale production of

source of green chemistry based tech-

metal nanoparticles. The biomolecules

niques. The flower extract of _Nyctanthes_

found in the plant extract will help in the

_arbortristis_ (night jasmine) are used to

bioreduction of metal nano particles in an

synthesis spherical shaped Gold nanopar-

eco-friendly way. Several plants are act as

ticle (Das _et al_., 2011). _Coriandrum sa-_

a source for green synthesizing nano par-

_tivum_ (coriander) leaf extracts are used to

ticles. In present study the microbial

produce Au nanoparticles at different

routes and the plant extract that are used

shape ranging in size from 7 to 58 nm

for synthesising nanoparticles were re-

(Narayanan and Sakthivel, 2008) Accord-

viewed. And also the antibacterial mech-

ing to Poinern _et al_. (2013) _Eucalyptus_

anism of the NPs was discussed.

_macrocarpa_ leaf extract could be utilised

to synthesize Au nanoparticles as well as

**2. Green synthesis**

silver nanoparticle with in a size from 50

****

to 200 nm. Moreover variety of plants

sources such as the leaves and bark of _Fi-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 172

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ _cus carica_ (Singh and Bhakat, 2012),

be synthesised using stem latex of _Eu-_

_Sphaeranthus amaranthoides_ (Nellore _et_

_phorbia nivulia_ (Common milk hedge).

_al_., 2012) and _Putranjiva roxburghii_

Some green methods for synthesis of

(Badole and Dighe, 2012), plant extract of

copper nanoparticles are reported using

Mango (Yang _et al_., 2014); _Gymnocladus_

plant leaf extracts such as _Capparis_

_assamicus_ (Tamuly _et al_., 2013 _); Cacu-_

_zeylanica Linn_ (Saranyaadevi _et al_.,

_men platycladi_ (Wu _et al_., 2013); _Po-_

2014), tamarind, lemon juice (Sastry _et_

_gestemon benghalensis_ (Paul _et al_.,

_al_., 2013); Ocimum sanctum as capping

2015) _; Nerium oleander_ (Tahir _et al_.,

agents (Kulkarni and Kulkarni 2013);

2015 _); Butea monosperma_ (Patra _et al_.,

_Magnolia kobus_ leaf extract (Lee _et al_.,

2015 _);_ Pea nut (Raju _et al_., 2014); _Hibis-_

2013); Syzygium aromaticum (cloves)

_cus cannabinus_ (Bindhu _et al_., 2014);

aqueous extract (Subhankari _et al_., 2013)

_Sesbania grandiflora_ (Das and Velusamy,

and _Nerium oleander_ (Gopinath _et al_.,

2014).

2014). _Brassica juncea_ , _Medicago sativa_

and _Helianthus annus_ and _Tridax pro-_

_3.2. Silver nanoparticle_

_cumbens_ (Abdul and Samarrai, 2012;

Normaly silver (Ag) nanoparticles

Asim _et al_., 2012), _Lantana camara_ (Ma-

ranged in size from 15 to 65 nm with an

jumder, 2012), _Zingiber officinale_ (Ipsa

average size of 34 nm and cuboidal, rec-

and Nayak, 2013) and _Ocimum sanctum_

tangular in shape. The medicinally im-

(Vasudev and Pramod, 2013). Copper na-

portant plants like _Boerhaavia diffusa_

noparticles could be directly used for ad-

(Vijaykumar _et al_., 2014), _Aloe vera_

ministration/ _in vivo_ delivery of nanoparti-

(Chandran _et al_., 2006), _Terminalia_

cles for cancer therapy (Valodkar _et al_.,

_chebula_ (Edison and Sethuraman 2012),

2011).

_Catharanthus roseus_ (Mukunthan _et al_.,

__

2011), _Ocimum tenuiflorum_ (Patil _et al_.,

_3.4. Copper oxide nanoparticles_

2012) _Azadirachta indica_ (Tripathi _et al_.,

According to Padile _et al_. (2013)

2009), _Emblica officinalis_ (Ankamwar _et_

_Sterculia urens_ (Karaya gum) synthesizes

_al_., 2005) _Cocos nucifera_ (Roopan _et al_.,

Cuprous Oxide (CuO at a range of 4.8 nm

2013), common spices _Piper nigrum_

size). Jayalakshmi and Yogamoorthi,

(Shukla _et al_., 2010), _Cinnamon zeylan-_

(2014) tried out the copper oxide nano-

_icum_ (Satishkumar _et al_., 2009a) have al-

particles synthesis using flower extract of

so been used for Ag NP's synthesis _._

_Cassia alata_. Rinkesh _et al_. (2016) use

Au-Ag bimetallic nanoparticles also syn-

Floral extract of _Caesalpinia pulcherrima_

thesize successfully by plants include

for the synthesis of CuO in an eco-

_Azadirachta indica_ (neem) (Shankar _et_

friendly method.

_al_., 2004), _Anacardium occidentale_ (cash-

ew nut) (Sheny _et al_., 2011), _Swietenia_

_3.5. Palladium nanoparticles_

_mahagony_

(West

Indies

mahogany)

Palladium nanoparticles were syn-

(Mondal _et al_., 2011) and cruciferous

thesised using an extract of _C. Zeylanicum_

vegetable extracts (Jacob _et al_., 2012).

taken from (cinnamon) bark Satishkumar

_et al_. (2009b) and also using _Annona_

_3.3. Copper nanoparticles_

_squamosa_ (Custard apple) peel extract

Many variety of plant extracts

with the size ranging from 75 to 85 nm

have been used for the synthesize of Cop-

(Roopan _et al_., 2011). The leaf extract of

per (Cu) and copper oxide (CuO) nano-

soybean ( _Glycine max_ ) have been able to

particles. Magnolia leaf extract and

synthesise nanoparticles with a mean size

_Syzygium aromaticum_ (Clove) extracts is

of 15 nm (Kumar _et al_., 2012). According

used to Cu nanoparticles with the size

to Nadagouda _et al_. (2008) _Coffea arabi-_

ranging from 40 to 100 nm (Subhankari

_ca_ (Coffee) and _Camellia sinensis_ (Tea)

and Nayak, 2013). Cu nanoparticles can

extracts have been utilised to synthesise

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 173

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ palladium nanoparticles at 20 to 60 nm in

_Eclipta prostrata_ leaf extracts can able to

size. _Gardenia jasminoides_ (Cape jas-

produce titanium particles ranging in size

mine) act as a good source for synthesis

from 36 to 68 nm (Rajakumar _et al_.,

of NPs. Many plant leaf extract are used

2012; Zahir _et al_., 2015). TiO2 nanoparti-

for the synthesis of palladium nano parti-

cles was found to be biologically synthe-

cle such as _Pulicaria glutinosa_(Mujeeb  _et_

sized by using _Catharanthus roseus_ leaf

_al_., 2014) _; Anogeissus latifolia_ (Kora _et_

extract ranged in size from 25 up to 110

_al_., 2015) _; Cinnamomum camphora_

nm Velayutham _et al_. (2011). Various

(Yang _et al_., 2010) _; Curcuma longa_

plants are used for the synthesis of TiO2

(Sathishkumar _et al_., 2009c) _;_ _Gardenia_

such

as

Psidium

guajava

_jasminoides_ (Jia _et al_., 2009) _; Glycine_

(Thirunavukkarasu  _et al_., 2014); _Aloe_

_max_ (Petla _et al_., 2012) _; Musa paradisica_

_Vera_ plant extract (Ganapathi _et al_.,

(Bankar _et al_., 2010) _;_ _Pinus resinosa_

2015); Nyctanthes, _Annona squamosapeel_

(Coccia _et al_., 2012); _Pulicaria glutinosa_

extract, (Roopan, 2012) and _Ecliptapros-_

(Khan _et al_., 2014) and _Moringa oleifera_

_trata_ (Gong _et al_., 2007), _Azadirachta_

(Anand _et al_., 2016) __ have been reported.

_indica_ (Siegel _et al_., 1999); _Azadirachta_

_indica_ (Anbalagan _et al_., 2015).

_3.6. Platinium nanoparticles_

Song _et al_. (2010) was first report-

_3.8. Zinc oxide nanoparticles_

ed the leaf extract of _Diospyros kaki_ (Per-

ZnO nanoparticles have been syn-

simmon) were utilized for the synthesis of

thesized using _Aloe vera_ extract (Sang-

platinium nanoparticles n this

eetha _et al_., 2011) in spherical shape. In

tinu i ns we e nve te int

addition, _Physalis alkekengi_ extract was

e i ss t within the range of 2

used to synthesis crystalline poly-

to 12 nm. Platinum nano particle can also

dispersed. ZnO nanoparticles with size

synthesized by using _Ocimum sanctum_

range of 72.5 nm (Qu _et al_., 2011a) and

(Holy basil) leaf extract within a range of

were pseudo-spherical shape and with a

23 n t (Soundarrajan _et al_.,

size of 53.7 nm from _Sedum alfredii_ (Qu

2012). Recently, very few reports availa-

_et al_., 2011b). Pragati _et al_. (2016) was

ble for the synthesis of Pt NPs using sev-

first to report the synthesis of zinc oxide

eral plant extracts including _Cacumen_

nanoparticles using flower extract of _Nyc-_

_platycladi_ , _Prunus yedoensis_ , _Azadirachta_

_tanthes arbor-tristis_. _Mimosa pudica_

_indica_ , _Cochlospermum gossypium_ , honey

leaves extract and coffee powder extract

(Zheng _et al_., 2013; Velmurugan _et al_.,

were utilized for the synthesis of ZnO

2016; Thirumurugan _et al_., 2016; Vinod

nano particle. Various plants are used for

_et al_., 2011; Venu _et al_., 2011). Similarly

the synthesis of ZnO nanoparticles such

Diopyros kaki plant (Jae _et al_., 2010);

as _Solanum nigrum_ (Ramesh _et al_., 2015);

_Lantana camara_ (Musthafa _et al_., 2016);

_Ocimum Tenuiflorum_ (Sagar _et al_., 2015);

_Quercus glauca_ (Qg); _Azadirachta indi-_

_Hibiscus subdariffa_ (Niranjan _e_ _t al_., 2015 _)_ ; _ca_ (Thirumurugan _et al_., 2016); _Ocimun_

_Cassia fistula_ (Vidya _et al_., 2013); __

_sanctum_ (Soundarrajan _et al_., 2012 _); Pi-_

_Agathosma betulina_ (Thema _et al_., 2015).

_nus resinosa_ (Manikandan _et al_., 2016).

__

_3.9. Indium oxide nanoparticles_

_3.7. Titanium dioxide nanoparticles_

Indium Oxide (In2O3) nanoparti-

According to Roopan _et al_. (2012)

cles were synthesized by utilizing the leaf

TiO2 nanoparticles was effectively syn-

extracts from _Aloe vera_ (Aloe barbadensis

thesize by _Annona squamosa_ peel. Sun-

Miller) in spherical shaped with the size

drarajan and Gowri (2011) found that

range from 5 to 50 nm (Maensiri _et al_.,

spherical sized titanium oxide with a

2008). _Astragalus gummifer_ (Katira Gum)

range of 100 to 150 nm was synthesized

is used for the synthesis of Indium oxide

by utilizing _Nyctanthes arbor-tristis_ leaf.

nano particle (Kanchana _et al_., 2016).

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_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ _3.10. Iron nanoparticles_

of Fe3O4-NPs using brown seaweed

Fe nanoparticles was biologically

( _Sargassum muticum_ ) extract.

synthesiszed using aqueous extract of

sorghum bran at room temperature (Njagi

_3.12. Lead nanoparticles_

_et al_., 2011). According to Pattanayak _et_

Spherical shape Pb nanoparticles

_al_. (2013), _Azadirachta indica_ (Neem)

with the size of 10 to 12 nm were able by

were utilized to synthesis Fe nanoparticles

utilizing the latex from _Jatropha curcas_

with the size range from 100 nm. Shah _et_

by Joglekar _et al_. (2011). Delma _et al_.

_al_. (2014) able to utilize stem extract of

2016 tried out the green synthesis of Lead

_Euphorbia milii_ and leaf extracts of

in association with copper nanoparticles

Cymbopogon citrates to synthesise Fe

by utilizing _Zingiber officinale_ stem ex-

nano particle with the range of 43-42 nm.

tract.

Additionally Fe nanoparticle can also syn-

thesized using _Euphorbia milii_ , _Tridax_

_3.13. Selenium nanoparticles_

_procumbens_ , _Tinospora cordifolia_ , _Datu-_

Recently, Sasidharan _et al_. (2014)

_ra innoxia_ , _Calotropis procera_ and

were able to synthesise spherically shaped

_Cymbopogon citratus_ (lemon grass tea).

particles Selenium (Se) nanoparticles us-

Plant parts like Mango leaves, Clove

ing the extracts taken from the peel of cit-

buds, Black Tea, Green tea leaves, Coffee

rus reticulata to produce with a mean par-

seeds, Rose leaves, Cumin seeds, Origano

ticle size of 70 nm. Similarly Garima _et_

leaves, Thymol seeds and Curry leaves

_al_. (2014) approach is to utilize dried _Vitis_

for synthesising Fe nano particle (Monali-

_vinifera_ (raisin) extracts for biosynthesize

sa and Nayak, 2013). Iron nano particle in

selenium nanoparticles (Se-Nps) using.

association with silver can be able to syn-

Fenugreek seed is used to synthesis sele-

thesis by utilizing aqueous sorghum ex-

nium nanoparticle (Ramamurthy _et al_.,

tract (Eric _e_ _t al_., 2011).

2013). Various plants are used for synthe-

__

sis selenium nano particle such as _Vitis_

_3.11. Iron oxide nanoparticles_

_vinifera_ (raisin) extracts (Sharma _e_ _t al_., Iron oxide was successfully syn-2014); _Clausena dentata_ (Sowndarya _et_

thesized by (Yen _et al_., 2016) using Sea-

_al_.,

2016);

_Leucas_

_lavandulifolia_

weed _Kappaphycus alvarezii._ Latha and

(Kirupagaran _et al_., 2016) and _Capsicum_

Gowri, (2014) found that caricaya papaya

_annuum_ (Shikuo _et al_., 2007).

leaves extract were able to synthesis

Fe3O4 nanoparticles at room temperature.

**4. Antimicrobial properties**

_Eucalyptus globulus_ leaf extract was uti-

lized for the synthesis of Iron oxide by

In the field of biotechnology, bi-

adding the extract into the aqueous solu-

ominearlization, bioremediation and mi-

tion of Ferric chloride (Matheswaran, _et_

crobial corrosion, Metal microbes interac-

_al_., 2014). Makarov _e_ _t al_. (2014) reported

tion plays an important role (Prabhu _et al_.,

that aquous extract of monocotyledonous

2012). Researchers found that metal oxide

( _Hordeum vulgare_ ) and dicotyledonous

nanoparticles have good antimicrobial

( _Rumex acetosa_ ) were utilized for the

activity against fungi, virus and bacteria.

synthesis of iron oxide with the size rang-

Antimicrobial NPs have nanosized carrier

ing from 10 to 40 nm. Iron oxide magnet-

for efficient delivery of antibiotics. This

ic nanoparticles (Fe3O4-MNPs) were syn-

can prove the effectiveness for treating

thesized using the aqueous extract of

infectious diseases (Huh and Kwon,

White tea ( _Camelia sinensis_ ) (Sara and

2011). The susceptibility or tolerance of

Mahnaz, 2016). Mahnaz _et al_. (2013)

bacteria against Np differs among gram +

work focused on the development of a

ve and gram –ve. The mechanisms of NP

biosynthetic method for the production

toxicity depend on composition, surface

modification and intrinsic properties.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 175

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ The ionic silver released by the silver na-sised green NPs have a good potential as

noparticles inactivates bacterial enzymes

an antimicrobial agent against different

by interacting with thiol group. It inhibits

microorganism. However, further re-

bacterial DNA replication and damage the

search is required to tap the potential.

bacterial cytoplasm membranes thereby

depleting the level of ATP and inhibit

**References**

DNA unwinding, which leads to cell

****

death. (Parveen _et al_., 2012). In cause of

**Abdul, H.M. and Al-Samarrai (2012).**

copper NPs Metallic and ionic forms of

Nanoparticles as Alternative to Pes-

copper produce hydroxyl radicals that

ticides in Management Plant Dis-

damage essential proteins and DNA

eases-A

Review,

_International_

thereby inhibit the growth (Wang _et al_.,

_Journal of Scientific and Research_

2011). In case of Au, oxidation of Au +

3

_Publications,_ **2(4), 1- 4.**

and decarboxylation of citrate generate

**Akl, M. Awwad and Nida, S.M. (2012).**

free radicals in the presence of light. This

Green synthesis of silver nanoparti-

will damage the DNA and essential pro-

cles by mulberry leaf extract. _Nano-_

tein of bacterial growth (Santo _et al_.,

_science_

_and_

_Nanotechnology._

2008). The ZnO NPs induce frame shift

**2(4), 125-128**.

mutation in the bacteria and cause cell

**Anand, K. Tiloke, C. Phulukdaree, A.**

death. TiO2 NPs peroxidise the phospho-

**Ranjan, B. Chuturgoon, A. Singh,**

lipid component of the lipid membrane

**S. and Gengan, R.M. (2016).** Bio-

thereby disturb the cell respiration and

synthesis of palladium nanoparticles

cause cell death. The exact mechanism of

by using _Moringa oleifera_ flower

action of CdS Nps is not known. But it

extract and their catalytic and bio-

has antibacterial activity against _E. Coli_

logical properties. _Photochem Pho-_

(Sarita, 2010) Fe2O3 nanoparticles to in-

_tobiol_.

teract closely with microbial membranes,

**doi:10.1016/j.jphotobiol.2016.09.0**

damaging their structure and inactivate

**39**

bacteria.

**Anbalagan, K, Mohanraj, S. and Puga-**

****

**lenthi ,V. (2015).** Rapid phytosyn-

**5. Concluding remarks**

thesis of nano-sized titanium using

leaf extract _of Azadirachta indica._

This review has summarized the

_International Journal of ChemTech_

recent research work in the field of green

_Research._ **8 (4), 2047-2052.**

synthesis of nanoparticles using plants.

**Ankamwar, B. Damle, C. Ahmad, A.**

Plants are able to reduce metal ions faster

**and Sastry, M. (2005).** Biosynthe-

than fungi or bacteria. Hence it is evident

sis of Gold and Silver Nanoparticles

that the metal nanoparticles produced by

Using _Emblica officinalis_ Fruit Ex-

plants are more stable while compared

tract, Their Phase Transfer and

with nanoparticles produced by microbes.

Transmetallation in an Organic So-

The capacity of plants in reducing ions

lution. _Journal of Nanoscience_ _and_

depends on the presence of polyphenols,

_Nanotechnology_ **. 5, 1665-1671**.

enzymes, and other chelating agents (in

**Anuj, S.A. and Ishnava, K.B. (2013).**

the plants). This could have the critical

Plant Mediated Synthesis of Silver

effects on the amounts of nanoparticle

Nanoparticles Using Dried Stem

production.

Nanoparticles

are

also

Powder of _Tinospora cordifolia_ , Its

demonstrated to have interesting antimi-

Antibacterial Activity and Its Com-

crobial activity against toxic pathogens.

parison with Antibiotics. _Interna-_

Usually, antibacterial activities of NPs

_tional Journal of Pharmacy_ _and Bi-_

depend on its physicochemical properties

_ological Sciences_. **4, 849-863.**

and type of targeted bacteria. The synthe-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 176

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ **Armendariz, V. Herrera, I. Peralta-Chandran,**

**S.P.**

**Chaudhary,**

**M.**

**Videa, J.R. Jose-Yacaman, M.**

**Pasricha, R. Ahmad, A. and**

**Troiani, H. Santiago, P. and**

**Sastry, M. (2006)**. Synthesis of

**Gardea-Torresdey, J.L. (2004).**

Gold Nanotriangles and Silver Na-

Size controlled gold nanopartilce

noparticles Using _Aloe vera_ Plant

formation by Avena sativa biomass:

Extract. _Biotechnology Progress_.

Use of plants in nanobiotechnology.

**22, 577-583.**

_Journal of Nanoparticle. Research._

**Chen, X. and Schluesener, H.J. (2008).**

**6, 377–382.**

Nanosilver: A nanoproduct in medi-

**Asim, U. Shahid, N. and Naveed, R.**

cal applications. _Toxicol. Lett._ **176,**

**(2012).** Selection of a Suitable

**1–12.**

Method for the Synthesis of Copper

**Coccia, F. Tonucci, L. Bosco, D. Bres-**

Nanoparticles. _NANO: Brief Reports_

**san, M. and d'Alessandro, N.**

_and Reviews_. **7(5), 1- 18.**

**(2012).** One pot synthesis of lignin-

**Badole, M.R. and Dighe, V.V. (2012).**

stabilized platinum and palladium

Synthesis of Gold Nano particles us-

nanoparticles and their catalytic be-

ing

Putranjiva

roxburghiiWall.

haviours in oxidation and reduction

leaves Extract _. Int. J. Drug Discov._

reactions. _Green Chem_. **14, 1073–**

_Herb. Res_. **2, 275–278.**

**1078.**

**Badole, M.R. and Dighe, V.V. (2012).**

**Colvin, V. L. Schlamp, M. C. and Alivi-**

Synthesis of Gold Nano particles us-

**satos, A.P. (1994).** Light-emitting

ing _Putranjiva roxburghii_ Wall

diodes made from cadmium selenide

leaves Extract. _Int. J. Drug Discov_.

nanocrystals and a semiconducting

_Herb. Res._ **2, 275–278.**

polymer. _Nature_. **370, 354–357.**

**Bankar, A. Joshi, B. Kumar, A.R. and**

**Das, J. and Velusamy, P. (2014).** Cata-

**Zinjarde, S. (2010).** Banana peeled

lytic reduction of methylene blue

extract mediated noval route for the

using biogenic gold nanoparticles

synthesis of palladium nanoparti-

from _Sesbaniagrandiflora L. J. Tai-_

cles. _Mater. Lett_ . **64, 1951–1953.**

_wan Inst. Chem. Eng_. **45, 2280-**

**Beveridge, T.J. and Murray, R.G.E.**

**2285.**

**(1980).** Sites of metal deposition in

**Das, R.K. Gogoi, N. and Bora, U.**

the cell wall of _Bacillus subtilis_. _J._

**(2011).** Green synthesis of gold na-

_Bacteriol_. **141, 876–887.**

noparticles using _Nyctanthes ar-_

**Bindhu,**

**M.R.**

**VijayaRekha,**

**P.**

_bortristis_ flower extract. _Bioprocess._

**Umamaheswari, T. and Umadevi,**

_Biosyst. Eng_. **34, 615–619.**

**M. (2014).** Antibacterial activities

**Delma, B.T. Vijila, B. and Jaya Rajan,**

of

_Hibiscus_

_cannabinus_

stem-

**M. (2016).** Green Synthesis of Cop-

assisted silver and gold nanoparti-

per and Lead Nanoparticles using

cles. _Mater. Lett._ **131, 194-197.**

_Zingiber Officinale_ stem extract. _In-_

**Brayner, R. Ferrari-Iliou, R. Brivois,**

_ternational Journal of Scientific and_

**N. Djediat, S. Benedetti, M. and**

_Research Publications_. **6, 11.**

**Fiévet, F. (2006).** Toxicological

**Diva, B. Lingappa, K. and Dayanand.**

impact studies based on _Escherichia_

**A. (2012).** Antibacterial activity of

_coli_ bacteria in ultrafine ZnO nano-

nano gold particles synthesezd by

particles colloidal medium. _Nano._

_Bacillus_

sps _._

_Journal-_

_Lett._ **6, 866–870.**

_ecobiotechnology_. **4 (1), 43-45.**

**Cai, W. Gao, T. Hong, H. and Sun, J.**

**Edison, T.J.I. and Sethuraman, M.G.**

**(2008).** Applications of gold nano-

**(2012).** Instant Green Synthesis of

particles in cancer nanotechnology _._

Silver Nanoparticles Using _Termi-_

_Nanotechnol. Sci. Appl_. **1, 17–32.**

_nalia chebula_ Fruit Extract and

Evaluation of Their Catalytic Ac-

__
__

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 177

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ tivity on Reduction of Methylene

**Gowramma, B. Keerthi, U. Mokula, R.**

Blue. _Process Biochemistry_. **47, __**

**and Rao, D.M. (2015).** Biogenic

**1351-1357.**

silver nanoparticles production and

**Eric, C.N.** **Hui, H.Lisa, S.Hom** **er,**

characterization from native stain of

**G.Hu** **go, M.G.John, B.C.G** **eorge,**

_Corynebacterium_ species and its an-

**E. H.** **andSteven, L. S. ****(2011).**

timicrobial activity. _Biotech_. **5, 195–**

Biosynthesis of Iron and Silver

**201.**

Nanoparticles at Room Temperature

**Hoffman, J. Mills, G. Yee, H. and**

Using _Aqueous_

_sorghum_ Bran

**Hoffmann, M. (1992).** Q-Sized

Extracts. _Langmuir_. _**27**_ **(1). 264–**

CdS: Synthesis, Characterization,

**271.**

and Efficiency of Photoinitiation of

**Ganapathi, R.K, Ashok, C.H. and Ven-**

Polymerization of Several Vinylic

**kateswara R.K. Shilpa C.C.H. and**

Monomers. _Journal of Physical_

**Pavani, T. (2015).** Green Synthesis

_Chemistry_. **96(13), 5546–5552.**

of TiO2 Nanoparticles Using _Aloe_

**Huang, X. Jian, P.K. El-Sayed, I.H. and**

_Vera_ Extract. _International Journal_

**El-Sayed, M.A. (2006).** Determina-

_of Advanced Research in Physical_

tion of the minimum temperature

_Science_ **. 2, 1A, 28-34.**

required for selective photothermal

**Garima, S. Ashish, R.S. Riju, B. Jong-**

destruction of cancer cells with the

**bong, P. Bilguun, G. Ju-Suk, N.**

use of immune-targeted gold nano-

**and Sang-Soo, L. (2014).** Biomole-

particles. _Photochem. Photobiol_. **82,**

cule-Mediated Synthesis of Seleni-

**412–417.**

um Nanoparticles using Dried _Vitis_

**Huh, A. J. and Kwon, Y.J. (2011).**

_vinifera_ (Raisin) extract. _Molecules._

Nanoantibiotic: a new paradigm for

**19, 2761-2770.**

treating infectious diseases using

**Geetha, N. Geetha, T.S. Manonmani, P.**

nanomaterials in the antibiotics re-

**and Thiyagarajan, M. (2014).**

sistant era. _Journal of Controlled_

Green Synthesis of silver nanoparti-

_Release_. **156(2), 128–145.**

cles using _Cymbopogan Citratus_

**Ipsa, S. and Nayak, P.L. (2013).** Anti-

(Dc) Stapf. Extract and its antibacte-

microbial Activity of Copper Nano-

rial activity. _Aust. J. Basic Appl. Sci._

particles Synthesised by Ginger

**8, 324–331.**

( _Zingiber officinale_ ) extract. _World_

**Ghosh, S.K. and Pal, T. (2007).** Inter-

_Journal of Nano Science & Tech-_

particle coupling effect on the sur-

_nology_. **2(1), 10-13.**

face plasmon resonance of gold na-

**Iravani, S. (2011).** Green Synthesis of

noparticles: From theory to applica-

Metal Nanoparticles Using Plants.

tion. _Chem. Rev_. **107, 4797–4862.**

_Green Chemistry_ , **13, 2638-2650**.

**Gong, P. Li, H. He, X. Wang, K. Hu, J.**

**Jacob, J. Mukherjee, T. and Kapoor, S.**

**and Zhang, S. (2007).** Preparation

**(2012).** A simple approach for facile

and antibacterial activity of Fe3O4,

synthesis of Ag, anisotropic Au and

Ag nanoparticles. _Nanotechnology,_

bimetallic (Ag/Au) nanoparticles us-

**18(28), 604-611.**

ing cruciferous vegetable extracts.

**Gopinath, M. Subbaiya, R. Selvam,**

_Mater. Sci. Eng. C._ **32, 1827–1834.**

**M.M. and Suresh, D. (2014).** Syn-

**Jae, Y.S. Eun-Yeong, K. and Beom,**

thesis of copper nanoparticles from

**S.K. (2010).** Biological synthesis of

_Nerium oleander_ leaf aqueous ex-

platinum nanoparticles using _Diopy-_

tract and its antibacterial activity.

_ros kaki_ leaf extract. _Bioprocess Bi-_

_International Journal of Current_

_osyst. Eng_. **33, 159–164.**

_Microbiology and Applied Science._

**Jayalakshmi, and Yogamoorthi, A.**

**3(9), 814–818.**

**(2014).** Green synthesis of copper

oxide nanoparticles using aqueous

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 178

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ extract of flowers of _Cassia alata_

and their catalytic activity towards

and particles characterisation. _Inter-_

the Suzuki coupling reaction _. Dal-_

_national Journal of Nanomaterials_

_ton Trans._ **43, 9026.**

_and Biostructures._ **4(4), 66-71.**

**Kirupagaran, R. Saritha, A. and Bhu-**

**Jia, L. Zhang, Q. Li, Q. and Song, H.**

**vaneswari, S. (2016).** Green Syn-

**(2009).** The biosynthesis of palladi-

thesis of Selenium Nanoparticles

um nanoparticles by antioxidants

from Leaf and Stem Extract of _Leu-_

in _Gardenia jasminoides_ Ellis: Long

_cas lavandulifolia_ Sm. and their

lifetime

nanocatalysts

for

p-

Application. _Journal of Nanoscience_

nitrotoluene hydrogenation. _Nano-_

_and Technology._ **2(5), 224–226.**

_technology._ **20,385601.**

**Kora, A.J. and Rastog,i L. (2015).**

**Joglekar, S. Kodam, K. Dhaygude, M.**

Green synthesis of palladium nano-

**and Hudlikar, M. (2011).** Novel

particles using gum ghatti ( _Anogeis-_

route for rapid biosynthesis of lead

_sus latifolia_ ) and its application as

nanoparticles using aqueous extract

an antioxidant and catalyst. _Arab. J._

of _Jatropha curcas L._ latex. _Mater._

_Chem. _**doi; 2015.06.024**

_Lett._ **65, 3170–3172.**

**Korbekandi, H. Iravani S. and Abbasi,**

**Kanchana, L.C. Aparna, Y. Ramchan-**

**S. (2009).** Production of nanoparti-

**der, M. Ravinder, D. and Jaipal,**

cles using organisms. _Critical Re-_

**K. (2016).** Synthesis and Character-

_views in Biotechnology._ **29, 279–**

isation of In2O3 Nanoparticles from

**306.**

_Astragalus gummifer_. _Advances in_

**Kulkarni, V.D. and Kulkarni, P.S.**

_Nanoparticles_. **5, 114-122.**

**(2013).** Green Synthesis of Copper

**Karthik, R. Sasikumar, R. Shen-Ming,**

Nanoparticles Using _Ocimum Sanc-_

**C. Govindasamy, M. Vinoth Ku-**

_tum_ Leaf Extract. _International_

**mar, J. and Muthuraj, V. (2016).**

_Journal of Chemical Studies_. **1(3),**

Green Synthesis of Platinum Nano-

**1–4.**

particles Using _Quercus Glauca_ ex-

**Kumar, P.R. Vivekanandhan, S. Misra,**

tract and Its Electrochemical Oxida-

**M. Mohanty, A. and Satyana-**

tion of Hydrazine in Water Samples.

**rayana, N. (2012).** Soybean ( _Gly-_

Int. _J. Electrochem. Sci._ **11, 8245 –**

_cine max_ ) leaf extract based green

**8255.**

synthesis of palladium nanoparti-

**Khalil, K.A. Fouad, H. Elsarnagawy, T.**

cles. _J. Biomater. Nanobiotechnol._

**and Almajhdi, F.N. (2013).** Prepa-

**3, 14–19**.

ration and characterization of elec-

**Latha, N. and Gowri. M. (2014).** Bio

trospun PLGA/silvercomposite nan-

Synthesis and Characterisation of

ofibers for biomedical applications.

Fe3O4 Nanoparticles Using _Carica-_

_Int J Electrochem Sci ._ **8, 3483–93.**

_ya Papaya_ Leaves Extract. _Interna-_

**Khalil, M.M.H. Ismail, E.H. El-**

_tional Journal of Science and Re-_

**Baghdady, K.Z. and Mohamed, D.**

_search_. **3, 11.**

**(2013).** Green synthesis of silver

**Lee, H.J. Song, J.Y. and Kim, B.S.**

nanoparticles using olive leaf extract

**(2013).** Biological synthesis of cop-

and its antibacterial activity. _Arabi-_

per nanoparticles using _Magnolia_

_an Journal of Chemistry_. **7, 1131–**

_kobus_ leaf extract and their antibac-

**1139.**

terial activity. _Journal of Chemical_

**Khan, M. Khan, M. Kuniyil, M. Adil,**

_Technology_

_and_

_Biotechnology_.

**S.F. Al-Warthan, A. Alkhathlan,**

**88(11), 1971–1977.**

**H.Z. Tremel, W. Tahir, M.N. and**

**Maensiri, S. Laokul, P. Klinkaewna-**

**Siddiqui, M.R.H. (2014).** Biogenic

**rong, J. Phokha, S. Promarak, V.**

synthesis of palladium nanoparticles

**and Seraphin, S. (2008).** Indium

using _Pulicaria glutinosa_ extract

oxide (In2O3) nanoparticles using

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 179

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ Aloe vera plant extract: Synthesis

and spices extract. _International_

and optical properties. _J. Optoelec-_

_Journal of Plant, Animal and Envi-_

_tron. Adv. Mater_. **10, 161–165.**

_ronmental Sciences_. **3, 1.**

**Mahnaz, M. Farideh, N. Mansor, B.A.**

**Mondal, S. Roy, N. Laskar, R.A. Sk, I.**

**and Rosfarizan, M. (2013).** Green

**Basu, S. Mandal, D. and Begum,**

Biosynthesis and Characterization

**N.A. (2011).** Biogenic synthesis of

of Magnetic Iron Oxide (Fe3O4)

Ag, Au and bimetallic Au/Ag alloy

Nanoparticles

Using

Seaweed

nanoparticles using aqueous extract

( _Sargassum muticum_ ) Aqueous Ex-

of mahogany ( _Swietenia mahogani_

tract. _Molecules_. **18(5), 5954-5964.**

JACQ) leaves. _Colloids Surf._ _B_. **82,**

**Majumder, B.R. (2012).** Bioremediation:

**497–504.**

Copper Nanoparticles from Elec-

**Mujeeb, K.Merajuddin, K. ****Mufsir, K.**

tronic-waste, _International Journal_

**Syed, F.A.** **Abdulrahman, A.W.**

_of Engineering Science and Tech-_

**Hamad, Z. A.** **Wolfgang, T.** **Mu-**

_nology_ , **4.**

**hammad, N.T.** **andMohammed, **

****

**R.H.S.** **(2014).** Biogenic synthesis of **Makarov, V.V. Love, A.J. Sinitsyna,**

palladium nanoparticles using _Pu-_

**O.V. Makarova, S.S. Yaminsky,**

_licaria glutinosa_ extract and their

**I.V. Taliansky, M.E. and Kalini-**

catalytic activity towards the Suzuki

**na, N.O. (2014).** Green nanotech-

coupling reaction. _Dalton Transac-_

nologies: Synthesis of metal nano-

_tions._ **24.**

particles using plants. _Acta Naturae._

**Mukherjee, P. Ahmad, A. Mandal, D.**

**6, 35–44.**

**Senapati, S. Sainkar, S.R. Khan,**

**Makarov, V.V.** **Makarova, S.S. ****Love, **

**M.I. Ramani, R. Parischa, R.**

**A.J.** **Sinitsyna, O.V.** **Dudnik,**

**Ajayakumar, P.V. and Alam, M.**

**A.O.** **Yaminsky, I.V.** **Taliansky, **

**(2001).** Bioreduction of AuCl4 ions

**M.E.** **Kalinina, N.O.** **(2014).** Bio-by the fungus, Verticillium sp. and

synthesis of Stable Iron Oxide Na-

surface trapping of the gold nano-

noparticles in Aqueous Extracts of

particles formed. _Angew. Chem. Int._

_Hordeum vulgare_ and _Rumex ace-_

_Ed._ **40, 3585–3588.**

_tosa_ plants. _J. Langmuir_. **30 (20),**

**Mukunthan, K.S. Elumalai, E.K. Patel,**

**5982-5988.**

**E.N. and Murty, V.R. (2011).**

**Manikandan, V. Velmurugan, P. Park,**

_Catharanthus roseus_ : A Natural

**J.H. Lovanh, N. Seo, S.K. Jayan-**

Source for Synthesis of Silver Na-

**thi, P. Park, Y.J. Cho, M. and Oh,**

noparticles. _Asian Pacific Journal of_

**B.T. (2016).** Synthesis and antimi-

_Tropical Biomedicine_. **1, 270-274.**

crobial activity of palladium nano-

**Musthafa, O.M. Sudip, C. Tasneem, A.**

particles from _Prunus_ x _yedoensis_

**Shahid, A. and Abbasi, A. (2016).**

leaf extract. _Materials Letter._ **185,**

Clean-Green Synthesis of Platinum

**335–338.**

Nanoparticles Utilizing a Pernicious

**Matheswaran, B. (2014).** Synthesis of

Weed Lantana ( _Lantana Camara_ ).

Iron Oxide Nanoparticles by Using

_American Journal of Engineering_

_Eucalyptus globulus_ Plant Extract.

_and Applied Sciences._ **9 (1), 84-90.**

_J. Surf. Sci. Nanotech_. **12, 363-367**.

**Nadagouda, M.N. and Varma, R.S.**

**Mittal, A.K. Chisti, Y. and Banerjee,**

**(2008).** Green synthesis of silver

**U.C. (2013).** Synthesis of metallic

and palladium nanoparticles at room

nanoparticles using plants. _Biotech-_

temperature using coffee and tea ex-

_nol. Adv_. **31, 346–356.**

tract _. Green Chem_. **10, 859–862.**

**Monalisa, P. and Nayak P.L. (2013).**

**Narayanan, K.B. and Sakthivel, N.**

Eco-friendly green synthesis of iron

**(2008).** Coriander leaf mediated bi-

nanoparticles from various plants

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 180

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ osynthesis of gold nanoparticles.

cles from _Cassia auriculata_ leaf ex-

_Mater. Lett_. **62, 4588–4590.**

tract and in-vitro evaluation of an-

**Nellore, J. Pauline, P.C. and Am-**

timicrobial activity. _International_

**arnath, K. (2012).** Biogenic synthe-

_Journal of Applied Biology and_

sis of _Sphearanthus amaranthoids_

_Pharmaceutical Technology_ **. 3(2),**

towards the efficient production of

**222-228.**

the biocompatible gold nanoparti-

**Patil, R.S. Kokate, M.R. and Kolekar,**

cles. Dig. _J. Nanomater. Biostruct_.

**S.S. (2012).** Bioinspired Synthesis

**7, 123–133.**

of Highly Stabilized Silver Nano-

**Niranjan, B.** **Saha,** **S.Chakraborty, ****M. **

particles using _Ocimum tenuiflorum_

**Maiti,** **M. Das, ****S.Basu, ****R. and** Leaf Extract and Their Antibacterial

**Nandy,** **P. (2015).** Green synthesis of

Activity. _Spectrochimica Acta Part_

zinc oxide nanoparticles using _Hibis-_

_A_ : _Molecular and_ _Biomolecular_

_cus subdariffa_ leaf extract: Effect of

_Spectroscopy_. **91, 234-238.**

temperature

on

synthesis,

anti-

**Patra, S. Mukherjee, S. Barui, A.K.**

bacterial activity and anti-diabetic ac-

**Ganguly, A. and Sreedhar, B.**

tivity. _RSC Advances_. **5, 4993-5003.**

**(2015).** Green synthesis, characteri-

**Njagi, E.C. Huang, H. Stafford, L.**

zation of gold and silver nanoparti-

**Genuino, H. Galindo, H.M. and**

cles and their potential application

**Collins, J.B. (2011).** Biosynthesis

for cancer therapeutics. _Mater. Sci._

of iron and silver nanoparticles at

_Eng. C._ **53, 298-309.**

room temperature using aqueous

**Pattanayak, M. and Nayak, P.L. (2013).**

sorghum bran extracts. _Langmuir._

Green synthesis and characterization

**27, 264–271.**

of zero valent iron nanoparticles

**Paciotti, G.F. Mayer, L. Weinreich, D.**

from the leaf extract of _Azadirachta_

**Goia, D. Pavel, N. McLaughlin,**

_indica_ (Neem). _World J. Nano_

**R.E. and Tamarkin, L. (2006).**

_Sci.Technol._ **2, 06–09.**

Colloidal gold: A novel nanoparticle

**Paul, B. Bhuyan, B. DharPurkayastha,**

vector for tumour directed drug de-

**D. Dey, M. and Dhar, S.S. (2015).**

livery. _Drug Deliv._ **11, 169–183.**

Green synthesis of gold nanoparti-

**Padil, V.V.T. and Cerník, M. (2013).**

cles using Pogestemonbenghalensis

Green synthesis of copper oxide na-

(B) O. Ktz. leaf extract and studies

noparticles using _Gum karaya_ as a

of their photocatalytic activity in

biotemplate and their antibacterial

degradation of methylene blue. _Ma-_

application. _Int. J. Nanomed._ **8,**

_ter Lett_ . **148, 37-40.**

**889–898.**

**Petla, R.K. Vivekanandhan, S. Misra,**

**Panigrahi, S. Kundu, S. Ghosh, S.**

**M. Mohanty, A.K. and Satyana-**

**Nath, S. and Pal, T. (2004).** Gen-

**rayana, N. (2012).** Soybean ( _Gly-_

eral method of synthesis for metal

_cine max_ ) leaf extract based green

nanoparticles _. Journal of Nanopar-_

synthesis of palladium nanoparti-

_ticle Research_. **6(4), 411–414.**

cles. _J. Biomater Nanobiotechnol._ **3,**

**Parak,W.J. Gerion, D. Pellegrino, T.**

**14–19.**

**Zanchet, D. Micheel, C. Williams,**

**Poinern, G.E.J. Shah, M. Chapman, P.**

**S.C. Boudreau, R. Le Gros, M.A.**

**and Fawcett, D. (2013).** Green bio-

**Larabell, G.A. and Alivisatos,**

synthesis of silver nanocubes using

**A.P. (2003).** Biological applications

the leaf extracts from _Eucalyptus_

of colloidal nanocrystals. _Nanotech-_

_macrocarpa_. _Nano Bull_. **2, 1–7.**

_nology._ **14, 15–27.**

**Prabhu S. and Paulose E. (2012).** Silver

**Parveen, A. Ashish, S.R. and R.**

nanoparticles mechanism of antimi-

**Srinath. (2012).** Biosynthesis and

crobial action, synthesis medical

characterization of silver nanoparti-

applications and toxicity effects. _In-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 181

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ _ternational Journal of Nano Letters._

/ Copper Oxide Nanoparticles in

**2.**

Eco-Friendly and NonToxic Manner

**Pragati, J. Poonam, K. and Rana J.S.**

from Floral Extract of Caesalpinia-

**(2016).** Green synthesis of zinc ox-

pulcherrima. _International Journal_

ide nanoparticles using flower ex-

_on Recent and Innovation Trends in_

tract of _Nyctanthes arbor_ -tristis and

_Computing and Communication._ **4,**

their antifungal activity _Journal of_

**4.**

_King Saud University – Science._

**Roopan, S.M. Bharathi, A. Kumar, R.**

**doi.2016.10.002** ****

**Khanna, V.G. and Prabhakarn,**

**Qu, J. Luo, and C. Hou, J. (2011b).**

**A. (2011).** Acaricidal, insecticidal,

Synthesis of ZnO nanoparticles

and larvicidal efficacy of aqueous

from Zn hyper-accumulator ( _Sedum_

extract of _Annona squamosa_ L peel

_alfredii Hance_ ) plants. _Micro Nano_

as biomaterial for the reduction of

_Lett._ **6, 174–176.**

palladium salts into nanoparticles.

**Qu, J. Yuan, X. Wang, X. and Shao, P.**

_Colloids Surf. B Biointerfaces._ **92,**

**(2011a).** Zinc accumulation and

**209–212.**

synthesis of ZnO nanoparticles us-

**Roopan, S.M. Bharathi, A. Prab-**

ing Physalis alkekengi L _. Environ._

**hakarn, A. Rahuman, A.A. Ve-**

_Pollut._ **159, 1783–1788.**

**layutham, K. and Rajakumar, G.**

**Rajakumar, G. Abdul-Rahuman, A.**

**(2012).** Efficient phyto-synthesis

**Priyamvada, B. Gopiesh-Khanna,**

and structural characterization of ru-

**V. Kishore-Kumar, and D. Sujin,**

tile TiO2 nanoparticles using _An-_

**P.J. (2012).** _Eclipta prostrata_ leaf

_nona squamosa peel_ extract. _Spec-_

aqueous extract mediated synthesis

_trochim Acta. A Mol. Biomol. Spec-_

of titanium dioxide nanoparticles.

_trosc._ **98, 86-90.**

_Mater. Lett._ **68, 115–117.**

**Roopan, S.M. Rohit, M.G. Rahuman,**

**Raju, D. Vishwakarma, R.K. Khan,**

**A.A. Kamraj, C. Bharathi, A. and**

**B.M. Mehta, U.J. and Ahmad, A.**

**Surendra, T.V. (2013).** Low-Cost

**(2014).** Biological synthesis of cati-

and Eco-Friendly Phyto-Synthesis

onic gold nanoparticles and binding

of Silver Nanoparticles Using _Coos_

of plasmid DNA. _Mater Lett_. **129,**

_nucifera_ Coir Extract and Its Larvi-

**159-161.**

cidal Activity. _Industrial Crops and_

**Ramamurthy, C.H. Sampath, K.S.**

_Products_. **43, 631-635.**

**Arunkumar, P. Suresh, K.M.**

**Sagar, R. Thorat, P.V. and Rohini, T.**

**Sujatha, V. Premkumar, K. and**

**(2015).** Green Synthesis of Zinc Ox-

**Thirunavukkarasu,**

**C.**

**(2013).**

ide (ZnO) Nanoparticles Using

Green synthesis and characterization

Ocimum Tenuiflorum Leaves. _In-_

of selenium nanoparticles and its

_ternational Journal of Science and_

augmented cytotoxicity with doxo-

_Research (IJSR)_. **4, 5.**

rubicin on cancer cells. _Bioprocess_

**Sangeetha, G. Rajeshwari, S. and**

_Biosyst. Eng._ **DOI 10.1007/s00449-**

**Venckatesh, R. (2011).** Green syn-

**012-0867-1**

thesis of zinc oxide nanoparticles by

**Ramesh, M.** **Anbuvannan, M. ****and** aloe barbadensis miller leaf extract:

**Viruthagiri, G.** **(2015).** Green Structure and optical properties.

synthesis of ZnO nanoparticles

_Mater. Res. Bull._ **46, 2560–2566.**

using _Solanum nigrum_ leaf extract

**Santo, C.E. Taudte, N. Nies D. H. and**

and their antibacterial activity.

**Grass G. (2008).** Contribution of

 _Spectrochim Acta A Mol Biomol_

copper ion resistance for survival of

 _Spectrosc._ **136, 864-70.**

_Escherichia coli_ on metallic copper

**Rinkesh, V.K. Sandesh, J. and Abhi-**

surfaces. _Applied and Environmen-_

**jeet, S. (2016).** Synthesis of Copper

_tal Microbiology._ **74(4), 977-986.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 182

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ **Sara, S. and Mahnaz, M.S. (2016).**

**Schmid, G. (1992).** Large clusters and

Green synthesis and characterization

colloids. Metals in the embryonic

of iron oxide magnetic nanoparticles

state. _Chemical Reviews_. **92(8),**

using Shanghai White tea ( _Camelia_

**1709–1727.**

_sinensis_ ) aqueous extract. _Journal of_

**Shah, S. Dasgupta, S. Chakraborty, M.**

_Chemical and Pharmaceutical Re-_

**Vadakkekara, R. and Hajoori, M.**

_search._ **8(5), 138-143.**

**(2014).** Green synthesis of iron na-

**Saranyaadevi, K. Subha, V. Ravindran,**

noparticlesusing plant extracts. _Int._

**R.E. and Renganathan, S. (2014).**

_J. Biol. Pharm. Res._ **5, 549–552.**

Synthesis and characterization of

**Shankar, S.S. Rai, A. Ahmad, A. and**

copper nanoparticle using _Capparis_

**Sastry, M. (2004).** Rapid synthesis

_zeylanica_ leaf extract. _International_

of Au, Ag, and bimetallic Au core-

_Journal of Chemistry, Technology_

Ag shell nanoparticles using Neem

_and Research_. **6(10), 4533–4541.**

( _Azadirachta indica_ ) leaf broth. _J._

**Sarita. (2010).** Synthesis, Characteriza-

_Colloid Interface Sci_. **275, 496–502.**

tion and applications of CdO, CdS

**Sharma, G.** **Sharma, A.R.** **Bhavesh, R.** ****

nanoparticles and nanocomposites ,

**Park, J.** **Ganbold, B. ****Nam, J.S. ******

M.Phil.Thesis, Shoolini University

**and Lee, S.S. ****(2014).** Biomolecule-of Biotechnology, **57-58.**

mediated synthesis of selenium

**Sasidharan, S. Sowmiya, R. and Bala-**

nanoparticles using dried _Vitis_

**krishnaraja, R. (2014).** Biosynthe-

_vinifera_

(raisin)

extract.

sis of selenium nanoparticles using

 _Molecules._ **19(3), 2761-70.**

citrus reticulata peel extract. _World_

**Sheny, D.S. Mathew, T. and Philip, D.**

_J. Pharm. Res_. **4, 1322–1330.**

**(2011).** Phytosynthesis of Au, Ag

**Sastry, A.B.S. Karthik, A.R. Linga,**

and Au-Ag bimetallic nanoparticles

**P.R. and Murthy, B.S. (2013).**

using aqueous extract and dried leaf

Large-scale green synthesis of Cu

of _Anacardium occidentale_. _Spec-_

nanoparticles. _Environmental chem-_

_trochim. Acta A_. **79, 254–262.**

_istry letters_. **11(2), 183–187.**

**Shikuo, L. Yuhua, S. Anjian, X.**

**Satishkumar, M., Sneha, K., Won,**

**Xuerong, Y. Xiuzhen, Z. Liang-**

**S.W., Cho, C.W., Kim, S. and**

**bao, Y. and Chuanhao, L. (2007)**

**Yun, Y.S. (2009a).** _Cinnamon_

Rapid, room-temperature synthesis

_zeylancium_ Bark Extract and Pow-

of

amorphous

selenium/protein

der Mediated Green Synthesis of

composites using _Capsicum annuum_

Nano-Crystalline Silver Particles

_L_ extract. _Nanotechnology_.  **18,** **40.**

and Its Antibacterial Activity. _Col-_

**Shukla, V.K. Singh, R.P. and Pandey,**

_loids and_ _Surfaces B_ : _Biointerfaces_ **.**

**A.C. (2010).** Black Pepper Assisted

**73, 332-338.**

Biomimetic Synthesis of Silver Na-

**Sathishkumar, M. Sneha, K. Kwak, I.S.**

noparticles. _Journal of Alloys and_

**Mao, J. Tripathy, S.J. and Yun,**

_Compounds_. **507, L13-L16.**

**Y.S. (2009b).** Phyto-crystallization

**Siegel, R.W. Hu, E. and Roco, M.C.**

of palladium through reduction pro-

**(1999).** Nanostructure Science and

cess using _Cinnamon zeylanicum_

Technology: R & D Status and

bark extract. _J. Hazard Mater._ **doi:**

Trends in Nanoparticles, Nanostruc-

**171:404–404.**

tured Materials, and Nanodevices.

**Sathishkumar, M. Sneha, K. Yun, and**

Springer Science & Business Media,

**Y.S. (2009c).** Palladium nanocrys-

_Kluwer Academic Publishers, Bos-_

tals synthesis using _Curcuma longa_

_ton_ , **336.**

tuber extract. _Int. J. Mater. Sci_ . **4,**

**Simon-Deckers, A. Loo, S. Mayne-**

**11–17.**

**L'hermite, M.N. Herlin-Boime, N.**

**Menguy, N. Reynaud, C. Gouget,**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 183

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ **B. and Carrière, M. (2009).** Size

conjugated _Clausena dentata_ plant

composition and shape dependent

leaf extract and their insecticidal

toxicological impact of metal oxide

potential against mosquito vectors.

nano-particles and carbon nano-

_Artificial cells, Nanomedicine and_

tubes toward bacteria. _Environ. Sci._

_Biotechnology_. **1-6.**

_Technol._ **43, 8423–8429.**

**Subhankari, I. and Nayak, P.L. (2013).**

**Singh, A. Jain, D. Upadhyay, M.K.**

Antimicrobial Activity of Copper

**Khandelwal, N. and Verma, H.N.**

Nanoparticles Synthesised by Gin-

**(2010).** Green synthesis of silver

ger _(Zingiber officinale_ ) Extract.

nanoparticles using _Argemone mexi-_

_World Journal of Nano Science & _

_cana_ leaf extract and evaluation of

_Technology_. **2(1), 10–13.**

their antimicrobial activity. _Dig. J._

**Sundrarajan, M. and Gowri, S. (2011).**

_Nanomater. Biostruct_. **5, 483–489.**

Green synthesis of titanium dioxide

**Singh, P.P. and Bhakat, C. (2012).**

nanoparticles by nyctanthes arbor-

Green synthesis of gold nanoparti-

tristis leaves extract. _Chalcogenide_

cles and silver nanoparticles from

_Lett_. **8, 447–451.**

leaves and bark of _Ficus carica_ for

**Suresh, D. Nethravathi,** **P.CUdaya-**

nanotechnological applications. _Int._

**bhanu,** **Rajanaika, ****H. Nagabhu-**

_J. Sci. Res. Publ._ **2, 1–4.**

**shana,** **H. andSharma, ****S.C.**

**Singh, R. Gautam, N. Mishra, A. and**

**(2015).** Green synthesis of multi-

**Gupta, R. (2011).** Heavy metals

functional zinc oxide (ZnO) nano-

and living systems: An overview.

particles using _Cassia fistula_ plant

_Indian J. Pharmacol_. **43, 246–253.**

extract and their photodegradative,

**Sondi, I. and Salopek-Sondi, B. (2004).**

antioxidant and antibacterial activi-

Silver nanoparticles as antimicrobial

ties. _Materials science in semicon-_

agent: A case study on _E. coli_ as a

_ductor processing,_ **31, 446-454.**

model for Gram-negative bacteria.

**Tahir, K. Nazir, S. Li, B. Khan, A.U.**

_J. Colloid Interface Sci._ **275, 177–**

**and Khan, Z.U.H. (2015).** _Nerium_

**182.**

_oleander_ leaves extract mediated

**Song, J.Y. Kwon, E.Y. and Kim, B.S.**

synthesis of gold nanoparticles and

**(2010).** Biological synthesis of plat-

its antioxidant activity. _Mater Lett._

inum nanoparticles using _Diopyros_

**156, 198-201.**

_kaki_ leaf extract. _Bioprocess. Bio-_

**Tamuly, C. Hazarika, M. and Bordoloi,**

_syst. Eng_. **33, 159–164.**

**M. (2013).** Biosynthesis of Au na-

**Sotiriou, G.A. and Pratsinis, S.E.**

noparticles by _Gymnocladu sas-_

**(2010).** Antimicrobial activity of

_samicus_ and its catalytic activity.

silver nanosilver ions and particles.

_Mater Lett._ **108, 276-279.**

_Environ. Sci. Technol_. **44, 5649–**

**Tan, M. Wang, G. Ye Z. and Yuan, J.**

**5654.**

**(2006).** Synthesis and characteriza-

**Soundarrajan,**

**C.**

**Sankari,**

**A.**

tion of titania-based monodisperse

**Dhandapani, P. Maruthamuthu,**

fluorescent europium nanoparticles

**S. Ravichandran, S. Sozhan, G.**

for biolabeling. _Journal of lumines-_

**and Palaniswamy, N. (2012).** Rap-

_cence_. **117(1), 20–28.**

id biological synthesis of platinum

**Thema,** **F.T. Manikandan, ****E. Dhlamini, **

nanoparticles using _Ocimum sanc-_

**M.S. and Maaza, ****M. (2015).** Green

_tum_ for water electrolysis applica-

synthesis of ZnO nanoparticles

tions. _Bioprocess Biosyst Eng._

via _Agathosma betulina_ natural ex-

**35,827–833.**

tract.  _Materials Letters._ **161(15)** **,**

**Sowndarya,** **P. Ramkumar, ****G. and** **124–127.**

**Shivakumar, M.S. (2016).** Green

**Thirumurugan,** ****

**A.**

**Aswitha,** ****

**P.**

synthesis of selenium nanoparticles

**Kiruthika,** **C. and Nagarajan, ****S.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 184

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ **(2016).**A. Nancy Christy Green

_Journal of Advanced Research._ **1(9),**

synthesis of platinum nanoparticles

**307-313.**

using _Azadirachta indica_ — an eco-

**Velayutham, K. Rahuman, A.A. Raja-**

friendly approach. _Mater. Lett._ **170,**

**kumar, G. Santhoshkumar, T.**

**175–178.**

**Marimuthu, S. Jayaseelan, C.**

**Thirunavukkarasu, S. Abdul, A.R.**

**Bagavan, A. Kirthi, A.V. Kama-**

**Chidambaram, J. Govindasamy,**

**raj, C. and Zahir, A.A. (2011).**

**R. Sampath, M. Arivarasan, V.K.**

Evaluation of Catharanthus roseus

**Kanayairam, V. (2014).** Green syn-

leaf extract-mediated biosynthesis

thesis of titanium dioxide nanoparti-

of titanium dioxide nanoparticles

cles using _Psidium guajava_ extract

against _Hippobosca maculata_ and

and its antibacterial and antioxidant

_Bovicola ovis_. _Parasitol. Res_. **111,**

properties. _Asian Pacific Journal of_

**2329–2337.**

_Tropical Medicine_. **968-976.**

**Velmurugan, P. Shim, J. Kim, K. and**

**Tripathi, A. Chandrasekaran, N. Rai-**

**Oh, B.T. (2016 _)._** _Prunus × yedoen-_

**chur, A.M. and Mukherjee, A.**

_sis_ tree gum mediated synthesis of

**(2009).** Antibacterial Applications

platinum nanoparticles with anti-

of Silver Nanoparticles Synthesized

fungal activity against phytopatho-

by Aqueous Extract of _Azadirachta_

gens, _Materials letters._ **174, 61-65.**

_indica_ (Neem) Leaves. _Journal of_

**Venu, R. Ramulu, T.S. Anandakumar,**

_Biomedical Nanotechnology_. **5, 93-**

**S. Rani, V.S. and Kim, C.G.**

**98.**

**(2011).** Bio-directed synthesis of

**Valentin, V.M.** **Svetlana, S.M.** **Andrew,**

platinum nanoparticles using aque-

**J.L.** **Olga, V.S.** **Anna, O.D.** **Igor,**

ous honey solutions and their cata-

**V.Y.** **Michael, E.T.** **andNatalia, **

lytic applications. _Colloids and Sur-_

**O.K.** **(2014).** Biosynthesis of Stable

_faces A: Physicochemical and Engi-_

Iron

Oxide

Nanoparticles

in

_neering Aspects_. **384(1-3), 733- 738.**

Aqueous Extracts of _Hordeum_

**Vidya, C. Shilpa, H. Chandraprabhab,**

_vulgare_ and _Rumex acetosa_ Plants.

**M.N. Lourdu Antonyraja, M.A.**

_Langmuir_. **(20), 5982–5988.**

**Indu, V.G. Aayushi, J. and Kokil,**

**Valodkar,**

**M.**

**Jadeja,**

**R.N.**

**B. (2013).** Green synthesis of ZnO

**Thounaojam, M.C. Devkar R.V.**

nanoparticles by _Calotropis Gigan-_

**and S. Thakore (2011).** Biocom-

_tea_. _International Journal of Cur-_

patible synthesis of peptide capped

_rent Engineering and Technology_ , **1.**

copper nanoparticles and their bio-

**Vijaya, K. S. (2012).** Silver nanoparticles

logical effect on tumor cells. _Mate-_

synthesized by in-vitro derived

_rials Chemistry and_ Physics. **128(1-**

plants and _Callus_ culture of _Clitori-_

**2), 83–89.**

_aternatea_ ; evaluation of antimicro-

**Vasudev, K.B. and Pramod, K. (2013).**

bial activity. _Research in Biotech-_

Green Synthesis of Copper Nano-

_nology_. **3 (5), 26-38.**

particles Using _Ocimum Sanctum_

**Vijaykumar, P.P.N. Pammi, S.V.N.**

Leaf Extract. _International Journal_

**Kollu, P. Satyanarayana, K.V.V.**

_of Chemical Studies_. **1(3), 1-4.**

**and Shameem, U. (2014).** Green

**Veera, B. N. Jahnavi, A. Rama, K.**

Synthesis and Characterization of

**Manisha, R. D. Rajkiran, B. and**

Silver Nanoparticles Using _Boer-_

**Pratap, R.M.P. (2013).** Green syn-

_haavia diffusa_ Plant Extract and

thesis of plant mediated silver nano-

Their Antibacterial Activity. _Indus-_

particles using _Withania somnifera_

_trial_ _Crops and Products_. **52, 562-**

leaf extract and evaluation of their

**566.**

antimicrobial activity. _International_

**Vinod, V.T.P. Saravanan, P. Sreedhar,**

**B. Keerthi, D.D. and Sashidhar,**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 185

_Biotech Sustainability (2017)_

_A Review on Green Synthesis of Nanoparticles... Arumugam and Sharma_ **R.B. (2011).** A facile synthesis and

**He, N. and Jia, L. (2010).** Green

characterization of Ag, Au and Pt

synthesis of palladium nanoparticles

nanoparticles using a natural hydro-

using broth of _Cinnamomum cam-_

colloid gum kondagogu (Cochlo-

_phora_ leaf. _J. Nanopart Res_. **doi:**

spermum gossypium). _Colloids and_

**12:1589–1598.**

_Surfaces B: Biointerfaces._ **83(2),**

**Yen, P.Y. Kamyar, S. Mikio, M.**

**291-298.**

**Noriyuki, K. Nurul, B. Ahmad, K.**

**Wang, S. Lawson, R. Ray, P.C. and Yu,**

**Shaza,**

**E.**

**Mohamad,B.and**

**H. (2011).** Toxic effects of gold na-

**Kar,X.L (2016).** Green Synthesis of

noparticles on _Salmonella typhi-_

Magnetite (Fe3O4) Nanoparticles

_murium_ bacteria. _Toxicology and_

Using

Seaweed

( _Kappaphycus_

_industrial health_ **27(6), 547-554.**

_alvarezii_ )

Extract.

_Nanoscale_

**Wang, Y. and Herron, N. (1991).** Na-

_Research Letters._ **11** , **276**.

nometer-sized semiconductor clus-

**Zahir, A.A. Chauhan, I.S. Bagavan, A.**

ters: materials synthesis, quantum

**Kamaraj, C. Elango, G. Shankar,**

size effects, and photophysical

**J. Arjaria, N. Roopan, S.M. Ra-**

properties. The _Journal of Physical_

**human, A.A. and Singh, N. (2015).**

_Chemistry_. **95(2), 525–532.**

Green synthesis of silver and titani-

**Wu, W. Huang, J. Wu, L. Sun, D. and**

um dioxide nanoparticles using _Eu-_

**Lin, L. (2013).** Two-step size- and

_phorbia prostrate_ extract shows

shape-separation of biosynthesized

shift from apoptosis to G0/G1 arrest

gold nanoparticles. _Sep. Purif._

followed by necrotic cell death in

_Technol._ **106, 117-122.**

_Leishmania donovani_. _Antimicrob._

**Yang, N. WeiHong, L. and Hao, L.**

_Agents Chemother._ **59, 4782–4799.**

**(2014).** Biosynthesis of Au nanopar-

**Zheng, B. Kong, T. Jing, X. Wubah,**

ticles using agricultural waste man-

**T.O. Li, X. Sun, D. Lu, F. Zheng,**

go peel extract and its in vitro cyto-

**Y. Huang, J. and Li, Q. (2013) _._**

toxic effect on two normal cells.

Plant-mediated synthesis of plati-

_Mater. Lett_. **134, 67-70.**

num nanoparticles and its bioreduc-

**Yang, X. Li, Q. Wang, H. Huang, J.**

tive mechanism _Journal of Colloid_

**Lin, L. Wang, W. Sun, D. Su, Y.**

_and Interface Science_. **396, 138-**

**Opiyo, J.B. Hong, L. Wang, Y.**

**145.**

****

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 186

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P187-199_

**Production of Secondary Metabolites Using a**

**Biotechnological Approach**

****

**Produtur Chandramati Shankar1, * and Senthilkumar Rajagopal2**

****

_1Department of Biotechnology, Yogi Vemana University, Kadapa-516003, AP, India;_

_2Department of Biochemistry, Rayalaseema University, Kurnool-518002, AP, India;_

_email: senthilanal@yahoo.com; *Correspondence: pchandra20@gmail.com; Tel: +91-_

_8562-225426_

**Abstract** : Secondary metabolites produced by Medicinal plants are useful for the pro-

duction of life saving drugs. The use of natural phytochemicals has its own advantage

as it has no side effects. Because of sustainability, attention on _in vitro_ plant materials

as potential factories for secondary phytochemical products is increasing. More than

80% of the world"s population relies on traditional medicine as it has negligible side

effects for their primary health care needs. Annually, about 95% of the medicinal

plants" used as a raw material is growing at the rate of more than 40%. However, ma-

jority of medicinal plants are collected from their wild habitats, especially from forests.

Repeated use of these medicinal plants has resulted in depletion and extinction of many

important medicinal plant species from their natural habitats. The other most common

reason is poor regeneration capacity of plant under natural habitats due to improper en-

vironmental factors like weather conditions, seasonal change, soil erosion etc. Due to

these reasons, the need of hour is to protect and conserve valuable important medicinal

plants otherwise many of these plants will be lost from natural vegetation forever. In

this article, biotechnological approaches such as plant cell culture and hairy root culture

is described as alternative methods for the production of secondary metabolites. The

advantages of these methods and enhancements of secondary metabolites production by

different methods are also discussed.

_**Keywords**_ : Biotransformation; cell suspension culture; hairy root culture; immobiliza-

tion; medicinal plants

****

****

**1. Introduction**

major role in the adaptation of plants to

their environment. Many higher plants are

Plants produce a wide variety of

major sources of natural products used as

chemical molecules that play important

pharmaceuticals, agrochemicals, flavor

roles in its development and its adaptation

and fragrance ingredients, food addi-

to the environment. These molecules based

tives, and pesticides (Balandrin and

on their functions and role in plants devel-

Klocke, 1988). **** The search for new plant-

opment have been grouped into two types

derived chemicals should thus be a priori-

namely, primary metabolites such as car-

ty in current and future efforts toward sus-

bohydrates, lipids and amino acids etc and

tainable conservation and rational utiliza-

secondary metabolites which are low mo-

tion of biodiversity (Phillipson, 1990). **** It

lecular weight compounds which have no

is estimated that about 100,000 plant sec-

recognized role in the maintenance of fun-

ondary metabolites or natural products

damental life processes in the plants that

have been identified.

synthesize them but are known to play a

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 187

_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

Over one quarter of new drugs that

most secondary metabolites. The recent

have been approved in the last 30 years are

advances, new directions, and opportuni-

based on a lead from a molecule from

ties in plant cell-based processes are being

plant origin. Moreover, 9 of the top 20

critically examined. Such as biotransfor-

selling drugs are derived from knowledge

mation using an exogenous supply of bio-

of plant secondary metabolites (Harvey,

synthetic precursors, genetic manipulation,

2000; Tulp and Bohlin, 2002). **** Higher

and metabolic engineering may improve

plants are rich source of bioactive constit-

the accumulation of compounds. Use of

uents or phyto-pharmaceuticals used in

biotic and a biotic elicitors, can also be

pharmaceutical industry. Some of the plant

used for triggering the formation of sec-

derived natural products include drugs

ondary metabolites. The possible use of

such as morphine, codeine, cocaine, qui-

plant cell cultures for the specific biotrans-

nine etc; anti-cancer Catharanthus alka-

formation of natural compounds has been

loids, belladonna alkaloids, colchicines,

demonstrated (Cheetham, 1995; Scragg,

phytostigminine, pilocarpine, reserpine

1997; Krings and Berger, 1998; Ravishan-

and steroids like diosgenin, digoxin and

kar and Ramachandra Rao, 2000). In the

digitoxin. Many of these pharmaceuticals

search for alternatives for production of

are still in use today and often no useful

desirable medicinal compounds from

synthetic substitutes have been found that

plants, biotechnological approaches, spe-

possess the same efficacy and pharmaco-

cifically, plant tissue cultures, are found

logical specificity. Currently one fourth of

to have potential as a supplement to tradi-

all prescribed pharmaceuticals in industri-

tional agriculture and also in the industri-

alized countries contain compounds that

al production of bioactive plant metabo-

are directly or indirectly, via semi-

lites (Ramachandra Rao and Ravishankar,

synthesis, derived from plants. Studies on

2000). Cell suspension culture systems

plant secondary metabolites have been in-

could be used for large scale culturing of

creasing over the last 50 years.

plant cells from which secondary me-

There are three potential pathways

tabolites could be extracted. Due to these

for primary metabolism: the Embden

advances, research in the area of tissue

Meyerhof-Parnas Pathway (EMP), the

culture technology for production of plant

Entner-Dourdorof pathway, and the hexose

chemicals has bloomed beyond expecta-

monophosphate (HMP) pathway. Due to

tions. _****_ The oncogenic strains of _Agrobacte-_

rapid deforestation and depletion of genet-

_rium rhizogenes_ is used to transform a

ic stocks, various efforts were made to

range of plant species, which induces hairy

implement new methods for several plant

roots. The hairy roots induced from _Agro-_

species conservation and also yielding in

_bacterium rhizogenes_ transformation has

high amounts of secondary metabolites,

shown to have attractive properties for

with photosynthetic efficiency, pest re-

secondary metabolite production as com-

sistant and disease resistant plants. To

pared to differential cell cultures (Kim _et_

overcome these problem new improved

_al.,_ 2002). While research to date has suc-

methods of plant tissue culture and trans-

ceeded in producing a wide range of valu-

formation through molecular approaches

able secondary photochemical in unor-

presents an alternative option for multipli-

ganized callus or suspension cultures, in

cation, development and conservation of

other cases production requires more dif-

elite plant species. Biotechnological ap-

ferentiated micro plant or organ cultures

proaches such as plant tissue culture hold

(Davioud _et al.,_ 1989). In this chapter we

great promise for controlled production of

will be describing the popular types of bio-

useful secondary metabolites on demand.

technological approaches for production of

The current yield and productivity cannot

secondary metabolites.

fulfill the commercial goal of plant cell-

****

based bioprocess for the production of

****

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

****

**Figure 1:** Schematic diagram of secondary metabolite production process. ****

****

**2. Biotechnological approaches for pro-**

Plant cell and tissue cultures can be

**duction of secondary metabolites**

established routinely under sterile condi-

tions from explants, such as plant leaves,

The increased appeal of natural

stems, roots, and meristems for multiplica-

products for medicinal purposes coupled

tion and extraction of secondary metabo-

with the low product yields and supply

lites. The explants selected from high

concerns of plant harvesting has renewed

yielding mother plants are usually selected

interest in large-scale plant cell and hairy

as the starting material ((Figure 1)). Fol-

root culture technology. Secondary metab-

lowing the standard sterilization proce-

olites can be produced by using different

dures the cultures are inoculated on a suit-

biotechnological approaches (Table1). In

able plant tissue culture medium for induc-

this chapter, some techniques have been

tion of callus which is the base for all fur-

described.

ther production work (Figure 2). Callus

cultures, can be divided into one of two

_2.1. Plant cell culture_

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

**Table 1:** Bioactive secondary metabolites produced using plant tissue cultures (Table cour-

tesy: Vanisree and Tsay, 2004)

**Plant name**

**Active ingredient**

**Culture type**

**Reference**

_Agave amaniensis_

Saponins

Callus

Andrijany et al., 1999

_Ailanthus altissima_

Alkaloids

Suspension

Anderson et al., 1987

_Ailanthus altissima_

Canthinone alkaloids

Suspension

Anderson et al., 1986

_Allium sativum_ L _._

Alliin

callus

Malpathak and David,

1986

_Aloe saponaria_

Tetrahydroanthracene glucosides

suspension

Yagi et al., 1983

_Ambrosia tenuifolia_

Altamisine

Callus

Goleniowski and Trip-

pi, 1999

_Anchusa officinalis_

Rosmarinic acid

Suspension

De-Eknamkul and

Ellis, 1985

_Brucea javanica_ (L.) Merr. __

Canthinone alkaloids

Suspension

Liu et al., 1990

_Bupleurum falcatum_

Saikosaponins

Callus

Wang and Huang,

1982

_Bupleurum falcatum_ L. __

Saikosaponins

Root

Kusakari et al., 2000

_Camellia sinensis_

Theamine, γ-glutamyl derivatives

suspension

Orihara and Furuya,

1990

_Canavalia ensiformis_

L-Canavanine

Callus

Ramirez et al., 1992

_Capsicum annuum_ L. __

Capsaicin

Suspension

Johnson et al., 1990

_Cassia acutifolia_

Anthraquinones

Suspension

Nazif et al., 2000

_Catharanthus roseus_

Indole alkaloids

Suspension

Moreno et al., 1993

_Catharanthus roseus_

Catharanthine

Suspension

Zhao et al., 2001b

_Cephaelis ipecacuanha_ A.

Emetic alkaloids

Root

Teshima et al., 1988

Richard __

_Chrysanthemum cinerari-_

Pyrethrins

Callus

Rajasekaran et al.,

_aefolium_

1991

_Chrysanthemum cinerari-_

Chrysanthemic acid and pyethrins

Suspension

Kueh et al., 1985

_aefolium_

_Cinchona_ L. __

Alkaloids

Suspension

Koblitz et al., 1983

_Cinchona robusta_

Robustaquinones

Suspension

Schripsema et al.,

1999

_Cinchona_ spec. __

Anthraquinones

Suspension

Wijnsma et al., 1985

_Cinchona succirubra_

Anthraquinones

Suspension

Khouri et al., 1986

_Citrus_ sp. __

Naringin, Limonin

Callus

Barthe et al., 1987

_Coffea arabica_ L. __

Caffeine

Callus

Waller et al., 1983

_Cruciata glabra_

Anthraquinones

Suspension

Dornenburg and

Knorr, 1996

_Cryptolepis buchanani_

Cryptosin

Callus

Venkateswara et al.,

Roem. & shult __

1987

_Digitalis purpurea_ L. __

Cardenolides

Suspension

Hagimori et al., 1982

_Dioscorea deltoidea_

Diosgenin

Suspension

Heble and Staba, 1980

_Dioscorea doryophora_

Diosgenin

Suspension

Huang et al., 1993

Hance __

_Duboisia leichhardtii_

Tropane alkaloids

Callus

Yamada and Endo,

1984

_Ephedra_ spp. __

L- Ephedrine, D Pseudoephidrin

Suspension

O"Dowd et al., 1993

_Eriobotrya japonica_

Triterpenes

Callus

Taniguchi et al., 2002

_Eucalyptus tereticornis_ SM. __

Sterols and Phenolic compounds

callus

Venkateswara et al.,

1986

_Fumaria capreolata_

Isoquinoline alkaloids

Suspension

Tanahashi and Zenk,

1985

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

**Table 1:** _Continued..._

**Plant name**

**Active ingredient**

**Culture type**

**Reference**

_Gentiana_ sp. __

Secoiridoid glucosides

Callus

Skrzypczak et al.,

1993

_Ginkgo biloba_

Ginkgolide A

Suspension

Carrier et al., 1991

_Glehnia littoralis_

Furanocoumarin

Suspension

Kitamura et al., 1998

_Glycyrrhiza echinata_

Flavanoids

Callus

Ayabe et al., 1986

_Glycyrrhiza glabra_ var.

Triterpenes

callus

Ayabe et al., 1990

_glandulifera_

_Hyoscyamus niger_

Tropane alkaloids

Callus

Yamada and Hashimo-

to, 1982

_Isoplexis isabellina_

Anthraquinones

Suspension

Arrebola et al., 1999

_Linum flavum_ L. __

5-Methoxypodophyllotoxin

Suspension

Uden et., al 1990

_Lithospermum erythrorhizon_

Shikonin derivatives

Suspension

Fujita et al., 1981

_Lithospermum erythrorhizon_

Shikonin derivatives

Suspension

Fukui et al., 1990

_Lycium chinense_

Cerebroside

Suspension

Jang et al., 1998

_Mentha arvensis_

Terpenoid

Shoot

Phatak and Heble,

2002

_Morinda citrifolia_

Anthraquinones

Suspension

Zenk et al., 1975

_Morinda citrifolia_

Anthraquinones

Suspension

Bassetti et al., 1995

_Mucuna pruriens_

L-DOPA

Suspension

Wichers et al., 1993

_Mucuna pruriens_

L-DOPA

Callus

Brain, 1976

_Nandina domestica_

Alkaloids

Callus

Ikuta and Itokawa,

1988

_Nicotiana rustica_

Alkaloids

Callus

Tabata and Hiraoka,

1976

_Nicotiana tabacum_ L. __

Nicotine

Suspension

Mantell et al., 1983

_Ophiorrhiza pumila_

Camptothecin related alkaloids

Callus

Kitajima et al., 1998

_Panax ginseng_

Saponins and Sapogenins

Callus

Furuya et al., 1973

_Panax notoginseng_

Ginsenosides

Suspension

Zhong and Zhu, 1995

_Papaver bracteatum_

Thebaine

Callus

Day et al., 1986

_Papaver somniferum_ L. __

Alkaloids

Callus

Furuya et al., 1972

_Papaver somniferum_

Morphine, Codeine

Suspension

Siah and Doran, 1991

_Peganum harmala_ L. __

β-Carboline alkaloids

Suspension

Sasse et al., 1982

_Phytolacca americana_

Betacyanin

Suspension

Sakuta et al., 1987

_Picrasma quassioides_ Ben-

Quassin

Suspension

Scragg and Allan,

nett __

1986

_Podophyllum hexandrum_

Podophyllotoxin

Suspension

Uden et al., 1989

royle __

_Polygala amarella_

Saponins

Callus

Desbene et al., 1999

_Polygonum hydropiper_

Flavanoids

Suspension

Nakao et al., 1999

_Portulaca grandiflora_

Betacyanin

Callus

Schroder and Bohm,

1984

_Ptelea trifoliata_ L. __

Dihydrofuro [2,3-b] quinolinium

Callus

Petit-Paly et al., 1987

_Rauwolfia sellowii_

Alkaloids

Suspension

Rech et al., 1998

_Rauwolfia serpentina_ Benth. __

Reserpine

Suspension

Yamamoto and Yama-

da, 1986

_Rauvolfia serpentina_ x _Rha-_

3-Oxo-rhazinilam

Callus

Gerasimenko et al.,

_zya stricta_

2001

_Rhus javanica_

Gallotannins

Root

Taniguchi et al., 2000

_Ruta s_ p. __

Acridone and Furoquinoline alka-

Callus

Baumert et al., 1992

loids and cumarins

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

**Table 1:** _Continued..._

**Plant name**

**Active ingredient**

**Culture type**

**Reference**

_Salvia miltiorrhiza_

Lithospermic acid B and rosma-

Callus

Morimoto et al., 1994

rinic acid

_Salvia miltiorrhiza_

Cryptotanshinone

Suspension

Miyasaka et al., 1989

_Scopolia parviflora_

Alkaloids

Callus

Tabata et al., 1972

_Scutellaria columnae_

Phenolics

Callus

Stojakowska and

Kisiel, 1999

_Solanum chrysotrichum_

Spirostanol saponin

Suspension

Villarreal et al., 1997

(Schldl.) __

_Solanum laciniatum_ Ait __

Solasodine

Suspension

Chandler and Dodds,

1983a

_Silybum marianum_

Flavonolignan

Root

Alikaridis et al., 2000

_Solanum paludosum_

Solamargine

Suspension

Badaoui et al., 1996

_Tabernaemontana divarica-_

Alkaloids

Suspension

Sierra et al., 1992

_ta_

_Taxus_ spp. __

Taxol

Suspension

Wu et al., 2001

_Taxus baccata_

Taxol baccatin III

Suspension

Cusido et al., 1999

_Thalictrum minus_

Berberin

Suspension

Kobayashi et al., 1987

_Thalictrum minus_

Berberin

Suspension

Nakagawa et al., 1986

_Torreya nucifera_ var. _radi-_

Diterpenoids

Suspension

Orihara et al., 2002

_cans_

_Trigonella foenumgraecum_

Saponins

Suspension

Brain and Williams,

1983

_Withaina somnifera_

Withaferin A

Shoot

Ray and Jha, 2001

.

the cells are densely aggregated, whereas

in friable callus the cells are only loosely

associated with each other and the callus

become soft and breaks apart easily. Fria-

ble callus are a good source to establish a

cell-suspension cultures. The friability of

callus is improved by manipulating the

medium components or by frequent sub-

culturing or use of semi solid medium.

When friable callus is placed into a suita-

ble plant tissue culture liquid medium

(usually the same composition as the solid

medium used for the callus culture) and

then agitated, single cells and/or small

clumps of cells are released into the me-

dium. Under the correct conditions, these

released cells continue to grow and di-

vide,

eventually

producing

a

cell-

****

suspension culture. Use of large inoculum

**Figure 2:** Schematic diagram showing

for initiation of cell suspensions cultures

the process of callus induction and cell

release more cell numbers into the medium

suspension culture establishment.

quickly. Cell suspensions can be main-

tained just simply as batch cultures in con-

types depending on the nature of callus

ical flasks. They are continually cultured

i.e., compact or friable. In compact callus

by repeated sub culturing into fresh medi-

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

um. This results in dilution of the suspen-

lites. The secondary metabolites produced

sion and the initiation of another batch

by hairy roots arising from the infection of

growth cycle. The degree of dilution dur-

plant material by _A. rhizogenes_ are the

ing subculture should be determined em-

same as those usually synthesized in intact

pirically for each culture. Too great a de-

parent roots, with similar or higher yields.

gree of dilution will result in a greatly ex-

This feature, together with genetic stability

tended lag period or, in extreme cases,

and generally rapid growth in simple me-

death of the transferred cells. After subcul-

dia lacking phytohormones, makes them

ture, the cells divide and the biomass of

especially suitable for biochemical studies

the culture increases in a characteristic

not easily undertaken with root cultures of

fashion, until nutrients in the medium are

an intact plant. The hairy roots are normal-

exhausted and/or toxic byproducts build up

ly induced on aseptic, wounded parts of

to inhibitory levels this is called the "sta-

plants by inoculating them with _A. rhi-_

tionary phase". If cells are left in the sta-

_zogenes_.

tionary phase for too long, they will die

and the culture will be lost. Therefore,

_2.3. Biotransformation using precursors_

cells should be transferred as they enter the

Commercial production of second-

stationary phase. It is therefore important

ary metabolites requires a reproducible and

that the batch growth-cycle parameters are

standardized protocol for cultivation of

determined for each cell-suspension cul-

plant cells/organs on a large scale. Em-

ture. Strain improvement, methods for the

ploying precursor feeding, transformation

selection of high-producing cell lines, and

methods, and immobilization techniques.

medium optimizations can lead to an en-

The treatment of plant cells with biotic

hancement in secondary metabolite pro-

and/or abiotic elicitors has been a useful

duction. Compared to whole plant cultiva-

strategy to enhance secondary metabolite

tion system cell culture system has the ad-

production in cell cultures (Karuppusamy,

vantages as follows:

2009). The most frequently used elicitors

i.

Useful compounds can be pro-

are fungal carbohydrates, yeast extract,

duced under controlled conditions

Methyl Jasmonate (MJ) and chitosan. MJ,

independent of climatic change or

a proven signal compound, is the most ef-

soil conditions;

fective elicitor of taxol production in _Tax-_

ii.

Cultured cells will be free of path-

_us chinensis_ Roxb (Wink _et al.,_ 2008) and

ogens and pests;

gonsenoside production in _P. ginseng_ C.A.

iii.

Less space is required and produc-

Meyer cell/organ culture (Xu _et al.,_ 2008;

tion is uniform.

Yamanaka _et al.,_ 1996).

_2.2. Hairy root culture_

_2.4. Immobilization of cells for secondary_

__

_Agro bacterium rhizogenes_ a gram

_metabolite production_

negative soil borne bacteria belonging to

One of the major limiting factors in

the family Rhizobiaceaae, induces hairy

the development of a commercial produc-

root formation at the site of infection

tion system using plant cell culture has

(Mugnier, 1988). Hairy roots are adventi-

been the production cost of phytopharma-

tious roots with lateral branching, growing

ceuticals. The use of high biomass levels

rapidly and showing plagiotrophic growth

for extended periods would be one method

with high branching and independent of

of increasing productivity and hence re-

plant hormones in the medium, these roots

ducing the costs. This can be achieved by

often posses the capacity to grow even

the immobilization of plant cells. Immo-

when removed from the mother plant. The

bilization is the newest culture technology

transformed roots generated show high

of plant cell, and considered as to be the

differentiation and can cause stable and

most "natural". It has been defined as a

extensive production of secondary metabo-

technique, which confines to a catalytical-

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

ly active enzyme or to a cell within a reac-

iv.

Mixing with suitable materials,

tor system and prevents its entry into the

changing their consistency with

mobile phase, which carries the substrate

temperature (embedding).

and product. The first successful immobi-

v.

Physical

retention

within

the

lization of plant cells was reported by

framework of diverse pore size and

Brodelius _et al.,_ (1979) and they entrapped

permeability (entrapment, micro

_Catharathus_ _roseus_ and _Daucus carota_

encapsulation).

cells in alginate beds. Following success

with enzymatic and microbial process,

_2.4.1.1. Selection of immobilization system_

immobilization has been suggested as a

The choice of a suitable immobili-

strategy to enhance the overall productivi-

zation system is determined by the follow-

ty of secondary metabolite in plant cell

ing requirements.

culture. The ability to immobilize plant

i.

The polymer material used for im-

cells has been reported for a large number

mobilization must be available in

of plant cells and protoplasts by using a

large quantities; it must be inert,

variety of polymers. Immobilization of

non-toxic and cheap.

plant cells has been used for a wide range

ii.

It must be able to carry large quan-

of reactions, which can be divided into

tities of biomass and its fixing po-

three groups. (1) Biotransformation or bio-

tential must be high.

conversion, (2) synthesis from precursors

iii.

The immobilization process must

and (3) the _De Novo_ synthesis of com-

not diminish enzymatic activity of

pounds. **** Some of the advantages of immo-

biological catalyst.

bilization are, retention of biomass enables

iv.

Manipulation of the biological cat-

its continuous reutilization as a production

alyst must be as simple as possible.

system. The immobilization of cells allows

the use of a higher biomass level compared

_2.4.2. Methods for immobilization_

to cell suspension culture, Separation of

****

cells from medium and the product is extra

_2.4.2.1. Gel entrapment by polymerization_

cellular, which will simplify downstream

A monomer or a mixture of mon-

processing compared to extract from tis-

omers is polymerized in the presence of a

sue. Immobilization allows a continuous

cell suspension, which is entrapped inside

process,

which

increase

volumetric

the lattice of the polymer.

productivity and allows the removal of

metabolic inhibitors. Reduces problems

_2.4.2.2. Gel entrapment by ionic net work_

such as aggregate, growth and foaming

_formation_

.Some of the disadvantage is the microen-

In this method, polymerization of

vironment favoring optimal production can

polyelectrolyte is achieved by addition of

be unfavorable for released secondary me-

multivalent ions. The most common meth-

tabolites and cause their degradation or

od is the entrapment in calcium alginate.

metabolization.

This is a non-toxic process in which sodi-

um alginate solution containing the cell

_2.4.1. Different types of immobilization_

suspension is dropped into a mixture of

i.

Direct intracellular binding due to

counter ion solution such as calcium chlo-

natural affinity (adsorption, adhe-

ride. A uniform, spherical and highly mi-

sion and agglutination).

crospores structure results, which retains

ii.

Covalent coupling on otherwise in-

the

cell.

ert matrices.

iii.

Intracellular connection via bi or

_2.4.2.3. Gel entrapment formation by pre-_

poly functional reagent (cross-

_cipitation_

linking).

Gels may be formed by precipita-

tion of some natural and synthetic poly-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 194

_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

mers by changing one or more parameters

Metabolic engineering involves the

in the solution, such as temperature, salini-

targeted and purposeful alteration of meta-

ty or PH of solvent. Several materials can

bolic pathways found in an organism to

be used for entrapment. The examples in-

achieve better understanding and use of

clude methods involving thermal treat-

cellular pathways for chemical transfor-

ment. Some disruption of viability can oc-

mation, energy transduction, and supramo-

cur naturally.

lecular assembly (Lessard, 1996). This

technique applied to plants will permit en-

_2.4.2.4. Entrapment in preformed struc-_

dogenous biochemical pathways to be ma-

_tures_

nipulated and results in the generation of

Hollow fiber reactors can be used

transgenic crops in which the range, scope,

to immobilize plant cells by entrapment.

or nature of a plant's existing natural prod-

The cells are placed on the shell side of the

ucts are modified to provide beneficial

hollow fibre cartridge and nutrient medium

commercial, agronomic, and/or posthar-

is rapidly re-circulated through the fibers.

vest processing characteristics (Kinney,

This may have important applications in

1998).

large-scale.

Several genes in the biosynthetic

pathways for scopolamine, nicotine, and

_2.4.2.5. Surface immobilization_ ****

berberine have been cloned, making the

Surface immobilization may occur on both

metabolic engineering of these alkaloids

natural and other matrices. Examples of

possible. Expression of two branching-

natural matrices are deeper callus layers

point enzymes was engineered: putrescine

and cellulose, while synthetic one includes

_N_ -methyltransferase (PMT) in transgenic

nets of steel and nylon.

plants of _Atropa belladonna_ and _Nicotiana_

_sylvestris_

and

( _S_ )-scoulerine

9- _O_ -

_2.4.2.6. Immobilization by embedding_

methyltransferase (SMT) in cultured cells

The temperature dependent solubility of

of _C. japonica_ and _Eschscholzia californi-_

macromolecules like agarose, agar and

_ca_. Over expression of PMT increased the

carrageenan or the differing solubility of

nicotine content in _N. sylvestris_ , whereas

the sodium and calcium salts in the case of

suppression of endogenous PMT activity

alginate are utilized to form polymeric gels

severely decreased the nicotine content

or gel combination. Insoluble are formed

and induced abnormal morphologies. Ec-

under cold conditions (Agar) or in aqueous

topic expression of SMT caused the accu-

CaCl2 solutions (Alginate). Their structure

mulation of benzylisoquinoline alkaloids

is non-uniform, with differing pore diame-

in _E. californica_ (Sato _et al.,_ 2001). ****

ters at the surface and in deeper layers.

__

The size and form of the beds can be de-

**3. Few commercial products obtained**

termined in part by stirring speed and us-

**by tissue cultures**

ing alginate, by the viscosity of the solu-

****

tion and dropping aperture.

Some of the commercial products

obtained by plant tissue culture are listed

_2.4.2.7. Types of bioreactors used for im-_

below:

_mobilization of plant cells_

The following types of reactors are

_3.1. Taxol_

generally used for immobilized plant cell:

__

Taxol (paclitaxel), a complex

(1) **** Packed bed reactors (2) Well mixed

diterpene alkaloid found in the bark of the

reactor (3) Fluidized bed reactors (4)

_Taxus_ tree, is an anticancer agent. At pre-

Membrane reactors

sent; production of taxol by various _Taxus_

species cells in cultures has been one of

_2.5. Metabolic engineering and production_

the most extensively explored areas of

_of secondary metabolites_

plant cell cultures.

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_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

reticulated polyurethane foam can increase

_3.2. Morphine and codeine_

production approximately 100-fold.

Latex from the opium poppy, _Pa-_

_paver somniferum_ , is a commercial source

_3.6. Camptothecin_

of the analgesics, morphine, and codeine.

Campothecin, a potent antitumor

Callus and suspension cultures of _P. som-_

alkaloid, was isolated from _Camptotheca_

_niferum_ are being investigated as an alter-

_acuminate_ (Padmanabha _et al.,_ 2006; **** Sa-

native means for the production of these

kato and Misawa, 1974) induced _C. acu-_

compounds Biotransformation of co-

_minata_ callus on MS medium containing

deinone to codeine with immobilized cells

0.2 mg/l 2, 4-D and l mg/l kinetin and de-

of _P. somniferum_ has been reported by Fu-

veloped liquid cultures in the presence of

ruya _et al.,_ (1972). The conversion yield

gibberellin, l-tryptophan, and conditioned

was 70.4%, and about 88% of the codeine

medium, which yielded camptothecin at

converted was excreted into the medium.

about 0.0025% on a dry weight basis.

When the cultures were grown on MS me-

_3.3. l-Dopa_

dium containing 4 mg/l NAA, accumula-

L-3, 4-dihydroxyphenylalanine, is

tion of camptothecin reached 0.998 mg/l

an important intermediate of secondary

(Thengane _et al.,_ 2003).

metabolism in higher plants and is known

as a precursor of alkaloids, betalain, and

_3.7. Berberine_

melanine, isolated from _Vinca faba_ ,

Berberine is an isoquinoline alka-

(Daxenbichler _et al.,_ 1971) _Mucuna, Bap-_

loid found in the roots of _Coptis japonica_

_tisia_ , and _Lupinus_ (Brain and Lockwood,

and cortex of _Phellondendron amurense_.

1976).

This antibacterial alkaloid has been identi-

fied from a number of cell cultures, nota-

****

bly those of _C. japonica_ , (Vanisree and

_3.4. Diosgenin_

Tsay, 2004; Breuling _et al.,_ 1985; Sato _et_

Tal _et al._ (1983) reported on the

_al.,_ 2001) _Thalictrum_ spp., (Nakagawa _et_

use of cell cultures of _Dioscorea deltoidea_

_al.,_ 1984; Suzuki _et al.,_ 1988) and _Berberis_

for the production of diosgenin. They

spp. (Breuling _et al.,_ 1985). The productiv-

found that carbon and nitrogen levels

ity of berberine was increased in cell cul-

greatly influenced diosgenin accumulation

tures by optimizing the nutrients in the

in one cell line. Ishida (1988) established

growth medium and the levels of phyto-

_Dioscorea_ immobilized cell cultures, in

hormones (Sato and Yamada 1984; Nak-

which reticulated polyurethane foam was

agawa _et al.,_ 1986; Morimoto _et al.,_ 1988).

shown to stimulate diosgenin production,

By selecting high-yielding cell lines, Mit-

increasing the cellular concentration by

sui group produced berberine on a large

40% and total yield by 25%.

scale with a productivity of 1.4 g/l over 2

weeks. Other methods for increasing

_3.5. Capsaicin_

yields include elicitation of cultures with a

Capsaicin, an alkaloid, is used

yeast polysaccharide elicitor, which has

mainly as a pungent food additive in for-

been successful with a relatively low-

mulated foods (Ravishankar _et al.,_ 2003).

producing _Thalictrum rugosum_ culture

It is obtained from fruits of green pepper

(Funk _et al.,_ 1987).

( _Capsicum_ spp.). Capsaicin is also used in

pharmaceutical preparations as a digestive

**4. Concluding remarks**

stimulant and for rheumatic disorders

(Sharma _et al.,_ 2008). Suspension cultures

Industrial compounds obtained and

of _Capsicum frutescens_ produce low levels

prepared from plant sources are in high

of capsaicin, but immobilizing the cells in

demand. This is because it is safer with no

side effects and cheaper compared to syn-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 196

_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

thetic products. Many lead molecules in

of L-DOPA, _Journal of MEdicinal_

drugs are from plant origin; hence, large

_Chemistry_ **14, 463-465.**

amount of natural resources are destroyed

**Funk, C., Gugler, K., and Brodelius, P.**

for the production of these compounds.

**(1987).** Increased secondary product

The alternate means of production of in-

formation in plant cell suspension

dustrially important compounds is needed

cultures after treatment with a yeast

for the sustainability of natural resources

carbohydrate preparation (elicitor)

and preserve the diversity in the ecosys-

_Phytochemistry_ **26, 401-405.**

tem. There are many advanced approaches

**Harvey,**

**A.**

**(2000).**

Strategies

for

which can be used for the production of

discovering drugs from previously

secondary metabolites; but, only limited

unexplored natural products, _Drug_

methods have been highlighted in this arti-

_Discovery Today_ **5, 294-300.**

cle. Biotechnology can play an important

**Karuppusamy, S. (2009).** A review on

role in production of useful secondary me-

trends in production of secondary

tabolites and will help in sustained devel-

metabolites from higher plants by _in_

opment of plant diversity.

_vitro_ tissue, organ and cell cultures,

_Journal of Medicinal Plant Research_

**References**

**3, 1222-1239.**

****

**Kim, Y., Wyslouzil, E., and Pamela, J.**

**Balandrin, M., and Klocke, J. (1988).**

**(2002).** Secondary metabolism of

_Medicinal, aromatic and industrial_

hairy root cultures in bioreactors, _In_

_materials from plants_ , Vol. 4,

_vitro Cell Developmental Biology_

Springer-Verlag, Berlin, Heidelberg.

_and Plant_ **38, 1-10.**

**Brain, K., and Lockwood, G. (1976).**

**Kinney, A. (1998).** Manipulating flux

Hormonal control of steroid levels in

through plant metabolic pathways,

tissue cultures

from

_Trigonella_

_Current Opinion in Plant Biology_ **1,**

_foenumgraecum_ , _Phytochemistry_ **15,**

**173-178.**

**1651-1654.**

**Krings, U., and Berger, R. (1998)**.

**Breuling, M., Alfermann, A., and**

Biotechnological

production

of

**Reinhard, E. (1985).** Cultivation of

flavours and fragrances, _Applied_

cell cultures of Berberis wilsonae in

_Microbiology and Biotechnology_ **49,**

20l airlift bioreactors, _Plant Cell_

**1-8.**

_Reproduction_ **4, 220-223.**

**Lessard,**

**P.**

**(1996).**

****

Metabolic

**Brodelius, P., Deus, B., Mosbach, K., & **

engineering: the concept coalesces,

**Zenk, M. H. (1979).** Immobilized

_Nature Biotechnology_ _**14**_ **, 1654-**

plant cells for the production and

**1655.**

transportation of natural products.

**Morimoto, T., Hara, Y., Kato, Y.,**

_Febs Letters_ **103(1), 93-97.**

**Hiratsuka, J., Yoshioka, T., and**

**Cheetham, P. (1995)**. Biotransformations:

**Fujita,**

**Y.**

**(1988).**

Berberine

new routes to food ingredients,

production

by

cultured

_Coptis_

_Chemistry and Industry_ **4, 265-268**.

_japonica_ cells in one-stage culture

**Davioud, E., Kan, C., Hamon, J.,**

using medium with a high copper

**Tempe, J., and Husson, H. (1989).**

concentration,

_Agricultural_

Production of indole alkaloids by in

_Biological chemistry_ **52, 1835-1836.**

vitro

root

cultures

from

**Mugnier, J. (1988).** Establishment of new

_Catharanthus_

_trichophyllus_ ,

axenic hairy root lines by inoculation

_Phytochemistry_ **28, 2675–2680.**

with

_Agrobacterium_

_rhizogenes_ ,

**Daxenbichler,**

**M.,**

**VanEtten,**

**C.,**

_Plant Cell Reproduction_ **7, 9-12.**

**Hallinan, E., Earle, F., and**

**Nakagawa, K., Konagai, A., Fukui, H.,**

**Barclay, A. (1971).** Seeds as sources

**and Tabata, M. (1984).** Release and

crystalization of berberine in the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 197

_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

liquid medium of _Thalictrum minus_

_Coptis_

_japonica_

cells,

cell suspension cultures, _Plant Cell_

_Phytochemistry_ **23, 281-285.**

_Reproduction_ **3, 254-257.**

**Sato, F., Hashimoto, T., Hachiya, A.,**

**Nakagawa, K., Fukui, H., and Tabata,**

**Tamura,**

**K.,**

**Choi,**

**K.,**

**and**

**M. (1986).** Hormonal regulation of

**Morishige, T. (2001).** Metabolic

berberine

production

in

cell

engineering

of

plant

alkaloid

suspension cultures of _Thalictrum_

biosynthesis, _Proceedings of Natural_

_minus_ , _Plant Cell Reproduction_ **5,**

_Academy of Sciences USA_ **2, 367-**

**69-71.**

**372.**

**Padmanabha, B., Chandrashekar, M.,**

**Scragg, A. (1997)**. _The production of_

**Ramesha, B., Hombe Gowda, H.,**

_aromas by plant cell cultures_ , Vol.

**Rajesh, P., and Suhas, S. (2006).**

55, Springer-Verlag, Berlin.

Patterns

of

accumulation

of

**Sharma, A., Kumar, V., Giridhar, P.,**

camptothecin,

an

anti-cancer

**and**

**Ravishankar,**

**G.**

**(2008).**

alkaloids

in

_Nothapodytes_

Induction of _in vitro_ flowering in

_nimmoniana Graham_ , in the Western

_Capsicum_

_frutescens_

under

the

Ghats,

India:

Implications

for

influence of silver nitrate and cobalt

identifying high-yielding sources of

chloride and pollen transformation,

the alkaloid, _Current Science_ **90, 95-**

_Plant Biotechnology Journal_ **11, 1-8.**

**100.**

**Suzuki, M., Nakagawa, K., Fukui, H.,**

**Phillipson, J. (1990)**. _Plants as source of_

**and Tabata, M. (1988).** Alkaloid

_valuable products_ , Clarendon Press,

production

in

cell

suspension

Oxford. **Ramachandra Rao, S., and**

cultures of _Thalictrum flavum_ and _T._

**Ravishankar,**

**G.**

**(2000).**

_dipterocarpum_ ,

_Plant_

_Cell_

Biotransformation of protocatechuic

_Reproduction_ **7, 26-29.**

aldehyde and caffeic acid to van illin

**Thengane, S., Kulkarni, D., Shrikhande,**

and capsaicin in freely suspended

**V., Joshi, S., Sonawane, K., and**

and immobilized cell cultures of

**Krishnamurthy,**

**K.**

**(2003).**

Capsicum frutescens, _Journal of_

Influence of medium composition on

_Biotechnology_ **76, 137-146.**

callus induction and camptothecin(s)

**Ravishankar, G., and Ramachandra**

accumulation

in

_Nothapodytes_

**Rao, S. (2000).** Biotechnological

_foetida_ , _Plant Cell Tissue and Organ_

production of phyto-pharmaceuticals,

_Culture_ **72, 247-251.**

_Journal_

_of_

_Biochemistry_

_and_

**Tal, B., Rokem, J.S., Gressel, J.,**

_Molecular Biology and Biophysics_ **4,**

**Goldberg, I. (1983).** Diosgenin

**73-102.**

production by Dioscorea deltoidea

**Ravishankar, G., Suresh, B., Giridhar,**

cell suspension culture. _IAPTC_

**P., Rao, S., and Johnson, T. (2003).**

_Newslett_ **40, 15.**

_Biotechnological_

_studies_

_on_

**Tulp, M., and Bohlin, L. (2002)**.

_capsicum for metabolite production_

Unconventional natural sources for

_and plant improvement_ , Harwood

future

drug

discovery,

_Drug_

Academic

Publishers,

United

_Discovery Today_ **9, 450-458**.

kingdom.

**Vanisree, M., and Tsay, H. (2004).** Plant

**Sakato, K., and Misawa, M. (1974).**

Cell Cultures - An Alternative and

Effects of chemical and physical

Efficient Source for the Production

conditions

on

growth

of

of Biologically Important Secondary

_Camptotheca_

_acuminata_

cell

Metabolites, _International Journal of_

cultures,

_Agricultural_

_Biological_

_Applied Science and Engineering_ **2,**

_chemistry_ **38, 491-497.**

**29-48.**

**Sato, F., and Yamada, Y. (1984).** High

**Wink, M., Alfermann, A., Franke, R.,**

berberine producing cultures of

**Wetterauer, B., Distl, M., and**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 198

_Biotech Sustainability (2017)_

_Production of Secondary Metabolites Using a Biotechnological Approach Shankar and Rajagopal_

**Windhovel, J. (2008).** Sustainable

_Society and Applied Biological_

bioproduction of phytochemicals by

_Chemistry_ **51, 349-351.**

plant _in vitro_ cultures: anticancer

**Yamanaka,**

**M.,**

**Ishibhasi,**

**K.,**

agents, _Plant Genetic Resources_ **12,**

**Shimomura, K., and Ishimaru, K.**

**113-123.**

**(1996).** Polyacetylene glucosides in

**Xu, H., Kim, Y., Suh, S., Udin, M., Lee,**

hairy root cultures of _Lobelia_

**S., and Park, S. (2008).** Deoursin

_cardinalis_ , _Phytochemistry_ **41, 183-**

production from hairy root culture of

**185.**

_Angelica gigas_ , _Journal of Korean_

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 199

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P200-211_

**Potential of Marine Algae Derived Extracts as a Natural**

**Biostimulant to Enhance Plant Growth and Crop**

**Productivity**

****

**Lakkakula Satish and Manikandan Ramesh** * ****

__

_Department of Biotechnology, Science Campus, Alagappa University, Karaikudi, Tamil_

_Nadu, India; *Correspondence: pandu.pine@gmail.com _ _/ mrbiotech.alu@gmail.com; Tel:_ __

_+91 4565 225215_

__

__

**Abstract:** Photobioreactor (PBR) is a reactor which exploits a light supply to farm photo-

trophic microorganisms which can generate biomass using light, water and carbon dioxide.

Macro- and microalgae are developed naturally in fresh water as well as marine water

(highly economical) or brackish water. Universal production of marine algal monocultures

is basically limited to a selective species such as _Scenedesmus spp._ , _Nannochloropsis spp_.,

and _Chlorella spp._ , and some extremophiles like _Arthrospira spp_., and _Dunaliella spp_.,.

Owing to their inborn resistance to predators and competitors, algal species can be cultivat-

ed naturally in open systems akin to ponds, stirred tanks and bubble columns. Nowadays,

numerous research groups are focusing on PBRs (viz., selective closed type or open sys-

tems) to produce marine algae (cyanobacteria or seaweeds) through utilizing a light source

with lavish biomass accumulation. The cultivation of marine algae has distinguished a new

extension in numerous fields crucial for humanity, especially energy, food, health and envi-

ronment. Currently, marine algae and algae-based extracts remain predominantly unex-

ploited despite their huge potential applications. This chapter highlights the present and fu-

ture prospects of marine plants and their derived aqueous extracts for crop improvement

and enhancement of plant biomass production.

_**Keywords**_ **:** Crop protection; cyanobacteria; marine seaweed; photobioreactor; plant bio-

stimulants; plant growth hormones

**1. Introduction**

fresh water and marine environments

across a wide range of habitats. Micro

As per previously published data

algae, comprise of cyanobacteria and

about micro algae biotechnology, the

seaweeds (macroalgae) are a resource of

foremost concern of external mass farm-

valuable bioactive compounds including

ing has been planned at obtaining suc-

vitamins, pigments, secondary metabo-

cessful consumption of prominent light

lites, plant growth hormones and other

potency (Masojidek _et al.,_ 2003). The

food appurtenances representing extreme-

word algae cover a wide choice of diverse

ly discriminatory pharmacological activi-

organisms which can be normally illus-

ties (Masojidek _et al.,_ 2009; Satish _et al.,_

trated as eukaryotic protests (a complicat-

2015, 2016, Rency _et al.,_ 2017). Marine

ed group to describe), that are diverse

algae (macro and micro) is the vital re-

from plants but are naturally aquatic and

source for bio-fuel production, since they

photosynthetic. They can also be micro-

can collect a large quantity of lipid con-

scopic single celled micro algae or larger,

tent within their cells and contain very

more complex multi cellular seaweeds.

huge biomass productive capacity (Wu

They can be found all-inclusive in both

and Merchuk, 2004; Yang _et al.,_ 2014). In

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 200

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ case of filamentous micro algae, a variety

population over the foregoing few dec-

of strains are recognized to make intra- or

ades. In the year 2012, an incredible 90.4

extracellular metabolites by dissimilar

million tonnes of marine based food and

biological activities (Glombitza and

beverages were farmed and this amount is

Koch, 1989). However, numerous re-

expected to increase until about 2030, at

search groups focusing on photobioreac-

which moment it is indefinite that capture

tors (PBRs) for making of prominent al-

fisheries and aquaculture will distribute

gal biomass production, is not nearly as

equal quantities (FAO, 2013; Taelman _et_

superior as requisite by the worlds de-

_al.,_ 2015). Nevertheless, an entire control

sires, particularly in the pharmaceutical,

on culture conditions is sufficient only

for human utilization as well as fish and

through closed systems, only PBR.

animal feed, and for field level agricultur-

Commonly, PBR is a bioreactor

al applications. Still, there is inert ques-

which uses a light source to grow photo-

tion as to whether algal based cultures are

trophic microorganisms which can pro-

able to make plant growth stimulants and

duce biomass through light and CO2 and

concerning their mechanisms of action

include plants, macro and micro algae,

towards elevated yielding, biotic and abi-

cyanobacteria, purple bacteria and moss-

otic stress resistance and high biomass

es. The first step was taken by Richmond

production of crops in agriculture through

and group in 1978 as the introduction of

regulation of growth succession such as

external algal biotechnology in Israel

cell division and in the sensitivity of envi-

(Richmond

and

Vonshak,

1978;

ronmental changes.

Masojidek _et al.,_ 2003, 2009). Compared

to open culture systems like ponds and

**2. Recent developments for cultivation**

raceways, PBR systems have several ad-

**of marine algae using photobioreac-**

vantages viz., reproducible farming with

**tors**

regard to environmental changes, mainte-

nance of dissolved oxygen concentration,

The majority of marine algae use

sufficient mixing of the culture with the

photosynthesis to confine light energy to

appropriate flow rate, the opportunity of

change inorganic substances into useful

temperature regulation with low CO2

sugars and then other molecules which is

losses and making it feasible to focus and

similar to plants. The limitations of de-

regulate the irradiance for augmentation

velopment forced by the constraint and

of algal cultures with reduced risk of cul-

saturation of light and results have im-

ture contamination (Masojidek _et al.,_

pelled algal biotechnologists to come up-

2009). Rorrer _et al._ (2004) showed the

on for resolution to convince this outcome

bioprocess engineering method, especial-

in outdoor cultures (Masojidek _et al.,_

ly for the marine algae process through

2003). Due to the significance of light

the development of cell and tissue culture

saturation, information on the behavior of

systems and his group described a diversi-

cyanobacteria cultures showed to very

ty of techniques to grow phototropic sus-

high illumination is comparatively sparse

pension cultures appropriate for the culti-

and most of it has been acquired in labor-

vation in PBR systems. Also, the same

atory experiments. The possible devel-

group compared three foremost PBR con-

opment of algal biotechnology is moving

figurations for macroalgal suspension cul-

towards the field of high charge commod-

ture systems viz., bubble-column or air-

ities grown in well controlled cultivation

lift, tubular recycle and stirred tank, and

conditions, such as temperature, nutrient

considered the major factors that limit

supply, pH, light, and CO2 (Masojidek _et_

their development and cultivation perfor-

_al.,_ 2009). Aquaculture has turned into

mance (Rorrer _et al.,_ 2004). Later on, a

extremely important in the salvation of

novel tubular PBR with linear Fresnel

food and nutrition for the growing world

lenses depends on solar concentrators dis-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 201

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ covered and studied the correlation be-energy and environment (Muller-Feuga _et_

tween changes in physiological and pho-

_al.,_ 2012). Greater attention to several

tochemical characters in central Europe

enterprises and businesses has to be the

(Masojidek _et al.,_ 2009). Nevertheless, a

reality that there is plenty of other value

number of guideline ideologies about the

added product opportunities that exist

best possible design of PBRs have been

based on algae. The recombinant pharma-

established (refer for recent reviews, Pulz,

ceutical protein production began to be

2001; Carvalho _et al.,_ 2006). But, still the

improved more than 25 years before and

worldwide monoculture of algae is large-

today above 300 protein commodities are

ly limited to some unique species, includ-

in the market or in late clinical stages

ing extremophiles, easy and quick grow-

(Decker and Reski, 2008). Though, a few

ers and this slow growing spp., have to be

years back, the moss _Physcomitrella pat-_

cultivated using PBR systems in order to

_ens_ were suggested and merchandised as

certify their control, although the cultiva-

substitute production host that convene

tion of subtle spp., needs unusual protec-

this concern through providing a special

tion to keep away from forces which

amenability for accurate genetic engineer-

could give stress to the cells, centrifugal

ing together with economic cultivation

forces, mainly surface and shear stress

(Decker and Reski, 2008). However, the

(Masojidek _et al.,_ 2009).

production cost of algae in large scale is

not yet competitive, predominantly be-

**3. A long history of algal use**

cause of the prevalence of the PBR tech-

nology worldwide. As well the major

Over the past two decades a great

progress in classic PBR design, a number

covenant of literature has been focused on

of novel configurations have been antici-

the PBR potential of algal commercial

pated in the last two decades to advance

applications, due to space limitations we

their management expressed in terms of

account a few of them in this chapter.

biomass productivity, photosynthetic effi-

Kelps are cost-effectively precious and

ciency, light absorption and light to bio-

primary producers, as a result, numerous

mass acquiesce.

studies on algal cultivation through breed-

****

ing have considered enhancing its quality

**4. Advantages of PBRs in algal culti-**

and productivity (Sato _et al.,_ 2017). How-

**vation and exploring the power of**

ever, most cultivation tests have been per-

**algae**

formed in the ocean, thereby limiting the

development of new cultivars. Macroal-

The novel advantages of PBR

gae was being eaten at least 1,500 years

have been recommended in the last two

ago in Japan and it remains an important

decades as a substitute to the open type

food in many cultures where it is valued

cultures and they are widely used in food,

for its high mineral content (i.e., Nori and

cosmetics and pharmaceutical industries

Laverbread). Closer to home in Europe,

to produce algal biomass in large scale. In

kelp was farmed extensively from the 17th

another way, the cost of nutrient media

to 19th centuries for processing into soda

for algal (cyanobacteria) cultivation is

for the linen industry and into iodine for

greatly cheaper than to heterotrophic bac-

medicinal purposes. Micro algae have

teria and the inorganic nutrients at low

been used for decades as a food supple-

concentration limit the contamination

ment (i.e., spirulina) and as a feedstock

through other microorganisms (Chetsu-

for farmed shellfish and finfish. Com-

mon _et al.,_ 1994). The harvesting cost can

pounds extracted from both micro- and

be controlled through the production of

macroalgae today find their way into eve-

bioactive substances from the marine al-

ryday foods, health, cosmetics and phar-

gae cultivated by PBRs. An evidence of

maceutical industries, agricultural fields,

the rising interest in PBR applications is

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_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ the large number of research and review

consumption (Ngo _et al.,_ 2011; Taelman

articles published over the past few years

_et al.,_ 2015).

showing

novel

PBR

developments

(Olivieri _et al.,_ 2014; reviewed in this

**5. Extraction technology of bioactive**

chapter). An interesting application of

**compounds from marine algae**

micro algal cultures in PBRs concerns the

welfare of space mission teams. A PBR

Extraction technology is critical

intended to produce algae protein along

for the complete use of marine algae

with oxygen and to remediate devastate

(seaweeds and cyanobacteria) raw materi-

and CO2 can form a closed ecological life

als to reducing time, with high yields at

support system where it is elemental for

low costs and with low consumption of

astronauts concerned in long term inves-

solvents (Caamal-Fuentes _et al.,_ 2013).

tigation task and on space stations on oth-

Natural bioactive composite consists of

er planets like the Moon and Mars

an extensive range of functionalities and

(Olivieri _et al.,_ 2014). Recently, Cao _et al._

structures which give an outstanding pool

(2012) patented a method for micro algal

of molecules for the assembly of func-

biomass cultivation by 100% CO2 to pro-

tional foods, nutraceuticals and food addi-

long the life support system in such a

tives. A few of those natural compounds

harsh environment.

can be originated in nature at an elevated

Algal products are again being

level eg., polyphenols but other com-

commercially explored and developed

pounds can only be available at very

with the help of a growing global industry

small concentrations, so that enormous

using the latest algal biotechnologies.

harvesting is essential to acquire adequate

This involves the mass cultivation of mi-

amounts. The structural complexity of the

cro- and macroalgae and conversion of

compounds makes chemical synthesis un-

the harvested biomass into a range of val-

successful and the intrinsic complications

ue-added products. The EnAlgae project

in producing and screening of these com-

aims to develop technologies that will be

pounds include led to the improvement of

both economically-viable and environ-

advanced technologies (Gil-Chavez _et al.,_

mentally-friendly ways so that the pro-

2012). Marine algae derived bioactive

duction of algal biomass can be rolled out

compound production may be regulated

on industrial scales. Since macroalgae

by the assortment of suitable cultivation

generates energy through photosynthesis,

conditions and making these algae accu-

algae biomasses are situated in the aquatic

rate natural bioreactors (Ibanez _et al.,_

euphotic zone upper layers. Algal photo-

2012). For extraction of novel compounds

synthetic systems are comparable to that

from algae, it is essential to estimate how

of plants growing on terrestrial lands,

efficient ingredients are acquired. In this

however, usually, they are highly capable

view, there is a need to unite relevant,

of converting sunlight into biomass since

cost-effective, choosy and environmental-

less complex cellular structure and their

ly friendly extraction methods with the

direct access to water, nutrients and CO2

permitting requirements about the use of

(Kilinc _et al.,_ 2013; Taelman _et al.,_ 2015).

food grade reagents/solvents and progres-

Chetsumon _et al._ (1994) produced antibi-

sions. The application of nature friendly,

otic through the immobilized cyanobacte-

advanced and clean extraction methods

rium, _Scytonema_ spp., in a seaweed type

allows for the accomplishment of the

PBR. European Union imports of

aimed compounds of significance with

macroalgae have conventionally been

high efficient extraction techniques,

used as the products for agriculture or in

whereas, at the similar moment in time,

the food, pharmaceutical and cosmetic

reducing the utilize of organic toxic sol-

industries for their useful extracts and are

vents.

very less frequently used for direct human

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 203

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ The "Green Chemistry" associa-synthesized from marine algae have been

tion has been investigating traditions to

industriously illustrated in literature for

ease the risk of harmful chemical contact

their biological and physico-chemical

to the environment and humans. Ibanez _et_

characteristics in various models (Caillot

_al._ (2012) showed a schematic diagram

_et al.,_ 2012). To give an instance, a linear

which stated about the changes in the

_β_ -1,3-glucan polymer known as laminarin

waste prevention hierarchy. The innova-

derived from brown algae provoked the

tion and progress of marine algae based

development of antifungal compounds in

bioactive compounds is a quite new area

alfalfa ( _Medicago sativa_ ) cotyledons

when compared to the invention of bioac-

(Kobayashi _et al.,_ 1993) and a number of

tive compounds from other global re-

defense responses in rice ( _Oryza sativa_ )

sources. As a result, in the growth of this

(Inui _et al.,_ 1997), tobacco ( _Nicotiana_

field, novel, eco-friendly and sustainable

_tabacum_ )

cell

suspension

cultures

trends should be followed (reviewed by

(Klarzynski _et al.,_ 2000), and grapevine

Ibanez _et al.,_ 2012).

( _Vitis vinifera_ ) (Aziz _et al.,_ 2003). The

effectiveness of two different algal sac-

**6. Application of algal extracts for**

charides, glucuronan and oligoglucu-

**plant growth and development**

ronans against postharvest gray mold

caused by _Botrytis cinerea_ and blue mold

Worldwide, the policy drivers

caused by _Penicillium expansum_ on apple

supporting the application of agricultural

fruit, and the associated defense responses

biostimulants derived marine algae in ag-

implicated were assessed (Abouraicha _et_

riculture and are also emphasised in crop

_al.,_ 2015, 2016). Ulvan (algae derived

management through soil drenches, seed

glyco product) activated a large set of re-

priming, hydroponic treatments and foliar

lated defense enzymes and accumulated a

sprays (Sharma _et al.,_ 2014). The majori-

variety of resistance substances induced

ty of the research works associated to

the resistance to anthracnose caused by

crop improvement and protection has at-

_Colletotrichum lindemuthianum_ in beans

tracted in recent times an extraordinary

( _Phaseolus vulgaris_ ) (Paulert _et al.,_ 2009;

awareness in order to widen safer and

de Freitas and Stadnik, 2012), and pro-

new control methods as an alternative of

tected from Fusarium wilt in seedlings of

these methods depending on chemical

tomato ( _Lycopersicon esculentum_ ) (El

pesticides. One approach might be the

Modafar _et al.,_ 2012). Pre-treatment of

generation of the self-defenses in plants

wheat ( _Triticum aestivum_ ) and barley

through natural extracts (Terry and Joyce,

( _Hordeum vulgare_ ) plants with ulvan ob-

2004; Walters _et al.,_ 2005). Marine mi-

tained from green macroalgae _Ulvan fas-_

cro- and macroalgae are the abundant for

_ciata_ significantly reduced the symptom

several bioactive compounds, including

severity of _Blumeria graminis_ infection

algal polysaccharides, and the composi-

(Paulert _et al.,_ 2010).

tion and structure of these compounds

The application of algae derived

have greatly contributed to their potential

agricultural biostimulants on crop plants

on activating signaling pathways and en-

produced numerous benefits in the midst

hancing defense mechanisms in a variety

of reported effects including enhanced

of plants (El Modafar _et al.,_ 2012; Arman

seed germination, increased shooting pro-

and Qader, 2012; Abouraicha _et al.,_ 2015;

liferation, enhanced rooting (Hernandez-

de Freitas and Stadnik, 2015). In the late

Herrera _et al.,_ 2013; Satish _et al.,_ 2015,

1940's, the first seaweed liquid extract for

2016), higher crop and fruit yields, en-

agricultural utilize has been urbanized

hanced photosynthetic activity, salinity,

and sold as Maxicrop (Satish _et al.,_ 2015).

drought and freezing tolerance, and re-

All through the past two decades, _β_ -(1,4)-

sistance to various bacteria, fungi and vi-

d-polyglucuronic

acids

(glucuronans)

ruses (Sharma _et al.,_ 2014). We examined

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_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ the various marine macroalgae extracts in

spp., through increased chlorophyll con-

bioassays of _Solanum trilobatum_ and _El-_

tent, enhanced nutrient uptake, augmented

_eusine_

_coracana_

seed

germination,

flower and fruit set leading to elevated

growth assays and _in vitro_ regeneration

yields, delayed senescence and longer

including _Bacopa monnieri_ (Satish _et al.,_

shelf life of fruits (Briceno-Dominguez _et_

2015, 2016; Rency _et al.,_ 2017). In the

_al.,_ 2014). In addition, the chemical

past, marine algae extracts have been suc-

breakdown of marine algae and their bio-

cessfully affianced as biostimulatns in

active compounds has made known the

numerous plants, such as _Arabidopsis tha-_

standpoint of a wide range of substances

_liana_ (Khan _et al.,_ 2011) and _Nicotiana_

such as gibberellins, auxins and cytokin-

_tobaccum_ (Sanderson and Jameson, 1986)

ins which are stimulating plant shoot and

and _Glycine max_ (Stirk and Van Staden,

root growth, maturation and production

1997) for the detection of cytokinin-like

(Hernandez-Herrera _et al.,_ 2013; Satish _et_

activity. Marine algae or their substances

_al.,_ 2015). The supplementation of algae

are applied successfully for the improve-

based extracts showed positive response

ment of vegetables like _Brassica oleracea_

to the growth of various vegetables, fruits

(Abetz and Young, 1983), _Lactuca sativa_

and other food crops since micro (Cu, Zn,

(Abetz and Young, 1983; Crouch _et al.,_

B, Mn, Co and Mo), macro (Ca, K and P)

1990), _Beta vulgaris_ (Featonby-Smith and

nutrients, amino acids, organic acids, vit-

Van Staden, 1983b), _Cucumis sativus_

amins, antioxidants and complex minerals

(Nelson and Van Staden, 1984; Jayara-

are available in their extracts (Hernandez-

man _et al.,_ 2011), _Daucus carota_ (Jayaraj

Herrera _et al.,_ 2013; Briceno-Dominguez

_et al.,_ 2008), _Lycopersicon esculentum_

_et al.,_ 2014; Satish _et al.,_ 2015, 2016;

(Featonby-Smith and Van Staden, 1983a;

Rency _et al.,_ 2017). More or less, around

Finnie and Van Staden, 1985; Kumari _et_

15 million metric tons of marine plant

_al.,_ 2011; Zadope _et al.,_ 2011; Basher _et_

products are emerging every year (FAO,

_al.,_ 2012; Vinoth _et al.,_ 2012, 2014; Her-

2006), a substantial percentage of which

nandez-Herrera _et al.,_ 2013; Briceno-

distributed for nutrient supplementation

Dominguez _et al.,_ 2014), _Brassica rapa_

and as biostimulants or biofertilizers in

(Chinese cabbage) (Sharma _et al.,_ 2012),

worldwide agriculture. At present, the

_Abelmoschus esculentus_ (Papenfus _et al.,_

advanced crop growing is paying atten-

2013) and _Brassica napus_ plants (Ferreira

tion for existing biotechnologies where it

and Lourens, 2002). Other than this, puls-

would consent for a concession in the ap-

es such as _Phaseolus vulgaris_ (Featonby-

plication of chemicals without toxic ef-

Smith and Van Staden, 1984; Beckett _et_

fects on crop growth and yield as well as

_al.,_ 1994), _Phaseolus acutifolius_ (Beckett

the farmers' earnings (Hernandez-Herrera

_el al.,_ 1994), _Vigna sinensis_ (Sivasankari

_et al.,_ 2013). These biostimulants can be

_et al.,_ 2006), _Glycine max_ (Rathore _et al.,_

useful as an alternative to herbs and pesti-

2009) and _Vigno mungo_ (Sharma _et al.,_

cides, or used in conjunction through syn-

2012; Selvam and Sivakumar, 2013; Bri-

thetic/artificial crop protection goods and

ceno-Dominguez _et al.,_ 2014), in cereals

as plant growth stimulants (Satish _et al.,_

like _Zea mays_ (Jeannin, 1991), _Hordeum_

2015, 2016), and they play a role in sus-

_vulgare_ (Steveni _et al.,_ 1992) and _Triti-_

taining the crop production levels, high

_cum aestivum_ (Beckett and Van Staden,

yield, health and quality (Sharma _et al.,_

1989; Kumar and Sahoo, 2011), horticul-

2014), and marine algae or their deriva-

tural crops like _Fragaria ananassa_ (Spi-

tives are remaining largely unexploited

nelli _et al.,_ 2010) and in _Malus domestica_

universally.

(Spinelli _et al.,_ 2009) seaweed extracts

****

applied successfully in different forms.

**7. Future prospects**

Marine algae extracts elicit a wide

range of responses in the different plants

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 205

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ Although a little information on

gappa University Bioinformatics Infra-

the effects of glucuronan and its oligomer

structure Facility (funded by Department

is already available on biological activi-

of Biotechnology, Government of India;

ties in plants (El Modafar _et al.,_ 2012;

Grant No.BT/BI/25/001/2006).

Caillot _et al.,_ 2012; reviewed above) there

is a lack of studies regarding plant-

**References**

pathogen models (Abouraicha _et al.,_

****

2017). Research outputs to decrease use

**Abetz, P. and Young, C. L. (1983).** The

of synthetic fertilizers and chemical pesti-

effect of seaweed extract sprays

cides for growing agricultural crops

derived from _Ascophyllum no-_

through the invention of novel natural

_dusum_ on lettuce and cauliflower

compounds is immediately required in

crops. Botanica Marina **26, 487–**

order to assemble the needs of sustainable

**492.**

agriculture and to counter to the increas-

**Abouraicha, E. F., Alaoui-Talib, Z.,**

ing demand for pesticide-free food

**Boutachfaiti, R. E., Petit, E.,**

(Abouraicha _et al.,_ 2017). Worldwide en-

**Courtois, B., Courtois, J. and**

vironmental demonstration in relation to

**Modafar, C. E. (2015).** Induction

the exhaustion of natural resources and

of natural defense and protection

industrialized pollution had led to the im-

against _Penicillium expansum_ and

provement of more renewable resources

_Botrytis cinerea_ in apple fruit in re-

such as algal biomass, which is translated

sponse to bioelicitors isolated from

in elevated aquaculture assembly rates of

green algae. Scientia Horticulturae

6.4 million tonnes in 2000 where it was

**181, 121–128.**

20.8 million tonnes fresh weight in 2012.

**Abouraicha, E. F., Alaoui-Talibi, Z. E.,**

Since the potential ecological and eco-

**Tadlaoui-Ouafi, A., Boutachfaiti,**

nomic payment becomes evident, the re-

**R. E., Petit, E., Douira, A., Cour-**

search institutions, government and in-

**tois, B., Courtois, J. and Modafar,**

dustries have been showing special inter-

**C. E. (2017).** Glucuronan and oli-

est towards developing the algal biomass

goglucuronans isolated from green

(Kilinc _et al.,_ 2013; Taelman _et al.,_ 2015).

algae activate natural defense re-

For the past few years, PBR sys-

sponses in apple fruit and reduce.

tems have been improved through adopt-

Journal of Applied Phycology **29,**

ing photosynthesis in marine algae and it

**471–480.**

is a positive application of a photovoltaic

**Arman, M. and Qader, S. A. U. (2012).**

PBR system mimicking a plant. The effi-

Structural analysis of kappacarra-

ciency and miniaturization of algal PBRs

geenan isolated from _Hypneamusci-_

are a different and novel challenge and

_formis_ (red algae) and evaluation as

the PBR culture systems are extraordinar-

an elicitor of plant defense mecha-

ily scaled up with respect to the open cul-

nism. Carbohydrate Polymers **88,**

tures.

**1264–1271.**

**Basher, A. A., Mohammed, A. J. and**

__
__

## Acknowledgements

**Teeb, A. I. H. (2012).** Effect of

seaweed and drainage water on

The author L. Satish sincerely

germination and seedling growth of

thanks the University Grants Commis-

tomato ( _Lycopersicon_ spp.). Euphra-

sion, New Delhi, India for financial sup-

tes Journal of Agriculture Science **4,**

port in the form of UGC-BSR SRF (UGC

**24–39.**

order no. F.4-1/2006 (BSR)/7-326/2011-

**Beckett, R. P. and Van Staden, J.**

BSR). Also the authors gratefully

**(1989).** The effect of seaweed con-

acknowledge the computational and bio-

centrate on the growth and yield of

informatics facility provided by the Ala-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 206

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ potassium stressed wheat. Plant Soil

**chi, T. (1994).** Antibiotic produc-

**116, 29–36.**

tion by the immobilized cyanobacte-

**Beckett, R. P., Mathegka, A. D. M. and**

rium, Scytonema sp. TISTR 8208,

**Van Staden, J. (1994).** Effect of

in a seaweed-type photobioreactor.

seaweed concentrate on yield of nu-

Journal of applied phycology, **6(5),**

trient-stressed

tepary

bean

**539-543.**

( _Phaseolus acutifolius_ Gray). Jour-

**Crouch, I. J., Beckett, R. P. and Van**

nal of Applied Phycology **6, 429–**

**Staden, J. (1990).** Effect of sea-

**430.**

weed concentrate on the growth and

**Briceno-Dominguez, D., Hernandez-**

mineral nutrition of nutrient-stressed

**Carmona, G., Moyo, M., Stirk, W.**

lettuce. Journal of Applied Phycolo-

**and Van Staden, J. (2014).** Plant

gy **2, 269–272.**

growth promoting activity of sea-

**de Freitas, M. B. and Stadnik, M. J.**

weed liquid extracts produced from

**(2012).** Race-specific and ulvan-

_Macrocystispyrifera_ under different

induced defense responses in bean

pH and temperature conditions.

( _Phaseolus vulgaris_ ) against _Colle-_

Journal of Applied Phycology **26,**

_totrichum lindemuthianum_. Physio-

**2203–2210.**

logical and Molecular Plant Pathol-

**Caamal-Fuentes, E., Chale-Dzul, J.,**

ogy **78, 8–13.**

**Moo-Puc, R., Freile-Pelegrin, Y.**

**de Freitas, M. B. and Stadnik, M. J.**

**and Robledo, D. (2013).** Bio-

**(2015).** Ulvan-induced resistance in

prospecting of brown seaweed

_Arabidopsis thaliana_ against _Alter-_

(Ochrophyta) from the Yucatan

_naria brassicicola_ requires reactive

Peninsula: cytotoxic, antiprolifera-

oxygen

species

derived

from

tive, and antiprotozoal activities.

NADPH oxidase. Physiological and

Journal of Applied Phycology **26,**

Molecular Plant Pathology **90, 49–**

**1009–1017.**

**56.**

**Caillot, S., Rat, S., Tavernier, M. L.,**

**Decker, E. L. and Reski, R. (2008).**

**Michaud,**

**P.,**

**Kovensky,**

**J.,**

Current achievements in the produc-

**Wadouachi, A., Clement, C.,**

tion of complex biopharmaceuticals

**Baillieul, F. and Petit, E. (2012).**

with moss bioreactors. Bioprocess

Native and sulfated oligoglucu-

and Biosystems Engineering **31, 3–**

ronans as elicitors of defence-

**9.**

related responses inducing protec-

**El Modafar, C., Elgadda, M., Elbou-**

tion against _Botrytis cinerea_ of _Vitis_

**tachfaiti, R., Abouraicha, E.,**

_vinifera_. Carbohydrate Polymers **87,**

**Zehhar, N., Petit, E., El Alaoui-**

**1728–1736.**

**Talibi, Z., Courtois, B. and Cour-**

**Cao, G., Concas, A., Corrias, G., Li-**

**tois, J. (2012).** Induction of natural

**cheri, R., Orru, R. and Pisu, M.**

defence accompanied by salicylic

**(2012).** A process for the production

acid-dependant systemic acquired

of useful materials to sustain

resistance in tomato seedlings in re-

manned space missions on Mars

sponse to bioelicitors isolated from

through in-situ resources utilization.

green algae. Scientia Horticulturae

Patent **PCT/IB2012/053754**

**138, 55–63.**

**Carvalho, A. P., Meireles, L. A. and**

**FAO. (2012).** Yearbook of fishery and

**Malcata, F. X. (2006).** Microalgal

aquaculture statistics, in: FAO (Ed.),

reactors: a review of enclosed sys-

Dataset global aquaculture produc-

tem designs and performances. Bio-

tion

1950–2012.

technology Progress **22, 490–1506.**

http://www.fao.org/fishery/statistics

**Chetsumon, A., Maeda, I., Umeda, F.,**

/global-aquaculture-

**Yagi, K., Miura, Y., and Mizogu-**

production/query/en.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 207

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ **FAO. (2013).** Agriculture and environ-nology. Longman Scientific &

mental services discussion paper 03,

Technical, New York. **pp.** **161–238.**

in: FAO (Ed.), Fish to 2030 pro-

**Hernandez-Herrera, R. M., Santacruz-**

spects for fisheries and aquaculture,

**Ruvalcaba, F., Ruiz-Lopez, M. A.,**

World Bank report number 83177-

**Norrie,**

**J.**

**and**

**Hernandez-**

GLB (Washington DC, United

**Carmona, G. (2013).** Effect of liq-

States).

uid seaweed extracts on growth of

**Featonby-Smith, B. C. and Van**

tomato seedlings ( _Solanum lycoper-_

**Staden, J. (1983a).** The effect of

_sicum_ L.). Journal of Applied Phy-

seaweed concentrate on the growth

cology **26, 619–628.**

of tomato plants in nematode infect-

**Ibanez, E., Herrero, M., Mendiola, J.**

ed soil. Scientia Horticulturae **20,**

**A. and Castro-Puyana, M. (2012).**

**137–146.**

Extraction and characterization of

**Featonby-Smith, B. C. and Van**

bioactive compounds with health

**Staden, J. (1983b).** The effect of

benefits from marine resources:

seaweed concentrate and fertilizer

Macro and micro algae, cyanobacte-

on the growth of _Beta vulgaris_.

ria, and invertebrates. Hayes M.

Zeitschrift für Pflanzenphysiologie

(Ed.) Marine bioactive compounds:

**112, 155–162.**

Sources, characterization and appli-

**Featonby-Smith, B. C. and Van**

cations. **pp. 55–98.**

**Staden, J. (1984).** The effect of

**Jayaraj, J., Wan, A., Rahman, M. and**

seaweed concentrate and fertilizer

**Punja, Z. K. (2008).** Seaweed ex-

on growth and endogenous cytokin-

tract reduces foliar fungal diseases

in content of _Phaseolus vulgaris_.

on carrot. Crop Protection **27, 1360–**

South African Journal Botany **3,**

**1366.**

**375–379.**

**Jayaraman, J., Norrie, J. and Punja,**

**Ferreira, M. I. and Lourens, A. F.**

**Z. K. (2011).** Commercial extract

**(2002).** The efficacy of liquid sea-

from the brown seaweed _Ascophyl-_

weed extract on the yield of canola

_lum nodosum_ reduces fungal diseas-

plants. South African Journal of

es in greenhouse cucumber. Journal

Plant and Soil **19, 159–161.**

of Applied Phycology **23, 353–361.**

**Finnie, J. F. and Van Staden, J. (1985).**

**Jeannin, I., Lescure, J. C. and Morot-**

Effect of seaweed concentrate and

**Gaudry, J. F. (1991).** The effect of

applied hormones on in vitro cul-

aqueous seaweed sprays on the

tured tomato roots. Journal of Ap-

growth of maize. Botanica Marina

plied Phycology **3, 215–222.**

**34, 141–145.**

**Gil-Chavez, G. J., Villa, J. A., Ayala-**

**Khan, W., Hiltz, D., Critchley, A. T.**

**Zavala, J. F., Heredia, J. B.,**

**and Prithiviraj, B. (2011).** Bioas-

**Sepulveda, D., Yahia, E. M. and**

say to detect _Ascophyllum nodosum_

**Gonzalez-Aguilar, G. A. (2013).**

extract-induced cytokinin-like activ-

Technologies for extraction and

ity in _Arabidopsis thaliana_. Journal

production of bioactive compounds

of Applied Phycology **23, 409–414.**

to be used as nutraceuticals and

**Kilinc, S., Cirik, B., Turan, G.,**

food ingredients: An overview.

**Tekogul, H. and Koru, E. (2013).**

Comprehensive Reviews in Food

Seaweeds for food and industrial

Science and Food Safety **12, 5–23.**

applications, in: Innocenzo Muz-

**Glombitza, K. W. and Koch, M.**

zalupo (Ed.), Agricultural and bio-

**(1989).** Secondary metabolites of

logical sciences, food industry. In

pharmaceutical potential. In Cress-

Tech. ISBN: 978-953-51-0911-2

well RC, Rees TAV, Shah N (Eds),

**Kumar, G. and Sahoo, D. (2011).** Ef-

Algal and cyanobacterial biotech-

fect of seaweed liquid extract on

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 208

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ growth and yield of _Triticum aes-tions, operation strategies and appli-_

 _

_tivum_ var. Pusa Gold. Journal of

cations. Journal of Chemical Tech-

Applied Phycology **23, 251–255.**

nology and Biotechnology **89, 178–**

**Kumari, R., Kaur, I. and Bhatnagar,**

**195.**

**A. K. (2011).** Effect of aqueous ex-

**Papenfus, H. B., Kulkarni, M. G.,**

tract of _Sargassum johnstonii_ Setch-

**Stirk, W. A., Finnie, J. F. and Van**

ell & Gardner on growth, yield and

**Staden, J. (2013).** Effect of a com-

quality of _Lycopersicon esculentum_

mercial seaweed extract (Kelpak®)

Mill. Journal of Applied Phycology

and

polyamines

on

nutrient-

**23, 623–633.**

deprived (N, P and K) okra seed-

**Masojidek, J., Papacek, S., Sergejevo-**

lings. Scientia Horticulturae **151,**

**va, M., Jirka, V., Cerveny, J.,**

**142–146.**

**Kunc, J., Korecko, J., Verboviko-**

**Paulert, R., Talamini, V., Cassolato, J.,**

**va, O., Kopecky, J., Stys, D. and**

**Duarte, M., Noseda, M., Smania,**

**Torzillo, G. (2003).** A closed solar

**A. J. and Stadnik, M. (2009).** Ef-

photobioreactor for cultivation of

fects of sulfated polysaccharide and

microalgae under supra-high irradi-

alcoholic extracts from green sea-

ance: basic design and performance.

weed _Ulva fasciata_ on anthracnose

Journal of Applied Phycology **15,**

severity and growth of common

**239–248.**

bean ( _Phaseolus vulgaris_ L.). Jour-

**Masojidek, J., Sergejevova, M., Rott-**

nal of Plant Diseases and Protection

**nerova, K., Jirka, V., Korecko, J.,**

**116, 263–270.**

**Kopecky, J., Zatkova, V., Torzillo,**

**Pulz, O. (2001).** Photobioreactors: pro-

**G. and Stys, D. (2009).** A two-stage

duction systems for phototrophic

solar photobioreactor for cultivation

microorganisms. Applied Microbi-

of microalgae based on solar con-

ology and Biotechnology **57, 287–**

centrators. Journal of Applied Phy-

**293.**

cology **21, 55–63.**

**Rathore, S. S., Chaudhary, D. R.,**

**Muller-Feuga, A., Lemar, M., Vermel,**

**Boricha, G. N. and Ghosh, A.**

**E., Pradelles, R., Rimbaud, L. and**

**(2009).** Effect of seaweed extract on

**Valiorgue, P. (2012).** Appraisal of a

the growth, yield and nutrient up-

horizontal two-phase flow photobio-

take of soybean ( _Glycine max_ ) un-

reactor for industrial production of

der rainfed conditions. South Afri-

delicate microalgae species. Journal

can Journal of Botany **75, 351–355.**

of Applied Phycology **24, 349–355.**

**Rency, A. S., Satish, L., Pandian, S.,**

**Nelson, W. R. and Van Staden, J.**

**Rathinapriya, P. and Ramesh, M.**

**(1984).** The effect of seaweed con-

**(2017).** In vitro propagation and ge-

centrate on growth of nutrient-

netic fidelity analysis of alginate-

stressed

greenhouse

cucumbers.

encapsulated

_Bacopa_

_monnieri_

Hortscience **19, 81–82.**

shoot tips using _Gracilaria salicor-_

**Ngo, D. H., Wijesekaraa, I., Voa, T. S.,**

_nia_ extracts. Journal of Applied

**Van Taa, Q. and Kima, S. K.**

Phycology **29, 481–494.**

**(2011).** Marine food-derived func-

**Richmond, A. and Vonshak, A. (1978).**

tional ingredients as potential anti-

_Spirulina_ culture in Israel. Biologi-

oxidants in the food industry: an

cal constraints in algal biotechnolo-

overview. Food Research Interna-

gy **11, 274–279.**

tional **44, 523–529.**

**Rorrer, G. L. and Cheney, D. P.**

**Olivieri, G., Salatino, P. and Mar-**

**(2004).** Bioprocess engineering of

**zocchella, A. (2014).** Advances in

cell and tissue cultures for marine

photobioreactors for intensive mi-

seaweeds. Aquacultural Engineering

croalgal

productions:

configura-

**32, 11–41.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 209

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ **Sanderson, K. J. and Jameson, P. E.**

**R., McCormack, R. and Mellon,**

**(1986).** The cytokinins in a liquid

**R. (2012).** Biostimulant activity of

seaweed extract: could they be ac-

brown

seaweed

species

from

tive ingredients?. Acta Horticulturae

Strangford Lough: compositional

**179, 113–116.**

analyses of polysaccharides and bi-

**Satish,**

**L.,**

**Rameshkumar,**

**R.,**

oassay of extracts using mung bean

**Rathinapriya, P., Pandian, S.,**

( _Vigno mungo_ L.) and pakchoi

**Rency, A. S., Sunitha, T. and**

( _Brassica rapachinensis_ L.). Journal

**Ramesh, M. (2015).** Effect of sea-

of Applied Phycology **24, 1081–**

weed liquid extracts and plant

**1091.**

growth regulators on in vitro mass

**Sivasankari, S., Venkatesalu, V., An-**

propagation of brinjal ( _Solanum_

**antharaj, M. and Chandrasekar-**

_melongena_ L.) through hypocotyl

**an, M. (2006).** Effect of seaweed

and leaf disc explants. Journal of

extracts on the growth and biochem-

Applied Phycology **27, 993–1002.**

ical constituents of _Vigna sinensis_.

**Satish, L., Rathinapriya, P., Rency, A.**

BioresourceTechnology **97, 1745–**

**S., Ceasar, S. A., Pandian, S. and**

**1751.**

**Rameshkumar, R. (2016).** Somatic

**Spinelli, F., Fiori, G., Noferini, M.,**

embryogenesis and regeneration us-

**Sprocatti, M. and Costa, G.**

ing _Gracilaria edulis_ and _Padina_

**(2009).** Perspectives on the use of a

_boergesenii_ seaweed liquid extracts

seaweed extract to moderate the

and genetic fidelity in finger millet

negative effects of alternate bearing

( _Eleusine coracana_ ). Journal of Ap-

in apple trees. The Journal of Horti-

plied Phycology **28, 2083–2098.**

cultural Science and Biotechnology

**Sato, Y., Yamaguchi, M., Hirano, T.,**

**84, 131–137.**

**Fukunishi, N., Kawano, T. and**

**Spinelli, F., Fiori, G., Noferini, M.,**

**Kawano, S. (2017).** Effect of water

**Sprocatti, M. and Costa, G.**

velocity on _Undaria pinnatifida_ and

**(2010).** A novel type of seaweed ex-

_Saccharina japonica_ growth in a

tract as a natural alternative to the

novel tank system designed for

use of iron chelates in strawberry

macroalgae cultivation. Journal of

production. Scientia Horticulturae

Applied Phycology **29, 1429–1436.**

**125, 263–269.**

**Selvam, G. G. and Sivakumar, K.**

**Steveni, C. M., Norrington-Davies, J.**

**(2013).** Effect of foliar spray from

**and Hankins, S. D. (1992).** Effect

seaweed liquid fertilizer of _Ulva re-_

of seaweed concentrate on hydro-

_ticulate_ (Forsk.) on _Vigna mungo_ L.

ponically grown spring barley.

and their elemental composition us-

Journal of Applied Phycology **4,**

ing SEM – energy dispersive spec-

**173–180.**

troscopic analysis. Asian Pacific J

**Stirk, W. A. and Van Staden, J. (1997).**

Reproduction **2, 119–125.**

Comparison of cytokinin- and aux-

**Sharma, H. S. S., Fleming, C., Selby,**

in-like activity in some commercial-

**C., Rao, J. R. and Martin, T.**

ly used seaweed extracts. Journal of

**(2014).** Plant biostimulants: a re-

Applied Phycology **8, 503–50.**

view on the processing of macroal-

**Taelman, S. E., Champenois, J., Ed-**

gae and use of extracts for crop

**wards, M. D., Meester, S. D. and**

management to reduce abiotic and

**Dewulf, J. (2015).** Comparative en-

biotic stresses. Journal of Applied

vironmental life cycle assessment of

Phycology **26, 465–490.**

two seaweed cultivation systems in

**Sharma,**

**S.**

**H.**

**S.,**

**Lyons,**

**G.,**

North West Europe with a focus on

**McRoberts, C., McCall, D., Car-**

quantifying sea surface occupation.

**michael, E., Andrews, F., Swan,**

Algal Research **11, 173–183.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 210

_Biotech Sustainability (2017)_

_Potential of Marine Algae Derived Extracts Lakkakula and Manikandan_ **Terry, L. A. and Joyce, D. C. (2004).**

ing the efficacy of resistance elici-

Elicitors of induced disease re-

tors. Phytopathology **95, 1368–**

sistance in postharvest horticultural

**1373.**

crops: a brief review. Postharvest

**Wu, X. and Merchuk, J. C. (2004).**

Biology and Technology **32, 1–13.**

Simulation of algae growth in a

**Vinoth, S., Gurusaravanan, P. and**

bench scale internal loop airlift reac-

**Jayabalan, N. (2012).** Effect of

tor. Chemical Engineering Science

seaweed extracts and plant growth

**59, 2899–2912.**

regulators on high-frequency in

**Yang, Z., del Ninno, M., Wen, Z. and**

vitro mass propagation of _Lycoper-_

**Hu, H. (2014).** An experimental in-

_sicon_

_esculentum_

L

(tomato)

vestigation on the multiphase flows

through double cotyledonary nodal

and turbulent mixing in a flat-panel

explant. Journal of Applied Phycol-

photobioreactor for algae cultiva-

ogy **24, 1329–1337.**

tion. Journal of Applied Phycology

**Vinoth, S., Gurusaravanan, P. and**

**26, 2097–2107.**

**Jayabalan, N. (2014).** Optimization

**Zadope, S. T., Gupta, A., Bhandari, S.**

of somatic embryogenesis protocol

**C., Rawat, U. S., Chaudhary, D.**

in _Lycopersicon esculentum_ L. using

**R., Eswaran, K. and Chikara, J.**

plant growth regulators and seaweed

**(2011).** Foliar application of sea-

extracts. Journal of Applied Phycol-

weed sap as biostimulant for en-

ogy **26, 1527–1537.**

hancement of yield and quality of

**Walters, D., Walsh, D., Newton, A. and**

tomato ( _Lycopersicon esulentum_

**Lyon, G. (2005).** Induced resistance

Mill.). Journal of Scientific and In-

for plant disease control: maximiz-

dustrial Research **70, 215–219.**

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 211

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P212-222_

**Biotransformation of Various Wastes into a Nutrient**

**Rich Organic Biofertilizer - a Sustainable Approach**

**towards Cleaner Environment**

****

**Geetha Karuppasamy1, Michael Antony D'Couto1, 2, Sangeetha Baskaran1, 3 and**

**Anant Achary1, ***

_1Department of Biotechnology, Centre for Research, Kamaraj College of Engineering and_

_Technology, K.Vellakulam-625701, Near Virudhunagar, Madurai District, Tamil Nadu,_

_India; 2JLL-Jones Lang LaSalle, TVH Belicia Towers, Thandavarayan Street, Manda-_

_velipakkam, Raja Annamalai Puram, Chennai-600028, Tamil Nadu, India; 3Department of_

_Biotechnology, St. Joseph's College Of Engineering, Old Mamallapuram Road, Sem-_

_mencherry, Kamaraj Nagar, Semmancheri, Chennai-600119, Tamil Nadu, India; * Corre-_

_spondence: achyanant@yahoo.com; Tel: 91-4549-278-171_

**Abstract:** Environmental degradation is one of the main threats confronting the world and

the widespread use of chemical fertilizers contributes essentially to the degeneration of the

environment through exhaustion of fossil fuels, generation of carbon dioxide (CO2) and

contamination of water bodies. Excessive use of fertilizers has adversely affected agricul-

tural productivity causing soil degradation. Biotransformation through vermicomposting

implies the production of nutrient-rich excreta of worms. After earthworms digest organic

matter, they excrete a high-nutrient product known as Castings. Sustainable development

can be achieved by providing adequate quantity of food for which agricultural land man-

agement is an important aspect. Earthworms are known to consume all types of organic

wastes including vegetable waste, wastes generated by pulse and rice processing industries

and other organic wastes. The food passes through the digestive tract and the worms secrete

chemicals that break down organic matter into sustainable nutrition. Vermicompost is a

peat like material consisting of excellent porosity, structure, aeration and moisture holding

capacity that makes it a good organic manure for growing plants. Another important proper-

ty of vermicompost is its vast surface area that provides strong absorbability and nutrient

retention ability. Vermicompost increases soil fertility, enhance plant growth and suppress

the population of plant pathogens and pests. As a soil conditioner, vermicompost is healthi-

er to traditional compost for its capability to improve and enhance soil configuration and its

water-holding capacity. Thus vermicomposting can be proposed as a cost effective and en-

vironment friendly method for efficient utilization of various organic wastes. This will

promote healthy plant growth and aid in sustainable management of agricultural lands with

cleaner environment.

****

_**Keywords**_ **:** Biotransformation; earthworms; organic waste; vermicomposting; vermiwash

**1. Introduction**

sector and industrial sector, the disposal

of wastes is still a prime concern. There is

With the increase in the popula-

an estimate that India produces approxi-

tion there has been a tremendous increase

mately 3000 million tons of wastes per

in generation of a variety of wastes. Alt-

annum and among that more than 60% is

hough various measures are in place to

found to be decomposable. Vegetable

handle the wastes generated by domestic

wastes are one of the major sources of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 212

_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

municipal wastes. The disposal of bio-

of the total waste produced in countries. It

degradable solid wastes from domestic,

has also been noted that 60% of food pro-

agricultural and industrial sources has

cessing industry waste belongs to organic

caused ever-increasing environmental and

matter.

economic problems (Garg _et al_., 2006).

Global urbanization has led to in-

Moreover, an additional key threat

crease in the volume of solid wastes. Ac-

is the environmental degradation due to

cording to a survey conducted in 1990,

the extensive use of chemical fertilizers

about 1.3 billion metric tons of municipal

that has led to deterioration of the envi-

solid waste was generated globally

ronment through generation of carbon

(Beede and Bloom, 1995). In today"s sce-

dioxide (CO2), exhaustion of fossil fuels,

nario, the generation of solid waste per

and water resources being contaminated.

year equals to 1.6 billion metric tons ap-

Agricultural productivity also has taken a

proximately. A significant amount of cap-

heavy toll due to disproportionate use of

ital is being invested into managing such

fertilizers leading to soil degradation.

huge volumes of solid waste suggesting

Recycling of wastes through bio-

that solid waste management (SWM) has

transformation of various biodegradable

become a large, complex and costly ser-

wastes can reduce the problem of non-

vice.

utilization of wastes. Locally available

The solid waste and its manage-

organic wastes of anthropogenic nature,

ment at various stages are chiefly affected

domestic waste and agricultural lignocel-

by the ever growing population. The

lulosic waste products can be used as bio-

numbers of households owing to the in-

fertilizer as an alternative to chemical fer-

crease in population also has an important

tilizers.

Vermicomposting

employing

role in generation and collection of the

earthworm as decomposers, for degrada-

solid waste. Solid waste is mainly collect-

tion and recycling, may be used to en-

ed by municipalities and the uncollected

hance the production of crops which are

waste, approx. 31% to 49%, is left on

free from pollution and health hazard

street or road corners, open spaces like

(Bakthvathsalam

and

Ramakrishnan,

vacant plots that pollute the environment

2004). Preserving the quantity and quality

on continuous basis. The collected waste

of soil is one of the main objectives of

is usually disposed of within or outside

current efforts to make agriculture more

the municipal limits into low lying areas

"sustainable".

like ponds etc, without any treatment ex-

cept separation of recyclable waste by

**2. Current waste disposal practices & **

scavengers.

**its impact**

Extensive research around the

world today is being carried out to find a

Currently, waste is a major con-

solution for utilization of various agricul-

cern worldwide becoming exceedingly

tural residues as energy sources. A simple

significant in developing countries like

example is the sugarcane processing in-

India, China as well as in Europe. Waste

dustries. Sugarcane bagasse and sugar-

can be classified into industrial, agricul-

cane agriculture residues are the two main

tural, sanitary and solid urban residues on

residues of sugar and ethanol production

the basis of their origin. There might be a

process. Sugarcane bagasse is the fibrous

significant change in their distribution

waste that remains after recovery of sugar

depending on the country. Waste generat-

juice via crushing and extraction. It is one

ed by various food processing industries

of the principal fuels used around the

is a good example of globally generated

world in the sugarcane agro-industry be-

waste on a large scale. Waste of this type

cause of its well-known energy proper-

has become a great source of concern as

ties. However, the bagasse management

in certain cases it is found to be over 50%

and disposal practices employed by the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 213

_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

sugar agro-industry have, in most cases,

of pesticides, heavy metals, and patho-

remained the same as those used back in

gens polluting the soil via poultry wastes

the early 19th century leading to enor-

are the central environmental issues at the

mous amount of bagasse being disposed

present time.

(Faria _et al_., 2012). This bagasse can be

an excellent material for biotransfor-

**3. Role of chemical fertilizers in envi-**

mation by vermicomposting. A similar

**ronmental degradation**

scenario is also found in industries deal-

ing with pulses and grains. The amount of

_3.1. Nutrient requirements for plant_

waste generated as husk and bran is

_growth_

enormous. Although the husk and bran

For the survivability and growth

are being widely used in various industri-

of a plant, 16 essential nutrients are re-

al applications like industrial fuel, acti-

quired including carbon, hydrogen, oxy-

vated carbon, as pet food fiber, substrate

gen, nitrogen, phosphorous, potassium,

for various fermentation processes etc,

magnesium, calcium and sulphur, iron,

there is still lot of waste that is left un-

zinc, copper, manganese, boron, chlorine

used.

and molybdenum. Air, water and sunlight

Consumption of fruits and vegeta-

provide the necessary oxygen, carbon,

bles has radically increased in the various

hydrogen and energy.

nations by more than 30% during the past

few years. It is also projected that approx-

_3.2. Chemical fertilizers – a threat_

imately 20% of all the fruits and vegeta-

Current practices mainly use the

bles produced is lost each year due to

chemical fertilizer to accomplish the nu-

spoilage. Also, vegetable markets in vari-

trients requirement of a growing plant

ous cities & towns are known to produce

which has a serious impact on the soil and

significant amount of non-edible vegeta-

water bodies. Excessive utilization of

ble wastes. As mentioned earlier, solid

chemical fertilizer leads to increased sa-

waste management of spoilt fruits and

linity of soil which is one of the major

vegetables is one of the biggest problems

threats causing lower productivity in the

faced today by all the cities including col-

soil. Some of the serious impacts of using

lection, transportation and disposal of the

chemical fertilizers are:

waste. These wastes are usually discarded

 Pollution of ground & surface wa-

in the market itself and allowed to rot.

ter

However, this discarding leads to produc-

 Soil fertility is reduced leading to

tion of hazardous ecological impacts

reduced food production

(Kumari, 2013).

 Depletion in the soil microbial

The poultry industry is one of the

ecosystem

biggest and fastest growing livestock pro-

 Ground water pollution and de-

duction systems in the world (Edwards

struction of the aquatic life

and Daniel, 1992). As per the reports of

 Loss of terrestrial and aquatic bio-

Foreign Agricultural Service in 1992, in-

diversity

ternationally, approximately 40 million

 Depletion of the Ozone leading to

metric tons of poultry meat and 600 bil-

global warming

lion eggs were produced. Although rea-

sonably flourishing, the poultry industry

Exhaustive cropping involving

is at present facing a challenging envi-

continuous use of high levels of chemical

ronmental problem. From an agricultural

fertilizers (CF) often leads to nutritional

standpoint, the role of poultry wastes in

disparity in soil and decline in crop

the contamination of soil and groundwa-

productivity (Nambiar, 1994). Numerous

ter, the eutrophication of surface waters

properties characterizing the status of soil

by nitrogen and phosphorus, and the fate

microbial biomass, activity and nutrient

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 214

_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

content have been suggested as indicators

C



6H12O6

3CO2 + 2CH4 + 393kJ ... 1

of soil quality (Doran and Parkin, 1994).

The drawback of this technique is that it

Although microbial biomass only forms a

cannot be used for mixed domestic waste

small fraction of soil organic matter, it

composting.

contributes to agricultural sustainability

because its high turnover rate is responsi-

ble for nutrient release and therefore pro-

motes plant uptake (Smith _et al_., 1993).

Conventional

agro-ecosystems

have been characterized by high input of

chemical fertilizer instead of organic

amendments, leading to deterioration of

soil quality due to reductions in soil or-

ganic matter. With increasing global con-

cerns about energy crisis and environ-

mental protection, it is becoming more

**Figure 1:** Schematic representation of

important to rely on locally abundant ag-

anaerobic digestion process for compost-

ricultural bioresources than on chemical

ing of agricultural wastes.

fertilizers. Recent studies have focused on

re-considering

traditional

fertilization

**ii) Aerobic digestion:** Aerobic di-

practices to enhance soil organic input by

gestion may be defined as a biological

amendment of organic fertilizers like

degradation process where vigorous hu-

vermicompost.

mification and pasteurization of organic

****

residues takes place. The process requires

**4. Biological decomposition of wastes**

air breathing microbes like bacteria, fun-

gi, actinomycetes, mesophilic-exothermic

The breakdown of raw organic

microbes and thermophilic microorgan-

materials to a finished compost - a pro-

isms that flourish in elevated temperature

cess known as decomposition - is a com-

of greater than 60°C. Mineralization of

plex gradual process wherein both chemi-

biodegradable organic matter takes place

cal and biological processes take place in

leading to release of carbon dioxide, wa-

order to mineralize the organic matters.

ter and energy (Figure 2). Conversion of

Generally, biological degradation of or-

****

ganic material takes place through two

distinct pathways:

**i) Anaerobic digestion:** Anaero-

bic digestion may be defined as the

breakdown of organics in the absence of

oxygen under controlled conditions. Here,

fermentation of waste results in the for-

mation of ammonia-like substances and

hydrogen sulfide. Organic wastes with

high degradability are easily degraded

through anaerobic digestion. Bacterial

****

species capable of degrading targeted or-

**Figure 2:** Schematic representation of

ganics are involved in this process along

aerobic digestion process for composting

with thorough mixing for efficient sub-

of agricultural wastes.

strate conversion (Figure 1). The carbon

content released as biogas containing me-

residual organic components to humic

thane, carbon dioxide and energy is repre-

acids results in stabilization of the pro-

sented using the equation:

cess. As the end result, the heterogeneous

waste is transformed into a homogeneous

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_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

and valuable organic fertilizer that is rich

cess is the conversion of important plant

in humus.

nutrients into a more soluble state (Nair _et_

C



6H12O6 + 6O2

6CO2 + 6H2O + 2840kJ

_al_., 2006).

... 2

Several parameters can be used

Compared to chemical fertilizers,

for the evaluation of the vermicomposting

compost is an excellent product that re-

process, like, survival of the worms, bio-

tains most of the original nutrients benefi-

mass growth, and increase in worm popu-

cial for the increase of soil"s organic and

lation. Since earthworms are known to

nutrient constituents. Application of com-

feed on the pathogens present in the waste

post can improve structure and fertility of

used, vermicomposting process guaran-

the soil. There are basically four im-

tees pathogen removal. Hence, as per

portant parameters that must be consid-

USEPA, Vermicomposting has been rec-

ered for the evaluation of quality of com-

ognized as "Class A" stabilization process

post and process performance. They are:

(Eastman _et al_., 2001). Another advantage

volatile solids, respiration rate, germina-

of biotransformation of waste through

tion tests and pathogen indicators.

vermicomposting is the loss of moisture

to yield a drier product. High concentra-

**5. Vermicomposting**

tion of moisture in the compost can lead

to process failure. In vermicomposting,

Vermicomposting is an ecological

the worm burrows act as channels for air

stabilization process involving the break-

passage, hence, this can support higher

down of organic waste by the joint action

humidity.

of earthworms and mesophilic microor-

The main problems encountered

ganisms. Earthworms require an envi-

with composting of many organic wastes

ronment conducive for microbial degrada-

are their high moisture content, need of

tion and maintenance of biochemical pro-

bulking substrate and components unde-

cesses for enhanced microbial decomposi-

sirable for worm consumption. As a re-

tion. Various intestinal microflora of

sult, composting of raw organic wastes

earthworms are transferred to the compost

requires constant monitoring of moisture

matrix along with their gut enzymes that

level, composition of the waste, C/N ratio

play an important role (Whiston and Seal,

and the composting period. This has been

1988). Furthermore, earthworms are also

overcome by the process of pre-digestion

known to augment the microbial activities

of organic waste before it can be utilized

by improvement of the environment nec-

for vermicomposting. Geetha _et al_.,

essary for their growth (Syers _et al._ , 1979;

(2016) and Nair _et al.,_ (2006) have car-

Mulongoy and Bedoret, 1989). Through

ried out pre-digestion of the non-edible

the process of vermicomposting, wastes

vegetables waste and kitchen wastes re-

are converted to a better homogenized,

spectively, in order to achieve a better

nutrient rich and well stabilized product.

quality of vermicompost. Pre-digestion

Several studies reveal that vermicompost-

prior to vermicomposting was helpful in

ing can be used as an effective technique

pH, moisture and waste stabilization. Pre-

for the treatment of wastes rich in patho-

digestion was also found to effectively

gens (Eastman _et al._ , 2001). Vermicom-

reduce the pathogen load from the ver-

posting gives a better end product than

micompost.

composting due to the enzymatic and mi-

crobial activity that takes place during the

_5.1. Earthworms_

process (Bajsa _et al.,_ 2003). Various stud-

Earthworms belong to kingdom

ies indicate that vermicomposting can at-

Animalia, phylum Annelida, class Clite-

tain harmless pathogen levels which may

latta and subclass Oligochaetae. They are

be aided by the microbial and enzymatic

known as soil forming organisms inhabit-

activity. An added advantage of this pro-

ing almost all part of the earth. They can

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_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

be found in different groups of varying

excrete. The following steps are involved

size, shape, colouration, life span, feeding

in vermicomposting process (Abbasi _et_

habit and depth in the soil where they are

_al_., 2008):

found. The shape of the worm is cylindri-

_i._ Ingestion and breaking down of the in-

cal with segmented body tapering off at

gested organic waste by the action of

both ends. The segments are separated by

earthworm"s gizzard, located next to

fluid-filled compartment that surrounds a

the mouth of the worm.

central digestive tract.

_ii._ Digestion of the broken down particles

More than 1800 known species of

by the action of enzymes and microbes

earthworm have been found all over the

while it passes through the earth-

world (Minnich, 1977) and they can be

worm"s body.

subdivided

in

three

basic

groups:

_iii._ Exit of the digested matter as "Ver-

_Epigeics, Endogeics_ and _Anecics_. Alt-

micast" after few hours of ingestion.

hough there are probably many species of

earthworms available, only a few have

Vermicomposting bins (Figure 3)

been utilized for biotransformation of or-

are set up using Vermitech pattern

ganic waste processing. The most com-

(Geetha _et al.,_ 2016). The plastic or ceme-

monly used species of earthworms in-

clude _Eisenia foetida_ (Red wiggler),

_Lumbricus rubellus_ (Red worm), _Eisenia_

_Andrei_ (Red tiger), _Perionyx excavates_

(Blue worm), _Eudrilus eugeniae_ (African

night crawler), _Enchytraeids_ (White

worm), _Dendroba enaveneta_ and _Peri-_

_onyx hawayana_. Although all the above

mentioned species may be used for ver-

micomposting, _Eisenia foetida_ and _Lum-_

_bricus rubellus_ are the most commonly

used as it is easy to replicate the suitable

environmental condition for their growth

and regeneration. Among the two species,

_Eisenia foetida_ has been proved best for

vermicomposting of any organic waste

(Edwards and Bater, 1992) because the

growth and reproduction of these worms

is quite rapid. _Eisenia foetida,_ commonly

**Figure 3:** Schematic representation of

known as Red wiggler, is an epigenic

design of vermicompost bin (Geetha _et_

worm that favours living in organic ma-

_al_., 2016).

nure or compost. It has the ability to pro-

cess large quantities of organic matter as

-nt bins are placed in a shaded elevated

under ideal conditions; it is known to

area on a pedestal of bricks for effective

consume food as much as its body weight

water drainage. The basal layer of the

each day.

vermibed comprises of broken bricks and

stones above which a layer of sand up to

_5.2. Vermicomposting process_

the height of 10 cm is set up to ensure

The process of vermicomposting

proper filtration and drainage. This is fol-

can be carried out either in batch or in

lowed by first a 10 cm high layer of cow

continuous modes. The time taken for the

dung (3kg) and 5cm high layer of pre-

completion of decomposition process may

digested organic waste. A 6~8 cm layer of

vary depending on the time taken by the

straw is added to cover the bedding mate-

earthworms to ingest the feed, digest and

rial that helps in retaining moisture. Ap-

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_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

proximately 60-75 earthworms are inocu-

in various enzymes, high quality organics

lated in the composting bins. The ver-

(humus) and plant growth regulators.

micomposting units are kept in shade and

covered with a mesh. Water is sprinkled

**6. Application of biotransformed waste**

to maintain sufficient moisture. For op-

**for better agricultural productivity**

erative collection of wormy wash, a small

hole is drilled near the base of compost-

One of the biggest environmental

ing bin and a tube is attached to it. Turn-

challenges that the world is facing today

ing of the vermicomposting pile must be

is solid waste management. A sustainable

carried out periodically to ensure proper

approach towards management of this

aeration.

problem may be the use of biotransfor-

mation technique to treat and convert or-

_5.3. Advantages of waste management by_

ganic waste into vermicompost. Compost-

_vermicomposting_

ing is the most cost-effective and ecologi-

Composting is known as an effec-

cal option for management of various or-

tive biotransformation technique for treat-

ganic wastes since it is easily operable

ing organic wastes, following nature"s

and can be carried out in a small scale

way of recycling.

Vermicomposting

level with proper management of the pro-

requires very less resources like water,

cess to obtain a good quality product. A

energy and land/space required for treat-

list of various wastes converted to ver-

ment of per unit of bio-waste as compared

micompost is given in Table 1. Although,

to aerobic composting. It is a rapid, cost

husk is abundantly available as lignocel-

effective and sustainable alternative for

lulosic waste, the problem with using

transformation of organic waste, carried

husks as vermicomposting feedstock is its

out by earthworms, leading to formation

high initial carbon (lignin and cellulose)

of a product rich in plant nutrients and

contents, which impedes the composting

humic acids. The compost thus generated

process (Kumar _et al_., 2013). Hence, the

has the ability to hold nutrients for a

substrate requires amendments to achieve

longer period without unfavorably im-

an optimum range of C/N ratio to attain

pacting the environment. No curing is

optimal process efficiency (Goyal _et al_.,

necessary for vermicompost as it is highly

2005). This can be achieved with the help

rich in beneficial microorganisms. The

of pre-digestion of husks along with other

overall time required for processing of

organic wastes (such as non-edible vege-

waste is therefore reduced to a great ex-

table waste) as amendment, prior to ver-

tent, resulting in non-toxic by-products.

micomposting. This will help in conver-

Gardeners, all over the world, highly pre-

sion of husk into biofertilizer within few

fer the application of vermicompost over

months.

chemical fertilizers; hence, vermicompost

Integrated use of fly ash along

is finding a significant market value

with organic wastes has been effectively

(Sudhakar _et al_., 2002).

used in increasing the yield of crops when

Application of vermicompost has

compared to continuous use of chemical

been proved to improve the texture, struc-

fertilizers alone (Rautaray _et al_., 2003).

ture, aeration, fertility and water holding

This may be due to beneficial effects on

capacity of soil. This way most of the

rice and residual effects on mustard. Ac-

valuable nutrients that are taken out of the

cording to Rautaray _et al_. (2003) greater

soil during crop cultivation are replen-

crop yield was related to higher uptake of

ished. Also, vermicompost addition is

nutrient. Further to the yield advantage, a

known to enhance plant-root develop-

better soil chemical properties were also

ments that help in control of soil erosion.

noted namely pH, organic carbon and

Vermicompost are also known to be rich

available N, P and K as compared to the

soils where chemical fertilizers were con-

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_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

tinuously used. When fly ash was used in

ability of macro and micronutrients from

combination with organic waste during

vermicompost and wormy wash (Erich _et_

vermicomposting, the end product ob-

_al.,_ 2002). Vermicompost and wormy

tained gave better plant growth than with

wash augments crop growth and yield

chemical fertilizers alone.

when added to soil (Lalitha _et al_., 2000).

_Vigna radiata_ (Green gram) when

According to the studies conducted by

grown in soil amended with a combina-

Atiyeh _et al._ (2001), and Suthar (2009),

tion of vermicompost and wormy wash

addition of vermicompost in bedding me-

were found to develop nodes and new

dia enhanced seed germination and

leaves at a significantly higher rate when

growth leading to overall increase in plant

compared to control (Geetha _et al.,_ 2016).

productivity.

This might be due to increase in bioavail-

****

**Table 1:** List of organic wastes converted to vermicompost

**No.**

**Organic**

**Earthworm em-**

**Outcome**

**Reference**

**waste**

**ployed**

1.

Non-edible

_Eisenia foetida_

Enhanced growth of _Vigna_

Geetha _et_

vegetables

_radiata_ (green gram)

_al.,_ 2016

from market

2.

Kitchen

_Eisenia fetida_ &

Effect of thermocomposting

Nair _et al_.,

waste

_Lumbricus rubellus_

prior to vermicomposting

2006

3.

Industrial

_Eisenia foetida_

Removal of the heavy metals

Shaymaa _et_

sludge

from electronic industrial

_al_., 2010

sludge

4.

Vegetable

_Eisenia foetida_

Composting with minimum

Sibi and

wastes

resources leading to zero waste Manpreet,

generation

2011

5.

Farm garbage _Eisenia fetida, Eu-_

Odourless product with high

Indrajeet _et_

_drilus eugeniae_ & __

nutrient status.

_al_., 2010

_Peronyx excavates_

__

6.

Agriculture

_Eisenia Foetida_ &

Simplest, scientific, economic

Mane and

waste from

_Eudrilus euginiae_

and environmental friendly

Raskar,

market yard

way to transform waste mate-

2012

rials into compost

through vermicomposting by

using an exotic species of

earthworm

7.

Fruit waste

_Eisenia Foetida_ &

Degradation strategy of organ-

Seetha _et_

_Eudrilus euginiae_

ic waste

_al_., 2012

8.

Coconut

_Eudrilus euginiae_

Efficient method to convert

Tahir and

waste

coconut waste into valuable

Hamid,

by-product

2012

9.

Rice husk

_Eudrilus eugeniae_

Bio-transforming RH into val-

Lim _et al_.,

ue-added material,

2012

10.

Water hya-

_Eisenia fetida_

Nutrient rich vermicompost

Ansari and

cinth and

Rajpersaud,

Grass clip-

2012

pings

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 219

_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

**7. Concluding remarks**

**Atiyeh, R. M., Arancon, N. Q., Ed-**

**wards, C. A. and Metzger, J. D.**

Environmental degradation is a

**(2001).** The influence of earthworm-

major cause of concern challenging the

processed pig manure on the growth

world. Owing to improper waste man-

and productivity of marigolds. _Biore-_

agement amenities and treatment, discard-

_source Technology_ **81(2), 103-108.**

ing of organic wastes from various sectors

**Bajsa, O., Nair, J., Mathew, K. and Ho,**

like domestic, agricultural and industrial

**G. E. (2003).** Vermiculture as a tool

sources has become a source of distress

for domestic wastewater manage-

causing serious environmental complica-

ment. _Water Science and Technology_

tions. As a result of fast growing popula-

**48(11–12), 125–132.**

tion, there is also significant increase in

**Bakthvathsalam, R. and Ramakrish-**

the generation of various wastes all over

**nan, G. (2004).** Culture of earth-

the world. Vermicomposting, being an

worm using different organic wastes

environmental-friendly technique, is an

of agricultural importance. _Environ-_

attractive method for conversion of waste

_ment & Ecology_ **22(Spl-2), 386-391.**

into wealth. In today"s world of organic

**Beede, D. N. and Bloom, D. E. (1995).**

products, people are becoming increas-

The Economics of Muni- cipal Solid

ingly aware of the importance of convert-

Waste. _World Bank Research Ob-_

ing our waste into valuable product that

_server_ **10(2), 113-150.**

will not only solve the problem of waste

**Doran, J. W. and Parkin, T. B. (1994).**

disposal but also help the agriculturalists

Defining and assessing soil quality.

in acquiring a safe and cost-effective crop

_In:_ Defining Soil Quality for a Sus-

promotion technique.

tainable Environment. Doran, J.W.,

Coleman, D. C., Bezdicek, D.F. and

___
___

## Acknowledgements

Stewart, B.A. (eds.). SSSA, Inc.,

Madison, Wisconsin, USA.

The authors wish to thank Man-

**Eastman, B. R., Kane, P. N., Edwards,**

agement and Department of Biotechnolo-

**C. A., Trytek, L., Gunadi, B.,**

gy, Kamaraj College of Engineering and

**Stermer, A. L. and Mobley, J. R.**

Technology, Virudhunagar, Tamil Nadu,

**(2001).** The effectiveness of ver-

India, for their constant support and en-

miculture in human pathogen reduc-

couragement.

tion for USEPA biosolids stabiliza-

tion. _Compost Science and Utiliza-_

**References**

_tion_ **9(1), 38-49.**

****

**Edwards, C. A. and Bater, J. E. (1992).**

**Abbasi, T., Gajalakshmi, S. and Ab-**

The use of earthworms in environ-

**basi, S. A. (2009).** Towards model-

mental management. _Soil Biology_

ing and design of vermicomposting

_and Biochemistry_ **24(12), 1683-1689.**

systems: Mechanisms of compost-

**Edwards, D. R. and Daniel, T. C.**

ing/vermicomposting and their impli-

**(1992).** Environmental Impacts of

cations. _Indian Journal of Biotech-_

On-Farm Poultry Waste Disposal - A

_nology_ **8, 177-182.**

Review. _Bioresource Technology_ **41,**

**Ansari, A. A. and Rajpersaud, J.**

**9-33.**

**(2012).** Physicochemical Changes

**Erich, M. S., Fitzgerald, C. B. and Por-**

during Vermicomposting of Water

**ter, G. A. (2002).** The effect of or-

Hyacinth ( _Eichhornia crassipes_ ) and

ganic amendments on phosphorus

Grass Clippings. _International Schol-_

chemistry in a potato cropping sys-

_arly Research Network, ISRN Soil_

tem. _Agriculture, Ecosystems & En-_

_Science_ **2012, 1-6.**

_vironment_ **88, 79-88.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 220

_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

**Faria, K. C. P., Gurgel, R. F. and Hol-**

**Mane, T. T. and Raskar Smita, S.**

**anda, J. N. F. (2012).** Recycling of

**(2012).** Management of Agriculture

sugarcane bagasse ash waste in the

Waste from Market yard through

production of clay bricks. _Journal of_

Vermicomposting. _Research Journal_

_Environmental Management_ **101, 7-**

_of Recent Sciences_ **1, 289-296.**

**12.**

**Minnich, J. (1977).** The Earthworm

**Garga, P., Guptaa, A. and Satya, S.**

book. Rodale press. Emmaus, P. A.

**(2006).** Vermicomposting of different

**pp. 372.**

types of waste using _Eisenia foetida_ :

**Mulongoy, K. and Bedoret, A. (1989).**

A comparative study. _Bioresource_

Properties of worm casts and surface

_Technology_ **97(3), 391-395.**

soil under various plant covers in the

**Geetha, K., Michael A. D. & Anant A. **

humid tropics. _Soil Biology and Bio-_

**(2016).** Bioconversion of Non Edible

_chemistry_ **21, l97-203.**

Vegetables from Market into Biofer-

**Nair, J., Sekiozoic, V. and Anda, M.**

tilizer for Crop Improvement. _Jour-_

**(2006).** Effect of pre-composting on

_nal of Agricultural Science_ **8(6), 71-**

vermicomposting of kitchen waste.

**83.**

_Bioresource Technology_ **97, 2091–**

**Goyal, S., Dhull, S. K. and Kapoor, K.**

**2095.**

**K. (2005).** Chemical and biological

**Nambiar, K. K. M. (1994).** Soil Fertility

changes during composting of differ-

and Crop Productivity under Long-

ent organic wastes and assessment of

term Fertilizer Use in India. _ICAR_

compost maturity. _Bioresource Tech-_

_Publication_ , New Delhi.

_nology_ **96, 1584–1591.**

**Rautaray, S. K., Ghosh, B. C. and Mit-**

**Indrajeet, Rai, S. N. and Singh, J.**

**tra, B. N. (2013).** Effect of fly ash,

**(2010).** Vermicomposting of Farm

organic wastes and chemical fertiliz-

Garbage in different combination.

ers on yield, nutrient uptake, heavy

_Journal of Recent Advances in Ap-_

metal content and residual fertility in

_plied Sciences_ **25, 15-18.**

a rice–mustard cropping sequence

**Kumar, S., Sangwan, P., Dhankhar, R.,**

under acid lateritic soils. _Bioresource_

**Mor, V. and Bidra, S. (2013).** Utili-

_Technology_ **90, 275–283.**

zation of Rice Husk and Their Ash:

**Seetha Devi, G., Karthiga, A., Susila, S.**

A Review. _Research Journal of_

**and Muthunarayanan, V. (2012).**

_Chemical and Environmental Scienc-_

Bioconversion Of Fruit Waste Into

_es_ **1(5), 126-129.**

Vermicompost By Employing _Eu-_

**Kumari, S. (2013).** Solid Waste Man-

_drillus eugenia_ e And _Eisenia fetida_.

agement by Vermicomposting. _Inter-_

_International Journal of Plant, Ani-_

_national Journal of Scientific & En-_

_mal and Environmental Sciences_

_gineering Research_ **4(2), 1-5.**

**2(4), 245-252.**

**Lalitha, R., Fathima, K. and Ismail, S.**

**Shaymaa, M., Ahmed, H., Norli, I.,**

**A. (2000).** Impact of biopesticides

**Morad, N. and Hakimi, I. M.**

and microbial fertilizers on produc-

**(2010).** Removal of Aluminium,

tivity and growth of _Abelmoschus es-_

Lead and Nickel from Industrial

_culentus_. _Vasundhara the Earth_ ,

Sludge via Vermicomposting Pro-

**1 &2, 4-9. **

cess. _World Applied Sciences Journal_

**Lim, S. L., Wu, T. Y., Sim, E. Y. S.,**

**10(11), 1296-1305.**

**Lim, P. N. and Clarke, C. (2012).**

**Sibi, G. and Manpreet, K. (2011).** Man-

Biotransformation of rice husk into

agement of vegetable waste by Ver-

organic fertilizer through vermicom-

micomposting Technology. _Ameri-_

posting. _Ecological Engineering_ **41,**

_can-Eurasian Journal of Agricultural_

**60-64.**

_& Environmental Sciences_ **10(6),**

**983-987.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 221

_Biotech Sustainability (2017)_

_Conversion of Wastes into Organic Biofertilizer for Sustainability Karuppasamy et al._

**Smith, D. H., Wells, M. A., Porter, D.**

composition rate. _Ecological Engi-_

**M. and Fox, F. R. (1993).** Peanuts.

_neering_ **35, 914-920.**

_In:_ Nutrient deficiencies & toxicities

**Syers, J. K., Sharpley, A. N. and Keen-**

in crop plants, Bennett, W. F. (eds.).

**ey, D. R. (1979).** Cycling of nitrogen

St. Paul, M. N. The American Phyto-

by surface-casting earthworms in a

pathological Society. **pp. 105-110.**

pasture ecosystem. _Soil Biology and_

**Sudhakar, G., Lourduraj, A. C.,**

_Biochemistry_ **11, 181-185.**

**Rangasamy, A., Subbian, P. and**

**Tahir, T. A. and Hamid, F. S. (2012).**

**Velayutham, A. (2002).** Effect of

Vermicomposting of two types of co-

Vermicompost Application On The

conut wastes employing _Eudrilus eu-_

Soil Properties, Nutrient Availability,

_geniae_ : a comparative study. _Interna-_

Uptake And Yield Of Rice - A Re-

_tional Journal of Recycling of Organ-_

view. _Agricultural Reviews_ **23(2),**

_ic Waste in Agriculture_ **2, 1-7.**

**127 – 133.**

**Whiston, R. A. and Seal, K. J. (1988).**

**Suthar, S. (2009).** Vermicomposting of

The occurrence of cellulases in the

vegetable-market solid waste using

earthworm _Eisenia foetida_. _Biologi-_

Eisenia fetida: Impact of bulking ma-

_cal Wastes_ **25, 239-242.**

terial on earthworm growth and de-

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 222

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P223-247_

**Bacterial Endophytes as Biofertilizers and Biocontrol**

**Agents for Sustainable Agriculture**

****

**Amrutha V. Audipudi1, *, Bhaskar V. Chakicherla2 and Shubhash Janardhan Bhore3**

_1Department of Microbiology, Acharya Nagarjuna University, Nagarjuna Nagar 522510,_

_Andhra Pradesh, India; 2Department of Botany, V.R College, Affiliated to V.S. University,_

_Nellore 524002, A.P, India; 3Department of Biotechnology, Faculty of Applied Sciences,_

_AIMST University, Bedong-Semeling Road, 08100 Bedong, Kedah Darul Aman, Malaysia;_

_*Correspondence: audipudiamrita@gmail.com; _ _Tel.: +91 9440995842_

**Abstract:** Plant health and development promoted through microbial interactions have been

the main motif for sustainable agriculture. To find new and beneficial endophytic microor-

ganisms from plants of different ecosystems is highly considerable because endophytic bac-

teria are not restricted to a specific species but showed a wide range of host diversity. En-

dophytes colonize an ecological niche similar to that of phytopathogens and baffle disease

development through endophyte-mediated de novo synthesis of novel compounds and anti-

fungal metabolites. Seedling emergence, plant growth and plant's establishment under ad-

verse conditions can be accelerated by endophytes. Endophytic _Pseudomonas_ _sp._ and _Bacil-_

_lus sp._ recorded a significant improvement in morphological characters and ISR in the

plantlets. Endophyte fortifies plant cell wall strength, alters host physiology and metabolic

responses thereby enhance the defense mechanism by the synthesis of different metabo-

lites such as phenolic compounds, pathogenicity related protein (PR-1, PR-2, PR-5), oxida-

tive stress enzymes (chitinases, peroxidases, polyphenyoxidase, phenyl alanine ammonia

lyase, Oxidase and/or chalcone synthase, phytoalexins etc. The contribution of endophytes

as biological fertilizers is highly significant because of their metabolic acclimatization in

the host plant with mutualism. Endophytic microbes must be properly selected, combined

and formulated with respect to the environmental conditions for development of efficient

endophytic biofertilizers that can very much contribute to the enhanced food production in

the world. This review aims to provide an overview on bacterial endophytes and their po-

tential application for sustainable agriculture.

****

_**Keywords**_ **:** Agriculture; biocontrol; endophytes; endophytic bacteria; induced systemic re-

sistance; phytopathogens; plant growth promotion; sustainable development

****

**1. Introduction**

1999). Plant–microbe interactions that

promote plant health and development

Agriculture and agri-food sector is

have been the subject of considerable

expected to move towards environmental-

study for sustainable agriculture. A re-

ly sustainable development by increasing

newed interest in the internal colonization

the productivity and protecting the natural

of healthy plants by non- rhizobium bac-

resource base for future generations.

teria and exploitation of their potential in

Greater productivity and competitiveness

agriculture becomes apparent (Fahey _et_

are anticipated to come from increased

_al_., 1991; Kloepper _et al_., 1992; Turner _et_

efficiency through the acquisition and

_al_., 1993). Rhizosphere bacteria which

management of new biotechnologies and

can easily colonize the internal roots and

crop production strategies (Stutz _et al_.,

stems are major source of endophytes

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_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

(Figure 1) and often phyllosphere bacteria

Siciliano, 2001; Zinniel _et al_., 2002; Ses-

may also be a source of endophytes

sitsch _et al_., 2002; Dent _et al_., 2004; Sun

(Hallmann _et al_., 1997; Germaine _et al_.,

_et al_., 2008). More than 200 bacterial

2004). Though each individual plant ex-

genera from 16 phyla have been reported

ists on the earth is a host to one or more

as endophytes since the first report of en-

endophytes (Strobel _et al_., 2004), only a

dophytic bacteria (Samish _et al_., 1963a)

few of these plants have ever been com-

and include both culturable and uncul-

pletely studied to their endophytic biolo-

turable bacteria (Sun _et al_., 2008; Berg

gy and to find novel beneficial endophytic

and Hallmann, 2006; Mengoni _et al_.,

microorganisms.

2009; Manter _et al_., 2010; Sessitsch _et al_.,

2012). Most predominantly studied endo-

**2. Endophytic bacterial diversity in the**

phytes belong to three major phyla ( _Ac-_

**host plants**

_tinobacteria_ , _Proteobacteria_ and _Firmicu-_

_tes_ ) and include members of _Streptomyces_

Endophytic bacteria were isolated

(Suzuki _et al_., 2005), _Azoarcus_ (Krause _et_

from both monocotyledonous and dicoty-

_al_., 2006), _Acetobacter_ (renamed as _Glu-_

ledonous plants (Table 1) ranging from

_conobacter_ ) (Bertalan _et al_., 2009), _Pseu-_

woody trees to herbaceous crops such as

_domonas_ , _Serratia_ , _Stenotrophomonas_

prairie plants, agronomic crops, tuberous

(Ryan _et al_., 2009), _Enterobacter_ (Ped-

crops

and

grasses

(McInroy

and

rosa _et al_., 2011). Bacteria which are

Kloepper, 1995; Gutiérrez-Zamora and

ubiquitous in the soil/ rhizosphere repre-

Martínez-Romero, 2001; Germida and

sent the main source of endophytic

**Figure 1:** Plant colonization routes by endophytic bacteria.

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**Table 1:** Monocotyledonous and dicotyledonous plants that harbor bacterial endophytes

(Sudhir and Audipudi, 2014) ****

Plant species

Endophytes

Reference

Rice _(Oryza sativa_ L.)

_Rhizobium leguminosarum,_

Yanni _et al.,_ 1997,

_Pseudomonas_

_Azorhizobium caulinodans,_

Engelhard _et al.,_ 2000,

_Sphingomonas paucimobilis_

_Chromobacterium violaceum_ ,

Phillips _et al.,_ 2000,

_Sphingobacterium_ sp _._

_Bradyrhizobium japonicum_

Chantreuil _et al.,_ 2000

_Serratia marcescens_

Gyaneshwar _et al.,_ 2001

_Serratia_ sp.

Sandhiya _et al.,_ 2005

_Agrobacterium, Azorhizobium,_

Reddy _et al_., 1997;

_Azospirillum, Bacillus,_

Stoltzfus _et al_., 1997

Potato

_(Solanum_

_tu-_

_Actinomyces, Agrobacterium,_

Hollis, 1951; de Boer _I_

_berosum_ L.) tuber __

_Alcaligenes, Arthrobacter,_

Copeman, 1974;

_Bacillus, Capnocytophaga,_

Sturz, 1995; Sturz

_Cellulomonas, Clavibacter,_

& Matheson, 1996;

_Comamonas, Corynebacterium,_ Sturz _et al_., 1998

_Curtobacterium, Deleya,_

Reiter _et al_., 2003

_Enterobacter, Erwinia,_

Asis and Adachi, 2003

_Flavobacterium, Kingella,_

_Klebsiella, Leuconostoc, Mi-_

_crococcus, Pantoea, Pasteurel-_

_la, Photobacterium, Pseudomo-_

_nas, Psychrobacter, Serratia,_

_Shewanella, Sphingomonas,_

_Vibrio, Xanthomonas, Sinorhi-_

_zobium meliloti, Paenibacillus_

_odorifer, Enterobacter asburiae_

Red clover

_(Trifolium_

_Acidovorax, Agrobacterium,_

Sturz _et al_., 1997

_pra_ tense L.)

_Arthrobacter, Bacillus, Bor-_

Sturz _et al_., 1998

_detella, Cellulomonas, Coma-_

_monas,_ _Curtobacterium, De-_

_leya,_ _Enterobacter, Escherich-_

_ia, Klebsiella,_ _Methylobacte-_

_rium, Micrococcus,_

_Pantoea, Pasteurella,_

_Phyllobacterium, Pseudomo-_

_nas, Psychrobacter, Rhizobium,_

_Serratia, Sphingomonas, Vario-_

_vorax, Xanthomonas_

Rough

lemon

_(Citrus_

_Achromobacter, Alcaligenes_

Feldman _et al_., 1977;

_jambhiri_ Lush.)

_Moraxella, Acinetobacter,_

Gardner _et al_., 1982

_Actinomyces, Arthrobacter, Ba-_

_cillus, Citrobacter, Corynebac-_

_terium, Enterobacter, Flavo-_

_bacterium, Klebsiella, Provi-_

_dencia,_

_Pseudomonas, Serratia, Vibrio,_

_Yersinia, Rickeltsia-like_

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_**Table 1:** Continued... _

Grapevine _(Vitis spp_.)

_Bacillus, Clavibacter, Coma-_

Bell _et al_., 1995a; 1995b

_monas, Curtobacterium, Enter-_

_obacter, Klebsiella, Moraxella,_

_Pantoea, Pseudomonas, Rah-_

_nella, Rhodococcus, Staphylo-_

_coccus,_

_Xanthomonas_

Soybean

_Enterobacter sakazakii_

Kuklinsky-Sobral _et al_.,

_Enterobacter agglomerans_

2004

_Erwinia_ sp.

_Klebsiella oxytoca_

_Klebsiella pneumoniae_

_Pseudomonas citronellolis_

Maize

_Burkholderia pickettii_

McInroy and Kloepper,

_Enterobacter_ spp.

1995

_Arthrobacter globiformis_

Chelius and Triplett, 2000a

_Microbacterium testaceum_

Zinniel _et al_., 2002

Citrus plants

_Bacillus spp._

Araujo _et al_., 2001, 2002

_Curtobacterium flaccumfaciens_

_Nocardia_ sp.

_Methylobacterium mesophili-_

_cum_

Carrot

_Pseudomonas putida_

Surette _et al_., 2003

_Pseudomonas fluorescens_

_Staphylococcus saprophyticus_

_Klebsiella terrigena_

Sugar cane _(Saccharum_

_Herbaspirillum rubrisulbalbi-_

Olivares _et al_., 1996

_officinarum_ L.)

_cans, Acetobacter, Herbaspiril-_

Cavalcante & Döbereiner,

_lum_

1988;

__

Gillis _et al_., 1989;

Boddey _et al_., 1991;

Dong _et al_., 1994;

Ohvares _et al_., 1997

Corn _(Zea mays_ L.)

_Bacillus, Burkholderia,_

Lalande _et al_., 1989;

_Corynebacterium, Enterobac-_

Fisher _et al_., 1992;

_ter, Klebsiella, Pseudomonas_

Mclnroy & Kloepper, 1995;

Palus _et al_., 1996

Wheat

_Streptomyces_

Coombs and Franco, 2003a

Alfalfa _(Medicago sativa_

_Erwinia-likc, Pseudomonas_

Gagne _et al_., 1987

L.)

Coffee _(Coffea arabica_

_Acetobacter_

Jimenez-Salgado _et al_.,

L.);

Cameroon

grass

1997; Reis _et al_., 1994

_(Pennisetum purpureum_

Schumach)

Cotton _(Gossypium hirsu-_

_Agrobacterium, Bacillus,_

Misaghi & Donndelinger,

_tum_ L.)

_Burkholderia, Clavibacter_

1990;

_Erwinia, Serratia,_

Mclnroy & Kloepper,

_Xanthomonas_

1995

Cucumber _(Cucumis sa-_

_Agrobacterium, Arthrobacter,_

Mclnroy & Kloepper,

_tivis_ L.)

_Bacillus, Burkholderia,_

1995

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_**Table 1:** Continued..._

__

_Chryseobacterium, Enterobac-_

_ter,_

_Pseudomonas, Stenotrophomo-_

_nas_

Hybrid spruce _{Picea_

_Bacillus, Pseudomonas,_

O'Neill _et al_., 1992;

_glauca_ x _Engelmannii)_

_Phyllobacterium, actinomy-_

Chanway _et al_., 1994

_cetes,_

_Staphylococcus_

Kallar grass _(Leptochloa_

_Azoarcus_

Reinhold _et al_., 1986;

_fusca_ [L.] Kunth) root

Reinhold-Hurek _et al_.,

1993

Lodgepole pine _(Pinus_

_Bacillus_

Shishido _et al_., 1995

_contorta_ Dougl. Ex Loud)

root __

_Sorghum_

_bicolor_

L.

_Herbaspirillum_

James _et al_., 1997

Moench shoot

Sugar beet (Beta _vulgaris Bacillus, Corynebacterium, Er-_

Jacobs _et al_., 1985

L.) __

_winia,_

_Lactobacillus; Pseudomonas,_

_Xanthomonas_

Teosinte _(Zea luxurians_

_Klebsiella_

Palus _et al_., 1996

Itins

and Doebley) stem

Banana

_Azospirillum brasilense_

Weber _et al_., 1999

_Citrobacter_ sp.

Martínez _et al_., 2003

Marigold

_Kocuria varians_

Sturz and Kimpinski, 2004

_Microbacterium esteraromati-_

_cum_

Banana, pineapple

_Azospirillum amazonense_

Weber _et al_., 1999

Sugarcane, coffee

_Gluconacetobacter diazo-_

Cavalcante and Döbereiner,

_trophicus_

1988; Jiménez-Salgado _et_

_al_., 1997

Scots pine, citrus plants

_Methylobacterium extorquens_

Araujo _et al_., 2002; Pirttilä

_et al_., 2004

Carrot, rice

_Rhizobium_ ( _Agrobacterium_ )

Surette _et al_., 2003

_radiobacter_

Kallar grass, rice

_Azoarcus_ sp _._

Reinhold-Hurek _et al_., 1993,

Engelhard _et al_., 2000;

Yellow

lupine,

citrus

_Burkholderia cepacia_

Araujo _et al_., 2001; Barac _et_

plants

_al_., 2004

Banana, pineapple, rice

_Burkholderia_ sp.

Araujo _et al_., 2001; Barac _et_

_al_., 2004

Sugarcane, rice, maize,

_Herbaspirillum seropedicae_

Olivares _et al_., 1996; Weber

sorghum, banana

_et al_., 1999

Banana, rice, maize, sug-

_Klebsiella variicola_

Rosenblueth _et al_., 2004.

arcane

Citrus plants, sweet pota-

_Pantoea agglomerans_

Araujo _et al_., 2001, 2002;

to

Asis and Adachi 2003

Rice, soybean

_Pantoea_ sp.

Kuklinsky-Sobral _et al_.,

2004; Verma _et al_., 2004

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_**Table 1:** Continued..._

__

Marigold ( _Tagetes_ spp.),

_Pseudomonas chlororaphis_

Sturz and Kimpinski, 2004;

carrot

Surette _et al_., 2003

Alfalfa, carrot, radish,

_Salmonella enterica_

Cooley _et al_., 2003; Guo _et_

tomato

_al_., 2002; Islam _et al_., 2004

Dune grasses ( _Ammophila_

_Stenotrophomonas_

Dalton _et al_., 2004

_arenaria_ and

_Elymus mollis_ )

Maize,

carrot,

citrus

_Bacillus megaterium_

Araujo _et al_., 2001; McInroy

plants

and Kloepper, 1995; Surette

_et al_., 2003

Grass _Miscanthus sinen-_

_Clostridium_

Miyamoto _et al_., 2004

_sis_

Wheat, Scots pine

_Mycobacterium_ sp.

Conn and Franco 2004; Prit-

tilä _et al_., 2005

Citrus plants, maize

_Enterobacter cloacae_

Araujo _et al_. 2002; Hinton _et_

_al_., 1995

Lettuce

_Escherichia coli_

Ingham _et al_., 2005

Wheat, sweet potato, rice

_Klebsiella_ sp.

Engelhard _et al_., 2000;

Iniguez _et al_., 2004;

colonizers (Hallmann and Berg, 2006).

_et al_., __ (2008) isolated 77 endophytic bac-

Earlier studies reported that endophytic

teria from roots, stems and leaves of _So-_

population of _Bacillus polymyxa_ inside

_lanum nigrum_ grown in two different na-

the root is highly specific and less diverse

tive habitats of Jena and Germany. Ara-

than the root surface population, though

vind _et al_., __ (2009) isolated 80 endophytic

there are different populations of _Bacillus_

bacteria from different varieties of _Piper_

_polymyxa_ in soil, rhizosphere, and rhi-

_nigrum_ L. cultivated in different regions

zoplane of wheat field. It clearly indicates

of India. Bhore and Sathisha (2010) iso-

that endophytes appear to be originated

lated 115 putative cultivable endophytic

from rhizosphere or rhizoplane (Mavingui

bacteria from leaves of 72 different plant

_et al_., 1992; Germida _et al_., 1998).

species collected from northern part of

Endophytic genera isolated from

Peninsular Malaysia. Magnani _et al_.,

the interior of ginseng roots cultivated in

(2010) isolated 32 endophytic bacteria

three different areas showed marked dif-

from Brazilian sugarcane. A total of 264

ferences in microbial community (Cho _et_

colonies of endophytic bacteria were re-

_al_. 2007). Ryan _et al_., (2008) reported

ported from leaves and roots of young

that endophytic bacteria in a single plant

radish (Seo _et al_., 2010).

host not restricted to a single species but

Pereira _et al_., __ (2011) investigated

comprise several genera and species. En-

endophytic bacterial diversity associated

dophytic bacteria belong to one genera

with the roots of maize through culture-

was not only restricted a specific host but

dependent and culture-independent meth-

also showed wide range of host diversity.

ods. _Enterobacter_ , _Erwinia_ , _Klebsiella_ ,

Bacteria belonging to the genera _Bacillus_

_Pseudomonas_ , and _Stenotrophomonas_

and _Pseudomonas_ were identified as pre-

genera belong to _γ_ -Proteobacteria were

dominantly occurring endophytes (Segh-

reported as predominant group. Based on

ers _et al_., 2004).

culturable component of the bacterial

Jha and Kumar (2007) isolated

community genus _Bacillus_ belong to Fir-

and characterized 10 endophytic diazo-

micutes was identified as another pre-

trophic bacteria from surface-sterilized

dominant group, while _Achromobacter_ ,

roots and culm of _Typha australis._ Long

_Lysinibacillus_ , and _Paenibacillus_ genera

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_Biotech Sustainability (2017)_

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were rarely found in association with the

Internal colonization of plant tissues

roots.

by bacteria is primarily intercellular and

Yang _et al_. (2011) isolated 45

xylem vessels act as reservoirs of internal

strains from stems and 27 strains from

populations of bacteria (Gardner _et al_.,

leaves of tomato as endophytic bacteria

1982; Jacobs _et al_., 1985; Sumner, 1990;

and reported that endophytic efficiency of

Frommel _et al_., 1991; Kloepper _et al_.,

bacteria in stem was higher than leaves.

1992; Bell _et al_., 1995; Sprent and James

Out of 72 endophytic bacteria isolated

1995; Reinhold-Hurek and Hurek 1998a).

from tomato on of the W4 was identified

Intracellular endophytic bacteria have al-

as _Brevibacillus brevis_ W4 on the bases

so been found in the cytoplasm and vacu-

of 16S rDNA gene analysis and Biolog

oles of epidermal cells (Quadt - Hallman

systemic analysis. Patel _et al_. (2012) iso-

and Kloepper, 1996), root hairs (Vance,

lated and characterized bacterial endo-

1983), and parenchyma cells (Jacobs _et_

phytes from root and stem of _Lycopersi-_

_al_., 1985).

_con esculentum._ Out of 18 endophytic

bacteria selected from tomato only one

**3. Endophytes in agriculture**

isolate HR7 was identified as _Pseudomo-_

****

_nas aeruginosa_ by 16S rDNA analysis.

The endophyte-plant interaction is one

Fisher _et al_. (1992) found that endo-

of the least studied biochemical systems

phytic bacteria appear to be preferentially

in nature. Plants host to one or more en-

located in the lower part of the stems of

dophytic microorganisms include fungi,

corn, with a declining gradient running

bacteria and actinomycetes. Endophytes

from the base to the top of the plant.

reside in the tissues beneath the epidermal

Roots and other below ground tissues

cell layers (Stone _et al_., 2000) are transi-

tend to yield the highest numbers of CFU

ently symptomless and inconspicuous

of bacteria compared with above-ground

with several beneficial effects on plants

tissues is an indicative of the upward path

(Hallmann _et al_., 1997). More than thou-

of bacteria (Table 2) from the roots into

sand endophytic bacteria were reported in

the stem during plant development

the last decade (Table 3). Like plant

(McInroy and Kloepper, 1994; Sturz _et_

growth promoting Rhizobacteria (PGPR),

_al_., 1997a; Rosenblueth and Martínez-

Romero, 2004; Gagné _et al_. (1987).

****

**Table 2:** Population densities of endophytic bacteria in host tissues

___
___

## Part of the plant

**Colony forming Unit (CFU)**

**Reference**

Alfalfa xylem tis-

6.0 × 10 3 to 4.3 × 104 per g

Gagné _et al_., 1987

sue

Cotton xylem tissue 1 × 102 to 11 × 103 per g

Misaghi and Donndelinger, 1990

Sugar beet tissue

3.3 × 103 to 7.0 × 105 per g

Jacobs _et al_., 1985

Potato tubers

0 to 1.6 × 104 per g

De Boer and Copeman, 1974

****

****

**Table 3:** Endophytic strains reported in between 2001-2007 (Sudhir, 2014) ****

Source

No of endophytes

Reference

Indian sugarcane

81 endophytic bacterial strains Suman _et al_., 2001

agronomic crop species

853 endophytic bacteria

Lodewyckx _et al_., __ 2002

prairie plant species

27 endophytes

Zinniel _et al_., 2002

_Daucus carota_

360 endophytic strains of Surette _et al_., 2003

and _Agrobacterium_

_Pseudomonas_ and _Staphylo-_

_coccus_

soybean

_35_ endophytic bacteria

Hung _et al_., 2007

__

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endophytes also influence the growth of

adjustment, stomatal regulation, modifi-

plant directly by the production of plant

cation of root morphology, enhanced up-

growth promoting traits such as IAA pro-

take of minerals and alteration of nitrogen

duction, Phosphate solubilization, sidero-

accumulation and metabolism. Adhikari

phore production, ammonia production,

_et al_.( 2001) reported that endophytic bac-

nitrogen fixation antagonism against phy-

terial strains are potential in controlling

topathogens and indirectly by induced

the seedling disease of rice and promote

systemic resistance (ISR). Endophytic

growth in rice. Application of endophytic

bacteria colonize an ecological niche sim-

bacterial strains significantly increased

ilar to that of phytopathogens, which

the growth parameters _viz_., pseudostem

makes them suitable as biocontrol agents

height, girth, number of leaves and physi-

(Berg _et al_., 2005a). Endophytic microor-

ological parameters _viz_., chlorophyll sta-

ganisms control plant pathogens (Sturz &

bility index, stomatal resistance and tran-

Matheson, 1996; Duijff _et al_., 1997;

spiration in banana plants both under

Krishnamurthy

&

Gnanamanickam,

greenhouse and field conditions (Harish,

1997), insects (Azevedo _et al_., 2000) and

2005).

nematodes (Hallmann _et al_., 1997, 1998)

through endophyte-mediated de novo syn-

_4.1. IAA production_

thesis of novel compounds and antifungal

According to earlier studies IAA

metabolites. Endophytes can also acceler-

production by endophytes can vary

ate seedling emergence, promote plant

among different species and isolates and

establishment under adverse conditions

it is also influenced by culture condition,

(Chanway, 1997) and enhance plant

growth stage and substrate availability.

growth (Bent & Chanway, 1998).

Out of 65 endophytes of soybean, 15 iso-

Bacterial endophytes stimulate the

lates were positive for IAA production

growth of host plant by nitrogen fixation,

produced more than 25 μg/ml of IAA and

enhancement in the availability of miner-

_Acetobacter diazotrophicus_ and _Her-_

als and the production of phytohormones

_baspirillum seropedicae_ found to produce

(Hurek _et al_. 2002; Iniguez _et al_. 2004;

IAA in chemically defined culture media.

Sevilla _et al_. 2001). Endophyte mediated

Seven out of 10 endophytic isolates of __

_de novo_ synthesis of antifungal or anti-

_Typha australis_ were positive for IAA

bacterial metabolites, siderophores and

production (Hung and Annapurna 2004;

competition for nutrients induce system-

Chen _et al_., 1998; Jha and Kumar, 2007).

atic resistance in the host to check the

Two bacterial endophytes of _Capsicum_

progress of disease (Sessitsch _et al_.

_annuum_ L. also reported to show plant

2002a; Sturz _et al_. 2000). ****

growth promotion and defense against

phytopathogens along with IAA produc-

**4. Endophytic bacteria as bio fertilizers**

tion. Long _et al_. (2008) reported 1.1 to

154μg/ml of IAA production by the en-

Research has been revealed that

dophytic bacteria isolate from _Solanum_

endophyte increase plant growth through

_nigrum_. Gangawar and Kaur (2009) re-

the improved cycling of nutrients and

ported 15 endophytic isolates of sugar-

minerals such as phosphate solubilisation

cane produced 4 to 19.3 μg/ml of IAA.

(Verma _et al_., 2001; Wakelin _et al_.,

Chilli endophytes are observed to be more

2004), indole acetic acid production (Lee

potential in IAA production than sugar-

_et al_., 2004) production of siderophore

cane and similar to _Solanum nigrum_ re-

(Costa and Loper, 1994) and supply of

ported (Sudhir 2014). Amrutha _et al_.

essential vitamins to plants (Pirttila _et al_.,

(2012) reported that _Pseudomonas aure-_

2004). Compant _et al_. (2005) reported

_ginosa_ CEFR3, _Bacillus sp_ CEFR19,

that endophytes also influence other ben-

_Curtobacterium_

_oceanosedimentum_

eficial effects of host include osmotic

AVSCE3 and _Bacillus cereus_ AVSCE 5

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_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

isolated from different parts of chilli pro-

_Pseudomonas_ spp. Isolated from Serbia

duced 23µg/ml, 21µg/ml, 111.5µg/ml

able to solubilize TCP (Stajković _et al_.,

and 125μg/ml of IAA, respectively. It

2011; Djuric _et al_., 2011; Josic _et al_.,

was much higher than that of found in

2012b). Amrutha _et al._ (2012) reported

other reports (Long _et al_. 2008).

that _Pseudomonas aureginosa_ CEFR3

Harish _et al_., (2008) assessed the

(198ppm/ml),

_Bacillus_

_sp_

CEFR19

plant growth promotion efficacy of 45

(1354ppm/ml) isolated from ripened fruit

endophytic bacteria isolated from corm

and isolated from green fruit and _Bacil-_

and root of banana. 12 strains isolated

_lus cereus_ AVSCE 5(137ppm/ml) isolat-

from gingseng plant, endophytic _P. fluo-_

ed from leaf of chilli are able to solubil-

_rescens_ WCS365 as __ biocontrol s bacteria

ize inorganic phosphate efficiently. In-

isolated from _Lycopersicon esculentum_

crease in the yield of canola by endophyt-

produced significant amounts of IAA

ic _Bacillus sp_. was reported by de Freitas

(Thamizh Vendan _et al_., 2010, Patel _et_

_et al_. (1997). Sundara _et al_. (2002) report-

_al_., 2012). Three strains isolated from

ed that enhancement in available phos-

sugar beet roots produced indole-3-acetic

phorus and yield of sugarcane by applica-

acid (IAA) promote plant growth signifi-

tion endophytic bacteria. _Pseudomonas_

cantly increased plant height, fresh and

_spp_. are able to increase the growth and

dry weights and number of leaves per

phosphorus content of maize by endo-

plant (Long _et al_., 2008; Yingwu Shi _et_

phytes (Vyas and Gulatti, 2009).

_al_., 2009). Vetrivelkalai _et al_., __ 2010 also

reported significant enhancement in the

_4.3. Siderophore_

germination percentage, shoot and root

Researchers reported that endophytic

length and vigour index of bhendi seed-

fungal siderophore have lower affinity

lings by seed bacterization.

than bacteria to sequester iron and deprive

pathogenic fungi (Whipps, 2001; Loper

_4.2. Phosphate solubilization_

and Henkels 1999). Endophytic bacterial

Earlier reports revealed endophyt-

isolates associated with hyacinth and

ic bacteria also solubilize phosphate from

Genseng plants produce siderophore

organic or inorganic bound phosphates

(Jafra _et al_., 2009; Thamizh Vendan _et_

and type of organic acid produced during

_al_., 2010). Rajkumar _et al_., (2010) report-

phosphate solubilizaton depends on the

ed that the siderophore play a pivotal role

carbon source utilized as substrate. High-

in nitrogen fixation under iron deficiency.

est P solubilization has been observed

****

when glucose, sucrose or galactose has

_4.4. Nitrogen fixation_

been used as sole carbon source in the

According to earlier studies en-

medium (Khan _et al_., 2009; Park _et al_.,

dophytic bacteria better express their ni-

2010). Endophytic bacteria able to solu-

trogen fixation potential inside plant tis-

bilize inorganic phosphate and extracel-

sues due to the lower competition for nu-

lular __ tricalcium phosphate effectively in

trients and protection against high levels

presence of glucose (Kuklinsky - Sobral

of O2 present on the root surface. Many

_et al_., 2004; Long _et al_., 2008; Thamizh

diazotrophic bacteria are able to establish

Vendan _et al_., 2010; Patel _et al_., 2012).

a symbiotic relationship with plants for

Endophytic

_Bacillus,_

_Pseudomonas,_

biological nitrogen fixation (Robson _et_

_Klebsiella and Acinetobacter_ are also re-

_al_., 1986; Chisnell _et al_., 1988; Dekas _et_

ported as potential phosphate solubilizers

_al_., 2009). Earlier studies reveled that en-

(Huang _et al_., 2010). Endophytic _Bacillus_

dophytic diazotrophs constitute only a

_cereus and B. megaterium_ isolated __ of

small proportion of total endophytic bac-

Ginseng plant also showed significantly

teria include _Azospirillum lipoferum_ ,

high P solubilization (ThamizhVendan _et_

_Klebsiella pnemoniae_ and _Azorhizobium_

_al_., __ 2010). Endophytic and rhizosphere

_caulinadans_ (Schloter _et al_., 1994; Bar-

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raquio _et al_ ,. 1997; Martínez _et al_., 2003).

and studied growth response of _A. thali-_

Unlike symbiotic diazotrophes, endophyt-

_ana_. Each strain showed significant vola-

ic bacteria are unable to form nodules.

tile-mediated plant growth modulation

_Gluconacetobacter diazotrophicus_ was

and also reported that _Burkholderia pyr-_

identified as first N2-fixing endophytic

_rocinia_ as significant plant growth-

bacteria associated with sugarcane stem

promoter. The volatiles indole, 1-hexanol

(Cavalcante and Dobereiner, 1988) and

and pentadecane promotes growth only

confirmed by other scientists in USA,

under stress conditions

UK, and Germany and two more N2-

****

fixing endophytes _Herbaspirillum sero-_

**5. Endophytes as biological control**

_pedicae and H. rubrisubalbicans_ were

**agents (BCA)**

reported by Boddey _et al_., (1995). James

(2000) reported _Herbaspirillum sp_. as en-

Endophytes are potential biocon-

dophytic diazotroph in sugarcane and

trol agents like other biocontrol agents

rice. _Azoarcus sp_. in rice and Kallar grass

such as associative nitrogen fixing PGPB

and endophytic _Klebsiella sp_. Kp342

on sugarcane (Boddy, 2003) or _Burkhold-_

strain of wheat identified as nitrogen fix-

_eria phytofirmans_ PsJN, non-symbiotic

ers (Iniguez _et al_., 2004). Silva-Froufe

endophytic bacteria (Sharma and Nowak,

(2009) reported _Glucanoacetobacter dia-_

1998) endophyte holds potential of BCA

_zotrophicus_ as endophytic diazotrophic

may be due to self-perpetuating nature of

bacteria in sugarcane, sweet potato, and

endophytes inside the host by coloniza-

pineapple. Endophytic _Bacillus_ species of

tion and being transfer to progeny (Bod-

soybean nodule showed potential N fixing

dy, 2003). According to Backman _et al_.

and reported to improve root growth and

(1997), the effectiveness of endophytes as

function, (Bai _et al_., 2002; Asis _et al_., __

biological control agents (BCA) is de-

2004; Matiru and Dakora, 2004). 23 en-

pendent on many factors. These factors

dophytic bacteria are identified as poten-

include: host specificity, the population

tial ammonia producers in chilli (Amrutha

dynamics, pattern of host colonization,

_et al_., 2012) and reported that _Pseudomo-_

ability to move within host tissues and the

_nas,_ _Curtobacterium_ and _Bacillus_ sp iso-

ability to induce systemic resistance. Cer-

lated from chilli were found to be maxi-

tain endophytic bacteria trigger induced

mum producers of Ammonia.

systemic resistance (ISR) which is pheno-

****

typically similar to systemic-acquired re-

_4.5. Volatile compounds_

sistance (SAR). SAR develops when

Earlier literature reveled that en-

plants successfully activate their defense

dophytic bacteria can produce a wide

mechanism in response to primary infec-

range of volatiles. Biological function of

tion by a pathogen and induces a hyper-

most of these volatiles is not yet under-

sensitive reaction in the form of a local

stood. It is assumed that volatile com-

necrotic lesion of brown desiccated tissue

pounds involved in a number of processes

(van Loon _et al_., 1998). ISR is effective

including cell-cell signaling, inter-species

against different types of pathogens bu

signaling, promote plant growth and act

differs from SAR because in ISR the in-

as microbial inhibiting agents (Wheatley,

ducing bacterium does not cause visible

2002; Vesperman _et al_., 2007; Kai _et al_.,

symptoms on the host plant (van Loon _et_

2009). Ryu _et al_., (2003) reported that

_al_., 1998).

_Bacillus sp_. produce 2, 3 butanediol and

The first record of an endophyte

acetoin and promote plant growth in _Ara-_

affecting a plant disease was reported by

_bidopsis thaliana_. 38 volatile compounds

Shimanuki (1987) who showed that _Phle-_

were reported from rhizobacteria (Farag

_um pratense_ plants infected with the

_et al_., 2006). Blom _et al_. (2011) screened

_Epichloe typhina_ were resistant to the

42 strains in four different growth media

fungus _Cladosporium phlei_. In some cas-

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es, endophytes also accelerate seedling

_Serratia marcescens_ 90-166 reduced Cu-

emergence and promote plant establish-

cumber Mosaic Virus (CaMV) in toma-

ment under adverse conditions and en-

toes and cucumbers (Raupach _et al_.,

hance plant growth and development (Pil-

1996), anthracnose and _Fusarium_ wilt in

lay and Nowak, 1997). Several endophyt-

cucumber (Liu _et al_., 1995).

ic bacterial species including _Achromo-_

Sixty one (61) endophytic bacteria

_bacter_ sp., _Acinetobacter baumannii_ , _A._

isolated from potato stem tissues were

_lwoffii_ , _Alcaligenes, Moraxella_ sp., _Alcal-_

identified as effective biocontrol agents

_igenes_ sp., _Arthrobacter_ sp., _Bacillus_ sp.,

against _Clavibacter michiganensis_ subsp.

_Burkholderia cepacia_ , _Citrobacter freun-_

_Sepedonicus_ (Sturz _et al_., 1999). _Bacillus_

_dii_ , _Corynebacterium_ sp., _Curtobacterium_

_mycoides_ BacJ and _Bacillus pumilis_ 203-7

_flaccumfaciens_ , _Enterobacter cloacae_ , _E._

isolates from different host plants sup-

_aerogenes_ , _Methylobacterium extorquens_ ,

pressed _Cercospora_ leaf spot in sugar

_Pantoea agglomerans_ , _Pseudomonas ae-_

beet (Bargabus _et al_., 2002: 2004). Araujo

_ruginosa_ , and _Pseudomonas sp_. isolated

_et al_. (2002) reported _Curtobacterium_

from the xylem of lemon roots ( _Citrus_

_flaccumfaciens,_ citrus endophyte help

_jambhiri_ ) have been reported as antago-

citrus plants to better resist against the

nistic bacteria against root phytopatho-

pathogenic infection __ of _Xylella fastidi-_

gens (Araújo _et al_., 2001; Lima _et al.,_

_osa_. Endophytes isolated from potato

1994).

plants produce antibiotics and siderophore

Cabbage treated with endophytes

and showed antagonismic against fungal

did not reach the economic threshold for

pathogens (Berg _et al_., 2005a; Sessitsch

the disease approximately 50 days after

_et al_., 2004) and bacterial pathogens _Er-_

inoculation with _Xanthomonas campestris_

_winia_ and _Xanthomonas_ (Sessitsch _et al_.,

_pv. Campestris_ due to the induction of

2004). Of 2,648 bacterial isolates from

defense mechanisms (Jetiyanon, 1994).

the

rhizosphere,

phyllosphere,

en-

Several bacterial endophytes have been

dosphere, and endorhiza, only _Serratia_

reported to support growth and improve

_plymuthica_ root endophyte was a highly

the health of plants (Hallmann _et al_.,

effective against _Xanthomonas sp_. in

1997). _Erwinia caratovora_ , for example,

Brassica seeds (Berg _et al_., 2005b).

is inhibited by numerous endophytic bac-

Endophytic actinobacteria also

teria, including several strains of _Pseu-_

show effective antagonism against the

_domonas_ sp. _, Curtobacterium luteum_ , and

pathogenic

fungus

_Gaeumannomyces_

_Pantoea agglomerans_ (Sturz _et al_. _,_ 1999).

_graminis_ of wheat (Coombs _et al_. 2004)..

Wilhelm _et al_. (1997) demonstrat-

A number of endophytic actinobacteria

ed that _Bacillus subtilis_ strains isolated

isolated by culture dependent methods

chestnut trees exhibit antifungal effects

belong to the genera of _Streptomyces_ , _Mi-_

against _Cryphonectria_ _parasitica_ causing

_crobispora_ , _Micromonospora_ , and _No-_

chestnut blight. The ability of endophytic

_cardioides_ (Coombs and Franco, 2003a)

bacteria to inhibit pathogen has been de-

were capable of suppressing _Rizoctonia_

creased in potato tubers due to deep colo-

_solani_ , _Pythium_ spp., and _Gaeumannomy-_

nization interior to the plant host (Struz _et_

_ces graminis_ var _tritici_ , fungal pathogens

_al_., 1999). Barka _et al_. (2002) demon-

of wheat _in vitro_ and _in planta_ , (Coombs

strated the ability endophytes to colonize

_et al_. _,_ 2004).

in divergent hosts. _Pseudomonas sp._ an

Aravind _et al_. (2009) reported against.

onion endophyte colonized in grape vine

Native endophytes IISRBP 35, IISRBP 25

inhibited _Botrytis cinerea_ Pers. and pro-

and IISRBP 17 isolated from black pep-

moted growth in grapevines. Coloniza-

per exhibited 70% disease suppression of __

tion of multiple hosts has also been ob-

_Phytophthora capsici_ in black pepper in

served with other species of endophytes

greenhouse trials. The intimate relation-

such as _Pseudomonas putida_ 89B-27 and

ship between endophytic bacteria and

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their hosts make the endophytes as natural

ammonia lyase, , oxidase and/or chalcone

candidates for selection as biocontrol

synthase) and phytoalexins to protect the

agents with high level of competition

host plant from future infections (Nowak

(Chen _et al_., 1995; Van Buren _et al_.,

and Shulaev, 2003). Elicitors include lip-

1993).

opolysaccharides, flagella, siderophore,

Twenty two (22) endophytic bac-

antibiotics, VOCs or quorum-sensing sig-

teria isolated from different parts of chilli

nals produced by endophytic bacteria

plant showed antagonism against _Colle-_

elicit ISR in plants (Van Loon, 2007).

_totrichum capsici_ , _C. gloeosporioides_ and

ISR activated by PGPB is mediated by

_C. acutatum_ (Amrutha _et al_., 2014). An-

jasmonate or ethylene in majority of

tagonism of endophytic bacteria against

plants. Thickening of the outer tangential

_Clavibacter_

_michiganensis_

_subsp._

and outermost part of the radial side of

_sepedonicum_ cause rot on tomato(Van

the first layer of cortical cell walls was

Buren _et al_., 1993), _Pseudomonas chlo-_

also noticed in tomato roots with the

_roraphis, P. fluorescens, P. graminis, P._

treatment of endophytic _P. fluoresces_

_putida, P. tolaasii_ and _P. veronii agaist_

WCS417 (Duijff _et al_.,1997).

bacterial pathogens (Chen _et al_., 1995;

Application of endophytic bacteria

Adhikari _et al_., 2001) Invitro inhibition of

by stem injection in cotton plants reduced

various fungal pathogens by _Bacillus sub-_

the root rot caused by _Rhizoctonia solani_

_tilis_ ME488nd (Chung _et al_., 2008) hace

and vascular wilt caused by _F.oxysporum_

also been reported.

f. sp. _vasinfectum_ (Chen _et al_., 1995).

Pleban _et al_. (1995) reported that endo-

**6. Induced systemic resistance (ISR)**

phytic bacteria move upward and down-

ward from the point of application before

Endophytes have a natural and in-

colonizing the internal tissues and check

timate association with plants because

the entry of _F. solani_ in cotton and _Scle-_

the internal tissues of plant provide rela-

_rotium rolfsii_ in beans. Application of

tively uniform and protected environment

_Pseudomonas florescence_ prevented the

and favors endophytic bacteria to be a

entry _Pythium ultimum_ in the roots of pea, __

potential agent of ISR (Chen _et al_. 1995).

_P. florescence_ restrict the growth of

Viswanathan (1999) reported that applica-

_Fusarium oxysporum_ f. sp. pisi and

tion of endophytic strain is more benefi-

_P.ultimum_ in pea plant (Benhamou _et al_.,

cial in vegetatively propagated crops like

1996b, 1998 ). _Pseudomonas_ strain 63-28

banana, sugarcane and tapioca for induc-

also induced resistant in tomato against _F._

ing systemic resistance and also reported

_oxysporum_

f.

sp.

_radicislycopersici_

that endophytic _P.fluorescens_ strain EP1

(M'Piga _et al_., 1997). Inoculation of en-

isolated from stalk tissues of sugarcane

dophytic

_Pseudomonas_

_denitrificans_

induced systemic resistance against red

strain 1-15 and _P.putida_ strain 5-48 pro-

rot caused by _Colletotrichum falcatum_

tected the oak tree against _Ceratocystis_

(Viswananthan and Samiyappan ,1999a).

_fagacearum_ (Brooks _et al_., 1994) _._ Recent

van Loon (2007) reported that the phe-

reports have been reviewed mechanisms

nomenon of ISR has been noted to be ex-

of ISR induced by endophytic _Psuedomo-_

hibited both associative and endophytic

_nas_ (Jankiewicz and Koltonowicz, 2012).

bacteria. ISR triggered by endophytes for-

_Bacillus cereus_ AR156 in _A.thaliana_

tifies plant cell wall strength, alters host

(Niu _et al_., 2011) and different endophyt-

physiology and metabolic responses and

ic _bacillus sp._ from vegetable crops in-

enhance synthesis of plant defense chem-

duced systemic resistance . Combination

icals such as phenolic compounds, patho-

of three endophytic isolates resulted in

genicity related protein (PR-1, PR-2, PR-

significant growth promotion and ISR in

5), ROS enzymes (chitinases, peroxidas-

tomato, bell pepper, cucumber and tobac-

es, polyphenyoxidase, phenyl alanine

co (Kloepper _et al_., __ 2004). Mixed formu-

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lation of _B. subtilis_ GB03, _B. amylolique-_

principle behind the plant and endophytic

_faciens_ IN937a and _B. subtilis_ IN937b

interaction to elicit ISR or plant growth

together with chitosan in tomato and dif-

promotion and development of endophyt-

ferent endophytic _bacillus sp._ isolated

ic bioformulations will be an index of

from vegetable crops induced systemic

growing scientific knowledge in agricul-

resistance in _Theobroma cacao_ seedlings.

ture and horticulture.

Harish _et al_. (2008) reported mixed for-

mulation of rhizobacterial and endophytic

**8. Concluding remarks**

bacterial (EPB5 + EPB22 + Pf1 + CHA0)

****

was significantly effective in suppressing

According to the research con-

Banana Bunchy top virus under field con-

ducted so far, the use of chemical fertili-

ditions.

zation is necessary because biological fer-

tilization has not yet proven to be good

**7. Perspectives**

enough in providing complete plant nutri-

ent requirements. It has been indicated

Microorganisms with phytopatho-

that about less than 50% of chemical fer-

genic antagonism act as Biocontrol agents

tilizers is absorbed by plant and the rest

(BCA). Most of the biocontrol agents

would not be accessible by plant as it is

have not fulfilled their initial promise be-

subjected to leaching, runoff, and emis-

cause of poor rhizosphere competence.

sion from the soil surface. Hence, the use

The failure of BCA being attributed the

of biological fertilizers as supplementary

difficulties in long-term culture. It would

fertilization to chemical fertilization is

obviate the need for selecting bacterial

necessary with the above mentioned ad-

types with high levels of rhizosphere

vantages. Accordingly, the right and

competence and successful seed or root

proper application of chemical and bio-

bacterization treatments before or at

logical fertilization is very much depend-

planting (Schroth _et al_., 1984; Weller,

ent on realizing the interactions between

1988). The intimate relationship between

soil, plant and microorganisms. Endo-

endophytic bacteria and their hosts make

phytic bacteria a big help to plant and the

the endophytes as natural candidates as

environment as they own great abilities

biocontrol agents with high level of com-

that collectively enhance plant growth

petence (Chen _et al_., 1995; Van Buren _et_

and ISR. Among such abilities, enhanced

_al_., 1993).

nutrient uptake by plant is also of great

Selection of endophytic bacteria that

importance; in the presence of soil mi-

can elicit plant growth promotion and ISR

crobes, plant absorbs higher amounts of

in plants and research on such endophytes

nutrients and less risk of environmental

in understanding plant responses that oc-

pollution is likely. Some of the most im-

cur during the signal transduction path-

portant functions of endophytic bacteria

ways that culminate in disease protection

were reviewed in this article. However,

is essential to delineate the pathosystems.

the particular emphasis has been on the

In many cases, elicitation of ISR by en-

use of endophytic bacteria for biological

dophytic bacilli is associated with in-

fertilization and biological control of phy-

creased plant growth, and the relationship

topathogens. PGPR bio-fertilization is a

between ISR and growth promotion

very common method because of its rapid

should be further investigated. Elucida-

effects on plant growth and yield produc-

tion of specific bacterial determinants that

tion. But there are issues regarding the

account for elicitation of ISR is just be-

use of PGPR fertilizers, as their compe-

ginning, and further work is needed to

tence in soil and interaction with plant

understand why one strain of a given bac-

vary with the rhizosphere environment.

terial species can elicit ISR while another

Since the endophyte is metabolically ac-

strain of the same species cannot. The

climatized to the host plant with mutual-

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_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

ism indicate the contribution of endo-

production of disease free plant-

phytes as biological fertilizer is better op-

lets of black pepper ( _Piper nigrum_

tion in comparison to other fertilizers. For

L.). _Arch. Phytopathol. Plant Pro-_

the development of efficient endophytic

_tection_., **48,1–12.**

bacterial biofertilizers, the microbes must

**Arshad,M. and Frankenberger, W. T.**

be properly selected, combined and for-

**(1991)**.Microbial production of

mulated with respect to the agricultural

plant hormones. In: Keister BD,

and environmental conditions.

Cregan PB (eds) The rhizosphere

****

and plant growth. _Kluwer Aca-_

**Acknowledgement**

_demic Publishers_ , Dordrecht, The

Netherlands, **327-334.**

Authors are thankful to UGC New

**Asis C. A.J. R., Adachi, K. and**

Delhi, India for financial assistance for

**Shoichiro Akao. (2004)**.N2 fixa-

research project, F. No. 40-132-2011(SR).

tion in sugarcane and population

Authors are also thankful to the central

of N2 fixing endophytes in stem

instrumentation centre of Acharya Nagar-

apoplast solution. _Philippine J._

juna Univeristy, Guntur, Andhra Pradesh,

_Crop Sci_., **29, 45-58.**

India.

**Astchul, S. F., Gish, W., Miller, W.,**

****

**Myers, E. V. and Lipman, D.**

**References**

**(1990)**. Basic lcal alignment tooll.

****

_J. Mol. Biol_., **215, 403-410.**

**Adhikari, T. B., Joseph, C. M., Yang,**

**Azevedo,J.L., Maccheroni, J. Jr., Pe-**

**G., Phillips, D. A. and Nelson,**

**reira, O. and Ara, W. L. (2000)**.

**L. M**. **( 2001)**. Evaluation of bac-

Endophytic microorganisms: a re-

teria isolated from rice for plant

view on insect control and recent

growth promotion and biological

advances on tropical plants".

control of seedling disease of rice.

_Electr. J. Biotech._ , **3, 40-65**.

_Can. J. Microbiol_ **., 47, 916-924**.

**Backman,P.A.,Wilson, M. and Mur-**

**Agrios, G. N. (2005)**. Plantpathology. 5th

**phy, J. F. (1997).** Bacteria for bio-

ed. New York, Academic Press.

logical control of plant diseases.

**Alschul, S. F., Gish, W., Miller, W.,**

_Environmentally Safe Approaches_

**Myers, E. W. and Lipman, D. J.**

_to Plant Disease Control_ , CRC/

**(1990)**. Basic local alignment

Lewis Press, Boca Raton, F. L.,

search tool. _J. Mol. Biol_., **215,**

**95-109**.

**403-410**.

**Bacon, C. W. and White, J. F.**

**Amrutha V. Audipudi, Sudhir, A.,**

**(2000).** Microbial

endophytes.

**Pradeep Kumar, N. and Chow-**

_Marcel Dekker Inc_ , New York,

**dappa, P. (2014).** Plant growth

USA, **199.**

promoting potential of a novel en-

**Bacon, C.W., Yates, I. E., Hinton, D.**

dophytic _Curtobacterium_ CEG:

**M. and Meredith, F. (2001)**. Bio-

Isolation, evaluation and formula-

logical control of _Fusarium monili-_

tion. _Annals of Biological Re-_

_forme_ in maize. _Environ. Health_

_search_ **, 5, 15-21.**

_Perspect_., **109, 325-332.**

**Aneja, K. R**. ( **2006** ) Experiments in

**Bai, Y. M., D'Aoust, F., Smith, D. L.**

Microbiology". _Plant Pathology_

**and**

**Driscoll,**

**B.T.**

_and Biotechnology_ , 4th Edition,

**(2002)**.Isolation of plant growth

New Delhi **, 245-275**.

promoting _Bacillus_ strains from

**Aravind,R., Kumar,A. and Eapen,**

soybean root nodules. _Can. J. Mi-_

**S.J. (2012).** Pre-plant bacteriza-

_crobiol_., **48, 230-238**.

tion: a strategy for delivery of

Bailey, J. A. **1987**. "Phytoalexins: A

beneficial endophytic bacteria and

genetic view of their signifi-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 236

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

cance". In day P.R, Jellis, G. J

**15-24.**

(Eds), _Genetics and plant patho-_

**Benhamou, N., Bea langer, R. R. and**

_genesis_ , Black well oxford, 13-26.

**Paulitz, T. C. (1996a)** Ultrastruc-

**Bailey, J. A. and Jeger, M. J. (1992)**.

tural and cytochemical aspects of

_Colletotrichum_ : Biology, Pathology

the interaction between _Pseudo-_

and Control". _Common wealth My-_

_monas fluorescens_ and Ri T-DNA

_cological Institute_ , Wallingford,

transformed pea roots: host re-

**388** .

sponse to colonization by _Pythium_

**Bano,**

**N.and**

**Musarrat,J.(2004).**

_ultimum_ Trow. _Planta_ , **199, 105-**

Characterization of a novel carbo-

**117.**

furan degrading _Pseudomonas_ sp.

**Benhamou, N., Bea langer, R. R. and**

withcollateral biocontrol and plant

**Paulitz, T. C. (1996b)**. Induction

growth

promoting

potential.

of differential host responses by

_FEMS Microbiol. Lett_., **231, 13-**

_Pseudomonas fluorescens_ in Ri-T

**17.**

DNA transformed pea roots upon

**Bargabus, R. L., Zidack, N. K.,**

challenge with _Fusarium ox-_

**Sherwood, J. E. and Jacobsen,**

_ysporum_ f. sp. pisi and _Pythium_

**B. J. (2002)** Characterization of

_ultimum_.

_Phytopathology_ ,

**86,**

systemic resistance in sugar beet

**1174-1185**.

elicited by a nonathogenic, phyl-

**Benhamou, N., Kloepper, J. W. and**

losphere colonizing _Bacillus my-_

**Tuzun, S. (1998).** Induction of

_coides_ biological control agent.

resistance against _Fusarium_ wilt

_Physiol. Mol. Plant Pathol_., **61,**

of tomato by combination of chi-

**289-298**.

tosan with an endophytic bacterial

**Bargabus, R. L., Zidack, N. K.,**

strain: ultrastructure and cyto-

**Sherwood, J. E. and Jacobsen,**

chemistry of the host response.

**B. J.( 2004)**. Screening for the

_Planta_ , **204, 153-168.**

identification of potential biologi-

**Bent, E. and Chanway, C. P. (1998)**.

cal control agents that induce sys-

The growth promoting effects of a

temic acquired resistance in sugar

bacterial endophyte on lodgepole

beet. _Biol. Control_., **30, 342-350**.

pine are partially inhibited by the

**Barka, E. A., Gognies, S., Nowak, J.,**

presence of other rhizobacteria.

**Audran, J. C. and Belarbi, A.**

_Can. J. Microbiol._ , **44,980-988.**

**(2002)**.Inhibitory effect of endo-

**Berg, G., Krechel, A., Ditz, M., Si-**

phytic bacteria on _Botrytis cinerea_

**kora, R. A., Ulrich, A.and**

and its influence to promote the

**Hallmann, J. (2005a)**. Endophyt-

grapevine growth. _Biol. Control_.,

ic and ectophytic potato associat-

**24, 135-142**.

ed bacterial communities differ in

**Barraquio,W.L., Revilla, L. and**

structure and antagonistic function

**Ladha, J. K. (1997)**. Isolation of

against plant pathogenic fungi

endophytic diazotrophic bacteria

_FEMS Microbiol. Ecol._ , **51, 215-**

from wetland rice. _Plant Soil_ , **194,**

**229.**

**Berg, G. T., Tesoriero, L. and Hail-**

Sieber, T. N., (eds) Microbial

**stones, D. L. (2005b).** PCR-based

Root

Endophytes.

_Springer-_

detection of _Xanthomonas cam-_

_Verlag_ , Berlin, **53-69**.

_pestris_ pathovars in _Brassica_ seed.

**Bertalan, M., Albano, R., de Padua, V.,**

_Plant Pathol_., **54, 416-427**.

**Rouws, L., Rojas, C., Hemerly, A.**

**Berg, G. and Hallmann, J. (2006)**.

**and Teixeira,K. ( 2009)**. Com-

Control of plant pathogenic fungi

plete genome sequence of the

with bacterial endophytes. In:

sugarcane nitrogen fixing endo-

Schulz, B. J. E., Boyle, C. J. C.,

phyte _Gluconacetobacter_

_diazo-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 237

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

_trophicus_ Pal5 _BMC Genomics_ , **10,**

**Brunel, B., Périssol, C., Fernandez,**

**450.**

**M., Boeufgras, J. M. and Le**

**Bhore, S. J. and Sathisha, G. (2010)**.

**Petit, J. (1994).** Occurrence of

Screening of endophytic colo-

_Bacillus_ species on evergreen oak

nizing bacteria for cytokinin-like

leaves. _FEMS Microbiol. Ecol_.,

compounds: crude cell-free broth

**14, 331-342.**

of endophytic colonizing bacteria

**Byung K. K., Ok-Sun, K., Eun**

is unsuitable in cucumber cotyle-

**Young, M. and Jongsik, C.**

don bioassay. _World J. Agri. Sci._ ,

**(2009)**. Proposal to transfer _Fla-_

**6, 345-352.**

_vobacteriumoceanosedimentum_

**Bhutto, M., Aqeel, Dahot M. and**

CartyandLitchceld1978 to the ge-

**Umar. (2010).** Effect of Alterna-

nus _Curtobacterium_ as _Curtobac-_

tive Carbon and Nitrogen Sources

_terium oceanosedimentum_ comb.

on Production of Alpha-amylase

Nov. _FEMS Microbiol_. _Lett_., **296,**

by _Bacillus megaterium_. _World_

**137- 141.**

_Applied Sciences Journal_ ( _Special_

**Cavalcante, V. A. and Dobereiner, J.**

_Issue of Biotechnology & Genetic _

**(1988).** A new acid tolerant nitro-

_Engineering_ ), **8, 85- 90**.

gen-fixing bacterium associated

**Blom, D., Fabbri, C., Connor, E. C.,**

with sugar cane. _Plant Soil_ , **108,**

**Schiestl, F. P., Klauser, D. R.,**

**23-31.**

**Boller,**

**T.,Eberl,**

**L.and**

**Chanway, C. P. (1997)**. Inoculation of

**Weisskopf, L. (2011)**. Production

tree roots with plant growth pro-

of plant growth modulating vola-

moting soil bacteria: an emerging

tiles is widespread among rhizo-

technology for reforestation. _For-_

sphere bacteria and strongly de-

_est Sci_., **43, 99-112.**

pends on culture conditions. _Envi-_

**Chen, C., Bauske, E. M., Musson, G.,**

_ron. Microbiol.,_ **13, 3047-3058.**

**Rodriguez**

**Kabana,**

**R.and**

**Boddey, R. M., Oliveira, O. C., Ur-**

**Kloepper. J. W. (1995)**. Biologi-

**quiaga, S., Reis, V. M., Olivares,**

cal control of _Fusarium_ wilt on

**F. L., Baldani, V. L.D. and**

cotton by use of endophytic bacte-

**Dobereiner, J. (1995).** Biological

ria. _Biol. Control_., **5, 83-91**.

nitrogen fixation associated with

**Chen, R., Hilson, P., Sedbrook, J.,**

sugar cane and rice: Contributions

**Rosen, E., Caspar, T. and Mas-**

and prospects for improvement.

**son, P.H. (1998)**. The _Arabidop-_

_Plant Soil_ , **174, 195-209.**

_sis thaliana Agravitropic_ gene en-

**Boddey, R. M., Urquiaga, S., Alves,**

codes a component of the polar-

**B. J. R. and Reis, V.( 2003)**. En-

auxin- transport efflux carrier.

dophytic nitrogen fixation in sug-

_Proc. Natl. Acad. Sci_., USA **, 95,**

arcane: present knowledge and fu-

**15112-15117.**

ture applications. _Plant Soil_ , **249,**

**Cho, K. M., Hong, S. Y., Lee, S. M.,**

**165- 179.**

**Kim, Y. H., Kahng, G. G., Lim,**

**Bosland, P. W. and Votava, E. J.**

**Y. P., Kim H. and Yun H.D.**

**(2003)**. Peppers: Vegetable and

**(2007)**.Endophytic

bacterial

Spice _Capsicums_. _CAB Interna-_

communities in ginseng and their

_tional_ , England, **233.**

antifungal activity against patho-

**Brooks, D. S., Gonzalez, C. F., Appel,**

gens. _Microbial Ecol_., **54, 341-**

**D. N. and Filer, T. H. (1994)**.

**351**.

Evaluation of endophytic bacteria

**Chung, B. S., Aslam, Z., Kim, S. W.,**

as potential biological control

**Kim, G. G., Kang, H. S., Ahn, J.**

agents for oak wilt. _Biol. Control_.,

**W. and Chung,**

**4, 373-381.**

**Y.R. (2008)**. A bacterial endophyte,

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 238

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

_Pseudomonas_

_brassicacearum_

_vulgaris_ subsp. _vulgaris_ (sugar

YC5480 isolated from the root of

beet). _J. Microbiol. Methods_ , **56,**

_Artemisia_ sp. producing antifun-

**17-26.**

gal and phytotoxic compounds.

**Djuric, S., Pavic, A., Jarak, M., Pav-**

_The Plant Pathology Journal_ , **24,**

**lovic, S., Starovic, M., Pivic, R.**

**461-468**.

**and Josic, D. (2011)**. Selection of

**Compant, S., Reiter, B., Sessitsch, A.,**

indigenous fluorescent pseudo-

**Nowak, J., Clément, C. and Ait**

monad

isolates

from

maize

**Barka, E. (2005).** Endophytic

rhisospheric soil in Vojvodina as

colonization of _Vitis vinifera_ L. by

possible PGPR. _Romanian Bio-_

plant growth promoting bacterium

_technological Letters_ , **16, 6580-**

_Burkholderia_ sp. strain PsJN.

**6590**.

_Appl. Environ. Microbiol_., **71,**

**Elbeltagy, A., Nishioka, K., Suzuki,**

**1685-1693**.

**H., Sato, T., Sato, Y. I., Morisa-**

**Coombs, J. T. and C. M. M. Franco.**

**ki,**

**H.,**

**Mitsui,**

**H.**

**and**

**(2003a)**. Isolation and identifica-

**Minamisawa, K. (2000).** Isola-

tion of actinobacteria isolated

tion and characterisation of endo-

from

surface-sterilized

wheat

phytic bacteria from wild and tra-

roots. _Appl. Environ. Microbiol_.,

ditionally cultivated rice varieties.

**69, 5303-5308.**

_Soil Sci. Plant Nutrition_. **46, 617-**

**Coombs, J. T. and Franco, C. M.**

**629.**

**M. ( 2003b)**. Visualization of an

**Fahey,**

**J.**

**W.,**

**Dimock,**

**M.**

endophytic

**B.,Tomasino, S. F., Taylor, J.**

_Streptomyces_ species in wheat seed. _Appl._

**M. and Carlson, P. S. (1991)**.

_Environ. Microbiol_., **69, 4260-4262.**

Genetically

engineered

endo-

**Coombs, J. T., Michelsen, P. P.and**

phytes as biocontrol agents : a

**Franco, C. M. M. (2004)**. Evalu-

case study from industry. In: _Mi-_

ation of endophytic actinobacteria

_crobial Ecology of Leaves_. An-

as antagonists of _Gaeumannomy-_

drews, J. H., and Hirano, S. S.,

_ces graminis_ var. _tritici_ in wheat.

Eds., _Springer-Verlag_ , London,

_Biol. Control_ **, 29, 359-366.**

UK, **401-411.**

**Costa, J. M. and Loper, J. E. (1994)**.

**Farag, M. A., Ryu, C. M., Summer,**

Characterization of siderophore

**L. W. and Pare, P. W. ( 2006)**.

production by the biological con-

GC-MS SPMEprofiling of rhizo-

trol agent _Enterobacter cloacae_.

bacterial volatiles reveals prospec-

_Mol. Plant Microbe. Interact_., **7,**

tive inducers of growth promotion

**440- 448.**

and induced resistance in plants.

**Datta, S., Kim, C. M. and Pernas, M.**

_Phytochem._ , **67, 2262-2268.**

**(2011)**. Root hairs: development,

**Fritze, D. (2004)**. Taxonomy of the ge-

growth and evolution at the plant

nus Bacillus and related genera:

soil interface. _Plant Soil_ , **346, 1-**

the aerobic endospore-forming

**14**.

bacteria.

_Phytopathology_ ,

**94,**

**Dekas, A. D., Poretsky, R. S. and Or-**

**1245-1248**.

**phan, V. J.( 2009)**. Deep-sea ar-

**Gangwar, M. and Kaur, G. (2009)**.

chaea fix and share nitrogen in

Isolation and characterization of

methane-consuming

microbial

endophytic bacteria from endorhi-

consortia. _Science_ , **326, 422-426.**

zosphere of sugarcane and rye

**Dent, K. C., Stephen, J. R. and Finch-**

grass. _Internet. J. Microbiol._ , **7**.

**savage, W. E. (2004).** Molecular

**Germaine, K., Keogh, E., Garcia-**

profiling of microbial communi-

**Cabellos, G., Borremans, B.,**

ties associated with seeds of _Beta_

**van Der-Lelie D., Barac, T.,**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 239

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

**Oeyen, L., Vangronsveld, J.,**

**Harish, S., Kavino, M., Kumar, N.,**

**Moore, F. P., Moore, E. R. B.,**

**Balasubramanian,**

**P.**

**and**

**Campbell, C. D., Ryan,D. and**

**Samiyappan, R.( 2009)**. Induc-

**Dowling, D. N. (2004)**. Colonisa-

tion of defense-related proteins by

tion of poplar trees by _gfp_ ex-

mixtures of plant growth promot-

pressing bacterial endophytes.

ing endophytic bacteria against

_FEMS Microbiol. Ecol._ , **48, 109-**

Banana bunchy top virus. _Biol._

**118.**

_Control._ , **51, 16-25.**

**Germida, J. and Siciliano, S. (2001)**.

**Huang, Z. J., Yang, R. Y., Guo, Z. Y.,**

"Taxonomic diversity of bacteria

**She, Z. G. and Lin, Y. C. (2010)**.

associated with the roots of mod-

A new naphtho-γ- pyrone from

ern, recent and ancient wheat cul-

mangrove

endophytic

fungus

tivars". _Biol. Fertil. Soil_. **33, 410-**

ZSU-H26. _Chem. Nat. Compd.,_

**415**.

**46, 15-18.**

**Gnanamanickam, S. S. (2006)**. Plant-

**Hung, P. Q., Senthil Kumar, M., Go-**

Associated

Bacteria.

Springer

**vindsamy, V. and Annapurna,**

www.springer.com, 195-218.

**K. (2007).** Isolation and charac-

**Gutierrez-Zamora, M. L. and Mar-**

terization of endophytic bacteria

**tínez-Romero, E. (2001)**. Natural

from wild and cultivated soybean

endophytic association between

varieties. _Biol. Fertil. Soils_., **44,**

_Rhizobium etli_ and maize ( _Zea_

**155-162**.

_mays_ L.). _J. Biotechnol._ **91, 117-**

**Husen, E. (2003)**. Screening of soil bac-

**126**.

teria for plant growth promoting ac-

**Hallmann, J., Quadt-Hallmann, A.,**

tivities _in vitro._

**Mahaffee, W. F. and Kloepper,**

Indones . _J. Agric. Sci._ , **4, 27-31**.

**J. W. (1997).** Bacterial endophytes

**Idris, R., Trifonova, R., Puschen-**

in agricultural crops. _Can. J. Mi-_

**reiter, M. and Wenze, L. W. W.**

_crobiol._ , **43, 895-914.**

**(2004)**. Bacterial communities as-

**Hallmann, J., Quadt-Hallmann, A.,**

sociated with flowering plants of

**Rodriguez-Kabana,**

**R.**

**and**

the Ni hyperaccumulator _Thlaspi_

**Kloepper, J. W. (1998)**. Interac-

_goesingens_. _Appl. Environ. Mi-_

tions between _Meloidogyne in-_

_crobiol._ , **70, 2667-2677**.

_cognita_ and endophytic bacteria in

**Idriss, E. E., Makarewicz, O., Farouk,**

cotton and cucumber. _Soil Biol._

**A., Rosner, K., Greiner, R.,**

_Biochem._ , **30, 925-937**.

**Bochow, H., Richter,T and Bor-**

**Hallmann, J. and Berg, G. (2006)**.

**riss,**

**R.**

**(2002)**.

Extracellular

Spectrum and population dynam-

phytase activity of _Bacillus amylo-_

ics of bacterial root endophytes".

_liquefaciens_ FZB45 contributes to its

In: Microbial root endophytes.

plant-growth-promoting effect. _Mi-_

Schulz, B., Boyle, C., Sieber, T.,

_crobiol._ , **148, 2097-2109.**

eds. Springer, Heidelberg, **15-31**.

**Igual, J. M., Valverde, A., Cervantes,**

**Harish, S., Kavino, M., Kumar, N.,**

**E. and Velaquez, E. (2001)**.

**Saravanakumar,**

**D.,**

**Soo-**

Phosphate solubilizing bacteria as

**rianathasundaram,**

**K.**

**and**

inoculants for agriculture: use of

**Samiyappan, R. (2008)**. Bio-

updated molecular techniques in

hardening with plant growth pro-

their study. _Agronomie.,_ **21, 561-**

moting rhizosphere and endophyt-

**568**.

ic bacteria induces systemic re-

**Iniguez, A. L., Dong, Y. and Triplett,**

sistance against banana bunchy

**E. W. (2004).** Nitrogen fixation in

top virus. _Appl. Soil Ecol._ , **39,**

wheat provided by _Klebsiella_

**187-200**.

_pneumoniae_ 342. _Mol. Plant Mi-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 240

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

_crobe Interact_., **17, 1078-1085**.

_Chem. Lett._ , **7, 1- 19.**

**Jafra, S., Przysowa, J., Gwizdek-**

**Kim, K. Y., Jordan, D. and McDon-**

**Wisniewska, A. van Der-wolf, J.**

**ald, G. A. (1998)**. _Enterobacter_

**M. (2009)**. Potential of bulb asso-

_agglomerans_ , phosphate solubiliz-

ciated bacteria for biocontrol of

ing bacteria and microbial activity

hyacinth soft rot caused by _Dick-_

in soil: effect of carbon sources.

_eya zeae J. Appl. Microbiol._ ,

_Soil Biol. Biochem._ , **30, 995-1003**.

**106,268-277.**

**Kloepper, J. W. (1992)**. Plant growth

**James, E. K. (2000)**. Nitrogen fixation

promoting rhizobacteria as bio-

in endophytic and associative

logical control agents". In: _Soil_

symbiosis. _Field Crops Res._ **65,**

_Microbial Ecology: Applications_

**197-209**.

_in Agricultural and Environmen-_

**Jha, P. N. and Kumar, A. (2007)**. En-

_tal Management,_ Metting, F. B.,

dophytic colonization of _Typha_

Jr., Ed., Marcel Dekker, New

_australis_ by a plant growth pro-

York, 255-274.

moting bacterium _Klebsiella oxy-_

**Kloepper, J. W., Ryu, C. M. and**

_toca_ strain GR-3. _J. Appl. Micro-_

**Zhang, S. (2004).** Induced sys-

_biol_., **103, 1311-1320.**

temic resistance and promotion of

**Josic, D., Delic, D., Rasulic, N., Staj-**

plant growth by _Bacillus_ sp _. Phy-_

**kovic, O., Kuzmanovic, D.,**

_topathology_ , **94, 1259-1266**.

**Stanojkovic, A. and Pivic, R.**

**Krause, A., Ramakumar, A., Bartels,**

**(2012b).**

Indigenous

_Pseudo-_

**D., Battistoni, F., Bekel, T.,**

_monads_ from Rhizosphere of

**Boch, J. and Bohm, M. (2006)**.

Maize grown on Pseudogley Soil

Complete genome of the mutual-

in Serbia. _Bulgarian Journal of_

istic, N2 fixing grass endophyte

_Agricultural Science_ , **18, 197-206**.

_Azoarcu_ s sp. strain BH72. _Nature_

**Josic, D., Pivic, R., Miladinovic, M.,**

_Biotech._ , **24, 1385-1391.**

**Starovic,**

**M.,**

**Pavlovic,**

**S.,**

**Krishnamurthy, K. and Gnanaman-**

**Duric, S. and Jarak, M. (2012a)**.

**ickam, S. S. (1997)**. Biological

Antifungal activity and genetic

control of sheath blight of rice:

diversity of selected _Pseudomonas_

induction of systemic resistance in

spp. from maize rhizosphere in

rice by plant associated _Pseudo-_

Vojvodina. _Genetika_ , **44, 377-**

_monas_ sp. _Curr. Sci_., **72, 331-334.**

**388.**

**Lalande, R., Bissonnette, N., Coutlee,**

**Jung, H. W., Kim, W., Hwang, B. K.**

**D. and Antoun, H. (1989).** Iden-

**(2003)**. Three pathogen inducible

tification of rhizobacteria from

genes encoding lipid transfer pro-

maize and determination of their

tein from pepper are differentially

plant growth promoting potential .

activated by pathogens, abiotic,

_Plant Soil_ , **115, 7-11.**

and environmental stresses. _Plant_

**Lee, S., Flores-Encarnacion, M., Con-**

_Cell Environ_., **26, 915-928.**

**treras-Zentella,**

**M.,**

**Garcia-**

**Kai, M., Haustein, M., Molina, F., Pe-**

**Flores, L., Escamilla, J.E. and**

**tri, A., Scholz, B. and Piechulla,**

**Kennedy, C.(2004).** Indole-3-acetic

**B. (2009)**. Bacterial volatiles and

acid biosynthesis is deficient in

their action _Appl. Microbiol. Bio-_

_Gluconacetobacter diazotrophicus_

_technol._ , **81, 1001-1012.**

strains with mutations in cyto-

**.Khan, M. S., Zaidi, A., Wani, P. A.**

chrome C biogenesis genes. _J. Bac-_

**and Oves, M. (20090.** "Role of

_teriol_., **186, 5384-5391.**

plant growth promoting rhizobac-

**Lipton, D. S., Blanchar, R. W. and**

teria in the remediation of metal

**Blevins, D. G. (1987)**. Citrate,

contaminated soils". _Environ._

malate and succinate concentra-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 241

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

tion in exudates from P sufficient

water stress in tomatoes and pep-

and P stressed _Medicago sativa_ L.

pers. _Plant Sci_., **166, 525-530.**

seedlings. _Plant Physiol_., **85,315-**

**McInroy, J. A. and Kloepper, J.W.**

**317.**

**(1994)**. Studies on indigenous en-

**Liu, L., Kloepper, W. and Tuzun, S.**

dophytic bacteria of sweet corn

**(1995)**. Induction of systemic re-

and cotton. In: Molecular ecology

sistance in cucumber against

of rhizosphere microorganisms:

_Fusarium_ wilt by plant growth

Biotechnology and the release of

promoting rhizobacteria. _Phyto-_

GMOs. F. O'Gara, D. N.

_pathology_ , **85, 695-698**.

Dowling, and B. Boesten eds.

**Long, H. H., Schmidt, D. D. and**

VCH, New York, **19-28**.

**Baldwin, I. T. (2008)**. Native

**M'Piga, P., Belanger, R. R., Paulitz,**

bacterial endophytes promote host

**T. C. and Benhamou, N. (1997)**.

growth in a species specific man-

Increased resistance to _Fusarium_

ner phytohormone manipulations

_oxysporum_ f. sp. _radicislycopersi-_

do not result in common growth

_ci_ in tomato plants treated with

responses. _PLoS ONE_ , **3, 2702.**

the endophytic bacterium _Pseu-_

**Lugtenberg, B. and Kamilova, F.(**

_domonas fluorescens_ strain 63-28.

**2009).** Plant rowth promoting rhi-

_Physiol. Mol. Plant Pathol_., **50,**

zobacteria. _Annu. Rev. Microbiol_.,

**301-320.**

**63, 541-556.**

**Naik, P. R., Raman, G., Narayanan,**

**Manandhar, J. B., Hartman, G. L.**

**K. B. and Sakthivel, N. (2008)**.

**and**

**Wang,**

**T.**

**C.**

Assessment of genetic and func-

**(1995).** Anthracnose development

tional diversity of phosphate solu-

on pepper fruits inoculated with

bilizing

fluorescent

pseudo-

_Colletotrichum_

_gloeosporioide_.

monads isolated from rhizospher-

_Plant Disease_ , **79, 380- 383.**

ic soil". _BMC Microbiology_ , **8,**

**Manter, D. K., Delgado, J., Holm, D.**

**230.**

**G. and Stong, R. (2010)**. Pyrose-

**Ngoma, L., Babalola, O. O. and Ah-**

quencing reveals a highly diverse

**mad, F. (2012)**. Ecophysiology of

and cultivar specific bacterial en-

plant growth promoting bacteria.

dophyte community in potato

_Science Research Essays_ , **7, 4003-**

roots. _Microb. Ecol_., **60, 157-166**.

**4013**.

**Marques, A. P. G., Pires, C., Moreira,**

**Niu, D., Liu, H., Jiang, C., Wang, Y.,**

**H., Rangel, A. O. S. S. and Cas-**

**Jin, H. and Guo, J. (2011)**. The

**tro, P. M. L. (2010)**. Assessment

plant growth promoting rhizobac-

of the plant growth promotion

terium _Bacillus cereus_ AR156 in-

abilities of six bacterial isolates

duces systemic resistance in Ara-

using _Zea mays_ as indicator plant.

bidopsis thaliana, by simultane-

_Soil Biology Biochemistry_ , **42,**

ously activating salicylate and

**1229-1235.**

jasmonate/ethylene-

dependent

**Martinez, C., Michaud, M., Belanger,**

signaling pathways. _Mol_. _Plant_.

**R. R. and Tweddell, R. J.**

_Microbe_., **24, 533-542**.

**(2002)**. Identification of soils

**Nowak, J. and V. Shulaev. (2003)**. Prim-

suppressive

against

_Helmin-_

ing for transplant stress resistance in

_thosporium solani_ , the causal

_in vitro_ propagation. _In Vitro Cell._

agent of potato silver scurf. _Soil_

_Dev. Biol. Plant_., **39, 107-124.**

_Biol. Biochem,_ **34, 1861-1868**.

**Park, K. H., Lee, O. M., Jung, H. I.,**

**Mayak, S., Tirosh, T. and Glick, B. R.**

**Jeong, J. H., Jeon, Y. D.,**

**(2004)**. Plant growth-promoting

**Hwang, D. Y., Lee, C. Y. and**

bacteria that confer resistance to

**Son, H. J. (2010).** Rapid solubili-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 242

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

zation has of insoluble phosphate

**Rajkumar, M., Prasad, M. N. V. and**

by a novel environmental stress-

**Freitas, H. (2010)**. Potential of

tolerant

Burkholderia

viet-

siderophoreproducing bacteria for

namiensis M6 isolated from gin-

improving heavy metal phytoex-

seng rhizospheric soil. _Appl. Mi-_

traction. _Trends Biotechnol_., **28,**

_crobiol. Biotechnol_., **86, 947-955**.

**142-149**.

**Patel, H. A., Patel, R. K., Khristi, S.**

**Ramamoorthy, V. and Samiyappan,**

**M., Parikh, K. and Rajendran,**

**R. (2001)**. Induction of defense

**G. (2012)**. Isolation and charac-

related genes in _Pseudomonas_

terization of bacterial endophytes

_fluorescens_ treated chilli plants in

from _Lycopersicon esculentum_

response to infection by _Colleto-_

plant and their plant growth pro-

_trichum capsic_ ". _Journal of My-_

moting characteristics . _Nepal J._

_cology and Plant Pathology_. **31,**

_Biotech_., 2, 37-52.

**146-155**.

**Payne, S. M. (1994)**. Detection, isola-

**Ramamoorthy, V., Viswanathan, R.,**

tion and characterization of sider-

**Raguchande, Tr., Prakasam, V.**

ophores, in Methods Enzymology.

**and Samiyappan, R. 2001.** In-

edited by Clark, V. L. and Bovil,

duction of systemic resistance by

P. M. _Academic press New York,_

plant growth promoting rhizobac-

**235, __****329-344.**

teria in crop plants against pests

**Pereira, P., Ibanez, F., Rosenblueth,**

and diseases. _Crop Protection_ , **2,**

**M., Etcheverry, M. and Mar-**

**1-11.**

**tinez-Romero, M. (2011)**. Analy-

**Raupach, G.S., Liu, L., Murphy, J.F.,**

sis of the bacterial diversity asso-

**Tuzun, S. and Kloepper, J.W.**

ciated with the roots of maize

**(1996)**. Induced systemic re-

( _Zea mays_ L.) through culture-

sistance in cucumber and tomato

dependent

and

culture-

against cucumber mosaic cu-

independent methods. _ISRN Ecol-_

cumovirus using plant growth

_ogy,_ doi:10.5402/2011/938546.

promoting rhizobacteria (PGPR)".

**Pillay, V. K. and Nowak, J. (1997)** ,

_Plant Dis_., **80, 891-894.**

Inoculum density, temperature

**Robson, R. L., Eady, R. R., Richard-**

and genotype effects on in vitro

**son, T. H., Miller, R. W., Haw-**

growth promotion and epiphytic

**kins, M. and Postgate,J. R. 1986.**

and endophytic colonization of

The alternative nitrogenase of _Azo-_

tomato ( _Lycopersicon esculentum_

_tobacter chroococcum_ is a vanadi-

L.) seedlings inoculated with a

um enzyme. _Nature_ , **322, 388-390.**

pseudomonad bacterium. _Can. J._

**Rodriguez, H., Fraga, R., Gonzalez, T.**

_Microbiol_., **43, 354-361**.

**and Bashan, Y. (2006)**. Genetics of

**Pleban, S., Ingel, F. and Chet, I.**

phosphate solubilisation and its po-

**(1995).** Control of _Rhizoctonia_

tential applications for improving

_solani_ and _Sclerotium rolfsii_ in

plant growth promoting bacteria.

the greenhouse using endophytic

_Plant Soil_ , **287, 15-21**

_Bacillus_ sp. _Eur. J. Plant Pathol_.,

**Rosenblueth,**

**M.**

**and**

**Martinez**

**101, 665 - 672.**

**Romero, E. (2004)**. _Rhizobium_

**Provorov, N. A., Borisov, A. Y. and**

_etli_ maize populations and their

**Ikhonovich, I. A. (2002).** Devel-

competitiveness for root coloniza-

opmental genetics and evolution

tion. _Arch. Microbiol._ , **181, 337-**

of symbiotic structures in nitrogen

**344.**

fixing nodules and arbuscular my-

**Ryan, R. P. Germaine, K., Franks, A.,**

corrhiza". _J. Theor. Biol_., **214,**

**Ryan, D. J. and Dowling, D. N.**

**215-232.**

**(2008)**. Bacterial endophytes: re-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 243

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

cent developments and applica-

_Plant Sci_., **21, 323-378.**

tions. _FEMS Microbiol. Lett_. **278,**

**Sessitsch, A., Reiter, B. and Berg, G.**

**1-9.**

**(2004).**

Endophytic

bacterial

**Ryan, R. P., Monchy, S., Cardinale,**

communities of field- grown pota-

**M., Taghavi, S., Crossman, L.,**

to plants and their plant growth

**Avison, M. B., Berg, G., van der**

promoting and antagonistic abili-

**Lelie, D. and Dow, J. M. (2009)**.

ties. _Can. J. Microbiol_., **50, 239-**

The versatility and adaptation of

**249**.

bacteria from the genus _Steno-_

**Sessitsch, A., Hardoim, P., Doring, J.,**

_trophomona_. _Nat. Rev. Microbiol_.,

**Weilharter,**

**A.,**

**Krause,**

**A.,**

**7, 514-525.**

**Woyke, T. and Mitter,B.(2012)**.

**Samish, Z. and Etinger-Tulczynska,**

Functional characteristics of an en-

**R. (1963b)**. Distribution of bacte-

dophyte community colonizing rice

ria within the tissue of healthy

roots as revealed by metagenomic

tomatoes. _Appl. Environ. Micro-_

analysis. _Mol. Plant Microbe Inter-_

_biol._ , **11, 7-10**.

_act_., **25, 28-36.**

**Schloter, M., Kirchhof, G., Heinz-**

**Sharma, V. K. and Nowak, J. (1998)**.

**mann, U., Dobereiner, J. and**

Enhancement of verticillium wilt

**Hartmann, A. (1994)**. Immuno-

resistance in tomato transplants by

logical studies of the wheat root

in vitro co culture of seedlings

colonization by the _Azospirillum_

with a plant growth promoting

_brasilense_ strains Sp7 and Sp 245

rhizobacterium ( _Pseudomonas_ sp.

using strain specific monoclonal

strain PsJN). _Can. J. Microbiol_.,

antibodies". _In_ : Hegazi, N., Fayez,

**44: 528-536.**

M., and Monib, M., (eds.) Nitro-

**Sharma, P. N., Kaur, M., Sharma,**

gen Fixation with nonlegumes.

**O.P., Sharma, P. and Pathania,**

_The American University in Cairo_

**A. (2005)**. Morphological, Patho-

_Press_ , Cairo, Egypt, **290-295.**

logical and molecular variability

**Schloter, M. and Hartmann, A.**

in _Colletotrichum capsici_ the

**(1998)**. Endophytic and surface

cause of fruit rot of chillies in the

colonization of wheat roots ( _Triti-_

substropical region of north west-

_cum_

_aestivum_ )

by

different

ern India. _J. Phytopathol_., **153,**

_Azospirillum brasilense_ strains

**232-237.**

studied with strain- specific mon-

**Sharma, A., Wray, V. and Johri, B.**

oclonal antibodies. _Symbiosis_., **25,**

**N. (2007).** Molecular characteri-

**159-179.**

zation of plant growth promoting

**Schroth, M. N. and Hancock, J. G.**

rhizobacteria that enhance peroxi-

**(1981)**. Selected topics in biologi-

dase and phenylalanine ammonia

cal control". _Annu. Rev. Microbi-_

lyase activities in chile ( _Capsicum_

_ol_., **35, 453-476.**

_annuum_ L.) and tomato ( _Lycoper-_

**Seghers, D., Wittebolle, L., Top, E.**

_sicon esculentum_ Mill.). _Arch Mi-_

**M., Verstraete, W. and Sicili-**

_crobiol_., **188, 483-494.**

**ano, S. D. (2004)**. Impact of agri-

**Shimanuki, T. (1987)**. Studies on the

cultural practice on the _Zea mays_

mechanisms of the infection of

L. endophytic community. _Appl._

timothy with purple spot disease

_Environ. Microbiol_., **70, 1475-**

caused by _Cladosporium_ (Grego-

**1482**.

ry) de Vries. _In: Res. Bull. Hok-_

**Sessitsch, A., Howieson, J. G., Perret,**

_kaido Natl. Agric. Exp. Sta._ , **148,**

**X., Antoun, H. and Martinez-**

**1-56.**

**Romero, E. (2002).** Advances in

**Silva, F. S. (2009)**. Quantification of

_Rhizobium_ research. _Crit. Rev._

natural

populations

of

_Glu-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 244

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

_conacetobacter_

_diazotrophicus_

**Sturz, A. V., Christie, B. R. and**

and _Herbaspirillum sp._ in sugar

**Nowak, J. (2000)**. Bacterial en-

cane ( _Saccharum_ sp.) using dif-

dophytes: Potential role in devel-

ferent polyclonal antibodies. _Braz._

oping sustainable systems of crop

_J. Microbiol._ , **40, 4**.

production. _Crit. Rev. Plant Sci._ ,

**Stajkovic, O., Delic, D., Josic, D., Kuz-**

**19, 1-30.**

**manovic, D., Rasulic, N. and**

**Sudhir, A., and Amrutha V. A.**

**Knezevic Vukcevic, J. (2011)**.

**(2014)**. Ph D thesis entitled Bio-

Improvement of common bean

formulations

from

endophytic

growth by co-inoculation with Rhi-

bacteria isolated from capsicum

zobium and plant growth promoting

fruitescence US341 against an-

bacteria. _Romanian Biotechnologi-_

thracnose caused by Colleto-

_cal Letters_ , **16, 5919-5926.**

trichum sp .Acarya Nagarjuna

**Stone, J. K., Bacon, C. W. and White,**

University ,Guntur ,AP INDIA ****

**J. F. (2000).** An overview of en-

**Sudhir, A., Pradeep Kumar, N. and**

dophytic microbes: Endophytism

**Amrutha V. A. (2014)**. Isolation,

defined. In: C. W. Bacon, and J.

Biochemical and PGP characteri-

F. White (eds.) _Microbial Endo-_

zation of endophytic _Pseudomo-_

_phytes_. Marcel Dekker, New

_nas aeruginosa_ isolated from chil-

York, **3, 30**.

li red fruit antagonistic against

**Strobel, G., Daisy, B., Castillo, U. and**

chilli

anthracnose

disease.

**Harper, J. (2004)**. Natural products

_Int.J.Curr.Microbiol.App.Sci_ **.**

**3,**

from endophytic microorganisms. _J._

**318-329.**

_Nat. Prod_., 67, 257-268.

**Suman, A., Shasany, A. K., Singh, M.**

**Sturz, A. V. and Christie, B. R.**

**and Shahi, H. N. (2001)**. Mo-

**(1995)**. Endophytic bacterial sys-

lecular assessment of diversity

tems governing red clover growth

among endophytic diazotrophs

and development. _Ann. Appl. Bi-_

isolated from subtropical Indian

_ol_., **126, 285-290.**

sugarcane. _World J. Microbiol._

**Sturz, A. V. and Matheson, B. G.**

_Biotechnol._ , **17, 39-45**.

**(1996).** Populations of endophytic

**Surette, M. A., Sturz, A. V., Lada, R.**

bacteria which influence host-

**R. and Nowak, J. (2003).** Bacte-

resistance to _Erwinia_ induced bac-

rial endophytes in processing car-

terial soft rot in potato tubers.

rots ( _Daucus carota_ L. var. _sa-_

_Plant Soil_., **184, 265-271.**

_tivus_ ): their localization, popula-

**Sturz, A. V., Christie, B. R., Mathe-**

tion density, biodiversity and their

**son, B. G. and Nowak, J. (1997)**.

effects on plant growth. _Plant_

Biodiversity of endophytic bacte-

_Soil_., **253, 381-390.**

ria which colonize red clover

**Suzuki, T., Shimizu, M., Meguro, A.,**

nodules, roots, stems and foliage

**Hasegawa, S., Nishimura, T.**

and their influence on host

**and Kunoh, H. (2005).** Visuali-

growth. _Biol. Fertil. Soils_ , **25, 13-**

zation of infection of an endo-

**19**.

phytic Actinomycete _Streptomy-_

**Sturz, A. V., Christie, B. R., Mathe-**

_ces galbus_ in leaves of tissue cul-

**son, B. G., Arsenault, W. J. and**

tured _Rhododendron_. _Actinomyce-_

**Buchanan, N. A. (1999)**. Endo-

_tologica_ , **19, 7-12**.

phytic bacterial communities in

**Thamizh Vendan, R., Yu, Y. J., Lee,**

the epiderm of potato tubers and

**S. H. and Rhee, Y. H. (2010)**.

their potential to improve re-

Diversity of endophytic bacteria

sistance to soil borne plant patho-

in ginseng and their potential for

gens. _Plant Pathol_., **48, 360- 369.**

plant growth promotion. _J. Mi-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 245

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

_crobiol._ **48, 559-565.**

and colonization ability of endo-

**Than, P. P., Jeewon, R., Hyde, K. D.,**

phytic diazotrophs from deep wa-

**Pongsupasamit, S., Mongkolporn,**

ter rice. _J. Biotechnol._ **91, 127-**

**O. and Taylor, P.J.( 2008a)**. Char-

**141.**

acterization and pathogenicity of

**Vesperman,**

**A.,**

**Kai,**

**M.**

**and**

_Colletotrichum_ species associated

**Piechulla, B. (2007).** Rhizobacte-

withanthracnose disease on chilli

rial volatiles affect the growth of

( _Capsicum_ spp.) in Thailand. _Plant_

fungi and Arabidopsis thaliana.

_Pathology_ **, 57, 562-572.**

_Appl. Environm. Microbiol_., **73,**

**Ting, A. S. Y., Meon, S., Kadir, J.,**

**5639-5641**.

**Radu, S. and Singh, G. (2008)**.

**Vetrivelkalai, P., Sivakumar, M. and**

Endophytic microorganisms as

**Jonathan, E. I.( 2010).** Biocon-

potential growth promoters of ba-

trol potential of endophytic bacte-

nana. _BioControl_ , **53, 541-553**.

ria on _Meloidogyne incognita_ and

**Turner, J. T., Kelly, J. L. and Carl-**

its effect on plant growth in bhen-

**son, P. S. (1993)**. Endophytes: an

di. _J. Biopest._ 3, 452-457.

alternative genome for crop im-

**Viswanathan, R. and Samiyappan,**

provement". In: International

**R.( 1999a).** Induction of systemic

Crop Science I. _International_

resistance by plant growth pro-

_Crop Science Congress_ , Ames,

moting rhizobacteria against red

Iowa, July 14-22, 1992. Buxton,

rot disease in sugarcane. _Sugar_

D. R., Shibles, R., Forsberg, R.

_Tech_ , **1, 67-76**.

A., Blad, B. L., Asay, K. H.,

**Viswanathan, R. and Samiyappan, R.**

Paulsen, G. M. and Wilson, R. F.,

**(1999b)**. Identification of antifun-

Eds., _Crop Science Society of_

gal chitinase from sugarcane. _IC-_

_America_ , Madison, WI, **555-560**.

_AR News_ , **5, 1-2.**

**van Buren, A. M., Andre, C. and**

**Vyas, P. and Gulati, A. (2009).** Organ-

**Ishimaru, C. A. (1993)**. Biologi-

ic acid production _in vitro_ and

cal control of the bacterial ring rot

plant growth promotion in maize

pathogen by endophytic bacteria

under controlled environment by

isolated from potato. _Phyto-_

phosphate solubilizing fluorescent

_pathology_ , **83, 1406.**

_Pseudomonas_. _BMC Microbiol._ ,

**van Loon, L. C. (2007)**.Plant responses

**9, 174-189**.

to plant growth-promoting rhizo-

**Wakelin, S., Warren, R., Harvey, P.**

bacteria. _Eur. J. Plant Pathol._ ,

**and Ryder, M. (2004)**. Phosphate

**119, 243-254.**

solubilization by

**van Loon, L. C., Bakker, P. A. and**

_Penicillium_ sp. closely associated with

___
___

**Pieterse, C. M. J. (1998)**. Sys-

wheat roots. _Biol. Fertil. Soils_ , **40,**

temic resistance induced by rhizo-

**36-43**.

sphere bacteria. _Ann. Rev. Phyto_.,

**Weller, D. M. (1988)**. Biological con-

**36, 453-483.**

trol of soil borne plant pathogens

**van Peer, R., Punte, H. L. M., de We-**

in the rhizosphere with bacteria.

**ger, L. A. and Schippers, B.**

_Ann. Rev. Phytopathol_., **26,379-**

**(1990)**. Characterization of root

**407.**

surface

and

endorhizosphere

**Wheatley, R. E. (2002)**. The conse-

pseudomonads in relation to their

quences of volatile organic com-

colonization of roots, _Appl. Envi-_

pound mediated bacterial and

_ron. Microbiol_., **56,2462-2470.**

fungal interactions. _Antonie Van_

**.Verma, S. C., Ladha, J. K. and**

_Leeuwenhoek_ , **81, 357-364.**

**Tripathi, A.K. (2001)**. Evalua-

**Whipps, J. M. (2001)**. Microbial inter-

tion of plant growth promoting

actions and biocontrol in the rhi-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 246

_Biotech Sustainability (2017)_

_Bacterial Endophytes as Bio fertilizers and Bio control Agents... Audipudi et al._

zosphere. _J. Exp. Bot_., **52, 487-**

**337.**

**511**.

**Yang, C., Xang, Z., Shi, G., Zhao, H.,**

**Whitesides, S. K. and Spotts, R. A.**

**Chen, L., Tao, K. and Hou, T.**

**(1991)**. Frequency, distribution,

**(2011)**. Isolation and identifica-

and characteristics of endophytic

tion of endophytic bacterium W4

_Pseudomonas syringe_ in pear

against tomato _Botrytis cinerea_

trees. _Phytopathology_ , **81, 453-**

and antagonistic activity stabil-

**457**.

ity". _African J. Microbiol_., **5, 131-**

**Wilhelm, E., Arthofer, W. and**

**136**.

**Schafleitner, R. (1997)**. _Bacillus_

**Zinniel, D. K., Lambrecht, P., Harris,**

_subtilis_ , an endophyte of chestnut

**N. B., Feng, Z., Kuczmarski, D.,**

( _Castanea sativa_ ), as antagonist

**Higley, P., Ishimaru, C. A.,**

against

chestnut

blight

**Arunakumari, A., Barletta, R.**

( _Cryphonectria parasitica_ ), _In_ A.

**G. and Vidaver, A. K. (2002)**.

C. Cassells (ed.), _Pathogen and_

Isolation and characterization of

_microbial contamination man-_

endophytic colonizing bacteria

_agement in micropropagation._

from agronomic crops and prairie

Kluwer

Academic

Publishers,

plants. _Appl. Environ. Microbiol._ ,

Dortrecht, The Netherlands, **331-**

**68, 2198-2208**.

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 247

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P248--261_

**Microbial Metabolic Engineering: A Key Technology to**

**Deal with Global Climate and Environmental Challenges**

****

**Meerza Abdul Razak1, Pathan Shajahan Begum2, Senthilkumar Rajagopal3, ***

_1Department of Biotechnology, Rayalaseema University, Kurnool, Andhra Pradesh,_

_India; 2Department of Zoology, KVR Women's degree college, Kurnool, Andhra Pra-_

_desh, India; 3Department of Biochemistry, Rayalaseema University, Kurnool, Andhra_

_Pradesh, India;*Correspondence: senthilanal@yahoo.com; Tel: +91 9566860390_

****

**Abstract:** Global climate change and green house effects are very serious and contro-

versial problems that have severe negative impacts on environment, society, energy in-

dustry and government policies and sustainability. Global environment and climate

challenges are directly connected to the accumulation of green house gases which has

caused concerns related to the usage of traditional and fossil fuels as the key energy re-

source. To mitigate climate and environment changes, one solution is to utilize the po-

tential of metabolic engineering of microbes for biofuels production from renewable

sources. For long-term economic sustainability of the energy, industries and transporta-

tion sectors should adopt renewable and sustainable fuels produced by metabolically

engineered microorganisms. The biofuels produced from renewable sources by meta-

bolically engineered microbes carry good energy contents with minimal emission of

greenhouse gases and causes minimal impact on the environment, food chain, water

supply and land use. Toxic organic and inorganic chemicals are also one of the main

reasons for environment contamination and also present major risk for climate change.

Avoiding of upcoming contamination from these chemicals poses a huge technical

challenge. Currently, metabolically engineered microbes have been explored only for

selective and high capacity bioremediation of heavy toxic metals and chemicals. This

chapter will shed light on current trend and developments in metabolic engineering of

microbes for biofuel and bio-based chemicals production from renewable resources.

This chapter also highlights the potential of metabolically engineered microbes for bio-

remediation, a possible futuristic solution for sustainable development for energy and

reduction of global climate change and green house effects. ****

_**Keywords**_ : Biofuels; bioremediation; metabolic engineering; synthetic biology; systems

biology ****

****

****

**1. Introduction**

level and weakening of thermohaline cir-

****

culation. The atmospheric carbon dioxide

****

****

Since the past some decades,

concentration is 400 parts per million

constantly increasing greenhouse gases in

(ppm) and the carbon dioxide released

environment such as carbon dioxide, ni-

from fossil fuels worldwide is 7 Gt of

trous oxide, methane have been linked to

carbon per year (Pacala and Socolow,

global environmental and climate con-

2004; Lewis and Nocera, 2006). If the

cerns (O'Neill and Oppenheimer, 2002;

present upward trend of carbon dioxide

Stocker, 2013). Some of the effects of

continues, by the end of year 2050 the

global climate and environment change

carbon dioxide release rate will be dou-

are abolition of coral reef, rising of sea

bled. It is estimated that the carbon diox-

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_Biotech Sustainability (2017)_

_Microbial Metabolic Engineering for Sustainability Meerza et al._

ide concentration will reach to 500 ppm

in combination with rising global crises

by doubling of the carbon dioxide emis-

provides the platform for microbial meta-

sion rate without remediation. The carbon

bolic engineering applications and inno-

dioxide concentration of 500 ppm will

vation on a large scale. Scientists from

lead to a global warming of around 2°C

industries and academics strongly believe

above the level in year 1900 (Pacala and

that microbial metabolic engineering in

Socolow, 2004). This level of increase in

combination with other technologies such

temperature would raise the threat of dis-

as synthetic biology and systems biology

integration of the West Antarctic Ice

can help to solve the growing concerns of

Sheet (WAIS) along with other negative

climate and environment (Zhang _et al_.,

effects. It is estimated that increase in

2011).

temperature by 2°C would lead to disrup-

From the beginning microbial

tive rise of sea level by 4–6 meters

metabolic engineering had aimed the pro-

(Stocker, 2013).

duction of fuels and chemicals as chief

The atmospheric CO2 concentra-

goals of the rising field. The metabolic

tion, CO2 emission and global tempera-

engineering of yeast _Saccharomyces_

ture are having severe negative effects on

_cerevisiae_ and _E. coli_ are the classic ex-

the environment, climate, economy and

amples of metabolic application in the

society and we need to deal with it. To

field of biofuel production (Kuyper _et al_.,

avoid the rise in the temperature, it is

2003, Bro _et al_., 2006, Ingram _et al_.,

necessary that we should reduce the car-

1987). Microbial metabolic engineering

bon emission from the fossil fuel usage

applications have expanded in the past

and increase the sources of renewable

few years because of the growing atten-

energy and remove the toxic chemicals

tion on biofuels and chemical production

and metals present in the environment.

through biomass conversion. There are

When compared with different renewable

many reports stating that the combination

energy sources, biofuels are well-suited

of microbial metabolic engineering along

with present infrastructure and have an

with combinatorial approaches supported

advanced energy density. Thus there has

by high throughput systems had given

been much curiosity in establishing biore-

good results in biofuel production. The

fineries for the production of fuels and

key advantage of utilizing microorganism

chemicals from renewable resources.

for the production of biofuels from re-

We are in the modern age of

newable sources is the metabolic diversity

microbial metabolic engineering which

of fungi, algae and bacteria facilitate us

comprises of progressively more efforts at

the use of diverse substrates as the start-

cell, and pathway design. One of the rea-

ing point for biofuel generation.

sons for the more success of microbial

Bioremediation is a very cost

metabolic engineering is development of

effective and eco-friendly method and is

additional "omics" tools which provide

steadily making inroads for environmen-

both temporally and spatially analyzing

tal clean-up applications. Bioremediation

opportunity for cellular systems at the

depends on enhanced detoxification and

level of protein, metabolite, RNA and

degradation of toxic metals and degrada-

DNA (Peralta-Yahya _et al_., 2012). Mi-

tion of toxic pollutants either through en-

crobial metabolic engineering have been

zymatic transformation or intracellular

evolved to solve crucial international

accumulation to less or non-toxic com-

problems such as global warming, biore-

pounds. There are several physio-

mediation, food and human health. If

chemical processes for treating toxic pol-

properly utilized microbial metabolic en-

lutants in environment, but these process-

gineering can play an important role in

es are non specific, very costly and some-

facing the global challenges. By devel-

times they may introduce secondary con-

opment of novel technological innovation

tamination. Microbes naturally have the

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_Microbial Metabolic Engineering for Sustainability Meerza et al._

capability to transform, degrade and che-

_um_ _beijerinckii_ and _Thermoanaerobacte-_

late several toxic chemicals. But the mi-

_rium thermosaccharolyticum_ has been

crobial bioremediation process is having

used for the hydrogen production. The

relative slow transformation rates. By

drawback of these microbes is low pro-

metabolically engineering microbes it is

duction of hydrogen (Cai _et al_., 2011, Oh

possible to remove the toxic inorganic

_et al_., 2011). Kim _et al_. overcome the ma-

and organic chemicals from the environ-

jor obstacle of low hydrogen production

ment (Shailendra _et al_., 2008). The better

by metabolically engineered _E. coli_

understanding of microbes' natural trans-

strains (Kim _et al_., 2009). In one of the

formation ability at genetic level and ad-

investigation a high volumetric productiv-

vance of novel genetic tools are very es-

ity of 2.4 H2/L/h was produced using

sential for metabolic engineering of mi-

immobilized cells of a metabolically en-

croorganism for bioremediation. There

gineered _E. coli_ which had deletion muta-

are several metabolically engineered mi-

tion (Seol _et al_., 2011). Even though, bio-

croorganisms with superior biotransfor-

logical hydrogen production was consid-

mation capacity and more accumulation

erably increased by metabolically engi-

of toxic wastes. In this chapter we discuss

neered _E. coli_ strain, several vital obsta-

metabolic engineering strategies and suc-

cles involved in the productivity, yield

cessful examples of metabolically engi-

and metabolic robustness are still not up

neered microorganisms for production of

to the mark that would permit commer-

biofuels and chemicals and for bioreme-

cialization.

diation (Brar _et al_., 2006).

__

_2.2. Bioethanol production_

**2. Biofuels production by metabol-**

Bioethanol is the major renewable

**ically engineered microorgan-**

liquid energy source comprising 90% of

**isms**

the global world Biofuel market. It is es-

timated that annual production of bioeth-

With the increasing costs of

anol throughout the world is more than

energy and the challenges of global

105 billion liters. Most of the Bioethanol

warming that arise due to the usage of

production is by yeast and it is based on

petroleum based feedstock, the scientific

the sugarcane and starch, this type of pro-

community throughout the globe is

duction competes with feed and food

searching for energy substitutes without

(Geddes _et al_., 2011b). In one of the study

adding up to the existing carbon footprint.

_E. coli_ was metabolically engineered to

Biofuels can be an exciting substitute to

efficiently convert glycerol to ethanol.

solve both the climate and environmental

This strain was able to convert 40 g/L

issues since they are produced from the

glycerol to ethanol in 48 h with 90% of

renewable resources. Microbial produc-

the ethanol yield (Trinh and Srienc,

tion of hydrogen as future fuel is a hope-

2009). By introducing the _adh_ B and _pdc_

ful possibility as an alternative for petro-

genes from _Zymomonas mobilis_ which

leum based fuels. Hydrogen is a more

encodes alcohol dehydrogenase and py-

energy dense source and its conversion to

ruvate decarboxylase into _E. coli_ redi-

power or heart is very simple and clean.

rected the carbon flux into the ethanol

Hydrogen when combusted with oxygen

production and the obtained metabolically

only H2O is formed without the formation

engineered _E. coli_ produced ethanol upto

of toxic pollutants (Kim and Lee 2010,

1.28% (v/v) using xylose as carbon

Panagiotopoulos _et al_., 2009).

source within 36 hour fermentation (San-

ny _et al_., 2010). Metabolic engineering

_2.1. Hydrogen production_

approaches were implemented to engineer

Large number of microbes such as

_E. coli_ for ethanol production from mixed

_Sporoacetigenium mesophilum, Clostridi-_

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_Biotech Sustainability (2017)_

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sugars, glucose and xylose (Sanny _et al_.,

oped an _E. coli_ strain which can fermenta-

2010, Wang _et al_., 2008).

tively produce 1- butanol by transferring a

__

group of six genes from _C. acetobutyli-_

_2.3. Isopropanol production_

_cum_ 1- butanol pathway and removal of

_E. coli_ do not have some of the

the competing pathway. This metabolical-

necessary pathways which are very much

ly engineered _E. coli_ strain produced 552

necessary pathways for the production of

mg/L of 1- butanol under semi-anaerobic

advanced fuels, metabolic engineering

conditions from a rich medium (Atsumi

has provided the chance to produce non-

and Liao, 2008a). 1- Propanol is univer-

traditional biofuels by the construction of

sal solvent with several industrial applica-

non native biosynthesis pathways (Atsumi

tions which can be converted to propylene

_et al_., 2008b). The microorganisms such

and diesel and it is a promising gasoline

as Clostridium can naturally produce Iso-

substitute. The wild strain organisms can-

propanol which is one of the secondary

not produce the 1-propanol is considera-

alcohols. Isopropanol has several diverse

ble amounts (Shen and Liao, 2008). 2-

applications. In Clostridium several at-

ketobutyrate is a precursor of 1-propanol

tempts have been made to enhance the

and isoleucine. It is also can be converted

production ability, but product inhibition

to 2-methy 1-butanol and 1- butanol

and low titer. As a result, metabolic engi-

through several multi steps enzymes reac-

neering of _E. coli_ for Isopropanol produc-

tions. _E. coli_ was metabolically engi-

tion become a promising substitute for

neered to produce 1-propanol and 1-

industrial production of Isopropanol.

butanol through 2-ketobutyrate (Shen and

Metabolically engineered strain of _E. coli_

Liao, 2008).

was constructed that produced a 13.6 g/L

Metabolically

engineered

isopropanol from glucose under vigorous

strain was developed by overexpres-

aerobic culture conditions (Jojima _et al_.,

sion of the genes such as thrAfbBC,

2008). Isobutanol has same physical

leuABCD, ilvA from _E. coli_ , kivd

properties like isopropanol, except it has

from _L. lactis_ and adh2 from _S._

higher ocatane number. Metabolic engi-

_cereviasiae_ followed by deletion of

neering of _E. coli_ resulted in the produc-

the competing genes such as adhE,

tion of isobutanol using non-fermentative

ilvB, metA and tdh. This engineered

pathways through 2-ketoisovalerate as

strain produced 2 g/L propanol and

precursor.

butanol in 1: 1 ratio. The drawback

__

this developed strain is it production

_2.4. 1- butanol and 1- Propanol pro-_

of unwanted fermentative by prod-

_duction_

ucts. In another study, a more direct

1-Butanol is very attractive and

pathway to produce 2-ketobutyrate

alternative biofuel with high energy den-

through citramalte pathway was engi-

sity and good compatibility with the abil-

neered in _Methanococcus jannaschii_

ity to completely substitute gasoline.

by directed evolution to produce 1-

Generally, _C. acetobutylicum_ produces 1-

butanol and 1- propanol. CimA spe-

butanol along with butyrate, ethanol and

cific activity was enhanced by the

acetone. But due to absence of genetic

DNA shuffling and error prone PCR

information, complex physiology and un-

in _E. coli_ strain. This _E. coli_ strain

availability of genetic tools the _C. aceto-_

with enhanced CimA activity pro-

_butylicum_ had not been improved to the

duced more than 524 mg/L and

level of usage in large scale industrial

3.5g/L propanol. The production of

production. The complete information of

other side products is drawback of

the 1-butanol metabolic pathway, it be-

this strain.

come possible to construction of butanol

pathway in _E. coli_. Atsumi _et al_. devel-

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_2.5. 2-Methyl-1-butanol and 3-methyl-_

the less toxic dialkyl phosphates and p-

_1-butanol production_

nitrophenol is by metabolic engineering

The five carbon alcohols such as

of microbes with the required degradation

3-methyl-1-butanol

and

2-Methyl-1-

pathways (Cook _et al_., 1980; de la Pena

butanol have characteristics like high en-

Mattozzi _et al_., 2006).

ergy density and lower vapor pressure.

The p-nitrophenol can be degrad-

When compared with ethanol, 3-methyl-

ed by metabolically engineered _P.putida_

1-butanol and 2-Methyl-1-butanol are

and _Moraxella_ species. In _Moraxella_ spe-

more suitable to replace gasoline and

cies organophosphate hydrolase was ex-

more well-suited for the present fuel in-

pressed on the cell surfaces and this strain

frastructure. 3-methyl-1-butanol and 2-

showed to degrade paraoxon, parathion

Methyl-1-butanol can be naturally pro-

and methyl parathion and their hydrolysis

duced by as by- products and some meta-

product p-nitrophenol. In _P. putida_

bolic approaches have been tried to

strain, natural p-nitrophenol degrading

enhance the production of 2-Methyl-1-

operon was introduced. A synthetic oper-

butanol in _S. cerevisiae_ (Abe and

on for expression of alkaline phosphatase

Horikoshi, 2005). Table 1 illustrates the

(PhoA), phosphodiesterase (Pde) and or-

biofuels production by metabolically en-

ganophosphate hydrolase was introduced

gineered _E. coli_.

to improve the diethyl phosphate (DEP)

mineralization and hydrolysis of oragan-

**3. Bioremediation by metabolical-**

ophosphates. The resulting metabolically

**ly engineered microorganisms**

engineered _P.putida_ strain was able to

****

totally degrade p-nitrophenol in 78 hours,

_3.1. Bioremediation of organic pollu-_

paraoxon in 24 hours and diethyl phos-

_tants_

phate within 142 hours (de la Pena Mat-

****

****

The extensive use of extremely

tozzi, _et al_., 2006). This research study

toxic

compound

oraganophosphates

presents an effective novel approach to

(OPs) in agriculture as pesticide had led

create an artificial metabolite pathway for

to a serious environmental pollution. Ora-

total mineralization.

ganophosphates are used in the insecti-

The present hydrodesulfurization

cides; generally oraganophosphates are in

method for decreasing the level of organic

the form of phosphoric acid. The bacteria

sulfur compounds is not capable to please

growing in the soil had naturally acquired

the strict government regulations. The

the ability to degrade oraganophosphates

present advances in desulfurization have

with the help of enzyme called organo-

therefore been focused on biological pro-

phosphate hydrolase (McDaniel _et al_.,

cesses using microbes. Dibenzothiophene

1988; Kulkarni and Chaudhari 2006). P-O

which is a popular organosulfur com-

linkage is hydrolysed by the organophos-

pound is not removed by the hydrodesul-

phate hydrolase, releasing p-nitrophenol

furization from fossil fuels. The desulfu-

as leaving group. As the toxicity of ora-

rization of dibenzothiophene requires a

ganophosphates is extensively diminished

group of enzymes (Kilbane, 2006).

by hydrolysis of phoshoester bonds, sev-

Dibenzothiophene

monooxygenase

eral scientists have concentrated on the

(DszC) is the first enzyme that converts

primary hydrolysis by organophosphate

dibenzothiophene to dibenzothiophene

hydrolase. Even though oraganophos-

sulfone. The dibenzothiophene sulfone is

phates are transformed into dialkyl phos-

converted

into

2-hydroxybiphenyl-2-

phates and p-nitrophenol (PNP) by initial

sulfinate by the catalysis of dibenzothio-

hydrolysis. Still these degraded products

phene-5,5-dioxide monooxygenase. 2-

are resistant to biodegradation and they

hydroxybiphenyl-2-sulfinate is converted

are toxic. One solution to remove even

****

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_Microbial Metabolic Engineering for Sustainability Meerza et al._

**Table 1:** Biofuels produced by metabolically engineered _E. coli_ ****

**Biofuels**

**Fermentation**

**Engineering**

**Carbon Titer and Industrial**

**process**

**strategy**

**source**

**yield**

**applications**

Hydrogen

Immobilized

Deletion of nega-

Formate 1.0 mol

Fuel and en-

recombinant

tive regulator and

H2/mol

ergy carrier

cells

competing carbon

formate;

metabolic path-

2.4 l

ways

H2/L/ha;

formate

Bioethanol

10-L bioreactor

Minimized meta-

Xylose

38.81 g/L; Fuel, solvent,

batch anaerobic

bolic functionality and

0.49 g/g

food, bever-

cultivation

for conversion of

glucose

glucose

age

xylose and glu-

or xylose

cose into ethanol

by multiple-gene

knockout

1-Propanol

Shake flasks and Improving specif-

Glucose 3.5 g/L;

Gasoline ad-

IPTG induction

ic activity and

0.049

ditive,

releasing feedback

g/gb

allround sol-

inhibition of the

vent

key enzyme by

directed evolution

1-Butanol

1-L Bioreactor

Construction of

Glucose 30 g/L;

Bulk material,

with aerobic–

modified clostrid-

88% of

gasoline addi-

anaerobic dual

ial 1-butanol

the

tive or fuel

phase fermenta-

pathway in _E. coli_

theoretical

tion

strain and en-

yield

hancement of

driving forces

3-Methyl-

Shake flask with Random muta-

Glucose 9.5 g/L;

Advanced

1-butanol

two-phase fer-

genesis and selec-

0.11 g/g

fuel, alterna-

mentation

tion combined

glucose

tive gasoline

with overexpres-

sion of key genes

Isopropanol Fed-batch fer-

Heterologous ex-

Glucose 143 g/L;

Biodiesel,

mentation, gas-

pression of target

67.4%

precursor of

stripping-based

product pathway

(mol/mol) polypropylene

recovery process from various

sources in _E. coli_

host

Isobutanol

Screw-cap coni-

Introducing non-

Glucose 20 g/L;

Gasoline

cal flasks and

fermentative

86% of

blend stock,

IPTG induction

synthetic pathway

the theo-

precursor of

in _E. coli_ and

retical

butenes

elimination of

maximum

pathways compet-

ing for pyruvate

and cofactors

into 2-hydroxybiphenyl (HBP) and sulfite

sulfinate sulfinolyase (Reichmuth _et al_.,

by the enzyme2-hydroxybiphenyl- 2-

2004). By mutating at the 50 untranslated

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region of dibenzothiophene monooxygen-

resulted in 20 fold higher heavy metal

ase and overexpressing it enhanced the

accumulation (Sauge-Merle, _et al_., 2003).

desulfurization rate by 9 fold increase

Metallothionein and phytochelatins have

when compared with the unmutated

restriction such as non selective binding

dibenzothiophene

monooxygenase

to diverse heavy metals. Specific heavy

(Reichmuth _et al_., 2004).

metal transporters support to enhance up-

Nitrotoluene and nitrobenzene

take and accumulation of particular toxic

which are extensively used as pesticides,

heavy metals. By expression of Cd trans-

some polymers dyes and explosives are

porter MntA, selective Cd accumulation

one of the most commonly seen pollu-

can be achieved (Kim, _et al_., 2005). The

tants. Naturally microorganisms can

Fucus Metallothionein which is obtained

transform nitroaromatic compounds into

from the arsenic resistant marine alga _Fu-_

amines with the help of redox enzymes.

_cus vesiculosus_ is used in metabolic engi-

But the degradation is very slow because

neering to create superior strains of _E._

of electron loosing effect of nitro groups

_coli_. The co-expression of specific arsenic

and toxicity. Through metabolic engi-

transporter and Fucus Metallothionein in

neering and site directed mutagenesis at

_E. coli_ resulted in 45 fold increase in ar-

the position of 258 of 2-nitrotoluene di-

senic accumulation. It is possible that the

oxygenase which is responsibe for the

same strategies can be applied for other

oxidation of nitrotoluene to 3-methyl cat-

heavy metals also. Table 2 elucidates the

echol and nitrite changed the enantiospec-

environmental pollution creating inorgan-

ificity and resulted in more degradation of

ic heavy metals and radionuclides.

nitroaromatic compounds (Lee _et al_.,

The over expression of phyto-

2005). By metabolic engineering of

chelatin synthase responsible for enzy-

_Sphingobium chlorophenolicum_ ATCC

matic phytochelatins synthesis by enzy-

39723 the rapid degradation of another

matic method in symbiotic Rhizobia bac-

toxic pesticide pentachlorophenol was

terium showed good results. From this

achieved. Three rounds of genome shuf-

study it was demonstrated that phyto-

fling in _Sphingobium chlorophenolicum_

chelatins can be successfully used for

resulted in a strain with more resistance to

heavy metal accumulation, but the limita-

pentachlorophenol, increased growth and

tions in this approach are the minimum

rapid pentachlorophenol removal (Dai

supply of the precursor glutathione in

and Copley, 2004).

phytochelatin production and other heavy

metal accumulation. This limitation can

_3.2. Bioremediation of inorganic pol-_

be overcome by co-expression of the en-

_lutants_

zyme phytochelatin synthase and the en-

****

****

Generally microorganisms when

zymes responsible for precursor glutathi-

they come in contact with heavy metals,

one production Sriprang, _et al_., (2002).

they synthesize metal binding peptides

The co-expression of phytochelatin syn-

such as metallothionein and phytochela-

thase with feedback resistant glutathione

tins. These peptides which are rich in thi-

synthase resulted in 10 fold increases in

ol group bind to different heavy metals

phytochelatin production. The over-

and by sequestration they reduce the tox-

expression of Cadmium transporter in-

icity. Moreover, these peptides produced

creased the final cadmium accumulation

in different sub cellular locations of mi-

by 31.6 mmol/g dry weight (Kang _et al_.,

crobes have been very useful for them to

2007).

enhance their metal accumulation ability.

_E. coli_ was metabolically engineered by

_3.3. Bioremediation of radionuclide_

over expression of phytochelatin synthase

****

****

In addition to organic and inor-

of _Arabidopsis thaliana_ which is respon-

ganic pollutants, radionuclide contamina-

sible for synthesis of phytochelatins, this

tion occurred through nuclear weapons or

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**Table 2:** Environmental pollution creating inorganic heavy metals and radionuclides

**Contaminant**

**MCLG**

**MCL**

**Potential health effects Sources of contami-**

**(mg/L)**

**(mg/L)**

**from ingestion of wa-**

**nant in drinking**

**ter**

**water**

Arsenic

0

0.010

Skin damage or prob-

Erosion of natural

lems with circulatory

deposits; runoff from

systems, and may

orchards, runoff from

have increased risk of

glass and electronic

getting cancer

production wastes

Cadmium

0.005

0.005

Kidney damage

Corrosion of galva-

nized pipes; erosion

of natural deposits;

discharge from metal

refineries; runoff

from waste batteries

and paints

Lead

0

0.015

Infants and children:

Corrosion of house-

delays in physical or

hold plumbing sys-

mental development;

tems; erosion of nat-

children could show

ural deposits

slight deficits in atten-

tion span and learning

abilities. Adults: kidney

problems; high blood

pressure

Mercury

0.002

0.002

Kidney damage

Erosion of natural

(inorganic)

deposits; discharge

from refineries and

factories; runoff from

landfills and

croplands

Radium

0

5 pCi/L

Increased risk of cancer

Erosion of natural

226/228

deposits

Uranium

0

30 mg/L

Increased risk of cancer, Erosion of natural

kidney toxicity

deposits

MCLG: maximum contaminant level goal; MCL: Maximum contaminants limit; Source:

EPA safe water

nuclear plant leakage is one of the major

metabolically engineered bacteria _Dein-_

environmental problems. Naturally occur-

_ococcus geothemalis_ is able to decrease

ring bacteria which are more resistant to

mercury at higher temperature and ioniz-

radiation are ideal metabolic engineering

ing radiation, it is also able to reduce

candidates for enhanced radionuclide re-

Cr(VI), U(VI) and Fe(III). This study re-

moval. Metabolic engineering of thermo-

ported the possibility of using metaboli-

philic bacterium _Deinococcus geothe-_

cally engineered microorganisms for re-

_malis_ was done by over expressing of mer

moval of versatile radioactive wastes at

operon from _E. coli_ coding for Hg2+ re-

high temperatures. In _P. aeruginosa_ , radi-

duction, which resulted in radiation re-

onuclide precipitation can be achieved as

sistant bacterium (Brim _et al_., 2003). The

metal phosphate by over expressing of

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exopolyphosphatases and polyphosphate

possibility of production of the desired

kinase (Renninger _et al_., 2004). In radia-

biochemical or product in the non-native

tion resistant bacterium _D. radiodurans_ ,

organism. The genes can be isolated from

non-specific phosphatases _phoN_ was ex-

the native organisms which can produce

pressed which resulted bioprecipitation of

the required product and these genes can

uranium from dilute nuclear waste (Ap-

be expressed in another non-native organ-

pukuttan _et al_., 2006). The enzymes ura-

ism (heterologous host organisms). It is

nyl reductases and chromate are subjected

mandatory that the required substrate

to directed evolution and these engineered

should be available in the non-native or-

enzymes are used for metabolic engineer-

ganism. Multiple genes representing the

ing of _P.putida_ and _E. coli_. These meta-

group of enzymes of particular pathway

bolically engineered strains of _P.putida_

can be expressed in the non-native host.

and _E. coli_ showed more resistant against

Expressing the group of genes encoding

radiation and further enhanced the radio-

the most capable enzymes from diverse

nuclide precipitation efficiency (Barak _et_

organisms is another way to obtain the

_al_., 2006).

product which is produced in low quanti-

ties or not produced (Patil _et al_., 2005).

**4. Metabolic engineering strategies**

Figure 1 explains the strategies for meta-

****

bolic engineering for the production of a

****

****

Generally the metabolic engineer-

desired chemical.

ing strategies are based on genetic engi-

neering techniques. Some of the essential

**5. Tools for metabolic engineering**

requirements for metabolic engineering

****

are 1. Complete information of the bio-

****

****

Metabolic engineers use different

synthetic pathway of the interested chem-

protein engineering techniques and di-

ical. 2. Particular of the genes coding re-

rected proteomic techniques to get useful

lated enzyme. 3. Regulating factors of

information about protein levels in the

enzymes involved in pathway. 4. Expres-

microorganisms. Metabolic engineers be-

sion or deletion of required enzyme in

lieve that protein engineering and targeted

host bacteria. 5. Gene mutation effects on

proteomics are very valuable tools that fill

the enzyme properties 6. Assembly of

the gap to engineer novel metabolic

group of genes and their co-expression.

pathways for microorganisms. Synthetic

Now a day's along with bacteria and

biology had its application in the metabol-

yeast, plant cell, animal cells and fungi

ic engineering for the alteration of mi-

are also used for metabolic engineering

crobes for the biorenewable production of

(Kell _et al_., 2005).

biofuels and bioremediation (James, _et_

Some of the strategies of metabol-

_al_., 2016). Through synthetic biology we

ic engineering for the achievement of

can design or redesign and construct new

production of required biochemical are

biological components such as cells, en-

discussed below. 1. One of the most

zymes, proteins and pathways. The main

commonly used strategies is over express-

goal of systems biology is the explaining

ing of the gene encoding the rate-limiting

the cell physiology and function through

enzyme of the biosynthetic pathway of

the integrated use of broad physiological

the required end product. 2. In this way,

and genomic data. Directed evolution and

we can achieve the overproduction of the

genetic engineering are the main tools of

desired product by inhibiting or deleting

metabolic engineering which are very

the genes responsible for the competing

much responsible for the its success

metabolic reactions which use the same

(Leber and Da Silva, 2014, Meerza _et al_.,

substrate. Through this way the substrate

2016). The tools of metabolic engineer-

is metabolically channeled particularly

ing are shown in Figure 2.

towards the desired chemical. 3. There is

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 256

_Biotech Sustainability (2017)_

_Microbial Metabolic Engineering for Sustainability Meerza et al._

**1**

**2**

**Wild type strain**

**Wild type strain**

**A**

**B**

**C**

**A**

**B**

**C**

**Metabolically**

**Metabolically**

**engineered strain**

**engineered strain**

**A**

**B**

**C**

**A**

**B**

**C**

**D**

**Wild type strain**

**3**

****

**C**

****

**A**

**B**

****

****

**D**

**Metabolically**

**Figure 1:** Strategies for metabolic engineer-

**engineered strain**

ing for the production of a desired chemical;

(1) overexpression of the rate-limiting en-

**C**

zyme; (2) inhibition of the competing path-

way; (3) engineering a novel enzyme for the

**A**

**B**

production of non-natural chemical.

**D**

**SYSTEMS BIOLOGY**

**SYNTHETIC BIOLOGY**

**Metabolic models**

**Heterologous expression of**

**\- Omics analysis**

**natural pathways**

**- _de novo_** **pathway design**

**METABOLIC**

**DIRECTED**

**ENGINEERING**

**EVOLUTION**

**PROTEIN**

**GENETIC ENGINEERING**

**ENGINEERING**

**Deletion or silencing**

**Random mutagenesis**

**\- Transcriptional tuning**

**\- Rational modification**

**\- Translational tuning**

****

**Figure 2:** Overview of tools for metabolic engineering.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 257

_Biotech Sustainability (2017)_

_Microbial Metabolic Engineering for Sustainability Meerza et al._

**6. Perspectives**

_erichia coli._ _Appl Environ Mi-_

****

_crobiol_ , **74, 7802–8.**

****

Unquestionably, microbial meta-

**Atsumi, S. Hanai, T. and Liao, J. C.**

bolic engineering is the main tool for

**(2008b).**

Non-fermentative

production of biofuels and for bioremedi-

pathways

for

synthesis

of

ation. The scientific and technological

branched-chain higher alcohols

progress in metabolic engineering has

as biofuels. _Nature_ **451, 86–9.**

been estimated to make a good contribu-

**Barak, Y. Ackerley, D. F. Dodge, C.**

tion to promote green and sustainable en-

**J. Banwari, L. Alex, C. Fran-**

ergy by biofuels production. The key ad-

**cis, A. J. and Martin, A.**

vantages of biofuels produced by the

**(2006).** Analysis of novel soluble

metabolically engineered microbes over

chromate and uranyl reductases

petroleum based fuels are they are very

and generation of an improved

environmentally friendly, produced from

enzyme by directed evolution.

the renewable feedstocks and very low

_Appl Env Microbiol_ **72, 7074-**

emissions of carbon. From the microbial

**7082**. ****

metabolic engineering standpoint, the

**Brar, S. K. Verma, M. Surampalli,**

discovery and design of novel pathways

**R.Y. Misra, K. Tyagi, R. D.**

and enzymes for metabolic engineering

**Meunier, N, and Blais, J. F.**

of microorganisms coupled with efficient

**(2006).** Bioremediation of haz-

innovative and low cost processing tech-

ardous wastes: a review. _Pract_

nologies will contribute significantly to

_Periodical Hazard, Toxic Radio-_

the success of production of biofuels and

_active_

_Waste_

_Manag_ ,

bioremediation through which we can

**10, 59-72.**

meet the global challenges of environ-

**Brim,**

**H.**

**Venkateshwaran,**

**A.**

ment and climate change. Metabolically

**Kostandarithes,**

**H.**

**M.**

engineered microbes could be used as

**Fredrickson, J. K. and Daly,**

platforms towards green energy and bio-

**M. J. (2003).** Engineering _Dein-_

chemical production, another crucial area

_ococcus geothermalis_ for biore-

of application promises to the human

mediation of high-temperature

health.

radioactive waste environments.

_Appl Environ Microbiol_ , **69,**

**References**

**4575-4582.**

****

**Bro, C. Regenberg, B. Foerster, J.**

**Abe, F. and Horikoshi, K. (2005).**

**and Nielsen, J. (2006).** In silico

Enhanced production of isoamyl

aided metabolic engineering of

alcohol and isoamyl acetate by

_Saccharomyces cerevisiae_ for

ubiquitination-deficient _Saccha-_

improved bioethanol production.

_romyces cerevisiae_ mutants. _Cell_

_Metab. Eng_ **8, 102–111.**

_Mol Biol Lett_ **10,383–8.**

**Cai, G. Jin, B. Monis, P. and Saint,**

**Appukuttan, D. Rao, A. S. and Apte,**

**C. (2011).** Metabolic flux net-

**S. K. (2006).** Engineering of De-

work and analysis of fermenta-

inococcus radiodurans R1 for bi-

tive hydrogen production. _Bio-_

oprecipitation of uranium from

_technol Adv_ **29:375–87**. ****

dilute nuclear waste. _Appl Envi-_

**Cook, A. M. Daughton, C. G. and**

_ron Microbiol_ **72, 7873-7878.**

**Alexander, M. (1980).** Desul-

**Atsumi, S. and Liao, J. C. (2008a).**

furation of dialkyl thiophosphor-

Directed evolution of _Methano-_

ic acids by a _Pseudomonad_. _Appl_

_coccus jannaschii_ citramalate

_Environ Microbiol,_ **40, 463-465**. ****

synthase for biosynthesis of 1-

**Dai, M. H. and Copley, S. D. (2004).**

propanol and 1-butanol by _Esch-_

Genome shuffling improves deg-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 258

_Biotech Sustainability (2017)_

_Microbial Metabolic Engineering for Sustainability Meerza et al._

radation of the anthropogenic

fossil fuels. _Curr Opin Biotech_

pesticide pentachlorophenol by

**17, 305-314.**

_Sphingobium chlorophenolicum_

**Kim, M. S. and Lee, D. Y. (2010).**

ATCC

39723.

_Appl_

_Env_

Fermentative hydrogen produc-

_Microbiol_ **70, 2391-2397.**

tion from tofu processing waste

**de la Pena Mattozzi, M. Tehara, S.**

and anaerobic digester sludge us-

**K. Hong, T. Keasling, J. D.**

ing microbial consortium. _Biore-_

**(2006).**

Mineralization

of

_sour Technol_ **101, 48–52.**

paraoxon and its use as a sole C

**Kim, S. K. Lee, B. S. Wilson, D. B.**

and P source by a rationally de-

**and Kim, E. K. (2005).** Selec-

signed catabolic pathway in

tive cadmium accumulation us-

_Pseudomonas putida_. _Appl Envi-_

ing

recombinant

_Escherichia_

_ron Microbiol_ , **72, 6699-6706.**

_coli_. _J Biosci Bioeng_ **99, 109-**

**Geddes, C. C. Nieves, I. U. and In-**

**114.**

**gram, L. O. (2011b).** Advances

**Kim, S. Seol, E. Oh, Y. K. Wang, G.**

in ethanol production. _Curr Opin_

**and Park, S. (2009).** Hydrogen

_Biotechnol_ **22, 312–9.**

production and metabolic flux

**Ingram, L. O. Conway, T. Clark, D.**

analysis of metabolically engi-

**P. Sewell, G.W. and Preston,**

neered _Escherichia coli_ strains.

**J.F. (1987).** Genetic engineering

_Int J Hydrogen Energy_ **34,**

of ethanol production in _Esche-_

**7417–27.**

_richia coli_. _Appl. Environ. Mi-_

**Kulkarni, M. and Chaudhari, A.**

_crobiol_ **53, 2420–2425.**

**(2006).** Biodegradation of p-

**James, C. Luo, M. Sammy, P. and**

nitrophenol by P. putida. _Biore-_

**Shanshan, P. (2016).** LuoFuel-

_source Technol_ **97, 982-988**. ****

ling the future: microbial engi-

**Kuyper, M. Harhangi, H. R. Stave,**

neering for the production of

**A. K. Winkler, A. A. Jetten, M.**

sustainable biofuels. _Nature re-_

**S. M. De Laat, W. T. A. M.**

_views_ **14** , **288-304.**

**Den Ridder, J. J. J., Op den**

**Jojima, T. Inui, M. and Yukawa, H.**

**Camp, H. J. M. Van Dijken, J.**

**(2008).** Production of isopropa-

**P. and Pronk, J. T. (2003)**.

nol by metabolically engineered

High-level functional expression

_Escherichia coli_. _Appl Microbiol_

of a fungal xylose isomerase: the

_Biotechnol_ **77, 1219–24.**

key to efficient ethanolic fermen-

**Kang, S. H. Singh, S. Kim, J. Y.**

tation of xylose by _Saccharomy-_

**Lee, W. Mulchandani, A. and**

_ces cerevisiae_?. _FEMS Yeast_

**Chen, W, (2007).** Bacteria met-

_Res_. **4, 69–78.**

abolically engineered for en-

**Leber, C. and Da Silva N. A. (2014).**

hanced phytochelatin production

Engineering of _Saccharomyces_

and

cadmium

accumulation.

_cerevisiae_ for the synthesis of

_Appl_

_Environ_

_Microbiol_

short chain fatty acids. _Biotech-_

**73, 6317-6320.**

_nol Bioeng_ **111, 347-358.** ****

**Kell, D. B. Brown, M. Davey, H. M.**

**Lee, K. S. Parales, J. V. Friemann,**

**Dunn, W. B. and Spasic, I.**

**R. and Parales, R. E. (2005).**

**(2005).**

Metabolic

Active site residues controlling

footprinting and systems biolo-

substrate

specificity

in

2-

gy: The medium is the message.

nitrotoluene dioxygenase from

_Nat Rev Microbiol_ **3, 557-565.**

_Acidovorax_ sp. strain JS42. _J Ind_

**Kilbane, J. J. (2006).** Microbial bio-

_Microbiol Biotechnol_ **32, 465-**

catalyst developments to upgrade

**473.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 259

_Biotech Sustainability (2017)_

_Microbial Metabolic Engineering for Sustainability Meerza et al._

**Lewis, S. N. and Nocera, G. D.**

**Peralta-Yahya, P. P. Zhang, F.Z. del**

**(2006).** Powering the planet:

**Cardayre, S.B. and Keasling,**

chemical challenges in solar en-

**J. D.** **(2012).** Microbial engineer-

ergy utilization. _Proc. Natl Acad._

ing for the production of ad-

_Sci. USA_ **103, 15729–15735.** ****

vanced biofuels. _Nature_ **488,**

**McDaniel, C. S. Harper, L. L. and**

**320-328.**

**Wild, J. R. (1988).** Cloning and

**Reichmuth, D. S. Blanch, H. W. and**

sequencing of a plasmid-borne

**Keasling, J. D. (2004).** Diben-

gene (opd) encoding a phos-

zothiophene

biodesulfurization

photriesterase. _J Bacteriol_ **170,**

pathway improvement using di-

**2306-2311**. ****

agnostic GFP fusions. _Biotechnol_

**Meerza, A. R. Pathan, S. B. Rao D.**

_Bioeng_ **88, 94-99.**

**M. and Viswanath, B. (2016).**

**Renninger, N. Knopp, R. Nitsche,**

Metabolic Engineering to Im-

**H. Clark, D. S. and Keasling,**

prove the Activity of Aspartate

**J. D. (2004).** Uranyl precipita-

kinase for Biotechnological Pro-

tion by _Pseudomonas aeruginosa_

duction of L-lysine by _Coryne-_

via controlled

polyphosphate

_bacterium glutamicum_ ATCC

metabolism. _Appl Environ Mi-_

13032. _American Journal of Bio-_

_crobiol_ **70, 7404-7412**. ****

_chemistry and microbiology_ **6,**

**Sanny, T. Arnaldos, M. Kunkel, S.**

**33-44**. ****

**A. Pagilla, K. R. and Stark, B.**

**O'Neill, B. C. and Oppenheimer, M.**

**C. (2010).** Engineering of etha-

**(2002).**

Dangerous

climate

nolic _E. coli_ with the _Vitreoscilla_

impacts and the Kyoto Protocol.

hemoglobin gene enhances etha-

_Science_ **296** , **1971–1972**. ****

nol production from both glucose

**Oh, Y. K. Raj, S. M. Jung, G. Y.**

and xylose. _Appl Microbiol Bio-_

**and Park, S. (2011).** Current

_technol_ , **88,1103–1112.**

status of the metabolic engineer-

**Sauge-Merle, S. Cuine, S. Carrier,**

ing of microorganisms for bio-

**P. Lecomte-Pradines, C. Luu,**

hydrogen production. _Bioresour_

**D. T. and Peltier, G. (2003).**

_Technol_ , **102, 8357–67.**

Enhanced toxic metal accumula-

**Pacala, S. and Socolow, R. (2004)**.

tion in engineered bacterial cells

Stabilization wedges: solving

expressing _Arabidopsis thaliana_

the climate problem for the next

phytochelatin synthase. _Appl En-_

50

years

with

current

_viron Microbiol_ **69, 490-494.**

technologies. _Science_ **305, 968–**

**Seol, E. Manimaran, A. Jang, Y.**

**972**. ****

**Kim, S. Oh, Y. K. and Park, S.**

**Panagiotopoulos, I. A. Bakker, R. R.**

**(2011).** Sustained hydrogen pro-

**Budde, M. A. de Vrije, T.**

duction from formate using im-

**Claassen, P. A. and Koukios,**

mobilized recombinant _Esche-_

**E. G. (2009).** Fermentative hy-

_richia coli_ SH5. _Int J Hydrogen_

drogen production from pretreat-

_Energy_ **36, 8681–8686**. ****

ed biomass: a comparative study.

**Shailendra, S. Seung, H. K. Ashok,**

_Bioresour Technol_ **100, 6331-**

**M. and Wilfred, C. (2008).** Bio-

**6338**. ****

remediation:

environmental

**Patil, K. R. Rocha, I. Forster, J.**

clean-up through pathway engi-

**and Nielsen, J. (2005).** Evolu-

neering. _Current Opinion in Bio-_

tionary programming as a plat-

_technology_ **19, 437–444.**

form for in silico metabolic en-

**Shen, C. R. and Liao, J. C. (2008).**

gineering. _BMC Bioinformatics_

Metabolic engineering of Esche-

**6, 308.**

richia coli for 1-butanol and 1-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 260

_Biotech Sustainability (2017)_

_Microbial Metabolic Engineering for Sustainability Meerza et al._

propanol production via the keto-

**Trinh, C. T. and Srienc, F. (2009).**

acid pathways. _Metab Eng_ **10,**

Metabolic engineering of _Esche-_

**312–320**. ****

_richia coli_ for efficient conver-

**Sriprang, R. Hayashi, M. Ono, H.**

sion of glycerol to ethanol. _Appl_

**Takagi, M. Hirata, K. and**

_Environ Microbiol_ **75, 6696–**

**Murooka, Y. (2002).** Enhanced

**705.**

accumulation of Cd2+ by a

**Wang, Z. Chen, M. Xu, Y. Li, S. Lu,**

_Mesorhizobium sp_. transformed

**W. and Ping, S. (2008).** An eth-

with a gene from _Arabidopsis_

anol-tolerant recombinant _Esche-_

_thaliana_

coding

for

_richia coli_ expressing _Zymomo-_

phytochelatin synthase. _Appl En-_

_nas mobilis_ pdc and adhB genes

_viron Microbiol_ **69, 1791-1796.**

for enhanced ethanol production

**Stocker, T. F. In Climate Change**

from xylose. _Biotechnol Lett_ **30,**

**(2013).** The Physical Science

**657–63.**

Basis. Contribution of Working

**Zhang,**

**F.**

**Rodriguez,**

**S.**

**and**

Group I to the Fifth Assessment

**Keasling, J. D. (2011).** Metabol-

Report of the Intergovernmental

ic engineering of microbial

Panel on Climate Change (eds

pathways for advanced biofuels

Stocker, T. F. _et al_.) **pp** **13–115**. ****

production. _Curr Opin Biotech-_

_nol_ **22, 775-783.**

****

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 261

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P262-273_

**Biodiesel Production for Sustainability:** **An Overview**

****

**R. Meena Devi, R. Subadevi and M. Sivakumar***

_Energy Materials Lab, #120, School of Physics, Alagappa University, Karaikudi-630 004,_

_Tamil Nadu, India; *Correspondence: susiva73@yahoo.co.in; Tel: +91 4565 223304_

****

****

**Abstract:** Diminishing petroleum reserves and increasing environmental regulations are the

main driving forces to search for renewable fuel. Biodiesel is a renewable substitute fuel for

petroleum diesel fuel and produced by transesterification in which oil or fat is allowed to

react with a monohydric alcohol in the presence of a catalyst. This paper gives an overview

of the work carried out by researchers in the field of biodiesel production from different

types of oil. The different methods of biodiesel production techniques and factors affecting

biodiesel production are also highlighted.

_**Keywords** : Biodiesel, catalyst, transesterification, vegetable oil, viscosity_ ****

****

****

1. **Introduction**

is defined as the monoalkyl esters

derivative from lipid feedstocks, such as

The

demand

for

energy

is

vegetable oils or animal fats (Avhad and

increasing in the world due to the rapidly

Marchetti, 2016). The dominant biodiesel

growing

global

population

and

production

process,

namely

urbanization. The depletion of world

transesterification, typically involves the

petroleum

reserves

and

increased

reaction of an alkyl-alcohol with a long

environmental concerns has stimulated

chain ester linkage in the presence of a

the search for alternative renewable fuels

catalyst to yield mono-alkyl esters

that are capable of fulfilling an increasing

(biodiesel) and glycerol (Verma _et al_.,

energy demand in a sustainable manner

2016) _._

(Narasimharao _et al_., 2007). In recent

Due to the recent increased

decades,

research

concerning

and

awareness and development in this area,

knowledge about the external benefits of

the objective of this review is to give

renewable raw materials have intensified

fundamental insight into the production of

the efforts for sustainable energy sources.

biodiesel by different raw materials. Also,

The various alternative fuel

this paper, reviews the factors affecting

options tried in place of hydrocarbon oils

biodiesel production process such as

are mainly biogas, producer gas, ethanol,

temperature, reaction time, methanol to

methanol and vegetable oils. Out of all

oil molar ratio, type and amount of

these, biodiesel offers an advantage

catalyst, mixing intensity and fuel

because

of

their

comparable

fuel

properties of biodiesel.

properties with that of diesel. The

emissions produced from biodiesel are

2. **Merits and demerits of biodiesel**

cleaner compared to petroleum-based

diesel fuel. Biodiesel can be regarded as

Some of the advantages of using

an alternative diesel fuel.

biodiesel as a replacement for diesel fuel

Biodiesel is the colloquial name

are (Knothe, __ 2006; Romano _et al.,_ 2006):

for "fatty acid alkyl ester" (FAAE).

 Renewable fuel, obtained from

According to the American Society for

vegetable oils or animal fats.

Testing and Materials (ASTM), biodiesel

 Low toxicity, in comparison with

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 262

_Biotech Sustainability (2017)_

_Biodiesel Production for Sustainability Meena Devi et al._

diesel fuel.

injection systems. In consequence, the

 Degrades more rapidly than diesel

cleaning of tanks prior to filling with

fuel, minimizing the environmental

biodiesel is recommended. It must be

consequences of biofuel spills.

noted that these disadvantages are

 Lower emissions of contaminants:

significantly reduced when biodiesel

carbon monoxide, particulate matter,

is used in blends with diesel fuel.

polycyclic aromatic hydrocarbons,

****

aldehydes.

3. **History of biodiesel**

 Lower health risk, due to reduced

emissions of carcinogenic substances.

Dr.

Rudolf

Diesel

actually

 No sulfur dioxide (SO

invented the diesel engine to run on a

2) emissions.

 Higher flash point (100 °C minimum).

myriad of fuels including coal dust

May be blended with diesel fuel at

suspended in water, heavy mineral oil,

and, vegetable oil. Dr. Diesel's first

any proportion; both fuels may be

mixed during the fuel supply to

engine experiments were catastrophic

vehicles.

failures. But by the time he showed his

 Excellent properties as a lubricant.

engine at the World Exhibition in Paris in



1900, his engine was running on 100%

It is the only alternative fuel that can

peanut oil. Dr. Diesel was visionary. In

be used in a conventional diesel

1911, he stated that "the diesel engine can

engine, without modifications.



be fed with vegetable oils and would help

Used cooking oils and fat residues

considerably in the development of

from meat processing may be used as

agriculture of the countries which use it'.

raw materials.

In 1912, Diesel said, **"** The use of

vegetable oils for engine fuels may seem

There are certain disadvantages of

insignificant today. But such oils may

using biodiesel as a replacement for diesel

become in course of time as important as

fuel that must be also taken into

petroleum and the coal tar products of the

consideration:



present time" **.** No doubt, this statement

Slightly higher fuel consumption due

has come to stay. Since Dr. Diesel's

to the lower calorific value of

untimely death in 1913, his engine has

biodiesel.



been modified to run on the polluting

Slightly higher nitrous oxide (NOx)

petroleum fuel we now know as "diesel."

emissions than diesel fuel.

Nevertheless, his ideas on agriculture and

 Higher freezing point than diesel fuel.

his invention provided the foundation for

This may be inconvenient in cold

a society fueled with clean, renewable,

climates.

locally grown fuel. Today throughout the

 It is less stable than diesel fuel, and

world, countries are returning to using

therefore long-term storage (more

this form of fuel due to its renewable

than six months) of biodiesel is not

source

and

reduction

in

pollution

recommended.

(Owolabi _et al.,_ 2012). ****

 May degrade plastic and natural

****

rubber gaskets and hoses when used

4. **Oil crops in India**

in

pure form,

in

which case

replacement with Teflon components

The various oil sources are

is recommended.

classified as edible and non-edible. The

 It dissolves the deposits of sediments

edible sources like groundnut, peanut etc

and other contaminants from diesel

are primarily used to meet the food

fuel in storage tanks and fuel lines,

requirement. India is not using vegetable

which then are flushed away by the

oils derived from rapeseed & mustard,

biofuel into the engine, where they

soybean or oil palm for the production of

can cause problems in the valves and

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_Biotech Sustainability (2017)_

_Biodiesel Production for Sustainability Meena Devi et al._

biodiesel. It is because; India is not self-

biodiesel production (Agarwal, 2007).

sufficient in edible oils production and

The non-edible oil seed plant given in the

depends upon imports of palm oil and

above table has potential to produce oil

other vegetable oils in large quantities to

and subsequent conversion to biodiesel

meet the domestic demand. However,

apart from their uses for illumination,

utilization of non-edible seed oils

burning, soap making, candle making etc.

extracted from trees and forest sources

It is estimated that the potential

does not interfere with food security

availability of such oils in India is about 2

directly if the trees are grown on

million tons per year. The most abundant

marginal/waste land that does not

oil sources are Sal, Mahua, Neem,

compete with food production. Every year

Pongamia and Jatropha oil. Based on

around 1.2 million tonnes of tree borne

extensive

research,

Jatropha

and

non-edible seed oils are produced in the

Pongamia have been identified as the

country (Dwivedi _et al_., 2011). In India,

potential feed stocks for biodiesel

biodiesel is produced mostly from the

production in India.

non-edible oils extracted from the seeds

The future demand for biodiesel in

of plants like Jatropha, Pongamia, Mahua,

India is given in Table 2. The above table

Neem etc. Depending on climate and soil

indicates that by the year 2020–2021,

conditions, different nations are looking

about 24.61 MT of diesel could be saved

for different vegetable oils as substitute of

if B20 blend is utilized. This will ensure

diesel fuel for example soybean oil in

sustainable fuel availability with secured

USA, rapeseed and sunflower oils in

environmental conditions. As per the

Europe, palm oil in south East Asia and

report of the committee on biofuel, the

coconut oil in Philippines are being

estimated demand of diesel in 2011–2012

considered as substitutes for diesel.

was 64.19 MT, requiring 12.84MT of

Table 1 summaries the non-edible

biodiesel and plantation of _Jatropha_

oil producing plants that can be cultivated

_curcas_ over about 13.69 million hectare

for oil production on suitable land and

of land. As per Government of India

consequently the oil can be used for

survey, out of total land area, 124.7 mill-

**Table 1:** Production of non-edible oils in India

**Botanical**

**Local**

**Annual Productivity**

**No**

**Name**

**Name**

**(Tons)**

_1._

_Jatropha curcas_

Ratanjyot

45,000

_2._

_Pongamia pinnata_

Karanja

135,000

_3._

_Schleichera oleosa_

Kusum

25,000

_4._

_Azadirachta indica_

Neem

1,00,000

_5._

_Shorea robusta_

Sal

1,80,000

_6._

_Modhuca indica_

Mahua

1,80,000

**Table 2:** Projections of biodiesel demand and corresponding Jatropha area required for

meeting the blending targets in India (Area in Mha, Demand in Mt)

**For 5% blending**

**For 10 % blending**

**For 20 % blending**

**Diesel**

**Year**

**Biodiesel Jatropha Biodiesel Jatropha Biodiesel Jatropha**

**demand**

**demand**

**area**

**demand**

**area**

**demand**

**area**

2011-

64.19

3.21

3.42

6.42

6.85

12.84

13.69

12

2016-

92.15

4.61

4.91

9.21

9.83

18..43

19.66

17

2020-

123.06

6.15

6.56

12.31

___
___

13.13

24.61

26.25

21

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-ion hectare is classified as waste and

_curcas_ is the most known variety; it

degraded land (Dwivedi _et al.,_ 2014).

requires little water or additional care;

****

therefore, it is adequate for warm regions

_4.1. Typical oil crops useful for biodiesel_

with little fertility. Productivity may be

_production_

reduced by irregular rainfall or strong

The main characteristics of typical

winds during the flowering season. Yield

oil crops that have been found useful for

depends on climate, soil, rainfall and

biodiesel production are summarized in

treatment during sowing and harvesting.

the following paragraphs.

Jatropha plants become productive after 3

****

or 4 years, and their lifespan is about 50

_4.1.1. Castor seed_

years. Oil yield depends on the method of

The castor oil plant grows in

extraction; it is 28–32% using presses and

tropical climates, with temperatures in the

up to 52% by solvent extraction. Since the

range 20–30◦C; it cannot endure frost. It

seeds are toxic, jatropha oil is nonedible.

is important to note that once the seeds

The toxicity is due to the presence of

start germinating, the temperature must

curcasin (a globulin) and jatrophic acid

not fall below 12 ◦C. The plant needs a

(as toxic as ricin).

warm and humid period in its vegetative

****

phase and a dry season for ripening and

_4.1.4. Microalgae_

harvesting. It requires plenty of sunlight

Microalgae have great potential for

and adapts well to several varieties of

biodiesel production, since the oil yield

soils. The total rainfall during the growth

(in liters per hectare) could be one to two

cycle must be in the range 700–1,400

orders of magnitude higher than that of

mm; although it is resistant to drought,

other raw materials. Oil content is usually

the castor oil plant needs at least 5 months

from 20 to 50%, although in some species

of rain during the year. Castor oil is a

it can be higher than 70%. However, it is

triglyceride, ricinolenic acid being the

important to note that not all microalgae

main constituent (about 90%). The oil is

are adequate for biodiesel production.

non-edible and toxic owing to the

High levels of CO2, water, light, nutrients

presence of 1–5% of ricin, a toxic protein

and mineral salts are necessary for the

that can be removed by cold pressing and

growth

of

microalgae.

Production

filtering. The presence of hydroxyl groups

processes take place in raceway ponds

in its molecules makes it unusually polar

and photobiological reactors.

as compared to other vegetable oils.

****

5. **Biodiesel production techniques**

_4.1.2. Jojoba_

Although jojoba can survive

There are different processes which

extreme drought, it requires irrigation to

can be applied to synthesize biodiesel

achieve an economically viable yield.

such as direct use and blending, micro

Jojoba needs a warm climate, but a cold

emulsion

process,

thermal

cracking

spell is necessary for the flowers to

process and the most conventional way is

mature. Rainfall must be very low during

transesterification process (Gashaw _et al_.,

the harvest season (summer). The plant

2015).

reaches its full productivity 10 years after

****

planting. The oil from jojoba is mainly

_5.1._

_Direct use and blending_

used in the cosmetics industry; therefore,

The direct use of vegetable oils in

its market is quickly saturated.

diesel engine is not favorable and

****

problematic because it has many inherent

_4.1.3. Jatropha_

failings. Even though the vegetable oils

Jatropha is a shrub that adapts

have familiar properties as biodiesel fuel,

well to arid environments. _Jatropha_

it required some chemical modification

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before can be used into the engine. It has

proportions. The equipment for thermal

only been researched extensively for the

cracking and pyrolysis is expensive for

past couple of decades, but has been

modest biodiesel production particularly

experimented with for almost hundred

in developing countries. Furthermore, the

years. Although some diesel engine can

removal of oxygen during the thermal

run pure vegetable oils, turbocharged

processing

also

removes

any

direct injection engine such as trucks are

environmental benefits of using an

prone to many problems.

oxygenated fuel. Another disadvantage of

****

pyrolysis is the need for separate

_5.2._

_Microemulsion process_

distillation equipment for separation of

A micro emulsion is defined as the

the various fractions. Also the product

colloidal

equilibrium

dispersion

of

obtained is similar to gasoline containing

optically isotropic fluid microstructures

Sulphur which makes it less ecofriendly.

with dimensions generally in the range of

The pyrolyzed material can be vegetable

1–150 nm formed spontaneously from

oils, animal fats, natural fatty acids and

two normally immiscible liquids and one

methyl esters of fatty acids.

or more ionic or non-ionic. The problem

****

of the high viscosity of vegetable oils was

_5.4. Transesterification_

solved by micro-emulsions with solvents

Generally, biodiesel is produced

such as methanol, ethanol, and 1-butanol.

by

means

of

transesterification.

The components of a biodiesel micro-

Transesterification is the reaction of a

emulsion include diesel fuel, vegetable

lipid with an alcohol to form esters and a

oil, alcohol, surfactant and cetane

byproduct, glycerol. It is, in principle, the

improver

in

suitable

proportions.

action of one alcohol displacing another

Alcohols such as methanol and ethanol

from an ester, referred to as alcoholysis

are used as viscosity lowering additives,

(cleavage

by

an

alcohol).

In

higher alcohols are used as surfactants

Transesterification

mechanism,

the

and alkyl nitrates are used as cetane

carbonyl carbon of the starting ester

improvers. Microemulsions can improve

(RCOOR1) undergoes nucleophilic attack

spray

properties

by

explosive

by the incoming alkoxide (R O−) to give

2

vaporization

of

the

low

boiling

a tetrahedral intermediate, which either

constituents in the micelles. Micro-

reverts to the starting material, or

emulsion results in reduction in viscosity

proceeds to the transesterified product

increase in cetane number and good spray

(RCOOR2). Transesterification consists of

characters in the biodiesel. However,

a sequence of three consecutive reversible

continuous use of microemulsified diesel

reactions. The first step is the conversion

in engines causes problems like injector

of triglycerides to diglycerides, followed

needle sticking, carbon deposit formation

by the conversion of diglycerides to

and incomplete combustion. ****

monoglycerides,

and

finally

****

monoglycerides into glycerol, yielding

_5.3. Thermal cracking (pyrolysis)_

one ester molecule from each glyceride at

Pyrolysis is defined as the

each step. The reaction is represented in

conversion of one substance into another

equation 1.

by means of heat or heating with the aid

of a catalyst. Pyrolysis involves heating in

absence of air or oxygen and cleavage of

chemical bonds to yield small molecules.

The pyrolysis of vegetable oil to produce

biofuels has been studied and found to

produce alkanes, alkenes, alkadienes,

aromatics and carboxylic acids in various

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There

are

different

acid catalyzed transesterification process,

transesterification processes that can be

which converts the FFA to esters (Leung

applied to synthesize biodiesel: (a) base-

and Guo, 2006). ****

catalyzed transesterification, (b) acid-

****

catalyzed transesterification, (c) enzyme-

_5.4.3. Enzyme catalysts_

catalyzed transesterification, and (d)

Lipase enzymes can also catalyze

supercritical alcohol transesterification.

methanolysis of triglycerides. The most

****

promising results were obtained by using

_5.4.1. Catalysts: acid catalyst_

immobilized Candida Antarctica lipase

****

The use of an acid catalyst is

(Novozym 435). Shimada _et al.,_ (1999),

observed to be more effective than alkali

found that Novozym435 was inactivated

catalysts when the concentration of free

by shaking it in a mixture containing

fatty acids is high. Also the performance

more than 1.5 M eq. of methanol to oil.

of the acid catalyst is not strongly

Above this concentration, methanol is

affected by the presence of FFAs in the

partially present as small droplets in the

feedstock. In fact, acid catalysts can

oil phase. These droplets are believed to

simultaneously

catalyze

both

cause enzyme deactivation. Therefore,

esterification

and

transesterification.

methanol was added stepwise; after the

Thus, a great advantage with acid

addition of the third methanol equivalent,

catalysts is that they can directly produce

conversion to methyl esters was almost

biodiesel from low cost lipid feedstocks,

complete. The enzyme could be reused 50

generally associated with high FFA

times without loss of activity. The

concentrations (low-cost feedstocks, such

occurrence of free fatty acids did not

as used cooking oil and greases,

affect the enzyme catalyst. Before the

commonly have FFAs levels of >6%)3.

inlet of every reactor,1 M eq. was added

However, Homogeneous acid catalyzed

to the feed. Samukawa _et al._ (2000)

reaction is about 4000 times slower than

reported a dramatic increase of the lipase

the homogeneous base-catalyzed reaction.

efficiency when it was pretreated by a

Acids used in the catalysis of the

consecutive incubation in methyl ester

transesterification

of

biodiesels

are

and oil prior to reaction. The use of

usually either hydrochloric acid or

Novozym435

in

methanolysis

of

sulfuric acid. Though these two acids are

triglycerides

is

also

reported

in

the most common, any Bronsted acid can

supercritical carbon dioxide at 24.1 MPa

also be used in this reaction.

and 50 ◦C. High yields (90–95%) of fatty

acid methyl esters could be obtained

_5.4.2. Base catalyst_

when the reaction was carried out at

Transesterification reaction can be

molar methanol/oil ratios of 25:1. ****

catalyzed by both homogeneous (alkalies

****

and acids) and heterogeneous catalysts.

_5.4.4. Supercritical transesterification_

The used alkali catalysts are NaOH,

Saka and Kusdiana (2001) have

CH3ONa, and KOH for producing

developed a catalyst free method for

biodiesel (Wang _et al_., 2007). The alkali

biodiesel fuel production by employing

catalyzed transesterification of vegetable

supercritical methanol. The supercritical

oils proceeds faster than the acid

treatment at 350 ◦C, 43 MPa, and 240 s

catalyzed. But the use of base catalyzed

with a molar ratio of 42:1 in methanol is

transesterification is only limited to oil

the

optimum

condition

for

having low water and FFA content. This

transesterification of rapeseed oil to

reaction is the most widely used process

biodiesel fuel. The great advantage of this

for production of biodiesel worldwide. To

method was that free fatty acids present in

keep check on the water and FFA content

the oil could be simultaneously esterified

of the oil, they are first pretreated with an

in the supercritical solvent. Variables such

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as the molar ratio of alcohol to vegetable

found that reaction mixture containing

oil and reaction temperature were

65ml of methanol along with 2.4 g of

investigated during the transesterification

catalyst (KOH) took a good start in half

within this supercritical media. Increasing

an hour at30°C. In this reaction, amount

the reaction temperature within the

of glycerine removed as well as ester

supercritical regime resulted in increased

content

produced

was

considerably

ester conversion.

increased with rise in temperature of

****

mixture up to 70°C by extending time

**6. Previous work done on production of**

period (180-360 minutes). The removal of

**biodiesel from edible oil**

glycerine increased by two and half times

and ester content by four times,

Leung and Guo (2006) compared

respectively. When castor oil was

the transesterification reaction conditions

subjected to acid esterification, prior to

for fresh canola oil and used frying oil.

transesterification

(a

separate

Higher molar ratio (7:1, methanol/used

investigation), it was found that ester

frying oil), higher temperature (60° C)

contents up to 95% could be obtained.

and higher amount of catalyst (1.1 wt%

Hasan _et al_. (2013) produced biodiesel

NaOH) was maintained in used frying oil

from neem seeds, its properties were

when compared to fresh canola oil where

close to diesel. The methodology of

optimal conditions maintained were 315-

esterification process was selected and

318 K, 1.0 wt% NaOH and 6:1

carried out by 1000 ml raw neem oil,

methanol/oil molar ratio. However, less

300ml methanol and sodium hydroxide

reaction time (20 min) was observed for

on mass basis as a catalyst usually kept in

used frying oil when compared to fresh

oven to form methyl ester, and initially to

canola oil reaction time (60 min). Ying _et_

reach

equilibrium

condition

at

_al_. (2011), developed a new method

temperature 55-66°C. The ester and

catalyst,

benzyl

bromide-modified

glycerine were separated by stimulating

calcium oxide (CaO) for production of

continuously and allow settling under

biodiesel from rapeseed. The improved

gravity for 24 h. Thus the separated ester

catalytic activity was obtained by better

contains 3% to 6% methanol and soap

fat diffusion to the surface of the benzyl

agents. The methanol was removed by

bromide-modified CaO. Further, a 99.2%

vaporization. The biodiesel had some

yield of fatty acid methyl esters in 3h was

catalyst; it was removed by warm water

obtained in comparison to by better fat

mix with ester. Kinematic viscosity lay

diffusion to the surface of the benzyl

between 1.9 and 6.0 according to the

bromide-modified CaO. Wakil _et al._

ASTM D6751 specification. It was

(2012), chosen Cottonseed oil, Mosna oil

reported that, 0.95 L biodiesel was

and Sesame oil for producing biodiesel. ****

produced from 1 L neem oil. ****

****

****

**7. Previous work done on production of**

**8. Factors affecting biodiesel**

**biodiesel from non-edible oil**

**production**

****

Mohibbe _et al. (_ 2005), found that

The yield of biodiesel in the

FAME of _Jatropha curcas_ were most

process of transesterification is affected

suitable for use as bio- diesel and met the

by several process parameters which

major

specification

of

bio-diesel

include;

reaction

time,

reaction

standards of the European, Germany and

temperature, catalyst and molar ratio of

USA

Standards

Organization.

alcohol and oil and mixing intensity

Chakrabarti and Ahmad (2008) presented

(Gashaw _et al_., 2015).

work on extraction of oil from castor bean

****

and converting it into biodiesel. It was

_8.1. Temperature_

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Reaction

temperature

is

the

of alcohol and 1 mole of triglyceride are

important factor that will affect the yield

required for transesterification to yield 3

of biodiesel. For example, higher reaction

moles of fatty acid methyl/ethyl esters

temperature increases the reaction rate

and 1 mole of glycerol is used. In order to

and shortened the reaction time due to the

shift the reaction to the right, it is

reduction in viscosity of oils. However,

necessary to either use excess alcohol or

the increase in reaction temperature

remove one of the products from the

beyond the optimal level leads to decrease

reaction mixture. The second option is

of biodiesel yield, because higher reaction

usually preferred for the reaction to

temperature accelerates the saponification

proceed to completion. The reaction rate

of triglycerides and causes methanol to

is found to be highest when excess

vaporize resulting in decreased yield.

methanol is used (Gashaw and Lakachew,

Usually, the transesterification reaction

2014). Methanol, ethanol, propanol,

temperature should be below the boiling

butanol and amyl alcohol can be used in

point of alcohol in order to prevent the

the transesterification reaction, amongst

alcohol evaporation. The range of optimal

these alcohols methanol is applied more

reaction temperature may vary from 50°c

frequently as its cost is low and it is

to 60°c depends upon the oils or fats used.

physically and chemically advantageous

Therefore, the reaction temperature near

(polar and shortest chain alcohol) over the

the boiling point of the alcohol is

other alcohols. In contrast, ethanol is also

recommended for faster conversion by

preferred alcohol for using in the

various literatures. At room temperature,

transesterification process compared to

there is up to 78% conversion after 60

methanol since it is derived from

minutes, and this indicated that the

agricultural products and is renewable

methyl esterification of the FFAs could be

and biologically less offensive in the

carried

out

appreciably

at

room

environment. ****

temperature but might require a longer

****

reaction time.

_8.4. Type and amount of catalyst_

****

Biodiesel

formation

is

also

_8.2. Reaction time_

affected by the concentration of catalyst.

The increase in fatty acid esters

Most commonly used catalyst for

conversion observed when there is an

biodiesel production is sodium hydroxide

increase in reaction time. The reaction is

(NaOH) or Potassium hydroxide (KOH).

slow at the beginning due to mixing and

The type and amount of catalyst required

dispersion of alcohol and oil. After that

in the transesterification process usually

the reaction proceeds very fast. However

depend on the quality of the feedstock

the maximum ester conversion was

and

method

applied

for

the

achieved within < 90 min. Further

transesterification process. For a purified

increase in reaction time does not increase

feedstock, any type of catalyst could be

the yield product i.e. biodiesel/mono alkyl

used for the transesterification process.

ester. Besides, longer reaction time leads

However, for feedstock with high

to the reduction of end product (biodiesel)

moisture and free fatty acids contents,

due to the reversible reaction of

homogenous transesterification process is

transesterification resulting in loss of

unsuitable due to high possibility of

esters as well as soap formation.

saponification

process

instead

of

****

transesterification process to occur.

_8.3. Methanol to oil molar ratio_

****

One of the most important

_8.5. Mixing intensity_

parameters affecting the yield of biodiesel

Oils and alcohols are not totally

is the molar ratio of alcohol to

miscible, thus reaction can only occur in

triglyceride. Stoichiometrically, 3 moles

the interfacial region between the liquids

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and transesterification reaction is a

times include esterification of free fatty

moderately slow process. So, Mixing is

acids in sunflower oil and oleic acid

very important in the transesterification

(Berrios _et al_., 2007 and Kraai _et al_.,

process, adequate mixing between these

2008). Transesterification kinetics of

two types of feedstock is necessary to

soybean oil with five different catalysts

promote contact between these two feed

has also been studied (Singh and

stocks,

therefore

enhance

the

Fernando, 2007). ****

transesterification reactions to occur

****

(Jagadale and Jugulkar, 2012).

**10. Fuel properties of biodiesel**

****

**9. Thermo-kinetics of transesterificati-**

The fuel properties of biodiesel

**on**

are discussed below (Owolabi _et al._ ,

2012; Gopal and Karupparaj, 2015). The

The feasibility of a reaction is

limits of ASTM D 6751standard are listed

determined from the thermodynamic

in Table 3.

parameters. Since both reactants and

products are liquids, entropy change will

_10.1. Specific gravity and density_

tend to zero, hence equilibrium constant

Density is the mass of unit volume

will be low (Owolabi _et al_., 2012).

of a material at a specific temperature. A

Kinetics of transesterification reaction has

more useful unit used by the petroleum

at least 3 main reactions as shown in the

industry is specific gravity, which is the

equations stated below (Noureddini and

ratio of the weight of a given volume of a

Zhu, 1997).

material to the weight of the same volume

of

water

measured

at

the

same

temperature. Specific gravity is used to

calculate the mass of oils. Density

influences the efficiency of the fuel

atomization

for

airless

combustion

Activation energy for the reverse

system. It has some effect on the break-up

reaction is higher than that for the

of fuel injected into the cylinder. In

forward reaction, which again should

addition, more fuel is injected by mass as

confirm the low possibility of reverse

the fuel density increases.

reactions. Some of the few kinetics

studies that have been performed in recent

****

**Table 3:** ASTM D 6751 standard for biodiesel ****

**Parameters**

**Units**

**ASTM D 6751**

**ASTM D 6751**

**Limits**

**Methods**

Acid number

mg

0.50 max

D 664

KOH/g

FFA

%

\-

\-

Specific gravity

g/cm3

0.87 - 0.90

D 1250 - 08

Kinematic viscosity at

cSt

1.9 - 6

D 445

40ºC

Peroxide value

meq/kg

\-

\-

Calorific value

MJ/kg

\-

D 240 - 02

Sulphated ash

%

0.02

D 874

Water & Sediments

%

0.05

D 2709

Copper corrosion

\-

No. 3 max

D 130

Carbon residue

%

0.05

D 4530

Flash point

ºC

130 min

D 93

****

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_10.2. Lower calorific value_

****

It is a measure of the energy

_10.7. Pour point, cloud point_

produced when the fuel is burnt

The pour point of a crude oil or

completely which also determines the

product is the lowest temperature at

suitability of methyl ester as an

which oil is observed to flow under the

alternative to diesel fuel. The calorific

conditions of the test. Handling and

value of methyl ester is normally lower

transporting oils and heavy fuels is

than diesel due to oxygen content of

difficult at temperatures below their pour

methyl ester.

points .Often, chemical additives known

****

as pour point depressants are used to

_10.3. Iodine value_

improve the flow properties of the fuel.

Iodine value or iodine number is

The temperature at which wax crystals

defined as the number of grams of iodine

begin to form on the surface of the

taken up by 100 g of oil or fat. In this

biodiesel is the cloud point.

case, addition reaction takes place across

****

the double bonds of unsaturated fatty

_10.8. Kinematic viscosity_

acids present in the fat by the addition of

This is the resistance to flow of

a halogen, such as iodine. Thus, the

oil. Ease of starting depends on viscosity.

iodine number gives the indication of the

Glycerin

contamination

may

cause

degree of unsaturation of fats. ****

biodiesel viscosity to increase. It is the

****

most important fuel features and this

_10.4. Molecular weight_

factor affects the operation of fuel

The average molecular weight of

injection,

blending

formation

and

the methyl ester is calculated by

combustion process. The high viscosity

considering the weight percent of each

interferes with the injection process and

fatty acid and their corresponding

leads to insufficient atomization.

molecular weights.

****

****

**11. Concluding remarks**

_10.5. Cetane number_

It is the measure of the ignition

Biodiesel, of the family of biofuel,

quality of diesel fuel; higher this number

has been described in this review as a fuel

the easier it is to start a standard (direct-

with necessary potentials to replace fossil

injection) diesel engine. It denotes the

diesel in future. The trials biodiesel and

percentage (by volume) of cetane

its

blend

have

undergone

is

a

(chemical name Hexadecane) in a

confirmatory test to all advantages

combustible mixture (containing cetane

including environmental benefits accrued

and 1-Methylnapthalene) whose ignition

to it thereby plays a vital role in meeting

characteristics match those of the diesel

future fuel requirements. The availability

fuel being tested.

of major feedstock namely oil from

****

biosources

and

simplicity

of

the

_10.6. Flash point_

transesterification technology that ensures

It is the minimum temperature of

its conversion to biodiesel are added

the fuel at which the fuel gives flash when

advantage in terms of the future needs of

it comes to contact with testing flame. It

biodiesel. The use of inedible oil and

is an important parameter from the safety

waste frying/cooking oil has equally

point of view such as safe for transport,

assisted in establishing a balance between

handling, storage purpose and safety of

energy and food security. However,

any fuels. This is higher than petrol diesel

serious efforts have to be intensified on

which has flash point of 70°C. A fuel with

design of large scale bio-refineries for

high flash point may cause carbon

future biodiesel production.

deposits in the combustion chamber.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 271

_Biotech Sustainability (2017)_

_Biodiesel Production for Sustainability Meena Devi et al._

**References**

**(2012)**. Review of Various Reaction

Parameters

and

Other

Factors

**Agarwal, A.K. (2007)**. Biofuels (alcohols

Affecting on Production of Chicken

and biodiesel) applications as fuels

Fat Based Biodiesel. _Int. J. Mod._

for internal combustion engines,

_Eng. Res.,_ **2(2), 407–411.**

_Progress in Energy and Combustion_

**Knothe,G.(2006)**. Analyzing biodiesel:

_Science_ **33, 233–271.**

Standards

and

Other

Methods,

**Avhad, M.R. and Marchetti, J.M.**

_JAOCS.,_ **83, 823 – 833**.

**(2016)**.

Innovation

in

solid

**Kraai, G.N. Winkelman, J.G.M. De**

heterogeneous

catalysis

for the

**Vries, J.G. and Heeres, H.J (2008).**

generation of economically viable

Kinetic Studies on the Rhizomucor

and ecofriendly biodiesel: A review,

Miehei

Lipase

Catalyzed

_Catalysis Reviews_ **58, 2: 157-208**.

Esterification Reaction of Oleic Acid

**Berrios, M. Siles, J. Martin, M.A and**

with 1-Butanol in Biphasic System.

**Martin A.A (2007)**. Kinetic Study of

_Biochem Engg_. _J_. **41, 87–94.** ****

the Esterification of Free Fatty Acids

**Kusdiana, D. and Saka, S (2001)**.

(FFA) in Sunflower Oil. _Fuel_ **86,**

Biodiesel fuel from rapeseed oil as

**2383-2388**. ****

prepared in supercritical methanol. __

**Chakrabarti, M. and Rafiq Ahmad**

_Fuel_ **80, 225-231**. ****

**(2008)**. Transesterification studies on

**Leung,**

**Y.**

**Guo**

**(2006)**.

castor oil as a first step towards its

Transesterification of neat and used

use in bio diesel production. _Pakistan_

frying oil: Optimization for biodiesel

_Journal of Botany_ **40, 1153-1157**. ****

production.

_Fuel_

_Processing_

**Dwivedi, G. Sharma, M.P. and Jain, S.**

_Technology_ **87, 883-890**. ****

**(2011)**. Impact analysis of biodiesel

**Mohibbe, A.M. Waris, A. and Nahar,**

on engine performance- A Review.

**M. N (2005)**. Prospects and Potential

_Renewable and Sustainable Energy_

of Fatty Acid Methyl Esters of Some

_Review_ **15, 4633-4641.** ****

Non-Traditional Seed Oils for Use as

**Dwivedi, G. Sharma, M.P. Kumar, M**

Biodiesel in India. _J. Biomass and_

**(2014)**.

Status

and

Policy

of

_Bioenergy_ , **29, 293-302**. ****

Biodiesel Development in India.

**Narasimharao, K. Lee, A. and Wilson,**

_International Journal of Renewable_

**K. (2007).** Catalysts in Production of

_Energy Research_ **4 (2), 246-254.**

Biodiesel: A Review, _Journal of_

**Gashaw, A. and Lakachew, A (2014)**.

_Biobased Materials and Bioenergy_ **1,**

Production of biodiesel from non-

**1–12**. ****

edible

oil

and

its

Properties.

**Noureddini, H and Zhu, D (1997).**

_International Journal of Science,_

Kinetics of transesterification of

_Environment and Technology_ , **3(4),**

soybean oil. _J Am Oil Chem Soc_

**1544 – 1562.**

**74(11),1457–1463**. ****

**Gopal, K. and Karupparaj, R (2015).**

**Owolabi,**

**R.U. Adejumo, A.L.and**

Effect of pongamia biodiesel on

**Aderibigbe, A.F (2012)**. Biodiesel:

emission

and

combustion

Fuel for the Future (A Brief Review).

characteristics of DI compression

_International Journal of Energy_

ignition

engine,

_Ain_

_Shams_

_Engineering_ , **2(5), 223-231.**

_Engineering Journal_ **6, 297-305**.

**Samukawa,**

**T.**

**Kaieda,**

**M.**

**Hasan, Ali Md. Mashud, M. Rubel,**

**Matsumoto,T. Ban, K. Konda, A.**

**Md. R. and Ahmad R.H. (2013)**.

**Shimada,**

**Y.**

**Noda,**

**H.**

**and**

Biodiesel from Neem oil as an

**Fududa, H (2000).** Pretreatment of

alternative fuel for Diesel engine.

immobilized

Candida

antarctica

_Procedia Engineering_ **56, 625- 630.** ****

lipase for biodiesel fuel production

**Jagadale,S.S.**

**and**

**Jugulkar,**

**L.M**

from plant oil, **** _Journal of Bioscience_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 272

_Biotech Sustainability (2017)_

_Biodiesel Production for Sustainability Meena Devi et al._

_and Bioengineering_ **90 (2), 180–183.**

**Rahman, Md. Hasibur, and Md.**

**Shimada, Y. Watanabe, Y. Samukawa,**

**Arifuzzaman (2012)**. Study on fuel

**T. Sugihara, A. Noda, H. Fukuda,**

properties of various vegetable oil

**H. and Tominaga, Y (1999)**.

available

in

Bangladesh

and

Conversion of vegetable oil to

biodiesel production. _International_

biodiesel using immobilized Candida

_Journal of Mechanical Engineering_

antarctica lipase. _J Am Oil Chem Soc_

**2(5),10-17**. ****

**76(7),789–793.**

**Wang, Y. Ou, S. Liu, P. and Zhang, Z**

**Singh, A. K. and Fernando, S.D (2007)**.

**(2007)**. Preparation of biodiesel from

Reaction Kinetics of Soybean Oil

waste cooking oil via two-step

Transesterification

using

catalyzed

process.

_Energy_

Heterogenous Metal Oxide Catalysts.

_Conversion_

_and_

_Management_

_Chem. Eng. Technol._ **30, 1716– 1720**. ****

**48,184–188**. ****

**Verma, D. Raj, J. Pal, A. and Jain M.**

**Ying, T. Gang, C. Zhang, J. and Lu, Y**

**(2016)**.

A

critical

review

on

**(2011)**. Highly active CaO for the

production of biodiesel from various

transesterification

to

biodiesel

feedstocks, _Journal of Scientific and_

production

from

rapeseed

oil.

_Innovative Research_ **5, (2): 51-58.**

_Bulletin of the Chemical Society of_

**Wakil, Md. Abdul, Ahmed, Z.U.**

_Ethiopia_ **25(1), 37-42.**

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 273

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P274--286_

_**In vitro**_ **Cell Bioassays in Pollution Assessment**

**Narayanan Kannan1, *, Poorani Krishnan2 and Ahmad Zaharin Aris3**

__

_1Postgraduate Research and Innovation and Strategic Development, Taylor's Uni-_

_versity (Lakeside Campus), No. 1, Jalan Taylor's, 47500, Subang Jaya, Selangor_

_Darul Ehsan, Malaysia; 2Department of Medical Microbiology and parasitology,_

_Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Ser-_

_dang, Selangor, Malaysia; 3Faculty of Environmental studies, Department of Envi-_

_ronmental Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malay-_

_sia;*Correspondence: Kannan.Narayanan@taylors.edu.my; Tel: +603 5629 5463_

**Abstract:** Most fast developing countries in Asia face severe pollution problems due to in-

dustrialization and modernization. Analytical chemical measurements are common in pollu-

tion monitoring. However, the ecological (biological) effects of those chemicals are diffi-

cult to understand. The latest generation of bioanalytical tools, such as _in vitro_ transactiva-

tion bioassays, show great promise as water and sediment quality monitoring tools. A bat-

tery of _in vitro_ cell bioassays has been developed in recent years that are effective, specific,

fast, less expensive and easy to handle. One example is, Aryl hydrocarbon Receptor (AhR)

mediated CYP1A1 protein expression that has been utilized effectively as a biomarker of

pollution stress to marine biota. Hence, AhR binding potential, subsequent induction, geno-

toxic expression and cytotoxic potential of pollutants are reviewed. The list of _in vitro_ cell

bioassays reviewed will hopefully be utilized in ecotoxicological studies in developing

countries in Asia.

****

_**Keywords**_ **:** Developing countries; ecotoxicology; _in vitro_ cell bioassays; monitoring; policy;

pollution ****

****

****

**1. Introduction**

they are not very fast, at times, cumber-

some and usually expensive, as new

There is a growing concern over persis-

chemicals keep entering the aquat-

tent organic pollutants (POPs) that are

ic/terrestrial environment. Moreover, this

ubiquitous, persistent and toxic (Kannan

approach does not address unknown

_et al_. 1988, 1989 a.b,c, 1997, 2016; Kaw

chemicals such as photolytic/biological

and Kannan 2016). Hence, monitoring

transformation products or complex

these chemicals in the environment is im-

chemical mixtures that sentinel species of

portant. Conventional monitoring of tar-

interest are exposed to.

geted chemicals relies on analytical

Thus, chemical measurement of priori-

methods for measurements in the matrix

ty pollutants alone is not sufficient, as

of interest (i.e. water or sediment). Moni-

their biological impacts need to be as-

toring can also be based on surveys on the

sessed. Toxicity end-points such as sur-

health of individuals and populations of

vival and mortality of an organism are

sentinel species (e.g. indigenous fish); or

very useful indicators of biological im-

at a larger scale, the integrity of ecosys-

pacts. However, most of the contaminants

tem health by measuring community

occur at low concentrations for any possi-

composition, diversity and or function.

ble toxicological investigation. High

Though these approaches are effective,

throughput cell bioassay techniques have

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 274

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

recently been shown to effectively screen

tants that exist in environmental extracts

environmental contaminants based on

is made possible. Moreover, in compari-

their biological mode of action. In partic-

son to the limited identification of pollu-

ular, a number of _in vitro_ cell bioassays

tants provided through instrumental anal-

have been adopted to measure the inte-

ysis, _in vitro_ techniques provide an as-

grated response of bioactive contami-

sessment of the total biological impact

nants, such as estrogens, in recycled and

exerted by complex mixtures of environ-

surface waters as well as in wastewater

mental contaminants that are mediated

effluents (van der Linden _et al_. 2008;

through a common mechanism of action.

Leusch _et al_. 2010; Escher _et al_. 2014;

Various bioassays have been developed

Mehinto _et al_. 2015). These emerging bi-

over the years to evaluate the toxic poten-

oanalytical tools can indicate which clas-

cy of environmental extracts with refer-

ses of chemicals are of concern, thus nar-

ence to a specific target receptor. Bioas-

rowing the field of targeted chemical

says are reliable tools to measure the re-

analysis needed and toxicity endpoints to

sponse of a cell in terms of protein ex-

evaluate (Brack _et al_. 2015; Maruya _et al_.

pression and enzyme activity when bio-

2016). In general, _in vitro_ bioassays are

logical organisms are exposed to envi-

good markers of biological toxicity as

ronmental pollutants. These responses are

they are sensitive, quick, less expensive

reportedly triggered by specific genes that

(Mehinto _et al_. 1993; Qiao _et al_., 2006).

mediate the transcription of the particular

The advantage of effect-based bioassay

target protein. Cell lines developed from

measurements is that they determine the

mammals and fish utilizing CYP1A1 in-

integrated toxic potency of the complex

duction as a biomarker of exposure to

mixture of micro contaminants in the en-

Poly aromatic hydrocarbons (PAHs), Pol-

vironment. In addition, bioassays may

ychlorinated biphenyls (PCBs) and other

detect mixture effects of compounds,

Halogenated

aromatic

hydrocarbons

even when the individual constituents of

(HAHs) are effective _in vitro_ tools for

the mixture are present at concentrations

cumulative impact of sediment extracts

too low to cause an effect or to be detect-

(Jung _et al_., 2012; Schnell _et al_. 2013).

ed by chemical analysis (Hamers _et al_.,

Thus, various cell lines developed from

2010). The impact of unmonitored con-

mammals (He _et al_., 2011; Willet _et al_.,

taminants (contaminants of emerging

1997 a, b) and fish (Fernandes _et al_.,

concern) remains largely uncharacterized.

2014; Schnell _et al_. 2013; Fent, 2001) in-

Considering these points, one can safely

ducing CYP1A monooxygenases enzyme

conclude that _in-vitro_ bioassays are com-

belonging to the cytochrome P450 family

prehensive and holistic and can act as ear-

have been utilized. ****

ly warning signals of environmental deg-

****

radation.

**3. Aryl hydrocarbon Receptor (AhR)**

****

**mediated toxic response**

**2. Significance of cell _in vitro_** **bioassays**

****

The most potent inducer of Aryl hydro-

_In vitro_ cell bioassays utilizing either

carbon Receptor (AhR) is 2,3,7,8-

wild type cells or genetically engineered

tetrachlorodibenzo-p-dioxin

(TCDD).

eukaryotic cells providing an assessment

Thus chemicals that elicit response simi-

on the potency of contaminants extracted

lar to TCDD are generally clustered as

from environmental matrices. Applica-

dioxin-like chemicals. The ability of these

tions of _in vitro_ cell bioassays are highly

chemicals to cause hepatotoxicity, em-

effective in terms of cost and time. A no-

bryotoxicity, teratogenicity, immunotoxi-

table advantage is that through _in vitro_

city, dermal toxicity, lethality, carcino-

bioassays the detection and assessment of

genesis, and tumor promotion in many

toxicity of a complex mixture of pollu-

different species at low concentrations

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_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

have generated much concern (Giesy _et_

AhR signaling pathway through the ex-

_al_ ; 2002; Ahlborg _et al_., 1992; Peterson _et_

pression of the protein CYP1A and its

_al_., 1993). These chemicals are largely

enzymatic activity has been developed

mediated through AhR dependent mecha-

and constantly improved with modifica-

nism of action in biological system (Po-

tions to evaluate and applied in environ-

land and Knutson., 1982; Willet _et al_.,

mental assessment.

1997, Yoo _et al_ , 2006). Several reviews

****

have been published on the potency of

**4. AhR active compounds**

dioxin-like environmental xenobiotics

****

(Gillesby and Zacharewski, 1998; Ankley

AhR ligands are made by hydropho-

_et al_., 1998; van den Berg _et al_., 1998;

bic compounds such as polychlorinated

Giesy _et al_ , 2002).

dibenzo-p-dioxins

and

dibenzofurans

AhR is described as a ligand dependent

(PCDDs and PCDFs), chlorinated azo-

transcription factor located in the cytosol,

benzenes and azoxybenzenes, polychlo-

chaperoned with heat shock proteins

rinated biphenyls (PCBs), several polycy-

(Giesy _et al_., 2002). Increased toxicity,

clic aromatic hydrocarbons (PAHs), poly-

enhanced gene transcription and enzyme

chlorinated naphthalenes (Giesy _et al._ ,

activity are indicators of the binding

2002; Blankenship _et al_., 2000; Jung et

strength of congeners to the AhR (Giesy

al., 2012) and various halogenated aro-

_et al_., 2002; Safe, 1995). The AhR medi-

matic hydrocarbon (HAHs) (Willet _et al_.,

ated mechanism is initiated once the lig-

1997). Relatively weak AhR ligand has

ands bind to cytosolic AhR. The receptor

been identified for natural and synthetic

ligand complex is then translocated into

compounds (Giesy _et al_., 1998; Denison

the nucleus leading to the dissociation of

and Heath-Pagliuso, 1998).

heat shock proteins followed by for-

mation of a dimer by binding to Ah Re-

**5. Dioxin responsive (DR) CALUX bio-**

ceptor Nuclear Traslocator (ARNT) pro-

**assay**

tein. The heteromeric ligand AhR:ARNT

complex then binds to dioxin-responsive

The CALUX (chemically activated

element (DRE) a specific DNA sequenc-

luciferase expression) is a reporter-gene

es. This binding in turn will stimulate

based cell bioassay that is increasingly

transcriptional activation of adjacent re-

applied in screening of dioxin and dioxin

sponsive genes leading to production of

like chemicals in environmental matrices

specific protein such as CYP1A1 (Giesy

and in food materials (Tsutsumi _et al_.,

_et al_., 2002; Denison and Heath-Pagliuso,

2003; Cederberg _et al_., 2002). To perform

1998; Hankinson, 1995; Celander _et al_.,

this assay, recombinant mammalian cell

1996). CYP1A1, belonging to the super-

lines that have been stably transfected

family of cytochrome P450 plays a signif-

with one of two different AhR-responsive

icant role in the biotransformation of xe-

luciferase reporter gene plasmids that

nobiotic such as PAHs and PCBs in or-

responds to dioxin and related chemicals

ganisms. Induction of CYP1A has been

are utilized (Denison _et al_., 2004;

the most useful biomarker for environ-

Garrison _et al_., 1996). Briefly, these cells

mental contamination and had been ap-

contains AhR, that when bound to

plied in various pollution monitoring pro-

activating ligands will innitiate the

grams (Bucheli and Fent, 1995; Celander

transcription of the luciferase genes and

_et al_., 1996).

the induction of the luciferase activity that

Biotransformation is a complex process

will

be

determined

by

measuring

of excretion of hydrophobic substrate by

luminescence. Induction of luciferase

converting them to hydrophilic metabolite

activity is directly proportional to the

through monooxygenation (Czeka, 2000).

amount and potency of inducing chemical

Bioassays that measure the activation of

to which the cells are exposed (Han _et al_.,

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_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

2004; Denison _et al_., 2004). However, the

provides a highly responsive and sensitive

results obtained will vary significantly

bioassay system for the detection and

depending on the type of cell lines used.

relative quantitation of very low levels of

This is due to the species and tissues

dioxin-like chemicals in sample extracts

specific differences and functionality of

(He _et al_., 2011). This bioassay is an

the AhR as well as the antagonistic and

essential assay to characterize AhR active

synergistic effects of some compounds

compounds in environmental matrices as

that are cell line dependent.

it accounts for the total AhR mediated

The analysis of the luminescence

activities in the test samples including

measured through this assay is converted

non-dioxin-like chemicals such as PAHs

to CALUX – TEQ toxic equivalents. The

and HAHs. Several advantages of

luminescence value given by a sample is

CALUX assay are discussed by Windal

compared to the dose response curve of a

_et al_. (2005) such as the reliability of

reference standard such as 2,3,7,8-

CALUX assay to analyzes the overall

tetrachlorodibenzo-p-dioxin. Briefly, the

biological activity of all AhR ligands

sample

concentration

that

induce

(agonists and antagonists) present in an

luciferase to 25% of the TCDD-induced

extract, in comparison to chemical

maximum luciferase activity is designated

analyses that only focuses on a selected

as the EC25 TCDD for that sample. The

number of compounds. Besides, for

TEQ concentrations were calculated as;

enviromental screening CALUX and

chemical analysis are complimentary

reflecting the mangitude of dioxin like

activity induced by other compounds that

were undetected in chemical analaysis;

particularly, when large numbers of envi-

ronmental samples are to be screened.

H4IIE-luc cells are among the most

CALUX ensures a rapid and cost effec-

common cells utilised in luciferse based

tive analysis for dioxin-like chemicals.

assay to characterize the induction

****

potency of AhR active compounds such

**6. EROD Assay**

as halogenated aromatic hydrocarbon

(HAH)

mixtures

containing

Induction

of

7-ethoxyresorufin- _O_ -

polychlorinated

biphenyls

(PCBs),

deethylase (EROD) activity has been ap-

dibenzo-p-dioxins

(PCDDs)

in

plied in numerous environmental toxicol-

environmental matrices (Yoo _et al_., 2006;

ogy studies as to indicate pollution stress

Giesy _et al_., 2002; Whyte _et al_., 2004;

(Fernandes _et al_., 2014; Kim _et al_., 2013;

Willet _et al_., 1997 a,b; Schmitz _et al_.

Schnell _et al_., 2013; Huuskonen _et al_.,

1995). H4IIE-luc cells have been studied

2000). EROD activity is based on the

and

suggested

as

an

alternative

deethylation of the synthetic model sub-

bioanalytical tool to the wild-type cells

strate 7-ethoxyresorufin by CYP1A1, an

for the detection of AhR agonists in

enzyme that belong to the phase-1 group

environmental samples (Sanderson e _t al_.,

of biotransformation enzymes. This reac-

1996).

tion will produce a fluorescent product

Progressive studies in this particular

resorufin that is quantified fluorometrical-

assay lead to the development of a third

ly and normalized to the total protein con-

generation (G3) CALUX cell bioassays.

tent. In biological system, the induction of

In

such

studies

mouse

hepatoma

CYP1A1 is triggered when dioxin-like

(hepa1c1c7) cells transfected with G3

chemicals such as PAHs and HAHs bind

CALUX

plasmids

with

increased

to the AhR. As an immediate response, a

numbers of dioxin response elements

binding enhanced gene expression of

(DREs) are utilized. This development

CYP1A1 protein occurs to detoxify the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 277

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

system from the xenobiotics. A study

IEQDetected = Total Concentration of mix-

conducted

on

ethoxyresorufin-

_O_ -

ture × IEF of mixture

(4)

deethylase (EROD) activity of 19 PAHs

(4)

(Jung _et al_., 2001)

in the fish hepatoma cell line PLHC-1 has

shown that it is a useful tool for the eco-

By relating the experimentally detected

toxicological evaluation of landfill leacha-

EC50 value of the mixture to reference

tes (Fent and Batscher, 2000). Previously,

compound and multiplying this value by

EROD assay was conducted mostly to

the total concentration of compounds pre-

study the traditional environmental pollu-

sent in the mixture, an IEQdet is deter-

tants such as PAHs, PCBs and HAHSs.

mined and compared to the IEQcalc (Jung

Recently, EROD assay is being utilized

_et al_., 2001). The IEQ values are ex-

for studying various new kinds of com-

pressed in mg reference substance equiva-

pounds/pollutants such as heterocyclic

lents (Reference substance equivalents

aromatic compounds containing nitrogen,

/L).

sulfur or oxygen (NSO-HET) (Hinger _et_

The 2,3,7,8-tetrachlorodibenzo-p-dioxin

_al_., 2011). EROD assay is also reported to

(TCDD), the most potent ligand to the

be used in determining the CYP1A induc-

aryl hydrocarbon receptor and the strong-

tion potencies of Nitrated polycyclic aro-

est inducer of EROD activity in most test

matic hydrocarbons (NPAHs) and N-

systems has been used as a reference sub-

heterocyclic

aromatic

hydrocarbons

stance in many studies (Huuskonen _et al_.,

(azaarenes) in fish hepatoma for the first

2000; Celender _et al_., 1996), however,

time (Jung _et al_., 2001).These compounds

recent

studies

suggest

that

β-

are commonly found PAH-contaminated

naphthoflavone is proved to be the most

environmental samples.

suitable reference for the routine _in vitro_

To reflect the induction potency in eco-

EROD assay (Heinrich _et al_., 2014; Fer-

toxicological evaluation, a concept of in-

nandes _et al_., 2014; Schnell _et al_., 2013).

duction equivalency factors (IEFs) was

Many other alternatives have been de-

developed based on half maximal effect

veloped to analyze the data generated

concentration (EC50). EC50 is the concen-

through EROD assay. A formula devel-

tration of substance that induces 50% of

oped by Sprague and Ramsay (1965) are

the maximum induction level. IEF values

also utilized in which the EROD activity

are calculated as follows;

is converted to toxic units (Schnell _et al_.,

2013).

As for the analysis involving binary

mixtures, the EC50 of both single com-

pounds and mixtures were determined

and the IEF of the compounds in relation

to a reference substance is determined.

Induction equivalents (IEQs) for a mix-

ture are then calculated to show the addi-

****

tive interactions of independent com-

**7. Enzyme-linked immunosorbent as-**

pound in a mixture.

**say (ELISA)**

In the determination of CYP1A en-

zymatic activity through EROD assay,

quantification of CYP1A protein induc-

tion level provides important and com-

plementary information about the regula-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 278

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

tion of CYP1A and mechanism of toxic

ronmental sample. Comet assay (single

action. Enzyme-linked immunosorbent

cell gel electrophoresis) is a technique of

assay (ELISA) is a useful technique in

measuring DNA damage in eukaryotic

immunologically detecting CYP1A pro-

cells or disaggregated tissues when ex-

tein. ELISA provides complementary in-

posed to hazardous agents (Azqueta and

formation on the induction of CYP1A in

Collins, 2013). In this assay the DNA is

the presence of inducing substances. Pre-

drawn out towards the anode through

viously quantification of CYP1A protein

electrophoresis, forming a comet-like im-

content was made possible through the

age that is observed with fluorescence

preparation of subcellular fractions in-

microscopy (Azqueta and Collins, 2013).

volving scrapping of cells from culture

In general, this assay refers to the relaxa-

flask, followed by sonication and centrif-

tion of supercoiled DNA in agarose-

ugation and eventually quantification

embedded nucleoids (the residual bodies

through western blotting (Hahn _et al_.,

remaining after lysis of cells with deter-

1993). This technique is rather time con-

gent and high salt), which then allows the

suming. Hence, a much rapid and sensi-

DNA to be drawn out towards the anode

tive detection of CYP1A protein was de-

under electrophoresis, forming comet-like

veloped in which the detection of the

images as seen under fluorescence mi-

amount of CYP1A protein extracted was

croscopy. DNA break frequency is indi-

measured directly in the microwells (Bru-

cated through the relative amount of DNA

schweiler _et al_., 1995). This technique

in the comet tail (Azqueta and Collins,

allows a semiquantitave determination of

2013). The extent of DNA migration are

the amount of immunoreactive CYP1A

determined as a percentage of DNA in the

protein directly in the microwells in

tail (% tDNA) using an image analysis

which the cells are cultured. The data of

system. For statistical analysis, the induc-

absolute absorption values are presented

tion factor (IF) was calculated using the

as the percent of maximal induction of

following equation:

CYP1A and EC50 is determined. Studies

have utilized ELISA to quantify CYP1A

protein to provide a complementary quan-

(6)

tification on CYP1A based EROD activi-

ty (Herrero and Castel, 1994; Jung _et al_.,

(Šrut _et al_., 2011)

2001).

(6) (Šrut _et al_., 2011)

****

**8. Genotoxicity assay**

Concentration dependent induction

__

factor (CDI) index is then calculated by

_8.1. Comet assay_

integrating all the important information.

Genotoxicity is of great interest be-

This forms the basis for a general com-

cause in the case of chronic exposure sit-

parison of the genotoxic potential of sam-

uation, the possibility of delayed conse-

ples in the Comet assay. CDI is calculated

quences at the population level is high

according to the following equation:

(Devaux _et al_., 2011). As such, the appli-

cation of Comet assay provides an early

and universal genotoxicity endpoint re-

vealing the primary DNA damage oc-

curred due to the exposure to environ-

mental pollutants (Frenzilli _et al_., 2009).

Genotoxicity assay such as Comet assay

is a biomarker assay providing valuable

Recent advancement in this particular

information on the presence of potential

assay suggests that digestion with lesion-

carcinogens and mutagens in the envi-

specific enzymes such as Formamidopyri-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 279

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

midine DNA glycosylase (FPG), provides

tact metabolism ensures the existence of

a greater yield for strand breaks and in-

full biotransformation capacity within the

creases the sensitivity of the assay in gen-

cells that is being exposed to the samples.

eral (Azqueta and Collins, 2013). In the

Recent research suggests that the results

case of low level of strand breaks at non-

of _in vitro_ EROD assay to be presented by

cytotoxic concentrations of genotoxic

reference to a truly physiological cytotox-

chemical, the yield of breaks was greatly

icity assay, the 3-(4,5-dimethylthiazol-2-

enhanced after incubation with (FPG)

yl)

2,5-diphenyltetrazo-lium

bromide

(Azqueta _et al_., 2013).

(MTT) test replacing protein normaliza-

This is a sensitive, simple and versatile

tion which will enable an optimized _in_

assay performed to visualize the single

_vitro_ EROD protocol to a reference com-

strand break in nuclear DNA of single

pound (Heinrich _et al_., 2014).

cells (Wilkening _et al_., 2003). Besides

providing the overall level of damage in

**9. Conclusion and recommendations**

the cells being analysed, comet assay also

provides the data on how the individual

Pollution is a major setback in devel-

cells respond to the xenobiotic. Comet

oping countries with heavy emphasis on

assay has been applied in studies on

industrialization and modernization. Con-

PLHC-1 fish cell line (Šrut _et al_., 2011)

stant and consistent pollution monitoring

and characterization of the genotoxicity

is crucial in order to maintain pollution

of sediment extracts from the Baltic and

level in control. In pursuit of the effort,

North Sea on EPC ( _epithelioma papu-_

extensive environmental toxicology stud-

_losum cyprini_ ) fish cell line (Kammann _et_

ies are essential because they provide the

_al_., 2001, 2004).

actual impact situation before the expen-

sive instrumental analysis is conducted.

_8.2. Cytotoxicity assay_

Ecotoxicological studies in Malaysia are

To assess the acute toxicity of pollu-

extremely rare. Hence, there is a need for

tants on biological organisms, various cy-

immediate exploration and application of

totoxicity tests are usually performed.

these techniques and this will aid the rel-

Among them, tetrazolium salt reduction

evant authorities to improve the environ-

(MTT) assay is widely used as an end-

mental status monitoring.

point for the cytotoxicity measurement of

chemicals/pollutants in monolayer cell

**Acknowledgement**

cultures (Vakharia _et al_., 2001; Bru-

schweiler _et al_., 1995; Heinrich _et al_.,

This work was supported by grants-in-

2014). The MTT assay detects the reduc-

aid from Research University Grant

tion of soluble MTT tetrazolium salt to a

Scheme by University Putra Malaysia

blue insoluble MTT formazan product by

(9331400) to Prof. Ahmad Zaharin Aris.

mitochondrial succinate-dependent dehy-

drogenase (Bruschweiler _et al_., 1995).

**References**

The percentage of viability was calculated

relative to DMSO treatment control wells

**Ahlborg, U. G., Brouwer, A., Fin-**

from triplicate observations that are des-

**gerhut, M. A., Jacobson, J. L.,**

ignated as 100% viable cells. Only cells

**Jacobson, S. W., _et al_** **. (1992).** Im-

with active mitochondria are able to cata-

pact of polychlorinated dibenzo-p-

lyze this reaction. Cellular stress determi-

dioxins, dibenzofurans, and bi-

nation is crucial when analyzing complex

phenyls on human and environ-

samples such as environmental samples

mental health, with special empha-

because enzymatic assays such EROD

sis on application of the toxic

assay requires an intact metabolism for

equivalency factor concept. _Euro-_

the _in-vitro_ live cell approaches. This in-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 280

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

_pean Journal of Pharmacology_

**Burg, B. (2008).** Detection of multiple

**228, 179–199.**

hormonal activities in wastewater

**Ankley, G., Mihaich, E., Stahl, R., Til-**

effluents and surface water, using a

**litt, D., Colborn, T., _et al._** **(1998).**

panel of steroid receptor CALUX

Overview of workshop on screen-

bioassays. _Environmental Science_

ing methods for detecting potential

_and Technology_ **42, 5814-5820.**

(anti-)

estrogenic/androgenic

**Cederberg, T., Laier, P., Vinggaard, A.**

chemicals in wildlife. _Environmen-_

**M. (2002).** Screening of food sam-

_tal Toxicological Chemistry_ **17,**

ples for dioxin levels Comparison

**68–87.**

of GC-MS determination with the

**Azqueta, A., Arbillaga, L., Lopez de**

CALUX bioassay. _Organohalogen_

**Cerain, A., Collins, A. (2013).**

_Compounds_ **58, 409-412.**

Enhancing the sensitivity of the

**Celander, M., Hahn, M. E., Stegeman,**

comet assay as a genotoxicity test,

**J. J. (1996).** Cytochromes P450

by combining it with bacterial re-

(CYP) in the _Poeciliopsis lucida_

pair enzyme FPG. _Mutagenesis_ **28,**

Hepatocellular

Carcinoma

Cell

**271-277.**

Line (PLHC-1): Dose and Time-

**Azqueta, A., and Collins, A. R. (2013)**.

Dependent Glucocorticoid Potenti-

The essential comet assay: a com-

ation of CYP1A Induction without

prehensive guide to measuring

Induction of CYP3A. _Archives of_

DNA damage and repair. _Archives_

_Biochemistry and Biophysics._ **329,**

_of Toxicology_ **87, 949–968.**

**113–122.**

**Blankenship, A. L., Kannan, K.., Villa-**

**Czeka, P. (2000).** Phenobarbital-induced

**lobos, A., Falandysz, J., Giesy, J.**

expression of cytochrome P450

**P. (2000).** Relative potencies of

gene. _Acta Biochimica Polonica_ 47 ****

halowax mixtures and individual

**(4), 149–159.**

polychlorinated

naphthalenes

**Denison, M. S., Zhao, B., Baston, D. S.,**

(PCNs) in H4IIe-Luc cell bioassay.

**Clark, G. C., Murata, H., Han,**

_Environmental Science and Tech-_

**D. H.** **(2004).** Recombinant cell bi-

_nology_ **34, 3153–3158.**

oassay systems for the detection

**Brack, W., Altenburger, R., Schüür-**

and relative quantitation of halo-

**mann, G., Krauss, M., Herráez,**

genated dioxins and related chemi-

**D. L., _et al_** **. (2015).** The SOLU-

cals. _Talanta_ **63, 1123-1133**.

TIONS project: Challenges and re-

**Denison, M. S., Heath-Pagliuso. (1998).**

sponses for present and future

The Ah Receptor: a regulator of the

emerging pollutants in land and

biochemical and toxicological ac-

water resources management. _Sci-_

tions of structurally diverse chemi-

_ence of the Total Environment_ **503-**

cals. _Bulletin of Environmental_

**504: 22–31.**

_Contamination_

_and_

___
___

_toxicology_

**Bruschweiler, B. J., Wurgle, F.E., Fent.**

**61(5), 557-68.**

**K. (1995).** An Elisa Assay For Cy-

**Devaux, A., Fiat, L., Gillet, C., Bony, S.**

tochrome P4501A in Fish Liver

**(2011).** Reproduction impairment

Cells. _Environmental Toxicology_

following paternal genotoxin ex-

_and Chemistry,_ **15 (4), 592–596.**

posure in brown trout ( _Salmo trut-_

**Bucheli, T. D., Fent K. (1995).** Induction

_ta_ ) and Arctic char ( _Salvelinus al-_

of cytochrome P450 as a biomarker

_pinus_ ). _Aquatic Toxicology_ **101,**

for environmental contamination in

**405–411.**

aquatic ecosystems. _Critical Re-_

**Escher, B. I., Allinson, M, Altenburger,**

_views in Environmental Science_

**R., Bain, P. A, Balaguer, P., _et al_** **.**

_and Technology_ **25, 201–268.**

**(2014).** Benchmarking Organic

Micropollutants in Wastewater,

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 281

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

Recycled Water and Drinking Wa-

effects of polychlorinated biphen-

ter with In Vitro Bioassays . _Envi-_

yls (PCBs): implications from risk

_ronmental Science and Technology_

assessment. _Critical Review in Tox-_

**48 (3), 1940–1956.**

_icology_ **28, 511–569.**

**Fent, K., and Batscher, R. (2000).** Cyto-

**Gillesby, B. E., Zacharewski, T. R.**

chrome P4501A induction poten-

**(1998).** Exoestrogens: Mechanisms

cies of Polycyclic aromatic hydro-

of action and strategies for iden-

carbons in a fish Hepatoma cell

tification and assessment. _Envi-_

line: Demonstration of additive in-

_ronmental Toxicological Chemistry_

teractions. _Environmental Toxicol-_

**17, 3–14.**

_ogy and Chemistry_ **19(8), 2047–**

**Hahn, M. E., Lamb, T. M., Schultz,**

**2058.**

**Roxanna, M. E., Smolowitz, R.**

**Fent, K. (2001).** Fish cell lines as versa-

**M., Stegeman, J. J. (1993).** Cyto-

tile tools in ecotoxicology: assess-

chrome P4501A induction and in-

ment of cytotoxicity, cytochrome

hibition

by

3,3',4,4'-

P4501A induction potential and es-

tetrachlorobiphenyl in an Ah recep-

trogenic activity of chemicals and

tor- containing fish hepatoma cell

environmental samples. _Toxicology_

line (PLHC-1). _Aquatic Toxicology_

_in Vitro_ **15, 477–488.**

**26 185-208.**

**Fernandes, D., Pujol, S., Albaladejo, E.**

**Hamers, T., Leonards, P. E. G., Legler,**

**P., Tauler, R., Bebianno, M. J.,**

**J., Vethaak, A. D, Schipper, C.**

**Porte, C. (2014).** Characterization

**A. (2010).** Toxicity Profiling: An

of the environmental quality of

integrated effect-based tool for

sediments from two estuarine sys-

site-specific sediment quality as-

tems based on different in-vitro bi-

sessment. Integrated Environmen-

oassays. _Marine Environmental_

tal Assessment and Management

_Research_ **96, 127-135.**

6:761-773.

**Frenzilli, G., Nigro, M., Lyons, B. P.**

**Han, D., Nagy, S. R., Denison, M. S.**

**(2009).** The Comet assay for the

**(2004).** Comparison of recombi-

evaluation of genotoxic impact in

nant cell bioassays for the detection

aquatic environments. _Reviews in_

of Ah receptor agonists. _Biofactor_

_Mutation Research_ **681, 80–92.**

**20, 11-22.**

**Garrison, P. M., Tullis, K., Aarts, J. M.**

**Hankinson, O. (1995).** The aryl hydro-

**M. J. G., Brouwer, A., Giesy, J.**

carbon receptor complex. _Annual_

**P. and Denison, M. S. (1996).**

_Review of Pharmacology and Toxi-_

Species-specific recombinant cell

_cology_ **35, 307–340.**

lines as bioassay systems for the

**He, G., Tsutsumi, T., Zhao, B., Baston,**

detection

of

2,3,7,8-

**D. S., Zhao, J., _et al_** **. (2011).**

tetrachlorodibenzo-p-dioxin-like

Third-generation

Ah

receptor-

chemicals. _Fundamentals of Ap-_

responsive

luciferase

reporter

_plied Toxicology_ **30, 194–203.**

plasmids: amplification of dioxin-

**Giesy, J.P., Hilscherova, K., Jones, P.**

responsive elements dramatically

**D., Kannan, K., Machala, M.**

increases CALUX bioassay sensi-

**(2002).** Cell bioassays for detection

tivity and responsiveness. _Toxico-_

of aryl hydrocarbon (AhR) and es-

_logical Science_ **123(2):511-22.**

trogen receptor (ER) mediated ac-

**Heinrich, P., Diehl, U., Förster, F.,**

tivity in environmental samples.

**Braunbeck. T. (2014).** Improving

_Marine Pollution Bulletin_ **45, 3–**

the

_in-vitro_

ethoxyresorufin- _O_ -

**16.**

deethylase (EROD) assay with

**Giesy, J.P., Kannan, K. (1998).** Dioxin-

RTL-W1 by metabolic normaliza-

like and non-dioxin-like toxic

tion and use of β-naphthoflavone as

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 282

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

the reference substance. _Compara-_

genotoxicity testing of marine sed-

_tive Biochemistry and Physiology_ ,

iments with the Comet assay. _Mu-_

Part C **164, 27–34.**

_tation Research_ **498, 67–77.**

**Herrero, M. E., and Castell, J. V.**

**Kannan, K., Tanabe, S., Giesy, J.P.,**

**(1994).** Quantification of CYP1A1

**Tatsukawa, R. (1997).** Organo-

and 2B1/2 in rat hepatocytes cul-

chlorine pesticides and polychlo-

tured in microwells by immunolog-

rianted biphenyls in foodstuffs

ical methods. _Toxicology In Vitro_

from Asian and Oceanic countries.

**8(6), 1167-l 175.**

_Reviews on Environmental Con-_

**Hinger, G., Brinkmann, M., Bluhm, K.,**

_tamination and Toxicology_ **15, 1–**

**Sagner , A., Takner, H., _et al_** **.**

**55.**

**(2011).** Some heterocyclic aro-

**Kannan, N., Tanabe,S., Tatsukawa, R.**

matic compounds are Ah receptor

**(1988).** Toxic potential of mono-

agonists in the DR-CALUX assay

ortho and non-ortho coplanar PCBs

and the EROD assay with RTL-W1

in commercial PCB preparations:

cells. _Environmental Science and_

"2,3,7,8-T4CDD Toxicity Equiva-

_Pollution Research_ **18(8), 1297-**

lence Factors Approach". _Bulletin_

**1304.**

_of_ _Environmental Contamination_

**Huuskonen S. E, A. Tuvikene, M.**

_and Toxicology_ **41, 267-27.**

**Trapido, K. Fent, M. E. Hahn.**

**Kannan, N., Wakimoto, T., Tatsukawa,**

**(2000).** Cytochrome P4501A In-

**R. (1989a).** Possible involvement

duction and Porphyrin Accumula-

of frontier (π) electrons in the me-

tion in PLHC-1 Fish Cells Exposed

tabolism of polychlorinated bi-

to Sediment and Oil Shale Extracts.

phenyls (PCBs). _Chemosphere_ **18,**

_Archives of Environmental Con-_

**9-10.**

_tamination and Toxicology_ **38, 59–**

**Kannan, N., Tanabe, S., Okamoto, T.,**

**69.**

**Tatsukawa, R., Phillips, D. J. H.**

**Jung, D. K., Klaus, T., Fent, K. (2001).**

**(1989b).** Polychlorinated Biphen-

Some heterocyclic aromatic com-

yls (PCBs) in Sediments in Hong

pounds are Ah receptor agonists in

Kong: A Congener-Specific Ap-

the DR-CALUX assay and the

proach to the Study of Coplanar

EROD assay with RTL-W1 cells.

PCBs in Aquatic Ecosystems. _En-_

_Environmental_

_Toxicology_

_and_

_vironmental Pollution_ **62, 223-235.**

_Chemistry_ **20(1), 149-159**.

**Kannan, N., Tanabe, S., Ono, M.**

**Jung, J. H., Hong, S. H., Yim, U. H.,**

**Tatsukawa, R. (1989c)**. Critical

**Ha, S. Y., Shim, W. J., Kannan,**

evaluation of PCB toxicity in Ter-

**N. (2012).** Multiple In Vitro Bioas-

restrial and marne mammals: In-

say Approach in Sediment Toxicity

creasing impact of non-ortho and

valuation: Masan Bay, Korea. _Bul-_

mono-ortho coplanar PCBs from

_letin of_ _Environmental Contamina-_

land to ocean. _Archives of Envi-_

_tion and Toxicology_ **89(1):32-37.**

_ronmental Contamination and Tox-_

**Kammann, U., Biselli, S., Hühnerfuss,**

_icology_ **18, 850-857.**

**H., Reineke, N., Theobald, N., _et_**

**Kaw, H. Y., Kannan, N. (2016).** A re-

_**al**_ **. (2004).** Genotoxic and terato-

view on Polychlorinated Biphenyls

genic potential of marine sediment

(PCBs) and Polybrominated Di-

extracts investigated with Comet

phenyl Ethers (PBDEs) in South

assay and zebrafish test. _Environ-_

Asia with a focus on Malaysia. _Re-_

_mental Pollution_ **132, 279–287.**

_views of Environmental Contami-_

**Kammann, U., Bunke, M., Steinhart,**

_nation and Toxicology_ , **242, 153-**

**H., Theobald, N. (2001).** A per-

**181.**

manent fish cell line (EPC) for

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 283

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

**Keiter, S., Grund, S., Bavel, B.V., Hag-**

emerging concern in aquatic eco-

**berg, J., Engwall, M., _et al_** **.**

systems. _Integrated Environmental_

**(2008).** Activities and identifica-

_Assessment Management_ **12:540-**

tion of aryl hydrocarbon receptor

**547.**

agonists in sediments from the

**Mehinto, A. C, Jia, A., Snyder, S. A.,**

Danube River. Anals of Bioanal

**Jayasinghe, B. S., Denslow, N. D.,**

Chem 390: 2009–2019

_**et al**_ **. (1993).** Developmental and

**Kim, H. N., Park, C. I., Chae, Y. S.,**

reproductive toxicity of dioxins

**Shim, W. J., Addison, R. F., _et al_** **.**

and related compounds: Cross spe-

**(2013).** Acute toxic responses of

cies comparisons. _Critical Review_

the rockfish ( _Sebastes schlegeli_ ) to

_in Toxicology_ **23, 283–335.**

Iranian heavy crude oil: Feeding

**Mehinto, A. C., Jia, A., Snyder, S. A.,**

disrupts the biotransformation and

**Jayasinghe, B. S., Denslow N. D.,**

innate immune systems. _Fish and_

_**et al**_ **. (2015).** Interlaboratory com-

_Shellfish Immunology_ **35, 357-365.**

parison of in vitro bioassays for

**Krishnan, P., Kannan, N., Aris, A. Z.,**

screening of endocrine active

**Arulselvan,**

**P.,**

**Fakurazi**

**S.**

chemicals in recycled water. _Water_

**(2016).** Possible Application of

_Research_ **83, 303-9.**

bio-analytical assays in the biolog-

**Poland, A., Knutson, J. C. (1982).**

ical impact assessment of Persis-

2,3,7,8-Tetrachlorodibenzo-pdioxin

tent Organic Pollutants (POPs) in

and related halogenated aromatic

mangrove sediments in South East

hydrocarbons: examination of the

Asia with particular reference to

mechanism of toxicity. _Annual Re-_

Malaysia. In: _Persistent Organic_

_view of Pharmacology_ **22, 517–**

_Chemicals in the Environment:_

**554.**

_Status and Trends in the Pacific_

**Qiao, M., Chen, Y., Zhang, Q., Huang,**

_Basin Countries II_. ACS Symposi-

**S., Ma, M., _et al_** **. (2006).** Identifi-

um Series; American Chemical So-

cation of Ah receptor agonists in

ciety. Loganathan, B. G., Khim, J.

sediment of Meiliang Bay, Taihu

S., Prasada Rao S. Kodavanti, P. R.

Lake, China. _Environmental Sci-_

S., Masunaga, S. (eds.) Publication

_ence and Technology_ **40,1415–**

Date (Web): December 7, 2016 |

**1419.**

doi: 10.1021/bk-2016-1244.ch009

**Safe, S., Krishnan, V. (1995).** Cellular

**Lapa, N., Barbosa, R., Morais, J.,**

and molecular biology of aryl hy-

**Mendes, B., Méhu, J., _et al_** **.**

drocarbon (AH) receptor mediated

**(2002).** Ecotoxicological assess-

gene expression. _Archives of. Toxi-_

ment of leachates from MSWI bot-

_cology_. **7, 99–115.**

tom ashes. Waste Management 22,

**Sanderson J. T., Aarts, M. M. J. G.,**

583–593

**Brouwer, A., Froese, K. I., Den-**

**Leusch, F. D, de Jager, C., Levi, Y.,**

**ison, M. S., _et al_** **. (1996).** Compari-

**Lim, R., Puijker, L., _et al_** **. (2010).**

son of Ah Receptor-Mediated Lu-

Comparison of five in vitro bioas-

ciferase and EthoxyresorufinO-

says to measure estrogenic activity

deethylase Induction in H4IIE

in environmental waters. _Environ-_

Cells: Implications for Their Use as

_mental Science and Technology_

Bioanalytical Tools for the Detec-

**44:3853-3860.**

tion of Polyhalogenated Aromatic

**Maruya KA, Dodder NG, Mehinto AC,**

Hydrocarbons. _Toxicology and ap-_

**Denslow ND, Schlenk D, _et_**

_plied pharmacology_ **137, 316–325.**

_**al**_ **.(2016).** A tiered, integrated bio-

**Schmitz, H., Hagenmaier, A., Ha-**

logical and chemical monitoring

**genmaier, H., Bock, K. W.,**

framework for contaminants of

**Schrenk, D. (1995).** Potency of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 284

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

mixtures of polychlorinated bi-

and wildlife. _Environmental Health_

phenyls as inducers of dioxin re-

_Perspective_ **106, 775–790.**

ceptor-regulated CYP1A activity in

**Van der Linden, S. C., Heringa, M. B.,**

rat hepatocytes and H4IIE cells.

**Man, H. Y., Sonneveld, E., _et al_** **.**

_Toxicology_ **99, 47–54.**

**(2008).** Detection of multiple hor-

**Schnell, S., Olivares, A., Piña, B.,**

monal activities in wastewater ef-

**Erasun, B. E., Porte, C., _et al_** **.**

fluents and surface water, using a

**(2013).** The combined use of the

panel of steroid receptor CALUX

PLHC-1 cell line and the recombi-

bioassays. _Environmental Science_

nant yeast assay to assess the envi-

_and Technology_ **42(15), 5814-20.**

ronmental quality of estuarine and

**Whyte, J. J., Schmitt, C. J., Tillitt, D.**

coastal sediments. _Marine Pollu-_

**E. (2004).** The H4IIE cell bioassay

_tion Bulletin_ **77, 282–289.**

as an indicator of dioxin-like chem-

**Seitz, N., Böttcher, M., Keiter, S.,**

icals in wildlife and the environ-

**Kosmehl, T., Manz, W., _et al_** **.**

ment. _Critical Reviews in Toxicol-_

**(2008).** A novel statistical approach

_ogy_. **34, 1–83.**

for the evaluation of Comet assay

**Wilke, B. M., Riepert, F., Kich, C.,**

data. _Mutation Research_ **652, 38–**

**Kühne, T. (2008).** Ecotoxicologi-

**45.**

cal characterization of hazardous

**Sprague, J. B., Ramsay, B. A. (1965).**

wastes. _Ecotoxicology and Envi-_

Lethal effects of mixed copper and

_ronmental Safety_ **70, 283–293.**

zinc solutions for juvenile salmon.

**Wilkening, S., Stahl, F., Bader, A.**

_Journal of the Fisheries Research_

**(2003).** Comparison of primary

_Board of Canada_ **22, 425–432.**

human hepatocytes and hepatoma

**Šrut, M., Traven, L., Štambuk, A.,**

cell line HEPG2 with regard to

**Kralj, S., Roko , _et al_** **. (2011).**

their biotransformation properties.

Genotoxicity of marine sediments

_Drug Metabolism and Disposition_

in the fish hepatoma cell line

**31 (8) 1035-1042.**

PLHC-1 as assessed by the Comet

**Willett, K. L., Gardinali. P. R., Serica-**

assay. _Toxicology in Vitro_ **25, 308–**

**no, J. L., Wade, T. L., Safe, S. H.**

**314.**

**(1997a).** Characterization of the

**Tsutsumi, T., Amakura, Y., Nakamura,**

H4IIE rat hepatoma cell bioassay

**M., Brown, D. J., Clark, G. C., _et_**

for evaluation of environmental

_**al**_ **. (2003).** Validation of the

samples containing Polynuclear

CALUX bioassay for the screening

Aromatic Hydrocarbons (PAHs).

of PCDD/Fs and dioxin-like PCB

_Archives of Environmental Con-_

in retail fish. _Analyst_ **128, 486-492.**

_tamination and Toxicology_ **32,**

**Vakharia, D. D., Liu, N., Pause, R.,**

**442–448.**

**Fasco, M., Bessette E, _et al_** **.**

**Willett, K. L., Randerath, K., Zhou, G.**

**(2001).** Polycyclic Aromatic Hy-

**D., Safe, S. H. (1997b).** Inhibition

drocarbon/Metal Mixtures: Effect

of CYP1A1-dependent activity by

On PAH Induction Of CYP1A1 in

the Polynuclear Aromatic Hydro-

Human HepG2 Cells. _Drug Metab-_

carbons (PAH) Fuoranthene. _Bio-_

_olism and Disposal_ **29(7), 999-**

_chemical Pharmacology_ **55, 831–**

**1006.**

**839.**

**Van den Berg, D., Birnbaum, L.,**

**Windal, I., Denison, M. S., Birnbaum,**

**Bosveld, B. T. C., Brunstrom, B.,**

**L. S., Wouwe, N. V., Baeyens,**

**Cook, P., _et al_** **. (1998).** Toxic

**W., _et al_** **. (2005).** Chemically Acti-

equivalency factors (TEFs) for

vated Luciferase Gene Expression

PCBs, PCDDs, PCDFs for humans

(CALUX) cell bioassay analysis

for the estimation of Dioxin-like

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 285

_Biotech Sustainability (2017)_

_In vitro Cell Bioassays in Pollution Assessment Kannan et al._

activity: Critical parameters of the

say for characterization of Ah-R-

CALUX procedure that impact as-

active compounds and activities in

say results. _Environmental Science_

sediment from Korea. _Chemo-_

_and Technology_ **39, 7357-7364.**

_sphere_ **62, 1261–1271.**

**Yoo, H., Khim, J. S., Giesy, J. P. (2006).**

Receptor-mediated in vitro bioas-

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 286

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P287-294_

**Lipopeptide Biosurfactants from Bioagent, _Bacillus_** **as a**

**Weapon for Plant Disease Management**

**Sampath Ramyabharathi, Balaraman Meena*, Lingan Rajendran and Thiruvenga-**

**dam Raguchander**

__

_Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricul-_

_tural University, Coimbatore – 641-003, Tamil Nadu, India; *Correspondence:_

 _meepath@rediffmail.com;_ _Tel: +91 422 6611226_

__

**Abstract:** Biosurfactants are surface active biological compounds that are produced by

broad range of microorganisms. They are ecofriendly with low toxicity, no residual effects

with high biodegradable properties and known to suppress the growth of pathogenic fungi.

_Bacillus_ genus is considered microbial factories for the production of a huge number of bio-

logically active molecules that are inhibitory for plant pathogen growth. In rhizobacteria

_Bacillus subtilis,_ an average of 4 – 5% of its genome is dedicated to antibiotic synthesis and

has the possibility to produce more than two dozen of structurally diverse antimicrobial

compounds. Because of the surfactant properties, the antimicrobial peptide compounds or

cyclic lipopeptide antibiotics of the surfactin, iturin and fengycin families are well-

recognized for their potential applications in biotechnology. Different groups of lipopep-

tides can give an advantage to the _Bacillus_ strains in specific environmental niches. The

ability to induce systemic resistance in plants and their use in the spreading of the bacterial

cells that leads to rhizosphere colonization could open new fields of applications for their

use in phytopharmaceutical products. In this review article, we are highlighting the role and

functions of some major biocontrol lipopeptide biosurfactants present in the _Bacillus_ spe-

cies. ****

_**Keywords**_ **:** Biological control; _Bacillus_ species; lipopeptide biosurfactants

**1. Introduction**

The hydrophobic portion of lipopeptides

is made up of fatty acids and the hydro-

Biosurfactants are produced on the

philic portion is composed of peptides or

microbial cell surface and are capable of

polysaccharides (Georgiou _et al_., 1992).

lowering surface and interfacial tensions.

Hence the presence of hydrophobic and

They are produced extracellularly and

hydrophilic portions within a single mol-

thus are potential substitutes for widely

ecule, the biosurfactants tend to migrate

used synthetic surfactants. Biosurfactants

toward an interface with different degrees

are widely used in industries, pharmaceu-

of polarity and hydrogen bonding (Desai

tical, agriculture, food, cosmetics, oil

and Banat, 1997). In Gram-positive _Bacil-_

production industries and in bioremedia-

_lus subtilis_ IAM1213 the production of

tion process. Till date a broad range of

lipopeptide biosurfactants was first re-

structurally different biosurfactants have

ported (Arima _et al_., 1968) and the differ-

been identified that includes lipopeptides,

ent types of lipopeptide biosurfactants

polysaccharides, proteins, lipoproteins

with significant surface activity, antipath-

and glycolipids. Lipopeptide biosurfac-

ogenic (Ramyabharathi and Raguchander,

tants are composed of a lipid tail connect-

2014; Ramyabharathi _et al_., 2016),

ed to a short linear or cyclic oligopeptide.

antinematicidal (Ramyabharathi, 2015;

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_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

Sankari Meena _et al_., 2016) and anti-

amount, to the production of iturin A

microbial activity have been reported

(Tsuge _et al_., 2001). Gene clusters in-

from other _Bacillus_ strains.

volved in iturin A production have been

intensively investigated (Hiraoka _et al_.,

**2. _Bacillus_** **lipopeptides __**

1992, Huang _et al_., 1993, Kunst _et al.,_

__

__

1997, Yao _et al.,_ 2003). The iturin A op-

The lipopeptides in _Bacillus_ are

eron spans a region more than 38 kb long

classified into three families namely Itu-

and it contains four open reading frames

rin, Surfactin and fengycin family. There

_viz.,_ ituD, ituA, ituB and ituC (Tsuge _et_

are several _Bacillus_ strains isolated and

_al.,_ 2001). The ituD gene encodes a puta-

reported to produce all the three

tive malonyl coenzyme A transacylase,

families of lipopeptide biosurfactants

whose distraction results in deficiency of

simultaneously (Ramyabharathi and Ra-

iturin A production (Kunst _et al_., 1997).

guchander, 2014).

Ramyabharathi and Raguchander (2014)

reported that the _B. subtilis_ strain EPCO

_2.1. Iturin lipopeptide_

16 contains _ItuC, ItuD, BmyA, BacD,_

Iturins are a member of an anti-

_BacAB_ and _FenD_ genes involved in the

fungal lipopeptide group comprises iturin,

biosynthesis of Iturin, Bacillomycin,

bacillomycin and mycosubtilin which are

Bacilysin and Fengycin, respectively. Itu-

cyclic lipoheptapeptides and are linked by

rin and surfactin were detected in culture

beta amino acid residue. Iturin family

filtrates from isolate EPCO16 by thin lay-

contains iturins A-E, bacillomycins D, F,

er chromatography that showed tremen-

and L, and mycosubtilin. Members of this

dous control over _Fusarium oxysporum_

family have powerful antibiotic activity

pathogen infecting tomato.

but moderate surfactant activity and en-

Bacillomycin D which is a mem-

hanced swarming motility (Leclere _et al_.,

ber of the Iturin family along with myco-

2006). Hence the presence of iturin in

subtilin and iturin A, is made of one β-

_Bacillus_ is responsible for disease sup-

amino fatty acid and seven α-amino acids

pression and growth inhibition of wide

exhibits a strong antifungal activity

number of phytopathogens. _Bacillus sub-_

against a broad range of plant pathogenic

_tilis_ produces a diversity of antibiotics

fungi. Biosynthesis of bacillomycin D is

that are effective against phytopathogenic

independent of the ribosomal process and

fungi and bacteria (Phae _et al_., 1990). Itu-

the

enzymes

responsible

for

rin production seems to be constrained to

bacillomycin D production are complex

_B. subtilis_ and _B. amyloliquefaciens_

peptide synthetases. Bacillomycin D and

(Bonmatin _et al_., 2003). In control of

fengycin jointly contributed to the inhibi-

phyllosphere diseases, _Podosphaera fusca_

tion of conidial germination of _Monilinia_

infecting melon leaves the iturins and

_fructicola_ and fengycin played a major

fengycins

lipopeptides

produced by

role in suppressing mycelial growth of the

_B. subtilis_ contributed more in disease

fungal pathogen. __ Luo _et al.,_ 2011 per-

suppression. Vater (1986) reported that

formed bioassay of antifungal compound

the iturin is produced, during slow, sta-

bacillomycin against _Magnaporthe ory-_

tionary growth phase. Sandrin _et al_.

_zae,_ _Rhizoctonia solani_ and _Botrytis ci-_

(1990) reported that iturin A, one member

_nerea_. __ The hyphae of the pathogenic fun-

of the iturin group, shows a strong antibi-

gi treated with bacillomycin L showed

otic activity with a broad antifungal activ-

abnormal growth, and conidia produced

ity, making it an ideal potential biological

enlarged and constricted germ tube. When

control agent with the aim of reducing the

bacillomycin L used in high concentration

use of chemical pesticides in agriculture.

there is a possibility for cellular leakage.

The

antimicrobial

action

of

Bacillomycin

D

was

detected

in

_B. subtilis_ can be attributed, to a certain

_B. subtilis_ (Moyne _et al_., 2001; Ramara-

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_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

thnam

_et_

_al_.,

2007)

and

film formation of producing cells, swarm-

_B. amyloliquefaciens_ strains. _B. subtilis_

ing motility, and fruiting body formation.

AU195 exhibit antifungal and have an

However, surfactin also inhibits biofilm

amino acid sequence identical to bacillo-

formation of other bacteria by interfering

mycin D. There is higher effect of bacil-

with attachment of the cells to surfaces.

lomycin D than of fengycin against vari-

Surfactin or closely related variants such

ous phytopathogenic fungi. (Koumoutsi,

as lichenysin have been isolated from _B._

2006; Peypoux _et al_., 1984).

_coagulans_ , _B. pumilus_ and _B. licheniform-_

_is._

2.2. _Surfactin lipopeptide_

Surfactins are not toxic for fungal

The biosurfactant surfactin is an

pathogens by themselves but they main-

acidic cyclic lipopeptide produced by

tain some synergistic effect on the anti-

strains of _B. subtilis_. Among the lipopep-

fungal activity of iturin A. The mode of

tide antibiotics produced by _Bacillus_ spp

action of surfactin is act on the phospho-

surfactin and Iturin A was most common.

lipids and is able to form selective ionic

The specific surface and membrane active

pores in lipid bilayers of cytoplasmic

properties of the surfactin help bacteria to

membranes. Both surfactin and iturinA

form biofilm. It is thought to perform de-

are surfactants with a hydrophilic ring of

velopmental functions rather than disease

seven amino acids and a long, hydropho-

resistance mechanism in the environment.

bic hydrocarbon tail. Usually the amino

Surfactin also produces a sturdy mem-

acid end stays in the soil and the hydro-

brane-destabilizing action at concentra-

carbon tail penetrates inside the pathogen

tions even below its critical micellar con-

cell membranes. This action creates open-

centration and induces the arrangement of

ings in cell membranes and restricting the

ion channels in lipid bilayers. Surfactins

growth of many phytopathogens (Ohno _et_

have been reported to be powerful surfac-

_al_., 1995; Asaka and Shoda, 1996; Carril-

tants due to their outstanding surface ac-

lo _et al_., 2003; Ongena and Jacques,

tivities. Compared with conventional sur-

2008).

factants, surfactin also have significant

IturinA has antibiotic property

biological activities, such as antiviral and

while surfactin has extremely powerful

antibacterial activity. Phae _et al_., (1990)

surface-active property, making its sepa-

reported that Iturin A and surfactin pro-

ration much more difficult. Surfactin,

ducing _B. subtilis_ suppressed more than

which is produced early in the bacte-

23 types of plant pathogens _in vitro_.

rium's growth cycle, has a deep influence

Surfactin and lichenysin are struc-

on _B. subtilis_ colonization of the root sur-

turally related lipopeptides produced by

face. Surface motility can be increased in

_B. subtilis_ and _B. licheniformis._ Bonmatin

rapid manner and thereby surfactin accel-

_et al_., 2003 reported different forms of

erates the development of multicellular

surfactin with amino acid variation at po-

communities which brings colonization of

sition 2, 4, and 7. Surfactin bears power-

the bacteria referred to as biofilms (Bais

ful surfactant properties by declining the

_et al_., 2004; Nagorska _et al_., 2007; Ru-

surface tension of water from 72 to 27

drappa _et al_., 2008). Biofilm formation in __

mN/m at a critical micelle concentration

_B. subtilis_ added __ a distinct advantage over

(CMC) of 25–220 mg/L based on its vari-

many competing organisms in the rhizo-

ants and determined conditions. Surfactin

sphere soil. Poor root colonization by sur-

is an inhibitor of fibrin clot formation.

factin-deficient _B. subtilis_ strains is asso-

Surfactins (C12 to C16) were produced

ciated with lack of biocontrol activity

simultaneously to increase the antifungal

(Schippers _et al_., 1987; Bais _et al_., 2004).

activity of iturin A. Thus exhibits anti-

viral, anti-tumor, anti-microbial and he-

2.3. _Fengycin lipopeptide_

molytic properties. It is required for bio-

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_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

Fengycin is a biologically active

FZB42 have indicated that fengycin and

lipopeptide produced by several _Bacillus_

bacillomycin D act synergistically to in-

_subtilis_ strains. The third family of

hibit the growth of _Fusarium oxysporum_

lipopeptide

biosurfactants

includes

under _in vitro_ condition (Koumoutsi _et_

fengycins A and B, which are also called

_al_., 2004).

plipastatins. The structure is composed of

a β-hydroxy fatty acid linked to a peptide

**3. Biocontrol potential of lipopeptide**

part comprising 10 amino acids. It is an

**biosurfactants**

anti-fungal antibiotic that inhibits fila-

mentous fungi but is ineffective against

Antibiotic production by _B. sub-_

yeast and bacteria. Fengycin production

_tilis_ strains plays a major role in suppres-

was identified in _B. cereus_ and _B. thurin-_

sion of plant diseases (Kinsella _et al_.,

_giensis_ in addition to _B. subtilis_ and _B._

2009). _B. subtilis_ EPCO16 has lipopep-

_amyloliquefaciens_. It is also capable of

tide genes _viz., ItuC_ gene, _ItuD_ gene (Itu-

inhibiting phospholipase A2 and biofilm

rin); _BmyA_ gene (Bacillomycin A), _BacD_

formation of several bacteria. These types

gene (Bacillomycin D), _BacAB_ gene

of lipodecapeptides are produced by vari-

(Bacilysin) and _FenD_ gene (Fengycin).

ous strains of _Bacillus_ spp. and exhibit

The presence of lipopeptide antibiotics in

moderate surfactant activities. It shows

_B. subtilis_ EPCO16 inhibited the mycelia

antifungal activity and more specific for

growth (46.04%) of _F. oxysporum_ f. sp.

filamentous fungi.

_lycopersici_ (Fol) under _in vitro_ (Ramya-

Fengycin produced by _B. subtilis_

bharathi and Raguchander, 2014). _B. sub-_

has antifungal activity against the fila-

_tilis strain_ Bbv 57 is positive for Iturin

mentous fungus. The fungal cell mem-

( _ItuD_ gene), Surfactin ( _srfA_ gene; _sfp_

brane is the primary site for antimicrobial

gene), Bacilysin ( _bacAB_ gene; _bacD_

attack by antibiotics fengycin. Fengycins

gene), Bacillomycin D ( _bamD_ gene),

are fewer haemolytic than iturins and sur-

Fengycin ( _fenB_ gene), Ericin ( _eriB_ gene),

factins but maintain a strong fungitoxic

Mycosubtilin ( _mycC_ gene) and Subtilin

activity, exclusively against filamentous

( _spaB_ gene) lipopeptides (Ramyabharathi

fungi (Koumoutsi _et al_ 2004; Vanitta-

and Raguchander 2014a). The inhibition

nakom _et al_., 1986). Mechanistically, the

of _Fusarium oxysporum_ f. sp. _gerberae_

activity of fengycins is less well known

might be due to the production of antimi-

compared with other lipopeptides but they

crobial metabolites which are toxic to the

also readily interact with lipid layers and

pathogen. HPLC analysis for _B. subtilis_

to some extent hold the potential to alter

Bbv 57 showed 91.69 μg/μl of surfactin

cell membrane structure and permeability

with the retention time of 2.304 min and

in a dose-dependent manner (Deleu _et al_.,

0.453 μg/μl of Iturin with the retention

2005). The connection of iturins and

time of 8.739 min at 205nm whereas the

fengycins was shown in the antibiosis-

standard iturin and surfactin at 205nm

based biocontrol activity of _Bacillus_

recorded retention time of 8.5 min and 2.5

strains against various pathogens and in

min respectively.

different plant species. In the case of soil-

Crude lipopeptides extracted from

borne diseases, iturin A produced by _B._

the culture supernatant of _B. subtilis_

_subtilis_ RB14 is involved in damping-off

strain, Bbv 57 treatment revealed least

of tomato caused by _Rhizoctonia solani_

egg hatching of 7 juveniles / egg mass

(Asaka and Shoda, 1996). _Bacillus sub-_

with highest juvenile mortality of 87 per

_tilis_ S499 efficiently produces lipopep-

cent of _M. incognita_ with the 25 per cent

tides from the three families, and notably

concentration of the antibiotic after 72 h

produces a wide variety of fengycins

of exposure of nematodes in it. It also in-

(Jacques _et al_., 1999). Mutant analyses in

hibited the mycelial growth of _F. ox-_

_B. subtilis_ subsp. _amyloliquefaciens_ strain

_ysporum_ (28.20 %) at 10 micro litre con-

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_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

centrations. Presence of these diverse

ited

antifungal,

antimicrobial

and

genes plays a crucial role in biological

antinematicial activity, whereas surfactin

control of root knot nematode and

retained the antifungal effect of iturin A

_Fusarium_ in gerbera both in poly house

as a synergistic factor. The lipopeptides

and _in vitro_ through synergistic action of

are less toxic and helps in disease reduc-

antimicrobial peptide genes (Ramyabha-

tion with control of phytopathogens and

rathi, 2015). Among lipopeptides iturin

pathogenic nematodes than agrochemi-

have strong broad spectrum antifungal

cals. _B. subtilis_ seems to be a good bio-

and hemolytic activity. _B. subtilis_ isolate

control agent and a flourishing antagonist

ME488 showed positive reaction for PCR

with lipopeptide antibiotic production.

detection of antimicrobial peptide gene

Further research is needed to know the

_viz., ituC, tuD, bacA, bacD, mrsA_ and

stability of lipopeptide antibiotics subject

_mrsM_ (Chung _et al_. 2008). Ramarathnam

to field conditions. The biosurfactant

_et al._ (2007) detected lipopeptides antibi-

lipopeptides are used in food industry,

otics genes bacillomycin and fengycin

chemical industry, clinics, cosmetics and

using specific primers in _Bacillus_ spp.

used for cleaning oil spills by bioremedia-

Several strains of _Bacillus,_ has

tion approach. These lipopeptides may be

AMP biosynthetic genes _bmyB_ , _fenD_ ,

useful in various industries and agricul-

_ituC_ , _srfAA_ , and _srfAB_ responsible for the

ture as biosurfactants and biopesticides in

suppression of plant pathogens (Gonzalez

plant protection, respectively. However,

_et al_., 2010). Presence of antimicrobial

further research is needed to explore the

peptide (AMP) biosynthetic genes _srfA_

full potential of lipopeptide biosurfac-

(surfactin),

_bacA_

(bacylisin),

_fenD_

tants.

(fengycin), _bmyB_ (bacyllomicin), _spas_

(subtilin), and _ituC_ (iturin) in 184 isolates

**References**

of _Bacillus_ spp (Mora _et al_., 2011). Ca-

****

dena _et al_., 2008 reported that _B. amylo-_

**Arima, K., Kakinuma, A. and Tamura,**

_liquefaciens_ strain FZB42 produced

**G. (1968).** Surfactin, a crystalline

lipopeptides, surfactins, bacillomycin D,

peptidelipid surfactant produced by

and fengycins, which are secondary me-

_Bacillus subtilis_ : isolation, charac-

tabolites with mainly antifungal activity,

terization and its inhibition of fibrin

also decreased gall formation, egg mass

clot formation. _Biochemical and Bi-_

count and juvenile counts of _M. incognita_

_ophysical Research Communica-_

extracted from roots of tomatoes. Kou-

_tions_ **31, 488–494.**

moutsi _et al_. (2004) reported that mutant

**Asaka, O. and Shoda, M. (1996).** Bio-

analyses in _B. subtilis_ sub sp _. amylolique-_

control of _Rhizoctonia solani_ damp-

_faciens_ strain FZB42 with fengycin and

ing-off of tomato with _Bacillus sub-_

bacillomycinD act synergistically and in-

_tilis_ RB14. _Applied Environmental_

hibited the growth of _F. oxysporum in_

_Microbiololgy_ **62, 4081–4085.**

_vitro._ Cellular leakage was also observed

**Bais, H. P., Fall, R. and Vivanco, J. M.**

when bacillomycin L was used at higher

**(2004).** Biocontrol of _Bacillus sub-_

concentration.

_tilis_ against infection of _Arabidopsis_

roots by _Pseudomonas syringae_ is

**4. Concluding remarks**

facilitated by biofilm formation and

surfactin production. _Plant Physiol-_

_Bacillus_ strains do have the capa-

_ogy_ **134, 307–319.**

bility to produce a variety of lipopeptides

**Bonmatin, J. M., Laprevote, O. and**

with outstanding surface active proper-

**Peypoux, F. (2003).** Diversity

ties, disease reduction and biological ac-

among microbial cyclic lipopep-

tions. Among the three lipopeptides, itu-

tides: iturins and surfactins. Activi-

rin and fengycin family separately exhib-

ty-structure relationships to design

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_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

new bioactive agents. _Combinatori-_

responsible for the biosynthesis of

_al Chemistry & High Throughput _

the lipopeptide antibiotics iturin and

_Screening_ **6, 541–556.**

surfactin. _Journal of Fermentation_

**Cadena, M. B., Burelle, N. K., Kathy S.**

_and Bioengineering_ **74, 323–326.**

**L., Santen, E. V. and Kloepper J.**

**Huang, C. C., Ano, T. and Shoda, M.**

**W. (2008).** Suppressiveness of root-

**(1993).** Nucleotide sequence and

knot nematodes mediated by rhizo-

characteristics of the gene, lpa-14,

bacteria. _Biological Control_ **47, 55–**

responsible for biosynthesis of the

**59.** ****

lipopeptide antibiotics iturin A and

**Carrillo, C., Teruel, J. A., Aranda, F. J.**

surfactin from _Bacillus subtilis_

**and Ortiz, A. (2003).** Molecular

RB14. _Journal of Fermentation and_

mechanism of membrane permea-

_Bioengineering_ **76, 445–450.**

bilization by the peptide antibiotic

**Jacques, P., Hbid, C. and Destain, J.**

surfactin. _Biochimica et Biophysica_

**(1999).** Optimization of biosurfac-

_Acta – Biomembranes_ **161, 91–97.**

tant lipopeptide production from

**Chung, S., Kong, H., Buyer, J. S., Lak-**

_Bacillus subtilis_ S499 by Plackett-

**shman, D. K., Lydon, J., Dal Kim,**

Burman design. _Applied Biochemis-_

**S. and Roberts, D. P. (2008).** Isola-

_try and Biotechnology_ **77, 223–233.**

tion and partial characterization of

**Kinsella, K., Schulthess, C. P., Morris,**

_Bacillus subtilis_ ME488 for sup-

**T. F. and Stuart, J. D. (2009).** Rap-

pression of soilborne pathogens of

id quantification of _Bacillus subtilis_

cucumber and pepper. _Applied Mi-_

antibiotics in the rhizosphere. _Soil_

_crobiology and Biotechnology_ **80,**

_Biology and Biochemistry_ **41, 374-**

**115-123.**

**379.**

**Deleu, M., Paquot, M. and Nylander, T.**

**Koumoutsi, A., Chen, X.-H., Henne, A.,**

**(2005).** Fengycin interaction with

**Liesegang, H., Hitzeroth, G.,**

lipid monolayers at the air-aqueous

**Franke, P., Vater, J. and Borriss,**

interface – implications for the ef-

**R. (2004).** Structural and functional

fect of fengycin on biological mem-

characterization of gene clusters di-

branes. _Journal of Colloid and In-_

recting nonribosomal synthesis of

_terface Science_ **283, 358–365.**

bioactive cyclic lipopeptides in _Ba-_

**Desai, J. D. and Banat, I. M. (1997).**

_cillus_

_amyloliquefaciens_

strain

Microbial production of surfactants

FZB42. _Journal of Bacteriology_

and their commercial potential. _Mi-_

**186, 1084-1096.**

_crobiology and Molecular Biology_

**Koumoutsi, A. (2006).** Functional ge-

_Reviews_ **61, 47–64.**

nome analysis of the plant growth

**Georgiou, G., Lin, S. C. and Sharma,**

promoting bacterium _Bacillus amy-_

**M. M. (1992).** Surface-active com-

_loliquefaciens_ strain FZB42; charac-

pounds from microorganisms. _Bio-_

terizing its production and regula-

_technology_ **10, 60–65.**

tion of non-ribosomal peptide syn-

**Gonzalez, S. M. A., Perez-Jimenez, R.**

thetases. Dissertation, Humboldt-

**M., Pliego, C., Ramos, C., de Vi-**

Universitat, Berlin.

**cente, A. and Cazorla, F. M.**

**Kunst, F., Ogasawara, N. and Moszer,**

**(2010).** Biocontrol bacteria selected

**I. (1997).** The complete genome se-

by a direct plant protection strategy

quence of the Gram-positive bacte-

against avocado white root rot show

rium _Bacillus subtilis_. _Nature_ **390,**

antagonism as a prevalent trait.

**249–256.**

_Journal of Applied Microbiology_

**Leclere, V., Marti, R., Bechet, M.,**

**109, 65-78.**

**Fickers, P. and Jacques, P. (2006).**

**Hiraoka, H., Ano, T. and Shoda, M.**

The lipopeptides mycosubtilin and

**(1992).** Molecular cloning of a gene

surfactin enhance spreading of _Ba-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 292

_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

_cillus subtilis_ strains by their sur-

_nal of Fermentation and Bioengi-_

face-active properties. _Archives of_

_neering_ **69, 1-7.**

_Microbiology_ **186, 475–483.**

**Ramarathnam, R., Bo, S., Chen, Y.,**

**Luo, A. H., Pouya, T. F., Roy, A. W.,**

**Fernando, W. G. D., Xuewen, G.**

**Carl, R. L. and Gary, A. J. (2011).**

**and de Kievit, T. (2007).** Molecular

Linking Context with Reward: A

and

biochemical

detection

of

Functional Circuit from Hippocam-

fengycin- and bacillomycin D-

pal CA3 to Ventral Tegmental Area.

producing _Bacillus_ spp., antagonis-

_Science_ **333, 353-357.**

tic to fungal pathogens of canola

**Mora, I., Jordi, C. and Emilio, M.**

and wheat. _Canadian Journal of_

**(2011).** Antimicrobial peptide genes

_Microbiology_ **53, 901–911.**

in _Bacillus_ strains from plant envi-

**Ramyabharathi, SA and Raguchander,**

ronments. _International Microbiol-_

**T. (2014).** Characterization of anti-

_ogy_ **14, 213-223.**

fungal antibiotic synthesis genes

**Moyne, A. L., Shelby, R., Cleveland, T.**

from different strains of _Bacillus_

**E. and Tuzun, S. (2001).** Bacillo-

_subtilis. Journal of Pure and Ap-_

mycin D: an iturin with antifungal

_plied Microbiology_ **8, 2337-2344.**

activity against _Aspergillus flavus._

**Ramyabharathi. SA and Raguchander**

_Jounal of Applied Microbiology_ **90,**

**T. (2014a)**. Efficacy of Secondary

**622–629.**

Metabolites Produced by _Bacillus_

**Nagorska, K., Bikowski, M. and**

_subtilis_ EPCO16 against Tomato

**Obuchowski, M. (2007).** Multicel-

Wilt Pathogen _Fusarium oxysporum_

lular behavior and production of a

f.sp. _lycopersici. Journal of Mycol-_

wide variety of toxic substances

_ogy and Plant Pathology,_ **44(2):**

support usage of _Bacillus subtilis_ as

**148-153.**

a powerful biocontrol agent. _Acta_

**Ramyabharathi, SA. (2015).** Manage-

_Biochimica Polonica_ **54, 495–508**.

ment of _Fusarium_ wilt- root knot

**Ohno, A., Ano, T. and Shoda, M.**

nematode disease complex in gerbe-

**(1995).** Effect of temperature on

ra using lipopeptides producing

production of lipopeptide antibiot-

PGPR under protected cultivation.

ics, iturin A and surfactin, by a dual

PhD. Thesis, __ Tamil Nadu Agricul-

producer, _Bacillus subtilis_ RB14, in

tural University, Coimbatore, Tamil

solid-state fermentation. _Journal of_

Nadu, India.

_Fermentation and Bioengineering_

**Ramyabharathi, SA., Rajendran, L.,**

**80, 517–519.**

**Karthikeyan, G. and Raguchan-**

**Ongena, M. and Jacques, P. (2008).** Ba-

**der, T. (2016)**. Liquid formulation

cillus lipopeptides: versatile weap-

of endophytic bacillus and its stand-

ons for plant disease biocontrol.

ardization for the management of

_Trends in Microbiology_ **16, 115–**

Fusarium wilt in tomato. _Bangla-_

**125.**

_desh journal of botany_ **45(2), 283-**

**Peypoux, F., Pommier, M. T., Das, B.**

**290**

**C., Besson, F., Delcambe, L. and**

**Rudrappa, T., Biedrzycki, M. L. and**

**Michel, G. (1984).** Structures of ba-

**Bais, H. P. (2008).** Causes and con-

cillomycin D and bacillomycin L

sequences of plant associated bio-

peptidolipid antibiotics from _Bacil-_

films. _FEMS Microbiology Ecology_

_lus subtilis. Journal of Antibiotics ****_

**64, 153–166.**

**37, 1600-1604.**

**Sandrin, C., Peypoux, F. and Michel,**

**Phae, C. G., Shida, M. and Kubota, H.**

**G. (1990).** Coproduction of surfac-

**(1990).** Suppressive effect of _Bacil-_

tin and iturin A, lipopeptides with

_lus subtilis_ and its products on phy-

surfactant and antifungal properties,

topathogenic microorganisms. _Jour-_

by _Bacillus subtilis_. _Biotechnology_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 293

_Biotech Sustainability (2017)_

_Lipopeptide Biosurfactants as a Weapon for Plant Disease Management Ramyabharathi et al._

_and Applied Biochemistry_ **12, 370–**

eron _. Journal of Bacteriology_ **183,**

**375.**

**6265-6273.**

**Sankari Meena, K., Ramyabharathi,**

**Vanittanakom, N., Loeffler, W., Koch,**

**S.A. and Raguchander, T. (2016)**.

**U. and Jung, G. (1986).** Fengycin -

Biomanagement

of

nematode-

a novel antifungal lipopeptide anti-

fungus disease complex in tuberose

biotic produced by _Bacillus subtilis_

using plant growth promoting rhi-

F-29-3. _Journal of Antibiotics_ **39,**

zobacteria. _International journal of_

**888–901.**

_science and nature_. **7 (3), 1-9.**

**Vater, J. (1986).** Lipopeptides, an attrac-

**Schippers, B., Bakker, A. W. and Bak-**

tive class of microbial surfactants.

**ker, P. A. H. M. (1987).** Interac-

_Progress in Colloid & Polymer Sci-_

tions of deleterious and beneficial

_ence_ **72, 12–18.**

rhizosphere microorganisms and the

**Yao, S., Gao, X., Fuchsbauer, N., Hil-**

effect of cropping practices. _Annual_

**len, W., Vater, J. and Wang, J.**

_Review of Phytopathology_ **25, 339–**

**(2003).** Cloning, sequencing, and

**358.**

characterization of the genetic re-

**Tsuge, K., Akiyama, T. and Shoda, M.**

gion relevant to biosynthesis of the

**(2001).** Cloning, sequencing, and

lipopeptides iturin A and surfactin

characterization of the iturin A op-

in _Bacillus subtilis._ _Current Micro-_

_biology_ **47, 272–277.**

****

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 294

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P295-304_

**Biotechnology as a Tool for Conservation and Sustaina-**

**ble Utilization of Plant and Seaweed Genetic Resources**

**of Tropical Bay Islands, India**

****

**Pooja Bohra1,*, Ajit Arun Waman2 and Anuraj Anirudhan3**

_1Division of Horticulture and Forestry, ICAR- Central Island Agricultural Research Insti-_

_tute, Port Blair- 744105, Andaman and Nicobar Islands, India; 2Division of Horticulture_

_and Forestry, ICAR- Central Island Agricultural Research Institute, Port Blair- 744105,_

_Andaman and Nicobar Islands, India; 3Division of Fisheries Sciences, ICAR- Central Is-_

_land Agricultural Research Institute, Port Blair- 744105, Andaman and Nicobar Islands,_

_India;*Correspondence: poojabohra24@gmail.com; Tel.: +91-3192-250436_

**Abstract:** Andaman and Nicobar Islands are known to harbor large diversity of plant and

seaweed species, a number of them being endemic. Micropropagation could be used as an

effective tool for large scale multiplication of economically important plants and seaweeds.

The technique could also help in multiplying threatened species to conserve them. _In vitro_

production of pharmaceutical macromolecules could be a viable option for avoiding de-

structive harvesting of plant species. Somaclonal variation, _in vitro_ mutagenesis and trans-

genic could be useful in some cases. Molecular markers could help in assessment of genetic

diversity, DNA barcoding, marker assisted selection etc. The article highlights the im-

portance and relevance of various biotechnological tools in the management of biodiversity

of the fragile ecosystem of Andaman and Nicobar Islands. Various research activities un-

dertaken to conserve species of these islands are also highlighted.

_**Keywords**_ **:** Andaman and Nicobar Islands; Bay of Bengal; biodiversity; endemism; sustain-

able development

**1. Introduction**

the stresses posed by climate change. The

processes of industrialization, commercial

Natural resources including flora

synthetic farming, pollution, deforesta-

and fauna have been the major associates

tion, urbanization etc. are the direct or

of humankind since evolution. We are

indirect consequences of population ex-

largely dependent on these resources for

plosion, which have largely contributed in

our existence and leading a normal day to

misbalancing the resource utilization in a

day life. However, the increasing pres-

sustainable way. Considering the sensitiv-

sures of manmade and natural disasters

ity of these issues, concerted efforts are

have jeopardized these resources in such a

required to protect our valuable resources

way that every year the conservation sta-

so that they are available to the future

tus of a large number of species is pushed

generations too.

further in the _red_ list. On the other hand,

The tropical rainforests are known

our dependence on a few species for

to harbor wide array of unique floral di-

meeting most of our requirements has

versity and a number of mega biodiversity

worsened the situation by eliminating the

hotspots are located in these regions. The

so called non-useful types, which could

Andaman and Nicobar Islands in the Bay

be carrying potent genes for mitigating

of Bengal (a Union Territory of the India)

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 295

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

are strategically placed between two such

actives which are of great interest in the

biodiversity hotspots _viz_. Arakan Yoma

medical field. Andaman and Nicobar Is-

ranges of Myanmar and the Sumatra. This

lands, with 1/3rd of India's coastal line,

has resulted in unique confluence of flora

supports good diversity of seaweeds and

of both these regions in ANI (Pandey and

so far about 206 species including com-

Diwakar, 2008). The islands are charac-

mercially important agarophytes and al-

terized by lush green forests occupying

ginophytes have been reported from here.

about 81.8% of the total geographical ar-

Interestingly, different parts of

ea. There are more than 2,314 species of

these islands are inhabited by six native

flowering plants reported from these is-

tribes since centuries. Two Mongoloid

lands so far (Murugan _et al_., 2016) and

tribes, Shompen and Nicobarese, reside in

the number may still increase considering

the Nicobar groups of islands, while the

the larger unexplored areas. Furthermore,

tribes of Negrito origin i.e. Jarawa, Great

these islands are known to harbor a large

Andamanese, Onge and Sentinelese, are

number of endemic species in a relatively

residing in the Andaman islands. These

smaller geographical area of about 8,249

tribes differ in most of their cultures and

km2. So far, about 300 species of endemic

habits. Some of the local species are pres-

plants have been reported from ANI

ently being used by the native tribes for

(Murugan _et al_., 2016). Majority of the

food, medicine, fodder, fuel and other

diversity is still unexplored and consider-

purposes. Similarly, the settler population

able scope exists for utilizing these spe-

migrated from different parts of mainland

cies for the betterment of humankind.

India are utilizing these plants for variety

Since, horticulture has been the major

of purposes. Underutilized fruits, indige-

source of livelihood and nutritional secu-

nous leafy vegetables and tuber crops

rity for the island dwellers (Singh _et al_.,

have immensely contributed in the liveli-

2016), the present article focuses on the

hood and nutritional security of the island

management of genetic resources of hor-

dwellers. Further, a large number of wild

ticultural crops including their wild rela-

relatives of cultivated crop plants have

tives.

been reported to occur in these islands.

Similarly, seaweeds are important

This diversity needs to be assessed and

component of the diversity of these is-

utilized sustainably to strengthen our re-

lands. The macro algae mainly belonging

source base, while striking the fine bal-

to Chlorophtya, Phaeophtya and Rhodo-

ance between development and ecological

phyta are found attached to the substra-

soundness (Waman and Bohra, 2016).

tum in benthic zone. They are non-

Biotechnology could be an effec-

flowering plants with true roots, stem and

tive tool for achieving this target through

leaves, and are known to contribute sub-

the application of techniques namely mi-

stantially to the primary production in the

cropropagation, _in vitro_ production of

marine environment. Seaweeds have been

secondary metabolites, _in vitro_ mutation,

used for centuries as food either in raw or

_in vitro_ conservation, marker assisted se-

processed form in many of the South East

lection, genetic diversity assessment, de-

Asian countries and the trend is picking

velopment of trait specific markers _etc_.

up in the western countries as well. Sea-

(Waman _et al_., 2015; Waman and Bohra,

weeds are the only known natural sources

2016). Present chapter concerned explor-

of hydrocolloids viz. agar, algin and car-

ing the possibility of utilizing various bio-

rageenan. These multipurpose products

technological tools for management of

find application in industrial, pharmaceu-

biodiversity of the tropical Bay Islands of

tical and medicinal fields. Besides, sea-

India.

weeds are used as animal feed and biofer-

tilizers in crop production. The current

**2. Relevance of biotechnological ap-**

research on seaweeds is centering on bio-

**proaches and tools for island ecosystem**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 296

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

_2.1. Micropropagation and in vitro con-_

hardening has been emphasized in _low_

_servation_

_price-high value_ crops, especially medic-

Andaman and Nicobar Islands are

inal plants (Waman and Bohra, 2016).

home to many endemic species belonging

_In vitro_ conservation is a tech-

to different botanical families, which are

nique, wherein plant tissues are cultured

of potential economic/ ecological signifi-

_in vitro_ under sub-optimal growth condi-

cance and require timely attention for

tions in order to reduce the frequency of

their conservation. A number of species

sub-culturing. The technique has proven

belonging to rare, endangered and threat-

to be very efficient for short to medium

ened (RET) category are also found dis-

term storage of a number of species. Con-

tributed in these islands. Some of these

sidering vulnerability of the islands to

species have problems in natural regener-

natural disasters, the endemic species

ation owing to the damage caused by

could be conserved under _in vitro_ condi-

birds/animals, poor seed viability, anthro-

tions and copies of the same could be

pogenic pressure etc. For example, _Myris-_

maintained at other laboratories in main-

_tica andamanica_ is a vulnerable wild

land India. Some horticulturally important

nutmeg species endemic to the islands

endemic species of islands have been

and micropropagation could help to mul-

listed in Table 1.

tiply it in large number. Similarly, natu-

****

ral populations of an underutilized fruit

_2.2. In vitro production of secondary me-_

species – blood fruit ( _Haematocarpus_

_tabolites_

_validus_ ) are dwindling (Bohra _et al_.,

A large number of species are val-

2016a) and micropropagation could help

ued for their medicinal properties. How-

in saving the species from extinction from

ever, the yield of bioactive molecules is

the region. Experiments are in progress to

very low in most of the cases. At times,

standardize micropropagation protocol for

complete plants are destroyed for obtain-

this species.

ing the desired active ingredients. Such

Secondly, banana is a major crop

practice of destructive harvesting has

of the islands covering more than half of

been a cause of concern as it tends to

the area under fruit crops cultivation.

threaten the natural populations to a great

However, the islands are largely depend-

extent (Waman and Bohra, 2013). Bio-

ent on the planting material supplies from

technological tools could help in large

mainland India. This has probably result-

scale quality production of secondary me-

ed in inadvertent introduction of dreadful

tabolites under _in vitro_ conditions. Induc-

banana bunchy top virus in the pristine

tion of callus and extraction of active in-

islands. Developing protocols for _in vitro_

gredients from them has been suggested

multiplication of locally suitable varieties

as an important alternative for obtaining

would help in production of their quality

the desired molecules without disturbing

planting material. The importance of tis-

the wild populations (Waman _et al_.,

sue culture technology for the island

2015).

farmers has been emphasized earlier

(Bohra _et al_., 2016b). Considering this,

_2.3. Creation of variability_

experiments have been initiated at au-

There are a few species e.g. man-

thors' institute for optimization of proto-

gosteen ( _Garcinia mangostana_ ), which

cols for locally popular banana varieties

are economically important for the islands

of the islands. Other commercializable

but have narrow genetic base. For im-

crops of the islands include variety of or-

provement of such crops, creation of vari-

chids and ornamental plants, which need

ability is possible through the induction of

further attention. Use of low cost options

somaclonal variations in tissue culture or

including concurrent ex vitro rooting cum

using the technique of _in vitro_ mutation

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 297

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

**Table 1:** List of selected endemic species of horticultural importance reported from ANI

**Family**

**Species**

Anacardiaceae

_Mangifera andamanica, M. nicobarica, Semecarpus kurzii_

Apocyanaceae

_Carissa andamanensis_

Arecaceae

_Phoenix andamanensis_

Clusiaceae

_Garcinia andamanica, G. cadelliana, G. calycina, G. dhanikhariensis,_

_G. kingii, G. kurzii, G. microstigma_

Dilleniaceae

_Dillenia andamanica_

Dioscoreaceae

_Dioscorea vexans_

Musaceae

_Musa bulbisiana var. andamanica, M. indandamanensis, M. sabuana,_

_M. paramjitiana_

Myristicaceae

_Myristica andamanica, Knema andamanica_

Myrtaceae

_Syzygium andamanicum, S. manii_

Pandanaceae

_Pandanus lerum var. lerum_

Tiliaceae

_Grewia indandamanica_

Orchidaceae

_Vanilla andamanica, Eulophia nicobarica_

Zingiberaceae

_Kaempfaria siphonantha_

breeding. Being cornerstone of breeding

species, which play pivotal role in the

activity, variability created will also be

lives of island dwellers. The generated

useful in development of island suitable

information from such studies could be

varieties.

useful for selection of parents for carrying

out conventional crop improvement pro-

_2.4. Estimation of genetic diversity_

grammes.

The ANI is considered as a centre

of origin or diversity for a number of spe-

_2.5. Evolutionary studies_

cies e.g. wild populations of _Piper betle_

From time to time, a number of

are present in these islands. Both inter and

new species have been reported by vari-

intra specific diversity occurs for a num-

ous research workers in the ANI, howev-

ber of species of ecological and economic

er, absence of requisite scientific infor-

importance (Singh _et al_., 2016). This di-

mation about their genetic relationship

versity needs to be tapped in such a way

with their existing commercial counter-

that commercially viable types are identi-

parts could delay the process of their

fied for the benefit of island farmers. Se-

commercial utilization. Molecular phylo-

lection of such elite types needs systemat-

genetic studies could help in this regard.

ic characterization. Molecular characteri-

For example the presence of Indo-

zation is one of the most reliable methods

Myanmarese as well as Indonesian forms

of estimating such diversity. Further,

of

_Erianthus_

_arundinaceus_

(Retz.)

available diversity could also be com-

Jeswiet, a wild relative of sugarcane

pared with their counterparts occurring in

( _Saccharum officinarum_ ) was confirmed

mainland India or other parts of the

through the use of molecular markers

world. Through this information, the stud-

(Nair and Mary, 2006). They concluded

ied population could be categorized in a

that collections from North Andaman

way to pave the way for future breeding

were more similar to Indo-Myanmarese

programmes. A few attempts were initiat-

form, while that from Nicobar were of

ed at the authors' Institute or other organ-

Indonesian form. This report supports the

izations in country in this direction, which

fact that ANI harbor confluence of flora

have been summarized in Table 2. The

of two different regions. Similarly, genet-

similar technique could also be employed

ic diversity between the collections of

for diversity estimation and further stud-

_Musa balbisiana_ from mainland India and

ies in non-traditional and underutilized

the islands suggested that ANI is one of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 298

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

**Table 2:** Selected examples of application of molecular markers for diversity assessment in

various horticultural crops of ANI ****

**Species**

**Molecular**

**Salient findings**

**Reference**

**marker**

**used**

_Morinda citrifolia_

RAPD,

Distinct clustering of collections from Singh _et al_.,

ISSR

ANI islands

2012

_Morus laevigata_

RAPD,

Significant genetic divergence be- Naik _et al_.,

ISSR

tween collection from mainland India 2015

and Andaman Is.

_Carica papaya_

ISSR

Geographical clustering of collections Sudha _et al_.,

from various islands

2013

_Bouea oppositifo-_

SSR

Genetic similarity of 43% between Damodaran _et_

_lia,_

_Mangifera_

both the species of Anacardiaceae _al_., 2013

_andamanica_

family

_Cocos nucifera_

RAPD

Two distinct clusters based on mor- Sankaran

_et_

phological and nut parameters

_al_., 2012

SSR

High genetic diversity among the col-

Rajesh _et al_.,

lections, separate clustering of tall and 2008

dwarf types

_Syzygium cuminii_

RAPD,

Genotype collected from Car Nicobar Ahmad _et al_.,

ISSR

was distinct amongst 21 island geno- 2012

types and 2 mainland genotypes

_Colocasia_

_escu-_

RAPD,

Island collections were distinctly dif-

Singh _et al_.,

_lenta_

ISSR

ferent from 3 released varieties used 2012

as reference genotypes

_Mangifera indica_

SSR

Separate clustering of monoembryon- Damodaran _et_

ic, polyembryonic and wild mango _al_., 2012

species

Orchid species

RAPD

Distinct clustering of green orchid Singh and Sri-

species

vastava, 2010

_Costus speciosus_

RAPD

Intra-specific variations to the extent Mandal _et al_.,

of 35%

2007

the centres of diversity of the species

fied during early stages of development.

(Uma _et al_., 2005). This new piece of in-

In case of medicinal plants, identification

formation would go a long way in devis-

of markers linked to the presence of their

ing conservation strategy of _Musa_ spp.

active ingredients would be very useful.

from ANI.

Flowering behavior related markers in

mango have been identified, which could

_2.6. Development of trait linked markers_

be of practical utility (Damodaran _et al_.,

As previously mentioned, a num-

2006).

ber of perennial dioecious species are

known to occur in the islands. Important

**3. Applications of biotechnological in-**

being _Myristica andamanica, Knema an-_

**terventions in seaweeds**

_damanica, Horsfieldia glabra_ , _Piper_

_betle_ , _Carica papaya_ , _Garcinia_ _spp_.,

In the wake of increasing demand

_Momordica spp_. etc. Development of sex

for seaweeds for various applications, col-

linked markers would be a boon for the

lections from the natural habitats are not

conservation and utilization of these spe-

sufficient to meet the requirement. Artifi-

cies as the desired plants could be identi-

cial culture of commercially exploited

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 299

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

seaweeds still remains the major means of

be still in nascent stage. _In vitro_ derived

raw material supply to the industries.

calli of economically important seaweeds

However, on farm culture of seaweeds

have been commonly used for mainte-

using vegetative fragments generally re-

nance and clonal propagation of seed

sults in reduced growth rate and produc-

stock for mariculture (Dawes and Koch,

tivity over time. In order to overcome the-

1991; Reddy _et al._ , 2003; Rajakrishna _et_

se problems and to develop strains with

_al._ , 2004; Reddy _et al_ , 2008). Studies

improved growth and yield parameters,

suggested that growth and quality of car-

biotechnological techniques such as mi-

rageenan obtained from tissue cultured

cropropagation, transgenics and molecu-

_Kappaphycus alvarezii_ were superior

lar markers have been tried in seaweed

when compared with conventional vege-

breeding and genetic studies. Microprop-

tative fragments (Rajakrishna _et al._ ,

agation is a tool to produce large number

2007). Non-availability of standardized

of seeding material from explants of sea-

protocols for obtaining viable axenic cul-

weed possessing desirable traits. Among

tures from wild, lack of knowledge about

the three cellular organization types in

the role of culture incubation conditions,

seaweeds, most of the work on micro-

media supplements, explanting season

propagation has been reported on paren-

etc. on callus induction are the major lim-

chymatous and pseudo-parenchymatous

iting factors in the development of sea-

types (Aguirre-Lipperheide _et al._ , 1995).

weed micropropagation _._ Table 3 repre-

Although Gibor attempted to cultivate

sents list of selected seaweed species re-

seaweeds axenically in as early as 1950,

ported from ANI in which micropropaga-

the first successful attempt is considered

tion has been attempted elsewhere.

to be made during 1978 by Chen and Tay-

Molecular marker assisted breed-

lor in _Chondrus crispus_ (Yokoya and

ing based on quantitative trait loci (QTLs)

Valentin, 2011). Seaweed micropropaga-

offers several advantages over traditional

tion protocols have been developed in

phenotypic based breeding. Development

similar lines with that of higher plants and

of markers linked with genes of desirable

so far micropropagation protocols have

traits could increase accuracy and effi-

been developed in about 85 species (Red-

ciency of the breeding process. Several

dy _et al_ , 2008). Considering the diversity

molecular markers such as Random Am-

present and diversified applications of

plified Polymorphic DNA (RAPD), Inter

seaweeds, the technique is considered to

Simple Sequence Repeat (ISSR), Se-

****

**Table 3:** List of seaweed species reported from ANI in which cell and tissue culture has

been accomplished elsewhere ****

**Seaweed**

**Reference**

**Chlorophyta** __

_Boergesenia forbessi_

Enomoto and Hirose, 1972

_Bryopsis plumosa_

Tatewaki and Nagata, 1970

_E. intestinalis_

Polne-Fuller and Gibor, 1987; Russig and Cosson,

2001

**Rhodophyta** __

_Gracilaria corticata_

Subbaraju _et al._ , 1981; Rajakrishna _et al.,_ 2007

_G. verrucosa_

Gusev _et al.,_ 1987; Kaczyna and Megnet, 1993

_Gelidiella acerosa_

Rajakrishna _et al._ , 2004

**Phaeophyta** __

_Grateloupia filipina_

Huang and Fujita, 1997; Baweja and Sahoo, 2009

_Hypnea musciformis_

Rajakrishna _et al.,_ 2007

_Sargassum tenerri-_

Rajakrishna _et al._ , 2007

_mum_

_Turbinaria conoides_

Rajakrishna _et al._ , 2007

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 300

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

quence Characterized Amplified Region

**4. Perspectives**

(SCAR), Amplified Fragment Length

Polymorphism (AFLP), Sequence Tagged

The tropical islands in the Bay of

Site (STS), Microsatellites etc. have been

Bengal are known to harbor considerable

developed in seaweeds (Lin _et al.,_ 2012).

diversity of flowering plants as well as

Molecular markers have also been used

seaweed species. Most of these species

for DNA barcoding, which could be used

are ecologically important, while a large

for assessing genetic diversity and taxo-

number of species could be utilized for

nomic identification of seaweeds species,

their commercial potential. The diversi-

which lack reliable morphological charac-

fied applications of biotechnology could

ters for identification. For this purpose,

be helpful in carrying out the major activ-

RAPD, Restriction Fragment Length Pol-

ities related to management of this unique

ymorphism (RFLP), AFLP, Microsatel-

diversity including conservation, regener-

lites, Single Nucleotide Polymorphism

ation, characterization, utilization etc.

(SNP) etc. have been used (Liu and

Though some efforts have been initiated

Cordes, 2004). Further, molecular mark-

in this direction, there is vast scope for

ers could also be used for identification of

employing biotechnological tools for sus-

invasive seaweed species, which could

tainable development of such fragile eco-

pose threats to the pristine biodiversity of

system in near future.

these Islands. Transgenic technology has

been mainly focused on genetic engineer-

___
___

## Acknowledgements

### ing of economically important seaweeds

(Lin _et al.,_ 2012) like _Gracilaria, Kap-_

Authors are thankful to the Direc-

_paphycus, Porphyra_ and _Ulva_. As with

tor, ICAR-CIARI, Port Blair for provid-

any other transgenic technology, the safe-

ing necessary facilities for conduct of var-

ty issues associated with culture of genet-

ious studies and extending support and

ically modified seaweeds should be of

guidance.

prime consideration. Transgenic technol-

ogy has been attempted in Chlorophyta

**References**

(Huang _et al_., 1996), Rhodophyta (Wang

_et al.,_ 2010) and Phaeophyta (Zhang _et_

**Aguirre-Lipperheide,**

**M.,**

**Estrada-**

_al.,_ 2008). Production of valuable prod-

**Rodríguez, F. J. and Evans, L. V.**

ucts in an indoor cultivation system with

**(1995).** Facts, problems and need in

proper biosafety from transgenic kelp

seaweed tissue culture – an apprais-

sporophyte has been successfully demon-

al. _Journal of Phycology_ **31, 677-**

strated (Qin _et al._ , 2005).

**688.**

The general perception that com-

**Ahmad, I., Bhagat, S., Simachalam, P.**

pounds and chemicals produced from

**and Srivastava, R. C. (2012).** Mo-

natural resources are safe and pose less

lecular characterization of _Syzygium_

risk to health has brought seaweeds in the

_cuminii_ from A&N Islands. _Indian_

limelight for extracting environmental

_Journal of Horticulture_ **69, 306-**

friendly natural compounds and chemi-

**311.**

cals. The development of species-specific

**Baweja, P. and Sahoo, D. (2009).** Re-

_in vitro_ cell and tissue culture technology

generation studies in _Grateloupia_

is essential for use of other biotechnologi-

_filicina_ (J.V. Lamouroux) C. Agardh

cal tools for genetic improvement and

– an important Carrageenophyte and

production of high value compounds from

edible seaweed. _Algae_ **24, 163-168.**

seaweeds. With plethora of problems

**Bohra, P., Waman, A. A., Sakthivel, K.,**

faced in seaweed cultivation, the interven-

**Gautam, R. K. and Dam Roy, S.**

tion of biotechnological tools is essential

**(2016a).** Plant Tissue Culture: A vi-

for overall growth of this sector.

able technique for augmenting the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 301

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

productivity of banana in Andaman

phyte marine algae. _Marine Biology_

and Nicobar Islands. Technical Bul-

**95, 593–597.**

letin, ICAR-CIARI, Port Blair. **pp.**

**Huang, W. and Fujita, Y. (1997).** Callus

**1-12.**

induction and thallus regeneration

**Bohra, P., Waman, A.A. and Dam Roy,**

insome species of red algae. _Phyco-_

**S. (2016b).** Khoon phal ( _Haemato-_

_logia Research_ **45, 105–111.**

_carpus validus_ (Miers.) Bakh. ex F.

**Huang, X., Weber, J. C., Hinson, T. K.,**

Forman). Technical Bulletin, ICAR-

**Matheison, A. C. and Minocha, S.**

CIARI, Port Blair. **pp. 1-8.**

**C. (1996).** Transient expression of

**Damodaran, T., Ahmad, I. and Naga-**

the GUS reporter gene in the proto-

**rajan, B. (2013).** _Bouea oppositifo-_

plasts and partially digested cells of

_lia_ \- A fast disappearing native

_Ulva lactuca_ L. (Chlorophyta). _Bo-_

mango

genetic

resource

_tanica Marina_ **39, 467-474.**

from Andamans: morphological and

**Kaczyna, F. and Megnet, R. (1993)**. The

molecular evidences. _Indian Journal_

effects of glycerol and plant growth

_of Horticulture_ **70, 161-164.**

regulators on _Gracilaria verrucosa_

**Damodaran, T., Kannan, R., Ahmad,**

(Gigartinales, Rhodophyceae). _Hy-_

**I., Srivastava, R.C., Rai, R. B. and**

_drobiologia_ **268, 57–64.**

**Umamaheshwari, S. (2012).** As-

**Hanzhi L., Song Q. and Peng J. (2012).**

sessing genetic relationships among

Biotechnology of seaweeds: Facing

mango ( _Mangifera indica_ L.) acces-

the coming decade. _In_ : Se-Kwon

sions of Andaman Islands using in-

Kim (Eds), Handbook of marine

ter simple sequence repeat markers.

macroalgae: Biotechnology and Ap-

_New Zealand Journal of Crop and_

plied Phycology **pp. 424-430.**

_Horticultural Sciences_ **40, 229-240.**

**Liu, Z. J. and Cordes, J. F. (2004).**

**Damodaran, T., Medhi, R.P., Kapil**

DNA marker technologies and their

**Dev, G., Damodaran, V., Rai, R.**

applications in aquaculture genetics.

**B. and Kavino, M. (2006).** Identifi-

_Aquaculture_ **238, 1-37.**

cation of molecular markers linked

**Mandal,**

**A.B.,**

**Thomas,**

**V.A.,**

with differential flowering behavior

**Elanchezhian, R. (2007).** RAPD

of mangoes in Andaman and Nico-

Pattern of _Costus speciosus_ Koen

bar Islands. _Current Science_ **92,**

Ex. Retz., An Important Medicinal

**1054-1056.**

Plant from the Andaman and Nico-

**Dawes, C. J. and Koch, E. W. (1991)**.

bar Islands. _Current Science_ **93,**

Branch, micropropagules and tissue

**369-373.**

culture of the red algae _Eucheuma_

**Murugan, C., Prabhu, S., Sathiyasee-**

_denticulatum_ and _Kappaphycus al-_

**lan, R. and Pandey, R.P. (2016).** A

_varezii_ farmed in the Philippines.

checklist of plants ofAndaman and

_Journal of Applied Phycology_ **6,**

Nicobar islands (Eds. Paramjit

**21–24**.

Singh and Arisdason, W.). ENVIS

**Enomoto, S. and Hirose, H. (1972).** Cul-

Centre on Floral Diversity, Botani-

ture studies on artificially induced

cal Survey of India, Kolkata, India.

aplano spores and their development

**Naik, G. V., Dandin, S. B., Tikader, A.,**

in the marine alga _Boergesenia_

**Pinto, M. V. (2015).** Molecular Di-

_forbesii_ (Harvey) Feldmann (Chlo-

versity of Wild Mulberry ( _Mo-_

rophyceae, Siphonocladales). _Phy-_

_rus_ spp.) of Indian Subcontinent.

_cologia_ **11, 119–122.**

_Indian Journal of Biotechnology_ **14,**

**Gusev, M. V., Tambiev, A. H., Kiri-**

**334-343.**

**kora, N. N., Shelyastina, N. N. and**

**Nair, N.V. and Mary, S. (2006).** RAPD

**Aslanyan, R. R. (1987).** Callus

analysis reveals the presence of

formation in seven species of agaro-

mainland Indian and Indonesian

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 302

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

forms of _Erianthus arundinaceus_

_varezii_ (Gigartinales, Rhodophyta).

(Retz.) Jeswiet in the Andaman–

_Journal of Phycology_ **39, 610–616.**

Nicobar Islands. _Current Science_ **90,**

**Russig A M and Cosson J (2001)** Plant

**1118-1122.**

regeneration from protoplasts of _En-_

**Pandey, R.P. and Diwakar, P.G. (2008).**

_teromorpha intestinalis_ (Chloro-

An integrated check-list flora of

phyta, Ulvophyceae) as seedstock

Andaman & Nicobar Islands, India _._

for macroalgal culture. _Journal of_

_Journal of Economic and Taxonom-_

_Applied Phycology_ **13, 103–108.**

_ic Botany_ **32, 403–500.**

**Sankaran, M., Damodaran, V., Singh,**

**Polne-Fuller, M. and Gibor, A (1987)**

**D. R., Jaisankar, I. and Jerard, B.**

Callus and callus likegrowth in sea-

**A. (2012).** Characterization and Di-

weeds: induction and culture. _Pro-_

versity Assessment in Coconut Col-

_ceedings of International Seaweed_

lections of Pacific Ocean Islands

_Symposium_ **12, 131–138.**

and Nicobar Islands. _African Jour-_

**Qin, S. and Tseng, C. (2005).** Trans-

_nal of Biotechnology_ **11, 16320-**

forming kelp into a marine bioreac-

**16329.**

tor. _Trends in Biotechnology_ **23,**

**Singh, D. R. and Srivastava, R. C.**

**264-268.**

**(2010).** Genetic Diversity Analysis

**Rajakrishna Kumar, G., Reddy, C. R.**

among the Indigenous Orchids of

**K. and Jha, B. (2007).** Callus in-

Bay of Islands Using RAPD Mark-

duction and thallus regeneration

ers. _Indian Journal of Horticulture_

from the callus of phycocolloid

**13, 142-145.**

yielding seaweeds from the Indian

**Singh, D. R., Singh, S., Minj, D., An-**

coast. _Journal of Applied Phycology_

**bananthan, V., Salim, K. M., Ku-**

**19, 15–25.**

**mari, C. and Varghese, A. (2012).**

**Rajakrishna Kumar, G., Reddy, C. R.**

Diversity of _Morinda citrifolia_ L.

**K., Ganesan, M., Tiruppathi, S.,**

in Andaman and Nicobar Islands

**Dipakkore, S., Eswaran, K., Sub-**

(India) assessed through morpholog-

**ba Rao, P. V. and Jha, B. (2004).**

ical and DNA markers. _African_

Tissue culture and regeneration of

_Journal_

_of_

_Biotechnology_

**11,**

thallus from callus of _Gelidiella ac-_

**15214-15225.**

_erosa_

(Gelidiales,

Rhodophyta).

**Singh, S., Singh, D. R., Faseela, F.,**

_Phycologia_ **43, 596–602.**

**Kumar, N., Damodaran, V. and**

**Rajesh, M. K., Nagarajan, P., Jerard,**

**Srivastava, R. C. (2012).** Diversity

**B. A., Arunachalam, V. and**

of 21 taro ( _Colocasia esculenta_ (L.)

**Dhanapal. R. (2008).** Microsatellite

Schott)

accessions

variability of coconut accessions

of Andaman Islands. _Genetic Re-_

( _Cocos_

_nucifera_

L.)

_sources and Crop Evolution_ **59,**

from Andaman and Nicobar Islands.

**821-829.**

_Current Science_ **94, 1627-1631.**

**Singh, S., Waman, A. A., Bohra, P.,**

**Reddy, C. R. K., Jha, B., Fujita, Y. and**

**Gautam, R. K. and Dam Roy, S.**

**Ohno, M. (2008).** Seaweed micro-

**(2016).** Conservation and sustaina-

propagation techniques and their po-

ble utilization of horticultural biodi-

tentials: an overview. _Journal of_

versity in tropical Andaman and

_Applied Phycology_ **20, 609–617.**

Nicobar Islands, India. _Genetic Re-_

**Reddy, C. R. K., Raja Krishna Kumar,**

_sources and Crop Evolution_ **63,**

**G., Eswaran, K., Siddhanta, A. K.**

**1431–1445.**

**and Tewari, A. (2003).** _In vitro_ so-

**Subbaraju, D. P., Ramakrishna, T.,**

matic embryogenesis and regenera-

**Sreedhara, S. and Murthy, M.**

tion of somatic embryos from pig-

**(1981).** Effects of some growth reg-

mented callus of _Kappaphycus al-_

ulators on _Gracilaria corticata_ , an

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 303

_Biotech Sustainability (2017)_

_Genetic Resources Sustainability Bohra et al._

agarophyte. _Aquatic Botany_ **10, 75–**

Publications, New Delhi, India. **pp.**

**80.**

**141-156.**

**Sudha, R., Singh, D. R., Sankaran, M.,**

**Waman, A. A. and Bohra P. (2016).**

**Singh, S., Damodaran, V. and**

Sustainable development of medici-

**Simachalam, P. (2013).** Genetic di-

nal and aromatic Plants sector in In-

versity analysis of papaya ( _Carica_

dia: an overview. _Science and Cul-_

_papaya_

L.)

genotypes

_ture_ **82, 245-250.**

in Andaman Islands using morpho-

**Waman, A. A. and Bohra, P. (2013).**

logical and molecular markers. _Afri-_

Choice of explants- a determining

_can Journal of Agricultural Re-_

factor in tissue culture of Ashoka

_search_ **8, 5187-5192.**

( _Saraca indica_ L.). _International_

**Tatewaki, M. and Nagata, K. (1970).**

_Journal of Forest Usufructs Man-_

Surviving protoplasts _in vitro_ and

_agement_ **14, 10-17.**

their development in _Bryopsis_.

**Wang, J., Jiang, P., Cui, Y., Deng, X.,**

_Journal of Phycology_ **6, 401–403.** __

**Li, F., Liu, J. and Qin, S. (2010).**

**Uma, S., Siva, S. A., Saraswathi, M. S.,**

Genetic transformation in _Kap-_

**Durai, P., Sharma, T. V. R. S.,**

_paphycus alvarezii_ using micropar-

**Singh, D. B., Selvarajan, R., Sa-**

ticle bombardment: a potential strat-

**thiamoorthy, S. (2005).** Studies on

egy for germplasm improvement.

the origin and diversification of In-

_Aquaculture_

_International_

**18,**

dian wild banana ( _Musa balbisiana_ )

**1027–1034.**

using arbitrarily amplified DNA

**Yokoya,**

**N.**

**S.**

**and**

**Yoneshigue-**

markers. Journal _of Horticultural_

**Valentin, Y. (2011).** Micropropaga-

_Science and Biotechnology_ **80, 575-**

tion as a tool for sustainable utiliza-

**580.**

tion and conservation of populations

**Waman, A. A., Bohra, P., Sathyana-**

of Rhodophyta. _Brazilian Journal of_

**rayana, B. N. and Hanumantha-**

_Pharmacognosy_ **21, 334-339.**

**raya, B. G. (2015).** _In vitro_ ap-

**Zhang, Y. C., Jiang, P., Gao, J. T.,**

proaches in medicinal plants- a via-

**Liao, J. M., Sun, S. L. and Shen,**

ble strategy to strengthen the re-

**Z. L. (2008).** Recombinant expres-

source base of plant based systems

sion of rt-Pa gene (encoding Retep-

of medicines. _In:_ Biotechnological

lase) in gametophytes of the sea-

approaches for sustainable devel-

weed _Laminaria japonica_ (Laminar-

opment. Pullaiah, T. (ed.). Regency

iales, Phaeophyta). _Science China_

_Life Sciences_ **51, 1116-1120.**

****

****

__

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 304

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P305-321_

**Plantibodies for Global Health: Challenges and**

**Perspectives**

****

**Prasad Minakshi1,*, Basanti Brar1, Manimegalai Jyothi1, Ikbal1, Koushlesh Ranjan1,**

**Upendra Pradeep Lambe1 and Gaya Prasad2**

_1Department of Animal Biotechnology, LLR University of Veterinary and Animal Sciences,_

_Hisar 125004, Haryana, India; 2Sardar Vallabhbhai Patel University of Agriculture and_

_Technology, Meerut 250110, Uttar Pradesh, India; *Correspondence:_

_minakshi.abt@gmail.com; Tel: 09992923330_

**Abstract:** Antibodies are the important part of adaptive immune system. Plants do not natu-

rally make the antibodies; but, they can be produced in plants by introducing antibody-

coding genes from humans and animals. Plant derived antibodies are called as plantibodies

and known to work in the same way as mammalian antibodies. The plantibodies bioproduc-

tion offers several advantages over the production of antibodies using mammals. Plants

are more economic than all other forms of creating antibodies and the technology for ob-

taining and maintaining them is already present. Plantibodies are safer in use because,

plants reduce the chance of coming in contact with pathogens. Plantibodies can be made at

an affordable cost using plants as the genetic engineering methods are well established for

agricultural crops such as tobacco, tomato, potato, soyabean, alfalfa, rice, and wheat. Plan-

tibodies production is cost effective and safe. This review highlights the methods of produc-

tion and purification of plantibodies as well as the various types of pharmaceutical antibod-

ies produced in transgenic plants. ****

_**Keywords**_ **:** Antibody production; human and animal health **;** plants

****

**1. Introduction**

of plants and today it became the front-

runners among plant-derived pharmaceu-

Plantibody is an antibody that is

tical proteins. Plants offer many ad-

produced by plants that have been genet-

vantages and potential benefits for the

ically engineered with animal DNA. The-

production of recombinant proteins in

se plant produced antibodies, namely

terms of cost, safety and scalability (Sto-

plantibodies were first demonstrated by

ger _et al._ , 2014). For large-scale needs,

Hiatt _et al_. (1989) and Duering _et al_.

production of recombinant protein using

(1990). They demonstrated that plants can

transgenic plants as bioreactors is more

express and assemble functionally active

economical than alternative systems such

antibodies. Plants are used in this tech-

as cell culture based antibody production.

nology as antibody factories, to produce

The main anticipated advantage is cost

large amount of clinically viable proteins

saving, low cast biomass production using

by using the endomemebrane and secreto-

agriculture in a short time without any

ry systems of plants and later it will be

specialized equipment or expensive me-

derived from those plant tissue (Jain _et_

dia. Moreover, scale up process can be

_al._ , 2011). For more than a decade, vari-

achieved quickly and inexpensively by

ous kinds of antibody formats have been

cultivating more land (Stoger _et al.,_

produced and studied in different species

2014). Other important advantage is the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 305

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

ability of plant cells to correctly fold and

cells with the cost of plantibody produc-

assemble antibody fragments, single chain

tion in plants are substantially lesser

peptides and also full-length multimeric

(Oluwayelu _et al_., 2016). Different types

proteins and heterologous proteins such as

of plantibodies are produced from plants

antibodies accumulate in large amount to

(Figure 1).

plant cells. Protein synthesis, secretion

and folding as well as posttranslational

modifications like signal peptide cleav-

age, di-sulphide bond formation and the

initial stages of glycosylation are very

similar in plants and animals. There is

very low risk of product contamination by

mammalian viruses, bacterial toxins,

blood-borne pathogens and oncogenes

**Figure 1:** Different types of plantibodies

(Jain _et al.,_ 2011). Use of these plantibod-

are produced in plants. ****

ies avoids the ethical issue of producing

transgenic animals, elimination of purifi-

**2. Plants used as expression host for**

cation requirement when the plant con-

**plantibody production**

taining recombinant protein is edible and

production of disease resistant plants by

A range of different plant systems

raising antibodies in them. The plantibod-

have been developed for antibody produc-

ies bioproduction process offers several

tion (Table 1). The choice of expression

advantages over the conventional method

of antibody production in mammalian

**Table 1:** Pharmaceutical antibodies produced in transgenic plants ****

**Antigen**

**Plant**

**Antibody form Application**

**Reference**

Human chorion-

Tobacco

scFv, diabody,

Diagnos-

Kathuria _et al._ ,

ic Gonadotro-

chimeric, IgG1

tic/contraceptive

2002

phin ****

Glycophorin ****

Barley, po-

ScFv-fusion

Diagnostic (HIV)

Schunmann _et_

tato, tobac-

_al._ , 2002

co

Streptococcus

Tobacco

SigA/G

Therapeutic (topical Ma _et al._ , 2003

surface antigen

(CaroRx)

SAI/II ****

Sperm ****

Corn

IgG

Contraceptive (topi-

Cone and

cal)

Whaley, 2002

Rhesus D ****

Arabidop-

IgG

Diagnostic

Bouquin _et al._ ,

sis

2002

Human IgG ****

Alfalfa

IgG

Diagnostic

Khoudi _et al._ ,

1999

Rabies ****

Tobacco

IgG

Therapeutic

Ko _et al._ , 2003

Herpes simples

Soyabean,

IgG

Therapeutic (topi-

Zeitlin, 1998

virus ****

rice

cal)

CD40 ****

Tobacco

ScFv-

Therapeutic

Francisco _et al._ ,

cell culture

immunotoxin

1997

fusion

Herpes simplex

Algae

One-chain anti-

Therapeutic

Mayfield _et al._ ,

virus ****

chlamydo-

body

2003

monas

chloroplast

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_Plant Tissue Culture for Sustainability Minakshi et al._

_Table 1: Continued..._

**Antigen**

**Plant**

**Antibody form Application**

**Reference**

Colon cancer

Tobacco

IgG

Therapeutic

Verch _et al._ ,

antigen ****

/Diagnostic

1998

Carcinoembry-

Tobacco,

scFv, diabody,

Therapeu-

Stoger _et al._ ,

onic antigen

rice, wheat, chimeric, IgG1

tic/Diagnostic

2002

(CEA) ****

pea, tomato

Herpes simplex

Corn

sIgA

Therapeutic

Hood _et al._ ,

virus ****

2002

Non-Hodgkins

Tobacco

scFv

Personalized vac-

McCormick _et_

lymphoma idi-

Virus vec-

cines

_al._ , 2003

otypes ****

tor

Clostridium dif-

Corn

IgG

Therapeutic (oral)

www.epicyte.co

ficile ****

m

Hepatitis B vi-

Lettuce

IgG

Vaccine

Kapusta _et al._ ,

rus ****

1999

New castle dis-

Corn

Surface glyco-

Vaccine

Guerrero-

ease virus ****

protein F

Andrade _et al_ ,

2006

Cholera ****

Tomato

Cholera toxin B

Oral vaccine

Jiang _et al._ ,

subunit ( _ctb_

2007

gene)

Enterovirus ****

Tomato

Serum IgG VP1

Oral vaccine

Chen _et al._ ,

2006

Porcine repro-

Banana

IgG and IgA

Oral immunization

Chan _et al._ ,

ductive and res-

2013

piratory syn-

drome virus ****

system depends upon many factors such

crop several times in a year (Fischer _et_

as suitability for scale-up, storage and

_al.,_ 2003). Tobacco grows quickly and

downstream processes. Other considera-

has been shown to produce comparatively

tions attributed to host choice are antici-

large quantity of antibodies. Additionally,

pated production scale, the value and use

tobacco is a non-food/non-feed crop if

of the product, geographical area of pro-

grown separately; there is less chance of

duction, proximity of processing facility,

cross-contamination food chain by phar-

biosafety, intellectual property right and

maceuticals (Schillberg _et al.,_ 2002).

economical aspects (Twyman _et al.,_

Chloroplast engineering in tobacco, an

2003). Several works have shown that

alternative to nuclear transgenics, trans-

tobacco, potatoes, soya beans, corn, alfal-

plastomic plants are produced by intro-

fa and similar kind of crops are the alter-

ducing DNA into the chloroplast genome

native ways for production of recombi-

instead of nuclear genome, and this will

nant proteins (Hiatt _et al.,_ 1989; Mason

be achieved by particle bombardment

and Amtzen, 1995). Figure 2 showed that

(Daniell _et al.,_ 2002). Chloroplast trans-

many benefits of plants used for planti-

formation is more advantageous, includes

body production.

high transgene copy number, absence of

position effects and transgene silencing.

_2.1. Tobacco_

Combination of these properties leads to

Among leafy crops tobacco have

extraordinary levels of expression, ex-

the greatest biomass yield per hectare and

ceeding 25 percent of the total soluble

allow rapid scale up because they can

protein (Tregoning _et al.,_ 2003). Other

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benefit of chloroplast engineering in-

which was immunized with leaf extracts

cludes the ability to express several genes

conferred specific antibodies (Shao _et al.,_

as operons and the accumulation of re-

2008).

combinant protein in the chloroplast,

which reduces the toxicity of host plant.

_2.2. Alfalfa and other legumes_

Biologically active and structurally accu-

Another leafy crop used to pro-

rate Human growth hormone and serum

duce recombinant antibodies are alfalfa

albumin produced at high levels in tobac-

and soya bean. Alfalfa is a perennial crop

co chloroplasts (Staub _et al.,_ 2000; Fer-

can propagated easily and also have good

nandez _et al.,_ 2003). Recently tetanus tox-

biomass yield. The Biotechnology Com-

in fragment (Tregoning _et al.,_ 2003) and

pany Medicago selected it as a platform

cholera toxin B subunit (Daniell _et al.,_

technology. The strong advantage of this

2001) has been expressed in tobacco chlo-

crop is its tendency to synthesize homog-

roplast and was shown that tetanus toxin

enous N-glycans, which improves the

fragment induce protective levels of anti-

consistency of recombinant proteins batch

tetanus antibodies and cholera B subunit

to batch (Bardor _et al.,_ 2003). One of the

shows that plastids can fold and assemble

potential advantages of alfalfa is that re-

oligomeric proteins perfectly. Fischer _et_

combinant antibodies produced as a single

_al_. (1999) reported that recombinant pro-

glycoform rather than heterogeneous col-

teins can also be produced from tobacco

lection of different glycoforms that is

cell culture and several recombinant pro-

found in other plant systems. Pea, a grain

teins including antibody derivatives were

legume also a useful production crop, rea-

derived from a suspension cell line of to-

son that of its high protein content of

bacco strain BY-2. The expression of

seed. Although at present only low yield

classical swine fever virus E2 protein was

is possible with this species (Perrin _et al.,_

expressed in tobacco chloroplast elicited

2000).

protective immune response in mice

**Figure 2:** Benefits of using plants for plantibodies production.

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_2.3. Cereals_

_2.4. Fruits and vegetables_

The disadvantage of tobacco in

The benefit of fruits, vegetables,

recombinant protein production is its in-

and other leafy crops is that they can be

stability. The leafy tissue needs preserva-

consumed raw or partially processed,

tion such as freezing and drying for trans-

which make them particularly suitable for

portation (Ma _et al.,_ 2003). Cereals and

the production and expression of recom-

legumes produce less biomass when com-

binant antibodies for passive oral immu-

pared to leafy crops but the accumulation

notherapy. Storage organs of plants such

of recombinant antibodies in seeds allows

as tubers combine this advantage with a

long-term storage in ambient temperature

prolonged shelf-life when compared to

because the desiccated environment of the

leafy crops. Potato have been used widely

mature seeds reduces the exposure of

for the production of plant-derived vac-

stored proteins to non-enzymatic hydroly-

cines and been administered to humans in

sis and protease degradation. It has been

most of the clinical trials so far. Arts-

shown that antibodies expressed in rice

eanko _et al.,_ 1995 first demonstrated the

seeds remains stable in room temperature

potential of potato tubers for antibody

for years with no detectable loss of activi-

production and recently this crop has been

ty (Stoger _et al.,_ 2000). Seeds of cereals

investigated as a bulk-production system

lack phenolic substances, which are pre-

for antibodies (Wilde _et al.,_ 2002). Pota-

sent in tobacco leaves, so it increases the

toes were also used for the production of

efficiency of downstream process (Ma _et_

diagnostic

antibody-fusion

proteins

_al.,_ 2003). Several crops have been inves-

(Schunmann _et al.,_ 2002) and human milk

tigated for antibody production includes,

proteins (Chong and Langridge, 2000).

rice, wheat, barley, maize, legume pea

Tomatoes have outstanding properties for

and soya bean. Since the said seed crops

pharmaceutical protein production, such

are used as food, so the downstream pro-

as high biomass yield and advantage of

cessing steps may benefit from the food

contained growth in greenhouse, by con-

processing facilities. Maize is now the

sidering these potentials; tomatoes were

main commercial crop used for the pro-

used to produce the first plant-derived

duction of two technical proteins Avidin

rabies vaccine (Garvey _et al.,_ 1995; stoger

and β-glucoronidase by a commercial mo-

_et al.,_ 2000). Lettuce was used as a pro-

lecular farming venture called Prodigene,

duction host to produce edible recombi-

and Maize also reflects the advantageous

nant vaccine against Hepatitis B virus.

factors such as high biomass yield, ease

Kapusta _et al.,_ (1999) shown that mice

of scale-up, ease of transformation and _in_

fed with transgenic lupin tissue were de-

_vitro_ manipulation (Hood _et al.,_ 1997;

veloped significant levels of Hepatitis B

Witcher _et al.,_ 1998). Maize is also being

virus specific antibodies and also human

used for the production of recombinant

volunteers who fed with transgenic lettuce

antibodies, technical/pharmaceutical en-

plants expressing HBV surface antigen

zymes such as laccase, trypsin, and apro-

developed specific serum IgG response to

tinin (Hood _et al.,_ 2002a and 2002b). The

plant produced protein. Transgenic toma-

expression of antibodies in other cereal

to based developed edible vaccine ex-

crops has been explored and experiments

pressed cholera toxin B against cholera in

have been carried out in wheat and rice

the ripening tomato fruit under the control

(Stoger _et al.,_ 2000). The antibody level

of tomato fruit-specific E8 promoter using

of 150 µg g-1 was expressed in transgenic

_Agrobacterium_ -mediated transformation

barley was one of the most encouraging

(Jiang _et al.,_ 2007). The immunogenicity

result have far come from the expression

of the CTB protein expressed in tomato

of a diagnostic scFv fusion protein called

fruit was evaluated through determination

SimpliRED (Schunmann _et al.,_ 2002).

of the serum and mucosal anti-CTB anti-

body levels in experimental mice. A study

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conducted by Chen _et al_ (2006), proved

This is an economically important

that VP1 protein of enterovirus in trans-

method for producing plantibodies where

genic tomato plant provide both cellular

in plant cells in differentiated stages are

and humoral immunity in orally immun-

grown under controlled conditions hav-

ized mice, those fed with tomato fruit ex-

ing desired genes/ proteins and are har-

pressing VP1 protein. Additionally, serum

vested either in the form of biomass and

from mice fed with transgenic tomato

culture liquid or combination of both.

could neutralize the EV71 infection to

This method offers large amounts of re-

rhabdomyosarcoma cells, indicating that

combinant proteins production in a short-

tomato fruit expressing VP1 was success-

er time (Doran, 1999). This method offers

ful in orally immunizing mice. Pigs were

many advantages over conventional

immunized with recombinant GP5 protein

method of plantibodies production by

expressed in transgenic Banana leaves

overcoming the problem of extracting and

using _Agrobacterium_ -mediated transfor-

purifying proteins but this method has not

mation with ORF5 gene of porcine repro-

used for edible vaccines production. In

ductive and respiratory syndrome virus

this method no sexual reproduction is

envelope glycoprotein GP5. Pigs immun-

needed, so transgenic stability is in-

ized orally with GP5 protein showed a

creased because of absence of crossing

gradual dependent increase in the elicita-

over, segregation and recombination and

tion of serum and saliva anti-PRRSV IgG

provides more chances in plantibodies

and IgA was observed and significantly

production (Ferrante and David, 2001).

lower viraemia and tissue viral load were

recorded when compared to the pigs

_3.3. Breeding and sexual crossing_

which are fed with untransformed banana

An experiment on tobacco plant

leaves (Chan _et al.,_ 2013).

was established for its breeding and sexu-

al crossing as a method for the production

**3. Methods for plantibody production**

of plantibodies. In this experiment, trans-

formation was used to introduce kappa

Various techniques have been de-

chains of either light or heavy regions into

veloped to exploit plants as bioreactors

tobacco plants. Same way was done with

for the production of plantibodies. Some

gamma heavy chains. Upon crossing one

of the techniques are described below:

plant with kappa-chains and another with

gamma-chains, an antibody was produced

_3.1. Conventional method_

that expressed both chains (Hiatt _et al.,_

Once the desired DNA from the

1989; Whitelam _et al.,_ 1994). This meth-

transformed host cell is isolated and puri-

od provides an easy way to produce plan-

fied, it can be injected into the embryo of

tibodies without the need for double ferti-

a maturing plant, which we want to use

lization (Ferrante and David, 2001).

for plantibodies production. After inject-

ing the desired gene, followed by propa-

_3.4. Transgenic seeds_

gation of plant in open field allow large

Above mentioned methods have

scale production of plantibodies. Plant

certain limitations. Further restrictions are

tissue culture is most economic and time

found when plants used as storage system

saving method for antibody production

because plants cannot store antibodies for

from plants. In this method, plant cells in

a longer time. This is due to certain prote-

differentiated states are grown in bioreac-

ases degrade the protein piece by piece.

tors with foreign proteins harvested either

So, **s** ome researchers suggest that, use of

from biomass or culture liquid with less

transgenic seeds in place of green leafy

contaminant (Moffat, 1989).

plants, because seeds can store antibodies

for an extended period without degrada-

_3.2. In vitro cell and tissue cultures_

tion. Seeds contain low level of proteases

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_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

that allows proteins to be stored for longer

filteration, immunofluorescence, chroma-

period when compared to green plants

tography, diafilteration, polymer fusion

(Larrick _et al.,_ 1998; Fieldler and cornad,

and protein A-sepharose chromatography.

1995). So, seeds can be used as bioreac-

Some other techniques such as RIA (Ra-

tors and used as natural storage organs

dioimmunoassay), northern blot tech-

(Ferrante and David, 2001).

nique, ELISA (Enzyme linked immune

sorbent assay), western blot analysis and

_3.5. Targeting and compartmentalization_

immunofluorescence southern blot analy-

Antibodies can be targeted to

sis have been used of evaluation of plan-

some compartments by tagging with a

tibody.

small peptide sequence and this allows

antibodies to be protected from proteases

**5. Applications of plantibodies**

that present in the cytoplasm (Kusnadi _et_

****

_al.,_ 1997). Compartmentalization to be

_5.1. Bioreactors_

easily isolated organelles and makes easy

Antibodies produced in plants

purification procedure (Kusnadi _et al.,_

have applications such as production of

1997). Targeting, however, has to be spe-

vaccine antigens, protein for clinical di-

cifically controlled and this involves

agnosis, pharmaceutical and industrial

proper cleavage of targeted sequences.

proteins, carbohydrates, vitamins, miner-

als, biopolymer and food (Sharma and

**4. Purification techniques**

Sharma, 2009). These applications were

proved in basic agronomy research (Jae-

The main reason for raising anti-

ger _et al.,_ 2000). In recent years, many

bodies in plants is its easy purification

plant systems has been developed in order

and low downstream processing. Easy

to using plants as bioreactors for the pro-

purification of plantibodies makes bio-

duction of recombinant antibodies for

pharmaceutical production more econom-

many purposes (Stoger _et al.,_ 2002).Using

ic (Arntzen, 1998). Transgenic seeds as-

plants as a bioreactor, or as a factories or

sure excellent storage properties and thus

using them as antibody replacement for

added flexibility in processing manage-

microorganisms like bacteria to produce

ment and batch production. Separation of

human antibodies to communicate with

plantibody in seeds is less complicated

human health mainly due to two reasons.

because of limited range of endogenous

(i) When compared to prokaryotes, plants

proteins (Kusandi _et al.,_ 1997). Absence

are better for the production of antibodies

of human pathogens in plants eliminates

due to large scale production efficiency

expensive validation of virus removal

and low cost of production.

steps during purification. But the proba-

(ii) Process of post-translational modifica-

bility of presence of a diverse burden on

tion such as glycosylation that they are

plants grown outdoors in non-sterile con-

the kind of post-translational modifica-

ditions is high. So the process for elimina-

tions of proteins, in plants can be done

tion or minimization of contamination

more carefully than bacteria (Ma and

with endotoxin and mycotoxins will be

Hein, 1995).

necessary in all commercial process to

purify antibodies (Gegenheimer, 1990).

_5.2. Therapeutic applications_

Phenolics can interact with proteins in

CaroRx, the first plant derived an-

ways that can irreversibly alter the prop-

tibody created from tobacco (Fischer _et_

erties of proteins but most of the phenol-

_al.,_ 2006), is a Sig A secretory antibody.

ics released during extraction are small in

It is a clinically advanced anti _Streptococ-_

size, water soluble, and removable by ul-

_cus mutans_ secretory immunoglobulin, a

trafilteration steps. Main techniques used

plantibody that binds specifically to the

for the purification of plantibodies are

bacterium, thus protecting humans from

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_Plant Tissue Culture for Sustainability Minakshi et al._

dental carries (Larrick _et al.,_ 2001).

Treatment or cure for rabies through plan-

CaroRx is intended for regular topical

tibodies has been investigated by Ko _et al_.

preventive administration by both dental

2003. A plantibody based rabies vaccine

hygienists and patients allowing a thor-

produced in tobacco was experimentally

ough cleaning and intervention for any

administered in hamsters to check wheth-

existing decay. Plantibodies have been

er it could effectively target rabies. The

investigated for inflammatory disease and

plantibody proved to be safe and econom-

to induce tolerance (Jain _et al.,_ 2011). A

ically feasible alternative method com-

humanised antibody, another plantibody

pared to the current antibody production

with human medical application was ex-

in animal systems. Another study, tobac-

pressed in soya bean against herpes sim-

co-derived plantibodies were experimen-

plex virus (Zietlin _et al.,_ 1998). Anti-

tally administered in mice against the

tumour antibodies against Burkitt`s lym-

Lewis Y antigen found on tumour cells in

phoma was expressed in rice and wheat

mice and also in lung, breast, ovarian and

(Ghasempour _et al.,_ 2014). Antibodies

colorectal cancer. According to Brodzick

engineered to bind to _Bacillus anthracis_

_et al_ (2006), the plantibodies showed a

was extracted from transgenic strains of

definite positive effect on the cancer-

tobacco and tested in mice in a study con-

striken mice by preventing tumour for-

ducted by Hull _et al_ in 2005. The result of

mation in them (Figure 3).

this study showed that the antibodies were

effective in fighting _B. anthracis_ strain

_5.2.1. Immunization_

and bodes well for the future in any an-

One of the most interesting appli-

thrax epidemic will be a cheap and effec-

cations of this technology is production of

tive prevention against the disease.

edible vaccines or oral vaccines. Produc-

**Figure 3:** Use of plantibodies for cancer treatment.

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_Plant Tissue Culture for Sustainability Minakshi et al._

Potential proteins such as cytokines, hor-

progress has been made towards engineer-

mones, enzymes, epidermal growth fac-

ing residence against insects (Schillberg

tors, interferons, human protein C, and

_et al.,_ 2001; Jaeger _et al.,_ 2000). Im-

pharmaceutical foodstuff are produced,

munomodulation is a dynamic tool for

which are considered for oral immuniza-

altering the function of an antigen _in vivo_.

tion (Mason and Amtzen, 1995). Trans-

When an artificial abscisic acid sink was

genic plants that express antigens might

created by the production of an anti- ab-

be used as an inexpensive oral vaccine

scisic acid specific scFv in the endoplas-

production and delivery system, so im-

mic reticulum of potato and tobacco

munization might be possible through

plants, both physiological and morpholog-

consumption of an edible vaccine to pro-

ical changes were noticed (Conrad and

vide immunization. Due to all these rea-

Manteuffel, 2001). Moreover, agro-

sons transgenic plants are considered as

filtration of tobaco was used to produce a

better alternative for oral vaccines. This

diabody against carcinoembryonic antigen

offers convenient immunization strategies

(Vaquero _et al.,_ 2002). In addition, plan-

for implementing universal vaccination

tibodies may also prove useful as feed

programmes throughout the world (Tack-

additives or for phytoremediation in hu-

et _et al.,_ 1998). In human host the patho-

man health care (Mason and Arntzen,

gens usually attack mucosal sites in the

1995).

respiratory tract, gastro-intestinal tract or

genital tract. Stimulation of immune re-

**6. Pathogen resistance in plants**

sponses in these sites through mucosal

vaccines to protect against illness is desir-

Antibody mediated pathogen re-

able and this can be achieved by applying

sistance in plants is a novel strategy for

vaccine to the mucosal surface directly,

generating transgenic plants resistant to

inducing systemic and cellular immune

pathogens have been developed in many

responses as well as local immune re-

cases for therapeutic applications, for

sponses at the initial site of interaction

Immunomodulation.

Peschen

_et_

_al.,_

between the pathogen and host directly

(2004) demonstrated antibody-mediated

(Kusnadi _et al.,_ 1997). Oral vaccines

resistance against fungal pathogens and

must be protected during passage through

protecting plants against fungal diseases.

the hostile environment of the stomach

Schillberg _et al_ (2000), targeted anti-

and intestine to the sites of immune

TMV antibodies to the plasma membrane

stimulation.

in vivo (in planta) results in evolvement

of transgenic plants resistant to TMV in-

_5.2.2. Immunomodulation_

fection. A study conducted by Boonrod _et_

Immunomodulation is a technique

_al_ (2004), paved the way for engineering

that allows interference with cellular me-

broad-range virus resistance by expres-

tabolism, signal transduction or pathogen

sion of scFv antibodies in vivo that are

infectivity by the ectopic expression of

specific and highly conserved motifs in

gene encoding antibodies or antibody

viral replicase or polymerases. Boonrad

fragments (Jaeger _et al.,_ 2000). Applica-

and his colleagues demonstrated that

tions that are relaying on modulating an-

transgenic tobacco plants expressing scFv

tigen levels in vivo are dependent on ex-

antibodies against a conserved domain in

pression and accumulation in specific sub

plant viral RNA dependent RNA poly-

cellular compartments and specific tis-

merase either in the cytosol or endoplas-

sues. Development of crop resistance and

mic reticulum, showed high levels of re-

passive immunization of plants by expres-

sistance to four plant viruses from differ-

sion of pathogen-specific antibodies re-

ent genera. Malembic-Maher _et al._ (2005)

duces infection and symptoms caused by

studied the scFv 2A10 protein expression

viruses and mollicutes, and significant

in engineered tobacco plants and for their

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resistance to stobular disease. It was ob-

_na benthamiana_ , was used for the produc-

served that tobacco plants producing se-

tion of mAb (6D8) that protected animals

creted scFvs shown a short delay in symp-

from Ebola virus infection (Chen _et al_.,

tom appearance, reduction to pathogen

2002). An Ebola immune complex (EIC)

susceptibility and phyloplastoma multi-

was produced by fusing Ebola glycopro-

plication. Isolated antipectinase scFv an-

tein GP1 to the C-terminus of the heavy

tibodies directed against extracellular pro-

chain of humanized 6D8 mAb that binds

teins from _Rhizoctonia solani_ secluded

specifically to a linear epitope on GP1

from a page display library. Soluble scFv

using the geminivirus-based expression

antibodies shown to inhibit polygalac-

system and _Nicotiana benthamiana_ (Bhoo

turonase in the culture supernatants of a

_et al_., 2011). The recombinant immuno-

range of fungal pathogens such as asco-

globulins were produced in leaves of _Ni-_

mycetes, basidiomycetes and oomycetes.

_cotiana benthamiana,_ which was purified

This soluble antibody also inhibited mac-

by protein G affinity chromatography.

eration in potatoes (Manatunga _et al.,_

The

resultant

recombinant

antibody

2005). First time antibody mediated fun-

bound the the complement facto C1q, in-

gal resistance was demonstrated by Wu _et_

dicating immune complex formation.

_al_ in wheat and cereal grains. This fungal

Therefore subcutaneous immunization of

resistance in transgenic plants is mediated

mice with purified EIC showed high level

by generating specific antibodies against

of production of anti-Ebola virus antibod-

_Fusarium graminearum_ , a predominant

ies that provides protection against Ebola

fungal species infecting wheat and small

virus (Figure 4). This was the first pub-

cereal grains in china (Wu _et al.,_ 2005).

lished report of an Ebola virus candidate

vaccine to be produced in plants.

**7. Treatment of Ebola patients**

The Ebola virus disease outbreak

in West Africa has provided a great op-

Recently, antibodies against Ebola

portunity for the use of plantibodies in

virus have been explored in plants. A

resolving global human health challenges

high yielding geminivirus-based expres-

as

sion system in the tobacco plant, _Nicotia-_

**Figure 4:** Use of plantibodies for Ebola virus treatment.

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as two American medical aid workers

down streaming processess of plantbodies

who contracted the disease in Liberia

production such as extraction and purifi-

were successfully treated with an experi-

cation of plantibodies is an important

mental drug called ZMapp produced in

step, covers more than half of the total

the tobacco plant. The drug ZMapp con-

cost. Thus purification system during

tains combination of three humanized an-

plantibodies production is very expensive.

ti-Ebola virus mAbs and developed by

Currently purification system in plant sys-

Mapp biopharmaceutical incorporated,

tems, an affinity purification protocol re-

San Diego (Langreth _et al_., 2014). Alt-

quires protein A-based matrices is mainly

hough the drug ZMapp holds great prom-

used (Valdes _et al_., 2003). So it is neces-

ise for the future, the major limitation is

sary to use alternative economic methods

producing large quantity of anti-Ebola

that use oleosin or polymer fusions for the

virus antibody to meet the demand of

purification of plantibodies (Daniell _et al_.,

widespread outbreak requirement, multi-

2001). Till date, researches have had dif-

ple doses, direct delivery of highly pure

ficulty in achieving the high level of chlo-

antibody into the blood stream and more-

roplast gene expression. Each type of

over the drug is intended originally for

plants poses its own challenges and dos-

expression levels sufficient for animal

age of vaccines may be variable. Planti-

trial (Powell, 2014). ZMapp is yet to re-

bodies are not suitable for infants (Doshi

ceive the approval by the US food and

_et al_., 2013).

Drug Administration who have to certify

that the plant extraction process has not

**9. Future perspectives**

led to the contamination of the resulting

drug (Begley, 2014).

Plant-derived systems for produc-

tion of biopharmaceutics should meet the

**8. Advantages and challenges of planti-**

same standards of safety and performance

**bodies production**

as other production systems (Daniell _et_

_al_., 2001). Plantibodies production system

Plantibodies

production

from

have many advantages over animal sys-

plants has many potential advantages for

tems, such as well-established cultivation,

creating biopharmaceuticals related to the

quick scale-up, simple distribution by

medicine. First, the plant systems used for

seeds, oral delivery, can be used as raw

plantbodies production are more econom-

food or dry powder, desperate of cold

ical than industrial equipments using fer-

chain requirement, mucosal and serum

mentation system. Second, this system

immune responses, cost efficiency, ease

provides large amount of plant products.

of genetic manipulation, ease of produc-

Third, the system can be omitted the puri-

tion and scale-up, safer than conventional

fication step when the aim is to produce

vaccines, ideal to face bio-weapons and

edible vaccines. Fourth, plants can be

ideal for veterinary use as feed additive

directed to the desired proteins into com-

(Yoshida _et al_., 2004; Sala _et al_., 2003).

partments/organelles such as chloroplasts.

Fifth, the amount of proteins produced in

**10. Concluding remarks**

such an amount it can be suitable for in-

dustrial levels. Last, one of the important

Substantial progress has been

advantage, health risks from contamina-

made in recent years towards the produc-

tion with potential human pathogens are

tion of a wide range of antibodies in

minimized (Sala _et al.,_ 2003; Daniell _et_

plants and now it is a feasible and eco-

_al_., 2001).

nomical system of producing antibodies.

After lots of advantages, there are

These transgenic plants have been shown

some remaining challenges are associated

to be the most productive system in

with the plantibodies production. During

providing therapeutics and edible vac-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 315

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

cines, which are cheap and can be easily

**and Vezina, L.P. (2003).** Monoclo-

administered. Plants can be easily ex-

nal C5-1 antibody produced in trans-

plored by pharmaceutical industries and

genic alfalfa plants exhibits a N-

Indian climate helps for the production of

glycosylation that is homogenous

diversified crops. Engineering of in-

and suitable for glyco-engineering

creased pathogen resistance and alteration

into human compatible structures.

of phenotypes by immunomodulation

_Plant Biotechnology Journal_ **1 _,_** **451-**

provide a great platform to develop the

**462.**

prevention strategies. Various other strat-

**Begley, S. (2014).** Tobacco-derived 'plan-

egies have been developed to exploit

tibodies' enter the fight against Ebo-

plants as bioreactors for the production of

la.

pharmaceutical antibodies and many plant

http://www.reuters.com/article/2014/

produced antibodies are proved increased

08/06/healthebolatobaccoidUSL2N0

disease resistance. Recent developments

QB24N20140806.

focus on the detailed characterisation of

**Bhoo, S.H., Lai, H., Ma, J., Arntzen,**

recombinant products. Recent indications

**C.J., Chen, Q. and Mason, H.S.**

that tissue specific and physiological fac-

**(2011).** Expression of an immuno-

tors may have an impact on the quality

genic Ebola immune complex in _Ni-_

and glycosylation pattern of a plantibod-

_cotiana benthamiana_. _Plant Biotech-_

ies will perhaps lead to new insights and

_nology Journal_ **9 _,_** **807 -816.**

production strategies. Although some

**Boonrod, K., Galetzka, D., Nagy, P.D.,**

plant-derived antibody products have suc-

**Conrad, U. and Andkrczal, G.**

cessfully completed early phase clinical

**(2004).**

Single-chain

antibodies

trials, several issues including regulatory

against a plant viral RNA-dependent

guidelines and public acceptance must

RNA polymerase confers virus re-

still be resolved. Currently, more than 200

sistance. _Nature Biotechnology_ **22 _,_** ****

novel antibody-based potential products

**856-862.**

are in clinical trials worldwide, and mar-

**Bouquin, T., Thomsen, M., Nielsen,**

ket demand will certainly strain the capa-

**L.K., Green, T.H., Mundy, J. and**

bilities of existing production systems.

**Hanefeld, D. (2002).** Human anti-

Moreover, adoption of plants as bioreac-

rhesus D IgG1 antibody produced in

tors on a larger scale would reduce the

transgenic plants. _Transgenic Re-_

cost of antibody therapy and simultane-

_search_ **11 _,_** **115-122.**

ously increase the number of patients who

**Brodzick, R., Glogowska, M., Ban-**

access to these treatments.

**durska, K., Okulicz, M., Deka, D.**

**and Ko, K. (2006).** Plant-derived

**References**

Anti-Lewis Y mAbexhibits biologi-

****

cal activities for efficient immuno-

**Arntzen, C.J. (1998).** Pharmaceutical

therapy against human cancer cells.

food stuffs oral immunization with

_Proceedings of the National Acade-_

transgenic plants. _Nature Medicine_ **4 _,_** ****

_my of Sciences USA_ **103 _,_** **8804 –**

**502-503.**

**8809.**

**Artsaenko, O., Peisker, M., Zur-**

**Chan, H.T., Chia, M.Y., Pang, V.F.,**

**neieden, U., Fiedler, U., Weiler,**

**Jeng, C.R., Do, Y.Y. and Huang**

**E.W. and Muntz, K. (1995).** Ex-

**P.L. (2013).** Oral immunogenicity of

pression of a single-chain Fv anti-

porcine reproductive and respiratory

body against abscisic acid creates a

syndrome virus antigen expressed in

wilty phenotype in transgenic tobac-

transgenic banana. _Journal of Plant_

co. _The plant Journal_ **8 _,_** **745-750.**

_Biotechnology_ **11 _,_** **315–324.**

**Bardor, M., Loutelier-Bourhis, C., Pac-**

**Chen,**

**H.F.,** **Chang,**

**M.H.,** **Chiang,**

**calet, T., Cosette, P., Fitchette, A.**

**B.L.** **andJeng, S.T. ****(2006).** Oral _ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 316

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

immunization of mice using trans-

scope. _Journal of Pharmaceutical_

genic tomato fruit expressing VP1

_and scientific innovation_ **2 _,_** **1-6.**

protein

from

enterovirus

**Duering, K., Hippe, S., Kreuzaler, F.**

71. _Vaccine_ **24 _,_** **2944-2951.**

**and Schell, J. (1990).** Synthesis and

**Chen, Q., He, J., Phoolcharoen, W. and**

self-assembly of a functional mono-

**Mason, H.S. (2002).** Geminiviral

clonal antibody in transgenic _Nicoti-_

vectors based on bean yellow dwarf

_ana tabacum_. _Plant Molecular Biol-_

virus for production of vaccine anti-

_ogy_ **15 _,_** **281-293.**

gens and monoclonal antibodies in

**Fernandez, S., Millan, A., Mingo-**

plants. _Human Vaccines_ **7 _,_** **331-338.**

**Castel, A., Miller, M. and Daniell,**

**Chong, D.K.X. and Langridge, W.H.R.**

**H. (2003).** A chloroplast transgenic

**(2000).** Expression of full-length bi-

approach to hyperexpress and purify

oactive antimicrobial human lactofer-

human serum albumin, a protein

rin in potato plants. _Transgenic Re-_

highly susceptible to proteolytic deg-

_search_ **9 _,_** **71–78.**

radation. _Plant Biotechnology_ **1 _,_** **77–**

**Cone, R.A. and Whaley, K.J. (2002).**

**79.**

Topical application of antibodies for

**Ferrante, E and David S. (2001).** A re-

contraception and for prophylaxis

view of the progression of transgenic

against sexually transmitted diseases.

plants used to produce plantibodies

US patent **6 _,_** **355-235.**

for human usage. Biological & Bio-

**Conrad, U. and Manteuffel, R. (2001).**

medical Sciences **1 _,_** **1-6.**

Immunomodulation

of

phytohor-

**Fieldler, U. and Conrad, U. (1995).**

mones and functional proteins in

High-level production and long-term

plant cells. _Trends in Plant Science_

storage of engineered antibodies in

**6 _,_** **399-402.**

transgenic tobacco seeds. _Biotech-_

**Daniell, H., Khan, M.S. and Allison, L.**

_nology_ **13 _,_** **1090-1094.**

**(2002).** Milestones in chloroplast ge-

**Fischer, R., Emans, N., Schuster, F.,**

netic engineering: an environmental-

**Hellwig, S. and Drossard, J. (1999).**

ly friendly era in biotechnology.

Towards molecular farming in the fu-

_Trends in Plant Science_ **7 _,_** **84–91.**

ture: using plant-cell-suspension cul-

**Daniell, H., Lee, S.B., Panchal, T. and**

tures as bioreactors. _Biotechnology_

**Wiebe, P.O. (2001).** Expression of

_and_ _Applied Biochemistry_ **30 _,_** **109–**

the native cholera B toxin subunit

**112.**

gene and assembly as functional oli-

**Fischer, R., Richard, M.T. and Schill-**

gomers in transgenic tobacco chloro-

**berg, S. (2003).** Production of anti-

plasts. _Journal of Molecular Biology_

bodies in plants and their use for

**311, 1001-1009.**

global health. _Vaccines_ **21 _,_** **820-825.**

**Daniell, H., Streatfield, S.J. and**

**Fischer, R., Twyman, M.R., Hellwig, S.,**

**Wycoff, K. (2001).** Medical molecu-

**Drossard, J. and Schillberg, S.**

lar farming: production of antibodies,

**(2006).** Facing the Future with

biopharmaceuticals and edible vac-

Pharmaceuticals from Plants. Bio-

cines in plants. _Trends in Plant Sci-_

technology and Sustainable Agricul-

_ence_ **6, 219-226.**

ture and Beyond. Xu, Z., Li, J., Xue,

**Doran, P.M. (1999).** Foreign protein pro-

Y., Yang, W. (Eds.). Proceedings of

duction in plant tissue cultures. _Cur-_

the 11th IAPTC&B Congress. Bei-

_rent Opinon in Biotechnology_ **11 _,_** ****

jing, China. **pp. 13-32**

**199-204.**

**Francisco, J.A., Gawlak, S.L., Miller,**

**Doshi, V. Rawat, H. And Mukhergee,**

**M., Bathe, J., Russell, D. and Cha-**

**S. (2013).** Edible vaccines from GM

**ce, D. (1997).** Expression and char-

crops: current status and future

acterization of bryodin 1 and a bry-

odin 1- based single-chain immuno-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 317

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

toxin from tobacco cell culture. _Bio-_

**Hull, A.K., Criscuolo, C.J., Mett, V.,**

_conjugate Chemistry_ **8 _,_** **708-713.**

**Groen, H., Steeman, W. and Wes-**

**Garvey, P.B.,** **Hammond, J.,** **Dienelt, **

**tra, H. (2005).** Human-derived,

**M.M.,** **Hooper, D.C.,** **Fu, Z.F.,** **Die-**

plant-produced monoclonal antibody

**tzschold, B.,** **Koprowski, H.** **and** for the treatment of anthrax. _Vaccine_

**Michaels, F.H.** **(1995). ** Expression **23 _,_** **2082-2086.**

of the rabies virus glycoprotein in

**Jaeger, D.G., De Wilde, C., Eeckhout,**

transgenic tomatoes. _Biotechnology_

**D., Fiers, E. and Depicker, A.**

_New York_ **13 _,_** **1484-1487.**

**(2000).** The plantibody approach:

**Gegenheimer, P. (1990).** Preparation of

expression of antibody genes in

extracts from plants. _Methods in En-_

plants to modulate plant metabolism

_zymlogy_ **182 _,_** **174-193.**

or to obtain pathogen resistance.

**Ghasempour, H.R., Kahrizi, D. and**

_Plant Molecular Biology_ **43 _,_** **419-**

**Mehdiah, N. (2014).** Application of

**428.**

transgenic plants as factories for pro-

**Jain, P., Pandey, P., Jain, D. and**

ducing biopharmaceuticals. _Journal_

**Dwivedi, P. (2011).** Plantibody: an

_of Biodiversity and Environmental_

overview. _Asian Journal of Pharma-_

_Sciences_ **4, 58-74.**

_cy and Life Science_ **1 _,_** **87-94.**

**Guerrero-Andrade, O., Loza-Rubio, E.,**

**Jiang, X.L., He, Z.M., Peng, Z.Q., Qi,**

**Olivera-Flores,**

**T.,**

**Fehervari-**

**Y., Chen, Q. and Su, S.Y. (2007).**

**Bone, T. and Gomez-Lim, M.A.**

Cholera toxin B protein in transgenic

**(2006).** Expression of the Newcastle

tomato fruit induces systemic re-

disease virus fusion protein in trans-

sponse in mice _. Transgenic research_

genic maize and immunological stud-

**16 _,_** **169-175.**

ies. _Transgenic Research_ **15 _,_** **455–**

**Kapusta, J.,** **Modelska, A.,** **Figlerowicz,**

**463.**

**M.,** **Pniewski, T., ****Letellier, M., ******

**Hiatt, A., Cafferkey, R. and Bowdish,**

**Lisowa,**

**O.,** **Yusibov,**

**V.,** ****

**K. (1989).** Production of antibodies

**Koprowski, H.,** **Plucienniczak, A. **

in transgenic plants. _Nature_ **342 _,_** **76-**

**and Legocki, A.B. ****(1999).** A plant-78.

derived edible vaccine against hepa-

**Hood, E.E. (2002b).** From green plants to

titis B virus. _Federation of American_

industrial enzymes. _Enzyme Micro-_

_Societies for Experimental Biology_

_bial Technol_ ogy **30 _,_** **279–283.**

_Journal_ **13 _,_** **1796-1799.**

**Hood, E.E., Witcher, D.R., Maddock,**

**Kathuria, S., Sriraman, R., Nath, R.,**

**S., Meyer, T., Baszczynski, C. and**

**Sack, M., Pal, R. and Artsaenko,**

**Bailey, M. (1997).** Commercial pro-

**O. (2002).** Efficacy of plant pro-

duction of avidin from transgenic

duced recombinant antibodies against

maize: characterization of trans-

HCG. _Human Reproduction_ **17 _,_** ****

formant, production, processing, ex-

**2054-61.**

traction and purification. _Molecular_

**Khoudi, H., Laberge, S., Ferullo, J.M.,**

_Breeding_ **3 _,_** **291-306.**

**Bazin,**

**R.,**

**Darveau,**

**A.**

**and**

**Hood, E.E., Woodard, S.L. and Horn,**

**Castonguay, Y. (1999).** Production

**M.E. (2002).** Monoclonal antibody

of a diagnostic monoclonal antibody

manufacturing in transgenic plants–

in perennial alfalfa plants. _Biotech-_

myths and realities _. Current Opinion_

_nology and Bioengineering_ **64 _,_** **135-**

_in Biotechnology_ **13 _,_** **630-5.**

**43.**

**Hood, E.E., Woodard, S.L. and Horn,**

**Ko, K., Tekoah,Y., Rudd, P.M., Har-**

**M.E. (2002a).** Monoclonal antibody

**vey, D.J., Dwek, R.A. and Spitsin,**

manufacturing in transgenic plants-

**S. (2003).** Function and glycosyla-

myths and realities. _Current Opinion_

tion of plant-derived antiviral mono-

_in Biotechnology_ **13 _,_** **630–635.**

clonal antibody. _Proceedings of the_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 318

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

_National Academy of Sciences USA_

**Mason, H.S. and Arntzen, C.J. (1995).**

**100 _,_** **8013-8018.**

Transgenic plants as vaccine produc-

**Kusnadi, A.R., Nikolov, Z.L. and How-**

tion systems. _Trends Biotechnology_

**ard, J.A. (1997).** Production of re-

**13 _,_** **388-392.**

combinant proteins in transgenic

**Mayfield, S.P., Franklin, S.E. and Ler-**

plants: practical considerations. _Bio-_

**ner, R.A. (2003).** Expression and as-

_technology and Bioengineering_ **56 _,_** ****

sembly of a fully active antibody in

**473-484.**

algae. _Proceedings of the National_

**Langreth, R., Chen, C., Nash, J. and**

_Academy of Sciences USA_ **100 _,_** **438-**

**Lauerman, J. (2014).** Ebola drug

**442.**

made from tobacco plant saves U.S.

**McCormick, A.A., Reinl, S., Cameron,**

aid

workers.

Available

at:

**T.I., Vojdani, F., Fronefield, M.**

http://www.bloomberg.com/news/20

**and Levy, R. (2003).** Individualized

14-08-05/ebola-drug-made-from-

human scFv vaccines produced in

tobacco-plant-saves-u-said-

plants: humoral anti-idiotype re-

workers.html Accessed January 10,

sponses in vaccinated mice confirm

2015.

relevance to the tumor Ig. _Journal of_

**Larrick, J.W., Yu, L. and Chen, J.**

_Immunological Methods_ **278 _,_** **95-104.**

**(1998).** Production of antibodies in

**Moffat, A.S. (1989).** Exploring transgen-

transgenic plants. _Research in Immu-_

ic plants as a new vaccine source.

_nology_ **149 _,_** **603-608.**

_Science_ **268 _,_** **658-660.**

**Larrick, J.W., Yu, L., Naftzger, C.,**

**Oluwayelu, D.O. and Adebiyi, A.I.**

**Jaiswal, S. and Wycoff, K. (2001).**

**(2016).** Plantibodies in human and

Production of secretory IgA antibod-

animal health: a review. _African_

ies in plants. _Biomolecular Engineer-_

_Health Sciences_ **16 _,_** **640-645.**

_ing_ **18 _,_** **87–94.**

**Perrin, Y., Vaquero, C., Gerrard, I.,**

**Ma, J.K.C. and Hein, M. (1995).** Immu-

**Sack, M., Drossard, J., Stoger, E.,**

notherapeutic potential of antibodies

**Christou, P. and Fischer, R. (2000).**

produced in plants. _Trends in Bio-_

Transgenic pea seeds as bioreactors

_technology_ **13 _,_** **522–527.**

for the production of a single-chain

**Ma, J.K.C., Drake, P.M.W. and Chris-**

Fv fragment (scFV) antibody used in

**tou, P. (2003).** The production of re-

cancer diagnosis and therapy.  _Mo-_

combinant pharmaceutical proteins in

 _lecular Breeding_ **6 _,_** **345–352.**

plants. _Nature Review Genetics_ **4 _,_** ****

**Peschen, D., Li, H.P., Fischer, R.,**

**794-805.**

**Kreuzaler, F. and Liao, Y.C.**

**Malembic-Maher, S., Le Gall, F.,**

**(2004).** Fusion proteins comprising a

**Danet, J.L., Borne, F.D., Bove,**

Fusty-nun-specific antibody linked to

**J.M. and Garniersemancik, M.**

anti-fungal peptides protect plants

**(2005).** Transformation of tobacco

against fungal pathogens. _Nature Bi-_

plants for single-chain antibody ex-

_otechnology_ **22 _,_** **732-738.**

pression via apoplastic and sym-

**Powell, J.D. (2015).** From Pandemic Pre-

plasmic routes, and analysis of their

paredness to Biofuel Production: To-

susceptibility to stolbur phytoplasma

bacco Finds Its Biotechnology Niche

infection. _Plant Science_ **168 _,_** **349-**

in North America. _Agriculture_ **5 _,_** ****

**358.**

**901-917.**

**Manatunga, V., Sanati, H., Tan, P. and**

**Sala, F.M., Rigano, M., Barbante, A.,**

**Andobrien, P.A. (2005).** Maceration

**Basso,**

**B.,**

**Amanda**

**M.**

**and**

of plant tissue by fungi is inhibited

**Walmsley, S. Castiglione. (2003).**

by recombinant anti pectinase anti-

Vaccine antigen production in trans-

bodies. _European Journal of Plant_

genic plants: strategies, gene con-

_Pathology_ **112 _,_** **211-220.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 319

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

structs and perspectives. _Vaccine_ **21 _,_** ****

necks. _Current Opinion in Biotech-_

**803–808.**

_nology_ **13 _,_** **161-166.**

**Schillberg, S., Emans, N. and Fischer,**

**Stoger, E., Sack, M., Perrin, Y.,**

**R. (2002).** Antibody molecular farm-

**Vaquero, C., Torres, E. and Twy-**

ing in plants and plant cells. _Phyto-_

**man, R.M. (2000).** Practical consid-

_chemistry Reviews_ **1 _,_** **45-54.**

erations for pharmaceutical antibody

**Schillberg,**

**S.,**

**Zimmermann,**

**S.,**

production __ in different crop systems.

**Findlay, K. and Fischer, R. (2000).**

_Molecular Breeding_ **9 _,_** **149-158.**

Plasma membrane display of anti-

**Stoger, E., Vaquero, C., Torres, E.,**

viral single chain Fv fragments con-

**Sack, M., Nicholson, L. and**

fers resistance to tobacco mosaic vi-

**Drossard, J. (2000).** Cereal crops as

rus _. Molecular Breeding_ **6 _,_** **317-326.**

viable production and storage sys-

**Schillberg, S., Zimmermann, S., Zhang,**

tems for pharmaceutical scFv anti-

**M.Y. and Fischer, R. (2001).** Anti-

bodies. _Plant Molecular Biology_ **42 _,_** ****

body-based resistance to plant patho-

**583-590.**

gens. _Transgenic Research_ **10 _,_** **1-12.**

**Tacket, C.O., Mason, H.S., Losonsky,**

**Schunmann, P.H.D., Coia, G. and Wa-**

**G., Clements, J.D., Levine, M.M.**

**terhouse, P.M. (2002).** Biopharming

**and Arntzen, C.J. (1998).** Immuno-

the SimpliRED TM HIV diagnostic

genicity in humans of a recombinant

reagent in barley, potato and tobacco.

bacterial antigen delivered in a trans-

_Molecular Breeding_ **9 _,_** **113-121.**

genic potato. _Nature Medicine_ **4 _,_** ****

**Shao, H.B., He, D.M., Qian, K.X., Shen,**

**607-610.**

**G.F. and Su, Z.L. (2008).** The ex-

**Tregoning, J.S., Nixon, P., Kuroda, H.,**

pression of classical swine fever vi-

**Svab, Z., Clare, S., Bowe, F., Fair-**

rus structural protein E2 gene in to-

**weather, N., Ytterberg, J., Wijk,**

bacco chloroplasts for applying chlo-

**K.J., Dougan, G. and Maliga, P.**

roplasts as bioreactors. _Comptes_

**(2003).** Expression of tetanus toxin

_Rendus Biologies_ **331 _,_** **179–184.**

Fragment C in tobacco chloroplasts.

**Sharma, A. and Sharma, K.M.K.**

_Nucleic Acids Research_ **31 _,_** **1174-**

**(2009).** Plants as bioreactors, Recent

**1179.**

developments and emerging oppor-

**Twyman, R.M., Stoger, E., Schillberg,**

tunities. Biotechnology Advances **27 _,_** ****

**S., Christou, P. and Fischer, R.**

**811-832.**

**(2003).** Molecular farming in plants:

**Staub,**

**J.M.,** **Garcia,** **B., Graves, **

Host systems and expression tech-

**J.,** **Hajdukiewicz,** **P.T., Hunter, **

nology. _Trends Biotechnology_ **21 _,_** ****

**P.,** **Nehra,**

**N., Paradkar, **

**570-578.**

**V.,** **Schlittler,** **M., Carroll, **

**Valdes, R., Gomez, L., Padilla, S.,**

**J.A.,** **Spatola, L., Ward, D., Ye, G. **

**Britom J., Reyes, B., Alvarez, T.,**

**and Russell, D.A. ****(2000).** High-Mendoza, O., Herrera, O., Ferro,

yield production of a human thera-

**W,, Pujol, M., Leal, V., Linares,**

peutic protein in tobacco chloro-

**M., Hevia, Y., Garcia, C., Mila, L.,**

plasts.

_Nature_

_Biotechnology_ **18 _,_**

**Garcia, O., Sanchez, R., Acosta, A.,**

**333-338.**

**Geada, D., Paez, R., Luis Vega, J.**

**Stoger, E., Fischer, R., Moloney, M.,**

**& Borroto, C. (2003). **Large-scale

**Ma, J.K.C. (2014).** Plant molecular

purification of an antibody directed

pharming for the treatment of chronic

against hepatitis B surface antigen

and infectious diseases. _Annual Re-_

from transgenic tobacco plants. _Bio-_

_view Plant Biology_ **65,743–768.**

_chemistry and Biophysics Research_

**Stoger, E., Sack, M., Fischer, R. and**

_Community ****_**308 _,_** **94–100.**

**Christou, P. (2002).** Plantibodies:

**Vaquero, C., Sack, M., Schuster, F.,**

applications, advantages and bottle-

**Finnern, R., Drossard, J. and**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 320

_Biotech Sustainability (2017)_

_Plant Tissue Culture for Sustainability Minakshi et al._

**Schumann, D. (2002).** A carcinoem-

_β-glucuronidase_ (GUS): a model sys-

bryonic

antigen-specific

diabody

tem for the production of proteins in

produced in tobacco. _Federation of_

plants. _Molecular Breeding_ **4 _,_** **301-**

_American Societies for Experimental_

**312.**

_Biology_ **16 _,_** **408-410.**

**Wu, A.B., Lt, H.P., Zhao, C.S. and**

**Verch, T., Yusibov, V. and Koprowski,**

**Liao, Y.C. (2005).** Comparative

**H. (1998).** Expression and assembly

pathogenicity of _Fusarium gramine-_

of a full-length monoclonal antibody

_arum_ from China revealed by wheat

in plants using a plant virus vector.

coleoptile and floret inoculations.

_Journal of Immunological Methods_

_Mycopathologia_ **164 _,_** **75-83.**

**220 _,_** **69-75.**

www.epicyte.com ****

**Whitelam, G.C., Cockburn, W. and**

**Yoshida, K., Matsui, T. & Shinmyo, A. **

**Owen, M.R.L. (1994).** Antibody

**(2004).** The plant vesicular transport

production in transgenic plants. _Bio-_

engineering for production of useful

_chemical Society Transactions_ **22 _,_** ****

recombinant proteins. _Journal of Mo-_

**940-943.**

_lecular Catalysis B: Enzymatic_ **28 _,_** ****

**Wilde, D.C., Peeters, K., Jacobs, A.,**

**167-171.**

**Peck, I. and Depicker, A. (2002).**

**Zeitlin, L., Olmsted, S.S., Moench,**

Expression of antibodies and Fab

**T.R., Co, M.S., Martinell, B.J. and**

fragments in transgenic potato plants:

**Paradkar, V.M. (1998).** A human-

a case study for bulk production in

ized monoclonal antibody produced

crop plants. _Molecular Breeding_ **9 _,_** ****

in transgenic plants for immunopro-

**2871–282.**

tection of the vagina against genital

**Witcher, D., Hood, E.E., Peterson, D.,**

herpes. _Nature Biotechnology_ **16 _,_** ****

**Bailey, M., Bond, D. and Kusnadi,**

**1361-1364.**

**A. (1998).** Commercial production of

****

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

****

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 321

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P322-335_

**Renewable Energy from Agro-industrial Processing**

**Wastes: An Overview**

**Sudhanshu S. Behera1, Ramesh C. Ray2, * and S. Ramachandran3**

****

_1Department of Fisheries and Animal Resource Development, Government of Odisha, In-_

_dia; 2ICAR- Central Tuber Crops Research Institute (Regional Centre), Bhubaneswar_

_751019, India; 3Department of Biotechnology, Birla Institute of Technology and Science,_

_Pilani, Dubai campus, Dubai International Academic City , P. O. Box No: 345055, Dubai,_

_UAE; *Correspondence: rc.ray6@gmail.com; Tel: +91- 674- 2470528_

**Abstract:** The continued use of fossil-derived fuels has resulted in progressive depletion of

non-renewable energy resources and environmental deterioration that led to the develop-

ment of an alternative renewable source of energy for sustainability. Agricultural and horti-

cultural crops waste processing offer an option among alternatives for generating renewable

energy (bio-refinery) due to its potential sustainable supply and abundance. The conversion

of agro-industrial processing wastes typically contains mixed hexoses and pentoses with

significant proportions of polymeric sugars (lingo-celluloses and cellulose) which are diffi-

cult to degrade and require advances in the technology. This article is highlighting the ma-

jor developments in various biomass-based agro-industrial processing waste-based fuel-

generating processes and suggests a possible major solution to provide energy (bio-energy)

in an eco-friendly way for the reduction of greenhouse gases and pollution. ****

_**Keywords**_ **:** Agro-industry; bio-ethanol; environment; processing; renewable energy

**1. Introduction**

pounds which are main sources of air pol-

lution and global warming (Crutzen _et al_.,

The world is faced with a chronic

2016). Moreover, fossil-fuel exposes the

energy crisis that has resulted in the crip-

earth to changes in price of petroleum re-

pling of most sectors of the economy. It is

sources and political instability from the

estimated that over 80% (about 450-500

oil producing region of the world (Behera

EJ/year) of the world's energy demands is

and Ray, 2014; Aliyu _et al_., 2015).

met by the combustion of fossil fuels

The renewable sources of energy,

(Ioelovich, 2015). The main energy

such as solar, wind, and biofuel (bioetha-

sources of fossil fuels are coal, petroleum

nol,

bio-diesel,

bio-hydrogen)

have

and natural gas. Coal does provide about

gained huge attention from governments

28% of world's consumed energy. Crude

of many countries across the world. Re-

oil- petroleum, provides about 32% of

cently, government policies have planned

world's energy while natural gas provides

to replace the petroleum based fuels with

about 20% of the world's energy con-

renewable biomass fuels, which are de-

sumption (Ioelovich, 2015). However,

rived from agricultural residues such as

fossil-based fuels are limited and exces-

sugarcane, corn, switchgrass, algae, etc

sive exploitation of such fossil fuel in-

(Sarkar _et al_., 2012). These renewable

creases carbon footprints. The combus-

resources are indigenous, non-polluting

tion of these fuels also caused the emis-

and virtually inexhaustible. Globally,

sion of harmful gases, including CO,

more than 30% of the loss occurs from

CO2, NOx and sulfur-containing com-

agricultural/horticultural substrates/ wast-

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_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

es (rice husk, coffee wastes, sugar cane

losses occur at retail and consumer levels.

biogases, maize harvesting wastes, and

Among them, lignocellulosic wastes de-

bamboo cellulose pulp, etc.,) at the retail

rived from cereals, oil seeds, pulses and

and consumer levels, of which the post-

plantation crops, account for 222 million

harvest and processing level wastages ac-

tons in industrialized countries. Fruits,

count for the major share (Singh _et al_.,

vegetables and root crop processing

2012).The different residues/biomass re-

wastes alone are available to the extent of

sulting from the production of agricultural

12 million tons (Auer _et al_., 2017). How-

crops might contribute to the achieve-

ever, over 86 x 106 t of fruits and 162 x

ments of the renewable energy target as

106 t vegetables are produced annually in

proposed for further uses (Scarlat _et al_.,

India, contributing 12.6% and 14.0% of

2010). The use of biomass for transport

the total world production of fruits and

fuel, heat and electricity production will

vegetables respectively (Source FAO

have to increase substantially to meet the

website- February 2014-15; Horticulture

proposed binding target of renewable en-

Database, India-2015). Out of the total

ergy in the EU energy mix of 20% by

production, nearly 76% is consumed in

2020 (Scarlat _et al_., 2010). The chapter

fresh form, while wastage, and loss ac-

discusses the agricultural/horticultural

count for 20-22 percent. Of which only 2-

resources and food processing wastes for

4% is processed in the fruits and vegeta-

potential production of bioenergy.

bles processing industries. This is in sharp

contrast to the extent of processing of

**2. Agricultural and food processing**

fruits and vegetables in several other de-

**wastes**

veloping countries such as Brazil (70%),

Malaysia (83%), Philippines (78%), and

There are various forms of agricultural

Thailand (30%) (Horticulture Database,

and horticultural (agro-industrial) re-

India- 2015). Disposal of these putresci-

sources in the world. Among the agro-

ble fruits and vegetables processing

industrial wastes, horticulture processing

wastes (organic refuse) leads to environ-

industries form a major share throughout

mental and economic problems (Viswa-

the world. Horticultural wastes mainly

nath _et al_., 1992; Scano _et al_., 2014). The

generate from fruits and vegetable pro-

nutrients of food waste may be re-used in

cessing, starchy roots and tubers, coconut,

agriculture by composting or by biotrans-

olive and palm oil mills and fruit-based

formation of food waste into animal feed

fermentation industries (Panda and Ray,

and/or converted to biofuels (Table 1).

2015; Panda _et al_., 2017). The fermenta-

These wastes contain a high amount cel-

tion industries of solid waste include

lulose, hemi-cellulose, and pectin, which

items removed from fruits and vegetables

provide a suitable substrate for fermenta-

during cleaning, processing, cooking,

tion process (Khalid _et al_., 2011). These

and/or packaging. These items may in-

polymers can be hydrolyzed enzymatical-

clude leaves, peels, pomace, skins, rinds,

ly by cellulose, β-glucosidase and pa-

cores, pits, pulp, stems, seeds, twigs, and

tience to their corresponding soluble car-

spoiled fruits and vegetables (Bouallagui

bohydrates and subsequently to biofuels

_et al_., 2005; Panda _et al_., 2016). Globally,

(Ariunbaatar _et al_., 2014; Behera and Ray

more than billion tons of agro- industrial

2016).

and food processing solid wastes are

available. The losses in industrial coun-

_2.1.Bio-ethanol production_

tries are as high as in developing coun-

Bio-ethanol has been proved to be

tries, but in developing countries more

a most promising alternate energy source

than 40% of losses occur at post-harvest

with various added advantages (Manzano-

and processing levels, while in industrial-

Agugliaro _et al_., 2013). The worldwide

ized countries, more than 40% of the

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_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

**Table 1:** Current status of biofuel technologies utilizing agricultural and horticultural sub-

strates and wastes. (Lynd _et al_., 2015, modified) ****

**No Crop**

**Example**

**Industry status**

**category**

****

1.

Starch-rich

Maize, wheat,

About 50 billion L ethanol in the US based on

sorghum

maize

2.

Sugar -rich

Sugar cane, sug-

About 23 billion L ethanol in Brazil based on sug-

ar beet

ar cane

3.

Oil-rich

Rapeseed, soy,

About 23 billion L produced worldwide, mostly in

sunflower, palm

the EU, US and Brazil

oil

4.

Cellulosic

Grass trees, vari-

Liquid fuel capacity about 175 million L world-

ous horticultural wide

wastes

bio-ethanol production is increasing con-

fermentation processes (Lin and Tanaka,

stantly. The world bio-ethanol production

2006; Behera and Ray, 2016). However,

in 2006 was 39 billion liters and has

bio-ethanol from agro-residues could be a

grown to 100 billion liters in 2015 (Sarkar

promising technology that involves four

_et al_., 2012) and is expected to reach 180

processes of pre-treatment, enzymatic hy-

billion liters in 2020 (Ioelovich, 2015).

drolysis, fermentation and distillation

Brazil and USA are the two major ethanol

(Gupta and Verma, 2015). These process-

producers accounting for 62% of the

es have several challenges and limita-

world production (Sarkar _et al_., 2012). In

tions, such as biomass transport and han-

Brazil ethanol is completely produced

dling, and efficient pre-treatment process

from sugar cane. In USA, the production

for removing the lining from the lignocel-

of ethanol relies on corn starch (Ioelo-

lulosic

agro-residues.

Proper

pre-

vich, 2015). Conventional indigenous raw

treatment process may increase the con-

materials for bio-ethanol production in-

centrations of fermentable sugars after

clude sugarcane, molasses/starch and

enzymatic hydrolysis, thereby improving

corn-based material, although the amount

the efficiency of the whole process. Con-

of bio-ethanol produced can hardly meet

version of cellulose to ethanol requires

the current global demand (Sarkar _et al_.,

some new pre-treatment, enzymatic and

2012; Saggi and Dey, 2016). Hence, cel-

fermentation technologies, to make the

lulosic materials such as agro-residues are

whole process cost effective (Gupta and

attractive feedstock for bio-ethanol pro-

Verma, 2015). However, many agricul-

duction. The wastes from agro-residues

tural and horticultural wastes have low

being rich in polysaccharides (cellulose,

lignin content and lesser amounts of cel-

hemi-cellulose and lignin) have been sub-

lulose with more easily degradable poly-

jected to solid state fermentation to pro-

saccharides. This results in lower enzyme

duce bio- ethanol (Singh _et al_., 2012).

pretreatment or physio-chemical costs and

__

increased yields of fermentable sugars

_2.1.1. Waste pre-treatment_

(Sindhu _et al_., 2016).

The biomass from agro-industries

_****_

are the most abundant on the earth. These

_2.1.2. Microflora_

biomass/substrates include corn (maize),

Most

agricultural

bio-

wheat, oats, rice, potato, and cassava. On

mass/vegetable and fruit wastes (VFWs)

a dry basis, corn, wheat, sorghum (milo),

have been used as a potential substrate for

and other grains contain around 60-70%

the ethanol fermentation by microbial

(wt/wt) of starch, which are hydrolyzed to

processes. VFWs contain mainly starch,

hexose and offered a good resource in

cellulose, soluble sugars and organic ac-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 324

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

ids (Table 2). Microbes are well-suited

beet, molasses and fruits) converted into

natural agents for recycling of organic

ethanol directly. The starches (corn, cas-

wastes including VFWs (Zupancic and

sava, potatoes, and root crops) are initial-

Grilc, 2012; Panda _et al_., 2016). The mi-

ly hydrolyzed to fermentable sugars by

croorganisms such as _Bacillus_ , _Pseudo-_

the action of enzymes (Ray and Naskar,

_monas_ , _Rhizopus_ and _Trichoderma_ , well

2007; Ray _et al_., 2008). Celluloses (agri-

known for production of hydrolytic en-

cultural residues, wood, waste sulfite liq-

zymes, are employed for biodegradation

uor from pulp and paper mills) are con-

of VFWs (organic matter) to reduce the

verted into sugars by the action of mineral

biological oxygen demand and chemical

acids. The simple sugar so formed, can

oxygen demand of the liquid wastes

readily fermented to ethanol by the action

(Panda and Ray, 2015). More specifically,

of microbial enzymes (Lin and Tanaka,

microorganisms, such as fungi ( _Mucor_

2006; Monlauet _al_., 2013).

_indicus_ ), bacteria ( _Zymomonas mobilis_ ),

and yeasts ( _Candida utilis_ , _Kluyueromy-_

_2.2.1. Anaerobic digestion and methano-_

_ces marxianus_ ) have been used for etha-

_genesis_

nol production from VFWs. However,

Anaerobic digestion is a biochem-

yeast is most common microorganisms

ical degradation process that is widely

grown on VFWs as substrates (Stabniko-

used for the treatment and energy recov-

va _et al_., 2005).Among the yeast, _Can-_

ery from many kinds of biomass, espe-

_dida utilis_ is selected for cultivation of

cially agricultural products and agro-

concentrated effluents of the food indus-

industrial wastes (Scano _et al_., 2014).

try after its anaerobic acidogenic treat-

Hydrolysis and acidification are the ad-

ment (Stabnikova _et al_., 2005). In con-

vantages in two-phase in anaerobic diges-

trast, _Saccharomyces cerevisiae_ is the

tion processes. FVWs are characterized

most commonly used yeast in industrial

by a high percentage of moisture (>80%),

ethanol production from red beet (juice

high organic content (volatile solids>95%

and bagasse) (Jimenez-Islas _et al_., 2014).

of total solids), are readily biodegraded

Moreover, biotransformation of cellulose-

and are therefore suited to energy recov-

to-ethanol can be conducted by various

ery through anaerobic digestion (Jiang _et_

anaerobic thermophilic bacteria, such as

_al_., 2012). Scano _et al_. (2014) studied the

_Clostridium thermocellum_ , and some fil-

biogas production through an anaerobic

amentous fungi, including _Monilia_ sp., __

digestion pilot plant by using VFWs as

_Neurosprora_ sp., _Zygosaccharomyces_

single substrate. The experimental study

_rouxii_ , _Aspergillus_ sp., _Paecilomyces_ sp.,

was carried out using most suitable oper-

and _Trichoderma viride_ (Lin and Tanaka,

ating parameters and optimum organic

2006). Others efficient microbes and ge-

loading rate was reported at 2.5-3.0 kg

netically modified microbes may also en-

volatile solids/m3d with maximum biogas

hance the enzymatic hydrolysis. The ap-

production of 0.78 Nm3/kg volatile solids

proach of using depolymerizing enzymes

(CH4 content of 55%).

producing co-culture or construction of

engineered microorganisms are attractive

_2.3. Factors affecting biogas production_

for low capital cost and enhanced yield of

Various aspects/factors influence

fermentable sugars (Arora _et al_., 2015).

the biogas production from the feed

__

stocks. The C: N ratio, pH, temperature,

_2.2. Fermentation process_

total solid contents, organic loading rates,

The varied raw materials used in

hydraulic retention time, design of digest-

the fermentation are classified into three

er, inoculum quality and volatile fatty ac-

categories: sugars, starches, and cellulose

id content also regulate and impact biogas

materials (Mohapatra _et al._ 2017). The

production.

main sources of sugars (sugar cane, sugar

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 325

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

**Table 2:** Bio-energy production from lignocellulosic material by various microorganisms

**Microorganisms**

**Waste sub-**

**Feature of the**

**Bio-energy**

**References**

**strate**

**employed mi-**

**production**

**croorganism**

****

**Bacteria**

****

****

****

****

_Pichia stipites_

Sugar cane ba-

Ferment both

Bio-ethanol;

Buaban _et_

BCC15191

gasse

glucose and xy-

0.92g/g

_al_. (2010) ****

lose

_Pichia stipitis_ DSM

Wheat straw

\-

Bio-ethanol;

Bellido _et_

3651 ****

hydrolysates

0.41 g/g

_al_. (2013) ****

_Clostridium aceto-_

Rice straw

Ferment mono-,

ABE; 10.5 g/L

Amiri _et al_.

_butylicum_

poly-

(2014)

saccharides

_C. beijerinckii_

Wheat straw

Ferment hexose ABE; 11.44 g/L

Bellido _et_

hydrolysates

and pentose

_al_. (2014)

_Kluyveromyces_

Sunflower bio-

Cellulase, & β-

Bio-ethanol;

Camargo _et_

_marxianus_ ATCC

mass

glucosidase ac-

27.88 g/L

_al_. (2014)

36907 __

tivity

**Yeasts**

_Saccharomyces_

Spent sulfite

Ferment hexose

Bio-ethanol;

Novy _et al_.

_cerevisiae_

liquor

and pentose

0.31-0.44g/g

(2013)

IBB10B05 ****

_Saccharomyces_

Banana & or-

Improved cellu-

Bio-ethanol;

Singh _et al_.

_cerevisiae_

ange peels

lase activity

28.6-40.7 g/L

(2014)

**Co-culture strains**

_Saccharomyces sp._

Tuber meal of

Recombinant

Bio-ethanol;

Zhang _et al_.

_W0_ \+ _Pichia pastor-_

Jerusalem arti-

inulinase

0.319g/g

(2010)

_is_ X-33/pPICZaA-

choke

_INU1_

_Candida shehatae +_

Sugarcane ba-

Improved cellu-

Bio-ethanol;

Chandel _et_

_Saccharomyces_

gasse

lase activity

3.2g/L

_al_. (2013)

_cerevisiae_

_Bacillus subtilis_ and

Orange peel

Improved cellu-

Bio-ethanol;

Gomaa,

_Pseudomonas aeru-_

lase activity

82.7-92.2 g/L

(2013)

_ginosa_

_Trichoderma_

Oil palm EFB

Improved cellu-

Bio-ethanol;

Karim _et al_.

_reesei_ \+ _Saccharo-_

lase activity

4.6 mg/mL

(2014)

_myces cerevisiae_

**Filamentous fungi** __

_Fusarium ox-_

Glucose, birch-

Over-

Bio-ethanol;

Anasontzis _et_

_ysporum_ ****

woodxylan, corn

expression of

2.68-2.85 g/L

_al_. (2011)

cob or wheat

xylanase

bran

_Trichoderma_ sp. +

Sugarcane ba-

Over-produced

Bio-ethanol; 58

El-Bondkly

_Penicillium_ sp.

gasse and corn-

cellulases*

g/L

and El-

\+ _Aspergillus_ sp. __

cob

Gendy,

(2012)

**Recombinant (mi-**

**cro) organisms**

_Saccharomyces_

Corn stover

Ferment glu-

Bio-ethanol; 40

Lau and

_cerevisiae_ 424A

cose and xylose

g/L

Dale, (2009)

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_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

_**Table 2:** Continued.. _

(LNH-ST)

****

_Escherichia coli_

Wheat straw

Ferment hexose

Bio-ethanol;

Saha and

FBR5 __

hydrolysate

and pentose

8.9-17.3 g/L

Cotta,

(2011)

_Escherichia coli_

Lignocellulosic

Ferment hexose Bio-ethanol; 50

Cotta,

FBR5 __

biomass

and pentose

g/L

(2012)

**Immobilized (mi-**

**cro) organisms**

_Zymomonas mobilis_

Sugarcane mo-

Entrapped in

Bio-ethanol;

Behera _et al_.

MTCC 92

lasses

luffa sponge

58.7-59.1 g/L

(2012)

disc & Ca-

alginate beads

_Saccharomyces_

Molasses

Immobilized

Bio-ethanol;

Agnihotry _et_

_cerevisiae_ MTCC

sodium-alginate

7.6 g/L

_al_. (2015)

3090

_ABE_ : Acetone-butanol-ethanol; _EFB_ : Empty fruit bunches; _Luffa_ : _Luffa cylindrica_ L.; _Over_

_produced cellulase_ ***** : exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase ****

_2.3.1. Carbon to nitrogen (C: N) ratio_

ture has significant effect on the microbial

A C/N ration between 22 and 25

community, process kinetics and stability

seemed to be better for anaerobic co-

and methane yield (Patil and Deshmukh,

digestion of FVWs with its co-substrates.

2015). The high temperature cooking

The most significant factor for enhanced

(140-180oC) is very effective for fermen-

FVWs digestion performance is the im-

tation of ethanol from starchy materialin

proved organic nitrogen content provided

industrial-scale. The high temperature

by the additional wastes (Bouallagui _et_

enhances the efficiency of starch sacchar-

_al_., 2009).

ification and achieves high levels of etha-

nol under complete sterilization of harm-

_2.3.2. PH_

ful microorganisms (Krishania _et al_.,

The optimum pH and pH range

2013). However, to resolve the difficul-

differs with substrate and bio methanation

ties of high production costs and require-

technique. The optimum pH values for

ment of additional amounts of enzymes

the acidogenesis and methanogenesis

(amylase),

non-cooking

and

low-

stages are different. During acidogenesis

temperature cooking fermentation system

stage, lactic, propanoic, and acetic acids

have been recently developed (Ghimire _et_

are formed and thus, the pH falls. The low

_al_., 2015).

pH (about 6.4) can be toxic for methane-

_****_

forming bacteria (optimum between 6.6-

_2.3.4. Total solid content_

7.0). Most of digested carbohydrate sub-

Total solid content influences bio-

strates are acidic and developing pH of

gas production from fruits and vegetable

6.2 or less and thus becoming toxic

wastes. Abbassi-Guendouz _et al_. (2012)

(Chanakya and Malayil, 2012). A suitable

investigated the role of total solid content

amount of lime is added to neutralize the

in anaerobic digestion in batch reactors.

acid accumulated in the carbohydrate res-

idues of processing digesters.

_2.3.5.Organic loading rate /Volatile sol-_

_ids_

_2.3.3. Temperature_

The organic loading rate deter-

The temperature is an important

mines the input of organic matter per unit

parameter for bio methanation. Tempera-

___
___

## volume of digester capacity per day

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_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

measured of the biological conversion

gesters, high rate digesters or digesters

capacity of the anaerobic digestion sys-

with combination of different approaches

tem (Patil and Deshmukh, 2015). There is

for bioenergy (Ganesh _et al_., 2014).

an optimum feed rate for a size of digester

However, most commonly used tech-

is essential for optimum yield of biogas

niques of bio-hydrogen production, in-

(Patil and Deshmukh, 2015). Shen _et al_.

cluding direct bio-photolysis, indirect bio-

(2013) performed the anaerobic co-

photolysis, photo-fermentation and dark-

digestion of FVWs and food waste in sin-

fermentation and conventional or modern

gle-phase and two-phase digesters at var-

techniques (Mudroom _et al_., 2011).

ious organic loading rate (3.5-5.0kg. Vol-

atile solids. m-3. d-1) to investigate bio-

**3. Anaerobic digestion process from**

methane production (0.328-0.544m3. kg-1.

**fruit and vegetable wastes**

Volatile solids).

Normally, biogas is composed of

_2.3.6. Hydraulic retention time_

45-70% methane, 30-45% carbon dioxide,

The amount of time the feedstock

0.5-1.0% hydrogen sulfide, 1-5% water

stays in the digester is known as hydraulic

vapor, and a small amount of other gases

retention time the retention time must be

(hydrogen, ammonia, nitrogen, etc.).

sufficient to carry out the necessary de-

However, the composition varies with the

gree

of

biodegradation

(Patil

and

sources of biodegradable biomass. Bio-

Deshmukh, 2015). Bio methanation of

methane, obtained during anaerobic di-

banana peel and pineapple wastes studied

gestion by the microbial community of

at various hydraulic retention times

biodegradable agricultural and horticul-

showed a higher rate of gas production at

tural substrates/wastes (Singh _et al_.,

lower retention time (Velmurugan and

2012).

Ramanujam, 2011). The lowest possible

hydraulic retention time for banana peel

_3.1. Ensiling and methane generation_

was 25 days, resulting maximum rate of

Ensiling techniques is the process of bio

gas production of 0.76 vol/vol/day with

methanation using the storing of forage

36% substrate utilization, while pineap-

crops and various other agricultural

ple-processing waste digesters was oper-

commodities such as mango peel, orange,

ated at 10 days' hydraulic retention time,

lemon and lime peels, pineapple and to-

with a maximum rate of gas production of

mato processing wastes for a prolong pe-

0.93 vol/vol/day and 58% substrate utili-

riod (Kreuger _et al_., 2011; Panda _et al_.,

zation (Hosseini and Abdul Wahid,

2017). Effects of ensiling process, storage

2014). To maximize the yield of biogas

of biological/agricultural silage additives

and to improve its quality (high CH4 con-

are attributed to increases in organic acids

tent and low H2S content) different strate-

and alcohols contents and showed posi-

gies can be followed: 1) daily organic

tive effects on methane yield (Herrmann

loading rate must be kept constant 2) use

_et al_., 2011). Several processes have been

of well balanced mix of feeding sub-

developed for high rate bio-methanation.

strate/wastes 3) two stages process to sep-

The processes include: 1) up-flow anaer-

arate the hydrolysis and acidogenesis

obic sludge blanket, 2) expanded granular

phases from methanogenesis phase (Sca-

sludge bed, 3) fixed film, 4) fluidized bed

no _et al_., 2014).

and 5) plug flow. Fang _et al_. (2011) oper-

ated the up-flow anaerobic sludge

_2.3.7. Digester design_

blanketreactor using the potato juice for

Various kinds of digesters are

biogas production. The methane potential

used for anaerobic process such as one-

was determined at the highest organic

stage or two-stage digester, wet or dry

loading rates of 5.1 g COD. (L-reactor. d)

digesters, batch or continuous process di-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 328

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

with the methane yield of 240 mL-CH4/g

_genes eutrophus_ and _Bacillus licheniform-_

volatile solids-added.

_is_ when held under anoxic conditions, can

produce hydrogen from organic sub-

_3.2. Acetone-butanol-ethanol production_

strates/wastes (Sivagurunathan _et al_.,

There is also renewed interest in

2016).

reviving

the

acetone-butanol-

ethanolprocess through application of the

_3.4. Biodiesel_

recombinant strains (Lütke-Eversloh and

The technology implemented for

Bahl, 2011) and process development and

production of liquid biofuels is based on

using

cheaper

agricultural

transformation of food-grade biomass

wastes/substrates (Green, 2011). The bio-

(carbohydrates) into bioethanol and vege-

conversion

of

lignocellulosic

sub-

table oils into biodiesel fuel. The main

strate/wastes to monomeric sugars and its

sources of juices of sugar cane, sugar

consequent fermentation has been sug-

beet, and sweet sorghum, as well as

gested for economic production of ace-

starches of corn, wheat, potato, and some

tone-butanol-ethanol (Amiri _et al_., 2014).

other

agricultural

plants

(Ioelovich,

A variety of bacterial strains, such as

2015). Oil-seed crops are the largest

_Clostridium aurantibutyricum_ , _C. bei-_

sources of exploitable biomass to produce

_jerinckii_ and _C. butyricum_ participatein

liquid fuel, bio-diesel (i.e., fatty esters).

acetone-butanol-ethanol production and

Bio-diesel offers enhanced safety charac-

utilize a variety of substrates including

teristics as compared to diesel fuel, hav-

pentose, hexose, starch, and xylan but not

ing no emission of explosive air/fuel va-

cellulose (Bellido _et al_., 2014). Further,

pors (Bhuiya _et al_.,2014; Kumar and

development can be directed by manipu-

Sharma, 2015). Considerable research has

lating and controlling the fermentation

been progressed on the use of vegetable

conditions by reducing the toxic effect of

oils as diesel fuel. Vegetable oils such as

products (repression) on cell physiology

soybean oil, sunflower oil, coconut oil,

and promoting one dominant solvent

rapeseed oil, Tung oil, and palm oil are

product during production of acetone-

the best choice (Carlsson, 2009). The

butanol-ethanol.

most common way to produce bio-diesel

is by transesterification, which refers to a

_3.3. Microbial hydrogen production_

catalyzed chemical reaction of vegetable

Hydrogen is produced by several

oil and an alcohol to yield fatty acid alkyl

processes, such as electrolysis of water,

esters (i.e., biodiesel) and glycerol (Sha-

thermocatalytic reformation of hydrogen-

hid and Jamal, 2011). Indigenous to cen-

rich organic compounds, and biological

tral-south America, Jatropha was intro-

processes. Currently, biological produc-

duced to Africa a few centuries ago. It is

tion of hydrogen (bio-hydrogen) from

currently widely distributed throughout

horticultural residues, using microorgan-

these areas where rural inhabitants gener-

isms, is an exciting new area of technolo-

ally make extensive use of it. Oil from the

gy development (Levin _et al_., 2004).

seeds of jatropa is used as a bio-diesel

Asian countries possess significant poten-

substitute (Osseweijer _et al_., 2015).

tial for producing bio-hydrogen from crop

residues. Bio-hydrogen production by

**4. Challenges and further prospective**

culture of bacteria is highly attractive for

larger-scale applications (Kumar _et al_.,

The production of bioenergy and

2015). Microbes, including strict anaer-

food production is interrelated and is af-

obes (clostridia, ruminococci and ar-

fected by global change of atmospheric

chaea) and facultative anaerobes, includ-

(rising

CO2

and

tropospheric

ing _Escherichia coli_ and _Enterobacter_

ozone),climate (temperature and soil

_aerogenes_ and aerobes, including _Alcali-_

moisture), and land degradation (saliniza-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 329

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

tion, desertification, fertility loss) (Osse-

**Aliyu, A. S. Dada, J. O. and Adam, I.**

weijer _et al_., 2015).Recently, global ener-

**K. (2015).** Current status and future

gy crisis needs optimum yield of bioener-

prospects of renewable energy in Ni-

gy from advanced fermentation technolo-

geria. _Renewable and Sustainable_

gy converting residues/substrates from

_Energy Reviews_ **48, 336-346.**

agro-industries into ethanol, enzyme

**Amiri, H. Karimi, K. and Zilouei, H.**

technology for hydrolysis of lignocellulo-

**(2014).** Organosolv pretreatment of

sic materials, immobilization of microor-

rice straw for efficient acetone, buta-

ganisms in pilot-scale for production of

nol,

and

ethanol

produc-

bio-energy. Furthermore, C4-type crops

tion. _Bioresource_

_Technology_ **152,**

possess the features of high photosynthet-

**450-456.**

ic yield, high rate of CO2 fixation, pro-

**Anasontzis, G. E. Zerva, A. Stathopou-**

duce more biomass, and resistance to

**lou, P. M. Haralampidis, K. Dial-**

aridity when compared with C3 crops.

**linas, G. Karagouni, A. D. and**

Therefore, C4 type of crops are to be

**Hatzinikolaou, D. G. (2011).** Ho-

more investigated and need to be focused

mologous overexpression of xy-

for further bio-energy production (Koçar

lanase in Fusarium oxysporum in-

and Civaş, 2013).

creases ethanol productivity during

consolidated bioprocessing (CBP) of

**5. Concluding remarks**

lignocellulosics. _Journal of Biotech-_

_nology_ **152, 16-23.**

To date, bio-fuel has been evolved

**Ariunbaatar, J. Panico, A. Esposito, G.**

from first to fourth generation and they

**Pirozzi, F. and Lens, P. N. (2014).**

are mainly differed in feedstock and pro-

Pretreatment methods to enhance an-

duction technologies. The agricultural and

aerobic digestion of organic solid

horticultural residues based energy crops

waste. _Applied Energy_ **123, 143-156.**

are critical and needs to be investigated as

**Arora, R. Behera, S. and Kumar, S.**

raw materials for bio-fuels for today and

**(2015).**

Bioprospecting

thermo-

for the future demand. To attain the

philic/thermotolerant microbes for

highest sustainability in bio-fuel produc-

production of lignocellulosic ethanol:

tion, continuous research and develop-

A future perspective. _Renewable and_

ment on all sustainability-aspects is es-

_Sustainable Energy Reviews_ **51, 699-**

sential.

**717.**

****

**Auer, A. VandeBurgt, N.H. Abram, F.**

**References**

**Barry, G. Fenton, O. Markey, B.K.**

****

**Nolan, S. Richards, K. Bolton, D.**

**Abbassi-Guendouz, A. Brockmann,**

**De Waal, T. and Gordon, S.V.**

**D.Trably, E. Dumas, C. Delgenès,**

**2017.** Agricultural anaerobic diges-

**J. P. Steyer, J. P.and Escudié,**

tion power plants in Ireland and

**R.(2012).** Total solids content drives

Germany:

policy

and

prac-

high solid anaerobic digestion via

tice. _Journal of the Science of Food_

mass transfer limitation. _Bioresource_

_and Agriculture_ **97, 719-723.**

_Technology_ **111, 55-61.**

**Behera, S. and Ray, R.C. (2014).** Batch

**Agnihotry, H. Sharma, S. Kaur, R.and**

ethanol production from cassava

**Singh, H.(2015).** Ethanol fermenta-

( _Manihotesculenta_ Crantz) flour by

tion from molasses using free and

_Saccharomyces cerevisiae_ cells im-

immobilized cells of _Saccharomyces_

mobilized in calcium alginate. _An-_

_cerevisiae_ [MTCC3090]–a compara-

_nals_

_of_

__

_Microbiology_

(DOI:

tive study. _ACADEMICIA: An Inter-_

10.1007/s13213-014-0918-8)

_National Multidisciplinary Research_

**Behera, S.S. and Ray, R.C. (2016).** Sol-

_Journal_ **5, 215-223.**

id state fermentation for production

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 330

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

of microbial cellulases: Recent ad-

**preda, V. Pichyangkura, R. Reng-**

vances and improvement strategies.

**pipat,**

**S.**

**and**

**Eurwilaichitr,**

_International Journal of Biological_

**L.(2010).**

Bioethanol

production

_Macromolecule_ **86, 656–669.**

from ball milled bagasse using an on-

**Behera, S. Mohanty, R. C. and Ray, R.**

site produced fungal enzyme cocktail

**C. (2012).** Ethanol fermentation of

and xylose-fermenting _Pichia stipi-_

sugarcane molasses by _Zymom-_

_tis. Journal of Bioscience and Bioen-_

_onasmobilis_ MTCC 92 immobilized

_gineering_ **110, 18-25.**

in _Luffa cylindrica_ L. sponge discs

**Camargo, D. Gomes, S. D. and Sene, L.**

and Ca-alginate matrices. _Brazilian_

**(2014).** Ethanol production from sun-

_Journal of Microbiology_ **43, 1499-**

flower meal biomass by simultane-

**1507.**

ous saccharification and fermentation

**Bellido, C. González-Benito, G. Coca,**

(SSF) with _Kluyveromyces marxi-_

**M. Lucas, S. and García-Cubero,**

_anus_ ATCC 36907. _Bioprocess and_

**M. T. (2013).** Influence of aeration

_Biosystems Engineering_ **37, 2235-**

on bioethanol production from ozo-

**2242.**

nized wheat straw hydrolysates using

**Carlsson, A. S. (2009).** Plant oils as feed-

_Pichia stipitis_. _Bioresource Technol-_

stock alternatives to petroleum–A

_ogy_ **133, 51-58.**

short survey of potential oil crop

**Bellido, C. Pinto, M. L. Coca, M. Gon-**

platforms. _Biochimie_ **91, 665-670.**

**zález-Benito,**

**G.**

**and**

**García-**

**Chanakya, H. N. and Malayil, S.**

**Cubero, M. T. (2014).** Acetone–

**(2012).** Anaerobic digestion for bio-

butanol–ethanol (ABE) production

energy from agro-residues and other

by _Clostridium beijerinckii_ from

solid wastes—an overview of sci-

wheat straw hydrolysates: Efficient

ence, technology and sustainabil-

use of penta and hexa carbohy-

ity. _Journal of the Indian Institute of_

drates. _Bioresource Technology_ **167,**

_Science_ **92, 111-144.**

**198-205.**

**Chandel, A.K. Antunes, F.F. Anjos, V.**

**Bhuiya, M. M. K. Rasul, M. G. Khan,**

**Bell, M.J. Rodrigues, L.N. Singh,**

**M. M. K. Ashwath, N. Azad, A.**

**O.V. Rosa, C.A., Pagnocca, F.C.**

**K. and Hazrat, M. A. (2014).** Se-

**and Da Silva, S.S.(2013).** Ultra-

cond generation biodiesel: potential

structural mapping of sugarcane ba-

alternative to-edible oil-derived bio-

gasse after oxalic acid fiber expan-

diesel. _Energy Procedia_ **61, 1969-**

sion (OAFEX) and ethanol produc-

**1972.**

tion by _Candida shehatae_ and _Sac-_

**Bouallagui, H. Lahdheb, H. Romdan,**

_charomyces_

_cere-_

**E. B.Rachdi, B. and Hamdi, M.**

_visiae. Biotechnology for Biofuels_ **6,**

**(2009).** Improvement of fruit and

**4-12.**

vegetable waste anaerobic digestion

**Cotta, M. A. (2012).** Ethanol production

performance and stability with co-

from lignocellulosic biomass by re-

substrates addition. _Journal of Envi-_

combinant _Escherichia coli_ strain

_ronmental Management_ **90, 1844-**

FBR5. _Bioengineered_ **3, 197-202.**

**1849.**

**Crutzen, P. J. Mosier, A. R. Smith, K.**

**Bouallagui, H. Touhami, Y. Cheikh, R.**

**A. and Winiwarter, W. (2016).**

**B. and Hamdi, M. (2005).** Bioreac-

N2O release from agro-biofuel pro-

tor performance in anaerobic diges-

duction negates global warming re-

tion

of

fruit

and

vegetable

duction by replacing fossil fuels.

wastes. _Process_

_Biochemistry_ **40,**

_In_ : _Paul J. Crutzen: A pioneer on_

**989-995.**

_atmospheric chemistry and climate_

**Buaban, B. Inoue, H. Yano, S. Tanap-**

_change_

_in_

_the_

_anthropo-_

**ongpipat, S. Ruanglek, V. Cham-**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 331

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

_cene_. Springer International Publish-

crops. _Bioresource Technology_ **102,**

ing. **pp. 227-238.**

**5153-5161.**

**El-Bondkly, A. M.and El-Gendy, M. M.**

**Hosseini, S. E. and Abdul Wahid, M.**

**(2012).** Cellulase production from

**(2014).** The role of renewable and

agricultural residues by recombinant

sustainable energy in the energy mix

fusant strain of a fungal endophyte of

of Malaysia: a review. _International_

the marine sponge latrunculiacortica-

_Journal of Energy Research_ **38,**

ta for production of ethanol. _Antonie_

**1769-1792.**

_Van Leeuwenhoek_ **101, 331-346.**

**Ioelovich, M. (2015).** Recent findings

**Fang, C., Boe, K. and Angelidaki, I.**

and the energetic potential of plant

**(2011).** Biogas production from pota-

biomass as a renewable source of

to-juice, a by-product from potato-

biofuels–A review. _BioResources_ **10,**

starch processing, in upflow anaero-

**1879-1914.**

bic sludge blanket (UASB) and ex-

**Jiang, Y. Heaven, S. and Banks, C. J.**

panded granular sludge bed (EGSB)

**(2012).** Strategies for stable anaero-

reactors.

_Bioresource_

_Technolo-_

bic

digestion

of

vegetable

_gy_ **102, 5734-5741.**

waste. _Renewable Energy_ **44, 206-**

**Ganesh, R. Torrijos, M. Sousbie, P.**

**214.**

**Lugardon, A. Steyer, J. P. and**

**Jiménez-Islas, D.Páez-Lerma, J. Soto-**

**Delgenes, J. P. (2014).** Single-phase

**Cruz, N. O. and Gracida, J. (2014).**

and two-phase anaerobic digestion of

Modelling of ethanol production

fruit and vegetable waste: compari-

from red beet juice by _Saccharomy-_

son of start-up, reactor stability and

_ces cerevisiae_ under thermal and acid

process performance. _Waste Man-_

stress conditions. _Food Technology_

_agement_ **34, 875-885.**

_and Biotechnology_ **52, 93-99.**

**Ghimire, A. Frunzo, L. Pirozzi, F. Tra-**

**Karim, R. A. Hussain, A. S. and Zain,**

**bly, E. Escudie, R. Lens, P. N. and**

**A. M. (2014).** Production of bioetha-

**Esposito, G. (2015).** A review on

nol from empty fruit bunches cellu-

dark fermentative biohydrogen pro-

losic biomass and Avicel PH-101

duction from organic biomass: pro-

cellulose. _Biomass Conversion and_

cess parameters and use of by-

_Biorefinery_ **4, 333-340.**

products. _Applied Energy_ **144, 73-95.**

**Khalid, A. Arshad, M. Anjum, M.**

**Gomaa, E. Z. (2013).** Bioconversion of

**Mahmood, T. and Dawson, L.**

orange peels for ethanol production

**(2011).** The anaerobic digestion of

using _Bacillus subtilis_ and _Pseudo-_

solid organic waste. _Waste Manage-_

_monas aeruginosa_. _African Journal_

_ment_ **31, 1737-1744.**

_of Microbiology Research_ **7, 1266-**

**Koçar, G. and Civaş, N. (2013).** An

**1277.**

overview of biofuels from energy

**Green, E. M. (2011).** Fermentative pro-

crops: Current status and future pro-

duction of butanol—the industrial

spects. _Renewable and Sustainable_

perspective. _Current Opinion in Bio-_

_Energy Reviews_ **28, 900-916.**

_technology_ **22, 337-343.**

**Kreuger, E. Nges, I. A. and Björnsson,**

**Gupta, A. and Verma, J. P. (2015).** Sus-

**L. (2011).** Ensiling of crops for bio-

tainable bio-ethanol production from

gas production: effects on methane

agro-residues: a review. _Renewable_

yield and total solids determination.

_and Sustainable Energy Reviews_ **41,**

_Biotechnology for Biofuels_ **4, 44-50.**

**550-567.**

**Krishania, M. Kumar, V. Vijay, V. K.**

**Herrmann, C Heiermann, M. and**

**and Malik, A. (2013).** Analysis of

**Idler, C. (2011).** Effects of ensiling,

different techniques used for im-

silage additives and storage period

provement of biomethanation pro-

on methane formation of biogas

cess: A review. _Fuel_ **106, 1-9.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 332

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

**Kumar, G. Bakonyi, P. Periyasamy, S.**

**Mohapatra, S. Mishra, C. Behera, S. S.**

**Kim, S. H. Nemestóthy, N. and Bé-**

**and Thatoi, H. N. (2017).** Applica-

**lafi-Bakó, K. (2015).** Lignocellulose

tion of pretreatment, fermentation

biohydrogen: practical challenges

and molecular techniques for enhanc-

and recent progress. _Renewable and_

ing bioethanol production from grass

_Sustainable Energy Reviews_ **44, 728-**

biomass – A review. _Renewable and_

**737.**

_Sustainable Energy Reviews_ **78,**

**Kumar, M. and Sharma, M. P. (2015).**

**1007–1032.**

Assessment of potential of oils for

**Monlau, F. Barakat, A. Trably, E. Du-**

biodiesel production. _Renewable and_

**mas, C. Steyer, J. P. and Carrère,**

_Sustainable Energy Reviews_ **44, 814-**

**H. (2013).** Lignocellulosic materials

**823.**

into biohydrogen and biomethane:

**Lau, M. W. and Dale, B. E. (2009).** Cel-

impact of structural features and pre-

lulosic ethanol production from

treatment. _Critical Reviews in Envi-_

AFEX-treated corn stover using _Sac-_

_ronmental Science and Technolo-_

_charomyces cerevisiae_ 424A (LNH-

_gy_ **43, 260-322.**

ST). _Proceedings of the National_

**Mudhoo, A. Forster-Carneiro, T. and**

_Academy of Sciences_ **106, 1368-**

**Sánchez, A. (2011).** Biohydrogen

**1373.**

production and bioprocess enhance-

**Levin, D. B. Pitt, L. and Love, M.**

ment: a review. _Critical Reviews in_

**(2004).**

Biohydrogen

production:

_Biotechnology_ **31, 250-263.**

prospects and limitations to practical

**Novy,**

**V.**

**Krahulec,**

**S.**

**Longus,**

application. _International Journal of_

**K.Klimacek, M. and Nidetzky, B.**

_Hydrogen Energy_ **29, 173-185.**

**(2013).** Co-fermentation of hexose

**Lin, Y. and Tanaka, S. (2006).** Ethanol

and pentose sugars in a spent sulfite

fermentation

from

biomass

re-

liquor matrix with genetically modi-

sources: current state and pro-

fied

Saccharomyces

cere-

spects. _Applied Microbiology and_

visiae. _Bioresource Technology_ **130,**

_Biotechnology_ **69, 627-642.**

**439-448.**

**Lütke-Eversloh, T. and Bahl, H. (2011).**

**Osseweijer, P. Watson, H. K. Johnson,**

Metabolic engineering of _Clostridi-_

**F. X. and Batistella, M. (2015).** Bi-

_um acetobutylicum:_ recent advances

oenergy and food security. _Bioenergy_

to

improve

butanol

produc-

_& Sustainability: Bridging the Gaps _

tion. _Current Opinion in Biotechnol-_

_(eds Souza GM, Victoria R, Joly C,_

_ogy_ **22, 634-647.**

_Verdade L)(Chapter 4)_ **72, 779.**

**Lynd, L.R. Sow, M. Chimphango, A.F.**

**Panda, S. K. and Ray, R. C. (2015).** Mi-

**Cortez, L.A. Cruz, C.H.B. Elmissi-**

crobial processing for valorization of

**ry, M. Laser, M. Mayaki, I.A.**

horticultural

wastes.

**Moraes, M.A.Nogueira, L.A. and**

_In_ : _Environmental microbial bio-_

**Wolfaardt, G.M.(2015).** Bioenergy

_technology **.**_ Springer

International

and

African

transfor-

Publishing, Heidelberg. **pp. 203-221.**

mation. _Biotechnology for Biofuels_ **8,**

**Panda, S.K. Mishra, S.S.Kayitesi, E**

**18-28.**

**and Ray, R.C. (2016).** Microbial-

**Manzano-Agugliaro, F. Alcayde, A.**

processing of fruit and vegetable

**Montoya, F. G. Zapata-Sierra, A.**

wastes for production of vital en-

**and Gil, C. (2013).** Scientific pro-

zymes and organic acids: Biotech-

duction

of

renewable

energies

nology and scopes. _Environmental_

worldwide: an overview. _Renewable_

_Research_ **146, 161- 172.**

_and Sustainable Energy Reviews_ **18,**

**Panda, S.K. Ray, R.C.Mshra, S. S. and**

**134-143.**

**Kayitesi1, K. (2017).** Microbial pro-

cessing of fruit and vegetable wastes

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 333

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

into potential biocommodities: a re-

the availability of agricultural crop

view. _Critical Review in Biotech-_

residues in the European Union: Po-

_nology,_

tential and limitations for bioenergy

http://dx.doi.org/10.1080/07388551.20

use. _Waste Management_ **30, 1889-**

17.1311295 ****

**1897.**

**Patil, V. S.and Deshmukh, H. V. (2015).**

**Shahid, E. M. and Jamal, Y. (2011).**

A review on optimization of parame-

Production of biodiesel: a technical

ters for vegetable waste biomethana-

review. _Renewable and Sustainable_

tion. _International Journal of Cur-_

_Energy Reviews_ **15, 4732-4745.**

_rent Microbiology and Applied Sci-_

**Shen, F. Yuan, H. Pang, Y.Chen, S.,**

_ences_ **4, 488-493.**

**Zhu, B. Zou, D. and Li, X. (2013).**

**Ray, R.C. and Naskar, S.K. (2008).** Bio-

Performances

of

anaerobic

co-

ethanol production from sweet potato

digestion of fruit & vegetable waste

( _Ipomoea batatas_ L.) by enzymatic

(FVW) and food waste (FW): Single-

liquefaction and simultaneous sac-

phase vs. two-phase. _Bioresource_

charification and fermentation. _Dy-_

_Technology_ **144, 80-85.**

_namics Biotechnology, Process Bio-_

**Sindhu, R. Binod, P. and Pandey, A.**

_chemistry and Molecular Biology_ **2,**

**(2016).** Biological pretreatment of

**47- 49.**

lignocellulosic biomass–An over-

**Ray, R. C. Mohapatra, S. Panda, S.**

view. _Bioresource Technology_ **199,**

**and Kar, S. (2008).** Solid substrate

**76-82.**

fermentation of cassava fibrous resi-

**Singh, A. R. P. Singh, A. K. Charan, A.**

due for production of α- amylase,

**I.and Charan, A. A. (2014).** Produc-

lactic acid and ethanol. _Journal of._

tion of bioethanol by banana and or-

_Environmental Biology_ **29, 111-115.**

ange peel using _Saccharomyces_

**Saggi, S. K. and Dey, P. (2016).** An

_cerevisiae_. _Trends in Biosciences_ **7,**

overview of simultaneous saccharifi-

**18-21.**

cation and fermentation of starchy

**Singh, A. Kuila, A. Adak, S. Bishai, M.**

and lignocellulosic biomass for bio-

**and Banerjee, R. (2012).** Utiliza-

ethanol production. _Biofuels_ **4, 1-13.**

tion of vegetable wastes for bioener-

**Saha, B. C. and Cotta, M. A. (2011).**

gy

generation. _Agricultural_

_Re-_

Continuous ethanol production from

_search_ **1, 213-222.**

wheat straw hydrolysate by recombi-

**Sivagurunathan,**

**P.**

**Kumar,**

nant ethanologenic _Escherichia coli_

**G.Bakonyi, P. Kim, S.H. Koba-**

strain FBR5. _Applied Microbiology_

**yashi, T. Xu, K.Q. Lakner, G.,**

_and Biotechnology_ **90, 477-487.**

**Tóth, G.Nemestóthy, N. and Bélafi-**

**Sarkar, N. Ghosh, S. K Bannerjee, S.**

**Bakó, K.(2016).** A critical review on

**and Aikat, K. (2012).** Bioethanol

issues and overcoming strategies for

production from agricultural wastes:

the enhancement of dark fermenta-

an overview. _Renewable Energy_ **37,**

tive hydrogen production in continu-

**19-27.**

ous systems. _International Journal of_

**Scano, E. A. Asquer, C. Pistis, A. Ortu,**

_Hydrogen Energy_ **41, 3820-3836.**

**L.Demontis, V. and Cocco, D.**

**Stabnikova, O. Wang, J. Y. and Ding,**

**(2014).** Biogas from anaerobic diges-

**H. B. (2005).** Biotransformation of

tion of fruit and vegetable wastes:

vegetable and fruit processing wastes

Experimental results on pilot-scale

into yeast biomass enriched with se-

and preliminary performance evalua-

lenium. _Bioresource Technology_ **96,**

tion

of

a

full-scale

power

**747-751.**

plant. _Energy Conversion and Man-_

**Velmurugan, B. and Ramanujam, R.**

_agement_ **77, 22-30.**

**A. (2011).** Anaerobic digestion of

**Scarlat, N.Martinov, M. and Dalle-**

vegetable wastes for biogas produc-

**mand, J. F. (2010).** Assessment of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 334

_Biotech Sustainability (2017)_

_Renewable Energy from Agro-industrial Processing Wastes Behera et al._

tion

in

a

fed-batch

reac-

production from hydrolysates of inu-

tor. _International Journal of Emerg-_

lin and the tuber meal of Jerusalem

_ing Science_ **1, 478-486.**

artichoke by _o_ sp. W0. _Bioresource_

**Viswanath, P. Devi, S. S. and Nand, K.**

_Technology_ **101, 8166-8170.**

**(1992).** Anaerobic digestion of fruit

**Zupančič, G. D. and Grilc, V. (2012).**

and vegetable processing wastes for

Anaerobic treatment and biogas pro-

biogas

production. _Bioresource_

duction

from

organic

_Technology_ **40, 43-48.**

waste. _Management_

_of_

_Organic_

**Zhang, T. Chi, Z. Zhao, C. H. Chi, Z.**

_Waste_ **3, 1-28.**

**M. and Gong, F. (2010).** Bioethanol

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 335

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P336-343_

**Mitigation of Climatic Change by Organic Agriculture**

**Mohan Mani1, *, Manohar Murugan2, Ganesh Punamalai3 and**

**Vijayalakshmi Ganesan Singaravelu4**

_1Mahendra Engineering College(Autonomous), Namakkal, Tamil Nadu, India;_

_2Vivekananda College of Arts and Science (Autonomous), Elayampalayam, Namakkal Dt.,_

_Tamil Nadu, India; 3Department of Microbiology, Faculty of Science, Annamalai Universi-_

_ty, Chithamparam, Tamil Nadu, India; 4Former Professor of Environmental Biotechnology,_

_Manonmaniam Sundaranar University, Tirunelveli, Tamil Nadu, India;_

_*Correspondence: mohanrtt@gmail.com; Tel: +91-9486069246_

**Abstract:** Agricultural inputs and farming systems are playing vital role in the consumption

of fossil fuels and climate change. Organic and non-organic production in terms of energy

use is essential for understanding the energy inefficiencies of different agro systems and

their potential for minimizing energy consumption and mitigating environmental impacts

particularly climate change. Organic agriculture can provide a more energy efficient ap-

proach which leads to sustainable agricultural productions. Organic agro productions is

contributing less greenhouse gas emissions and have a superior potential to sequester car-

bon available in biomass than conventional agro production. The energy efficiency of or-

ganic agriculture in terms of bioenergy production and thereby renewable fuel source is to

reduce dependency of fossil fuel energy and mitigate environmental pollution caused by

emissions. The industrialized production of agro products is responsible for a heavy impact

on the environment and playing a major role in increasing global Green House Gas (GHG)

emissions in respect to the usage of synthetic fertilizers and pesticides. More than 480 mil-

lion tons of GHG is released to the atmosphere each year by the synthetic fertilizers and

pesticides factories. Climate change is a serious environmental issue and has broad impacts

on sustainable development and the future of our economy, health, and agricultural produc-

tion sector. Integrated farm management system is reducing and sequestering GHG emis-

sions. Organic farming is essential to ensure the future of our environment and food pro-

duction in a sustainable manner. This article highlights the importance of organic agricul-

ture and its role in mitigating climatic change.

****

_**Keywords**_ : Carbon sequestration; climatic change; greenhouse gases; organic farming; sus-

tainable production

**1. Introduction**

and agricultural practices highly depend-

****

ent on synthetic pesticides and fertilizers.

Agriculture in India is the promising

Equipment and machinery for cultivation

sector for millions of livelihood around

is mainly dependent on consumption of

two thirds of the work force in the coun-

fossil fuels. The modern agricultural

try. Most of the population is dependent

practices and industrialization of agro

on agriculture and allied sectors which

system has created adverse effects on the

contribute nearly 24 per cent of gross

environment and thereby emitting more

domestic product (GDP) of India (Ma-

Green House Gases (GHG).The main fac-

hadevan, 2003). Crop production has

tor in anthropogenic climate change is the

changed significantly in the past decades

increase in the concentration of carbon in

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 336

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

the atmosphere over time. This increased

the fermentation that takes place in the

concentration has been caused by the

digestive tract of ruminants observed in

emission of GHGs as a result of economic

livestock resulted the emission of 40% of

activities, including energy, industry,

CO2eq (Figure 1). Photosynthesis is the

transport, and land use, many of which

natural process in which the CO2 is con-

rely upon fossil fuels (Banuri and Op-

verted to organic carbon and it is convert-

schoor, 2007). Climatic change is influ-

ed to CO2 by respiration and decomposi-

encing the food caching behavior of a

tion. Carbon restoration to the soil is hap-

wide range of animals to store food for

pened by many agricultural practices in-

future use. (Sutton _et al_ , 2016). Migrat-

clude, conservation tillage, recycling ag-

ing animals are vulnerable to climate

ricultural residues, cover cropping and

change and it affects the migratory bird

crop rotations.Biological carbon seques-

movement. (Seebacher and Post, 2015).

tration is a process by which the green-

Mollusks, and coral reefs are observed

house gases generated during agricultural

negative impacts of climatic change

activities have been removed from the

(Tschakert 2015). Prolonged exposures to

atmosphere. Agriculture recorded from

elevated CO2 have affected structural pro-

10 to 12 percent of total global human

teins like actin (Ertl _et al.,_ 2016). The

caused emissions of greenhouse gases

core objective of the this chapter is to elu-

during 2005, according to the report by

cidate the importance of organic farming

Intergovernmental Panel on Climate

as the alternative to modern farming sys-

Change (IPCC, 2007). India is recorded

tem and leads to the sustainable agricul-

the second largest emitter of CO2 from

tural system to overcome the recent

total agricultural practices and by the ap-

changes in the environment and energy

plication of the synthetic fertilizers fol-

crisis faced by the present generation.

lowed by China (Figure 2 and 3).

****

**2. Greenhouse gas emissions from ag-**

**3. Organic agriculture and climatic**

**riculture**

**change**

****

Agricultural activities are respon-

Organic farming is the time immemorial

sible for the emission and sinks for

practice in India and it was a part of the

greenhouse gases. The industrial produc-

traditional farming systems. The ancient

tion of nitrogen based fertilizers, the

history of knowledge like the Vrik-

combustion of fossil fuels are the primary

shayurveda, Agnipurana, Brihat Samhita

sources of greenhouse gases released

and Arthasasthra is exemplifying the tra-

from agriculture. Enteric fermentation or

ditional Indian agriculture. Traditional

**Figure 1:** Average CO2 emission by sector observed during the year 2000 and 2014

(Source: FAOSTAT 2014).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 337

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

****

**Figure 2:** Top 10 CO2 emitters during agricultural production (Source: FAOSTAT 2014).

**Figure 3:** Top 10 CO2 emitters by the application of synthetic fertilizers (Source: FAO-

STAT 2014). ****

****

farmers who are doing organic agriculture

farming practices. The demand for food

were efficiently utilized the land resource

and renewable energy sources are in-

and appropriately utilizing the SOIL

creased with the increase of population.

(Soul Of Infinite Life).

The impacts of climate change by the us-

Organic agriculture is a holistic pro-

age of fossil fuel supported chemical fer-

duction management system that avoids

tilizers, herbicides and pesticides will cre-

use of synthetic fertilizers, pesticides and

ate deleterious issue on agricultural pro-

genetically modified organisms, minimiz-

duction.

es the pollution of air, soil and water and

Organic agriculture is proved to be a

optimizes the health and productivity of

remedy for climate change and the global

interdependent communities of plants,

food insecurity and potentially solve the

animals and people (Codex Alimentarius

issues related to vulnerability, unsustain-

Commission, 2001).

ability and social inequity of agricultural

Organic agriculture is playing a signif-

production. Climate change has its most

icant role in climate change and food se-

significant impacts on agriculture because

curity to the world's population.Climate

of its broad geographic dispersion and

change mitigation and food security are

highly dependent on climate and envi-

vital management mediated by organic

ronmental factors. Developing countries

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 338

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

are the least responsible for climate

work Convention on Climate Change

change, yet the most at risk from its ef-

(UNFCCC) held in Paris in December

fects (UNESCO, 2010).There is a need of

2015 focused to lower the greenhouse gas

a policies and practices to protect the ever

(GHG) by launching a voluntary action

changing environmental conditions and

plan named 4 per 1000 initiative - Soils

ecosystems that ensure the sustainable

for food security and climate (4o/oo Initi-

development. Organic agriculture is ef-

ative). It initiated to increase soil carbon

fectively retaining soil organic matter,

stock annually by 0.04 % through carbon

soil carbon and there by balancing of eco-

sequestration. The annual growth rate 4

system. Organic agriculture is the tradi-

parts per thousand of the soil carbon stock

tional practice that mitigates climate

would make it possible to stop the present

change, generate evergreen farming sys-

increase in atmospheric CO2. Food securi-

tems, eradicate poverty and augment the

ty is confirmed by providing proven tech-

level of food security. Organic agriculture

nologies to the farmers in developing

discharges very minimum levels of

countries to repair and sustain the soils.

greenhouse gases (GHG) and efficiently

To feed 9.5 billion in 2050, it is essen-

sequesters carbon in the soil. Organic ag-

tial to keep our soils alive. Figure 4 shows

riculture makes faming system and people

various steps to ensure soil organic matter

dependent on this sector more resilient to

by providing various ecosystem services

climate change due to its water use effi-

like capable of resisting the soil from ero-

ciency, tolerance to extreme weather con-

sion, retaining water, increasing soil fer-

ditions and lower or no risk of total crop

tility and proliferating soil biodiversity as

failure. Organic agriculture is extensively

per the initiation suggested by 4 per 1000.

practiced agro-ecological farming system

It can meet three fold challenges includ-

that accomplishes the main objectives of

ing food security, adaptation to food sys-

enabling people to thrive while improving

tem and people to climate change and

our eco-systems and their natural cycling

mitigation of anthropogenic emission.

process.

Soils under organic management will of-

ten increase the level of soil organic car-

**4. Soils for food security and climate -**

bon more than conventional management

**4 per 1000 initiative**

(FAO 2011 and 2011a).

****

The 21st Conference of the Parties

(COP21) of the United Nations Frame-

**Figure 4:** Ecosystem services by soil organic matter and its effects and challenges.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 339

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

**5. Climatic change – Indian scenario**

• National Mission on Sustainable Habi-

tat

India is holding 2.4% world sur-

• National Water Mission

face area, 17.5% of world's population

• National Mission on Sustainable Agri-

and is the fastest growing major economy

culture

in the world. It is the fourth largest

• National Mission for Sustaining the

greenhouse gas emitter, accounting for

Himalayan Ecosystem

5.8 % of global emissions. India's emis-

• National Mission for Green India

sions increased by 67.1 % between 1990

• National

Mission

on

Strategic

and 2012, and are projected to increase up

Knowledge for Climate Change

to 85 % by 2030(MoEF, 2014). Increase

• State Action Plan on Climate Change

in temperature beyond critical limits by

• Auto Fuel Vision and Policy 2025

increased industrialization resulted to re-

• Indian Network for Climate Change

ductions in rice, sorghum and maize yield

Assessment

(Saravanakumar, 2015). The climatic

change includes changes in the intensity

An Expert Committee was consti-

and distribution of rainfall and elevation

tuted by the Government of India and

of the level of oceans and a growing in-

have recommended a roadmap for im-

crease in the frequency and intensity of

proving auto fuel quality in India till 2025

extreme climatic phenomena (Escobar,

and provided vehicular emission norms

2009).

for various categories of vehicles. As a

India has abundance source of solar,

result, a roadmap for rolling out Bharat

wind, hydro power and more potential for

Stage-IV (BS-IV), equivalent of Euro-IV,

bioenergy production. Sustainable devel-

by 2017 and BS-V (Euro-V) auto fuels by

opment can be achieved by the integration

2020 in the entire country was recom-

of bioenergy sector with agricultural prac-

mended for implementation (MoEF,

tices. Various regulatory frameworks are

2014).

formed by India for the developmental

strategies for encouraging bioenergy and

**6. Sustainable development and cli-**

sustainable agriculture. Biomass poten-

**matic change**

tial in integration with agriculture for

****

power generation and climate change mit-

Sustainable development, defined

igation in Indian scenario is essential for

as " _development that meets the needs of_

the current situation (Kothari _et.al,_ 2015).

_the present without compromising the_

The contributions of our country

_ability of the future generations to meet_

will take in to account the imperatives for

_their own needs_ " (WCED, 1987). It in-

addressing the challenges of poverty erad-

volves a harmonious incorporation of a

ication, food security and nutrition, uni-

viable economy, responsible governance,

versal access to education and health,

people's empowerment, social consisten-

gender equality and women empower-

cy and ecological balance. Sustainable

ment, water and sanitation, energy, em-

development means economic develop-

ployment, sustainable cities and human

ment with strong correlation with the

settlement and last but not the least, the

improvement of environmental quality.

means of implementation for the follow-

Economic development and maintaining

ing enhanced action for achieving among

environmental quality are essential for

others sustainable development goals.

sustainable development.

• Jawaharlal Nehru National Solar Mis-

Sustainable development is an ef-

sion

fective tool for mitigation and adaptation

• National Mission for Enhanced Energy

from climate change, a major constraint

Efficiency

faced by recent years. Eriksen _et al._

(2011) have charted out four principles

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 340

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

that can guide adaptation responses in a

partment of Economic and Social

manner that supports sustainability. Sus-

Affairs

Working

Paper

No.

tainable adaptation should (1) recognize

**56**.ST/ESA/2007/DWP/56. **pp.** **1 -**

the context of vulnerability, including

**26.**

multiple stressors, (2) acknowledge that

**Codex**

**Alimentarius**

**Commission.**

different values and interests affect adap-

**(2001)**. Guidelines for the produc-

tation outcomes, (3) integrate local

tion, processing, labelling and mar-

knowledge into adaptation responses and

keting of organically produced

(4) consider potential feedbacks between

foods. First Revision. Joint Food

local and global processes.

and

Agriculture

Organization

(FAO) and World Health Organiza-

**7. Concluding remarks**

tion (WHO) Food Standards Pro-

****

gram, Rome, Italy **pp. 3.**

This chapter highlights that the

**Eriksen,S., Paulina, A., Chandrasekar,**

organic farming system is an efficient tool

**B., Rafael, D. M., John I. M.,**

for tackling the climatic change mitiga-

**Charles, N. K., O'Brien, Olorun-**

tion and energy efficient pathway for ful-

**femi, F., Park, J., Sygna, L. and**

fil the goal of food and energy for all.

**Ulsrud, K. (2011).** When not every

Both food security and energy efficiency

response to climate change is a

are consistently managed by organic agri-

good one: Identifying principles for

cultural practices. It is essential to miti-

sustainable adaptation. _Climate and_

gate the climate change and shift from

_Development_ **3, 7–20.**

modern agricultural practice to organic

**Ertl, N.G., O'Connor, W.A., Wiegand,**

agriculture is an alternative that can con-

**A.N and Elizur, A. (2016).** Molec-

serve energy with environment protection

ular analysis of the Sydney rock

without compromising the requirements

oyster ( _Saccostrea glomerata_ ) CO2

of the human beings. It is essential to

stress response. _Climate Change_

consider that organic agriculture will be

_Responses_ **3, 6. 1-19.**

the solution for the problem faced by the

**Escobar, J. C., Lora, E. S., Venturini,**

present agricultural practices. The global

**O. J., Yań˜ez b, E. E., Castillo,**

solution is necessary at this juncture to

**E. F. and Almazan, O. (2009).**

overcome the impact caused by climatic

Biofuels: Environment, technology

change. Adopting organic agricultural

and food security **.** _Renewable and_

practices globally for the sustainability

_Sustainable Energy Reviews_ **13,**

will boost the chances of achieving 2oC

**1275–1287.**

target and minimize the temperature be-

**FAO (2011).** Energy Smart Food for

low 1.5oC.

People and Climate. Food and Ag-

riculture Organization of the United

**Acknowledgement**

Nations, Rome. **pp. 1-78**.

**FAO (2011a).** Food and Agriculture Or-

The authors are glad to express their

ganization of the United Nations

deep sense of gratitude to Mr. R. Gopal

Natural Resources Management and

Sharma, Leading farmer at Kallidai-

Environment Department Rome,

kurichi for providing valuable suggestions

December 2011 - Organic Agricul-

and views when writing this chapter.

ture and Climate Change Mitigation

\- A Report of the Round Table on

**References**

Organic Agriculture and Climate

****

Change. **pp. 1-82**.

**Banuri, T. and Opschoor,H. (2007).**

**FAO (2014).** Asia and the Pacific Food

Climate Change and Sustainable

and Agriculture Statistical Year

Development United Nations De-

book **. pp. 152-168.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 341

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

**Gliessman, S. (2017).** Confronting cli-

Combating Climate Change. Brief-

mate change with agroecology in

ing Paper for UNFCCC COP 20

Mozambique. . _Agroecology and_

Lima, Peru. **pp. 1-28.**

_Sustainable Food Systems_ **41, 99-**

**Nicholls, R. J., Hanson, S., Herweijer,**

**100.**

**C., Patmore, N., Hallegatte, S.,**

**Heather, E. D., Robert, H. G., Jona-**

**Jan C. M., Jean, C. and Muir, W.**

**than, C. W., William, K. P., Ben-**

**R. (2007).** Ranking the World's Cit-

**jamin, Z., Guillermo, G., Ma-**

ies Most Exposed to Coastal Flood-

**ría, A. and William, C. (2014).** El

ing Today and in the Future. Execu-

Niño adversely affected childhood

tive Summary, Environment Work-

stature and lean mass in northern

ing Paper no. **1**. Paris: Organization

Peru. _Climate Change Responses_ **1,**

of Economic Cooperation and De-

**1-10.**

velopment. **pp. 1-10.**

**IEA** (International Energy Agency)

**Ortiz, R., Jarvis, A., Fox, P., Ag-**

**(2016).** World Energy Outlook

**garwal, P. K. and Campbell, B.**

OECD/IEA, Paris. **pp. 1-13**.

**M. (2014).** Plant genetic engineer-

**IPCC (2007).** Climate Change **:** Working

ing, climate change and food securi-

Group II: Impacts, Adaptation and

ty. CCAFS Working Paper no. **72**.

Vulnerability **. pp. 1-987.**

CGIAR Research Program on Cli-

**IPCC (2014)**. Climate Change: Impacts,

mate Change, Agriculture and Food

Adaptation and Vulnerability. Fifth

Security (CCAFS). Copenhagen,

Assessment Report, Intergovern-

Denmark. **pp.1-27.**

mental Panel on Climate Change,

**Osman, E. B. (2009).** Climate change

Working Group II. **pp. 1-207.**

impacts, adaptation and links to sus-

**Julia, I. B.**

**Chelsea, L. P.**

**An-**

tainable development in Africa.

**drew, J. F. and Ross, A. V. _(_** **2015)**.

_Unasylva_ **231/232, 12-16.**

Temperature sensitivity of mineral

**Ravenscroft, C. H., Raj, W. and Jason**

soil carbon decomposition in shrub

**D. F. (2015).** Rapid genetic diver-

and

Graminoid

Tundra,

West

gence in response to 15 years of

Greenland. _Climate Change Re-_

simulated climate change. _Global_

_sponses_ **3, 1-15.**

_Change Biology_ **21, 4165–4176**.

**Kothari, R. V., Pathak, A. K., Chopra,**

**Robinson, J. B. and Herbert, D. (2001).**

**S. A., Tanu, A. and Yadav, B. C.**

Integrating climate change and sus-

**(2015** ) **.** Developments in Bioenergy

tainable development. _International_

and Sustainable Agriculture Sectors

_Journal of Global Environmental_

for Climate Change Mitigation in

_Issues_ , **1, 130 -149.**

Indian Context: A State of Art. _Cli-_

**Saravanakumar, V. (2015).** Impact of

_mate Change and Environmental_

Climate Change on Yield of Major

_Sustainability_ **3, 93-103.**

Food Crops in Tamil Nadu, India -

**Lima Paris Agenda for Action (LPAA)**

South Asian Network for Develop-

**The 4o/ooInitiative (2015).** Minis-

ment and Environmental Economics

try of Agriculture agrifood and for-

(SANDEE) Working Paper No **. 91-**

estry COP21 Paris. **pp. 1-8**.

**15, pp.1-33**

**Mahadevan, R. (2003)** Productivity

**Seebacher, F. and Post, E. (2015).** Cli-

growth in Indian Agriculture: The

mate change impacts on animal mi-

role of globalization and economic

gration. _Climate Change Responses_

reform. _Asia-Pacific Development_

**2, 1-2.**

_Journal_ **10, 57-72**.

**Sudhakar, K., Rajesh, M. and Premala-**

**Ministry of Environment, Forests and**

**tha, M. (2012).** A Mathematical

**Climate Change. Government of**

Model to Assess the Potential of

**India, (2014)**. India's Progress in

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 342

_Biotech Sustainability (2017)_

_Mitigation of Climatic Change by Organic Agriculture Mani et al._

Algal Bio-fuels in India. _Energy_

tions Children's Fund 3 United Na-

_Sources_ , Part A, **34, 1114–1120**.

tions Plaza New York, NY 10017,

**Sutton, A.O., Strickland, D. and D.**

USA **pp.1-6.**

**Norris, D.R. (2016).** Food storage

**UNESCO (2010).** Climate Change Initia-

in a changing world: implications of

tive - Climate Change Education for

climate change for food-caching

Sustainable Development.7, Place

species. _Climate Change Responses_.

De Fontenoy 75253 Paris 07 SP

**3, 12. 1-25.**

France. **pp. 1-20.**

**Trevor, D. (2014).** Climate Change and

**WCED. (1987).** Our common future: Ox-

the Food Chain. Royal Irish Acad-

ford University Press.

emy. Climate Change Sciences

**Wyss, W. S. and Yim. (2008).** Carbon

Committee. Issue **12**. **pp. 1-6**.

Trading, Climate Change, Envi-

**Tschakert. (2015).** 1.5°C or 2°C: a con-

ronmental Sustainability and Saving

duit's view from the science-policy

Planet Earth. Paper no. **PA21A-**

interface at COP20 in Lima, Peru. __

**1292, 2007-2008**. ****

_Climate Change Responses_ **2, 1-11.**

**Yusuf Chisti. (2007).** Biodiesel from mi-

**UNICEF (2009).** Child Friendly Schools

croalgae _. Biotechnology Advances_

Manual, Climate Change and Envi-

**25,294–306.**

ronmental Education, United Na-

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 343

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P344-357_

**Application of Anti-vibrio and Anti-quorum Sensing**

**Technology for Sustainable Development in Shrimp**

**Aquaculture**

**Ramesh Kandasamy1,*, Amutha Raju2 and Manohar Murugan1**

_1Department of Microbiology, Vivekanandha College of Arts and Sciences for Women (Au-_

_tonomous), Elayampalayam - 637 205, Tiruchengode, Namakkal Dt., Tamil Nadu, India;_

_2Department of Biotechnology, Periyar University PG Extension Centre, Dharmapuri,_

_Tamil Nadu, India; *Correspondence: ksrames@gmail.com; Tel:+91 9943112125_

****

****

**Abstract:** Farming of various marine shrimp species has been developed and commercial-

ized in the last three decades. In the beginning, marine shrimp were cultivated in South-east

Asia by farmers who raised them as incidental crops in tidal fish ponds. Over the years,

enormous progress in developing shrimp culture techniques has been made. Shrimp culture

evolved from an extensive farm using tidal zones to a super-intensive one in the 2000's us-

ing ponds more inland. Outbreak of disease such as Vibriosis is considered as one of the

important constraint and challenge in aquaculture industry of the world.Conventional ap-

proaches such as the use of antibiotics, disinfectants and other antimicrobial drugs have

shown limited success in the disease prevention. In this context, there are sound reasons for

studying the beneficial eco-friendly actions of probiotics and phyto-medicine in boosting the

shrimp aquaculture production without or minimal adverse impacts on environment. This

chapter highlights the anti-vibrio and anti-QS nature of probionts and plants against the _Vib-_

_rio_ pathogen of luminous Vibriosis, a major bacterial disease in shrimp aquaculture.

****

_**Keywords**_ **:** Anti-vibrio; _Penaeus monodon;_ probiotcs; quorum sensing; _Vibrio harveyi_

****

****

**1. Introduction**

there are about 68 c ****

ountr **** ies having shrimp

farm operations. Among them, 22 coun-

Aquaculture is one which comprises

tries reported producing _Litopenaeus van-_

all forms of culturing aquatic animals and

_namei_ (white shrimp), while 23 countries

plants in fresh, brackish and marine envi-

are producing _P. monodon_ (black tiger

ronments. According to FAO statistics,

shrimp). In 2002, the global shrimp farm-

aquaculture is one of the fastest growing

ing industry produced an estimate of 1.6

food producing industries. It has increased

million metric tons of shrimp and produc-

at an average rate of 8.9% per year since

tion is projected to increase at a rate of 12-

1970. In developing countries, in addition

15% per year over the next several years

to agriculture the exploitation of aquatic

(Rosenberry, 2003). World shrimp aqua-

resources can provide additional animal

culture production has grown tremendous-

protein. Aquaculture can be an excellent

ly from a production of 200,000 tons in

complement to meet the food requirement

1985 to approximately 3.8 million metric

of growing population. Further, it is esti-

tons in 2012 (GOAL 2011 shrimp aqua-

mated that half of the world's seafood

culture survey). In 2002, the global shrimp

demand will be met by aquaculture in

farming industry produced an estimate of

2020. Shrimp aquaculture is widespread

1.6 million metric tons of shrimp and pro-

throughout the tropical world. Currently,

duction is projected to increase at a rate of

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_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

12-15% per year over the next several

growth of Juveniles.Adults prefer high

years (Rosenberry, 2003). The black tiger

salinity for reproduction; hence they mi-

shrimp, _Penaeus monodon_ and the Pacific

grate into deep shore where the mating

white shrimp, _Litopenaeus vannamei_ are

takes place. About 50,000 - 1,000,000

the most widely cultured species every-

eggs are laid by female per spawning

where. Currently, there are about 68 coun-

(Rosenberry, 1997). The eggs hatched out

tries having shrimp farm operations.

and release the first stage of larvae called

Among them, 22 countries reported pro-

nauplius. After a few days they develop

ducing _Litopenaeus vannamei_ (white

into the protozoeae and metamorphose

shrimp), while 23 countries are producing

into myses. The myses develop in to

_P. monodon_ (black tiger shrimp). But as

postlarvae (PLs), a stage of megalopas and

with many other industries, rapid growth

share most of the adult characters. As they

of this sector has brought with it the prob-

become larvae, migrate in to offshore

lem of environmental pollution. Though

plankton-rich surface water.PL reaches

the shrimp hatchery technology has ad-

330 mm or above in length and 25-30 g in

vanced over the decades, thehatchery pro-

weight within 3-4 months after stocking in

duction is frequently affected by viral and

culture ponds with wide range of salinity

bacterial disease inflicting huge loss.

(Lee and Wickins, 1992; Rosenberry,

1997). This rapid growth of _P. monodon_

**2. Life cycle of penaeid shrimp**

made to initiate many culturing industries

near the coastal area. Ultimately it leads to

Penaeid shrimp ( _Penaeus monodon_ ) is

generate crowding and environmental

otherwise called Giant black tiger shrimp

degradation that make the rearing animal

because of its huge size and tiger-striped

more susceptible to various diseases

band appearance in the tail. Penaeid

(Johnson, 1989).

shrimp belong to the largest phylum in the

animal kingdom, the Arthropoda. This

**3. Factors influencing shrimp health**

group of animal is characterized by the

presence of paired appendages and a pro-

Many factors influence shrimp

tective cuticle or exoskeleton that covers

health status such as the age of shrimp,

the whole animal. Presence of cephalotho-

management conditions, biotic and abiotic

rax with stiff rostrum and segmented ab-

stress and pathogens (Figure 1). Infectious

domen on the external of the animal is

disease is one of the major limiting factors

unique characters which distinguished

in shrimp farming. Shrimp can be threat-

from other species. Organs like heart, gills

ened by protozoan, fungal, bacterial and

and digestive tract are to be found in

viral pathogens but viral and bacterial dis-

Cephalothorax. In the head region, anten-

eases cause major troubles in shrimp

nules and antennae perform sensory func-

farming (Lightner, 1996). Shrimp farming

tions. The mandibles and the two pairs of

itself has got a significant effect on the

maxillae form the jaw-like structures that

environment such as loss of mangrove

are involved in food uptake. In the thorax

ecosystems, nutrient enrichment and eu-

region, the maxillipeds are the first three

trophication of coastal waters, develop-

pairs of appendages, modified for food

ment of antibiotic resistance in marine

handling and the remaining five pairs are

bacteria and accumulation of chemicals

the walking legs (pereiopods). Five pairs

and

toxicity

to

non-target

species

of swimming legs (pleopods) are found on

(Menasveta, 1997).

the abdomen.

Life cycle of a typical penaeid spe-

**4. Bacterial disease, vibriosis**

cies includes several stages in different

habitats. Mangrove estuaries and brackish

The reason of low level of aqua-

water provide suitable environment for the

culture production has been due to a

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_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

**Figure 1:** Factors influencing the susceptibility of shrimp to pathogens.

combination of both disease and pollution.

The disease produced by Vibrio in shrimp

While disease played the prominent role

culture is commonly called as vibriosis.

in declining aquaculture stocks, both fac-

The other names of bacterial vibriosis are

tors caused massive mortality in the lead-

luminescent vibriosis, penaeid bacterial

ing aquaculture countries. Among the

septicaemia and red-leg disease (Aguirre-

groups of microorganisms that cause seri-

Guzmán _et al.,_ 2004). With the rapid de-

ous losses, the best known are bacteria

velopments in aquaculture particularly in

because of the devastating economic ef-

Asia and South America, _V. harveyi_ and

fects they have on affected farms. Bacteri-

related bacteria have become recognized

al diseases mainly due to Vibriosis have

as a serious cause of disease (Austin and

been reported in Penaeid shrimp culture

Zhang, 2006). In many cases, _Vibrios_ are

systems implicating several species of

opportunists causing disease when the

Vibrios which includes _Vibrio harveyi_ ,

host organism is immune-suppressed or

_V.splendidus_ , _V. parahaemolyticus, V. al-_

otherwise physiologically stressed (Peddie

_ginolyticus, V. anguillarum, V. vulnificus,_

and Wardle, 2005). Even though all crus-

_V. campbelli, V. fischeri, V. damsella, V._

taceans are suffered by this bacterium,

_pelagicus, V. orientalis, V. ordalii, V._

most serious struggle was reported in Pe-

_mediterrani, V. logei_ etc. From the all

naeid shrimp culture (Austin and Zhang,

above species _V. harveyi_ is the main causa-

2006).

tive agent causing luminous vibrosis to

The ill effects of adult animal due to

larva in hatchery and pond cultivation

vibriosis includes reddening body with red

(Won and Park, 2008). Being an important

to brown gills, swimming lethargy and

etiological agent, it causes mass mortali-

reduced feeding behaviours (Nash _et al.,_

ties in penaeid shrimp culture and leads to

1992). Similarly infected PL showed emp-

huge economic losses.They continue to

ty gut and reduced motility and phototax-

cause chronic mortalities of up to 30%

is. Based on the syndrome,they are ex-

among _P. monodon_ larvae, post larvae and

pressed as localised and systemic vibri-

adult under stressful conditions (Le

osis, oral and enteric vibriosis, septic

Groumellec _et al.,_ 1996). In production

hepatopancreatitis and appendage and cu-

unit it causes 100% losses at a time due to

ticular vibriosis (Lightner, 1996). Lumi-

various virulence factors (Chythanya _et_

nescent _V. harveyi_ appears to release exo-

_al.,_ 2002).

toxins and may cause 80-100% mortality

The genus Vibrio is a gram negative

in _P. monodon_ hatcheries (Harris, 1995).

motile rod shaped gamma proteobacter.

_Vibrio_ species also cause red-leg disease

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_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

characterised by red colouration of the

Bassler, 2001). These molecules are con-

pleopods, periopods and gills in juvenile

stantly produced and received at a basal

to adult shrimps. Shrimps suffering vibri-

level by bacterial cells. With high popula-

osis may display localised lesions of the

tion density, there is a surplus of signal-

cuticle typical of bacterial shell disease,

ling molecules in the environment. These

localised

infections

from

puncture

signals diffuse back into the cell where

wounds, loss of limbs, cloudy muscula-

they facilitate the regulation of gene ex-

ture, localised infection of the gut or

pression (Hastings and Greenberg, 1999).

hepatopancreas and general septicemia

The quorum sensing signal molecules are

(Lightner, 1993). Virulence factors asso-

found to be involved in the regulation of

ciated with _V. harveyi_ pathogenicity is due

various physiological processes such as

to the production of bacteriocin-like sub-

the bioluminescence, biofilm formation,

stance, phage induced haemolytic activity,

pigment production, toxin production, ex-

extracellular products such as chitinases,

opolysaccharide production, motility and

cysteine protease, haemolysin, luciferase,

virulence factor production in many

metalloprotease, proteases, phospholipas-

Gram-negative bacteria including fish and

es, siderophores and proteinaceous exo-

shrimp pathogens like _V. harveyi_ (Bruhn

toxins.

_et al.,_ 2005; Kennedy _et al.,_ 2006).

High loads of either _V. parahae-_

In each QS system, the autoin-

_molyticus_ or _V. harveyi_ induced the round-

ducer attaches to a gene that is known as

ing up and detachment of epithelial cells

the transcriptional activator and this at-

from the basal lamina of the mid gut trunk

tachment can lead to alterations in DNA

(MGT) and can cause high mortality in

and activation of virulence factors (Waters

shrimp by eliminating two layers that pro-

and Bassler, 2005). Gram-negative bacte-

tect the shrimp from infections: the epithe-

ria use N-acyl homoserine lactones

lium and the peritrophic membrane. In

(AHLs) as autoinducers, while Gram-

addition, loss of the epithelium may affect

positive bacteria use oligopeptides to

the regulation of water and ion uptake into

communicate (Miller and Bassler, 2001).

the body. It is well-known that there are

The most extensively investigated inter-

significant differences between different

cellular signaling molecules are the AHLs.

_V. harveyi_ isolates in terms of pathogenic-

Quorum sensing in _V. harveyi_ is regulated

ity with some strains being highly virulent

via a multichannel phosphorylation /

and others being non-pathogenic (Austin

dephosphorylation cascade. This bacte-

and Zhang, 2006). Even if many virulence

rium produces and responds to two auto-

factors have been already reported in _V._

inducers namely (i) harveyi autoinducer 1

_hrarveyi,_ complete pathogenic mecha-

(HAI-1) and (ii) autoinducer 2 (AI-2),

nisms should be elucidated.

which regulate the expression of biolumi-

nescence

and

other

virulence

fac-

**5. Quorum sensing in _V. harveyi_** ****

tors.Miller and Bassler (2001) have thor-

oughly studied the mechanism of QS in _V._

The term 'Quorum Sensing' re-

_harveyi._ HAI-1 is an AHL and its biosyn-

fers to the process of bacterial cell-to-cell

thesis is catalysed by the luxM enzyme

communication. It is a population depend-

(Figure 2). AI-2 is a furanosyl borate

ent phenomenon first characterized in the

diester and its biosynthesis is mediated by

1970's in luminescent marine species of

the luxS enzyme. Both HAI-1 and AI-2

_Vibrio_ (Nealson _et al.,_ 1970). Through

are detected at the cell surface by the

this mechanism bacteria coordinate gene

LuxN and LuxP-LuxQ receptor proteins

expression in a density-dependent manner.

respectively. In the absence of the signals,

It is solely depends on the production, re-

LuxN and LuxQ autophosphorylate and

lease and detection of chemical signal

transfer phosphate to LuxO via LuxU. The

molecules called autoinducers (Miller and

phosphorylated luxO is an active repressor

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_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

**Figure 2** : Mechanism of QS in _V. harveyi_ (Miller and Bassler, 2001).

for the target genes. In the presence of the

occur between resistant bacteria and viru-

signal molecules, LuxN and LuxQ interact

lent pathogens that re-enter the culture

with their autoinducers and change from

facilities after the treatment. Thus, antibi-

kinases to phosphatases that drain phos-

otic-resistant strains of pathogenic _V. har-_

phate away from LuxO via LuxU. The

_veyi_ can evolve rapidly. In view of the in-

dephosphorylated LuxO is inactive. Sub-

discriminate use of antibiotics in aquacul-

sequently, transcription of the target genes

ture, it cannot be surprising that many re-

is activated by LuxR. Recently, a third QS

ports have mentioned multiple resistance

component, a _Vibrio cholerae_ -like autoin-

of _V. harveyi_ strains to several antibiotics

ducer CAI-1 was discovered and identi-

(Table 1).From all this, it might be clear

fied as (S)-3-hydroxytridecan-4-one in _V._

that the efficacy of antibiotics to treat lu-

_Harveyi_ (Henke and Bassler, 2004). Sev-

minescent vibriosis is very poor. The

eral investigations have been made to find

presence of residual antibiotics in com-

the effect of _V. harveyi_ quorum sensing on

mercialized aquaculture products consti-

the production of the virulence factors like

tutes another problem with respect to hu-

caseinase, gelatinase, lipase, hemolysin

man health as this can lead to an alteration

and phospholipase by determining their

of the normal human gut microbiota and

expression levels both _in vitro_ and _invivo_

can also generate problems of allergy and

during infection of gnotobiotic brine

toxicity (Cabello, 2006). Anbiotics resi-

shrimp (Natrah _et al.,_ 2011; Darshanee _et_

dues in animals are also regarded as haz-

_al.,_ 2011).

ardous for exporting quality. In order to

limit the use of antibiotics, many workers

**6. Antibiotics vs anti-vibrio probiotics**

have been exploring the use of new bioac-

tive compounds for controlling bacterial

Traditionally

antibiotics

have

diseases of shrimp particularly that of

been used in attempts to control bacterial

caused by _V. harveyi_. Various solutions

disease in aquaculture. When antibiotics

have been proposed such as the use of

are used, the resistant strains can multiply

probiotics, immunostimulation, vaccina-

rapidly because their (sensitive) competi-

tion, specific pathogen-free (SPF) and

tors get removed. Moreover, horizontal

specific pathogen-resistant (SPR) shrimp.

exchange of resistant determinants can

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****

**Table 1:** Multiple antibiotic resistances in _V. harveyi_ isolated from aquaculture facilities ****

**Multiple**

**Location**

**Antibiotic(s)**

**Reference**

**resistance**

India

Cotrimoxazole, chloramphenicol,

**+**

Karunasagar _et al.,_ 1994

erythromycinand streptomycin

Java

Tetracyclin, ampicillin and other β-

**+**

Teo _et al.,_ 2002

lactams

Mexico

Ampicillin, amikacin, carbenicillin,

**+**

Molina _et al.,_ 2002

cephalotin and oxytetracycline

Philippines Oxytetracycline, furazolidone, ox-

Tendencia and De La Peña,

**+**

olinic acid and chloramphenicol

2001

Philippines Kanamycin, gentamycin, carbenicil-

**+**

Nakayama _et al_., 2006

lin and ampicillin

Taiwan

Nitrofurantoin, novobiocin and sul-

**+**

Liu _et al.,_ 1997

phonamide

Thailand

Kanamycin and carbenicillin

**+**

Nakayama _et al.,_ 2006

The addition of beneficial bacte-

agement of shrimp culture practices

ria to exclude potential pathogens from

(Vaseeharan _et al.,_ 2004). Probiotics in

shrimp larviculture has been suggested as

contrast to antibiotics can be a safer eco-

early as 1991. Beneficial bacteria may en-

logical alternative tool for sustainable aq-

hance larval nutrition by supplying essen-

uaculture. In order to be considered as

tial nutrients, improving digestion through

biological control agents in aquaculture,

essential enzymes, mediating direct uptake

probiotics should be non-pathogenic and

of dissolved organic material and produc-

biochemically and physiologically well

ing substances which may inhibit the

characterized. It should be genetically sta-

growth of opportunistic pathogens (Brow-

ble. They should be normal inhabitants of

dy, 1998). The potential negative conse-

the host and able to survive and grow at

quences of using antibiotics in aquaculture

the site of application while exerting their

have led to the use of non-pathogenic bac-

beneficial effect. Finally, they should

teria as probiotic control agents (Vasee-

maintain their viability and activity

haran and Ramasamy, 2003). Probiotics

throughout the product manufacturing and

are defined as ''live microbial feed sup-

storage.

plement which when consumed in ade-

Balcázar _et al.,_ (2006) found that

quate amounts confer a health benefit for

_V. alginolyticus_ UTM102, _Bacillus sub-_

the host''. As antibiotics become less

_tilis_ UTM126, _Roseobacter gallaeciensis_

popular for controlling the aquatic micro-

SLV03 and _Pseudomonas aestumarina_

flora in hatcheries the use of probiotics in

SLV22 are effective probiotics in prevent-

qauaculture has become increasingly pop-

ing _V. parahaemolyticus_ infection in

ular. The probionts commonly used for

shrimp culture. Feed conversion ratio,

aquaculture are isolated from healthy lar-

specific growth rate and final production

vae and adults. However, some probionts

were higher in shrimp receiving a probi-

used for humans and terrestrial animals

otic mixture of five _Bacillus_ species ( _B._

have also shown promise in aquaculture

_subtilis_ , _B. licheniformis_ , _B. polymyxa_ ,

(Vine _et al.,_ 2006). Probiotic use in aqua-

_B. laterosporus_ and _B. circulans_ ) than in

culture is practiced throughout the world

control shrimp which had received no

and the results showed improved man-

probiotic (Ziaei _et al.,_ 2006). _Bacillus_ S11

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_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

bacterium provided protection in _P. mon-_

activity against luminous strain of _V. har-_

_odon_ shrimp when challenged with _V._

_veyi_ (Ramesh and Umamaheswari, 2011).

_harveyi_. After a 100 days feeding trial,

Possible modes of action that

shrimp in the treatment groups displayed

have been mentioned in literature for pro-

100% survival after challenge with _V._

biotics include: (1) Production of inhibito-

_harveyi_ whereas high mortality was ob-

ry compounds, (2) Competition for nutri-

served in the control group (Rengpipat _et_

ents, (3) Competition for adhesion sites in

_al.,_ 1998). Continuous addition of _Bacillus_

the gastrointestinal tract, (4) Enhancement

sp. as probiotic to tanks containing black

of the immune response, (5) Production of

tiger shrimp for over 160 days decreased

essential nutrients such as vitamins and

shrimp mortality caused by luminescent-

fatty acids and (6) Enzymatic contribution

pathogenic _Vibrio_ (Moriarty, 1998).

to digestion. A protocol for the develop-

Cell-free extract of _Bacillus sub-_

ment of probiotics as biocontrol agents in

_tilis_ BT23 showed greater inhibitory ef-

aquaculture was proposed by Verschuere

fects against the growth of _V. harveyi_.

_et al._ (2000) and later by Vine _et_

Their probiotic effect was tested by expos-

_al._ (2006). It involves the following major

ing _P. monodon_ larvae to _B. subtilis_ BT23

steps: (1) _In vitro_ screening, (2) Identifica-

before a challenge with _V. harveyi_. The

tion, (3) Pathogenicity or toxicity test, (4)

results showed 90% decrease in accumu-

_In viv_ o validation and (5) Cost-benefit

lated mortality (Vaseeharan and Rama-

analysis. In addition to this, bacteria that

samy, 2003). Decamp _et al_.(2008) report-

are able to improve the water quality by

ed some field data of the use of a. The ad-

removing toxic inorganic nitrogen or by

dition of the commercial mixture of _Bacil-_

mineralizing organic matter are also con-

_lus_ strains in Thai and Brazilian hatchery

sidered as probiotics.

water significantly improved the survival

of _P. monodon_ and _Litopenaeus vannamei_

**7. Plant based anti-vibrio**

larvae. There have been reports that _Pseu-_

_domonas_ species produce bioactive com-

Antibacterial compounds from

pounds with the ability to control vibrios

natural resources would be the alternative

such as _V. harveyi_ and _V. parahaemolyti-_

to overcome the resistance problem in

_cus_ and that have no effect on the shrimp

most of the pathogens. Screening of anti-

(Vijayan _et al.,_ 2006). The culture super-

bacterial activity of medicinal plants is

natant or culture filtrate of _Pseudomonas_

very important since vast number of me-

sp. W3 contain secreted secondary metab-

dicinal plants have been used for centuries

olites (anti-vibrio) that inhibited the path-

as remedies for human and animal diseas-

ogenic bacteria responsible for shrimp

es. Much interest is now directed towards

luminous vibriosis disease The active anti-

the vast untapped source of plant-based

vibrio compound produced by Pseudomo-

antimicrobials, many of which reduce the

nas sp. W3 is a small molecule with heat

side effects of synthetic antimicrobials.

stable, pH resistant and mostly tolerant to

Medicinal plant extracts have been used

a variety of enzymes such as lysozyme,

for centuries as remedies for animal dis-

protease, lipase and amylase (Rattana-

eases because they contain components of

chuay _et al_., 2010). A bioactive compound

therapeutic value. Various chemothera-

produced by Pseudomonas MCCB 102

peutic agents isolated from plants have

and 103 that inhibited _V. harveyi_ was

proved effective against drug-resistant

identified

as

N-methyl-1-

bacteria. The role of plants in the discov-

hydroxyphenazine, a phenazine antibiotic

ery of drugs has increased notably in re-

(Preetha _et al_., 2009). Cell free extracts of

cent years due to a substantial improve-

four _Bacillus_ species isolated from Penae-

ment in biological screening methods. Un-

id shrimp gut have shown the inhibitory

fortunately, the chemical nature of phyto-

compounds present in many plants are still

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_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

has to be elucidated. Though numerous

fied (Figure 3). Lactonases block signal

studies have reported the basic bioactive

reception by removing the lactone ring

nature of various herbals, only a small

from the AHL molecule resulting in just

percentage of that have been examined

the acylhomoserine. Acylases detach the

and explored thoroughly for their bioac-

nitrogen bond to form a fatty acid chain

tive potential. Many terrestrial and coastal

and homoserine lactone. By changing the

plants and marine seaweeds were reported

structure of the AHL molecules, lactonas-

to have promising antibacterial (anti-

es and aclyases keep AHLs from attaching

vibrio) activity against aquaculture patho-

to the transcriptional activator (Czajkow-

gens including _V. harveyi_. Three man-

ski and Jafra, 2008). Several AHL-

grove

species

_(Avicennia_

_mari-_

degrading enzymes identified in various

_na,Bruguiers cylindrical_ and _Acanthus_

bacteria have the potential to be used as

_ilicifolius)_ collected from the coast was

quorum quenchers. The first application of

extracted in methanol and tested for dif-

autoinducer quenching for the purpose of

ferent range of biological activities includ-

disease control involves aiiA [autoinducer

ing antimicrobial activity against five spe-

inactivation (aiiA)], a Bacillus gene en-

cies of fish and shrimp _Vibrio_ pathogens

coding AHL lactonase, which inactivates

(Manilal _et al.,_ 2009).

AHL by hydrolyzing its lactone bond

(Dong _et al.,_ 2001). AHL degradation

**8. Anti-quorum**

**sensing**

**(quorum**

protects aquatic animals from infection,

**quenching)**

hence AHL-degrading Bacillus sp. might

be interesting novel biocontrol strains for

Strategies to interfere with quorum

use in aquaculture. Similarly one of the

sensing provide new avenues to combat

first AHL acylases identified was AiiD

bacterial diseases in humans, animals and

from _Ralstonia eutropha_ (Lin _et al.,_

plants. These strategies are termed as

2003). The homologs of aiiA were later

quorum quenching which targets different

found in the closely related _Bacillus_ spe-

components of bacterial quorum-sensing

cies including _B. subtilis_ , _B. cereus_ , _B._

communication systems and disintegrates

_mycoides_ and many subspecies of _B. thu-_

quorum-sensing-dependent bacterial at-

_ringiensis_ (Dong _et al.,_ 2002; Lee _et al.,_

tacks. Due to the increase in antibiotic re-

2002; Pan _et al_., 2008; Uroz _et al.,_ 2003).

sistance, disruption of QS could lead to

Medicinal plants contain large

new pharmaceuticals and it can signifi-

varieties of chemical substances with im-

cantly decrease the virulence factor pro-

portant therapeutic properties that can be

duction in bacteria without interfering

effectively utilized in the treatment of an-

their growth. Hence, the disruption of

imal diseases like Vibriosis caused by lu-

quorum sensing (quorum quenching **)** has

minous _Vibrio_ pathogens. Because of their

been suggested as a new anti-infective

history of medicinal properties, many folk

strategy in aquaculture and several tech-

medicinal plants have been screened for

niques that could be used to disrupt quor-

anti-quorum sensing activities. Among all

um sensing have been investigated (Dong

the possibilities to inhibit QS activity, the

and Zhang, 2005). This can be done by

use of anti-QS (AHL analogue) com-

one of the three methods: degradation of

pounds may be of great interest to avoid

the enzyme that produces autoinducers,

bacterial infections. Therefore, screening

degradation of the autoinducers or degra-

of anti-QS compounds or QS inhibitors

dation of the gene that the autoinducer at-

(QSI) from natural resources have been

taches to and by autoinducers analog

used for centuries as remedies for various

(Roche _et al_., 2004). Acylases and lacto-

diseases. QSI compounds have been iden-

nases are two kinds of AHL-degrading

tified from a wide range of natural re-

enzymes also known as quorum sensing

sources particularly medicinal plants, edi-

inhibitors (QSI) which have been identi-

ble vegetables and fruits, marine sponges

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_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

****

**Figure 3:** Mode of action of AHL lactonase and AHL acylase.

and seaweeds (Kim _et al.,_ 2007; Skinder-

_C. violaceum_ CV017. Various fruits and

soe _et al.,_ 2008; Daglia _et al.,_ 2010;

herbs were shown to possess anti-QS ac-

Musthafa _et al.,_ 2010).

tivity in a _C. violaceum_ biomonitor strain

The QS interference from novel

and on the swarming motility of _E. coli_

sources may also be an important as the

and _P. aeruginosa_ (Vattem _et al_., 2007).

antibacterial effects. Anti-QS agents were

The subsequent discovery of com-

first characterized in the red marine alga

pounds that inhibit cell-to-cell communi-

_D. pulchura_ (Manefield _et al.,_ 1999).

cation, dubbed anti-quorum sensing (anti-

This alga was investigated for its anti-

QS) agents could provide a novel method

fouling properties and was found to con-

of combating infection. It is possible that

tain halogenated furanones which block

several terrestrial plants also produce

AHLs via competitive inhibition and de-

quorum signal mimics capable of control-

stabilization of LuxR (Manefield _et al.,_

ling bacterial quorum sensing (Gao _et al.,_

2002). _Delisea_ furanones have been

2003). Even bacteria themselves produce

shown to reduce light emission in _Vibrio_

QSI substances (Nithya _et al_., 2010).

species (Givskov _et al.,_ 1996), inhibit

Spices such as garlic, ginger and turmeric

pigment production in _C. violaceum_

have been reported for their QSI potential

(Martinelli _et al.,_ 2004) and attenuate exo-

(Vattem _et al.,_ 2007). Similarly, the essen-

enzyme production and swarming motility

tial oils of cinnamon (Niu _et al.,_ 2006) and

in _Serratia liquefaciens_ (Rasmussen _et al.,_

clove (Khan _et al.,_ 2009) are also known

2000). The quorum sensing-disrupting

to possess QSI potentials. Acyl-homo ser-

natural

furanone,

(5Z)-4-bromo-5-

ine lactone analogs and other quorum

(bromomethylene)-3-butyl-2(5H)-

sensing inhibitors (QSI) have been inves-

furanone was found to block autoinducer

tigated to determine their ability to pre-

2 quorum-sensing in _V. harveyi_ in a con-

vent expression of quorum sensing con-

centration-dependent way (Defoirdt _et al.,_

trolled genes. The complex of signalling

2006). Persson _et al.,_ (2005) reported that

molecules (AHLs) and receptor proteins

toluene extracts of garlic contained several

trigger the expression of specific genes

compounds with varying levels of QSI

responsible for bioluminescence in _V._

against Gram-negative transcriptional reg-

_harveyi_ (LuxM/N). Hence the disintegra-

ulators Lux R or Lux R. Sergey _e_ _t al.,_ tion of signals with receptor by plant de-

(2011) have evaluated 78 natural products

rived QSI prevents the bioluminescence

from chemical libraries containing com-

and other virulence factors in _V. harveyi_.

pounds from marine organisms (Sponges,

Several compounds have been identified

algae, fungi and cyanobacteria) and terres-

that have the ability to interfere with QS-

trial plants were screened for the inhibi-

mediated gene expression through com-

tion of bacterial QS using a reporter strain

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 352

_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

petitive inhibition thus reducing biofilm

**Balcàzar, J. L., de Blas, I., Ruiz-Zarzuela,**

thickness (Hentzer _et al.,_ 2002).

**I., Cunningham, D., Vendrell, D. and**

**Muzquiz, J.L. (2006).** The role of pro-

**9. Perspectives**

biotics in Aquaculture. _Veterinary Mi-_

_crobiology_ **114, 173-186**.

The oceans cover more than 70% of

**Browdy, C. L. (1998).** Recent developments

the earth's surface and they are a promis-

in penaeid broodstock and seed produc-

ing source of novel pharmacologically

tion technologies: improving the outlook

active compounds. Although macroorgan-

for superior captive stocks. _Aquaculture_

isms of the ocean have proved to be good

**164, 3-21.**

sources of novel bioactive metabolites,

**Bruhn, J. B., Dalsgaard, I., Nielsen, K. F.,**

large-scale productions of these bioactive

**Buchholtz, C., Larsen, J. L. and**

metabolites have been difficult. Microor-

**Gram, L. (2005)**. Quorum sensing sig-

ganisms isolated from marine sources

nal molecules (acylated homoserine lac-

have been reported to produce anti-

tones) in Gram-negative fish pathogenic

bacterial, anti-fungal, anti-viral and anti-

bacteria. _Disease of Aquatic Organism_

tumor substances. Several studies have

**65, 43-52**.

suggested that such marine bacteria can be

**Cabello, F. C. (2006).** Heavy use of prophy-

used as bio-control to combat epizootics

lactic antibiotics in aquaculture: a grow-

in aquaculture systems.During the past

ing problem for human and animal

two decades, the use of probiotics as an

health and for the environment. _Envi-_

alternative to antibiotics has shown to be

_ronmental Microbiology_ **8, 1137-1144.**

promising in aquaculture. Data about the

**Chythanya, R., Karunasagar, I. and**

impact of quorum sensing on virulence of

**Karunasagar, I. (2002).** Inhibition of

aquatic pathogens are still lacking. Few

shrimp pathogenic vibrios by a marine

reports that deal with disruption of quor-

_Pseudomonas_ I-2 strain. _Aquacul-_

um sensing of aquatic pathogens indicate

_ture_ **208, 1-10.**

that this new approach has potential in

**Czajkowski, R. and Jafra, S. (2008).**

fighting infections in aquaculture. The

Quenching of acyl-homoserine lactone-

furanones reported earlier as quorum

dependent quorum sensing by enzymatic

quenchers (QS inhibitors) are toxic and

disruption of signal molecules. _Journal_

chemically synthetic (non-degradable).

_of Acta Biochimica Polonica_ **56, 1-16.**

Consequently the invention of non-toxic,

**Daglia, M., Stauder, M., Papetti, A., Si-**

broad spectrum QS inhibitors is needed

**gnoretto, C., Giusto, G _._** **,Canepari, P.,**

for its successful exploitation against bac-

**Pruzzo, C. and Gazzani, G. (2010).**

terial infections due to their drug re-

Isolation of red wine components with

sistance.

anti-adhesion and anti-biofilm activity

****

against _Streptococcus mutans_. _Food_

**References**

_Chemistry_ **119, 1182-1188.**

****

**Darshanee, R. H. A., Bhowmick, P. P.,**

**Aguirre-Guzmán, G., Meija Ruíz, H. and**

**Karunasagar, I., Bossier, P. and**

**Ascencio, F. (2004)**. A review of extra-

**Defoirdt, T. (2011).** Quorum sensing

cellular virulence product of _Vibrio_ sp.

regulation of virulence gene expression

important in disease of cultivated

in _Vibrio harveyiin vitro_ and _in vivo_ dur-

shrimp. _Aquaculture Research_ **35, 1395-**

ing infection of gonotobiotic brine

**1404.**

shrimp larvae. _Environmental Microbi-_

**Austin, B. and Zhang, X. H. (2006).** _Vibrio_

_ology Report_ **3, 597-602.**

_harveyi_ : a significant pathogen of ma-

**Decamp, O., Moriarty, D. J. W. and Lav-**

rine vertebrates and invertebrates. _Letter_

**ens, P. (2008).** Probiotics for shrimp

_of Applied Microbiology_ **43, 119-124.**

larviculture: review of field data from

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 353

_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

Asia and Latin America. _Aquaculture_

of a curious phenomenon reveals a

_Research_ **39,334-338.**

common characteristic of bacteria. _Jour-_

**Defoirdt, T., Crab, R., Wood, T.K.,**

_nal of Bacteriolology_ **181, 2667-2668.**

**Sorgeloos, P. and Verstraete, W.**

**Henke, J. M. and Bassler, B. L. (2004).**

**(2006).**

Quorum

sensing-disrupting

Three parallel quorum-sensing systems

brominated furanones protect the gnoto-

regulate gene expression in _Vibrio har-_

biotic brine shrimp _Artemia franciscana_

_veyi_. _Journal of Bacteriology_ **186,6902–**

from pathogenic _Vibrio harveyi_ , _Vibrio_

**6914.**

_campbellii_ and _Vibrio parahaemolyticus_

**Hentzer, M., Riedel, K., Rasmussen, T.B.,**

isolates. _Appllied Environmental Mi-_

**Heydorn, A., Andersen, J.B.,Parsek,**

_crobiology_ **72, 6419-6423.**

**M. R., Rice, S. A., Eberl, L., Molin, S.,**

**Dong, Y. H. and Zhang, L. H. (2005).**

**Hoiby, N., Kjelleberg, S. and Givskov,**

Quorum sensing and quorum-quenching

**M. (2002).** Inhibition of quorum sensing

enzymes. _Journal of Microbiology_

in _Pseudomonas aeruginosa_ biofilm

**43,101-109.**

bacteria by a halogenated furanone

**Dong, Y. H., Gusti, A. R., Zhang, Q., Xu,**

compound. _Microbiology_ **148, 87-102.**

**J. L. and Zhang, L. H. (2002).** Identifi-

**Johnson, S. K. (1989).** Handbook of shrimp

cation

of

quorum-quenching

N-

diseases. Texas A and M University

acylhomoserine lactonases from _Bacillus_

Press Sea Grant, Texas. **pp. 27.**

species. _Appllied Environmental Micro-_

**Karunasagar, I., Pai, R., Malahti, G. R.**

_biology_ **68, 1754-1759.**

**and Karunasagar, I. (1994).** Mass mor-

**Dong, Y. H., Wang, L. H., Xu, J. L.,**

tality of _Penaeus monodon_ larvae due to

**Zhang, H. B., Zhang, X. F. and Zhang**

antibiotic-resistant _Vibrio harveyi_ infec-

**L. H. (2001).** Quenching quorum-

tion. _Aquaculture_ **128, 203-209.**

sensing-dependent bacterial infection by

**Kennedy,**

**B.,**

**Venugopal,**

**M.**

**N.,**

an N-acyl homoserine lactonase. _Nature_

**Karunasagar, I. and Karunasagar, I.**

**411, 813-817**.

**(2006).** Bacterial flora aassociated with

**Gao, M., Teplitski, M., Robinson, J. B. and**

the giant fresh water prawn _Macrobra-_

**Bauer, W. D. (2003).** Production of

_chium rosenbergii_ in the hatchery sys-

substances by _Medicago truncatula_ that

tem. _Aquaculture_ **261, 1156 -1167.**

affect bacterial quorum sensing. _Molecu-_

**Khan, M. S. A., Zahin, M., Hasan, S., Hu-**

_lar Plant Microbe Interaction_ **16, 827-**

**sain, F.M. and Ahmad, I. (2009).** Inhi-

**834.**

bition of quorum sensing regulated bac-

**Givskov, M., de Nys, R., Manefield, M.,**

terial functions by plant essential oils

**Gram, L., Maximilien, R., Eberl, L.,**

with special reference to clove oil. _Let-_

**Molin,**

**S.,Steinberg,**

**P.**

**D.**

**and**

_ters in Applied Microbiology_ **49, 354-**

**Kjellerberg, S. (1996).** Eukaryotic in-

**359.**

terference with homoserine lactone me-

**Kim, J.S., Kim, Y.H., Seo, Y.W. and Park,**

diated prokaryotic signalling. _Journal of_

**S. (2007).** Quorum sensing inhibitors

_Bacteriology_ **178, 6618-6622.**

from the red alga, _Ahnfeltiopsis flabelli-_

**Harris, L. (1995).** The involvement of toxins

_formis_. _Biotechnology and Bioprocess_

in the virulence of _Vibrio harveyi_ strains

_Engineering_ 1 **2, 308-311.**

pathogenic to the black tiger shrimp _Pe-_

**Le Groumellec, M., Goarant, C., Haffner,**

_naeus monodon_ and the use of commer-

**P., Berthe, F. and Costa, R. (1996).**

cial probiotics to reduce shrimp hatchery

Syndrome 93 in New Caledonia: Inves-

disease outbreaks caused by _Vibrio har-_

tigation of the bacterial hypothesis by

_veyi_ strains. CRC for aquaculture, Scien-

experimental infections with reference to

tific Conference abstract, Bribie Island,

stress induced mortality. SICCPPS book

Australia.

of abstracts, SEAFDEC, Iloilo City,

**Hastings, J. W. and Greenberg, E. P.**

Phillippines. **pp. 46.**

**(1999).** Quorum sensing: the explanation

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 354

_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

**Lee, C.F., Han, C. K. and Tsau, J. L.**

sensing through accelerated LuxR turn-

**(2004).** _In vitro_ inhibitory activity of

over. _Microbiology_ **148, 1119-1127.**

Chinese leech extract against _Campylo-_

**Manilal, A., Sujith, S., Kiran, G. S., Selvin,**

_bacter_ species. _International Journal of_

**J. and Shakir, C. (2009).** Biopotentials

_Food Microbiology_ **94, 169-174.**

of mangroves collected from the South-

**Lee, D. O. C. and Wickins, J. F. (1992).**

west coast of India. _Global Journal of_

Crustacean farming. Blackwell Scien-

_Biotechnology and Biochemistry_ **4, 59-**

tific Publications, The University Press,

**65.**

Cambridge, **pp. 392**

**Martinelli, D., Grossmann, G., Séquin, U.,**

**Lee, S. J., Park, S. Y., Lee, J. J., Yum,**

**Brandl,**

**H.**

**and**

**Bachofen,**

**R.**

**D.Y., Koo, B.T. and Lee, J. K. (2002).**

**(2004).** Effects of natural and chemically

Genes encoding the N-acyl homoserine

synthesized furanones on quorum sens-

lactone-degrading enzyme are wide-

ing in _Chromobacterium violaceum_.

spread in many subspecies of _Bacillus_

_BMC Microbiology_ **4, 1-10.**

___
___

_thuringiensis_. _Appllied Environmental_

**Menasveta, P. (1997).** Mangrove destruction

_Microbiology_ **68, 3919-3924.**

and shrimp culture systems. _World aq-_

**Lightner, D. V. (1993).** Diseases of cultured

_uaculture Society_ **28, 36-42.**

penaeid shrimp. In: Handbook of Mari-

**Miller, M. B. and Bassler, B. L. (2001).**

culture,

_Crustacean_

_Aquaculture_.

Quorum sensing in bacteria. _Annual Re-_

McVey, J.P. (ed.). CRC Press Inc., Boca

_view of Microbiology_ **55, 165-199.**

Raton, FL. **pp. 393-486.**

**Molina, A., Alejandra, G., Alberto, A. G.,**

**Lightner, D. V. (1996).** A Handbook of pa-

**Carmen, B., Ana, R. and Gomez, G.**

thology and diagnostic procedures for

**B. (2002).** Plasmid profiling and antibi-

diseases of Penaeid shrimp. _World Aq-_

otic resistance of _Vibrio_ strains isolated

_uaculture Society_ , Baton Rouge, Louisi-

from cultured Penaeid shrimp. _FEMS_

ana, USA, **pp. 304**.

_Microbiology Letter_ **213,7-12.**

**Lin, Y. H., Xu, J. L., Hu, J., Wang, L. H.,**

**Moriarty, D. J. W. (1998).** Control of lumi-

**Ong, S. L., Leadbetter, J. R. and**

nous _Vibrio_ species in penaeid aquacul-

**Zhang, L. H. (2003).** Acyl-homoserine

ture ponds. _Aquaculture_ **164, 351-358.**

lactone acylase for _Ralstonia_ strain

**Musthafa, K. S., Ravi, A.V., Annapoorani,**

XJ12B represents a novel and potent

**A., Packiavathy, I. S and Pandian, S.**

class of quorum-quenching enzymes.

**K. (2010).** Evaluation of anti-quorum-

_Molecular Microbiology_ **47, 849-860**

sensing activity of edible plants and

**Liu, P. C., Lee, K. K. and Chen, S. N.**

fruits through inhibition of the N-acyl-

**(1997).** Susceptibility of different iso-

homoserine lactone system in _Chromo-_

lates of _Vibrio harveyi_ to antibiotics.

_bacterium violaceum_ and _Pseudomonas_

_Microbiololgy_ **91, 175-180.**

_aeruginosa_. _Chemotherapy_ **56, 333-339.**

**Manefield, M., de Nys, R., Kumar, N.,**

**Nakayama, T., Ito, E. and Nomura, N.**

**Read, R., Givskov, M., Steinberg, P.**

**(2006).** Comparison of _Vibrio harveyi_

**and Kjelleberg, S. (1999).** Evidence

strains isolated from shrimp farms and

that halogenated furanones from _Delisea_

from culture collection in terms of tox-

_pulchra_ inhibit acylated homoserine lac-

icity and antibiotic resistance. _FEMS_

tone (AHL)-mediated gene expression

_Microbiology Letter_ **258, 194-199.**

by displacing the AHL signal from its

**Nash, G., Nithimathachoke, C., Tung-**

receptor protein. _Microbiololgy_ **145,**

**mandi, C., Arkarjarmorin, A., Prath-**

**283-291.**

**ampipat, P. and Ruamthaveesub, P.**

**Manefield, M., Rasmussen, T. B., Henzter,**

**(1992).** Vibriosis and its control in pond-

**M., Andersen, J. B., Steinberg, P.,**

reared _Penaeus monodon_ in Thailand.

**Kjelleberg, S. and Givskov M. (2002).**

In: Diseases in Asian Aquaculture. Shar-

Halogenated furanones inhibit quorum

iff, I.M., Subasinghe, R.P. and Arthur,

J.R. (eds.).International Fish Health Sec-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 355

_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

tion, Asian Fisheries Society, Manila,

_veyi_ isolated from Penaeid shrimp

**pp. 143-155.**

hatcheries. _Asian Fishery Science_ **24,**

**Natrah, F. M. I.,Kenmegne, M. M., Wiyo-**

**186-196**.

**to, W., Sorgeloos, P., Bossier, P. and**

**Rasmussen, T. B., Manefield, M., Ander-**

**Defoirdt, T. (2011).** Effects of micro-

**sen, J. B., Eberl, L., Anthoni,**

algae commonly used in aquaculture on

**U.,Christophersen, C., Steinberg, P.,**

acyl-homoserine lactone quorum sens-

**Kjelleberg, S. and Givskov, M (2000).**

ing. _Aquaculture_ **317, 53-57.**

How _Delisea pulchra_ furanones affect

**Nealson, K. H., Platt, T. and Hastings, J.**

quorum sensing and swarming motility

**W. (1970).** Cellular control of the syn-

in _Serratia liquefaciens_ MG1. _Microbio-_

thesis and activity of the bacterial lumi-

_lology_ **146, 3237-3244.**

nescent system. _Journal of Bacteriology_

**Rattanachuay, P., Kantachote, D., Tanti-**

**104, 313-322.**

**rungkij, M., Nitoda, T. and Kanzaki,**

**Nithya, C., Aravindraja, C. and Pandian,**

**H. (2010).** Inhibition of shrimp patho-

**S. K. (2010).** _Bacillus pumilus_ of Palk

genic vibrios by extracellular com-

Bay origin inhibits quorum-sensing-

pounds from a proteolytic bacterium

mediated virulence factors in Gram-

_Pseudomonas_ sp. W3. _Electronic Jour-_

negative bacteria. _Research in Microbi-_

_nal of Biotechnology_ **13, 1-11.**

_ology_ **161, 293-304.**

**Rengpipat, S., Phianphak, W., Piyati-**

**Niu, C., Afre, S. and Gilbert, E.S. (2006).**

**ratitivorakul, S. and Menasaveta, P.**

Subinhibitory concentrations of cin-

**(1998).** Effects of a probiotic bacterium

namaldehyde interfere with quorum

in black tiger shrimp _Penaeus monodon_

sensing. _Letter in Applied Microbiology_

survival and growth. _Aquaculture_ **167,**

**43, 489-494**.

**301-313.**

**Pan, J., Huang, T., Yao, F., Huang, Z.,**

**Roche, D. M., Byers, J. T., Smith, D.S.,**

**Powell, C. A., Qiu, S. and Guan, X.**

**Glansdorp, F.G., Spring, D.R. and**

**(2008).** Expression and characterization

**Welch, M. (2004).** Communications

of aiiA gene from _Bacillus subtilis_ BS-1.

blackout?

Do

N-acylhomoserine-

_Microbiological Research_ **163, 711-716.**

lactone-degrading enzymes have any

**Peddie, S. and Wardle, R. (2005).** Crusta-

role in quorum sensing?. _Micobiolology_

ceans: the impact and control of vibri-

**150, 2023-2028.**

osis in shrimp culture worldwide. _Aqua-_

**Rosenberry, B. (1997).** World Shrimp farm-

_culture Health International_ **2, 4-5.**

ing. In:Shrimp News International. San

**Persson, T., Hansen, T. H., Rasmussen, T.**

Diego, California, USA, **pp. 284.**

**B., Skindersoe, M. E., Givskov, M.**

**Rosenberry, B. (2003** ). World Shrimp farm-

**and Nielsen, J. (2005).** Rational design

ing. In: Shrimp News International.San

and synthesis of new quorum-sensing

Diego, California, USA.

inhibitors derived from cylated homoser-

**Sergey, D., Teplitski, M., Bayer, M., Gun-**

ine lactones and natural products from

**asekera, S., Proksch, P. and Paul, V.**

garlic.

_Organic_

_and_

_Biomolecular_

**J. (2011).** Inhibition of marine biofoul-

_Chemistry_ **3, 253-262**.

ing by bacterial quorum sensing inhibi-

**Preetha, R., Jose, S., Prathapan, S., Vi-**

tors. _Biofouling_ **27, 893-905**.

**jayan, K.K., Jayaprakash, N. S., Phil-**

**Skindersoe, M. E., Ettinger-Epstein, P.,**

**ip, R. and Singh, I.S. B. (2009).** An in-

**Rasmussen, T. B., Bjarnsholt, T., de**

hibitory compound produced by _Pseu-_

**Nys, R. and Givskov, M. (2008).** Quor-

_domonas_ with effectiveness on _Vibrio_

um sensing antagonism from marine or-

_harveyi_. _Aquaculture Research_ **41,**

ganisms. _Marine Biotechnology_ **10, 56-**

**1452-1461**.

**63.**

**Ramesh, K. and Umamaheswari, S. (2011).**

**Tendencia, E. and De La Peña, L.D.**

Inhibitory activity of antibiotics and an-

**(2001).** Antibiotic resistance of bacteria

ti-vibrio Probiotics against _Vibrio har-_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 356

_Biotech Sustainability (2017)_

_Sensing Technology for Shrimp Aquaculture Sustainability Kandasamy et al._

from shrimp ponds. _Aquaculture_ **195,**

**Verschuere, L., Rombaut, G., Sorgeloos, P.**

**193-204.**

**and Verstraete, W. (2000).** Probiotic

**Teo, J. W., Tan, T. M. and Chit, L. P.**

bacteria as biological control agents in

**(2002).** Genetic determinants of tetracy-

Aquaculture. _Microbiology Molecular_

cline resistance in _Vibrio harveyi_. _Anti-_

_Biology Review_ **64, 655-671.**

_microbial Agents and Chemotherapy_ **46,**

**Vijayan, K. K., Singh, I. S. B., Jayapra-**

**1038-1045.**

**kash, N. S., Alavandi, S.V.,Pai, S. S.,**

**Uroz, S., Angelo-Picard, D. C., Carlier, A.,**

**Preetha, R.,Rajan, J. J. S. and Santia-**

**Elasri, M., Sicot, C., Petit, A., Oger,**

**go, T. C. (2006).** A brackishwater iso-

**P., Faure, D. and Dessaux, Y. (2003).**

late of _Pseudomonas_ PS-102, a potential

Novel

bacteria

degrading

N-

antagonistic bacterium against pathogen-

acylhomoserine lactones and their use as

ic vibrios in penaeid and non-penaeid

quenchers of quorum-sensing-regulated

rearing systems. _Aquaculture_ **251, 192-**

functions of plant-pathogenic bacteria.

**200.**

_Microbiology_ **149, 1981-1989.**

**Vine, N.G., Leukes, W. D. and Kaiser, H.**

**Vaseeharan, B. and Ramasamy, P. (2003).**

**(2006).** Probiotics in marine larviculture.

Control of pathogenic _Vibrio_ sp. by _Ba-_

_FEMS Microbiology Review_ **30, 404-**

_cillus subtilis_ BT23, a possible probiotic

**427.**

treatment for black tiger shrimp _Penaeus_

**Waters, C. M. and Bassler, B. L. (2005).**

_monodon_. _Letters in Appllied Microbiol-_

Quorum sensing: Cell-to-cell communi-

_ogy_ **36, 83-87.**

cation in bacteria. _Annual Review of Cell_

**Vaseeharan, B., Lin, J. and Ramasamy, P.**

_and Development Biology_ 21 **, 319-346**.

**(2004).** Effect of probiotics, antibiotic

**Won, K. M. and Park, S. (2008).** Patho-

sensitivity, pathogenicity, and plasmid

genicity of _Vibrio harveyi_ to cultured

profiles of _Listonella anguillarum_ like

marine fishes in Korea. _Aquaculture_

bacteria isolated from _Penaeus monodon_

**285, 8-13.**

culture systems. _Aquaculture_ **241, 77–**

**Ziaei, N. S., Rezaei, M. H., Takami, G. A.,**

**91.**

**Lovett, D. L. and Mirvaghefi, A. R.**

**Vattem, D. A., Mihalik, K., Crixell, S. H.**

**(2006).** The effect of _Bacillus_ sp. bacte-

**and McClean, R. J. C. (2007).** Dietary

ria used as probiotics on digestive en-

phytochemicals as quorum sensing in-

zyme activity, survival and growth in the

hibitors. _Fitoterapia_ **78, 302-310**.

Indian white shrimp _Fenneropenaeus_

_indicus_. _Aquaculture_ **252, 516-524.**

© 2017 by the authors. Licensee,Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 357

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P358-375_

**Promiscuous Rhizobia: A Potential Tool to Enhance**

**Agricultural Crops Productivity**

**Ikbal1, Prasad Minakshi1,*, Basanti Brar1, Upendra Pradeep Lambe1, Manimegalai**

**Jyothi1, Koushlesh Ranjan2, Deepika3, Virendra Sikka4 and Gaya Prasad5**

_1Department of Animal Biotechnology, LUVAS, Hisar, Haryana, 125004, India;_

_2Department of Veterinary Physiology and Biochemistry, SVPUAT, Meerut, 250110, Uttar_

_Pradesh, India; 3Department of Botany and Plant physiology, CCSHAU Hisar, 125004,_

_Haryana, India; Department of Molecular Biology, Biotechnology & Bioinformatics , _

_CCSHAU Hisar, 125004, Haryana, India; 5SVPUAT, Meerut, 250110, Uttar Pradesh, In-_

_dia; *Correspondence: minakshi.abt@gmail.com / minakshi.abt@luvas.edu.in; Tel: +91_

_9992923330_

**Abstract:** _Rhizobium_ -legume symbiosis is a complex and regulated association between

plant and bacteria. This symbiosis is under the coordinated and tight regulation of several

species specific (symbiosis related) genes of bacterium and respective host plant. Thus, rhi-

zobia require action of several classes of specific genes for the formation of an effective

symbiosis and dictate the host range. Other _nod_ genes mediate the "decoration" of the core

signaling compounds with various substituents and make them host- specific. But, there are

some reports that highlight that the rhizobia can infect non-legume plants. The signaling

compounds are responsible for the effective symbiosis; however, there are several other

factors which influence symbiosis and needs to be discovered. Certain modifications in the

signaling molecules can cause changes in legume host range. Genetic exchange and rear-

rangement among heterologous _Rhizobium_ _spp_. leading to broadening of host range and

become promiscuous. Such type of rhizobia having broad host range and could be benefi-

cial for the agricultural practices; because, choosing the correct inoculant group for a par-

ticular legume host is difficult for effective nodulation. Most of the commercially available

strains are known to have a very narrow host range. Promiscuous _Rhizobium_ strains for

greater symbiotic association and ability to infect across strict host specificity would be of

greater importance for the farming community. Farmers can enhance Biological Nitrogen

Fixation by inoculating such correct rhizobia to their legume crops. The potential of this

system is appealing because the whole world is seeking to adopt the organic farming. This

could provide an alternate method to improve the soil fertility and could boost the agricul-

tural sustainability.

****

_**Keywords**_ **:** Biofertilizer; nitrogen fixation; promiscous; _Rhizobium_

****

**1. Introduction**

system ever studied, involves bacteria

( _Rhizobium_ ) and legume plants. The es-

Biological nitrogen fixation occurs

tablishment of symbiosis involves several

mainly through symbiotic association of

signaling molecules exchange between

plants with N2-fixing microorganisms

bacteria and host plant. These molecules

(Shiferaw _et al_., 2004). BNF supply ni-

are regulated by several _nod_ genes and

trogen more than 2x1013 g/year to the

work in coordinated manner (Cohn _et al.,_

world agriculture system (Falkowski,

1998; Long, 1996). In a successful sym-

1997). It is one of the most economically

biosis rhizobia colonize on roots of host

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

plants and elicit the nodule formation

ers. The Bio-fertilizers are bacteria, algae

where they colonize and differentiate into

and fungi and may broadly be classified

non-dividing

endocellular

symbionts.

into two categories _viz._ Nitrogen fixing

These symbionts convert atmospheric di-

like Rhizobium, Azotobactor, Azospiri-

nitrogen into NH3 through the induction

lum, Acetobacter, Blue Green Algae and

of the nitrogenase complex (Patriarca _et_

Azola

and

Phosphorous

solubilis-

_al_., 2002). _**** Rhizobium_ species have been

ers/mobilisers like PSM and Mycorrizae

successfully used worldwide as a bio-

(Figure 3). Rhizobia and legumes estab-

inoculant leading to effective establish-

lish a mutualistic symbiosis. Host speci-

ment of nitrogen fixing symbiosis with

ficity is an important characteristic of

leguminous crop plants (Miller _et al_.,

symbiosis, where specific species of rhi-

2007). Nitrogen applied as fertilizers usu-

zobia forms nodules on defined legumes

ally provides high yields to plants. There-

(Ampomah _et al_., 2008). Rhizobia cur-

fore efficient monitoring of biological ni-

rently consist of 61 species belonging to

trogen fixation and status of chemical fer-

13 different genera, namely _Rhizobium,_

tilizers are essential to balance the yield

_Bradyrhizobium, Mesorhizobium, Azorhi-_

of crops and need to minimize environ-

_zobium, Allorhizobium, Sinorhizobium,_

mental pollution, especially water and soil

_Methylobacterium,_

_Cupriavidus,_

quality (Jaynes _et al.,_ 2001). The role of

_Burkholdera, Devosia, Ochrobactrum,_

BNF, especially in legumes, is well estab-

_Herbaspirullum and Phyllobacterium._

lished and documented but Legume-

Some Rhizobia have a narrow host range

_Rhizobium_ symbiosis is not so extensively

and form nodules with specific legume.

studied the system of nitrogen-fixation.

For example _Azorhizobium caulinodans,_

Soil containing adequate and diverse

_Sinorhizobium_

_saheli_

and __

sesbaniae

communities (Figure 1) of rhizobia and

biovar of _Sinorhizobium terange_ nodulate

become less effective at nitrogen fixing.

only _Sesbania rostrata_ (Boivin _et al_.,

The application of sufficiently high

1997) and _Rhizobium galegae_ is the only

numbers of improved inoculant strains

symbiont of _Galega offcinalis_ and _Galega_

can successfully compete with established

_orientalis_ (Lindstrom, 1989). In contrast

soil rhizobia and replace them (Figure 2).

some rhizobia are capable to infect a

The aim is to increase, the percentage of

spectrum of legumes as they have broad

crops that are inoculated in terms of bio-

host range (various degree of promiscui-

mass yield and extra nitrogen in the soil.

ty). For example, _Sinorhizobium_ sp.

Improved _Rhizobium_ strains for greater

NGR234 and closely related _Sinorhizobi-_

symbiotic association and ability to infect

_um fredii_ USDA257 nodulate at least 112

across strict host specificity would be of

and 77 legumes from two different tribes,

greater importance for the farming com-

respectively (Pueppke and Broughton.

munity. Farmers can enhance BNF by in-

1999).

oculating such correct rhizobia to their

legume crops. Such promiscuous _Rhizo-_

**3. Symbiotic infection is a regulated**

_bium_ strains with improved efficiency to

**pathway**

fix nitrogen would acts as a single inocu-

lum for all the legumes and may add

Symbiosis is a developmental process

higher amount of nitrogen per unit area.

driven by bacteria but ultimately under

the control of host plant (Murray, 2011).

**2. Classification of biofertilizers as per**

The successful establishment of infection

**host specificity**

requires several factors such as nod fac-

****

****

tors and plant exudates (flavonoids). The-

Biofertiliser are the low cost source of

se flavonoids activate different kinds of

plant nutrients, eco-friendly and have

nitrogen fixing genes and the interaction

supplementary role with chemical fertiliz-

takes

place

between

bacteria

and

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

**Figure 1:** Diverse communities of microorganisms found in soil.

**Figure 2:** Atmospheric nitrogen fixation by microorganisms.

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

**Figure 3:** Classification of biofertilizers based on microorganisms.

plants. In positive interaction rhizobia

moves towards the localized sites of plant

Work on molecular basis of host

root and followed by formation of infec-

specificity began at the end of the last

tion pocket (Barbour _et al.,_ 1991; Brewin,

century. Experimental evidence suggests

2004). A bacterial colony established in

that the progression of invasive rhizobia

this infection pocket and become a new

towards nodule primordial is challenged

organ called nodule (Fournier _et al_., 2008

at various steps. The host range is deter-

and Oldroyd and Downie, 2004).

mined at early stages of the plant-

These pre infection responses ready the

bacterium interaction. During initial phas-

plant for infection by rhizobia. However

es of nodulation (bacterial entry), molecu-

some β-rhizobia use an alternative path-

lar signals are given by flavonoids and

way to initiate symbioses in some leg-

Nod factors (Martinez _et al.,_ 1988). In

umes, where a purine derivative plays a

this process, NodD proteins are the chief

key role in triggering nodule formation.

interlocutors of molecular traffic in the

The universality of the nod factor para-

rhizosphere (Perret _et al.,_ 2000). NodD

digm was recently overturned by some

shows specificity to certain flavonoid se-

bacteria that elicit root and stem nodules

creted by plants (Figure 4). Therefore,

on a particular group of plants lack the

NodD takes part in determining host spec-

canonical _nod_ ABC genes required for the

ificity (Miller _et al_., 2007). Although

synthesis of the Nod factor (Giraud _et al_.,

some host plants and rhizobia have the

2007). This indicates that a group of rhi-

ability to enter into symbiosis with more

zobia uses a NF-independent strategy to

than one companion, only certain combi-

enter into symbiosis. Madsen _et al_.,

nation of symbionts results in the for-

(2010) found that snf1/nfr1/nfr5 triple

mation of nitrogen fixing nodules. Several

mutants allowed rhizobia to invade

other studies have also shown that the

through an "intercellular" route (crack

length of the oligosaccheride backbone of

entry) but in this case rhizobial infection

LCOs determine the host specificity of

was not accompanied by the formation of

nodulation (Bec-Ferte _et al_., 1994; Felle

infection threads within root hairs. This

_et al_., 1995; Heidstra _et al_., 1994; Stok-

finding suggests that alternative pathway

kermans _et al_., 1995). These results

may facilitate the entry of the bacterium

demonstrate that _nodC_ contributes to the

in to the roots of diverse legumes.

host specificity of _rhizobium._

The amount of Nod factors released

**4. Specificity of symbiotic infection**

by rhizobia also play important role in

**between legume plant species and**

determining the host range. For instance,

**rhizobia**

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

**Figure 4:** Symbiotic interaction between legume plant species and rhizobia.

introduction of strain NGR234 _nod_ D1

hybrid genes it was concluded that a cen-

into _R. Meliloti_ increases Nod factor pro-

tral region determine different host rang-

duction by about two-fold and permits the

es. Louise _et al_. (2002) have mutagenised

nodulation of _V. Unguiculata,_ a non-host

_Rhizobium_ strains with transposon Tn5 to

(Relic _et al_., 1994). In _S. Meliloti_ these

determine if additional negatively-acting

NodD proteins respond to different

traits exist that can alleviate cultivar-

groups of flavonoids, suggesting that

specific nodulation failure. They reported

NodD redundancy allows the bacterium to

two new mutants, proficiently nodulate

infect multiple hosts secreting a wide

cv. Woogenellup. They suggested that

range of flavonoids (Maillet _et al.,_ 1990).

simple gene to gene interaction is not suf-

To demonstrate that _nod_ is a key determi-

ficient for symbiosis but there are at least

nant of host specificity, Melicent _et al._

two independent mechanisms which me-

(2006) expressed _nod_ genes from differ-

diated the cultivar-specificity. Although

ent species of rhizobia in a strain of _S._

much information is available on the in-

_Meliloti_ which was lacking endogenous

fluence of Nod factors on the host range

_nod_ D activity. They observed that _nod_

yet no strict correlation can be drawn the

gene expression was initiated in response

types of LCOs produced by rhizobia and

to distinct set of flavonoid inducers. Fur-

host plants.

thermore, data from several researches

suggest that nodD controls the response

**5. Molecular factors control the sym-**

of rhizobia to flavonoids in species-

**biosis**

specific manner (Hovath _et al_., 1987)

Herman _et al_. (1989) found that node

The nitrogen fixation and nodulation

product is the main factor that distinguish

by _Rhizobium_ strains is controlled at vari-

the host range for symbiosis. __ Hybrid _nod_ E

ous levels by certain factors (Hooykaas _et_

genes, which consist of a 5" part Rhizobi-

_al_., 1981). The importance of each indi-

um leguminosarum _nod_ E genes and a 3"

vidual step would depend on the specific

part of the _Rhizobium trifolii_ gene, were

legume– _Rhizobium_ combination and Nod

constructed. From the properties of these

factors __ (NF) in the rhizosphere (Figure 5).

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

**Figure 5:** Molecular factors which are involved in symbiosis (Okazaki _et al_., 2013).

Bacterial Nod factors functions as a key

a particular host and are involved in vari-

to opens the door of its host **** (Perret _et al_.,

ous modification of the chitin backbone

2000), and there is a high degree of strin-

(Gibson _et al_., 2008). The nodulation

gency for chemical structure of Nod fac-

gene expression is activated when bacte-

tor that determine whether the host allows

ria perceive flavonoids that are secreted

bacterial invasion to proceed. Nod factor

by plant roots (Perret _et al_., 2000). _nod_ D,

elicits significant changes in the expres-

gene is central to the regulation of nod

sion of host gene (D"Haeze and Holster,

box which activates other _nod_ gene ex-

2002; Oldroyd and Dowine, 2008). Nod

pression (Loh and Stacey, 2003). It in-

factors are complex signaling molecules

duces the transcription of nodulation

secreted from bacteria as a cocktail of β

genes involved in the synthesis of nod

1-4-linked N acetyle D-glucosamine

factors (Capela _et al_., 2005 and Peck _et_

(GlcNAc) trimers, tetramers or pentamers ****

_al_., 2006). In response of isoflavone sig-

(D"Haeze and Holster, 2002). The host-

nals which are produced by plants

rhizobia co-evolution involved modifica-

NodVW, positively regulate nod genes,

tions of Nod factor structure such as re-

thus these are thought to activate tran-

placement of fucosyl, which made the in-

scription via a series of phosphorylation

teraction more specific and increased af-

steps. Mutation in either of these two

finity between partners (Mario _et al_.,

genes results in the complete loss of

2006).

nodulation activity in certain plant hosts.

All the rhizobial species have

The proposed role of _nod_ V as a sensor of

common nod genes ( _nod_ A, B, and C),

plants signals adds another point of com-

which are capable of cross species com-

plexity of _nod_ gene regulation. In re-

plementation and responsible for the syn-

sponse of plant signal, phosphorylation of

thesis of nod factor backbone. These

NodV takes place which subsequently

genes confer specificity for nodulation of

activate NodW via the transfer of phos-

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

phoryl group to an aspartate residue in its

portant for induction of different compo-

receiver domain. The nodW play a key

nents of the nodulation pathway but also

role in the regulation of nod gene expres-

some bacterial cell surface components

sion and in the ability of B. Japonicum to

such as LPS, cyclic-β-glucans, EPS, cap-

infect the host plant (Sanjuan _et al_.,

sular proteins and K antigens recognised

1994). The phosphorylation of NodV and

by plants help to determine host specifici-

NodW is essential not only for nod gene

ty (Mathis _et al_., 2005). These additional

expression but also for the nodulation.

factors must have role in symbiotic de-

Nod factors permit rhizobia to enter their

velopment between rhizobia and plant.

hosts and certain additional factors proba-

bly act within plant (D"Haeze _et al_.,

**6. Broadening of host range (promis-**

1998). Thus a number of physiological

**cuity) of rhizobia**

responses to nod factor are observed

when rhizobium applied to the plant roots

The host specificity concept has

(Gibson _et al_., 2008).

now almost defunct, because many over-

Said _et al_., (1998) have found that _no-_

lapping host ranges have observed so the

_l_ O factor is the principal host range de-

concept of host specificity has been chal-

terminant. They have mobilized large

lenged. A single legume _e.g_. Acacia, Gly-

fragments of the symbiotic plasmid of

cine max or Leucaena can be associated

_Rhizobium_ _spp_. NGR234 into heterolo-

with genetically dissimilar symbionts.

gous rhizobia. The trans-conjugants nodu-

Closely related rhizobia infect legumes

late _V. unguiculata_ at low frequency and

from different tribes and distantly related

extended the host range _._ They have con-

rhizobia infect closely related legumes

firmed that the conjugation of _nolO_ into

(Quesada _et al.,_ 1997). The _Rhizobium_ _sp_.

Rhizobium fredii extends the host range

strain NGR234 is good examples of this

of the recipient to the non-hosts. Nod fac-

phenomenon. It has broad host range and

tors are essential to the nodulation and

nodulate legume species from 112 genera

their modification contributes to host

and the non-legume _Parasponia_ (Pueppke

specificity, thus these signaling molecules

and Broughton, 1999). Zhu _et al_., (2002)

probably one of the several elements

characterized the rhizobia that nodulate

specifying host range. It should also be

legume species of the genus Lespedeza by

noted that although _nod_ FE mutants of

analysing whole cell proteins, and cross-

_Rhizobium melilotii_ secrete nod factors in

nodulation with selected legume species.

which C16 unsaturated fatty acids are re-

They have observed that the strains iso-

placed by vaccenic acid, the mutant still

lated from S _esbania_ _spp._ and _Lespedeza_

form nodules on various Medicago culti-

_spp_. represent a cross-nodulating group of

vars (Ardourel _et al_., 1994). Several other

bacteria. Hernandez-Lucas _et al_., (1995)

examples contradict the dogma that Nod

also found two strains of _R. etli_ and three

factors determine host specificity. Despite

stains of _R. tropici_ and tested on 43 leg-

the fact that predominant nod factors se-

ume species. Out of these 22 of the tested

creted by _R. Leguminosarum bv. trifolii_

legume species were nodulated by three

and _R. Leguminosarum bv. viciae_ are

or more of these strains. These strains

identical yet these two bacteria have dis-

have broad host range and nodulate

tinct host ranges (Orgambide _et al_., 1995).

woody species also such as _Albizia_

In contrast, two rhizobia that secrete dif-

_lebbeck_.

ferent nod factor may nodulate the same

Setiyo Hadiwaluyo (2011) **** charac-

plant: _R. Tropici_ and _R. etli_ produce dif-

terized forty one cross inoculating rhizo-

ferent nod factors but both effectively

bial isolates from Java and Sumatra and

nodulate _Phaseolus vulgaris_ (Poupot _et_

these isolates were used to inoculate soy-

_al_., 1995). It would thus seem that nod

bean and mungbean plants. He found 19

factors in absolute level are not only im-

isolates from Java and 15 isolates from

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_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

Sumatra were promiscuous. Beatrix _et_

lates a host of different cross-inoculation

_al_., (1987) have studied _nod_ D gene from

group. Promiscuity is probably ancestral

wide

host

range

rhizobium

strain

to restricted host range. In this support

MPIK3030 to verify the _nod_ D function,

hypothesis comes from the observation

as well as the host-range extension ability

that NGR234 and USDA257, both nodu-

of _R. Meliloti._ The 2.9-kb _nod_ Dl region

late _Parasponia andersonii_ (van Rhijn _et_

was mobilized into _R. Meliloti_ and trans-

_al_., 1996). There are some other reports of

conjugants were tested for their nodula-

promiscuous strains that have broad host

tion phenotype on siratro and alfalfa. The

range and nodulate soybean as well as

double mutant _R. meliloti_ _nod_ DI12 PP659

many other legumes, including cowpea,

carrying the MPIK3030 _nod_ DI-region

pigeon pea and mungbean (Scholla and

(pBH264), showed a clear restoration of

Elkan 1984; Stowers and Eaglesham

nodulation on alfalfa and simultaneously

1984; Chamber and Iruthayathas 1988).

extended the host range of the _R. meliloti_

In view of these reports it was concluded

trans-conjugants to siratro. Similarly

that promiscuity is widely dispersed in

Transfer of _nod_ D1 of NGR234 into _R._

nature and not only associated with a par-

_meliloti_ results in host range extension to

ticular bacterial or plant taxonomic group.

_M. atropurpureum_ and _Vigna unguicula-_

In further studies, nodulation capacities of

_ta_ , whereas _R. meliloti_ _nod_ D1is incapable

large collections of rhizobia have been

of restoring the ability of an NGR234

evaluated by inoculation of numerous

_nod_ D1 mutant to nodulate _M. atropur-_

legumes.

_pureum_ (Relic _et al_., 1994). Conjugation

of NGR234 _nod_ D1 into _R. legumi-_

**7. Hydrolytic-cell wall degrading en-**

_nosarum_ bv. _Trifolii_ extends its host-

**zymes in rhizobial infection**

range to the non-legume _Parasponia an-_

_dersonii_ (NGR234 host) whereas _nod_ D1

In the development of the Rhizobium-

mutant of _R._ _trifolii_ did not regain the

legume symbiosis localized erosion of

ability to nodulate _Trifolium repens_ when

cellulosic plant wall is the central event

it was complimented with _nod_ D1 of _R._

through which the bacterial symbiont en-

_meliloti_ (Spaink _et al_., 1987).

ter into host plant and establish a nitrogen

Promiscuity is not only the characteristics

fixing, intracellular endosymbiotic state.

of the rhizobia, but some legumes also

Previous studies found that rhizobia pro-

harbor diverse rhizobia (Perret _et al_.,

duce hydrolytic enzymes capable of de-

2000). Several plants such as _Phaseoleae_

grading the cell wall polymers, but little is

are nodulated by _R. leguminosarum_ _bv._

known about their molecular mechanism

_Phaseoli_ as well as _Bradyrhizobium_ and

(Angle 1986). In considering the process

_Sinorhizobium_ species (Gaultieri and Bis-

of active penetration of plant cell wall by

seling, 2000). Arya K. Bal (1982) studied

_Rhizobium_ _sp_., McCoy (1932) was the

how a legume interacts with _Rhizobium_

first to investigate the involvement of hy-

species of two different cross inoculation

drolytic enzymes. Ljunggren and

groups. They have compared physiology

Fahraeus (1961) gave the "polyglac-

and morphology of root nodules induced

turonase hypothesis" which describes the

by two _Rhizobium_ species of different

involvement of pectolytic enzymes at the

cross inoculation groups. They have

site of nodule formation. The hypothesis

found that _Rhizobium sp._ 127E15 promis-

in essence proposes a physical penetration

cuously induce effective root nodules on

of the root hair cell wall. Callaham and

pole bean. Similarly, Shantharam and Pe-

Torrey in 1981 gave the strongest evi-

ter (1982) have shown that _R. phaseoli_

dence for the involvement of wall hydrol-

127K14 is capable of forming effective

ysis by _R. leguminosarum bv. trifolii_ in

nodules on different legume. They have

white clover infection process. Baker _et_

showed that _R. phaseoli_ 127K14 nodu-

_al_. (1989) also found that many cells of _R._

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 365

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

_leguminosarum bv. trifolii_ attached to the

hair wall of the host and making a local-

root surface of white clover and produce

ized hole of sufficient size to allow rhizo-

pit erosions in epidermal wall that follow

bial cell penetration. This leads to develop

the penetration of the bacterium, suggest-

more nodules for successful nitrogen-

ing that wall-degrading enzymes are in-

fixation.

volved in symbiosis. Vashishat _et al_.,

Hussain _et al._ (1995) studied the in-

(1985b) observed that _R. trifolii_ strains

volvement of hydrolytic enzymes in the

were capable of producing hydrolytic en-

nodulation of berseem ( _Trifolium alexan-_

zymes like _pectinase, hemicellulase_ and

_drinum_ ). They selected a single mutant

_cellulose_ and these enzymes play im-

hrt20m7 in wild type strain hrt20 of _R._

portant role in symbiosis. Considering

_trifolii_ by screening for reduction in activ-

these **** evidence **** Angle in 1986 tried to de-

ity of degradative enzymes, the relative

termine whether differences exists be-

activities shown by the mutant for _pecti-_

tween fast and slow growing soyabean

_nases_ and _cellulase_ were 33 and 4 per-

rhizobia to produce _pectinase_ and proteo-

cent, respectively of wild type strain. It

lytic enzymes. It was proposed that wide

was observed that mutants unlike its par-

spread production of proteolytic enzymes

ent failed to nodulate clover seedlings.

indicates indirect evidences for their in-

Aggarwal _et al_. (2000) found rhizobia be-

volvement in the invasion of host. Al-

having as super nodulating rhizobia. They

Mallah _et al_. (1990) pre-treated clover

suggest _cellulases_ over _pectinases_ in the

roots with an enzyme mixture of 1%

process of symbiotic infection of berseem

(w/v) _cellulase_ and 0.1% _pectolase_ before

by _R. leguminosarum bv. trifolii._ They

inoculating clover with _R. trifolii_. In-

derived rhizobia mutants showing better

crease in nodulation indicated the role of

growth on CMC and /or pectin _i.e._ behav-

_cellulase_ and _pectinase_ in nodulation pro-

ing as super-nodulating rhizobia. Emtiazi

cess of clover.

_et al_. (2007) also studied the _cellulase_ ac-

In 1992, Pedro _et al_., verify the pro-

tivities in nitrogen fixing _Paenibacillus_

duction of _R. leguminosarum bv. trifolii_

isolated from nitrogen free media. The

enzymes

that

deteriorate

polygalac-

_cellulase_ positive _Paenibacillus_ were se-

turonate and carboxymethyl cellulose

lected by reduction of congored color on

(CMC) as model substrates of plant cell

CMC medium. They have observed that

wall polymers. Similarly Mateos _et al_.

nitrogen fixing strains with _cellulase_ ac-

(1992) reported the production of en-

tivities grow well on nitrogen free media

zymes from _R. leguminosarum bv. trifolii_

with sucrose or manitol as the only

that degrade carboxymethyl cellulose and

sources of carbon. They have concluded

polypectate substrates. Their studies

that most plant associated microorganism

shows that _R. leguminosarum_ bv. _trifolii_

might have _cellulase_ activities for adop-

produces multiple enzymes that cleave

tion or establishment of a plant microbe

glycosidic bonds in the plant cell wall.

interaction. Egamberdieva _et al_. (2010)

Mateos _et al_., (2001) also found that _cel-_

determined the bacterial _cellulase_ activity

_lulase_ (Cel2) enzyme is important for

on media containing the substrate car-

symbiotic development because rhizobial

boxy-methylcellulose. They have found

symbionts require its activity to breach

that _cellulase_ producing bacteria were

the host barrier to establish nitrogen fix-

significantly increase nodule numbers and

ing association with legumes. Robledo _et_

nitrogen content of the plants. Fouts _et al_.

_al_. (2008) have purified cell bound _cellu-_

(2008) studied the complete genome se-

_lase_ (Cel2) isozymes and analysed its

quence of the nitrogen fixing broad host

symbiotic function by reverse genetics

range endophyte _Klebsiella pneumonia_

and plant microscopy approaches. These

342. They have found the gene related to

results provide compelling evidence that

carbohydrates, including pectins and cel-

this enzyme could erode the tip of root

lulosic compound degradation are essen-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 366

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

tial for Kp342 to form endophytic associ-

Better nitrogen fixation may be

ations. So, it was concluded that hydrolyt-

brought by manipulating both rhizobia

ic enzyme activity in the form of _cellulase_

and plant host by eventually creating an

and _pectinase_ is an essential property of

artificial rhizosphere. Schlaman _et al_.,

_Rhizobium_ for infection of white clover

(1998) observed nodulation and the levels

during symbiosis.

of nitrogen fixation can be significantly

higher when plants are infected with rhi-

**8. Improvement in symbiotic efficien-**

zobia

containing

the

hybrid

gene

**cy of rhizobia**

_nod_ D604, which activates the transcrip-

****

tion of _nod_ genes independent from fla-

****

The rhizobium-legume symbiosis

vonoids. For introduction of foreign DNA

accounts for a significant proportion of

into bacterial species electroporation is a

nitrogen available to leguminous plants.

novel approach (Chassy _et al_., 1988).

Thus there is a need to **** improve rhizobia

Garg _et al_. (1999) successfully carried out

to increase their symbiotic efficiency and

electro-transformation of _R. legumi-_

host range. The traditional method for ob-

_nosarum_ with 15.1kb plasmid, pMP154

taining _Rhizobium_ strains with improved

(Cmr), containing a _nod_ ABC- _lac_ Z fusion

properties has been the selection of natu-

by electroporation. __ Chitchanok _et al_.

rally occurring field isolates that best ex-

(2011) derived mutants from wild type

hibit the trait desired (Figure 6). An alter-

_Rhizobium sp._ 6-1C1 through 0.8 and 1.0

native approach is to construct improved

kGy gamma radiation. They have ob-

_Rhizobium_ strains by genetic transfer of

served that _Rhizobium meliloti nod_ H gene

symbiotically

favorable

determinants.

mutants result in a change of host range.

Genomic rearrangements have been re-

They infect vetch with this mutated strain

ported to occur frequently in _R. legumi-_

but fail to nodulate their normal host, al-

_nosarum phaseoli_ (Flores _et al_., 1988,

falfa. ****

Garg _et al_., 1999).

****

****

****

**Figure 6:** Improvement and large scale production of biofertilizes. ****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 367

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

The _nod_ Q on the other hand, are

pMH682 for increasing NF production),

able to infect both alfalfa and vetch.

and

the

_R._

_tropici_

(pGMI149)

Mostly mutations results in the alteration

(pGMI1962) hybrid strain and then tested

or extension of the host range (Faucher _et_

the ability of these NFs to form nodules

_al_., 1989 and Horvath _et al_., 1986). In _R._

on alfalfa. NFs produced by the _R. tropici_

_leguminosarum_ bv. _viciae_ and bv. _trifolii_ ,

hybrid strain were able to induce nodule

the nod product is the main factor that

formation. The presence of _R. meliloti_

distinguishes the host range for nodula-

nodulation genes therefore enables _R._

tion. In contrast to wild type, _R. legumi-_

_tropici_ to produce new NFs that can in-

_nosarum bv. Trifolii nod_ EF mutants nodu-

duce nodule formation. Their results show

late white and red clover poorly but have

that allelic variation of the common _nod-_

acquired the ability to infect peas. When

ABC genes is a genetic mechanism that

these _nod_ EF mutants harbour the _node_

plays an important role in signaling varia-

gene of _R. leguminosarum bv. Viciae,_

tion and in the control of host range. They

they have an extended host range to _vicia_

have also found that mutations in the reg-

and _lathyrus_ species __ (Spaink _et al_., 1989). ****

ulatory genes _nod_ D1 and _nod_ D3 did not

Transfer of _nod_ D1 gene of strain

result in a detectable decrease in nodula-

NGR234 to restricted host range rhizobia

tion.

extend the nodulation capacity of the re-

Falguni _et al_., (2009) amplified 2.4 kb

cipients to new hosts, including the non-

_feg_ A gene (encoding ferrichrome recep-

legume _Parasponia andersonii_ (Horvath

tor) along with its native promoter from

_et al_., 1987). Nonetheless, _nod_ D gene rep-

_Bradyrhizobium japonicum_ 61A152 and

resents a molecular interface between the

cloned in a broad host range plasmid vec-

bacterium and the plant. Plasmid transfer

tor pUCPM18. The plasmid construct pFJ

may increase nodulation or nitrogen fixa-

was transferred by conjugation into _Rhi-_

tion in _R. Leguminosarum bv. viciae_

_zobium sp._ ST1 to give trans-conjugant

strains (DeJonj _et al_., 1982), and there is

ST1pFJ12. Inoculation of pigeon pea

one report of a plasmid loss that improves

seedlings with trans-conjugant ST1pFJ12

symbiotic properties in _Rhizobium loti_

led to a marked increase in plant growth

(Pankhurst _et al_., 1986). In _R. meliloti_ , a

parameters as compared to plants inocu-

non-symbiotic plasmid enhances nodula-

lated with the parent strain ST1, Nodule

tion of the strains harboring it (Urban, J.

occupancy on pigeon pea plant when in-

1988). Esperanza and Monica (1990) ge-

oculated with the trans-conjugant was in-

netically modified _R. leguminosarum bv._

creased. Gene _feg_ A not only supports the

_phaseoli_ CFN42, through transfer of a

growth of the trans-formants rhizobia un-

225kb

plasmid

from

typeII

strain

der iron limited laboratory conditions, but

CFN299. They have observed that more

also increases its survivability under natu-

nodules were obtained with the transcon-

ral soil conditions, which led to higher

jugants on _P. vulgaris_. These strains also

nodulation on peanut plant. Yoshitake _et_

have a diminished competitive ability.

_al_., (2010) introduced _vkt_ A into _R. legu-_

Philippe _et al_., (1996) introduced an IncP

_minosarum_ cells and the strain with a re-

plasmid, pGMI149, carrying the main _R._

markably high _catalase_ activity was con-

_meliloti_ nodulation region into _R. tropici_.

structed. The _vkt_ A trans-formant was in-

The _R. tropici_ (pGMI149) transconjugants

oculated to the host plant _P. vulgaris_ and

poorly nodulate _M. sativa_. When a second

the nodulation efficiency was evaluated.

plasmid, of the IncQ group (compatible

The nitrogen-fixing activity of nodules

with pGMI149), carrying the _nod_ L gene

was increased 1.7 to 2.3 times as com-

(pGMI1962) was introduced into _R._

pared to the parent. Results show that the

_tropici_ (pGMI149), a better nodulation

increase of _catalase_ activity in rhizobial

was observed. They have also prepared

cells could be a valuable way to improve

NFs from _R. tropici, R. meliloti_ (with

the nodulation and nitrogen-fixing ability

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 368

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

of nodules. Therefore, it was concluded

**C., Dénarié, J. and Truchet, G.**

that some genetic determinants of rhizo-

**(1994).** _Rhizobium meliloti_ lipooli-

bia involved in host range infectivity have

gosaccharide nodulation factors:

been worked out leading to the extension

different structural requirements

of their infectivity but their effectivity in

for bacterial entry into target root

terms of _nitrogenase_ expression in en-

hair cells and induction of plant

larged host is still not achieved. _Rhizobi-_

symbiotic developmental respons-

_um_ symbiosis with non-legume host clear-

es. _Plant Cell_ **6, 1357-1374.**

ly indicates the ability of this symbiont to

**Arya, K. B., Shantharam, S. and Peter,**

nodulate legume as well as non-legume

**P. W. (1982).** Nodulation of Pole

(Louise _et al_., 2002).

Bean ( _Phaseolus vulgaris_ L.) by

_Rhizobium_ species of two cross-

**9. Perspective**

inoculation groups. _Applied and_

_Environment Microbiology_ **44,**

Considerable research efforts have

**965-971.**

been made through development of pro-

**Baker, D., Petersen, M., Robeles, M.,**

miscuous rhizobia for improving the effi-

**Chen, J., Squartini, A., Dazzo, F.**

ciency of biological nitrogen fixation; be-

**and Hubbell, D. (1989).** Pit ero-

cause, this process has the potential to

sion of root epidermal cell walls in

reduce our dependence on nitrogenous

the _Rhizobium_ -white clover symbi-

chemical fertilizers.

osis. 12th North American Symbi-

otic Nitrogen Fixation Conference,

**References**

Iowa State University Press, Ames.

****

**Barbour, W. M., Hatterman, D. R. and**

**Aggarwl, M., Sikka, V. K. and Vash-**

**Stacey, G. (1991).** Chemotaxis of

**ishat, R. K. (2000).** Symbiotic

_Bradyrhizobium japonicum_ to soy-

properties of _Rhizobium trifolii_ mu-

bean exudates. _Applied and Envi-_

tants altered for cell wall degrada-

_ronment Microbiology_ **57, 2635–**

tive ability. _Tropical Agriculture_.

**2639.**

**77, 109-111.**

**Beatrix, H., Christian, W. B., Bachem,**

**Al-Mallah, M. K., Davey, M. R. and**

**J. S. and Adam, K. (1987).** Host-

**Cocking, E. C. (1990).** Enzymatic

specific regulation of nodulation

treatment, PEG, biotin and manni-

genes in _Rhizobium_ is mediated by

tol stimulate nodulation of white-

a plant-signal, interacting with the

clover by _Rhizobium trifolii_. _J._

_nod_ D gene product _The EMBO_

_Plant. Physio_. 137: 15-19.

_Journal_ **6, 841-848.**

**Ampomah, O. Y., Ofori-Ayeh, E., Sol-**

**Bec-Ferte, M. P., Krishnan, H. B.,**

**heim, B. and Svenning, M. M.**

**Prome,**

**D.,**

**Savagnac,**

**A.,**

**(2008).** Host range, symbiotic ef-

**Pueppke, S. G. and Prome, J. C.**

fectiveness and nodulation compet-

**(1994).** Structures of nodulation

itiveness of some indigenous cow-

factors from the nitrogen fixing

pea bradyrhizobia isolates from the

soybean symbiont _Rhizobium fredii_

transitional savanna zone of Ghana.

USDA257. _Biochemistry_ **33 (39),**

_African Journal of Biotechnol_ ogy **7**

**11782–11788.**

**(8), 988-996.**

**Boivin, C., Ndoye, I., Lortet, G.,**

**Angle, J. S. (1986).** Pectic and pectinolyt-

**Ndiaye, A., De Lajudie, P. and**

ic enzymes produced by fast ad

**Dreyfus, B. (1997).** The Sesbania

slow growing soyabean rhizobia.

root symbionts _Sinorhizobium sa-_

_Soil Biocem_ istry **18, 115-116.**

_heli_ and _S. teranga_ bv. sesbaniae

**Ardourel, M., Demont, N., Debellé, F.,**

can form stem nodules on _Sesbania_

**Maillet, F., de Billy, F., Promé, J.**

_rostrata_ , although they are less

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 369

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

adapted to stem nodulation than

tion, respectively. _Molecular Plant_

_Azorhizobium caulinodans_. _Applied_

_Microbe Interact_ ion **11, 999-1008.**

_and Environment Microbiology_

**De Jonj, T. M., Brewin, N. J., Johnston,**

**63, 1040–1047.**

**A. W. B. and Phillips, D. A.**

**Brewin, N. J. (2004).** Plant cell wall re-

(1982). Improvement of symbiotic

modelling in the _Rhizobium_ legume

properties in _Rhizobium legumi-_

symbiosis. _Critical. Rev. Plant Sci._ ****

_nosarum_ by plasmid transfer. _Jour-_

**23, 293–316.**

_nal of Genetics and Microbiology_

**Callaham, D. and Torrey, J. (1981).** The

**128, 1829-1838.**

structural basis for infection of root

**Egamberdieva,**

**D.,**

**Berg,**

**G.,**

hairs of _Trifolium repens_ by _Rhizo-_

**Lindströmc, K. A. and Rasanen**

_bium_. _Canadian Journal of Botany_

**L. A. (2010).** Co-inoculation of

**59, 1647-1664.**

_Pseudomonas_ spp. with _Rhizobium_

**Capela, D., Carrere, S. and Batut, J.**

improves growth and symbiotic

**(2005).** Transcriptome-based iden-

performance of fodder galega

tification of the _Sinorhizobium mel-_

( _Galega orientalis_ L.) _European_

_iloti_ NodD1 regulon. _Applied and_

_Journal of Soil Biology_ **46, 269-**

_Environment Microbiology_ **71,**

**272.**

**4910–13.**

**Emtiazi, G., Pooyan, M. and Sha-**

**Chamber, M. A. and Iruthayathas, E.**

**malnasab, M. (2007).** _Cellulase_

**E. (1988).** Nodulation and nitrogen

activities in Nitrogen fixing _Paeni-_

fixation by fast and slow growing

_bacillus_ isolated from soil in N-free

strains of soybean on several tem-

media _World Journal of Agricul-_

perate and tropical legumes. _Plant_

_tural Science_ **3(5), 602-608.**

_Soil_ **112, 239 – 245.**

**Esperanza,**

**Martinez-Romero**

**and**

**Chassy, B. M., Mercenier, A. and Flick-**

**Monica R. (1990).** Increased Bean

**inger, J. (1988).** Transformation of

( _Phaseolus vulgaris_ L.) nodulation

bacteria by electroporation. _Trends_

competitiveness

of

genetically

_Biotechnol_ ogy **6, 303–309.**

modified _Rhizobium_ strains _**Ap-**_

**Chitchanok,**

**A.,**

**Rattasaritt,**

**P.,**

_**plied and Environment Microbi-**_

**Suthatip, S., Suphaporn, P., Nat-**

_**ology**_ **56(8), 2384-2388.**

**tayana, P. and Yanee, T. (2011).**

**Falguni, R. J, Dhwani, K., Desai, G. A.**

Improvement of vitamin B6 pro-

**and Anjana J. D. (2009).** En-

duction from _Rhizobium_ sp. 6-1C1

hanced Survival and Nodule Occu-

by random mutation. _KKU Re-_

pancy of pigeon pea nodulating

_search Journal_ **16(8), 911-918.**

_Rhizobium_ sp. ST1 expressing

**Cohn, J., Day, R. B. and Stacey, G.**

fegA Gene of _Bradyrhizobium ja-_

**(1998).** Legume nodule organogen-

_ponicum_ 61A152. _Journal of Bio-_

esis. _Trends in plant science_ **3,**

_logical Science_ **9(2), 40-51**

**105-110.**

**Falkowski and Paul, G. (1997).** Evolu-

**D'Haeze, W. and Holsters, M. (2002).**

tion of the nitrogen cycle and its in-

Nod factor structures, responses

fluence on the biological sequestra-

and perception during initiation of

tion of CO2 in the ocean. _Nature_.

nodule development. _Glycobiology_

**387, 272–275.**

**12, 79–105.**

**Faucher, C., Camut, H., Denarie, J.**

**D'Haeze, W., Gao, M. S., De-Rycke, R.,**

**and Truchet, G. (1989).** The _nodH_

**Van Montagu, M., Engler, G.,**

and _nodQ_ host range genes of _Rhi-_

**and Holsters M. (1998).** Roles for

_zobium meliloti_ behave as aviru-

azorhizobial Nod factors and sur-

lence genes in _R. leguminosarum_

face polysaccharides in intercellu-

bv. _viciae_ and determine changes in

lar invasion and nodule penetra-

the production of plant-specific ex-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 370

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

tracellular signals. _Molecular Plant_

**Heidstra, R., Geurts, R., Franssen, H.,**

_Microbe Interaction_ **2, 291–300.**

**Spaink, H. P., van Kammen A.**

**Felle, H. H., Kondorosi, E., Kondorosi,**

**and Bisseling, T. (1994).** Root hair

**A. and Schultze, M. (1995).** Nod

deformation activity of nodulation

signal induced plasma membrane

factors and their fate on _Vicia sati-_

potential changes in alfalfa root

_va_. _Plant Physiology_ **105, 787–797**

hairs are differentially sensitive to

**Herman, P. S., Jeremy, W., Michael,**

structural modifications of the

**A., Djordjevic, C., A., Wijf-**

lipochito-oligosaccharide.

_Plant_

**felman, R., Okker, J. H. and Ben**

_Journal_ **7, 939–947**

**J. J. L. (1989).** Genetic analysis

**Flores, M., Gonzalez, V., Pardo, M. A.,**

and cellular localization of the _Rhi-_

**Leija, A. Martinez, E. Romero,**

_zobium_

host

specificity-

**D., Piniero, D., Davila, G. and**

determining NodE protein. _The_

**Palacios R. (1988).** Genomic in-

_EMBO Journal_ **8(10), 2811-2818.**

stability in _Rhizobium phaseoli_.

**Hernandez, L., Segovia, I. L., Martinez,**

_Journal of Bacteriology_ **170, 1191-**

**R. and Steven, G. P. (1995).** Phy-

**1196.**

logenetic relationships and host

**Fournier, J., Timmers, A. C., Sieberer,**

range of _Rhizobium_ spp. that nodu-

**B. J., Jauneau, A., Chabaud, M.,**

late _Phaseolus vulgaris_ L. _Applied_

**and Barker, D. G. (2008).** Mecha-

_and Environment Microbiology_

nism of infection threads elonga-

**61(7), 2775–2779.**

tion in root hairs of _Medicago trun-_

**Hooykaas, P., Van Brussel, J. J., Den,**

_catula_ and dynamic interplay with

**A. A. N., Dulk-Ras, H., Van**

associated rhizobial colonization.

**Slogteren,**

**G.**

**M.**

**G.**

**and**

_Plant Physiology_ **148, 1985-1995.**

**Schilperoort, R. A. (1981).** Sym

**Fouts, D. E., Tyler, H. L., De Boy, R.**

plasmid of _Rhizobium trifolii_ ex-

**T., Daugherty, S. and Ren, Q.**

pressed in different rhizobial spe-

**(2008).** Complete Genome Se-

cies and _Agrobacterium tumefa-_

quence of the N2-fixing broad host

_ciens_. _Nature_. **291, 351-353.**

range endophyte _Klebsiella pneu-_

**Horvath, B., Bachem, C. W., Schell, J.**

_moniae_ 342 and virulence predic-

**and Kondorosi. A. (1987).** Host-

tions verified in Mice. _Genetics_

specific regulation of nodulation

**4(7), e1000141.**

genes in _Rhizobium_ is mediated by

**Garg, B., Dogra, R. C. and Sharma, P.**

a plant signal interacting with the

**K. (1999).** High efficiency trans-

_nod_ D gene product. _EMBO Journal_

formation of _Rhizobium legumi-_

**6, 841–848.**

_nosarum_ by electroporation. _Ap-_

**Horvath, B., Kondorosi, E., John, M.,**

_plied and Environment Microbiol-_

**Schmidt, J., Torok, I., Gyor-**

_ogy_ **65(6), 2802–2804.**

**gypal, Z., Barabas, I., Wieneke,**

**Gibson, K. E., Kobayashi, H. and**

**U., Schell, J. and Kondorosi, A.**

**Walker, G. C. (2008).** Molecular

**(1986).** Organization, structure and

determinants of a symbiotic chron-

symbiotic function of _Rhizobium_

ic infection. _Annual Review of Ge-_

_meliloti_ nodulation genes determin-

_netics_ **42, 413–441.**

ing host specificity for alfalfa. _Cell_.

**Giraud, E. (2007).** Legumes symbioses:

**46, 335–343.**

absence of Nod genes in photosyn-

**Hussain, Z., Sikka, V. K. and Vash-**

thetic bradyrhizobia. _Science_ **316,**

**ishat, R. K. (1995).** Symbiotic in-

**1307–1312.**

fection of clover by _Rhizobium tri-_

**Gualtieri, G. and Bisseling, T. (2000).**

_folii_ depends on degradative en-

The evolution of nodulation. _Plant_

zymes _cellulase_ and _pectinse_. _An-_

_Molecular Biolology_ **42, 181–194.**

_nals of Biology_ **11, 285-289.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 371

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

**Jaynes, D. B., Colvin, T. S., Karlen, D.**

**Manuel, M. (2006)** _Soil Biology_

**L., Cambardella, C. A., and**

_and Biochemistry_ **38, 573–586.**

**Meek, D.W. (2001).** Nitrate losses

**Martinez, E., Flores, M., Brom, S.,**

in subsurface drainage as affected

**Romero, D., Davila, G., and Pa-**

by nitrogen fertilizer rate. _Jornal of_

**lacios,**

**R.**

**(1988).**

_Rhizobium_

_Environment_ **30, 1305-1314.**

_phaseoli_ : a molecular genetics

**Lindstom,**

**K.,**

**(1989).**

_Rhizobium_

view. _Plant Soil_ **108, 179-184.**

galegae, a new species of root nod-

**Mateos, P. F. (2001).** Erosion of root ep-

ule bacteria. _International Journal_

idermal cell walls by _Rhizobium_

_of System Bacteriology_ **39, 365–**

polysaccharide-degrading enzymes

**367.**

as related to primary host infection

**Ljunggren, H., and G. Fahraeus.**

in the _Rhizobium_ –legume symbio-

**(1961).** The role of polygalac-

sis. _Canadian Journal of Microbi-_

turonase in root-hair invasion by

_ology_ **47,475–487.**

nodule bacteria. _J. Gen. Microbiol_.

**Mateos, P. F., Jimenez-zurdo, J. I.,**

**26,521-528.**

**Chen, J., Squartini, A. S., Haack,**

**Loh, J. and Stacey, G. (2003).** Nodula-

**S. K., Martinez-Molina, E., Hub-**

tion gene regulation in _Bradyrhizo-_

**bell, D. H. and Dazzo, F. B.**

_bium japonicum:_ a unique integra-

**(1992).** Cell associated pectinolytic

tion of global regulatory circuits.

and cellulolytic enzymes in _Rhizo-_

_Applied and Environment Micro-_

_bium leguminosarum_ bv _trifolii_.

_biology_ **69, 10-17.**

_Applied and Environment Micro-_

**Long, S. R. (1996).** _Rhizobium_ symbio-

_biology_ **58(6), 1816-1822.**

sis: Nod factors in perspective.

**Mathis, R., De Rycke, R., D'Haeze W.,**

_Plant Cell_ **8, 1885-1898.**

**Van Maelsaeke, E., Anthonio, E.,**

**Louise, F., Roddam, A., Wendy, R.,**

**Van Montagu, M., Holsters, M.,**

**Lewis, H. B. and Michael, A. D.**

**Vreecke, D. and Van Gijsegem,**

**(2002).** Two novel chromosomal

**F. (2005).** Lipopolysaccharides as a

loci

influence

cultivar-specific

communication signal for progres-

nodulation failure in the interaction

sion of legume endosymbiosis.

between strain ANU794 and sub-

_Proceeding of Natural Academy of_

terranean clover cv. Woogenellup

_Science USA._ **102(7), 2655-2660.**

_Funct. Plant Biololgy_ **29, 473–483.**

**McCoy, E. (1932).** Infection by _B._

**Madsen, L. H., Tirichine, L., Jurkie-**

_radicicola_ in relation to the micro-

**wicz, A., Sullivan, J. T., Heck-**

chemistry of the host's cell walls.

**mann, A. B., Bek, A. S. (2010)**.

_Proceeding of Royal Society Lon-_

The molecular network governing

_don_ **110,514-533.**

nodule organogenesis and infection

**Melicent, C. P., Robert, F. F. and Sha-**

in the model legume _Lotus japoni-_

**ron, R. L. (2006)** Diverse flavo-

_cus_. _Nature Communication_ **1,10.**

noids stimulate NodD1 binding to

**Maillet, F., F. Debelle, and J. Denarie.**

_nod_ gene promoters in _Sinorhizobi-_

**(1990).** Role of the nodD and syrM

_um meliloti._ _Journal of Bacteriolo-_

genes in the activation of the regu-

_gy_ **188(15), 5417–5427.**

latory gene nodD3 and of the

**Miller, S. H., Elliot, R. M., Sullivan, J.**

common and host-specific nod

**T. and Ronson, C. W. (2007).**

genes of Rhizobium meliloti. Mol.

Host specific regulation of symbi-

Microbiol. 4:1975–1984 .

otic nitrogen fixation in _Rhizobium_

**Mario, O., Aguilar, M., Veronica, L.,**

_leguminosarum_ biovar _trifolii._ _Mi-_

**Mariano, D., Belen, M., Eugenia,**

_crobial_. **153, 3184-3195.**

**M., Soria, D., Clemente, M., An-**

**Murray, J. D. (2011).** Invasion by Invita-

**tonio, G. S., Carolina, S. and**

tion: Rhizobial infection in leg-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 372

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

umes. _Molecular Plant- Microbe_

zymes

in

_Rhizobium_

_legumi-_

_Interactions_ **24(6), 631–639.** __

_nosarum_ biovar Trifolii. _Applied_

**Okazaki, S., Takakazu, K., Shusei, S.**

_and Environment Microbiology_

**and Kazuhiko, S. (2013).** Hijack-

1816-1822.

ing of leguminous nodulation sig-

Perret, X., Staehelin, C. and Broughton,

naling by the rhizobial type III se-

W. J. (2000). Molecular basis of

cretion system. _PNAS_ **110(42),**

symbiotic promiscuity _. Microbiol-_

**17131-17136.**

_ogy and Molecular Biology Review_ ****

**Oldroyd, G. E. and Downie, J. A.**

**64, 180–201.**

**(2008).** Coordinating nodule mor-

**Philippe, R., Fabienne, M., Claire, P.,**

phogenesis with rhizobial infection

**Frede, R. D., Myriam, F.,**

in legumes. _Annual Review of Plant_

**Georges, T., Jean-Claude, P. and**

_Biology_ **59, 519–46.**

**Jean De, N. (1996).** The common

**Oldroyd, G. E. D. and Downie, J. A.**

_nod_ ABC genes of _Rhizobium meli-_

**(2004).** Calcium, kinases and nodu-

_loti_ are host-range determinants

lation signalling in legumes. _Natu-_

_Proceeding in Natural Academy of_

_ral Cell Biology_ **5, 566–576.**

_Science USA_ **93, 15305–15310.**

**Orgambide, G. G., Lee, J. I., Hol-**

**Poupot, R., Martínez-Romero, E., Gau-**

**lingsworth, R. I., and Dazzo, F.**

**tier, N. and Prome, J. C. (1995)**

**B.** (1995). Structurally diverse chi-

Wild-type _Rhizobium etli_ , a bean

tolipooligosaccharide Nod factors

symbiont,

produces

acetyl-

accumulate primarily in mem-

fucosylated, N-methylated and car-

branes of wild-type _Rhizobium le-_

bamoylated

nodulation

factors.

_guminosarum_ biovar _trifolii_. _Bio-_

_Journal of Biological Chemistry_

_chemistry_ **34, 3832-3840.**

**270, 6050-6055.**

**Pankhurst, C. E., Macdonald, P. E. and**

**Pueppke, S. G. and Broughton, W. J.**

**Reeves, J. M. (1986).** Enhanced

**(1999).**

_Rhizobium_

sp.

strain

nitrogen fixation and competitive-

NGR234 and _R. fredii_ USDA257

ness for nodulation of _Lotus pe-_

share exceptionally broad, nested

_dunculatus_ by a plasmid-cured de-

host ranges. _Molecular Plant Mi-_

rivative of _Rhizobium loti_. _Journal_

_crobe Interction_ **12, __****293-318.**

_of Genetics and Microbiology_ **132,**

**Quesada, Vincen, D., Fellay, R., Nas-**

**2321-2328.**

**sim, T., Viprey, V., Burger, U.,**

**Patriarca, E. J., Tatè, R. and Iaccarino,**

**Promé, J. C., Broughton, W. J.,**

**M. (2002).** Key role of bacterial

**and Jabbouri, S.** (1997). _Rhizobi-_

NH +

4 metabolism in _Rhizobium_ -

_um_ sp. NGR234 NodZ protein is a

plant symbiosis. _Microbiology and_

fucosyltransferase. _Journal of Bac-_

_Molecular Biology Review ****_**66(2),**

_teriology_ **179, 5087-5093.**

**203-222.**

**Relic, B., Perret, X., Estrada-Garcia,**

**Peck, M. C., Fisher, R. F. and Long, S.**

**M. T., Kopcinska, J., Golinowski,**

**R. (2006).** Diverse flavonoids

**W., Krishnan, H. B., Pueppke, S.**

stimulate NodD1 binding to _nod_

**G., and Broughton, W. J., (1994).**

gene promoters in _Sinorhizobium_

Nod factors of _Rhizobium_ are a key

_meliloti_. _Journal of Bacteriology_

to the legume door. _Molecular Mi-_

**188, 5417–27.**

_crobiology_ **13, 171-178.**

**Pedro, F., Mateos, J. I., Jimenez, Z.,**

**Relic, B., Staehelin, C., Fellay, R., Jab-**

**Jin, C., Andrea, S., Squartini,**

**bouri, S., Boller, T., Broughton,**

**Sheridan, K., Haack, E., Mar-**

**W. J. (1994) Do Nod-factor levels**

**tinez, M., David, H., Hubbell and**

**play a role in host-specificity?** In

**Frank, B. D. (1992)** Cell associat-

Proceedings of the first European

ed pectinolytic and cellulolytic en-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 373

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

nitrogen

fixation

conference.

_Applied and Environmental Micro-_

_Press: Szeged, Hungary_ **69-75.**

_biology_ **43(3), 677-685.**

**Robledo, M., Jimenez-Zurdo, J. I., Ve-**

**Shiferaw, B., Bantilan, M. C. S. and**

**lazquez, Trujillo, E. M. E., Zur-**

**Serraj, R. (2004).** Harnessing the

**do-Pineiro,**

**J.**

**L.,**

**Ramírez-**

potential of BNF for Poor Farmers:

**Bahena, M. H., Ramos, B., Díaz-**

Technological Policy and institu-

**Mínguez, J. M., Dazzo, F., Mar-**

tional constraints and research

**tínez-Molina, E. and Mateos, P.**

need. _Symbiotic Nitrogen Fixation;_

**F. (2008).** _Rhizobium_ cellulase

_prospects for enhanced application_

CelC2 is essential for primary

_in tropical agriculture_. (ed.): R.

symbiotic infection of legume host

Serraj. Oxford and IBH publishing

roots. _Proceeding in National_

Co. Pvt. Ltd. New Delhi. **pp.3.**

_Academy of Science USA._ **105(19),**

**Spaink, H. P., Weinman, J., Djordjevic,**

**7064–7069.**

**M. A., Wijfelman, C. A., Okker,**

**Saı¨d, J., Biserka, R., Moez, H.,**

**J. H. and Lugtenberg, B. J. J.**

**Philippe, K., Ulrich, B., Danielle,**

**(1989).** Genetic analysis and cellu-

**Prome., Jean C. P. and William,**

lar localization of the _Rhizobium_

**J. B. (1998)** _nol_ O and _noe_ I (HsnIII)

host specificity-determining NodE

of _Rhizobium_ sp. NGR234 are in-

protein. _EMBO Journal_ **8, 2811–**

volved in 3-O-carbamoylation and

**2818.**

2-O-methylation of Nod factors.

**Spaink, H. P., Wijffelman, C. A., Pees,**

_The Journal of Biological Chemis-_

**E., Okker, R. J. H. and Lugten-**

_try_ **273(20), 12047–12055**

**berg, B. J. J.** **(1987).** _Rhizobium_

**Sanjuan, J., Gro, P., Gottfert, M.,**

nodulation gene _nodD_ as a deter-

**Hennnecke, H. and Stacey, G.**

minant of host specificity. _Nature_

**(1994).** NodW is essential for the

**328, 337-340.**

full expression of the common

**Stokkermans, T. J. W., Ikeshita, S.,**

nodulation genes in _Bradyrhizobi-_

**Cohn, J., Carlson, R. W., Stacey,**

_um japonicum_. _Molecular Plant_

**G., Ogawa, and Peters, N. K.**

_Microbe Interaction_ **7, 364-369.**

**(1995).** Structural requirements of

**Schlaman,**

**H.**

**R.,**

**Horvath,**

**B.,**

synthetic and natural product lipo-

**Vijgenboom, E., Okker, R. J. and**

chitin oligosaccharides for induc-

**Lugtenberg, B. J. J.** **(1998).** Sup-

tion of nodule primordia on Gly-

pression of nodulation gene expres-

cine soja. _Plant Physiol_ ogy **108,**

sion in bacteroids of _Rhizobium le-_

**1587–1595.**

_guminosarum_ biovar viciae. _Jour-_

**Stowers, M. D., Eaglesham, A. R. J.**

_nal of Bacteriology_ **173, 4277-**

**(1984).** Physiological and symbi-

**4287.**

otic characteristics of fast-growing.

**Scholla, M. H., and Elkan, G. H.**

_Plant Soil_ **77, 3-14.**

**(1984).** A fast-growing species that

**Urban _,_** **N. R. and Eisenreich, S.**

effectively nodulates soybeans. _In-_

**J. _(_** **1988) _._** _Nitrogen_ cycling in a

_ternational Journal of Systematic_

forested Minnesota bog. _Canadian_

_Bacteriology_ **34, 484-486.**

_Journal of Bot_ any **66, 435-449.**

**Setiyo, H. (2011)** Characterization and

**van Rhijn, P., Luyten E., Vlassak K.**

phylogenetic analysis of Soybean

**and Vanderleyden, J. (1996).** Iso-

rhizobial strains from Java and

lation and characterization of a

Sumatra _Microbiology Indonesia_

pSym locus of _Rhizobium_ sp.

**5(4), 170-181.**

BR816 that extends nodulation

**Shantharam, S. and Peter, P. (1982).**

ability of narrow host range

Recognition of leguminous hosts

_Phaseolus vulgaris_ symbionts to

by a promiscuous _Rhizobium_ Strain

_Leucaena leucocephala_. _Molecular_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 374

_Biotech Sustainability (2017)_

_Promiscuous Rhizobia and its Potential to Enhance Crops Productivity Ikbal et al._

_Plant Microbe Interaction_ **9, 74–**

**Zhu, Y. Y., Feng, L. K., En, T. W., Ge,**

**77.**

**H. W. and Wen, X. C. (2002).**

**Vashishat, R. K., Yadav, A. S. and**

Characterization of rhizobia that

**Chaudhary, K. (1985b).** Produc-

nodulate legume species of the ge-

tion of hydrolytic enzymes in non-

nus Lespedeza and description of

nodulating strains of _Rhizobium tri-_

_Bradyrhizobium legumingense_ sp.

_folii._ _Haryana Agricutural Univer-_

nov. _Inter. Journal of Systemic and_

_sity Journal Research_ **15, 403-405.**

_Evolution of Microbiology_ **52,**

**Yoshitake, O.,Yoshinobu, N., Takuji,**

**2219–2230.**

**O., Hidetoshi, O., Nobutoshi, I.,**

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 375

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P376-385_

**Organic Farming and Halalan Toyyiban Foods: An**

**Attempt to Relate Them**

**Quamrul Hasan1, 2, * and Zakirah Othman1**

__

_1Knowledge Science Research Lab., School of Technology Management and Logis-_

_tics, College of Business, Universiti Utara Malaysia, 06010 Sintok, Kedah, Malay-_

_sia; 2Japan Halal Research Institute for Products and Services (JAHARI), Kobe,_

_Japan;*Correspondence: quamrul@uum.edu.my_

**Abstract:** Everyone wants to consume safe and healthy food. Also, the producers want to

position their products according to the customers" demands. In the context of safe and

healthy foods, among others, there are two different terminologies, "Organic" and "Halalan

Toyyiban". However, our understandings on these two terminologies are not clear enough

especially when it comes to relate them. Therefore, this research work was undertaken to

better understand the terminologies - organic and halalan toyyiban, and find out the rela-

tionship between them, if any. The research methodology involves both primary data by

visiting an organic farm and face-to-face interviewing farmers, and secondary data. The

findings might help the consumers in selecting the produce/product and business people in

promoting their products. Research informants were farmer, volunteer, and intern at the Sri

Lovely Farm, a government-certified organic farm at Sik, Kedah, Malaysia. The research

reveals new insights on the relationship of characteristics of organic farming with halalan

toyyiban. The three commonly found characteristics are: 1) quality; 2) healthy; 3) environ-

mental friendly. Based on the findings, we are proposing a model on the relationship of or-

ganic farming with halalan toyyiban. This study is the first of its kind and undertaken as an

exploratory research; therefore, further study should be conducted to obtain more under-

standing and knowledge on this subject.

****

_**Keywords**_ **:** Environment; halal food; halalan toyyiban; organic farming; organic food

**1. Introduction**

Consumers are mainly concerned

about health issues, protection of the en-

In the last two decades, globaliza-

vironment and animal welfare besides

tion has significantly advanced leading to

food safety in terms of food processing

not only technological and economic ad-

methods, innovative food technologies,

vancement but also in agriculture, food

and presence of chemical substances in

production, food safety and security.

foods such as pesticides, toxins and food

While efforts to establish trade rules led

additives (Borin _et al_., 2011; Hansen _et_

by the World Trade Organization and fur-

_al_., 2011; Stanton _et al_., 2012). Literature

ther progress in global free trade are ad-

suggests that cultural diversity is an im-

vantageous to create new and mutually

portant criterion to expedite more sustain-

benefitted opportunities, such develop-

able food consumption patterns among

ments have highlighted several risks as-

society (Nicolaou _et al_., 2009; Schösler _et_

sociated with agriculture and food, which

_al_., 2012). The role of religion in shaping

were originally characterized by an un-

consumers food choice is rather vague

clear food chain (Huynen _et al_., 2005).

except where the impact of food con-

sumption depends on the religion itself

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 376

_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ (Bonne _et al_., 2007). Religion can influ-do respond positively to _'halal'_ food cer-

ence consumers" attitudes and behavior,

tification (Hasnah _et al._ , 2009).

including food purchasing decisions and

Besides the religious value, the oth-

eating habits (Pettinger _et al_., 2004). Eat-

er motives behind the _halalan toyyiban_

ing _'halal'_ food by the Muslim communi-

concept include: 1) preserve life, 2) safe-

ty strictly follows the Islamic values as a

guard future generations, and 3) maintain

reflection of obedience and adherence to

self-respect and integrity (Muhammad _et._

the religion"s beliefs and teachings

_al_., 2007). Today, the concept of _halalan_

(Bonne _et al_., 2007). Muslim consumers"

_toyyiban_ is beyond the religious value.

attitudes towards _'halal'_ food consump-

Now a day, the rising concern of food

tion are influenced by religious belief,

consumers is health which could be an

mass media and people around them

untapped opportunity for the _'halal'_ food

(Aiedah, 2014). Further, the appearance

producers. This is because the concern of

of a religious ( _halal_ ) logo on product

health due to food consumption basically

packaging helps Muslims to choose and

shares the same value with the _halalan_

justify their product purchases without

_toyyiban_ concept. Being healthy means,

hesitation guided by their religious beliefs

being watchful over food on the cleanli-

and laws (Bakar _et al_., 2013).

ness, the source, and the method of han-

Though _'halal'_ concept applies spe-

dling and preparing it. The most im-

cifically to the Muslim society (Alam and

portant thing is to ensure and minimize

Nazura, 2011), there is a huge potential to

any possible harmful effects to the body

tap this in to the non-Muslim community

from the food. There could be several de-

as well especially in case of food. The

terminants for the market acceptance of

fact that food is a common need for all

the _'halal'_ food. It is believed that con-

people, the market potential is even more

sumers accept a product when they have

promising though people from different

the true intention to use it, or have used

cultural backgrounds and religious faith

the product earlier and want to continue

do not have same perceptions and experi-

in using it. Generally, consumers respond

ences to food. In Muslim community, the

positively to the products with high quali-

increasing awareness and concern over

ty. In the case of food, quality is defined

health is the basis for acceptance of _'hal-_

mainly by its cleanliness and freshness.

_al'_ food as it covers the whole under-

To achieve this, the food processing

standing of consuming clean and hygienic

methods are the key in ensuring the clean-

food to promote better health. In general,

liness and freshness of the food, which

consumers are more conscious of their

can also affect the nutritional value and

health which influences their behavior

quality of the food. The food quality is

while selecting their food. They search

also critical to determine food safety.

for food with the benefits to keep them

Grunert _et al_. (1996) classified the food

healthy and improve their mental state

quality dimensions into: hedonic, health-

leading to quality of life. The role of food

related, and convenience related. They

in cultural practices and religious beliefs

explained: "Hedonic quality is related to

might be complex; but, it has a unified

sensory pleasure and is therefore mainly

understanding among Muslims. For in-

linked to taste, smell, and appearance.

stance, the halal logo or label helps to

Health-related quality is concerned with

convince Muslim consumers that the food

the ways in which consumption of the

product is suitable for their consumption.

product will affect consumers" physical

On the other hand, the non-Muslim con-

health. Convenience-related quality is re-

sumers understand that food items carry-

lated to the time and effort which has to

ing the halal logo are prepared in the most

be expended while buying, storing, pre-

hygienic way. Furthermore, it has also

paring and consuming the product"

been proven that non-Muslim consumers

(Grunert _et al_., 1996). These explana-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 377

_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ tions, too, relate to the food quality and

engineering, growth hormones, irradiation

safety as well. Furthermore, Rezai _et al_.

and antibiotics. Many kinds of agricultur-

(2011) stressed that the benefits of _'halal'_

al products can be produced organically.

food could be explained from the context

These include produce of grains, meat,

of food safety, which is also demanded by

dairy, egg and processed food products.

non-Muslims.

The term "organic" does not mean "natu-

Studies on the consumers" attitude

ral". There is no fixed definition as to

towards the use of chemicals in agricul-

what constitutes a "natural" food. Never-

ture were explored since 1960"s (Bearler

theless, the food industry uses the term

and Willits, 1968). It marked the begin-

"natural" to indicate that a food has been

ning of the era when human beings start-

minimally processed and is preservative-

ed to be more concerned and aware about

free. A natural food can be called as an

preserving the environment. The findings

organic food, but not all natural foods are

from earlier studies confirmed that con-

organic foods.

sumers showed positive attitudes towards

This exploratory study aims to un-

the products of organic farming where

derstand about meaning of organic farm-

one of the most commonly found reasons

ing and _halalan toyyiban_ foods and find

for choosing these products was - the

out the relationships between them based

products of organic farming were per-

on the common characteristics, if any.

ceived as healthier than the conventional

The key research questions to be ad-

counterparts (Chinnici _et al_., 2002; Har-

dressed in this study were: i. What is our

per and Makatouni, 2002). Consumers do

understanding about the organic farming

not necessarily buy sustainable products

and _halalan toyyiban_ foods? ii. What are

due to environmental concern, giving

the common characteristics to relate them,

benefit to the community, and personal

if any?

beliefs; but, mainly to give priority to

health (Vermeir and Verbeke, 2004). Re-

**2. Literature review**

searchers have shown that the consumers

of organic food are less likely to pay at-

For the better understanding of the

tention to the price as compared to those

concept of halalan tayyiban, the discus-

who do not purchase organic product

sion here starts with the two Arabic

(Yiride _et al._ , 2005).

words, " _halal'_ and " _haram'._ The " _halal'_

In the past two decades, the in-

means to set free, to let go, to dissolve

creased awareness about the environment

and to allow, or to exit from something

has had an effect on consumers" behavior,

that is not allowed ( _haram_ ) (Ibn Manzur,

resulting in to expansion of market of the

n. d). Alternatively, " _halal'_ can be defined

green product at a remarkable rate (Aini

as something that is allowed and the fol-

_et al_., 2003). As a result, there is a huge

lower cannot be punished if it is conduct-

increase in production and consumption

ed properly (Jayyib, 1998). In other

of organic products. It is believed that or-

words, " _halal'_ means anything which is

ganic products have lesser negative effect

not __ prohibited or lawful, especially for

to the environment. The National Organic

food and meat from permitted animal

Standards Board of the U.S. Department

which is ritually slaughtered (Cyril,

of Agriculture (USDA), established a na-

1989). The opposite of " _halal'_ is " _haram'_

tional standard for the term "organic" in

(Ibn Manzur, n. d.). It means prohibited,

December 2000. According to them, or-

forbidden, unlawful, restricted and or un-

ganic food is defined by how it cannot be

permitted (Mohammad, 1993). _'Haram'_

made rather than how it can be made. The

can be defined as something that must be

organic food must be produced without

avoided by the Muslims, and committing

the use of sewer-sludge fertilizers, most

the act of " _haram'_ is sinful and immoral

synthetic fertilizers, pesticides, genetic

for them (Ibn Hazm, 1983). __

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_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ Allah s.w.t. (God) commanded

development of good quality human capi-

specifically on the intake of " _halal'_ food,

tal. _Halalan tayyiban_ food should be

referring to the term " _al-tayyib'_ or " _al-_

viewed from the aspect of its complete

_tayyibat'_ and urging to eat " _halal'_ and

supply chain, beginning from the farm

good quality food and avoiding filthy

and reaching up to the dining table. This

food. The word " _al-tayyibat'_ came from

means, it is important to ensure that dur-

" _taba'_ which means good, tasty, delicious,

ing the whole process, the food should not

sweet, pure, clean, and free from any ma-

be contaminated by anything which may

terials which are _makruh_ (detested) (Ibn

be harmful to the human health.

Manzur, n.d; al-Ghazzali, n.d). Some Is-

As underlined by the Syariah law,

lamic scholars suggested to integrating

the term " _halalan toyyiban_ " refers to the

_tayyib_ and _halal_ (al-Qurtubi, al-Suyuti,

products which are safe to be consumed

Ibn "Ashur and Ibn Kathir). This was

(Omar _et al_., 2013). As Allah s.w.t. (God)

supported by Sazelin and Ridzwan

says in the Quran, "O mankind! Eat of

(2011). __

that which is lawful and good on the

The _halalan tayyiban_ concept co-

earth" (Surah Al Baqarah 2: 172). They

vers all the necessary factors (physical

ask you (O Muhammad SAW) what is

and spiritual) of the food for the human

lawful for them (as food) ... Lawful unto

being. In this connection, the _halalan tay-_

you are at Tayyibaat (all kind of _'halal'_

_yiban_ can be translated as the foods which

foods) (Surah Al Maidah 5: 4). As ex-

are permitted ( _halal_ ) for human intake for

plained, Islam requires that Muslims find

providing benefits to the human body and

_rizk_ (sustenance) and consume food that

mind as well. The food classified as the

is _halalan toyyiban_ because it ensures a

_halalan tayyiban_ should fulfill two crite-

healthy living that reflects good attitudes

ria: firstly, the food is _'halal'_ (and taken

and behaviors as well (Yousef, 2010). It

from a _halal_ source), and secondly, it is a

goes further by covering the concept of

quality food as it provides benefit to hu-

wholesomeness, which includes quality,

man. If the food misses these two criteria,

cleanliness, and safety of the food (Omar

it cannot be called as the _halalan tay-_

_et al_., 2013).

_yiban._ Hence, it must be avoided by the

The results from an earlier study

followers of Islam. __

suggest that non-Muslim consumers are

The _halalan tayyiban_ also indi-

aware of the existence of _'halal'_ food in

cates that the determination of _'halal'_

Malaysia.

In

general,

socio-

food encompasses both the tangible and

environmental factors such as socially

intangible aspects of the food: Before

mixing (of non-Muslims) with Muslims

consumption, the food must be ensured as

and the presence of advertised _'halal'_

_'halal',_ in good quality, hygienic and

food

significantly

influence

non-

safe. These preconditions are applicable

Muslims' understanding of the _'halal'_

from the initial sourcing and handling to

principle. These findings also suggest

the final stage (preparation, manufactur-

that non-Muslims understand that _'hal-_

ing, storage, distribution and serving).

_al'_ principle that addresses the issues of

The idea of _tayyiban_ does not limit the

food safety and environmental friendly-

food to be _'halal'_ , good, delicious, tasty

ness. In the study, at least 94 percent

and pure only. It goes further with the re-

non-Muslims agree that the _'halal'_

quirement of beneficial and not causing

principle is religious obligation, while

any harm to the body. Al-Ghazali said

90 percent and 71 percent agree that it

that "what is beneficial for the body is

is concerned with food safety and envi-

also beneficial for the mind and soul". In

ronmental

friendliness,

respectively

addition, Sazelin and Ridzwan (2011)

(Rezai _et al_., 2012). Therefore, these

stated that the good quality food bounded

suggest that a close relationship exists

by Islam, also has a relationship in the

among _halalan toyyiban_ food safety and

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 379

_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ environmental friendliness, which are

ic Practice. Therefore, implementing the

also the essential characteristics of the

_halalan toyyiban_ requirements (to obtain

products from organic farming. In an

_'halal'_ accreditation) should ensure to

earlier and related study, we have high-

produce higher quality food products

lighted that sustainable agriculture can

(Talib and Ali, 2009). Considering this,

be achieved through organic farming

the _'halal'_ values may become popular

(Othman and Hasan, 2016).

among non-Muslim consumers, if the so-

ciety at large is made to be more aware of

**3. Methodology**

issues concerning health, hygiene, safety,

environment, and animal welfare which

This study employed a qualitative

come along with the _'halal'_ ways of do-

research using the face-to-face interviews

ing the things.

and secondary data approaches. The in-

terviews were conducted with six re-

_4.1.2. Healthy_

spondents who also allowed the inter-

_Halalan toyyiban_ foods are those,

views to be recorded. Later, phone calls

which have been handled and prepared by

were made to the selected respondents to

following the strict hygiene, and the high

obtain clarity of the information from

standards of nutrition, cleanliness and

them. The location of this field study was

safety. In other words, the food must be

at Sri Lovely Farm, Sik, Kedah, Malaysia.

produced and handled by fulfilling the

The interviews were conducted with the

stringent requirements of the Islamic Die-

managing director (Farmer 1), his two

tary Law, which as a result guarantees

assistants (Farmer 2 and Farmer 3), a vol-

that the food is healthy. Since more and

unteer (Farmer 4), and two interns by vis-

more people are becoming health-

iting the farm on October 24, 2016.

conscious, the _halalan toyyiban_ principles

Traditional and computer-based quali-

of preparing food may no longer remain

tative methodologies were used to ana-

confined to the strictly religious need but

lyze the data and to compare and contrast

may become an alternative to non-

the observation. The method suggested by

Muslims for a healthy life.

Corbin and Strauss (1990) was used in the

data analysis. All data were first reviewed

_4.1.3. Clean_

and then categorized.

Allah"s s.w.t. (God) command to

select _halalan toyyiban_ food can be seen

**4. Results**

in the verses of the al-Quran, and among

__

several one is surah al-A"raf (7) verse

_4.1. Characteristics of halalan toyyiban_

157. In this, the word " _al-tayyibat_ " is in-

The characteristics of _halalan toy-_

terpreted as _'halal'_ (al-Qurtubi, n. d.; al-

_yiban_ can be divided into four: 1) Quality,

Tabari, n. d.; al-Suyuti, 1990); _'halal'_ and

2) Healthy, 3) Clean, and 4) Environmen-

not repugnant (Ibn "Ashur, 1984). One

tal friendly, which are further explained

more interpretation is: _'halal'_ is good,

below.

beneficial to the body and helpful in

****

terms of habits and the law of Islam (Ibn

_4.1.1. Quality_

Kathir, n. d.). Also, " _tayyib_ " is mentioned

The whole process of _'halal'_ ac-

in surah al-Baqarah (2) verse 168. Fur-

creditation is stringent; therefore, it has

thermore, al-Sharbini (n.d) explained that

some beneficial characteristics which can

the " _toyyiban_ " has four principal elements

also be enjoyed by non-Muslim consum-

as listed below:

ers. Its requirements meet many of the

i) Both the source and whole content of

conventional quality standards, like ISO,

food is _'halal'_ , no haram is included

Codex Alimentarius, Hazard Analysis and

ii) It is clean, therefore, does not contain

Critical Control Point, and Good Hygien-

any impurities

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_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_

****

**Table 1:** Key differences between conventional and organic farming-Sri Lovely Farm fo-

cusing on the seed used ****

**Key practices**

**Conventional**

**Sri Lovely Farm**

1. Seed preparation

Seed not selected

Seed selected: Seeds soaked for 24

hours prior to sowing to eliminate

non-viable ones

2. Quality of seed-

All kinds of seedlings

Only healthy seedlings transplanted

ling at transplant

iii) It does not cause any negative effect

hereditary which, when we planted the

upon intake

seeds can later use to return to replant-

iv) The food contents are nutritious;

ing"- (Farmer 4, personal communication,

therefore, beneficial to human.

October 24, 2016)

_4.1.4. Environmental friendly_

_4.2.2. Natural fertilizer_

Taking the paradigm shift into ac-

Fertilizers used in Sri Lovely

count which emphasizes on the need of

Farm are natural, from 100% natural in-

the green supply chain, the _'halal'_ princi-

gredients produced locally, and which are

ples are no more only for the Muslims of

organic entirely. Fertilizers produced in

slaughtering permitted animals in the Is-

Sri Lovely Farm are from rice straw, and

lamic way. It also emphasizes on the sus-

fruit waste collected.

tainability, environmental friendliness,

food safety and animal welfare. Hence,

_4.2.3. Quality soil_

the _'halal'_ standard implies the Halalness

The quality of the soil in Sri Love-

of the products to Muslims and it stands

ly Farm is monitored by the Department

for not only just and fair business transac-

of Agriculture, Malaysia in order to main-

tions but also caring for the environment,

tain soil quality of the farm land.

sustainability, and animal welfare.

"Our soil was often taken to be

used as a sample, enter the lab.

_4.2. Characteristics of organic farming_

From there, we could know the

The characteristics of organic

soil contains heavy metal or not" -

farming can be divided in to four: 1)

(Farmer 1, personal communica-

Healthy seed, 2) Natural fertilizer, 3)

tion, October 24, 2016)

Quality soil, and 4) Natural insect control.

These characteristics are further explained

The soil/land of high quality can

below.

produce rice of high quality, and also help

in balancing the ecosystem, which is good

_4.2.1. Healthy seed_

for the environment. Quality of soil is

The seeds being used in Sri Love-

maintained by the nutrients - carbon (C),

ly Farm are of a very good quality. As

hydrogen (H), oxygen (O), Potassium

depicted in the Table 1, only viable seeds

(K), calcium (Ca), magnesium (Mg), sul-

are selected after soaking in water for 24

fur (S), Phosphorus (P) and nitrogen (N).

hours followed by sowing in the container

Carbon, hydrogen, oxygen and nitrogen

for 4 days before planting on the ground.

can be obtained from the air whereas; po-

The seeds are free of GM seeds. The con-

tassium, calcium, magnesium and sulfur

cept of the healthy seed selection can be

are usually obtained through fertilizers.

related with the quality of produce (in this

Fertility of the soil is very important and

case rice). "Original seed instead of the

it is one of determining factors of the crop

modified seeds". The original seeds are

yield.

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_Biotech Sustainability (2017)_

_

__Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ ready know that some non-Muslim con-4.2.4. Natural insect control _

sumers are familiar with the _'halal'_ prin-

Insect control is being practiced

ciples and food products available in the

by Sri Lovely Farm in an ecofriendly way

market. To make it further successful,

to avoid the synthetic and dangerous

more awareness promotion about the _hal-_

chemicals.

_alan toyyiban_ food is needed by empha-

"We have own methods, so how

sizing that it"s not only about the religious

we want to control of caterpillars,

point of view but also about the common

we use 'ubi gadung', so, we use the

benefits for all such as the food safety,

traditional concept of back to na-

wholesomeness, hygiene, caring for ani-

ture" - (Farmer 1, personal com-

mal and environment. In this effort, the

munication, October 24, 2016)

Muslim consumers have a key role to

play by promoting and making their non-

According to the one of respond-

Muslim friends aware about the _halalan_

ents from the Sri Lovely Farm, the insect

_toyyiban_ principles of producing the food.

control is being carried out by using "ubi

To the best of our knowledge, this

gadung" ( _Dioscorea daemona_ ). ****

study was the first attempt to relate the

****

foods of _halalan toyyiban_ with organic

**5. Discussion**

farming. Therefore, enough information

was not available in the published form

This study was exploratory with

especially when it was about to establish

an aim to further understand about the

the relationship between the _halalan toy-_

food choices classified under "organic"

_yiban_ and organic farming. However, it

and " _halalan toyyiban_ ". To our surprise,

was possible to collect some materials

it was found that the key issues of envi-

relevant which were found separately un-

ronmental friendliness and food safety

der _halalan toyyiban_ and organic farming.

were addressed by the _'halal'_ principles

By combining our insights obtained from

as revealed by the non-Muslims. Consid-

both primary and secondary data, we have

ering about our future generations, we

been able to come up with three common

must put in our best effort in promoting

characteristics to show the significant in-

and maintaining a sustainable green envi-

ter-relationships between _halalan toy-_

ronment, these issues are very critical in

_yiban_ and organic (farming) foods. These

that sense. And the _halalan toyyiban_ food

are: 1) quality; 2) healthy; and 3) envi-

helps by providing a choice to consumers

ronmental friendly (natural). This is fur-

to meeting the sustainability goal. We al-

ther illustrated in the Figure 1.

**Relationship between**

**organic farming and**

**Characteristics of**

**halalan toyyiban foods** :

**organic farming** :

**Characteristics of**

\- _Quality_

**halalan toyibban** :

-Healthy seed

_-Healthy_

-Quality

-Natural fertilizer

_-Environmental_

-Healthy

-Quality soil

_friendly/Natural_

-Clean (safe)

-Natural insect control

_****_

-Environmental friendl y

****

****

**Figure 1:** A proposed model to show relationship between organic farming and _halalan_

_toyyiban_ foods ****

****

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_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ **6. Conclusion**

**Al-Ghazzali, Abu Hamid Muhammad**

**ibn Muhammad ibn Muhammad.**

We conclude that there is a signif-

**(n. d.).** _Ihya'_ " _Ulum al-Din_ (v. 2).

icant relationship between the integral

Beirut: Dar al-Ma"rifah.

part of the _halalan toyyiban_ principles

**Al-Qurtubi, Abi 'Abd Allah Muham-**

and practices of organic farming to pro-

**mad ibn Ahmad al-Ansari. (n. d.).**

duce food. At least three characteristics

_Tafsir_

_al_ \- _Qurtubi_

_al-Jami_ "

_li_

namely, "quality", "healthy" and "envi-

_Ahkamal-Qur'an_ (v. 7). Beirut: Dar

ronmental friendly (natural)" were identi-

al-Sha"bi. __

fied in common between _halalan toyyiban_

**Al-Sharbini, Shams al-Din Muhammad**

principles and practices of organic farm-

**ibn al-Khatib. (n. d.).** _Mughni al-_

ing. These insights might help in the val-

_Muhtaj ila Ma'rifat Ma_ " _ani Alfazal-_

ue proposition of the both kind of produce

_Minhaj_ (v. 4). Dimashq: Dar al-Fikr. __

and products to all consumers regardless

**Al-Suyuti, Jalal**

**al-Din 'Abd al-**

of their faith and religion. Furthermore,

**Rahman ibn Abu Bakr ibn Mu-**

Malaysia, as the pioneer and promoter of

**hammad ibn Sabiq al-Din. (1990).**

the _halalan toyyiban_ food, should be able

_al-Daral-Manthur, v. 3._ Beirut: Dar

to further promote and successfully enter

al-Kutub al-"Ilmiyyah **. __**

in to the emerging global market with its

**Al-Tabari, Abu Ja'far Muhammad ibn**

own produce and products by utilizing

**Jarir. (n. d.).** _Tafsir al-Tabari_ , v. 9.

insights reported in this article.

Mesir: Dar al-Ma"arif.

**Bakar, A., Lee, R. and Rungie, C.**

__
__

## Acknowledgements

**(2013).** The effects of religious sym-

bols in product packaging on Muslim

The authors would like to extend

consumer responses. _Australasian_

their gratitude to Captain Zakaria Kaman

_Marketing Journal_ , **21, 198-204.**

Tasha at Sri Lovely Farm; Noor Azian

**Bearler, R.C., and Willits, F.K. (1968).**

Mohamad and Siti Noor Ashikin Abd

Worries **** and non-worries among con-

Latif at UUM for their assistance and

sumers about farmers use of pesti-

support to complete this study.

cides. _Journal of consumer Affairs_ **, 2,**

**189.**

**References**

**Bonne, K., Vermeir, I., Bergeaud-**

****

**Blackler, F. and Verbeke, W.**

**Aiedah, A.K. (2014).** Young consumers"

**(2007).** Determinants of halal meat

attitude towards halal food outlets

consumption in France. _British Food_

and JAKIM"s halal certification in

_Journal_ , **109 (5), 367-386.**

Malaysia. _Procedia-Social and Be-_

**Borin, N., Cerf, D.C. and Krishnan, R.**

_havioural Sciences_ **121, 26-34.**

**(2011).** Consumer effects of envi-

**Aini, M.S., Fakhru'l-Razi., A. Laily, P.,**

ronmental impact in product label-

**and Jariah, M. (2003).** Environ-

ling. _Journal of Consumer Marketing_

mental concerns, knowledge and

**28 (1), 76-86.**

practices gap among Malaysian

**Chinnici, G., D'Amico, M., and Pecori-**

teachers. _International Journal of_

**no, B. (2002).** A multivariate **** statisti-

_Sustainability in Higher Education._

cal analysis on the consumers of or-

**4(4), 305 – 313.**

ganic products. _British Food Journal_.

**Alam, S.S, and Sayuti, N. M. (2011).**

**104, 187-199.**

Applying the Theory of Planned Be-

**Corbin, J. M. and Strauss, A. (1990).**

havior in Halal Food Purchasing. _In-_

Grounded theory research: Proce-

_ternational Journal of Commerce and_

dures, cannons, and evaluative crite-

_Management_ **21(1), 8-20.**

ria _. Qualitative Sociology_ , **13 (1), 3-**

**21.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 383

_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ **Grunert, K. G., Hartvig Larsen, H.,**

**Muhammad, N., Norhaziah, N., Nu-**

**Madsen, T. K., and Baadsgaard,**

**radli, R., and Hartini, M. (2007).**

**A. (1996).** Market orientation in food

Halal Branding: An Exploratory Re-

and agriculture. Kluwer, Boston, MA ****

search among consumers in Malay-

**Hansen, T., Mukherjee, A. and Thyra,**

sia. Available at _nuradli.com_

**U.T. (2011).** Anxiety and search dur-

**Nicolaou, M., Doak, C.M., van Dam,**

ing food choice: the moderating role

**R.M., Brug, J., Stronks, K. and**

of attitude towards nutritional claims.

**Seidell, J.C. (2009).** Cultural and so-

_Journal of Consumer Marketing_ **28**

cial influences on food consumption

**(3), 178-186.**

in Dutch residents of Turkish and

**Harper, G.C., and Makatouni, A.**

Moroccan origin: a qualitative study.

**(2002).** Consumer **** perception of or-

_Journal of Nutrition Education and_

ganic food productions and farm an-

_Behaviour_ **41 (4), 232-241.**

imal welfare. _British Food Journal_ ****

**Omar, E.N., H.S. Jaafar and M.R. Os-**

**104, 287-299.**

**man. (2013).** Halalan toyyiban **** sup-

**Hasnah, S., H., Dann, S., Annuar,**

ply chain of the food supply industry.

**M.K., and De Run, E.C. (2009).** In-

_Journal of Emerging Economics and_

fluence of the Halal Certification

_Islamic Research_ **1, 1-12.**

Mark in Food Product Advertisement

**Othman, Z. and Hasan, Q. (2016).** Sus-

in Malaysia. (Chapter 14: The New

tainable agriculture through organic

Culture of food. Marketing Opportu-

farming: A case in paddy farming in

nities from ethnic, religious and cul-

peninsular Malaysia. _Focus on Envi-_

tural diversity. Edited by Adam

_ronment_. AIMST University, Malay-

Lindgreen and Martin K. Hingleg),

sia. **pp. 38-50.**

Gower Publishing Limited, England. ****

**Pettinger, C., Holdsworth, M. and**

**Huynen, M.M.T.E., P. Martens and**

**Gerber, M. (2004).** Psycho-social in-

**H.B.M. Hildetink (2005).** The health

fluences on food choice in Southern

impacts of globalisation: A conceptu-

France and Central England. _Appe-_

al framework. _Globalization and_

_tite_ , **42 (3), 307-316.**

_Health_ **1, 1-12.**

**Rezai,**

**G.,**

**Mohamed,**

**Z.,**

**and**

**Ibn 'Ashur, M. al-T. (1984).** _Tafsir al-_

**Shamsudin, Nasir Mad (2012).**

_Tahrir wa al-Tanwir, v. 6._ Tunisia:

Non-Muslim consumers" understand-

al-Dar al-Tunisi.

ing of Halal principles in Malaysia.

**Ibn Hazm, A. M. (1983).** _al-Ihkam fi_

_Journal of Islamic Marketing_ **3 (3),**

_Usul al-Ahkam, v. 3._ Beirut: Dar al-

**35-46.**

Afaq al-Jadidah.

**Sazelin, A., and Ridzwan, A. (2011).**

**Ibn Manzur, J. al-Din M. ibn M. al-A.**

Food quality standards in developing

**(n. d.).** _Lisan al-'Arab, v. 4_. Mesir:

quality human capital: An Islamic

Dar al-Misriyyah li al-Ta"lif wa al-

Perspective. _African Journal of Busi-_

Tarjamah.

_ness Management **5**_ **(31), 12242-**

**Jayyib, S. A. (1998).** _al-Qamus al-Fiqhi_

**12248.**

_Lughatan wa Istilahan._ Beirut: Dar

**Schösler, H., de Boer, J. and Boersema,**

al-Fikr. ****

**J.J. (2012).** The organic food philos-

**Mohammad, M. H. (1993).** _Islamic Die-_

ophy: a qualitative exploration of the

_tary Concepts and Practices_. The Is-

practices, values, and beliefs of

lamic Food and Nutrition Council of

Dutch organic consumers within a

America (IFANCA), Chicago, IL.

cultural-historical frame. _Journal of_

**Mohani, A. , Hasanah, I., Haslina, H.**

_Agricultural and Environmental Eth-_

**and Juliana, J., (2009).** SMEs and

_ics_ 2 **6 (2), 439-460.**

halal certification. _China-USA Busi-_

**Stanton, J.L., Wiley, J.B. and Wirth,**

_ness Review_ 8 (4).

**F.F. (2012).** Who are the locavores?

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 384

_Biotech Sustainability (2017)_

_Organic Farming and Halalan Toyyiban Foods... Hasan and Othman_ _Journal of Consumer Marketing_ **29**

and update of the literature. _Renewa-_

**(4), 248-261.**

_ble Agriculture and Food System_ **20,**

**Taib, H. A., and Ali, K.A. (2009).** An

**193-205.**

overview **** of Malaysian food industry:

**Yousef, D. K. (2010).** UAE: Halal food

the opportunity and quality aspects.

numbers look tasty. **** Available at:

_Pakistan Journal of Nutrition_ **8 (5),**

http://tinyurl.com/jn9f8m7

**507-17.**

**Yunus, A. M., Yusof, W. M., Chik, W.,**

**Vermeir, I., and Verbeke, W. (2006).**

**and Mahani, M. (2010)** , The Con-

Sustainable food consumption: Ex-

cept of Halalan Toyyiba and Applica-

ploring

the

consumer

attitude-

tion in Product Marketing: A Case

behavioral intention gap. _Journal of_

Study at Sabasun HyperRuncit Kuala

_Agricultural and Environmental Eth-_

Terengganu, Malaysia. _International_

_ics_ **19 (2), 169-194.**

_Journal of Business and Social Sci-_

**Yiridoe, E.K., Bonti-Ankomah, S., and**

_ence_ **1(3),** **239-248.**

**Martin, R.C. (2005).** Comparison of

**Zeithaml, V., Parasuraman, A. and**

consumers perceptions and prefer-

**Berry, L. (1996).** The behavioral

ences toward organic versus conven-

consequences of service quality.

tionally produced foods: A review

_Journal of Marketing_ **60** , **31-46.**

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 385

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P386-397_

**Biotechnological Approaches: Sustaining Sugarcane**

**Productivity and Yield**

**Ashutosh Kumar Mall and Varucha Misra***

_ICAR-Indian Institute of Sugarcane Research, Lucknow- 226 002, Uttar Pradesh, India;_

_*Correspondence: Ashutosh.Mall@icar.gov.in / misra.varucha@gmail.com; Tel: +91 522-_

_2480726_

**Abstract:** Biotechnology is an important field of science which is playing a vital role in

agriculture and other domains. The idea of creating new hybrid varieties is not new; how-

ever, earlier this process was possible only in close species associated with each other. With

the use of biotechnological techniques, it is now possible even in species which are not

closely associated. Sugarcane crop is also not left untouched by this field of science. It has

paved a new way for improving the cane production and productivity. It even helps in en-

hancing the sucrose content of the crop. Sugarcane researchers have achieved success in

several aspects with the use of these techniques like developing high yielding cane varie-

ties; enhance accumulation of sucrose content in cane stalks, _etc_. Although there are still

certain constrains which have yet not be solved in this crop but the way field of biotechnol-

ogy is developing, it is not far that these constrains will also be overcome. In this article we

are highlighting the usefulness and potential of biotechnology approaches to boost the sug-

arcane productivity and yield for the sustainability. ****

_**Keywords**_ **:** Abiotic stress; high yielding; productivity; sugarcane

**1. Introduction**

this new field of science as a tool, re-

searches specialized in plant breeding can

Sugarcane is a crop that imparts

able to produce better crops. The technol-

sweetness to human's life. It is a major

ogies used in this field have the capability

sugar producing crop that contributes to

to transfer and alleviate a single gene/or

more than 70 per cent for production of

number of genes of desired trait rather

sugar. It covers an area of around 3.8 mil-

than thousand of genes from one species

lion hectares with an annual cane produc-

to the other one (Nel, 2009).

tion of around 270 mt. 2.8 per cent of the

Biotechnological approaches in

cultivated land area is occupied by this

the plant kingdom has been playing sig-

crop and in respect to agricultural produc-

nificant role from past many decades.

tion about 7.5 per cent is contributed by

This field of science had encompassed the

this crop to India. In India, 42.02 (%) and

magnificent genetic engineering devel-

57.98 (%) is contributed to sugarcane area

opments within itself in several folds. The

in tropical and sub-tropical zone, respec-

crops obtained from such methods have

tively, while in terms of production it is

been known to be the latest technological

48.58 (%) and 51.42 (%), respectively

approaches that had helped in boosting up

(Shukla _et al_., 2016). It is well known that

the production of food to a great extent.

this crop is a major source of food as well

These transgenic crops had many benefi-

as fuel production. The new field of bio-

ciary points like easier application of

technology has the power of improving

herbicide and that too in very low levels

cane production as well as yield. Using

as per the normal practices which in turn

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 386

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ helps in reducing the cost of production

Another most important constrain is the

as well as in overcoming the environmen-

time required for a new variety to develop

tal pollution (Baker and Pretson, 2003). In

and commercialize that generally takes a

case of sugarcane crop, on worldwide ba-

long time of 12-15 years. As mentioned

sis there is high pressure to augment cane

before that biotechnology helps in trans-

productivity for sustaining the profits of

ferring a desired trait of gene from one

sugar mills (Halon _et al_., 2000). In this

plant to other so in case of sugarcane

regards, various sugarcane researchers

crop, there is certain desired traits which

have been showing effort in developing

would not be able to introduced into it

new hybrid cane varieties that posses high

through the normal plant breeding meth-

yield and high sugar contents under con-

ods. The victory of improving the crop

ventional breeding programmes of sugar-

production by biotechnological tools lies

cane at different institutes. With the use

in the high levels of the trans-gene ex-

of these approaches, new cultivars are

pression. In this aspect, promoters have

being able to develop which possess high

been identified in driving the high levels

sugar content, better ability of ratooning

of gene expression in transgenic sugar-

as well as resistance towards various dis-

cane, particularly in stem and leaves. The

eases. These newer techniques and meth-

first identified promoter was obtained

odology have paved new way in the field

from Cestrum yellow leaf curling virus

of breeding for improving varieties and

which impels the elevated level of consti-

also helped in rapid multiplication of the-

tutive trans-gene expression significantly

se varieties. A common breeding con-

higher than the ones obtained by the

strain in developing new varieties is its

maize _polyubiquitin_ \- _1_ ( _Zm_ \- _Ubi1_ ) promot-

slow multiplication rate as well as its rap-

er (a well known benchmark). Another

id spread. This creates a problem in not

identified

promoter

was

the

fulfilling the seed requirement of the new-

maize _phosphonenolpyruvate_

_carbox-_

ly developed varieties, biotechnology in

_ylate_ promoter which facilitates the ex-

this aspect, had helped in faster multipli-

pression levels, particularly in the leaf

cation of new varieties (Source access:

region of sugarcane, compared to _Zm_ -

http://shodhganga.inflibnet.ac.in/bitstrea

_Ubi1._ By the process of gene modifica-

m/10603/42274/7/07_chapter%202.pdf,

tion, the transgenic expression was en-

3.05.2017).

hanced by approximately 50-fold for bet-

ter cane production (Kinkema _et al_.,

**2. Biotechnological achievements**

2014). Bower and Birch (1992) had

****

achieved success in the sugarcane trans-

_2.1. In improving cane production_

formation trailing with the development

Being a major food and fuel

of micro-projectile system. Some studies

source all over the world, biotechnology

had showed improved resistance in de-

in this regard has the power for improving

veloping a transgenic sugarcane crop to-

the economically important traits in this

wards micro-organisms acting as patho-

crop. The key approach in improving

gens (Joyce _et al_., 1998a, b; Ingelbrecht _et_

sugarcane production lies in the classical

_al_., 1999; Zhang _et al_., 1999; Gilbert _et_

plant breeding method but plant breeders

_al_. 2005;), towards pests like stem borer

always encounter difficulty in this regard

(Arencibia _et al.,_ 1999; Braga _et al_.,

as cane genome is highly complex and

2003) and towards herbicide (Gallo-

possess narrow genetic base (Roach,

Meagher and Irvine, 1996; Enriquez-

1989; Lima _et al_., 2002). The biotechno-

Obregon _et al_., 1998).

logical approaches had successfully im-

The use of techniques of genetic

proved cane production especially the in-

engineering in the past two decades by

ter-specific _Saccharum officinarium_ and

plant breeders had caused transmission of

_S._ _spontaneous_ hybrids (Usman, 2015).

noble gene into the plant for developing

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 387

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ better characteristics in them. The tech-raise and secondly cost of production will

nique involves insertion of foreign genes

be reduced (Shanthi, 2016).

into the parent plant, use of protoplasmic

cells or tissues for the development of

_2.3. Towards abiotic stress_

transgenic plant having normal physiolog-

For tolerance to a particular stress

ical and biological functions. Several ad-

or multiple stresses, biotechnology had

vances have been seen from past several

showed a new way for developing trans-

years in field of molecular biology as well

genic plants. The best short-term ap-

as genetic engineering pertaining to crops

proach for development of stress tolerant

(Jenes _et al_., 1993). With the use of a

cane variety lies on the base of selection

technology based on recombinant DNA it

and breeding wherein wide crosses are

is now even achievable to clone a gene,

being made. For the development of the

modify or mobilize it and even integrate it

stress tolerant variety certain steps have

in any other without any discrimination

been outlined (Figure 1) (Epstein and

from where the gene has been taken

Rains, 1987). Researchers had identified

(Chakrabarty _et al_., 2002). About 50 %

candidate genes in sugarcane for impart-

losses are being occurring by different

ing tolerance to various abiotic and biotic

types of borers but the use of _Bacillus_

stresses. In this view, some of the exam-

_thurigenesis_ by these technologies have

ples of candidate genes which have been

shown harmful effects towards these bor-

identified in case of drought/water deficit

ers. In the present scenario, by the use of

condition are DREB (dehydration respon-

cry genes, greater than 30 species of dif-

sive transcription factor), HSP (heat

ferent plants have been transformed

shock proteins), LEA (late embryo-

(Schuler _et al_., 1998).

genesis), RAB (responsive to abscisic ac-

id), osmotin, choline oxidase and annexin

_2.2. In developing high yielding sucrose_

(Nair, 2011), stress-related clusters show-

_cane varieties_

ing differential expression (>2-fold) dur-

Biotechnological

approaches

had

ing biotic and abiotic stress conditions

made possible in increasing the frequency

(Gupta _et al_., 2010), sugarcane ethylene-

of capability of parental clones possess

responsive factor, SodERF3 (Trujillo _et_

with very high sucrose content which in

_al.,_ 2009), up-regulation of genes regulat-

prevailing breeding programmes is at a

ing intracellular redox status (Prabu _et al.,_

very low level. In case of sugarcane crop,

2011) and presence of LEA (late embryo-

Sugarcane Breeding Institute had initiated

genesis abundance)-related proteins and

programme in this aspect. The target of

dehydrin (Iskandar, _et al_., 2011), accumu-

this programme is to develop genetic

lation of trehalose and proline (Molinari

stocks that consist of high sucrose by the

_et al_., 2007; Guimarães _et al_., 2008), oth-

use of recurring cycles of intensive cross-

er stress-inducible proteins (Jangpromma

ing and selection. In cycle I, about 5420

_et al_., 2010), early response to dehydra-

seedlings obtained from 30 bi-parental

tion protein 4 (ERD4) (McQualter _et al_.,

crosses were considered. Out of all the

2007). Wahid and Close (2007) had iden-

crosses performed, crosses between CoC

tified various expressions of genes or pro-

671 x CoT 8201, Co 86002 x Co 62198,

teins in sugarcane grown under tempera-

Co 85002 x CoT 8201, CoC 671 x Co

ture and salinity induced stress. Some of

94019, PR 1080 x Co 94008 and PR 1080

the instances of such gene expression un-

x CoT 8201 had shown high levels of su-

der stress are heat stress-induced DHNs

crose contents. The approach of biotech-

(Wahid and Close, 2007), genes encoding

nology in this aspect of developing high

for O-/OH- radicals and reduction of H2O2

sugarcane varieties have opened up a new

by peroxidase/ catalase under heat stress

option which will reward to sugar millers

(McQualter _et al.,_ 2007; Chagas _et al_.,

in two ways, firstly productivity will

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 388

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_

****

**Figure 1:** A diagrammatic sketch depicting the steps involved in using molecular ap-

proaches for the development of stress tolerant sugarcane variety (Source: Data from

Shrivastava _et al_., 2016).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 389

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ 2008; Shrivastava _et al_., 2012), cold-response to foliar application of sali-

inducible ESTs, PPDK and NADP-ME

cylic acid (Almeida _et al.,_ 2013).

proteins and dehydrin-like proteins which

iii. Identification and expression of genes

protect membranes against chilling stress

related to defense/ signaling sequenc-

(Nogueira _et al.,_ 2003), reduced activity

es in smut and eyespot disease inocu-

of sucrose phosphate synthase, _NADP_ -

lated cane plants- 62 differentially

MDH

and

pyruvate

orthophosphate

expressed genes having 19 transcript

dikinase to maintain photosynthesis under

derived fragments (TDFs) and a chi-

chilling damage (Du _et al_., 1999), induc-

tinase gene _ScChi_ which is concerned

tion of Galactional synthase (GolS) and

in interaction of host with pathogen

pyrroline-5-carboxylase

synthetase

(Borrás-Hidalgo _et al.,_ 2005; Que _et_

(P5CS) (McQualter _et al_., 2007) and os-

_al.,_ 2014).

molytes like proline and glycine betaine

iv. Identification and expression of EST

(Patade _et al.,_ 2008) during salinity-

clusters that are responsible in signal-

induced stress. Shaik _et al._ (2007) had

ing of reactive oxygen species (ROS),

mediated transformation in sugarcane

defense response and sugarcane innate

through a microorganism _Agrobacterium_

immunity against red rot __ infection

_tumifaciens_ with two plasmid LBA4404

(Sathyabhama _et al_., 2016).

pB1 121 construct GLY1 that bestowed

v. Development of drought tolerant

stress tolerance in crop. Another bacterial

transgenic sugarcane- PT Perkebunan

transformation of _A. tumifaciens_ imparted __

Nusantara in Indonesia, University of

tolerance to drought and salinity in sugar-

Jember (East Java) and Ajinomoto

cane using Arabidopsis Vascular Pyro-

Co., Inc., Japan had developed this

phosphatase (AVP1) gene (Kumar _et al_.,

transgenic plant by using _bet A_ gene

2014). ****

from the _Rhizobium meliloti_ that pro-

Some recent achievements in provid-

duces glycine-betaine. This product is

ing tolerance to abiotic stress in sugarcane

an osmo-protectant which imparts tol-

through biotechnological tools are as fol-

erance towards drought stress. This

lows:

GM transgenic cane produced 20-30

i. Identification of nearly 600 differen-

per cent more sugar in comparison to

tially expressed genes in cane grown

other cane varieties opted for drought

under low temperature for activity of

conditions (Marshall, 2014; Waltz,

the trans-membrane transporter with

2014). With the approval by the na-

an enhancement of ~2.5 fold in _Ssp-_

tional genetically Modified Products

_NIP2_ expression ( _Saccharum_ homo-

Bio-safety Commission of Indonesia

log of a NOD26-like major intrinsic

this has gained the first position of the

protein gene (Park _et al.,_ 2015).

world's first commercialized GM

ii. For enhancing tolerance power of

sugarcane (Anon, 2013).

cane towards drought and salinity,

over expression of PDH45 (a DEAD-

**3. Sugarcane production and produc-**

box helicase gene- a pea isolated

**tivity**

gene) in transgenic sugarcane. This

gene also exhibited an up-regulation

Biotechnology has paved a new

of

DREB2-induced

downstream

way for improving the cane production

stress-related genes (Augustine _et al_.,

and productivity. To increase the sucrose

2015). Another expression of genes in

content in the crop, genetic manipulation

response to drought in sugarcane, ex-

are being used which requires a complete

pression of a set of genes majorly ac-

knowledge and command in the processes

countable for synthesis or expression

involved in sucrose accumulation within

of trehalose 5-PO4 and sucrose-PO4 in

the cane stalks (the storage house of the

plant). Researchers have been successful

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 390

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ in identifying the enzymes that gives a

these small products which in turn con-

start to these processes, however, through

verts the complete cane plant into a bio-

the modern technique of genetic engineer-

factory. A number of high value products

ing these enzymes cane be hasten or

are being produced like therapeutic pro-

slowed down to attain more efficient stor-

teins (Wang _et al.,_ 2005) and biopolymers

age and accumulation of sucrose in cane

(Petrasovits _et al_., 2007; McQualter _et al._ ,

stalks. The application of genetic manipu-

2005). Another important production of

lation in cane stalks are being conducted

small product is production of isomaltose

one step at a time. Towards first step for

which was possible by insertion of a bac-

success South African scientists had

terial gene into the cane plant for produc-

knocked down a certain enzyme by genet-

tion of an enzyme responsible for conver-

ic means which enhanced the sucrose

sion of sucrose into iso-maltose, an alter-

content in young stalks of sugarcane

native sweetener (Wu and Birch, 2007).

(Groenewald and Botha, 2008). Another

Biotechnological efforts gave positive

perspective is making cellulosic bio-fuel

results in stream of characterization of

production easier. It is well known that

genome structure, specific traits mapping,

the sucrose is an essential component for

marker assisted selection in resistance

production of bio-ethanol, an alternative

towards insect/disease, variability of

for fossil fuels, through the process of

pathogens on molecular basis, transfor-

fermentation. Breeders are focusing now

mation, pathogen detection in precise

on enhancing the sucrose yield for in-

manner in plants and many more. The

creasing production of ethanol without

sharpness in sensitivity of the assays

compromising the sucrose content as food

made them more rapid for routine analy-

commodity. By the modern use of bio-

sis of plant pathogen detection and identi-

technology tools the cellulose content in

fication. Besides, the assay has become

cane leaves as well as bagasse are being

even more economical. The better ability

used for the production of ethanol thereby

to detect the infection at early or latent

not utilizing the main cane product, sugar.

stages help in improving the management

The intricate structure of cellulose can be

of disease as well as restricting the

broken down into simpler molecules of

movement of the various diseases and

carbohydrates by number of enzymes

even assist in solving the phylogenetic

which later can be used for production of

relationship amongst the various patho-

ethanol by fermentation nevertheless cel-

gens. This also assists in developing new-

lulosic structure is guarded by lignin. This

er strategies for enhancing the resistance

hard guarding material requires a costly

activity of the host by genetic transfor-

procedure for its removal. Brazilian scien-

mation methods. There is a need to im-

tists are taking initiatives through genetic

prove the technologies for development

engineering in modifying the cellulosic

of transgenic before making it a part of

structure so that it could be separated

the varietal developmental programmes of

from bagasse without any difficulty

sugarcane. There is still a challenge for

(http://agencia.fapesp.br/en/16756,

ac-

the researchers in developing a pathogen-

cessed on 03.04.2017). Adding to it, Aus-

ic free transgenic as this requires com-

tralian researchers developed transgenic

plete understanding of interaction of plant

canes by inserting genes capable of pro-

and pathogen, however, gene cloning, to

ducing the cellulose degrading enzymes

some extent, is making a new revolution

in leaves of mature plants (Harrison _et al_.,

in this aspect. There is still a need to iden-

2011). Besides production of small prod-

tify and characterize the genes responsi-

ucts through sugarcane bio-factory which ****

ble for the antifungal activity in case of

involves the tweaking of genetic mecha-

disease resistance. In grassy shoot disease

nism occurring in cells of sugarcane plant

of sugarcane and Sugarcane yellow leaf

that instructs them for the production of

syndrome, a common occurring disease

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 391

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ development of PCR diagnostic kits are

short time, however, a major problem in

needed for Phytoplasma.

this technique is its cost of production

For improving the crop quality as

(Usman, 2015) .

well as productivity and even providing

One of the most talked topics in

resistivity to pathogen, tissue culture is

biotechnology achievements is the devel-

playing an effective role. The use of cryo-

opment of transgenic varieties and the

therapy has enhanced the likelihood of

area under the transgenic obtained plants

attaining healthy plants. In case of attain-

has increased to approximately greater

ing virus resistant plants, micropropaga-

than 81 million hectares but there are cer-

tion is the best tool which requires the use

tain limitations too in developing it. Singh

of shoot apical meristem (acting as ex-

_et al_. (2013) showed that the drawbacks

plants). In minimizing the somaclonal

in developing cultivars are not even re-

variation in times to come, application of

moved by the process of transformation;

clonal propagation as well as research in

however, other methods for precise inte-

transgenic are needed. There is a need to

gration and control trans-gene expression

work on the structure of genomes so that

are still to be performed in sugarcane

identification of markers associated with

crop. Furthermore, in studies related to

important agronomic characters may be

sugarcane association, there is still need

performed. Transgene constructs that help

for developing high throughput markers

in lessening the hazardous effect of envi-

as well as producing more markers and

ronment and even bio-safety jeopardize of

even ensure the proper availability of the-

these plants are need of the upcoming

se markers. The newly developed markers

times. The way researchers are moving

will enhance the knowledge and mystery

towards the biotechnological techniques

of complex structure of sugarcane ge-

many mysteries will unravel (Tiwari _et_

nome. DNA-based molecular markers of

_al_., 2010).

progenitor plants have the potential to

show the prevailing genetic polymor-

**4. Biotechnological challenges in en-**

phism that may be helpful in case of this

**hancing the cane production and**

crop as parental genome is much less

**productivity**

complex in comparison to the hybrids

ones (Henry _et al_., 2012).

The use of biotechnology in sug-

****

arcane crop has drawn researchers as well

**5. Constrains in improving cane pro-**

as entrepreneurs towards itself but its ap-

**duction using biotechnological tools**

plication over this crop on commercial

basis had always be a regulatory chal-

Biotechnology

is

generating

lenge particularly in case of field cultiva-

enormous information which had played

tion. There is higher probability of trans-

an important role in transforming the

ferring of genes along with unwanted

world of science. Biotechnological inter-

genes from the source plant to other

ference in sugarcane crop provides a

plants used as a food commodity. There-

chance for sugar producers and cane

fore, the practicability of these techniques

farmers to enhance the production and

on commercial cane bio-factory will be

sustainability. These approaches act as a

dependent on how much amount of risk

proactive approach in alleviation of the

containment as compared to the non-food

troubles occurring in cane production

product plants, for example tobacco ****

(Usman, 2015). Though researchers are

(http://isaaa.org/resources/publications/po

attaining much success in improving the

cketk/45/default.asp, 27.3.2017). A com-

cane production and yield yet there are

monly used technique now-a-days is tis-

certain constrain in this regard using bio-

sue culture technique that helps in devel-

technological tools. Some of these con-

oping uniform disease free plantlets in

strains are: High-throughput sugarcane

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 392

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ transformation that involves efficiency of

**References**

the low transformation, inactivation of

****

trans-gene, soma-clonal variation, and the

**Aitken, K. S., McNeil, M. D., Hermann**

long time required for regeneration and its

**S., Bundock, P. C., Kilian, A., Hel-**

commercial release. In restricting the ac-

**ler Uszynska, K., Henry, R. J. and**

cess in exploiting gene technology, trans-

**Li, J. (2014).** A comprehensive ge-

formation and tissue culture-induced so-

netic map of sugarcane that provides

ma-clonal variation are the two things that

enhanced map coverage and inte-

remain important for sugarcane improve-

grates high-throughput diversity ar-

ment (Arencibia _et al_., 1997). The sub-

ray technology (DArT) markers.

stantial alteration in the current transfor-

_BMC Genomics_ **15, 152.**

mation systems are required to make sure

**Almeida, C. M. A., Donato, V.M.T.S.,**

of the clonal fidelity of transgenic culti-

**Amaral, D. O. J., Lima, G. S. A.,**

vars. Also as stated before the time lag of

**Brito, G. G., Lima, M. M. A., Cor-**

developing and releasing a cane variety is

**reia, M. T. S. and Silva, M. V.**

also a crucial challenge as well as con-

**(2013).** Differential gene expression

strain for cane breeders. At times the cane

in sugarcane induced by salicyclic

grown and developed by classical method

acid under water deficit conditions.

yields similar/or enhancing gains as com-

_Agricultural Science Research J._

pared to the ones developed through bio-

**3(1), 38-44.**

technological approaches. There are two

**Anonymous (2013).** Indonesia approves

major factors, _viz._ , polyploidy and aneu-

first GM sugarcane. _Crop Biotech_

polyploidy that cause molecular charac-

_Update_ (May 22, issue), International

terization of cane genome more difficult.

Service for the Acquisition of Agri-

Aitken _et al._ (2014) had reported that the

Biotech

Applications

recent available genetic maps and mark-

(http://www.isaaa.org/kc/cropbiotech

ers obtained from sugarcane provide par-

update/

arti-

tial information in aspect of genome or-

cle/default.asp?ID=10989).

ganization. The reason behind is the low

**Arencibia, A., Vazquez, R. I., Prieto,**

density of markers and its coverage. This

**D., Tellez, P., Carmona, E. R.,**

causes difficulty in allocating the markers

**Coego, A., Hernandez, L., Dela Ri-**

into linkage groups (Souza _et al_., 2011).

**va, G. A. and Selman, H. G. (1997).**

Transgenic sugarcane plants resistant

**6. Concluding remarks**

to stem borer attack. _Molecular_

_Breeding_ **3, 247–255**.

This chapter concludes that vari-

**Arencibia,**

**A.D.,**

**Carmona,**

**E.R.,**

ous successful achievement have been

**Cornide, M.T., Castiglione, S.,**

attained by plant breeders by using the

**O'Relly, J., Chinea, A., Oramas, P.**

biotechnological techniques in this crop

**and Sala, F. (1999)** Somaclonal var-

but the way, application of biotechnology

iation in insect-resistant transgenic

is spreading its hands in agriculture espe-

sugarcane (Saccharum hybrid) plants

cially in sugarcane crop, the left over

produced by cell electroporation.

mysteries could be unravelled much easi-

_Transgenic Res_. **8, 349–360**.

er in the times to come. Progress in tradi-

**Augustine, S. M., Narayan, J. A., Sya-**

tional breeding of sugarcane, a highly

**maladevi,**

**D.P.,**

**Appunu,**

**C.,**

polyploid and frequently aneuploid plant,

**Chakravarthi, M., Ravichandran,**

is impeded by its narrow gene pool, com-

**V., Tuteja, N. and Subramonian N.**

plex genome, poor fertility, and the long

**(2015).** Introduction of pea DNA hel-

breeding/selection cycle. These con-

icase 45 into sugarcane ( _Saccharum_

straints, however, make sugarcane a good

spp. hybrid) enhances cell membrane

candidate for molecular breeding.

thermostability and upregulation of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 393

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ stress-responsive genes leads to abi-Enríquez-Obregón,

**G.A.,**

**Vázquez-**

otic stress tolerance. _Molecular Bio-_

**padrón,**

**R.I.,**

**Prieto-sansonov,**

_technology_ **57, 475-488.**

**D.L., de la Riva, G.A. and Selman-**

**Baker J., Preston C. (2003). ** Predicting

**Housein,G. (1998).** Herbicide re-

the Spread of Herbicide Resistance

sistant sugarcane (Saccharum offici-

in Australian Canola Fields. _Trans-_

narum L.) plants by _Agrobacterium_ -

 _genic Research_ **12, 731-737**.

mediated

transformation.

_Planta,_

**Borrás-Hidalgo, O., Thomma, P.H.,**

**206, 20-27**

**Bart, J., Carmona, E., Borroto, C.**

**Epstein, E. and Rains, D. W. (1987).**

**J., Pujol, M., Arencibia, A. and**

Advances in salt tolerance. _Plant & _

**Lopez J. (2005).** Identification of

_Soil_ **99, 17- 29**.

sugarcane genes induced in disease-

**Gallo-Meagher, M. and Irvine J.E.**

resistant somaclones upon inocula-

**(1996).** Herbicide resistant transgenic

tion with _Ustilago scitaminea_ or _Bi-_

sugarcane plants containing the bar

_polaris sacchari. Plant Physiology_

gene. **_Crop Science_** **, 36 (5), 1367-**

_and Biochemistry_ **43, 1115-1121.**

**1374.**

**Bower, R. and Birch, R. G. (1992)**

**Gilbert, R. A., Gallo-Meagher, M.,**

Transgenic sugarcane plants via mi-

**Comstock, J.C., Miller, J.D., Jain,**

croprojectile bombardment. _Plant J._

**M. and Abouzid, A.** **(2005).** Agro-

**2, 409–416.**

nomic evaluation of sugarcane lines

**Braga, D. P. V., Arrigoni, E. D. B., Sil-**

transformed for resistance to Sugar-

**va-Filho M. C., Ulian, E. C. (2003).**

cane mosaic virus strain E. Crop Sci.

Expression of the Cry1Ab protein in

**45 , 2060–2067.**

genetically modified sugarcane for

**Groenewald, J. H. and Botha, F. C.**

the control of _Diatraea saccharalis_

**(2008).** Down-regulation of pyro-

(Lepidoptera: Crambidae). J New

phosphate: fructose 6-phosphate 1-

Seeds, **5, 209–222**. ****

phosphotransferase (PFP) activity in

**Chagas, R. M., Silveira, J. A. G., Ribei-**

sugarcane enhances sucrose accumu-

**ro, R. V., Vitorello, V. A. and Car-**

lation

in

immature

inter-

**rer H. (2008).** Photochemical dam-

nodes. _Transgenic Research_ **17, 85-**

age and comparative performance of

**92.**

superoxide dismutase and ascorbate

**Guimarães, E. R., Mutton, M. A., Mut-**

peroxidase in sugarcane leaves ex-

**ton, M. J. R., Ferro, M. I. T., Rav-**

posed to Paraquat-induced oxidative

**aneli, G. C. and Silva, J. A. (2008).**

stress. _Pesticide Biochemistry and_

Free proline accumulation in sugar-

_Physiology ****_**90, 181-188.**

cane under water restriction and spit-

**Chakrabarty, R., Viswakarma, N.,**

tlebug infestation. _Sci. Agric_. **65,**

**Bhat, S. R., Kirti, P. B., Singh, B.**

**628-633.**

**D.**

**and**

**Chopra,**

**V.**

**L.**

**Gupta V., Raghuvanshi, S., Gupta, A.,**

**(2002). ** Agrobacterium-mediated

**Saini N., Gaur, A., Khan, M. S.,**

transformation of cauliflower: opti-

**Gupta, R. S., Singh, J., Duttama-**

mization of protocol and develop-

**jumder, S. K., Srivastava, S., Su-**

ment of Bt-transgenic cauliflower. __

**man, A., Khurana, J. P., Kapur, R.**

 _Journal of Biosciences_ **27, 495-502**.

**and Tyagi, A. K. (2010).** The water-

**Du, Y. C., Nose, A. and Wasano, K.**

deficit stress- and red-rot-related

**(1999).** Thermal characteristics of C4

genes in sugarcane. _Functional Inte-_

photosynthetic enzymes from leaves

_grative Genomics_ **10 (2), 207-214.**

of three sugarcane species differing

**Hanlon,**

**D.,**

**MacMahon,**

**G.**

**G.,**

in cold sensitivity. _Plant Cell Physi-_

**McGuire, P., Beattie, R. N. and**

_ology_ **40,298-304.**

**Stringer, J. K. (2000).** Managing

low sugar prices on farms – short

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 394

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ term and long term strategies. In: D.

**Jaisil, P. P. and Thammasirirak S.**

M. Hogarth (ed.): Proceeding of

**(2010).** A proteomics analysis of

Australian Society of Sugarcane

drought stress-responsive proteins as

Technology, **22, pp. 1–8.**

biomarker for drought tolerant sugar-

**Harrison, M. D., Jason, Geijskes,**

cane cultivars. _American Journal of_

**Coleman, Heather, D., Shand, Ky-**

_Biochemistry & Biotechnology _**6 (2),**

**lie, Kinkema, Mark, Palupe, An-**

**89-102.**

**thony, Hassall, Rachael, Sainz,**

**Jenes, B., Moore, H., Cao, J., Zhang,**

**Manuel, Lloyd, Robyn, Miles, Sta-**

**W. and Wu, R. (1993)**. Techniques

**cy and Dale, L. James (2011).** Ac-

for gene transfer in Transgenic

cumulation of recombinant cellobio-

Plants. Kung S, Wu R (eds), San Di-

hydrolase and endoglucanase in the

ego: Academic Press Inc **1, 125-146.**

leaves of mature transgenic sugar

**Joyce P., McQualter R., Handley J.,**

cane. _Plant Biotechnology Journal_ **9,**

**Dale J., Harding R., Smith G.**

**884-896**

**(1998).** Transgenic sugarcane re-

**Henry, R. J., Edwards, M., Waters, D.**

sistant to sugarcane mosaic virus.

**L., Gopala Krishnan, S., Bundock,**

_Proceedings-Australian Society of_

**P., Sexton, T. R., Masouleh A. K,**

_Sugar Cane Technologists_ (Salis-

**Nock, J. C. and Pattemore, J.**

bury: Watson Ferguson and Compa-

**(2012).** Application of large-scale

ny), **204–210.**

sequencing to marker discovery in

**Kinkema,**

**M.** **, Geijskes, **

**J.** **, Delucca, **

plants. _Journal of Bioscience_ **37,**

**P.** **, Palupe, **

**A.** **, Shand, **

**829–841.**

**K.** **, Coleman, **

**H.**

**D.** **, Brinin, **

http://shodhganga.inflibnet.ac.in/bitstrea

**A.** **, Williams, B. ****, Sainz, M. ****, Dale, J. **

m/10603/42274/7/07_chapter%202.p

**L.** **(2014).** Improved molecular tools

df, 3.05.2017

for sugar cane biotechnology.  _Plant_

http://agencia.fapesp.br/en/16756,

 _Molecular Biology,_ **84(4-5), 497-**

03.04.2017

**508.**

http://isaaa.org/resources/publications/poc

**Kumar, T., Uzma, Khan, M. U., Abbas,**

ketk/45/default.asp, 27.03.2017

**Z. and Ali, G. M. (2014).** Genetic

http://isaaa.org/resources/publications/poc

improvement

of

sugarcane

for

ketk/45/default.asp, 27.3. 2017

drought and salinity stress tolerance

**Ingelbrecht I.L., Irvine J.E. and**

using _Arabidopsis_ Vascular pyro-

**Mirkov T.E. (1999).** Post trancrip-

phosphatase (AVPi) gene. _Molecular_

tional gene silencing in transgenic

_Biotechnol_ ogy **56(3), 199-209**.

sugarcane. Dissection of homology-

**Lima,**

**M.L.A.,**

**Garcia,**

**A.A.**

**F.,**

dependent virus resistance in a mon-

**Oliveira, K.M., Matsuoka, S. Ari-**

ocot that has a complez polyploid

**zono, H. et al., (2002).** Analysis of

genome. _Plant Physiol_ **. 119, 1187-**

genetic similarity detected by AFLP

**1198.**

and coefficient of parentage among

**Iskandar, H. M., Casu, R.E., Fletcher,**

genotypes of sugar cane (saccharum

**A. T., Schimdt, S. Xu, J. Maclean.**

spp.) _Theoretical and Applied Genet-_

**D.J., Manners, J. M. and Bonnett**

_ics,_ **104** , **30-38.**

**G.D.**

**(2011).**

Identification

of

**Marshall, A. (2014).** Drought tolerant

drought response genes and a study

varieties begin global march. _Nature_

of their expression during sucrose

_Biotechnology,_ **32, 308.**

accumulation and water deficient in

**McQualter,**

**R.**

**B.**

**and**

**Dookun-**

sugarcane culms **.** _BMC Plant Biol_ **.,**

**Saumtally, A. (2007).** Expression

**11 , 12**

profiling of abiotic-stress-inducible

**Jangpromma, N.,**

**Kitthaisong, S.,**

genes in sugarcane. _Proceeding of_

**Lomthaisong, K., Daduang, S.,**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 395

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ _Australian Society of Sugar Cane_

_ficinarum_ L.) callus cultures. _Plant_

_Technol_ ogy **29, pp. 878-888.**

_Growth Regulation_ **55,169-173.**

**McQualter, R. B., Chong, B. F., Meyer,**

**Petrasovits, L., Purnell A., Matthew,**

**K., Van, Dyk D. E., Oshea, M. G.,**

**P., Nielsen, K. Lars and Brumbley,**

**Walton, N. J., Vitanen, P. V. and**

**M. Stevens (2007).** Production of

**Brumbley, S. M. (2005).** Initial

polyhydroxybutyrate

in

sugar-

evaluation of sugarcane as a produc-

cane. _Plant Biotech Journal_ **5, 162-**

tion platform for a _p_ -hydroxybenzoic

**172.**

acid. _Plant Biotech Journal_ **2, 1-13**

**Prabu, G. R., Kawar, P. G., Pagariya,**

**Molinari, H. B. C., Marur, C. J., Daros,**

**M. C. and Prasad, D. Theertha**

**E., Campos, M. K. F., Carvalho, J.**

**(2011).** Identification of water deficit

**F. R. P., Bespalhok, J. C. Filho,**

stress upregulated genes in sugar-

**Pereira, L. F. P. and Vieira, L. G.**

cane. _Plant Molecular Biology Re-_

**E. (2007).** Evaluation of the stress-

_porter_ **29, 291-304**.

inducible production of proline in

**Que, Y., Su, Y., Guo, J., Wu, Q. and**

transgenic sugarcane ( _Saccharum_

**Xu, L. (2014).** A global view of tran-

spp.): Osmotic adjustment, chloro-

scriptome dynamics during _Sporiso-_

phyll fluorescence and oxidative

_rium scitamineum_ challenge in sug-

stress. _Physiology Plant_ **130, 218-**

arcane by RNAseq. _PloS One_ **9(8),**

**229.**

**e106476.**

**Nair, N.V. (2011).** Sugarcane varietal

doi:10.1371/journal.pone.0106476.

development programmes in India:

**Roach, B. T. (1989).** Origin and im-

An overview. _Sugar Tech_ **13(4), 275-**

provement of the genetic base of

**280.**

sugarcane. _Proceedings of the Aus-_

**Nel, S. (2009).** The potential of biotech-

_tralian Society of Sugar Cane Tech-_

nology in the sugarcane industry: are

_nology_ , 34–47.

you ready for the next evolution?

**Shaik, M.M., Hossain, M.A., Khaton,**

_Proceeding of Sugar African Sugar_

**M.M.**

**and**

**Nasiruddin,**

**K.M.**

_Technology Association_ **82, 225–**

**(2007).** Efficient Transformation of

**234.** __

Stress tolerance Glygene in Trans-

**Nogueira, F. T. S., Vicente, V. E. D.,**

genic Tissue of Sugarcane (Sac-

**Menossi, Jr. M., Ulian, E. C. and**

charum officinarum L.) _Mol. biol._

**Arruda, P. (2003).** RNA expression

_biotechnol. J._ , **5(1 &2): 37-40. **

profiles and data mining of sugar-

**Shanthi, R. M. (2016).** Pre-breeding for

cane response to low temperature.

developing high sucrose genetic

_Plant Physiology_ **132, 1811-1824.**

stocks in sugarcane. Winter School

**Park, J. W., Benatti, T. R., Marconi, T.,**

on Biotechnological and convention-

**Q.Yu, N. Solis-Gracia, V. Mora**

al approaches for biotic and abiotic

**and Silva, J. A. da (2015).** Cold re-

stress management in sugarcane, pp

sponsive gene expression profiling of

**Sathyabhama, M., Viswanathan, R.,**

sugarcane and _Saccharum spontane-_

**Malathi, P. and Sundar, A. R.**

_um_ with functional analysis of a cold

**(2016).** Identification of differential-

inducible _Saccharum_ homolog of

ly expressed genes in sugarcane dur-

NOD26- like intrinsic protein to salt

ing pathogenesis of _Colletotrichum_

and water stress. _PloS One_ **10(5),**

_falcatum_ by suppression subtractive

**e0125810**.

doi:10.1371/journal.

hybridization (SSH). _Sugar Tech_ **18,**

pone.0125810.

**176.**

**Patade, V. Y., Suprasanna, P. and**

**Schuler. T. H., Poppy, G. M., Kerry, B.**

**Bapat, V. A. (2008).** Effects of salt

**R. and Denholm, I. (1998). 01171-2)** Insect 01171-2)

stress in relation to osmotic adjust-

resistant transgenic plants. _Trends_01171-2)

ment on sugarcane ( _Saccharum of-_

 _Biotechnology_ **16,168-175**. 01171-2)

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 396

_Biotech Sustainability (2017)_

_Biotech Approaches for Sustainable Sugarcane Productivity Mall and Misra_ **Shrivastava, A. K. and Srivastava, S.**

**Trujillo, L. E., Menéndez, C., Och-**

**(2012).** Sugarcane: Physiological and

**ogavía, M. E., Hernández, I.,**

molecular approaches for improving

**Borrás, B., Rodríguez, R., Coll, Y.,**

abiotic stress tolerance and sustain-

**Arrieta, J.G., Banguela, A., Ramí-**

ing crop productivity. In Improving

**rez, R. and Hernández, L. (2009).**

Crop Resistance to Abiotic Stress

Engineering drought and salt toler-

(Tuteja N., S S. Gill, A.F. Tiburcio

ance in plants using SodERF3, a

and R. Tuteja, eds.) Vol. 2, Wiley-

novel sugarcane ethylene responsive

Blackwell, Germany, **pp. 885-922.**

factor. _Biotechnology Applicada_ **26**

**Shrivastava, A. K., Srivastava, T. K.,**

**(2),168-171.**

**Sr** i **vastava, K. Arun, Misra, Var-**

**Usman Shehu Inuwa (2015).** Biotech-

**ucha,**

**Shrivastava,**

**Sangeeta,**

nology interventions for production

**Singh, V. K. and Shukla, S. P.**

of good quality seed canes. _Interna-_

**(2016).** Climate change induced abi-

_tional Journal of Scientific Research_

otic stresses affecting sugarcane and

_and Innovative Technology_ **2(9), 96-**

their mitigation. ICAR-Indian Insti-

**104.**

tute of Sugarcane Research, Luck-

**Wahid, A and Close, T.J. (2005).** Ex-

now, **Pp.** **108**

pression of dehydrins under heat

**Singh, R. K., Kumar, P., Tiwari, N. N.,**

stress and their relationship with wa-

**Rastogi, J., Singh, S. P. (2013).**

ter relations of sugarcane leaves. **** _Bi-_

Current Status of Sugarcane Trans-

_ol. Plantarum_. **51, 104-109.**

genic: an Overview. _Advance Genet-_

**Waltz, E. (2014).** Beating the heat. _Na-_

_ic_

_Engeneering_

**2,112**.

doi:

_ture Biotechnology_ **32(7), 610-613**.

10.4172/2169-0111.1000112.

**Wang, M. L., Goldstein, C., Su W.,**

**Souza, G. M., Berges, H., Bocs, S.,**

**Moore, H. Paul, and Henrik, H.**

**Casu, R., D'Hont, A., Ferreira, J.**

**Albert (2005).** Production of biolog-

**E., Henry, R., Ming, R., Potier B.,**

ically active GM-CSF in sugarcane:

**Van Sluys M. S., Vincetz M. and**

a secure biofactory. _Transgenic Re-_

**Paterson A. H. (2011).** The sugar-

_search_ **14, 167-178.**

cane genome challenge: strategies for

**Wu, L. and Birch, R. G. (2007)** Doubled

sequencing a highly complex ge-

sugar content in sugarcane plant

nome. _Tropical Plant Biology_ **4,**

modified to produce a sucrose iso-

**145–156.**

doi:10.1007/s12042-011-

mer. _Plant Biotech Journal_ **5,109–**

9079-0.

**117.**

**Tiwari, A. K., Tripathi, Shivanand,**

**Zhang, L. Xu, J and Birch, R. G.**

**Lal, Madan and Rao, G. P. (2010).**

**(1999).** Engineered detoxication con-

Biotechnological approaches for im-

fers resistance against a path **o** genic

proving sugarcane crop with special

bacterium. _Nature Biotechnology,_ **17,**

reference to disease resistance. _Acta_

**1021-1024.**

_Phytopathologica et Entomologica_

_Hungarica_ **45(2), 235-349.**

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 397

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P398-416_

**Bioremediation: A Biotechnology Tool for Sustainability**

****

**Niharika Chandra, Ankita Srivastava, Swati Srivastava, Shailesh Kumar Mishra and**

**Sunil Kumar***

_Faculty of Biotechnology, Institute of Biosciences and Technology, Shri Ramswaroop_

_Memorial University, Barabanki, Uttar Pradesh, India;*Correspondence: ****_

_sunil.bio@srmu.ac.in / sunilsbt@gmail.com_

****

**Abstract:** The term bioremediation refers to the use of natural biological agents such as

microbes (bacteria, fungi, and yeast), plants or the enzymes released by them to return the

polluted natural environment to its original uncontaminated state. Bioremediation is a

novel, safe, cost effective, and ecologically feasible technology to detoxify accumulated

pesticides, toxic chemicals, fertilizers, aromatic compounds, xenobiotics, hydrocarbons (oil

spills), heavy metals in soil and water. Both _in situ_ and _ex situ_ bioremediation are being

used at a large number of sites all around the world with varying level of success. Although

bioremediation cannot degrade all the toxicity, particularly all the inorganic contaminants,

but it is still more eco-friendly and less expensive as compared to other remediation

methods like incineration, chemical treatment and thermal recovery of pollutants.

Furthermore, advances in bioremediation are being achieved by coupling this biological

method with molecular, genetic engineering, microbiology, pathway engineering, and

enzyme design and immobilization tools. In this chapter we have discuss the general

process for bioremediation, _in situ_ and _ex situ_ classes of bioremediation followed by the

types of bioremediation techniques being used till date. The role of bioremediation to deal

with different types of pollutions and comprehend the advantages, disadvantages and

sustainable use of its approaches is also highlighted.

****

_**Keywords**_ : _Ex situ_ bioremediation; GMOs; _in situ_ bioremediation; __ microbes; pollutants ****

****

****

**1. Introduction**

biodiversity and functional aspects such

as cycling of nutrients (Su _et al_., 2014).

A rapid increase in human

Similarly, increased pollution in aquatic

population

accompanied

with

ecosystem is resulting in decreased purity

technological advancements in fields

and content of ground water as well as

related to agriculture, industries and

surface fresh water (Zhang _et al_., 2011).

health has led to accumulation of various

Hence, we are in urgent need to seek a

toxic

chemicals

and

xenobiotic

feasible and efficient system to manage

compounds

in

our

environment.

such pollutants.

Indiscriminate use of fertilizers and

Bioremediation is the use of living

pesticides in agriculture, poor handling of

organisms, primarily microorganisms, to

wastewater and solid waste, untreated

degrade the environmental contaminants

release of polluted discharge from

into less toxic forms. The process

industries has led to shortage of clean

involves the degradation or detoxification

water sources and disturbances in soil

of hazardous substances, which are

content and quality (Kamaludeen _et al_.

harmful to human health and/or the

2003). Contamination of soil with heavy

environment, with the help of naturally

metals,

xenobiotic

compounds

and

occurring bacteria, fungi and plants.

municipal waste is responsible for loss of

Microorganism

which

perform

the

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 398

_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

function of bioremediation are known as

bacteria. Also oil spills can be treated by

Bioremediators (Kumar _et al_., 2011).

bacteria (Agarwal and Liu, 2015).

Bioremediation is a natural, effective and

Recently it has been noticed that

environment

friendly

alternative

to

the awareness of the dangers of many

previously used methods for degradation

chemicals used in our society has led to

of harmful contaminants, such as physical

research on formulation of products that

removal, absorbents, catalytic destruction,

are more easily degraded in the

incineration etc. which are high cost and

environment.

The

process

of

nonspecific methods (Head and Swannell,

bioremediation involves the degradation

1999; Gillespie and Philp, 2013)

of contaminants using microorganisms

Although

use

of

microbial

that have adverse impact on environment

consortia have proved their capability for

and humans. Bioremediation includes the

remediation, application of biotechnology

actions of several microorganisms that are

and genetic engineering tools is further

acting in parallel or sequence, in order to

improving the efficiency and decreasing

complete the process of degradation. In

the cost involved in treating toxic

this process, both _in situ_ as well as _ex situ_

substances . Bacteria, fungi, yeast and

remediation

are

used.

Hence,

algae along with several plants are being

bioremediation is a technology applied in

used for this purpose.

case of different environmental conditions

We will discuss the general

where the numerous and versatile

process for bioremediation, _in situ_ and _ex_

microbes degrades a vast array of

_situ_ classes of bioremediation followed by

pollutants (Majone _et al_., 2015).

the types of bioremediation techniques

On the basis of ecological point of

being used till date. The role of

view, the term bioremediation involves

microorganisms in bioremediation as well

the interactions between three factors that

as their genetic modification by the use of

is substrate (pollutant), organisms, and

recombinant DNA technology will be

environment, as shown in Figure 1. The

understood. Further we will discuss the

interactions between these three factors

role of bioremediation to deal with

primarily affect the biodegradability and

different

types

of

pollutions

and

bioavailability

of

pollutants,

and

comprehend

the

advantages,

physiological requirements of microbes,

disadvantages and sustainable use of

which plays a vital role in the assessment

bioremediation approaches.

of the feasibility of bioremediation.

Biodegradability defines whether any

**2. Bioremediation**

chemical can be degraded by microbes or

not, whereas bioavailability refers to the

Bioremediation is the branch of

availability of a pollutant to organisms

biotechnology

that

deals

with

the

that are capable of degrading it. For

solutions of problems related to the

instance,

the

substrate

has

low

environment.

Bioremediation

also

bioavailability if it is tightly bound to soil

involves the process of cleaning the

organic matter or it is trapped inside its

environment from different types of

aggregates. The

conditions

that

are

pollutants as well as contaminants by

required by microorganisms to carry out

using bacteria, fungi etc. Bacteria play a

the process of bioremediation include

vital role in the process of bioremediation

different factors like nutrient availability,

since it break down the dead materials

optimal pH, and availability of electron

into organic matter and nutrients. Several

acceptors, such as oxygen and nitrate etc.

types of contaminants such as chlorinated

are

referred

to

as

physiological

pesticides etc. can be easily digested by

requirements. __

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****

**Figure 1:** Bioremediation from an environment perspective.

**Table 1:** Summary of bioremediation approaches

**S.N. Approaches**

**About**

**Advantages**

**Disadvantages**

**Examples**

1.

_In situ_

At the

Non-invasive

Monitoring

Bioventing

site

Most cost efficient

difficulties

Bioaugmentation

Relatively passive

Extended

Biosparging

Treats soil and

treatment time

water

Environmental

Can be done on

constraints

site

2.

_Ex situ_

Away

Low cost

Space

Landfarming

from the

Time efficient

requirements

Biopiles

site

Need to control

Composting

abiotic loss

Bioavailability

limitation

Mass transfer

problem

3.

Bioreactors

Rapid degradation

High cost capital

Slurry reactors

kinetic

High operating

Aqueous

Enhances mass

cost

reactors

transfer

****

biosparging,

bioslurping

and

**3. Classes of bioremediation**

phytoremediation along with physical,

****

chemical, and thermal processes. This

_3.1. In situ bioremediation (ISB)_

class of bioremediation technology is

_In situ_ bioremediation (ISB) is the class of

beneficial because of its low cost, more

bioremediation that is performed at the

effective method as an alternative to the

original site of contamination. There is no

standard pump and treats methods that are

excavation or removal of polluted

used to clean up aquifers and soils

soil/ground water to any secondary

contaminated with organic chemicals

location for conduction of remediation

including fuel hydrocarbons, chlorinated

process. _In situ_ remediation includes

solvents. ISB has the potential to

techniques

such

as

bioventing,

provide advantages such

as

complete

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destruction of the contaminants, lower

metals

by

the

application

of

risk to site workers, and lower equipment

biotechnology (Li and Li, 2017). Such

or operating costs (Koning _et al_., 2000;

types of transgenic plants are being

Vidali. 2001).

developed which can remove more and

more contaminants from the environment.

_3.2. Ex-situ bioremediation (EXB)_

Sometimes it is also referred to as "green

_Ex-situ_ bioremediation (EXB) is

clean" which means _cleaning up of_

defined as a biological process which

_environment with the help of plants_. Some

involves excavation of polluted soil or

plants, most notably, the Chinese brake

pumping of groundwater that is placed in

fern ( _Pteris vittata_ ) has been reported to

a lined above-ground treatment area to

be suitable for arsenic phytoremediation

facilitate

microbial

removal

of

(Alkorta _et al_., 2004). An American

contaminants.

_Ex_

_situ_

remediation

patent registered in 1994 describes how

includes techniques such as Landfarming,

genetically altered members of the family

biopiling, and processing by bioreactors

Brassicaceae (family of scavengers) like

along with thermal, chemical, and

_Brassica juncea_ have shown tremendous

physical processes (Koning _et al_., 2000).

potential for clean-up of polluting metals

_Ex situ_ remediation is a more thorough

through their roots. The plants accumulate

remediation technique, but due to the

metals to levels between 30 and 1000

costs associated not only with the

times higher than their concentration in

remediation processes, but also with the

the surrounding soil. The metals absorbed

excavation and transportation of the soil,

by various members of the _Brassica_ plant

many people are looking towards _in situ_

family include antimony, arsenic, barium,

remediation techniques (Vidali, 2001) as

beryllium, cadmium, cesium, chromium,

depicted in Table 1.

cobalt, copper, gold, both stable and

unstable form of lead, manganese,

**4. Types of bioremediation**

mercury, molybdenum, nickel, palladium,

plutonium, selenium, silver, strontium,

For the better understanding of

uranium, vanadium, zinc etc.

bioremediation and for convenience of the

Dust is a major air pollutant and

study it may be divided into following

around 40% of total air pollution in India

types according to biological agents used

is contributed by dust pollution. Based on

for the treatment of toxicants:

extensive field surveys and experimental

studies, the following species have been

_4.1. Phytoremediation_

recommended by NBRI, Lucknow for

In this method plants are used to

raising green belts around industrial and

remove pollutants from the environment.

urban areas to reduce the dust load:

Phytoremediation

targets

include

_Ipomoea_

_fisstulosa,_

_Peltophorum_

contaminating

metals,

metalloids,

_pterocarpum, Tectona grandis, Ficus_

petroleum hydrocarbons, pesticides etc. In

_bengalensis, F. infectoria, Terminalia_

comparison to conventional purification

_arjuna._

technologies, phytoremediation is a cost

Following are examples of some plants

effective one. This technology is the main

tolerant to gaseous pollutants:

driving force of the researches done in

Plants

tolerant

to

SO2: _Polyalthia_

this area resulting into commercialization

_longifolia,_

_Terminalia_

_arjuna,_

_Acer_

of

the

technology.

Commercial

_platanoides, Thuja orientalis._

phytoremediation systems for clean-up of

Plants tolerant to Ozone (Bowler and

shallow

aquifers

and

water

borne

Fluhr, 2000): _Zinnia elegans, Gladiolus_

contaminants are now in the market.

_spp., Pelargonium graveolens._

Today, we know about the plants which

Plants tolerant to NOx: _Carrisa carandas,_

purify air by absorbing toxic gases and

_Codiaeum variegatum._

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Plants tolerant to PAN: _Acer platanoides,_

his research in this field in the early

_Chrysanthemum spp._

1950s.

__

In another breakthrough, Dr.

_4.2. Rhizofiltration_

Ananda Mohan Chakrabarty of the

In this form of phytoremediation

General

Electric

Company

(USA)

roots of plants act as _biofilters_ i.e. these

developed a genetically engineered oil

absorb

toxic

metals

from

the

eating bacterium ( _Pseudomonas putida_ ).

contaminated water and accumulate them.

The patent was registered to him in

Therefore, the water which passes

1980s. It was welcomed by the scientists"

through root zone becomes free from

community as an answer to pollution

pollutants (Macek _et al_., 2004). Later, the

problem. The Environment Protection

contaminants are removed from the

Agency

(EPA)

reported

that

system by harvesting root biomass.

bioremediation eliminated both soil and

Members of family _Brassicaceae_ (the

water borne oil contamination at about

family of scavengers) have shown

1/5th cost of previous method. Since then,

tremendous potential for the cleanup of

bioremediation has been increasingly

polluting metals. The plants roots

used to clean up oil pollution in United

accumulate metals from the surrounding

States and in other countries. In 1989, oil

soil giving a metal content as high as 30%

spilled from the Exxon Valdez tanker off

of the dry weight of the plant roots.

the coast of Alaska where these oil eaters

Alan Baker, a geobotanist from

helped in clean-up of oil. They degraded

University

of

Sheffield,

England,

the oil efficiently trapped between rocks

discovered a tree _Sebertia acuminate_ (the

and under gravel beaches where all other

nickel lover) which hyper-accumulates Ni

means had failed.

so much that when slashed, it bleeds a

****

jade green liquid. The tree is a native of

_4.4. Zooremediation_

New Caledonia and accumulates Ni as

It

is

the

process

of

high as 20% of its dry body weight.

decontamination of polluted environment

Besides,

_Acedium_

plant

absorbs

by using animals as bioagents. Animals so

Vanadium and some plants of family

far used for bioremediation purposes are

_papilionaceae_ (e.g. Pea) are known to

fish, different arthropods and other filter

absorb higher amounts of Molybdenum

feeders

in

aquatic

systems

and

from soil.

earthworms in solid organic waste

management systems. Use of animals in

_4.3. Microbial bioremediation_

bioremediation is not very encouraging

Microorganisms like bacteria are

except earthworms because there are so

proving very important tools for the

many limitations with them e.g. fish and

removal

of

pollutants

from

the

arthropods may bioaccumulate the toxic

environment and are thus continuously

compounds and metals which may be

doing

their

job

of

detoxification,

biomagnified in the food chain creating

tirelessly, with or without coming into the

many problems to the environment

attention of man, who is the only culprit

(Gifford _et al_., 2007).

of dumping more and more pollutants into

Solid organic waste generation is a

the environment. In the recent past,

major problem of cities which are facing

microbes were first used to treat industrial

the threat of being overrun by garbage

wastewater as early as 1930s. There was a

and the piled up waste and is adversely

significant movement in the field of

threatening our health, environment and

microbial

bioremediation

when

Dr.

wellbeing.

In

this

context,

Howard Worne (USA) first discovered

vermicomposting - waste degradation

phenol degrading microbes, when began

through earthworms has proved to be very

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**Figure 2:** _Ex-situ_ bioremediation by composting.

promising. The principles behind this are

grinding gizzard, an organ that all

relatively simple and related to those

earthworms possess. The earthworms

involved

in

traditional

composting

derive their nourishment from the

(Figure 2 summarizes composting as a

microorganisms that grow upon the

basic _Ex-situ_ bioremediation process). In

organic materials. At the same time they

general, vermicomposting consists of 4

promote further microbial activity in the

major phases: Phase I: Collection of the

residues so that the faecal matter or casts

waste,

separation

of

metal,

glass,

that they produce are much more

ceramics etc. from the organic waste, and

fragmented. During this process, the

storage of the organic waste.

important plant nutrients in the organic

Phase II: Earthworm beds are

material

particularly

nitrogen,

maintained and the earthworms are fed

phosphorus, potassium and calcium are

with the organic waste.

released and converted into forms that are

Phase III: After the organic waste

much more soluble and available to plants

has been worked over by the earthworms,

than those in the parent compounds.

the vermicompost, cocoons, earthworms

Worms can digest waste several times

and the undigested material are separated.

their own weight each day.

Phase IV: Packaging of the

vermicompost and reintroduction of

_4.5. Bioventing_

undigested material into the vermipits.

Bioventing is

a process

of

stimulating

the

natural

in

situ

Certain species of earthworms

biodegradation of contaminants in soil by

( _Eisenia fetida_ , _E. Andrei, Lumbricus_

providing air or oxygen to existing soil

_rubellus, L. hortensis, L. terristris_ etc.)

microorganisms. Bioventing uses low air

can consume organic residues very

flow rates to provide only enough oxygen

rapidly and fragment them into much

to sustain microbial activity in the vadose

finer particles by passing them through a

zone (Hinchee, 1994). This is an on-site

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or _in-situ_ bioremediation option for

their ores. It is one of several applications

reducing or eliminating contaminants in

within biohydrometallurgy and several

soil and water. With benefits that include

methods are used to recover Cu, Zn, Pb,

minimal site disturbance and lower cost

As, Ni, Mo,

Au, Ag, and Co.

compared

to

other

remediation

Heterotrophic bacteria are widely used in

technologies,

in-situ

bioremediation

the study of bacterial leaching of

continues to be researched and applied

manganese from manganese dioxide ores

with the goal of helping 'close out'

and glucose or other organic compounds

specific sites, that is, reducing toxins to

are used as a source of energy, rendering

safe and/or legally acceptable levels

their commercial utilization uneconomic

(Agency, 1995, Gibbs _et al_., 1999).

(Cornu _et al_. 2017). Coal, especially

Among the most promising of

brown coal from certain coal mines may

these technologies is soil bioventing, the

contain a certain amount of rare metals,

process

of

supplying

oxygen

to

such as Germanium (Ge) and Gallium

contaminated soil in hopes of stimulating

(Ga). The conventional process to recover

microbial degradation of contaminants. A

Ge from brown coal is a lengthy process

typical bioventing setup is appealingly

involving many steps, i.e. burning of the

simple: a blower or compressor connected

brown coal, recovering of Ge from ashes

to one or more air-supply wells and a

by sulphuric acid leaching, precipitating

series of soil-gas monitoring wells

of Ge with tannin, roasting of Ge-

(Sellers, 1999). The technology of choice

containing

tannin

to

produce

Ge

for remediating many petroleum wastes,

concentrate with the grade of 11%. This

bioventing may eventually be used to

process is complex and has a low

treat a wider variety of more recalcitrant

recovery of 60%, which is sure to bring

toxins (McCauley, 1999a). Bioventing

about a great waste of resource. Instead, a

has noteworthy remediation relatives,

novel process to recover Ge from brown

with distinct principles, goals and

coal in the presence of microorganism has

applications.

Air

sparging

forces

been developed where a germanium

compress air into saturated soil in hope of

recovery of up to 85.33% has been

promoting

biodegradation.

Unlike

achieved.

sparging, bioventing uses low-pressure air

and is generally focused on the vadose or

_4.7. Landfarming_

unsaturated zone of soil (McCauley,

Landfarming is a process in which

1999b). Bioslurping combines bioventing

the soil is excavated and mechanically

and

direct

vacuum

extraction

of

separated via sieving. In land farming,

contaminants. Soil vapor extraction or

which is performed in the upper soil zone

soil vacuum extraction (SVE) maximizes

or in biotreatment cells, contaminated

volatilization of contaminants and sucks

soils are mixed with soil amendments

them out of the soil. Bioventing began to

such as soil bulking agents and nutrients

mature as a technology after 1988, when

and then they are tilled into the earth.

researchers on a SVE operation at Hill Air

Contaminated soils, sediments, or sludges

Force Base, Utah, concluded that a

are incorporated into the soil surface and

significant proportion of contaminant

periodically turned over or tilled to aerate

decrease was not due to volatilization, but

the mixture. The material is periodically

biodegradation (Agency, 1995;  Litchfield,

tilled for aeration. Contaminants are

1993).

degraded, transformed, and immobilized

by microbiological processes and by

_4.6. Bioleaching_

oxidation. Soil conditions are controlled

This principle of bioleaching

to optimize the rate of contaminant

involves the use of living organisms like

degradation (Datta _et al_., 2016). Moisture

microbes in the metal extraction from

content, frequency of aeration, and pH are

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all conditions that may be controlled.

metals, and stones. The stones will then

Land Farming differs from composting

be crushed into smaller pieces and then

because

it

actually

incorporates

depending on the degree of contamination

contaminated soil into soil that is

will either be added to a pile or sent out

uncontaminated. Landfarming is most

for reuse. The soil is then homogenized,

successful

in

removing

polycyclic

meaning that the pollution concentration

aromatic

hydrocarbons

(PAH)

and

is averaged out across the entire soil

pentchlorphenol (PCP).

sample. Homogenization allows for

****

biopiling to be more effective (Schulz–

_4.8. Bioreactor_

Berendt, 2000). Once the soil is piled,

Bioreactors treat contaminated

nutrients, microbes, oxygen, and substrate

soils in both solid and liquid (slurry)

are added to start the biological

phases. The solid phase treatment process

degradation of the contaminants. The

mechanically decomposes the soil by

results of the initial laboratory tests

attrition and mixing in a closed container.

indicate to the operators which substrates

The objective of the mixing is to

such as bark, lime, or composts needs to

guarantee that the pollutants, water, air,

be added to the soil. Nutrients such as

nutrients, and microorganisms are in

mineral fertilizers may also be added.

permanent contact. An acid or alkalinity

Additionally, microorganisms such as

may also be added to control the pH (van

fungi, bacteria, or enzymes could be

Deuren and Lloyd, 2002). Infixed bed

added (Schulz-Berendt, 2000).

reactors,

compost

are

added

and

****

significantly increase the degradation rate.

_4.10. Bio-stimulation_

In rotating drum reactors, the drum has a

Bio

stimulation

could

be

screw like mechanism in the middle of it

perceived as including the introduction of

that rotates to mix and transport the soil.

adequate amounts of water, nutrients, and

The liquid phase treatment process uses

oxygen into the soil, in order to enhance

suspension bioreactors and treats as

the activity of indigenous microbial

slurry. The slurry feed enters the system

degraders. The concept of bio stimulation

and is rinsed through a vibrating screen to

is to boost the intrinsic degradation

remove debris. Sand is then removed

potential of a polluted matrix through the

using a sieve or hydrocyclone. If a

accumulation of amendments, nutrients,

hydrocyclone is used to remove the sand,

or other limiting factors and has been

the sand falls to the bottom of the cyclone

used for a wide variety of xenobiotics

and the fines remain on top. The fines are

(Kadian _et al_., 2008). Microorganisms do

then treated in a bioreactor. After the

extremely well in thriving on herbicide

treatment, the slurry must be dewatered

compounds in the soil by utilizing them as

and the water is then treated with standard

a supply of nutrients and energy. Many

wastewater techniques (Kleijntjens and

herbicides serve as good carbon and/or

Luyben, 2000).

nitrogen sources for soil microorganism

****

(Qiu _et al_., 2009). Evidence for their

_4.9. Biopiling_

remarkable range of degradative abilities

It is an _in situ_ process that is also

can be seen in the recycling rather than

known as the heap technique. The first

accumulation of vast quantities of

step in the biopiling process is to perform

biological materials that have been

laboratory tests that will determine the

produced throughout the history of life on

biological degradation capabilities of the

earth (Dua _et al_., 2002).

soil sample. The next step involves the

mechanical separation of the soil, which

_4.11. Bio-augmentation_

will homogenize the sample and remove

Bio

augmentation

is

the

any disruptive material such as plastics,

enhancement of biodegradation of waste

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and contaminants in the media by the

following key site characteristics are

__
__

## introduction

### of

adapted

competent

required to evaluate the likely success of

microbes and nutrients. Microorganisms

intrinsic remediation; the bioavailability

from _Geobacteraceae_ family due to their

of contaminants, levels of nutrients, the

physiological characteristics can play an

presence of minerals to buffer the pH of

important role in the bioremediation of

the matrix, adequate levels of electron

subsurface environments contaminated

acceptors (either oxygen, nitrate, ferric

with organic or metal contaminants

iron, or sulphate), and site specific

(Lovley _et al_., 2004). In some instances,

contamination

migration rates. This

the rate of biological degradation can be

approach deals with stimulation of

increased

through

the

addition

of

indigenous

or

naturally

occurring

microorganisms that have been shown to

microbial populations by feeding them

degrade the contaminants of concern at

nutrients and oxygen to increase their

high rates or are particularly well suited to

metabolic activity.

remain active under prevailing site

conditions. This process is referred to as

**5. Microbes involved in bioremediation**

bio augmentation. This can be useful if

the

contaminants

are

particularly

Microorganisms are responsible

recalcitrant to degradation or if site

for biodegradation in various diverse

conditions are extreme (for example: high

environmental

conditions.

These

concentrations

or

toxicity

of

microorganisms include: _Acinethobacter,_

contaminants). To be effective, the

_Actinobacter, Acaligenes, Arthrobacter,_

introduced organism(s) must become

_Bacillins, Berijerinckia, Flavobacterium,_

distributed throughout the contaminated

_Methylosinus,_

_Mycrobacterium,_

matrix and compete with the indigenous

_Mycococcus, Nitrosomonas, Nocardia,_

microorganisms for available nutrients. If

_Penicillium,_

_Phanerochaete,_

they are not distributed throughout the

_Pseudomonas,_

_Rhizoctomia,_

_Serratio,_

matrix the positive effect will be

_Trametes and Xanthofacter_. Individual

localized. On the other hand if the

microorganisms are not efficient in

introduced organisms compete poorly,

mineralization of harmful substances.

they will not persist and the treatment

Thorough mineralization results in a

effect will be short lived. The problems

progressive degradation by a group of

encountered using this approach include

microorganisms

(or

microbial

biofouling of equipment, injection wells

consortiums) and involves coaction and

and seepage beds. Adjustments to the

co-metabolism actions. Microorganisms

system, such as the use of new discharge

in various habitats have remarkable

areas, may be required to prevent this

physiological flexibility, so they are able

from

occurring.

This

approach

to

to make use of and often mineralize an

bioremediation must be evaluated on a

enormous number of organic molecules.

site specific basis.

Several other requirements for microbial

****

growth in biodegradation process are

_4.12. Intrinsic bioremediation_

listed in Table 2. Some microbes with

Often bioremediation can be

specific biodegradation capabilities are

accomplished without human intervention

discussed below.

by microorganisms that are naturally

_Pseudomonas putida_ : In context

found in the contaminated matrix. For

of bioremediation, it is a microorganism

this approach to be used, it is usually

found in farmland soil involving high

necessary for the rate of contaminant

impact

xenobiotics

including

degradation to exceed the rate of

organophosphate insecticides, petroleum

contaminant migration. Knowledge of the

hydrocarbons, and both monocyclic and

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**Table 2:** Requirements for microbial growth in bioremediation process (Source: Vidali,

2001)

**Requirement**

**Description**

Nutrients

The growth and activity of the microorganisms must be estimated by adequate

maintenance and supply of nutrients. These nutrients are the basic building

blocks of life and allow microbes to create the necessary enzymes to break down

the contaminants. Bio-stimulation usually involves the addition of nutrients and

oxygen to help native microorganisms. Nitrogen (ammonic, nitrate, or organic

nitrogen) and phosphorous (orthophosphate or organic phosphorous) are

commonly used as the limiting nutrients. In certain anaerobic systems, the use of

trace metals (e.g. iron, nickel, cobalt, molybdenum and zinc) is generally

preferred.

Carbon source

Carbon which is considered as the most basic element of living forms is required

in larger quantities than other elements. Carbon contained in many organic

contaminants may function as a carbon source for cell growth. If the organism

involved is an autotroph CO2 or HCO3 in solution is required. In some cases,

contaminant levels are too low to supply suitable levels of carbon to cell. In these

cases the addition of carbon sources may be required.

Electron

All respiring bacteria require a terminal electron acceptor. In some cases, the

acceptor

organic contaminant may serve in this capacity. The most common electron

acceptor in aerobic bioremediation processes is dissolved oxygen. Under

anaerobic conditions, NO3-, SO 3-

4 , Fe3+, and CO2 may serve as terminal electron

acceptors. Certain co-metabolic changes are carried out by fermentative and other

anaerobic organisms, in which terminal electron acceptors are not necessary.

Energy source

In the case of primary metabolism, the organic contaminant supplies energy

required for growth. This is not the case when the contaminant is metabolized

via secondary metabolism or co-metabolism or as a terminal electron acceptor. If

the contaminant does not serve as a source of energy, the addition of a primary

substrate(s) is required.

Soil moisture

Microbial growth and activity is also affected by moisture content. The water-

holding capacity suggested for bioremediation process may range from 25% –

28%.

Temperature

Temperature regulates the rates of growth and metabolic activity. Surface soils

are particularly susceptible to wide variations in temperature. Generally,

mesophilic conditions are appropriate for most applications (with composting

being a notable exception).

pH

A pH is another important factor that affects bioremediation process. If the soil is

acidic, it is possible to raise pH by adding lime. A pH fluctuating between 6.5

and 7.5 is generally considered optimal. The pH of most ground water (8.0–8.5)

is not considered inhibitory.

Absence of

Many contaminated sites contain a mixture of chemicals, organic and inorganic,

toxic metals

which may be inhibitory or toxic to microorganisms. Heavy metals and phenolic

compounds are of particular concerns.

Adequate

For contaminants to be available for microbial uptake it must be present in

contact

aqueous phase. Thus contaminants that exist as non-aqueous phase liquids or are

between

sequestered within a solid phase may not be readily metabolized. For degradation

microorganisms it is necessary that bacteria and the contaminants be in contact. This is not easily

and substrates

achieved, as neither the microbes nor contaminants are uniformly spread in the

soil. It is possible to develop the mobilization of the contaminant utilizing some

surfactants such as sodium dodecyl sulphate (SDS).

Time

Time is an important factor in the start-up of bioremediation systems. Even the

above mentioned parameters are met, lag phases are often observed prior to the

onset of activity. In some cases, the intense bacterial population shifts that are

required for bioremediation will increase periods of slow activity.

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_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

polycyclic aromatics (Iyer and Damania,

organisms like bacteria, plant, virus or

2016).

animal and thus these are also referred to

_Dechloromonas aromatic_ : This

as transgenic organisms (Ozcan _et al_.,

bacterium is involved in degradation of

2012).

aromatic compounds like benzene in

nitrate reducing conditions as well as

_6.1. Role of GMOs in environmental_

physiological

and

molecular

_management_

characterization in anaerobic mixed

Genetically modified organisms

cultures (Ulrich and Edwards, 2003).

can be used to clean up the environment

_Deinococcus radiodurans_ – In

by bioremediation. Effects of some

field of development of bioremediation

genetically modified microorganisms are

strategies, this bacterium plays a role as a

unstable and vary according to species,

radiation resistant organism. It is used for

changes in population structure and loss

the treatment of mixed radioactive wastes

of some functions, to the formation of

containing ionic mercury (Brim _et al_.,

toxic metabolites. Presence of high and

2000). The radioactive waste sites can be

active microorganisms makes the process

treatedby this strategy of bioremediation.

of bioremediation more operative and

_Methylibium_

_petroleiphilum_

–

they must adapt with the changing

Also known as PM1 strain that is

environmental conditions. _Deinococcus_

involved in methyl tert butyl ether

_radiodurans_

that

exhibit

toluene

(MTBE)

bioremediation.

MTBE

is

dioxygenase to clear-out toxic elements

degraded by this strain using the

that are found in radioactive waste sites

contaminants as source of carbon and

was used by Lange (1998) as a

energy (Hanson _et al.,_ 1999).

recombinant. _Deinococcus radiodurans_ is

_Alcanivorax borkumensis_ is a rod-

known to have two properties, first it is

shaped bacterium having capability of

resistant to radiation and secondly it can

consuming hydrocarbons and produces

degrade chlorobenzene in radioactive

carbon dioxide. Hence it can be used

environments (Lange _et al_., 1998). Then

readily in oil damaged environment

again, it can only be produced in an

(Santisi _et al_., 2015).

environment at temperatures less than

 _Phanerochaete chrysosporium_ _–_ It

39°C and as radioactive sites generally

is the first fungi involved in degradation

have high temperatures, so a bacterium is

of organic pollutants (Kadri _et al_., 2017).

required that can function at higher

****

temperatures.

Another

well-known

**6. Genetically modified organisms**

example for the application of GMOs in

the management of environmental issues

Bioremediation by means of

can be cited through certain bacteria that

microorganisms is not significant for

can yield biodegradable plastics and this

treatment of all types of pollutants. For

quality of bacteria were transferred to

example, heavy metals such as cadmium

microbes which were cultured in the

and lead are not freely absorbed or taken

laboratory and now a days they have

by organisms. The role of genetically

enabled the wide scale greening of plastic

modified organisms in the process of

industry.

bioremediation has emerged as a new tool

In the early 1990s, Zeneca, a

(Jafari _et al_., 2013). A genetically

British

company,

established

a

modified organism, or GMO, is an

microbially manufactured biodegradable

organism that has an altered DNA

plastic

called

Biopol

configuration made through genetic

(polyhydroxyalkanoate, or PHA). The

engineering. Most of the genetically

plastic was made using a GM bacterium,

modified

organisms

have

been

_Ralstonia eutropha_ , to transform glucose

transformed with DNA from other

and a variety of organic acids into a

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_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

flexible polymer (Perez-Pantoja _et al_.,

environment. Bioremediation is one of the

2008). GMOs which are able to

emerging biological strategies which is

metabolize oil and heavy metals through

applicable to the repair of damaged

their bacterially encoded ability may

environment.

prove effective for the bioremediation

The three main types of pollution

process.

Simultaneously,

genetically

(Soil, water and marine pollution) that are

engineered microorganisms (GEMs) have

controlled by bioremediation using a

shown possible uses for bioremediation in

variety of microorganisms which belong

soil, groundwater, and activated sludge

to different environments and are active

environments, due to the enriched

members of microbial associations are

degradative capabilities for extensive

discussed here.

range of contaminants. Recent advances

in molecular biology have unlocked new

_7.1. Marine pollution_

perceptions for the development of

The derivatives of petroleum are

engineering microorganisms with the

the most important source of energy for

purpose

of

performing

specific

industry and societies. The probable cause

bioremediation.

of oil spills in marine environment is

From the biological safety view it

mainly through the frequent transport of

has also been reported that not all

petroleum across the world. Moreover, it

naturally occurring bacteria are ideal as

is

broadly

known

that

petroleum

bioremediation agents. For instance,

hydrocarbons pollution has obstructed,

_Burkholderia cepacia_ would be both used

and spoiled the world oceans, seas and

as an agent for bioremediation and for

coastal zones and due to this, the Earth"s

biological regulator of phytopathogens.

health sustainability is at high risk. In

However, it causes cystic fibrosis in

marine environment too, bioremediation

humans and it is also found to be resistant

is considered as an economic and

to many antibiotics (Holmes _et al_., 1998).

ecological biotechnology tool for the

For these reasons, the US Environmental

handling of polluted wastes (Paniagua-

Protection Agency (EPA) has led to its

Michel

and

Rosales,

2015).

The

elimination

to

be

used

as

an

frequently

applied

bioremediation

environmental agent (Davison, 2005).

methods that can be used in marine

environments facing disturbance due to

**7. Types of pollution controlled by**

oil spills are (i) using the process of bio

**bioremediation**

augmentation by the addition of oil

****

degrading bacteria so as to grow or

The population explosion throughout the

improve the existing bacterial biota, and

world has led to an increase in the

(ii) use of composts (nutrients), to

polluted soil and water regions. As the

encourage and stimulate the growth of

number of people continues increasing

native oil degraders, which is called bio-

day by day it also results in the overuse of

stimulation. In the case of oil spills, the

natural resource like air, water and land

processes make use of the catabolic skill

resources. For these reasons, there occurs

of microorganism feeding on oil. Several

rapid expansion of industries, food, health

workers (Odu, 1978; Sloan, 1987; Ijah

care, vehicles, etc. but it is very

and Antai, 1988; Okpokwasili and

challenging to retain the quality of life

Okorie, 1988; Barnhart and Meyers,

with all these new expansions, which are

1989; Anon, 1990; Pritchard, 1991;

critical to the environment in which we

Pritchard and Costa, 1991; Hoyle, 1992;

live. Since the quality of life is very much

Ijah, 2002; and Ijah, 2003) have

linked to the overall quality of the

pronounced numerous application of

environment, worldwide measures are

microorganism in the bioremediation of

taken to sustain and preserve the

oil pollution with promising results.

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_7.2. Water pollution_

dibenzodioxins

(PCDD),

and

Water pollution is a subject of

polychlorinated dibenzofurans (PCDF)

great global concern, and it can be largely

(Koning _et al_., 2000). The biological

distributed into three main groups, that is,

processes of ex situ remediation involve:

contamination by organic compounds,

composting, landfarming, biopiling and

inorganic

compounds

(e.g.,

heavy

the use of bioreactors. Alternatively,

metals), and microorganisms. It has

bioventing, biosparging, bioslurping and

caused an irreparable damage to aquatic

phytoremediation along with physical,

ecosystems

through

environmental

chemical, and thermal processes are

contamination by heavy metals from

included in _in situ_ remediation techniques

anthropogenic and industrial activities.

for treating soil pollution.

Other sources of heavy metals comprise

the mining and smelting of ores, run-off

**8. Advantages of bioremediation**

from storage batteries and automobile

exhaust, and the manufacturing and

For successful bioremediation to

inadequate use of fertilizers, pesticides,

occur, the bioremediation methods rest on

and many others. The bioremediation

having the right microbes in the right

approach works on the high metal binding

place with the right environmental factors

ability of biological agents, which can

for

the

process

of

degradation.

eliminate heavy metals from polluted sites

Bioremediation

is

considered

more

with

high

efficacy.

Specimens

of

advantageous

over

conventional

microorganisms

studied

and

techniques

like

land

filling

or

advantageously used in bioremediation

incineration.

Microbes

capable

of

treatments for heavy metals include the

destroying the contaminants increase in

following: (i) Bacteria: _Arthrobacter spp.,_

number when the pollutant is present and

_Pseudomonas veronii, Burkholderia spp.,_

when the pollutant is degraded, the

_Kocuria flava, Bacillus cereus, and_

biodegradative population declines. The

_Sporosarcina ginsengisoli_(Gautam _et al_.,

remains for the treatment are generally

2011;  Cycon _et al_., 2017); (ii) fungi:

harmless products and include carbon

_Penicillium_

_canescens,_

_Aspergillus_

dioxide,

water

and

cell

biomass.

_versicolor, and Aspergillus fumigatus_ ;(iii)

Theoretically, bioremediation is useful for

yeast: _Saccharomyces cerevisiae and_

the thorough damage of a wide variety of

_Candida utilis;_ (iv) algae: _Cladophora_

contaminants. Many compounds that are

_fascicularis,_

_Spirogyra_

_spp._

_and_

officially considered to be unsafe can be

_Cladophora spp., and Spirogyra spp. and_

transformed

to

nontoxic

products.

_Spirullina spp._

Bioremediation also reduces the chance of

__

future problem associated with treatment

_7.3. Soil pollution_

and disposal of polluted waste (Rajwade

Decontamination of soil can be

 _et al_., 2015). Bioremediation that is

processed through both _ex situ_ and _in situ_

performed on site is often less expensive

remediation techniques. _Ex situ_ thermal

and site interruption is nominal, it

remediation processes are best for use for

removes waste permanently, reduces

the following contaminants: petroleum

long-term problem, and has better public

hydrocarbons (TPH), polycyclic aromatic

acceptance,

with

regulatory

hydrocarbons (PAH), benzene, toluene,

encouragement, and it can be tied with

ethylbenzene, xylenes (BTEX), phenolic

other physical or chemical treatment

compounds, cyanides, and chlorinated

methods.

compounds

like

polychlorinated

biphenyls (PCB), pentchlorphenol (PCP),

**9. Disadvantages of bioremediation**

chlorinated hydrocarbons, chlorinated

pesticides,

polychlorinated

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There are limitations to every

absorption or metabolism of the polluting

process and so is bioremediation which is

compound. If the xenobiotic compound

limited to those compounds that are

acts as a source of energy, carbon or any

biodegradable. It has been found that

nutrients then it is absorbed by the

compounds such as heavy metals,

bioremediators. Otherwise, it is co-

radionuclides

and

some

chlorinated

metabolised by a single organism or a

compounds are not prone to rapid and

group of different organisms" together

complete

degradation

through

acting as bioremediators. To enhance

bioremediation and there are cases where

bioremediation by these strategies an

microbial breakdown of contaminants has

adequate understanding of microbial

resulted in toxic metabolites. Like most

behaviour

is

of

prime

importance

of

the

biological

processes,

(Alvarez _et al_., 2017). Furthermore,

bioremediation is also highly specific.

advanced engineering techniques have

The site factors that are significant and

been

developed

to

stimulate

the

required for the success include the

microorganism involved in detoxification

presence of metabolically proficient

process. For instance, sparging of gaseous

microbial

populations,

proper

phase by enhanced mechanisms has led to

environmental growth conditions, and

complete utilization of bioremediation

appropriate levels of nutrients and waste

potential of several aerobic microbes.

products

(Tran

 _et_

 _al_.,

2015).

Research is also being carried out to

Bioremediation is logically a thorough

promote the availability of pollutants to

procedure which can be designed for site-

the microbes. Advance techniques such as

specific conditions, i.e. before proceeding

use of surfactants, solubilisation of

to cleaning of the sites, one has to do

pollutants by exposure to steam, heat or

treatment studies on a minor scale

heated water, and application of high

(Ramrakhiani _et al_., 2016). The process of

pressure are being used for this purpose

bioremediation often involves the time

(Rittmann, 1993; Shukla _et al_., 2014).

factor as it takes much more time than

The

approach

of

developing

other treatment options, such as diggings

designer microbes (GMOs) with the help

and removal of soil or incineration. A

of recombinant DNA technology has

second drawback to this technique in

already being discussed in detail. Several

contrast to other remediation techniques is

other innovative technologies such as

its relative sensitivity to environmental

transcriptome and proteome analysis,

factors for example temperature, pH, and

molecular profiling, pyro sequencing,

the presence of various other substances

metatranscriptomics and metaproteomics,

or organisms. There are many questions

mass spectrometry, microarrays, and

that should be answered before using

numerous bioinformatics applications are

bioremediation: Whether the contaminant

helping in realization of complete

is

biodegradable?

Is

biodegradation

potential of microbes for bioremediation

occurring in the natural site? Are

(Kulshreshtha, 2012;  Rittmann, 1993).

environmental conditions appropriate for

In

another

very

innovative

biodegradation? Where the waste will be

approach the toxic industrial, domestic

disposed if it is not degraded completely?

and agricultural waste can be converted

These questions can be answered by

into useful forms and products such as

doing site classification and also by

bioethanol, biogas, biofuels, single cell

treatability studies.

proteins

etc.,

(Kulshreshtha,

2012).

Recently, immobilization of microbial

**10. A current update on bioremediation**

cells or the enzymes released by them

through several mechanisms such as

The

existing

bioremediation

adsorption, electrostatic binding, covalent

strategies

are

based

upon

either

binding,

aggregation,

crosslinking,

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_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

entrapment, and encapsulation has been

involves an ecosystem conservation

used extensively for bioremediation. Such

approach. However, if large amounts of

immobilization of microbes improves the

soil are physically removed from the site

bioremediation process by facilitating

in _ex-situ_ operations the remediation itself

reuse of microbes or the catalyst involved,

might be a threat to the ecosystem. In

and also reduce the costs of the process

view of economic sustainability, a

(Dzionek _et al_., 2016).

number of organic by-products are used

which include lignocellulosic wastes such

**11. Bioremediation and sustainability**

as sugarcane, bagasse and sawdust, crop

residues such as coffee pulp and molasses

Sustainable

remediation

and whey, a by-product of the dairy

technology has been defined as the

industry. These are also identified to

cautious use of natural resources by using

increase the degradation of diverse toxic

a

combination

of

remedies

which

compounds. A good bioremediation

maximizes the net benefit on human

methodology will include the planned use

health and environment. Environmental

of all native microbes in an engineered

modification through bioremediation is a

way to accomplish the best possible

dominant part of bio economy and

purification levels. In summary, we can

sustainable development. In recent years,

conclude that although bioremediation

there has been an increase in the use of

appears to be a promising alternative for

biodiversity

as

raw

material

for

the remediation of contaminants in

environmental decontamination. On the

different

ecosystems

and

is

also

other hand volume and diversity of

contributing to sustainability of the

contaminated substrates (water, soil and

environment but it is still in the

air) are increasing due to anthropogenic

developmental phase.

and technogenic sources. Microorganisms

have occupied some of the most life-

**12. Future perspectives**

threatening environments on the earth and

some of them are capable of degrading

The

application

of

the pollutants that are produced through

microorganisms to increase the fertility of

our industries. Ecological engineering has

soil conditions and removing the soil

been suggested as a theoretical framework

contaminations through bioremediation

to project "sustainable ecosystems that

technology is extensively used in Europe

incorporate human society with its natural

and USA. In Asia particularly in India as

environment for the profit of both".

major agriculture dependent country,

Energy use is one of the most important

progress has been made in applying

sustainability concerns for conventional

microorganisms to the restoration of

remediation projects. _Ex-situ_ remediation

polluted soil through bioremediation

is typically too energy intensive to be

processes. However, the application of

considered ecological engineering. In

bioremediation

technology

in

the

sustainable

bioremediation

external

restoration

of

ecosystem

and

soil

energy input is preferably used only in the

management is used less compared to

initiation phase to start a process that is

Europe and USA. Hence, extensive

later driven by solar energy and the

research programs are needed to increase

exemplified chemical energy of the

the capabilities of bioremediation to deep,

pollutant itself. The engineer"s role is to

extensive, subsurface contamination due

help provide the proper conditions in

to chlorinated hydrocarbons and complex

which such a process can take place.

mixed wastes, including soils and

Since the objective of bioremediation

groundwater. Besides that, The American

projects is to eliminate pollution that

Academy of Microbiology (AAM) has

employs stress on the ecosystem, it

concluded that enough knowledge is now

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_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

available for field trials of bioremediation

**and C. Garbisu (2004).** Recent

technology for organic compounds and

findings on the phytoremediation of

further they emphasized that research is

soils

contaminated

with

needed for the following classes of

environmentally toxic heavy metals

environmental

pollutants:

metals,

and metalloids such as Zinc,

metalloids, radionuclides and complex

Cadmium, Lead, and Arsenic. _Rev._

polycyclic hydrocarbons. The on-going

_Reviews in Environmental Science_

microbial genomics studies will deliver

_and Bio/Technology,_ **3, 71 - 90**.

more

robust

technologies

for

the

**Alvarez, A., J. M. Saez, J. S. Davila**

bioremediation of metal – contaminated

**Costa, V. L. Colin, M. S. Fuentes,**

waters and land. Exciting developments

**S. A. Cuozzo, C. S. Benimeli, M.**

in the use of microorganisms for the

**A. Polti & M. J. Amoroso (2017). **

recycling of metal waste, with the

Actinobacteria: Current research

formation of novel biominerals with

and perspectives for bioremediation

unique properties are also predicted in the

of pesticides and heavy metals.

near future. Moreover, a wide diversity of

_Chemosphere,_ **166, 41-62.**

microbes with detoxification abilities is

**Bowler, C. and R. Fluhr (2000).** The

waiting to be explored. The inadequate

role of calcium and activated

knowledge about microbes and their

oxygens as signals for controlling

natural role in the environment could

cross-tolerance. _Trends in Plant_

affect the acceptability of their uses. The

_Science,_ **5, 241-6.**

understanding

of

the

diversity

of

**Brim, H., S. C. McFarlan, J. K.**

microbial community's in petroleum

**Fredrickson, K. W. Minton, M.**

contaminated environment is essential to

**Zhai, L. P. Wackett and M. J.**

get a better insight into potential oil

**Daly**

**(2000).**

Engineering

degraders and to understand their genetics

Deinococcus radiodurans for metal

and biochemistry that will result in

remediation in radioactive mixed

developing appropriate bioremediation

waste

environments.

_Nature_

strategies, thus, preserving the long-term

_Biotechnology,_ **18, 85-90.**

sustainability of natural terrestrial and

**Cornu, J. Y., D. Huguenot, K. Jezequel,**

marine ecosystems.

**M. Lollier and T. Lebeau (2017).**

Bioremediation

of

copper-

__
__

## Acknowledgements

contaminated soils by bacteria.

_World Journal of Microbiology and_

Authors are thankful to Shri

_Biotechnology,_ **33, 1-9.**

Ramswaroop

Memorial

University,

**Cycon,**

**M.,**

**A.**

**Mrozik**

**and**

**Z.**

Barabanki, Uttar Pradesh, India for

**Piotrowska-Seget**

**(2017).**

providing facility and space for this work.

Bioaugmentation as a strategy for

the

remediation

of

pesticide-

**References**

polluted

soil:

A

review.

_Chemosphere,_ **172, 52-71.**

**Agarwal, A. and Liu, Y. (2015).**

**Datta, S., J. Singh and S. Singh (2016).**

Remediation technologies for oil-

Earthworms,

pesticides

and

contaminated sediments. _Marine_

sustainable agriculture: a review.

_Pollution Bulletin_ **101, 483-90.**

_Environmental_

_Science_

_and_

**Agency, U. S. E. P. (1995).** Bioventing

_Pollution Research (international),_

Principles and Practice. _Bioventing_

**23, 8227-43.**

_Principles, Washington D.C,_ **1, 15-**

**Davison, J. (2005).** Risk mitigation of

**20.**

genetically modified bacteria and

**Alkorta, I., J. Hernández-Allica, J. M.**

plants designed for bioremediation.

**Becerril, I. Amezaga, I. Albizu**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 413

_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

_Journal of Industrial Microbiology_

**Hinchee, R. E. (1994).** Air sparging for

_and Biotechnology,_ **32, 639-50.**

site

remediation.

_CRC_

_Lewis_

**Dua, M., A. Singh, N. Sethunathan and**

_Publishers_.

**A. K. Johri (2002).** Biotechnology

**Holmes, A., J. Govan and R. Goldstein**

and bioremediation: successes and

**(1998).**

Agricultural

use

of

limitations. _Applied Microbiology_

Burkholderia

(Pseudomonas)

_and Biotechnology,_ **59, 143-52.**

cepacia: a threat to human health?

**Dzionek, A., D. Wojcieszyńska and U.**

_Emerging Infectious Diseases,_ **4,**

**Guzik (2016).** Natural carriers in

**221-7.**

bioremediation:

A

review.

**Iyer, R. and A. Damania (2016).** Draft

_Electronic_

_Journal_

_of_

Genome Sequence of Pseudomonas

_Biotechnology_ , **19,** **28 - 36.**

putida CBF10-2, a Soil Isolate with

**Gautam, V., L. Singhal and P. Ray**

Bioremediation

Potential

in

**(2011).**

Burkholderia

cepacia

Agricultural

and

Industrial

complex: beyond pseudomonas and

Environmental Settings. _Genome_

acinetobacter. _Indian Journal of_

_Announcements_ **4** , **4, e00670-16**

_Medical Microbiology,_ **29, 4-12.**

**Jafari, M., Y. R. Danesh, E. M.**

**Gibbs, J. T., B. C. Alleman, R. D.**

**Goltapeh and A. Verma (2013).**

**Gillespie, E. A. Foote, S. E.**

Bioremediation

and

Genetically

**McCall, F. A. Snyder, J. E. Hicks,**

Modified Organisms. _Springer-_

**R. K. Crowe and J. Ginn (1999).**

_Verlag_

_Berlin_

_Heidelberg_. **29,**

Bioventing

Nonpetroleum

**33811-3**

Hydrocarbons.

IN

Engineered

**Kadian, N., A. Gupta, S. Satya, R. K.**

Approaches

for

In

Situ

**Mehta and A. Malik (2008).**

Bioremediation

of

Chlorinated

Biodegradation

of

herbicide

Solvent

Contamination.

_Battelle_

(atrazine) in contaminated soil using

_Press, Columbus_. OH, USA. **pp 7-**

various

bioprocessed

materials.

**14**

_Bioresource Technology,_ **99, 4642-**

**Gifford, S., R. H. Dunstan, W.**

**7.**

**O'Connor, C. E. Koller and G. R.**

**Kadri, T., T. Rouissi, S. Kaur Brar, M.**

**MacFarlane**

**(2007).**

Aquatic

**Cledon, S. Sarma and M. Verma**

zooremediation: deploying animals

**(2017).**

Biodegradation

of

to remediate contaminated aquatic

polycyclic aromatic hydrocarbons

environments.

_Trends_

_in_

(PAHs) by fungal enzymes: A

_Biotechnology,_ **25, 60-5.**

review. _Journal of Environmental_

**Gillespie, I. M. and J. C. Philp (2013).**

_Sciences (China),_ **51, 52-74.**

Bioremediation, an environmental

**Kamaludeen, S. P., K. R. Arunkumar,**

remediation technology for the

**S.**

**Avudainayagam**

**and**

**K.**

bioeconomy.

_Trends_

_in_

**Ramasamy (2003).** Bioremediation

_Biotechnology,_ **31, 329-32.**

of

chromium

contaminated

**Hanson, J. R., C. E. Ackerman and K.**

environments. _Indian journal of_

**M. Scow (1999).** Biodegradation of

_experimental biology,_ **41, 972-85.**

methyl tert-butyl ether by a bacterial

**Kleijntjens, R. H. and K. C. A. M.**

pure

culture.

_Applied_

_and_

**Luyben (2000).** Bioremediation.

_Environmental Microbiology,_ **65,**

_Biotechnology_ , **11b, 329-347.**

**4788-92.**

**Koning, M., K. Hupe and R. Stegmann**

**Head, I. M. and R. P. Swannell (1999).**

**(2000).**

Thermal

Processes,

Bioremediation

of

petroleum

Scrubbing/Extraction,

hydrocarbon contaminants in marine

_Bioremediation and Disposal_. **11b,**

habitats.

Current

Opinion

in

**306 - 317.**

Biotechnology _,_ **10, 234-9.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 414

_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

**Kulshreshtha, S. (2012).** Current Trends

bioremediation:

barriers

and

in

Bioremediation

and

perspectives

at

European

Biodegradation.

_Journal_

_of_

contaminated

sites.

_New_

_Bioremediation_

_and_

_Biotechnology,_ **32, 133-46.**

_Biodegradation,_ **3:e114.**

**McCauley,**

**P.**

**(1999a).**

Land

**Kumar, A., B. S. Bisht, V. D. Joshi and**

Remediation and Pollution Control

**T. Dhewa (2011).** Review on

Division

(Treatment

and

Bioremediation

of

Polluted

Destruction branch), . _Personal_

Environment: A Management

_communication. Chemist, U.S. EPA_

Tool. _International Journal of_

_National Risk Management and_

_Environmental Sciences,_ **1, 1079-**

_Research Laboratory_. Web Site

**1093**

<http://www.cluin.org/products/newsltrs/t

**Lange, C. C., L. P. Wackett, K. W.**

trend/tt0899.htm#bioventing

**Minton and M. J. Daly (1998).**

**McCauley, P.** **(1999b).** Bioventing for

Engineering

a

recombinant

Enhanced Degradation of PAHs.

Deinococcus

radiodurans

for

_Tech Direct. U.S. EPA,_ _Web Site_

organopollutant

degradation

in

_< http://www.cluin.org/products/newsltrs/t_

radioactive

mixed

waste

_trend/tt0899.htm#bioventing > _

environments.

_Nature_

**Ozcan, F., C. T. Kahramanogullari, N.**

_Biotechnology,_ **16, 929-33.**

**Kocak, M. Yildiz, I. Haspolat and**

**Li, J. and R. Li (2017).** Current research

**E. Tuna (2012).** Use of genetically

scenario

for

microcystins

modified

organisms

in

the

biodegradation - A review on

remediation of soil and water

fundamental knowledge, application

resources. _Fresenius Environmental_

prospects and challenges. _Science of_

_Bulletin,_ **21, 3443 - 3447.**

_The Total Environment,_ **595, 615-**

**Paniagua-Michel, J. and A. Rosales**

**632.**

**(2015).** Marine Bioremediation - A

**Litchfield, C. D. (1993).** In situ

Sustainable

Biotechnology

of

Bioremediation : Basis and Practice

Petroleum

Hydrocarbons

_IN Biotreatment of Industrial and_

Biodegradation in Coastal and

_Hazardous Wastes, M.A. Levin and_

Marine Environments. _Journal of_

_Michael A Gealt (eds.)McGraw-_

_Bioremediationand Biodegradation, ****_

_Hill, Inc._ **34, 167 -197** __

**6,273**

**Lovley, D. R., D. E. Holmes and K. P.**

**Perez-Pantoja, D., R. De la Iglesia, D.**

**Nevin (2004).** Dissimilatory Fe(III)

**H. Pieper and B. Gonzalez (2008).**

and Mn(IV) reduction. _Advances in_

Metabolic

reconstruction

of

_Microbial Physiology,_ **49, 219-86.**

aromatic compounds degradation

**Macek,**

**T.,**

**K.**

**Francova,**

**L.**

from the genome of the amazing

**Kochankova, P. Lovecka, E.**

pollutant-degrading

bacterium

**Ryslava, J. Rezek, M. Sura, J.**

Cupriavidus necator JMP134. _FEMS_

**Triska, K. Demnerova and M.**

_Microbiology Reviews,_ **32, 736-94.**

**Mackova**

**(2004).**

**Qiu, Y., H. Pang, Z. Zhou, P. Zhang, Y.**

Phytoremediation:

biological

**Feng and G. D. Sheng (2009).**

cleaning of a polluted environment.

Competitive

biodegradation

of

_Reviews on Environmental Health,_

dichlobenil and atrazine coexisting

**19, 63-82.**

in soil amended with a char and

**Majone, M., R. Verdini, F. Aulenta, S.**

citrate. _Environmental Pollution,_

**Rossetti,**

**V.**

**Tandoi,**

**N.**

**157, 2964-9.**

**Kalogerakis, S. Agathos, S. Puig,**

**Rajwade, J. M., K. M. Paknikar and J.**

**G. Zanaroli and F. Fava (2015).** In

**V. Kumbhar (2015).** Applications

situ groundwater and sediment

of bacterial cellulose and its

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 415

_Biotech Sustainability (2017)_

_Bioremediation: A Biotechnology Tool for Sustainability Chandra et al._

composites in biomedicine. _Applied_

**Su, C., L. Jiang and W. Zhang (2014).**

_Microbiology and Biotechnology,_

A

review

on

heavy

metal

**99, 2491-511.**

contamination in the soil worldwide:

**Ramrakhiani, L., S. Ghosh and S.**

Situation, impact and remediation

**Majumdar**

**(2016).**

Surface

techniques. _Environmental Skeptics_

Modification of Naturally Available

_and Critics,_ **3, 24 - 38.**

Biomass for Enhancement of Heavy

**Tran, N. H., H. H. Ngo, T. Urase and**

Metal

Removal

Efficiency,

**K. Y. Gin (2015).** A critical review

Upscaling

Prospects,

and

on characterization strategies of

Management Aspects of Spent

organic matter for wastewater and

Biosorbents: A Review. _Applied_

water

treatment

processes.

_Biochemistry and Biotechnology,_

_Bioresource Technology,_ **193, 523-**

**180, 41-78.**

**33.**

**Rittmann, B. E. (1993).** In Situ

**Ulrich, A. C. and E. A. Edwards (2003).**

Bioremediation:

When does

it

Physiological

and

molecular

work? _National Academy Press_

characterization

of

anaerobic

_Washington, D.C. 1st Ed._

benzene-degrading mixed cultures.

**Santisi, S., S. Cappello, M. Catalfamo,**

_Environmental Microbiology,_ **5, 92-**

**G. Mancini, M. Hassanshahian, L.**

**102.**

**Genovese, L. Giuliano and M. M.**

**Van Deuren, J. and T. Lloyd (2002).**

**Yakimov (2015).** Biodegradation of

Remediation technologies screeing

crude oil by individual bacterial

matrix and reference guide. _Federal_

strains and a mixed bacterial

_Remediation_

_technologies_

consortium. _Brazilian Journal of_

_Roundtable,_ **4th Ed.**

_Microbiology,_ **46, 377-87.**

**Vidali, M. (2001).** Bioremediation: An

**Schulz**

**-**

**Berendt,**

**V.**

**(2000).**

overview.

_Journal_

_of_

_Applied_

Bioremediation

with

heap

_Chemistry_ , **73,1163 - 1172.**

technique, Biotechnology. **320-328.**

**Zhang, W., F. Jiang and J. Ou (2011).**

**11b, 2nd Ed.**

Global pesticide consumption and

**Shukla, A. K., S. N. Upadhyay and S.**

pollution: with China as a focus.

**K. Dubey (2014).** Current trends in

_International Academy of Ecology_

trichloroethylene biodegradation: a

_and Environmental Sciences,_ **1, 125**

review.

_Critical_

_Reviews_

_in_

**\- 144.**

_Biotechnology,_ **34, 101-14.**

****

****

© 2017 by the authors. Licensee, Editors and AIMST University,

Malaysia. This article is an open access article distributed under the

terms and conditions of the Creative Commons Attribution (CC BY)

license (http://creativecommons.org/licenses/by/4.0/).

****

****

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 416

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P417-443_

**Sea Urchin - A New Potential Marine Bio-resource for**

**Human Health**

****

**M. Aminur Rahman1,** * **, Fatimah Md. Yusoff1, 2, Kasi Marimuthu3 and Yuji Arakaki4**

_1Laboratory of Marine Biotechnology, Institute of Bioscience, Universiti Putra Malaysia,_

_43400 UPM Serdang, Selangor, Malaysia; 2Department of Aquaculture, Faculty of Agri-_

_culture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; 3Department_

_of Biotechnology, Faculty of Applied Sciences, AIMST University, 08100 Bedong, Kedah_

_Darul Aman, Malaysia; **** 4Department of Tourism, Faculty of International Studies, Meio _

_University, Nago, Okinawa-905-8585, Japan;_

_*Correspondence: aminur1963@gmail.com; Tel: +60 3-8947-2141_

**Abstract:** Sea urchin gonads usually called as "Sea urchin Roe" or "Uni", are important

food delicacies in different parts of the world. In Asian, Mediterranean and Western Hemi-

sphere countries, the roe of sea urchins is considered as a highly prized delicacy sea food

because of its tastes and also have long been utilized as extravagance foods in Japan. Peo-

ples of the Asian Pacific region have long been utilizing it for improving general body tone

and also treatment for a number of diseases. It has been reported that, sea urchin gonads are

found to be rich with high-quantities of bioactive compounds, such as polyunsaturated fatty

acids (PUFAs) and β-carotenes. The PUFAs, particularly eicosapentaenoic acid (EPA,

C20:5) (n-3)) and docosahexanoic acid (DHA C22:6 (n-3)), have profound significant ef-

fects on arrhythmia, cardiovascular diseases and cancer. β-carotene and some xanthophyll"s

have strong pro-vitamin activity and can be used to prevent tumor development and light

sensibility. The sea urchin fisheries in recent years have extended so impressively that the

natural populations of them have been overexploited to meet-up the increasing demand. Not

surprisingly, the continued high demand and the decrease in supply have headed towards a

pronounced interest for the commercial aquaculture of sea urchins. Global sea urchin har-

vesting, having peaks at 120,000 metric tons in 1995, are presently in the scale of around

82,000 metric tons. However, these declining arrays evidently mirror the overfishing of ma-

jor fishery grounds and focus the necessity for conservation measures, aquaculture devel-

opment and sustainable fisheries management. Once the natural stocks decrease, the higher

market demand for foodstuff, nutraceuticals, pharmaceuticals and cosmeceuticals, increases

the value of the manufactured goods and therefore, culturing seems to become economical-

ly feasible. As per this assessment exhibits, there have been intense progresses in the aqua-

culture protocols of sea urchins during the past 15-20 years, we can come to the end that

presently the main impediments to successful farming are actually managerial, cultural,

conservational and economical rather than biological and ecological. Expected that demand

is implausible to decline, the commercial value of future product will be increased. Hence,

the fortune of sea urchin is strictly connected to those fisheries, whose prospect would

eventually depend on the stock improvement, aquaculture production, fishery management,

roe enhancement and market forces that will play a significant role to give a structure of

this expanding industry.

_**Keywords:**_ Biology; culture; ecology; health; management; roe; sea urchin

****

****

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 417

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

**1. Introduction**

_al_., 2016b). Not astonishingly, the contin-

uous strong demand and the decline in

Sea

urchins

(Echinodermata:

supply headed to a pronounced increase

Echinoidea) are important marine bio-

in awareness for aquaculture of sea ur-

resources for conducting research in di-

chins, predominantly, in those parts

verse areas of ecology, biology, biodiver-

wherein their natural populations have

sity, aquaculture, conservation, taxonomy

been dwindled (Lawrence _et al_., 1997;

and evolution. Simultaneously, they are

Lawrence, 2007). The species of sea ur-

utilized as raw material to produce food-

chins whose gonads have high commer-

stuff, particularly, the product of pro-

cial values could be obtained from a

cessing gonads recognized as "Sea urchin

number of genera such as: _Tripneustes_ , __

Roe" (Kaneniwa and Takagi, 1986;

_Strongylocentrotus, Paracentrotus, Lox-_

Oshima _et al_., 1986; Ichihiro, 1993). It is

_echinus,_

_Echinus_ ,

_Centrostephanus_ ,

also one of the highly prized seafood del-

_Hemicentrotus_ , _Lytechinus_ , _Diadema_ ,

icacy owing to the high tastes in Asian,

_Arbacia_ , _Colobocentrotus_ , _Anthocidaris,_

Mediterranean and Western Hemisphere

_Psammechinus, Evechinus, Heliocidaris,_

countries, for instance, Chile and Barba-

_Echinometra, Toxopneustes_ , _Pseudocen-_

dos (Kaneniwa and Takagi, 1986; Law-

_trotus_ and _Pseudoboletia_ (Sloan, 1985;

rence et al., 1997; Lawrence _et al_., 1997;

Saito, 1992; Keesing and Hall, 1998;

Yurˈeva _et al_., 2003; Rahman and Yusoff,

Lawrence, 2007; Rahman _et al_., 2014b).

2010; Rahman _et al_., 2013a, 2014a, b;

Nevertheless, the majority of the

Parvez _et al_., 2016a, b). In Japan, sea ur-

sea urchins fisheries have followed the

chin gonads (either in the state of fresh or

same trends of quick expansion to an un-

processed foods) have long since been

maintainable top, followed by a corre-

consumed as high-quality luxury foods

spondingly speedy decline. Global sea

(Shimabukoro, 1991; Rahman _et al_.,

urchin harvesting reached to 20,000 met-

2014a,b, Parvez _et al_., 2016a,b) and the

ric tons in 1995, are presently in the state

roe can sell for as much as AU$450/kg

of around 82,000 metric tons with an

(Richard, 2004). Due to the increasing

alarming decreasing rate of 32% (FAO,

demands for sea urchins, Japan imports

2010; Carboni _et al_., 2012; Rahman _et al_.,

big amounts from USA, South Korea,

2014b; Parvez _et al_., 2016b) (Figure 1).

Thailand and other producers, thus has

However, the newly extended sea urchin

elevated concerns about overfishing, and

fishery ( _Loxechinus albus_ ) from Chile

hence, making it one of the valuable sea

covers half amounts of the world catch

foods in the world (Hagen, 1996; Rahman

(Rahman _et al_., 2014b; Parvez _et al_.,

_et al_., 2014a; Parvez _et al_., 2016a,b). Tra-

2016b). The other main sea urchin fisher-

ditionally, sea urchin gonads have long

ies, landed in tonnage, are in Japan,

been used by the peoples of the Asian Pa-

Maine and California (United States), and

cific Region, as a remedy for improving

British Colombia (Canada) (Andrew _el al_.,

general body tones, treatment for a num-

2002). In case of Europe, the commercial

ber of diseases and increasing the sexual

sea urchin ( _Paracentrotus lividus_ ) in Ire-

potency of middle-aged men (Seifulla _et_

land and France were overfished to sup-

_al_., 1995; Yurˈeva _et al_., 2003). However,

ply the French markets (Barnes _et al_.,

in the recent years, the fisheries of sea

2002). There have been reported the large

urchins have been expanded so highly

populations and abundances of edible ur-

that the natural population in Chile, Japan,

chins in Norway ( _Strongylocentrotus_

France, Canada and different parts of

_droebachiencis_ ) and Scotland ( _Echinus_

USA have been overexploited to meet up

_esculentus_ and _Psammechinus milaris_ ),

the great demand (Lawrence _et al._ , 2001;

However, these stocks are not suitable for

Andrew _et al_. _,_ 2002, 2004; Rahman _et al_.,

profitable fishing because their roe

2005, 2012a,b, 2013b, 2014b; Parvez _et_

amounts are either very small or too flex-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 418

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

**Figure 1:** Global production of sea urchin fisheries from 1950 to 2008 (FAO, 2010).

__

ible (Hagen, 2000; Kelly, 2000, 2005;

inhabiting in temperate zones mostly have

Sivertsen, 2004; Rahman _et al_., 2014b).

moderate longevity between 10 and 20

Nonetheless, the continuous declining

years, even though highest longevity of

patterns noticeably reveal the overexploi-

100 years has been reported. Growth and

tation of most of the fishery grounds and

production performances are flexible and

focus the necessity for proper conserva-

greatly dependent on quality food and nu-

tion policies, sustainable management

trition. In conditions with lower density,

strategies and appropriate aquaculture

growth rates are usually high, while at

practices.

high densities, growth rates are low.

However, the density of sea urchins is not

**2. Biology and ecology of major spe-**

the single factor that starts the construc-

**cies**

tion of barrens or flats. Inter-annual

****

events that trigger warming (e.g., El

The most parts of the world involved

Nino), are known to cause kelp dieback

in sea urchin production are linked with

which then leads to overgrazing and the

main sites of primary productivity. In the

maintenance of barrens in the extended

subtropical and tropical regions, these are

period. The exclusion of predators that

usually associated with seagrass beds,

would then cause moderate urchin densi-

while in the temperate regions, with kelp

ties may also endorse conditions leading

forests. Urchins commonly occur at lower

to overgrazing. Usually, densities of sea

densities within the kelp communities,

urchins are the highest in barrens, but in-

overgrazing on kelps and then lead to the

dividual growth performance and overall

establishment of flats dominated by en-

productivity is low owing to competition

crusting macroalgae (called as coralline

for food and perhaps due to the deficient

flats and barrens). Both of the communi-

nourishment.

ties (barrens and kelp forests) are often

The most efficient predator of sea ur-

found near to each other, making either a

chins, particularly the sea otter and its re-

mosaic of stable patches or strata.

establishment around the North is having

The biological features of sea urchins

main effects on sea urchin fisheries. In

vary largely among species. The species

areas where populations have been re-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 419

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

established, predation can far exceed fish-

_2.2. Red sea urchin (Strongylocentrotus_

ing pressures and urchin densities have

_franciscanus)_

been reduced to levels below that which

_Strongylocentrotus franciscanus_ usu-

is required for fishing to be profitable

ally referred to as red sea urchin, is the

(Andrew _et al_., 2002). Recruitment mor-

biggest echinoid in the world and com-

tality is also affected by different types of

monly occurs along the West Coast of

habitats. It has been reported that mortali-

North America, extending from Baja Cal-

ty of juveniles among a number of species

ifornia to the Aleutian Archipelago and

is higher in kelp forests because of the

the coast of Siberia and northern. The

occurrences of micro-predators in their

ranges of their habitats encompass north-

habitats (Tegner, 2001).

ern-wards up the west coast to Sitka and

Given the similar habitat types, a

Kodiak AK at 58oN (Tegner, 2001). This

number of edible sea urchin species share

species is also occurs in the subtidal zone

some similarities in their distribution,

to a depth of 50 m seawards and is in-

abundance and reproduction. Yet, they

tensely associated with kelp forests.

are differed in their fundamental biologi-

Growth performance in the earlier stage

cal characteristics such as growth, surviv-

of urchin is comparatively fast and the

al, production, maturity and longevity.

species shows the highest longevity. TD

at recruitment is about 90 mm, which is

_2.1. Green sea urchin (Strongylocentrotus_

usually attains in around 6-8 years of the

_droebachiensis)_

age. The maximum size is around 200

Green sea urchin **** has a circumpolar

mm and the individuals over 150 mm TD

distribution **** and occurs through the North

are older than 100 years (Tegner, 2001).

Atlantic to the North Pacific. The fisher-

Spawning commonly occurs over the

ies of _S_. _droebachiencis_ have been fo-

spring and summer months when the ur-

cused in Maine and the Canadian Mari-

chins attain sexual maturity at around 50

times but smaller fisheries are concentrat-

mm TD and the spawning usually over

ed in Alaska, British Colombia, Washing-

summer and spring months. _****_

ton and Iceland. The species is mostly

common in the intertidal zone to a depth

_2.3. Japanese green sea urchin (Strongy-_

of 50 m, where it is closely related with

_locentrotus intermedius)_

kelp beds (Scheibling and Hatcher, 2001).

_Strongylocentrotus intermedius ****_ com-

Two distinct growth rates have been iden-

monly known as Japanese green sea ur-

tified, but the overall growth rates are

chin is the 2nd most economically im-

found to be moderate. The fast growing

portant regular echinoid in Japan. This

form attains the minimum legal size be-

species is distributed along the Asian and

tween 4 and 6 years and can live for 16-

Siberian coast of the Pacific. This species

20 years. On the other hand, the slow

is common in intertidal shallow waters

growing one inhabits for 8-12 years and

around Hokkaido and is exploited com-

never attains the optimum legal size (An-

mercially from Aomori, Irate and Hok-

drew _et al_., 2002). In British Colombia,

kaido (Agatsuma, 2001a). It generally

the minimum legal size is not more than

occurs in shallower stony substratum and

55 mm TD (test diameter) and in most

is usually associated with kelp forests

cases, the time needed to grow to this size

(Agatsuma, 2001a). The matured adult of

is supposed to range from 4 to 8 years

_S. intermedius_ contains small reddish-

(Taylor 2004). This sea urchin attains

yellow gonads, which do have a good

sexual maturity within 1-2 years and the

taste and thus listed on Tsukiji as Japa-

first spawning occurs between mid-

nese. It is well-adapted to cold water and

winters to early spring when it reaches to

the growth restriction does not appear to

45-50 mm in TD. _****_

be related to the temperature limits

_****_

(Agatsuma, 2001a). Density and nutrition

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 420

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

are the two major factors influencing

closely associated with kelp beds and its

growth of urchins. In appropriate culture

growth is extremely flexible and reliant

conditions, the urchin attains 40 mm TD

on the availability of algae. The maxi-

within 2-4 years and the maximum size of

mum size has been recorded to be 100

55 mm TD at ages between 6 and 10

mm TD. _S. purpuratus_ usually attains

years. It obtains sexual maturity at the age

sexual maturity around 2 years of age,

of 2 years with the size of 30-35 mm TD

and spawns during their natural breeding

and the spawning usually occurs in au-

season, extending from January to March. ****

tumn and spring. _****_

_2.6. Purple crowned sea urchin (Cen-_

_2.4. Strongylocentrotus nudus_

_trostephanus rodgersii)_

This species __ has been recorded on

The purple crowned urchin experienc-

Tsukiji as Japanese and also considered as

es a subtropical distribution throughout

the most commonly harvested edible sea

the water areas of Australia and New Zea-

urchin in Japan and accounts for ~ 44% of

land, but most abundantly occurs in East-

the total commercial catch (Agatsuma

ern Australia. This species has also been

2001b). It is found on inter- and sub-tidal

reported to be extended into Bass Strait

rocky bottoms extending from Dalian,

and the East Coast of Tasmania (Rdger,

China northwards to Primorskyi Kray,

1999) and is possibly related with the

Russia and in Japan where it is found in

warming of coastal waters around the re-

the Pacific from Sagami Bay to Cape Er-

gion (Andrew and Byrne, 2001). It is one

imo on Hokkaido and in the Sea of Japan

of the large urchins with long dark purple,

from Omi Island in Yamaguchi to Soya

black to red spines that have iridescent

Cape northern Hokkaido. The urchins

blue/green sheen. _Centrostephanus rodg-_

generally reach the legal size (40 mm) in

_ersii_ is usually seen in large numbers and

2 to 4 years when feeding on perennial

plays an important ecological role in in-

Laminarians whereas they take 7 to 8

tertidal near shore rocky reefs by cleaning

years on coralline flats. It occurs in the

the areas of kelp. Growth performances

intertidal to subtidal rocky reefs and is

follow the medium trends and the indi-

strongly associated with kelp communi-

viduals attain 70-90 mm TD within the

ties. Juveniles recruit to coralline flats and

age between 4 and 10 years. The longest

move to adjacent kelp forests. In kelp for-

size (120 mm TD) achieved when the ur-

ests individuals reach 50 mm TD in 2 to 4

chin become 20 years of age. The adult

years, whereas 7 to 8 years reported in

urchin attains sexual maturity at the size

coralline flats (Agatsuma, 2001b). Maxi-

between 40 and 60 mm TD and spawning

mum longevity is reported as 14 to 15

occurs during the winter months. _****_

years. Sexual maturity is attained at 40 to

****

45 mm TD, and spawning takes place in

_2.7. Kina (Evechinus chloroticus)_

autumn. _****_

_Evechinus chloroticus_ **** well-known as

kina is a sea urchin endemic to New Zea-

_2.5. Purple sea urchin (Strongylocentro-_

land waters. The distribution of kina is

_tus purpuratus)_

intensely linked within kelp beds or ag-

The sea urchin _S. purpuratus_ , usually

gregating in nearby barrens. It is typically

recognized as purple sea urchin, inhabits

occurs from the intertidal area to a depth

along the eastern edge of the Pacific coast

of 14 m; even some are found in 60 m.

of North America, extending from British

Growth rate is moderate and the individu-

Columbia, Canada to Ensenada, Mexico.

als reach to a size of 50 mm within the

It occurs abundantly in lower intertidal

age of 4 years. However, _E. chloroticus_

and nearshore subtidal communities but

attains the maximum size of 80-100 mm

has been found to a maximum depth of

in 8-9 years of age. Depending on the site,

150 m (Tegner, 2001). This species is

the longevity of this urchin varies be-

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_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

tween 10 and 20 years (Barker, 2001). It

age of 3 years, this species can reach to a

is predominantly herbivorous, feeding

maximum size of 92 mm TD. Average

mainly on brown algae, red algae and en-

longevity ranged from 1 to 2 years. When

crusting substrate (Barker, 2007). Kina

the urchin attains a size of 40 mm TD, it

usually reaches sexual maturity within 3-

gets sexual maturity within a year after

4 years of age having a size range be-

metamorphosis. No reasonability of

tween 40 and 50 mm TD and spawns in

spawning was observed in this species. _****_

spring, extending from November to Feb-

ruary (Barker, 2007).

_2.10. Rock sea urchin (Paracentrotus_

_lividus)_

_2.8. Chilean red sea urchin (Loxechinus_

_Paracentrotus lividus_ is a species of

_albus)_

sea urchin belongs to the family Parechin-

The Chilean red sea urchin, _Loxechi-_

idae and commonly known as rock or

_nus albus,_ is one of the reasonably slow-

purple sea urchin. It occurs in the Medi-

growing urchins, commonly found around

terranean Sea and eastern Atlantic Ocean,

the Pacific coasts of South America from

extending from western Scotland and Ire-

Isla Labos de Afuera in Peru to the

land to the Azores, Canary Islands and

Southern tip of South and usually occurs

Morocco, and most common in the west-

within the depths ranged from the inter-

ern Mediterranean, the coasts of Portugal

tidal zone to a maximum of depth of 340

and the Bay of Biscay, where the water

m (Vasquez, 2001). This urchin attains

temperature in winter months varies with-

sizes up to 130 mm TD and can live until

in 10 to 15oC. This species usually inhab-

20 years of age (Andrew et al., 2002). It is

its in the shallow sub-littoral area to a

considered as one of the important com-

maximum depth of 20 m. _Paracentrotus_

mercial species along the south-west

_lividus_ is intensely related to the seagrass

coast of South America due to high taste

meadows and mainly existed on encrusted

qualities. The distribution of _L. albus_ is

rocky substratum where it makes perma-

mostly on rocky substrates and closely

nent burrows to live in. It experiences

related with the kelp beds. It is an herbi-

with moderate growth rates and the indi-

vore and seems most likely to feed on

viduals having 40 mm TD are usually 4-5

whatever species of alga grow nearby.

years old, and the adults with a size of

The urchin is comparatively slow-

70+ mm TD are older than 12 years. The

growing, attaining a maximum size of

largest size of 15 mm TD is reported by

130 mm TD. Spawning period differs de-

Boudouresque and Verlaque (2001). The

pending upon its destitution patterns;

species gets sexual maturity at the size

happening in spring to summer in the

range between 13 and 20 mm TD and the

north, summer in the south and spring in

spawning usually occurs during spring to

the extreme south.

the early summer months.

__

_2.9. Variegated sea urchin (Lytechinus_

_2.11. Collector sea urchin (Tripneustes_

_variegatus)_

_gratilla)_

The variegated sea urchin occurs in

_Tripneustes gratilla_ is commonly rec-

the shallow waters and widely distributed

ognized as collector, cake or Parson"s hat

throughout the tropics and subtropics of

sea urchin and has a circumtropical distri-

the western Atlantic, from Florida,

bution, encompassing to the subtropics.

through the Caribbean to Brazil and Pan-

This species is usually occurs in the Indo-

ama (Watts _et al_., 2001). _Lytechinus var-_

Pacific, Hawaii, the Red Sea and Baha-iegatus is usually inhabits on seagrass

mas, and is widely distributed from Red

beds and hard bottoms covered with

sea westward to Hawaii and Clarion Is-

macroalgae. It is a fast-growing urchin

land, eastward to Paumotu, as far south as

but the longevity is limited. Within the

Port Jackson, and at Shark"s Bay on the

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west coast of Australia. It is regarded as a

can live for more than 10 years (Sander-

shallow-water sea urchin and usually in-

son, 1995). It attains sexual maturity at

habits on a diversity of substrates and oc-

TD sizes of 40–50 mm within 5–6 years

curs in the depth range between 2 and 30

of age (Sanderson _et al_., 1996). The best

meters (Lawrence, 2007). _Tripneustes_

roe production was found to be 10-14%

_gratilla_ grazes continuously during day

during August–December and spawning

and night and its diet comprises of algae,

usually occurs between summer and au-

periphyton and seagrass. It has higher

tumn (Sanderson, 1994). _****_

growth rate with lower longevity. Within

the age of 4 to 5 years, the urchin attains a

_2.13. Shore sea urchin (Psammechinus_

maximum size of 160 mm TD. However,

_miliaris)_

it can grow to 75 mm TD in the first year

_Psammechinus miliaris_ is a species of

of age. The collector sea urchin has an

sea urchin under the family Parechinidae

annual reproductive cycle __ mediated by

and sometimes known as shore of green

seawater temperature, length of day and

sea urchin. It shows restricted distribu-

feeding activity. Spawning mainly occurs

tions in the southern and eastern waters of

during mid to later winter months, when

the North Sea, and the eastern Atlantic

water temperatures and day lengths be-

Ocean from Scandinavia south to Moroc-

come the lowest and each clutch contains

co, where it occurs from the low tide

approximately 2 million eggs. Similar to

mark down to a maximum depth of 100 m.

the other regular sea urchins, the fertilized

Its abundance is strongly associated with

eggs develop into pluteus larvae, which

the presence of _Laminaria_ kelp. This sea

then stay in the water column for almost

urchin is often found on or under _Saccha-_

30 days. They then settle on the sea floor,

_rina lastissima_ , a large brown seaweed

undergo metamorphic induction and then

with which it shares its range of distribu-

become the tiny young juveniles. This

tions and also occurs in a variety of other

species attains the sexual maturity at

habitats including under boulders and

around 2-5 years of age to become a

rocks, among seaweeds, on rough ground

complete reproducing adult. _****_

and on the rhizomes of _Zostera marina_ in

seagrass meadows. It is an omnivore and

_2.12. Purple sea urchin (Heliocidaris_

mainly feeds of macroalgae, diatoms, hy-

_erythrogramma)_

droids, worms, small crustaceans, mol-

****

_Heliocidaris erythrogramma_ **** or

lusks and detritus. Longevity is relatively

the purple sea urchin is usually distributed

short and the growth rates are observed to

in the shallower coastal communities, ex-

be moderate. It has been reported that in-

tending from intertidal to a maximum

dividuals can reach to a maximum size

depth of 35 m in the southern Australia.

(45 mm TD) with an age between 3 and 4

In the coastal waters of Tasmania, this

years. _Psammechinus miliaris_ gets sexual

species is commonly occurs in kelp com-

maturity in the first year at 6-7 mm TD

munities and barrens, where it feeds by

and usually spawns in the months of

grazing and capturing drift weeds. It can

spring and early summer.

also be occurred in high densities in asso-

_****_

ciation with sea grass beds (Keesing,

_2.14. White_

_sea_

_urchin_

_(Salmacis_

2001). Growth rates of this sea urchin

_sphaeroides)_

usually vary depending on food availabil-

The short-spined white sea urchin ( _S._

ity and nutrition, but are mostly moderate.

_sphaeroides_ ) belonging to the family

Individuals __ of _H. erythrogramma_ attain a

Temnopleuridae, is considered as one of

size of 40 mm TD within one year as well

the rare species under the group of regular

as a harvestable size (60 mm TD) at 3 to 5

Echinoids. It usually occurs in the tropical

years. It has been reported that individu-

Indo-West Pacific Ocean, extending from

als having maximum size of 122 mm TD

China to Solomon Islands and Australia

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including Singapore and Malaysia (Tan

small-bodied Echinoids, having the max-

and Ng, 1988; Schoppe, 2000; Miskelly,

imum size of 85 mm TD and can live for

2002; Rahman _et al_., 2012b, 2013b). This

8 to 10 years. They inhabit burrows and

species has almost white cloudy test of

crevices and thereby defend themselves

55-80 mm TD with plentiful small white

from strong wave action and predators.

spines, the size of which are between 10

_Echinometra_ spp. are active herbivorous

and 15 mm. Some individuals have white

in nature and without the presence of

spines with marron bands, some with all

predators, they can occur in densities that

maroon species, while the others with ma-

exceed the primary production potential

roon

and

green

bands.

_Salmacis_

(McClanahan and Muthiga, 2001). Breed-

_sphaeroides_ can be found in the depth,

ing takes place in any time throughout the

ranging from 0 to 90 m seawards, and al-

year but they usually spawn during sum-

so be occurred in shallow intertidal zone,

mer and autumn in warmer waters. Simi-

particularly among seagrass meadows,

lar to the other regular Echinoids, _Echi-_

coral reef substrates and in muddy sublit-

_nometra_ spp. release their eggs and

toral areas or washed ashore Schoppe,

sperms in the water column, where fertili-

2000). This species feeds different types

zation occurs externally and the plankton-

of seaweeds, bryzoans and detritus.

ic echinopluteus larvae are produced

Spawning is found to be around the year.

through the embryonic and early larval

Following fertilization in the water col-

stages. The time when the competent lar-

umn, the embryo develops into a blastula

vae gets suitable substratum, they first

in about 9 hours. A series of larval stages

settle on the seabed then undergo meta-

follows, in which the larvae acquire more

morphosis to produce juvenile urchins.

and more arms, and develops tube feet

and spines within the larval body. Up to

_2.16. Long-spined black sea urchin (Di-_

this point, the process takes about 35 days.

_adema setosum)_

Competent larvae swim near the surface

The tropical sea urchin, _Diadema se-_

of the substrate to determine a suitable

_tosum_ commonly referred to as long-

site for settlement. After attachment, lar-

spined black sea urchin, is a member of

val structures are either discarded or re-

regular Echinoids under the family Di-

sorbed, and adult features continue to de-

adimatidae. It is broadly distributed

velop in the juvenile (Rahman _et al_.,

throughout the Indo-Pacific region, from

2012b).

Australia and Africa to Japan and Red

_****_

Sea, extending to the Gulf of Aqaba, Gulf

_2.15. Rock boring sea urchin (Echi-_

of Suez and Arabian/Persian Gulf (Lessi-

_nometra spp.)_

os _et al_., 2001). This species has charac-

A number of recently diverged spe-

teristics long black spines land five white

cies of rock boring sea urchins belonging

spots on the aboral side. The distinctive

to the genus _Echinometra,_ are widely dis-

orange ring around its anal cone com-

tributed throughout the World"s marine

pletes the special visual features of this

ecosystems. They occur commonly within

species. It is usually an omnivorous scav-

and around coral reefs from central Japan

enger and detritus eater and scraps films

in the north to southwest Australia in the

of hard substrates. They are generally

south, from Clarion Island off Mexico in

found in coral reefs and shallow rocky

the east, and to the Gulf of Suez in the

habitats at depths from 1 to 6 m. This

west (Rahman _et al_., 2000; 2005). Vari-

species has a wide range of diets, which

ous species of _Echinometra_ exhibits cir-

includes microalgae, seaweeds, coral

cumtropical distribution and usually occur

polyps and encrusting animals (Grignard

in shallow intertidal habitats, however a

_et al_., 1996). Gametogenesis begins in

few has been recorded at a maximum

April–May, when the seawater tempera-

depth of 20. These species are usually

ture rises above 25oC in the Gulf of Suez

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and spawning take place between June

Sea urchin aquaculture has successfully

and September (Pearse, 1970). It has also

been accomplished on a large scale in Ja-

been known to spawn both seasonally and

pan for many decades. In order to en-

thought out the year depending upon the

hance the natural stocks, millions of juve-

locations and sites of the spawning adults.

nile urchins are being produced in hatch-

Temperature levels higher than 25oC have

eries, for releasing to the managed areas

been observed to be a potential spawning

of seafloor on the intertidal seashore are-

cue (Pease, 1974). Throughout the year,

as. The nationally co-ordinated reseeding

the equatorial populations are found to be

program has been developed to the extent

spawn without following any particular

that over 66 million juveniles were re-

times. This is eventually true for the Ma-

leased on the reefs within which, over

laysian and Philippine populations of _D._

80% were _S. intermedius_ (Agatsuma _et_

_setosum_ (Tuason and Gomez, 1979). In

_al_., 2004). The contribution of released

the Persian Gulf, spawning usually occurs

sea urchin juveniles to the overall catch

between the months of April and May

has been estimated to be between 62 and

(Alsaffar and Khalid, 2000). Some other

80%. There have also been much small-

cues, such as the moon phases have been

scale reseeding programs functioning in

found to influence the spawning of _D. se-_

South Korea and Luzon Islands in the

_tosum_. This urchin has also been observed

Philippines (Andrew _et al_., 2002). The

to trigger spawning activities in concord-

farm

entrepreneurs

and

researchers

ance with the entrance of a full moon

around the southern Ireland have been

(Lessios, 1981).

developing techniques for commercial sea

****

urchin ( _P. lividus_ ) cultivation for more

**3. Culture, management and stock**

than 20 years (Leighton, 1995), and com-

**enhancement**

paratively newly in France (Grosjean _et_

****

_al_., 1998). Culture of 3 commercially im-

_3.1. Aquaculture_

portant sea urchins ( _P. miliaris_ , _E. escu-_

Mostly, the edible sea urchins are

_lentus_ and _P. lividus_ ) has been conducted

regular Echinoids (Lawrence, 2007), ex-

in Scotland since 1995 and there are also

periencing separate male and female sex-

well-established research teams in the

es and are generally broadcast spawners.

east coast of North America including

At the onset of the breeding season, the

Florida, Alabama, Maine, New Hen-

sexually matured adults release their

isphere, New Brunswick and Newfound-

gametes in the water column where ferti-

land – working on _S. droebechiensis_ and

lization takes place. The pluteus larvae

_L. variegatus_ ; On the west coast of North

form through the embryonic and early

America, including California and British

larval development of the fertilized eggs,

Columbia ( _S. droebechiensis_ , _S. francis-_

which after a period of planktonic devel-

_canus_ , _S. purpuratus_ ); in Chile ( _L. albus_ );

opment, feed on unicellular diatom, settle

in New Zealand ( _Evechinus chloroticus_ ),

to a suitable substratum and undergo met-

Norway ( _S. droebachiensis_ ) and in Israel

amorphosis to produce small juvenile ur-

( _P. lividus_ ) (Kelly, 2005).

chins. At 26-28oC, almost 1 month is re-

Sea urchin brood stocks are regu-

quired to complete the larval life cycle,

larly collected from the wild stocks when

comprising the feeding or 4-armed stage,

they reach proper sexual maturity. Ma-

the 6 to 8-armed stages and finally com-

tured gametes are obtained by injecting

petent stage for settlement (Figure 2). The

0.5 M KCl into the coelomic cavity of

newly born metamorphosed juveniles

both female and male urchins. Sperms in

grow on macroalgae until attain the mar-

its most concentrated from are pipetted

ketable size (40–50 mm) within the age

off the genital pores, while eggs are col-

ranged from 1 to 3 years, depending upon

lected by inverting the female urchins

the species (Kelly, 2005).

over a glass beaker filled with filtered sea

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****

**Figure 2:** Breeding, development and complete life-cycle of ball-like white sea urchin

( _Salmacis sphaeroides_ ) (Rahman _et al_., 2012b).

water (FSW) (Rahman _et al_., 2000, 2001,

they are reared in glass bottles on a roll-

2004, 2005, 2012b). At limited sperm

ing roller keeping a larval density of 1-2

concentrations, fertilization is usually

individual/ml of medium. The unicellular

done by mixing a few drops of diluted

cultured diatoms ( _Chaetoceros calcitrans_ ,

sperm (10-4 dry sperm dilutions) with egg

_Isochrysis galbana_ ) are commonly used

suspensions in a petri dish and the result-

as supplementary larval food at the con-

ing embryos are reared. Hatching of ferti-

centrations of 5,000, 10,000 and 15,000

lized eggs usually takes 10-15 hours after

cells per ml of medium daily at 4-, 6- and

insemination, to develop into a ciliated

8-armed pluteus stages, respectively until

blastula. When the swimming larvae

attaining metamorphic competence within

achieve feeding stage (4-armed pluteus),

around 30-35 days post-fertilization

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_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

(Rahman _et al_., 2016) (Figure 3). In Japan,

diatom (50:50) in petri dishes containing

partial water exchange system (Sakai _et_

FSW (Rahman _et al_., 2012b). Within 1

_al_., 2004) and continuous flow-through

day after the settlement induction, majori-

system (Hagen, 1996) are used for the

ty of the competent larvae are found to

large scale cultivation. The most costly

metamorphose into young juvenile (Fig.

aspect of captive larval culture is the need

4A). They are then reared on the encrust-

for the concurring production of the

ing coralline algal rocks in the aerated

above microalgae as live food for larvae.

glass/plastic aquaria for at least three

Nevertheless, larvae of the variegated sea

months by which they attains appropriate

urchin ( _L. variegatus_ ) have just been con-

juvenile (hereafter referred to as sea ur-

firmed to be appropriate for culturing

chin seed) sizes (Fig. 4D) for stocking in

with artificial diets (Gorge _at al.,_ 2004).

grow-out aquaculture system. In the coun-

Settlement and metamorphic in-

tries like Japan, South Korea, Ireland,

duction are considered as the most crucial

Norway, Scotland, and in British Colom-

stages for the development and culture of

bia, Canada, sea urchin juveniles have

sea urchin larvae. The higher survival rate

been produced on a commercial or semi-

is always dependent on the larvae to be-

commercial

scale

by

some

well-

come competent to metamorphose and

developed hatcheries and nurseries. Al-

then responding to the exact settlement

most all the culturists use natural biofilm

cues. In small-scale culture, induction for

or a specially seeded diatom substratum

metamorphosis of competent larvae has

made from species locally isolated and

recently been performed on coralline red

then grown on a PVC wave plate. How-

algal extracts + _Chaetoceros calcitran_

ever, one of the most challenging areas of

****

**Figure 3:** Developmental stages for larvae of short-spined white sea urchin, _Salmacis_

_sphaeroides_ : A) 4-arm pluteus, B) 6-arm pluteus, C) 8-arm pluteus, D) Pre-competent larva,

E) Competent larva with complete rudiment growth, and F) Just newly metamorphosed ju-

venile with adult spines and tubefeets (Rahman _et al_., 2012b).

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research is needed to optimize diets for

2001, 2005). Seawater is partially

the early juveniles and/or replacement of

changed twice a month and replenished

diatom biofilms. The differences in size

with freshly filtered sea water. The meth-

and succeeding variation in growth rates

od in continued for up to three months, by

of post-larvae remain a bottleneck in the

which time the juveniles reach to 9.0–10

supply of hatchery-reared juveniles. The-

mm in TD. These 3-month-old juveniles

se juveniles are robust enough to survive,

(Figure 5A) are then transferred to

transfer to sea cages or other grow-out

glass/plastic aquaria (90 x 45 x 45 cm)

systems from a small size of 5 mm TD

provided with filtered seawater in the cul-

(Kelly, 2002; Sakai _et al_., 2004). At this

ture unit of the Laboratory of Marine Bio-

instant, they are weaned into other diets,

technology, Institute of Bioscience, Uni-

soft macroalgae or artificial diets, depend-

versiti Putra Malaysia. Stocking density is

ing on the grow-out culture system.

fixed at 20 juveniles in each replicate

In the small-scale indoor aquaria-

aquarium and the urchins are fed with red

rearing system, 1-day-old juveniles are

alga ( _Amphiroa fragilissima_ ), brown alga

reared in small aquaria (25 x 20 x 20 cm)

( _Sargassum polysystum_ ) and sea grass

with aerated FSW and pieces of dead cor-

( _Enhalus acoroides_ ). Juveniles in all the

al with coralline red algae are supplied as

treatments were fed ad libitum and sea-

food (Figure 4) (Rahman _et al_., 2000,

water from each rearing aquarium was

**Figure 4:** Juveniles of the white sea urchin, _Salmacis sphaeroides_ : A) 1-day-old juvenile,

B) 1-month-old juvenile, C) 2-minth-old juvenile, and D) 3-month-old juvenile for grow-

out culture (Rahman _et al_., 2012b).

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****

**Figure 5:** Stocking juveniles and cultured adults of _Salmacis sphaeroides_ : A) Three-month-

old juveniles for stocking in grow-out culture, B) Sexually matured adults after the culture

period of two years in captive aquaria-rearing system.

**Table 1:** Comparison of growth and production of _S. sphaeroides_ fed with different types

of algae. Mean ± SE, _n_ = 30

Parameters

Treatments

T1 (Red alga)

T2 (Brown alga)

T3 (Sea grass)

Initial length (cm)

10.04 ± 0.70a

10.04 ± 0.70a

10.04 ± 0.70a

Final length (cm)

46.49  1.01a

43.56  1.04b

38.67  0.35c

Initial weight (g)

0.49  0.11a

0.49  0.11a

0.49  0.11a

Final weight (g)

51.17  1.17a

31.91  1.42b

20.80  0.65c

Weight gain (g)

50.67  1.93a

31.39  1.44b

20.31  0.43c

Length gain (cm)

36.46  1.01a

33.85  0.66b

28.63  0.35c

SGR (%/day)

0.73  0.01a

0.65  0.01b

0.58  0.01c

DGR (%/day)

7.92  0.30a

4.91  0.22b

3.17  0.10c

Wet gonad weight (g) 6.01  0.37a

3.56  0.26b

2.32  0.10c

Gonad index (%)

18.26  0.51a

16.44  0.19b

14.84  0.25c

Survival (%)

88.89  1.93a

73.33  3.34b

56.67  5.77c

Means sharing the same superscripts within the same row are not significantly different

from each other ( _P_ > 0.05).

completely changed at every 2–3 months.

from the relocation from poor to good

After two years of culture, the urchins

feeding grounds (Moylan, 1997) to the

attain sexual maturity (Figure 5B) and

ranching of urchins caged on the seafloor

those fed red alga, performed the best

(Cuthbert _et al_., 1995). Juveniles reared in

over the brown- and sea grass-fed urchins

hatcheries have been grown under sus-

with regard to body growth and edible

pended culture condition (Kelly, 2002,

gonad production (Table 1; Rahman un-

2005) in closed recirculation systems

published data). On the contrary to the

(Grosjean _et al_., 1998) and in demand

Japanese culture system, where hatchery-

rock pools in southern Ireland. The sea-

reared juveniles are mainly released to

cage culture system of stacking baskets

managed seafloor (Hagen, 1996; Sakai _et_

suspended from a ladder-like structure

_al_., 2004; Kelly, 2005), scientists of other

over which a work barge or raft can oper-

countries have conducted research with a

ate has been developed by Norwegian re-

wide range of grow-out culture systems

searchers (Aas, 2004). The time taken for

for the juvenile and adult urchins, ranging

juveniles of most species to reach market-

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_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

able size is in the range from 1 to 3 years

• limited entry (moratoriums) followed

(Kelly, 2005, Rahman _et al_., 2014b).

by active programs to reduce latent ef-

fort,

_3.2. Management_

• resource surveys at various levels of

The sea urchin fisheries around the world

complexity,

have consistently shown their susceptibil-

• the use of annual Total Allowable

ity to overfishing and to the problems as-

Catches based on resource assessment,

sociated with readjusting effort following

• zoning and area management, which

a fishing-down phase. Through the late

may be developed to the point of rota-

1980s and 1990s, a number of fisheries

tional harvest,

have developed to cater for the expanding

• the use of minimum legal sizes.

demand of the lucrative Japanese market.

This has driven the development of new

Classical fisheries science was de-

fisheries off Chile, both coasts of North

veloped in consideration of offshore, open

America and Australia. These fisheries

access and industrial fishing situations

**A**

**B**

commenced on virgin stocks and as a re-

and the resulting management systems are

sult have needed to deal with the issues of

not well adapted, or particularly robust,

rising expectations during fishing-down

when applied to more complex spatial

phase and adjustment of the fishery for

structure of small scale, inshore fishery

long-term sustainability.

resources (Orensanz and Jamieson, 1998).

The current position of the various

The social significance of small scale in-

American fisheries provides the full range

shore fisheries is much greater, given the

of outcomes in dealing with this problem.

numbers of fishermen and other players

For instance, the fisheries of Canada,

involved, than the sometimes more pro-

Alaska and Washington have had active

ductive offshore fisheries, which general-

programs to readjust effort levels and are

ly involve larger enterprises, higher capi-

now managed on the basis of catch limits

tal investment and limited numbers of

based on sustainable harvest strategies

fishermen. As a result, much of the chal-

using regular population surveys. The

lenge in ensuring the sustainability of

Californian fishery has yet to adequately

shellfish fisheries lies in developing and

adjust levels of effort and there is evi-

applying appropriate utilization, assess-

dence that the fishery is now being over-

ment and management models. According

fished. The Chilean fishery has little ca-

to Orensanz and Jamieson (1998), the

pacity to readjust effort levels and it

management measures that explicitly

would appear that once the fish-down

acknowledge spatial structure of fishery

phase is completed there is the potential

resources, and are therefore the most suit-

for significant overfishing. Of the fisher-

able for these sorts of fisheries, include

ies reviewed here, there are numerous ex-

(but not limited to):

amples of fisheries that have collapsed to

i) territorial property and use rights in-

levels of one or two magnitudes below

cluding lease, traditional tenure sys-

their peak production. These include the

tems etc.;

fisheries of France (Mediterranean and

ii) harvest rotation coupled with pulse

Atlantic), Iceland, Ireland, South Korea

fishing and/or thinning;

and Philippines. Significant parts of the

iii) reproductive refugia and Marine Pro-

Chinese fishery have also collapsed.

tected Areas;

Those fisheries that are currently being

iv) experimental management with spa-

managed for long term sustainability

tial control, contrasting treatments and

share a number of characteristics. They

replication;

are:

v) localized enhancement including habi-

tat manipulation, seeding and predator

control.

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Systems that accelerate growth to

seeding did not have any benefit on the

market size sea urchins while producing a

subsequent urchin production and crabs

uniform size class would give an econom-

and sea stars are removed from the

ic advantage One probable way to obtain

grounds using baited traps prior to the

sustainable and environmentally friendly

release of the urchins to control mortality

systems for urchin culture is to further

in the immediate period after release.

examine their potential in integrated sys-

Government has taken considerable in-

tems. They have already been shown to

volvement in the management of coastal

thrive in polyculture with Atlantic salmon

fisheries, particularly in the provision of

(Kelly _et al_., 1998) and to have a role in

subsidies for enhancement and infrastruc-

land-based integrated systems (Shpigel _et_

ture development as well as management

_al_., 2004). Nevertheless, many species are

coordination. A couple of studies have

true omnivores, so the potential for their

been looked at the contribution of re-

integration into systems where natural

seeding or habitat enhancement to the ac-

prey items, for instance, mussels and

tual abundance of urchins in harvest areas

clams, are already produced should be

in a sort of round-about way at localized

explored.

sites around Hokkaido and estimated that

re-seeded urchins comprised 62%, 66%

_3.3. Stock enhancement_

and 80% of the total catch in 1994, 1995

Decline in world production and over-

and 1996, respectively (Agatsuma, 2004;

fishing have prompted increasing in en-

Rahman _et al_., 2014b).

hancement as a means of maintaining

Translocation of the urchins is

production. It is most developed in Japan

used for a number of related reasons in

where the 1974 Coastal Fishing Ground

Japan. In areas where kelp forest devel-

Improvement and Development Law pro-

opment is held back by excessive urchin

vide the basis for stock enhancement

densities, urchins are sometimes removed

(Agatsuma _et al_., 2004). The goal of this

and replaced with adult kelps to permit

program is to "develop and improve

rapid development of complex kelp for-

coastal fishing grounds systematically by

ests (Agatsuma, 2004). The urchins may

the construction of artificial reefs and the

then be placed into intensive sea ranching

release of seedlings". Enhancement can

pens where they are fed _ad libitum_ and

comprise a number of different activities

prepared for harvest some months down

including

direct

stock

enhancement

the road. Experiments have shown that

through seeding of hatchery-raised juve-

Green Sea Urchins at densities up to 35

niles, habitat improvement or restoration,

kg/m have recorded recovery increases

creation of artificial reefs, predator con-

from 6% to over 18% in 11 weeks on an

trol, thinning and/or roe enhancement

artificial diet (Aas, 2004), although fur-

through supplemental feeding to increase

ther finishing for about 6 weeks on a nat-

the product recoveries etc. Re-seeding has

ural kelp diet is still required to get an

been especially applied in Japan since late

acceptable taste profile at this point. The

1980's. The numbers have been fairly sta-

sea urchin _Evechinus chloroticus_ is wide-

ble since 1997 with about 70-85 million

ly distributed around New Zealand but

juveniles reared to about 5-10 mm TD

attempts to establish a commercial fishery

and released each year primarily in the

have, like Norway, not succeeded because

areas with the largest historical harvest.

of the poor product quality and low re-

_Strongylocentrotus intermedius_ accounts

coveries. Experiments with ponding over

for about 85% of the urchins released by

2 month periods have seen recoveries to

the Japanese in Hokkaido (Andrew _et al_.,

increase near 20% and produced other

2002). Predator removal is required as

quality improvements that promise well

excess predation by sea stars etc. has been

for the future but further research is still

implicated in the few cases where the re-

needed to reach an economic breakeven.

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There are a number of aquaculture sites

the fore in a number of countries (Robin-

around New Zealand which are currently

son, 2004).

considered marginal for mussel farming,

which would be suitable for urchin cul-

**4. Bioactive compounds and human**

ture (Barker and Fell, 2004). Multi-

**health benefits**

disciplinary approaches are therefore

needed for stock enhancement and both

Alike many other marine inverte-

scientific and user group advisors should

brates, sea urchins have been considered

be involved (Masuda and Tsukamoto,

as a source of biologically active com-

1998). One method of enhancement that

pounds with biomedical applications

apparently works very well with Green

(Kelly, 2005, Rahman _et al_., 2014b).

Sea Urchins simply requires the presence

However, the full potential of echinoids

of a salmon net pen. The urchins can ap-

as a source of biologically active products

parently settle out quite abundantly on

is largely unexplored (Bragadeeswaran _et_

such structures and grow quite nicely by

_al_., 2013). The marine environment is an

feeding on the fouling organisms on the

exceptional reservoir of natural bioactive

mesh. This has provided some opportuni-

compounds, many of which exhibit struc-

ties for Canadian fishermen in British Co-

tural and chemical features not detected in

lombia (BC) for some easy harvests.

terrestrial derived natural products. The

The current market system used

richness of diversity offers a great oppor-

for the urchin trade developed in tandem

tunity for the discovery of new bioactive

with the wild fishery but this will no

compounds. Modern technologies have

doubt change dramatically once cultured

opened huge extents of research for the

product is available in substantial quanti-

extraction of bioactive compounds from

ties. Cultured production is more tightly

seas and oceans to treat the fatal diseases.

controlled than from the wild fishery so

The number of natural products isolated

that, as with the cultured salmon, the con-

and purified from marine organisms in-

sistent availability of an invariably high

creases rapidly and currently surpass with

quality product throughout the year will

hundreds of new compounds being dis-

have a tremendous impact on the urchin

covered every year (Proksch and Muller,

markets throughout the world. Traditional

2006). The isolated secondary metabolites

harvesters of sea urchins do not generally

have numerous functions; it is likely that

know much about the potential of aqua-

some of them may be pharmacologically

culture (Robinson, 2004) and will likely

active compounds for humans and useful

tend towards obstructing its development

as medicines (Briskin, 2000). A number

as opposed to recognizing the available

of such compounds have been isolated

advantages and applying them to their

from echinoderms (Carballeria _et al_.,

own benefit. This will be unfortunate be-

1996). There has also been much valuable

cause if the wild and cultured urchin fish-

information available for new antibiotics

eries could be more closely integrated,

and give new insights into bioactive com-

both would stand to benefit. For example,

pounds in sea urchins. Sea urchins shells

the gonad size and quality are quite easy

are containing various polyhydroxylated

to manipulate and the economic yield of

naphtoquinone pigments, spinochromes

the roe can be dramatically and fairly eas-

(Anderson _et al_., 1969) as well as their

ily increased. This knowledge is probably

analogous compound echinochrome A, of

directly applicable to the wild fishery and

which was showed bactericidal effect as

could contribute to an increase in quality,

reported by Service _et al_. (1984). The

value and profitability. Already, fisheries

phenolic hydroxyl groups in these mole-

and aquaculture are blurring together with

cules also suggested that they could par-

respect to product (gonad) enhancement

ticipate in particular antioxidant activity

and re-seeding of juveniles is coming to

as was observed in other well-known an-

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_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

tioxidant polyphenols such as tea cate-

Based on the nutritional facts a

chins. Similar to the structured com-

100 g of sea urchin gonad, which is equal

pounds are also found in the shells of sea

to 3.5 ounces, contain 172 calories and

urchins and thus suggesting that they as

very little fat. In fact, the fat that a serving

well as echinochrome A would act as an-

sea urchin does contain is almost all un-

tioxidant substances, similar to other pol-

saturated fatty acids. For instance, there is

yphenolic antioxidants in edible plants

1.75 g of polyunsaturated fat content in a

(Chantaro _et al_., 2008). While, squaric

serving sea urchin. Consumption of poly-

acid ester-based methodology was used in

unsaturated fats instead of saturated fats,

a new synthesis of chinochrome A was

such as those found in a burger, can help

used in a new synthesis of echinochrome

in reducing the overall cholesterol level.

A, a polyhydroxylated napthoquinone

Sea urchins also contain omega-3 fatty

pigment, commonly isolated from sea ur-

acids, which can help in lowering blood

chin spines (Pena-Cabrera _et al_., 2002).

pressure and reducing the risk of an ab-

Gonads of sea urchins contain polyhy-

normal heat beat followed by heart attack

drxylated polyhydoxylated naphtoqui-

(Rahim and Nurhasan, 2012). In addition,

none pigments, echinochrome A, which

they serve as frequent model organism for

highly potential in antioxidant activity

developmental and immunological studies.

(Lebedev _et al_., 2001). In our recent

study, we found that the ovary extracts of

5. **Bioassays for coastal water quality**

long-spined black sea urchin ( _D. setosum_ )

**using sea urchin embryo-larva and**

has profound and thereby inhibit the

**adults**

growth of pathogenic microorganisms

(Marimuthu _et al_., 2015).

Coastal ecosystems are now mat-

Sea urchin gonads are also very

ter to the impact of numerous human ac-

rich in valuable bioactive compounds,

tivities that lead to the input of a range of

such as polyunsaturated fatty acids

pollutants of agricultural, urban, or indus-

(PUFAs) and _β_ -carotine (Dincer and

trial origin. Sea urchins have been exten-

Cakli, 2007). PUFAs, especially eicose-

sively used as bioindicators of marine

pentanoic acid (EPA, C20:5) (n-3)) and

pollution over the last several decades

docosahexaenoic acid (DHA C22:6 (n-3)),

(Kobayashi, 1971; Phillips, 1990; Flam-

have significant preventive effects on ar-

mang _et al_., 1997). The two key life stag-

rhythmia, cardiovascular diseases and

es of the sea urchin most commonly stud-

cancer (Pulz, 2004). _β_ -carotene and some

ies and used in testing are the embryo-

xanthophylls have strong pro-vitamin A

larval and adult stages. Specifically, the

activity and can be used to prevent tumor

early life stages of some different species

development and light sensitivity (Britton

of sea urchins have been demonstrated to

_et al_., 2004). The composition of these

be sensitive to metals (Kobayashi, 1973,

valuable components, however, varies

1980; Kobayashi and Fujinaga, 1976;

greatly among different urchin species

Plillips _et al_., 2003).

and is influenced by their natural diets as

Sea urchins are also useful indica-

well as physiological processes i.e., re-

tor species for environmental contamina-

productive stages (Fernandez, 1998; Law-

tions due to the fact that their sperm, em-

rence, 2007). On the other hand, the high

bryos and larvae are very sensitive to tox-

levels of AA and EPA recently detected

ins in the water (Nacci _et al._ , 1986; Pa-

in _D. setosum_ and _S. sphaeroides_ greatly

gano _et al_., 1986; Dinnel _et al_., 1989;

supported the development of aquaculture

Ghiradini _et al_., 2003; Ghiradini _et al_.,

of sea urchins (Chen _et al_., 2010), since

2005). They are also considered as an ex-

PUFAs are important for human nutrition

cellent research species because spawning

(Lawrence, 2007).

and gamete collection is reasonably sim-

ple, published literatures on echinoid em-

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_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

bryological development is plentiful, the

1989; Ablanedo _et al_., 1990; Flammang _et_

larvae develop quickly, animals are avail-

_al_., 1997). The sea urchins, _D. setosum_

able throughout the year and are easily

and _P. lividus_ have been used as bioindi-

maintained under laboratory conditions

cators for assessing heavy metal contami-

(Hinegardner, 1969; Dinnel _et al_., 1989;

nations in coral reef ecosystems of the

Ghirardini _et al_., 2005). The sea urchin, _S._

Indo-West Pacific Ocean and the north-

_sphaeroides_ and _D. setosum_ are readily

western Mediterranean Sea, respectively

available in the Indo-Pacific including

(Warnau _et al_., 1995; Flammang _et al_.,

Malaysia and recently have documented

1997). Both the embryo-larva and adult

embryonic and larval development stages

_D. antillarum_ were also found to be high-

(Rahman et al., 2012b; Rahman _et al_.,

ly sensitive bioindicators for metal pollu-

2015). Sea urchin embryo-larva develop-

tion in marine environments on the Car-

ment test is a standard chronic toxicity

ibbean and should be considered when

bioassay advocated to be a cost-effective

determining ecological risks in coral reef

and useful method for use in screening

environments (Bielmyer _et al_., 2005). __

the toxicity of specific pollutants, mix-

****

tures of these and natural matrices, and

**6. Livelihood development and income**

has regularly been used to assess the tox-

**generation**

icity of water sediments (Beiras _et al_.,

****

2003; Cesar _et al_., 2009). This test con-

Alike other commercially important ma-

sists of the study of teratogenic effects in

rine invertebrates, Sea urchins offer im-

early embryo to larval stages. The stand-

portant benefits to human beings due to

ardized or classical criterion for evaluat-

their use in scientific research and educa-

ing toxicity by means of this test involves

tion and also for food. In the economic

distinguishing between normal larvae, i.e.,

point of view, sea urchin gonad either in

pyramid-shaped larvae with skeletal rods

the form of fresh or processed food, is

that are half the length towards the long

considered as one of the most expensive

axis of the larvae, a differentiated gut and

and luxury seafood in the world (Richard,

incipient postoral arms, and deformed

2004). In Japan, for example, sea urchin

larvae, i.e., larvae that display blocked or

(known as "uni") and its processed roe

delayed embryonic development, undif-

can retail for as much as AU$ 450 per kg.

ferentiated or abnormal gut and absent or

In addition, scientists and researchers can

abnormal

skeleton

(USEPA,

1994;

learn much about animal reproduction,

Warmau, _et al_., 1996). However, obser-

fertilization, development and evolution

vation of only skeletal anomalies may be

by studying sea urchins, sea stars and oth-

more rapid, sensitive and ecologically rel-

er echinoderms as model species (Parvez

evant than use of the classical criterion

_et al_., 2016b).

(without considering skeletal abnormali-

The Bajau, Suluk, Kokos and

ties), which, moreover may be affected by

Ubian tribes of Sabah (Eastern Malaysia)

the determining role of food availability

harvest the sea urchins, particularly their

in the larval form, rate of growth of body

eggs, to be sold and eaten as a delicacy.

parts and timing of development (Strath-

This delicacy is usually prepared for spe-

mann _et al_., 1992).

cial events such as Lepa-Lepa Festival,

The accumulation of pollutants in

wedding ceremony and other cultural

adult sea urchins has been used to moni-

events and is being treated as valuable

tor contaminations of many coral reef

fishery resources in Malaysia (Rahim and

habitats (Phillips, 1990; Flammang _et al_.,

Nurhasan, 2011). The sea urchins having

1997). Several studies have demonstrated

long spines are known as "tayum" in Sa-

metal accumulation in sea urchins ade-

bah, while the shorter spined species are

quately reflects abundance and availabil-

called "tehe-tehe". Sea urchins are usually

ity in contaminated waters (Augier _et al_.,

sold in wet markets at different prices de-

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_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

pending on their type and location. Ta-

This is mainly the case for the world"s

yum eggs are usually eaten raw and the

largest sea urchins fishery in Chile, where

selling price is from RM 2 to RM 5 per

the risks of collapse cannot be discounted.

pack. Meanwhile, "tehe-tehe" is sold at

Given that this fishery alone contributes

RM 1 to RM 2 per plate i.e., containing

upwards of 55% of the global harvest, a

three urchins with their skin intact to be

significant decline in Chile"s fishery

cooked into oku-oku, a traditional Bajau

would likely lead to structural realign-

delicacy. In comparison, price of sea ur-

ment in the market and higher prices for

chin eggs is RM 36 to RM 60 for every

mid-range products until aquaculture pro-

80 g in America, while in Japan, an ur-

duction ramped up. There is also general

chin can cost as much as RM 18 (Parvez

agreement that some form of exclusive

_et al_., 2016b). Aesthetically, the diverse

access as a prerequisite condition to pro-

forms of the sea urchins, and their beauti-

mote meaningful enhancement and intel-

ful coloring, are often providing not only

ligent harvesting to maximize roe value

a source of joy and recreation but also

will provide the best hedge against uncer-

increase the additional revenue to humans

tainties in fisheries productivity and mar-

observing them. Thus, sea urchins play a

ket stability. In the short term it is likely

significant role in livelihood development

that global production of sea urchin roe

and income generation to the local coastal

from wild fisheries will decline, with the

communities.

major production being provided by those

****

fisheries that have supported active man-

**7. Concluding remarks**

agement strategies to readjust the effort

and contain catches to levels that provide

This paper has been presented as a back-

long-term sustainability. Given that de-

ground document and review of the

mand is unlikely to decline; future pro-

World"s sea urchin fisheries. To summa-

duction will be increasingly valuable. In

rize the views, reports and publications of

order to make sea urchins fisheries viable

other scientists/researchers, it is apparent

and profitable, the following actions are

that sea urchin fisheries have a poor rec-

suggested:

ord of sustainability, as evidenced by the

 Refinement of artificial diet formula-

declines recorded in Japan, Maine, Cali-

tions for juveniles and adults to max-

fornia and South Korea among others, as

imize the growth rates and survivor-

well as by the ad hoc and/or ineffective

ship and produce gonads of the de-

management in many sea urchin fisheries.

sired taste, texture, flavor and color.

Very few stocks have been formally as-

 Optimization of grow-out facilities for

sessed, meaning it is near impossible to

juveniles and adults either at sea (in

qualify declines as the fish-down of ac-

containers and "ranched") or land-

cumulated biomass, which does not arrest

based.

the productivity of the stock, or as a case

 Regulations regarding fishing meth-

of over-fishing in which case its produc-

ods, fishing areas and protection of

tivity may be forced into permanent de-

company investments need to be de-

cline. Small-scale management is men-

veloped.

tioned time and again as offering the most

 Better surveillance of sea urchin den-

promise for ensuring long term sustaina-

sity to guarantee a steady flow of raw

bility. The strong and consistent spatial

materials.

structure inherent in sea urchin stocks

 Areas need to be thinned out to get the

combined with excessive effort from mo-

best possible product for the market,

bile fleets and inappropriately large scale,

this is also necessary for the kelp for-

and therefore ineffective management all

est to grow back.

contribute to declining production in

 More capital needs to be directed to-

many of the world"s sea urchin fisheries.

wards investing in technology for

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_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

processing to reduce labor costs and

Kuwait (Northern Arabian Gulf).

preserve product quality.

_Bulletin of Marine Science_ **67(2),**

 Improved cooperation between fish-

**845–856.**

ermen and processors, when market-

**Anderson, A. H., Mathieson, J. W. and**

ing and selling the sea urchins.

**Thomson, R. H. (1969).** Distribu-

****

tion of spinochrome pigments in

__
__

## Acknowledgements

echinoids. _Comparative Biochem-_

_istry and Physiology_ **28, 333–345.**

We would like to express our

**Andrew, N. and Byrne, M. (2001).** The

grateful thanks and appreciations to the

ecology of _Centrostephanus rodg-_

Ministry of Science, Technology and In-

_ersii_. _In_ : Lawrence J.M. (ed.). Ed-

novation (MOSTI), Malaysia, for provid-

ible Sea Urchins: Biology and

ing financial supports through the Re-

Ecology, Elsevier, Amsterdam,

search Management Centre (RMC) of the

The Netherlands. **pp. 149–160.**

Universiti Putra Malaysia (UPM) under

**Andrew N. L. et al**. **(2002)**. Status and

the "ScienceFund" grant (Project No. 04-

management of world sea urchin

01-04-SF2227) for completing this work

fisheries. _Oceanography and Ma-_

successfully.

_rine Biology Annual Review_ **40,**

**343–425.**

**References**

**Andrew, N. L., Agatsuma, Y., Dewees,**

****

**C. M. and Stotz, W. B. (2004)**.

**Aas, K. (2004).** Technology for sea-based

State of sea-urchin fisheries 2003.

farming of sea urchins. _In_ : Law-

_In_ : Lawrence, J. M. and Guzmań,

rence J. M. and Guzmán O. (eds.).

O. (eds.). Sea Urchin Fisheries

Sea Urchins: Fisheries and Ecolo-

and Ecology. DEStech Publica-

gy, DEStech Publications, Inc.,

tions, Lancaster, PA, USA, **pp.**

Lancaster, USA. **pp. 366–373.**

**96–** 98.

**Agatsuma, Y. (2001a).** The ecology of

**Barker, M. F. (2001).** The ecology of

_Strongylocentrotus_

_intermedius_.

_Evechinus chloroticus_. _In_ : Law-

_In_ : Lawrence, J. M. (ed.). Edible

rence, J. M. (ed.). Edible Sea Ur-

Sea Urchins: Biology and Ecology,

chins: Biology and Ecology, Else-

Elsevier, Amsterdam, The Nether-

vier, Amsterdam, The Netherlands,

lands. **pp. 333–346.**

**pp. 245–260.**

**Agatsuma, Y. (2001b).** The ecology of

**Barker, M .F. (2007).** The ecology of ****

_Strongylocentrotus_

_nudus_.

_In_ :

_Evechinus chloroticus_. In: Law-

Lawrence, J. M. (ed.). Edible Sea

rence J. M. (ed.). Edible Sea Ur-

Urchins: Biology and Ecology,

chins: Biology and Ecology, Else-

Elsevier, Amsterdam, The Nether-

vier, Amsterdam, The Netherlands,

lands. **pp. 347–361.**

**pp. 319–338.**

**Agatsuma, Y., Sakai, Y. and Andrew,**

**Barker, M. F. and Fell, J. (2004).** Sea

**N. L. (2004).** Enhancement of Ja-

cage experiments on roe en-

pan"s sea urchin fisheries. _In_ :

hancement of New Zealand sea

Lawrence, J. M. and Guzman, O.

urchin _Evachinus chloroticus_. _In_ :

(eds.). Sea urchins: fisheries and

Lawrence, J. M. and Guzmán, O.

aquaculture, DEStech Publications,

(eds.). Sea Urchins: Fisheries and

Lancaster, Pennsylvania. **pp. 18–**

Ecology, DEStech Publications,

**36.**

Inc., Lancaster, **pp. 375–383.**

**Alsaffar, A. H. and Khalid P. L. (2000).**

**Barnes, D. K. A., Verling, E., Crook, A.,**

Reproductive cycles of _Diadema_

**Davidson, I, and O'Mahoney, M.**

_setosum_ and _Echinometra mathaei_

**(2002).** Local population disap-

(Echinoidea: Echinodermata) from

pearance follows (20 yr after) cy-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 436

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

cle collapse in a pivotal ecological

cological assessment of sediment

species. _Marine Ecology Progress_

in Portmán Bay (southeast Spain).

_Series_ **226, 311–313.**

_Ecotoxicology and Environmental_

**Beiras, R., Fernández, N., Bellas, J.,**

_Safety_ **72, 1832e1841.**

**Besada, V., González-Quijano,**

**Chantaro, P., Devahastin, S. and**

**A. and Nunes, T. (2003).** Integra-

**Chiewchan, N. (2008).** Produc-

tive assessment of marine pollu-

tion of antioxidant high dietary fi-

tion in Galician estuaries using

ber powder from carrot peels.

sediment chemistry, mussel bioac-

_LWT-Food Science and Technolo-_

cumulation, and embryo–larval

_gy_ **41, 1987–1994.**

toxicity bioassays. _Chemosphere_

**Chen, G. -Q., Xian, W. -Z., Lau, C. -C.,**

**52(7), 1209–1224.**

**Peng, J., Qiu, J. -W., Chen, F.**

**Boudouresque, C.-F. and Verlaque, M.**

**and Jiang, Y. (2010).** A compara-

**(2001).** The ecology of _Paracen-_

tive analysis of lipid and carote-

_trotus lividus_. _In_ : Lawrence, J. M.

noid composition of the gonads of

(eds.). Edible Sea Urchins: Biolo-

_Anthocidaris_

_crassispina_ ,

_Di-_

gy and Ecology, Elsevier, Am-

_adema setosum_ and _Salinacis_

sterdam, The Netherlands, **pp.**

_sphaeroides_.

_Food_

_Chemistry_

**177–216.**

**120(4), 973–977.**

**Bragadeeswaran, S., Sri Kumaran, N.,**

**Cuthbert, F. M., Hooper, R. G. and**

**Prasath Sankar P. and Praba-**

**McKeever T. (1995).** Sea urchin

**har, R. (2013).** Bioactive poten-

aquaculture phase I: sea urchin

tial of sea urchin _Temnopleurus_

feeding and ranching experiments.

_toreumaticus_ from Devanampat-

Report of Project AUI-503. Cana-

tinam, Southeast coast of India.

dian Centre for Fisheries Innova-

_Journal of Pharmacy and Alterna-_

tion, Government of Newfound-

_tive Medicine_ **2(3), 9–17.**

land.

**Briskin, D. (2000).** Medicinal Plants and

**Dincer, T. and Cakli, S. (2007).** Chemi-

Phyto medicines. Linking plant

cal composition and biometrical

biochemistry and physiology to

measurements of the Turkish Sea ur-

human health. _Plant Physiology_

chin ( _Paracentrotus lividus_ , Lamarck,

**124, 507–514.**

1816). _Critical Reviews in Food Sci-_

**Britton, G., Liaaen-Jensen, S. and**

_ence and Nutrition_ **47(1), 21–26.**

**Pfander, H. (2004).** Carotenoids

**Dinnel, P. A., Link, J. M., Stober, Q. J.,**

handbook,

Birkhauser

Verlag,

**Letourneau, M. W. and Roberts,**

Boston, USA.

**W. E. (1989).** Comparative sensi-

**Carboni, C., Addis, P., Cau, A. and**

tivity of sea urchin sperm bioas-

**Atach, T. (2012).** Aquaculture

says to metals and pesticides. Ar-

could enhance Mediterranean sea

chives of _Environmental Contam-_

urchin fishery, expand supply.

_ination and Toxicology_ **18(5),**

_Global_

_Aquaculture_

_Advocate_

**748–755.**

**15(3), 44–45.**

**Edgar, G. (1999). Tasmania.** _In_ : Andrew,

**Carballeria, N. M., Cruz, C. and Sostre,**

N. L. (ed.). **** Under Southern Seas:

**A. (1996).** Identification of the

The Ecology of Australia"s Rocky

novel 7-methyl-6 octadecenoic ac-

Reefs, University of New South

id in _Holothuria mexicana_. _Jour-_

Wales Press, Sydney, **pp. 30-39.**

_nal of Natural Products_ **59(11),**

**FAO. (2010).** The State of World Fisher-

**1076–1078.**

ies and Aquaculture 2010. Food

**Cesar, A., Marín, A., Marin-Guirao, L.,**

and Agriculture Organization of

**Vita, R., Lloret, J. and DelValls,**

the United Nations, Rome, Italy.

**T.A. (2009).** Integrative ecotoxi-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 437

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

**Fernandez, C. (1998).** Seasonal changes

**Ichihiro, K. (1986).** Breeding, processing

in the biochemical composition of

and sale, Hokkai Suisan Shinbun-

the edible sea urchin _Paracentro-_

sha, Sappro, Japan. ****

_tus lividus_ (Echinodermata: Echi-

**Kaneniwa, M. and Takagi, T. (1986).**

noidea) in a lagoonal environment. ****

Fatty acids in the lipid of food

_Marine Ecology_ **19, 1–11.**

products from sea urchin. _Bulletin_

**Flammang, P., Warnau, M., Temara,**

_of the Japanese Society of Scien-_

**A., Lane, D. J. W. and Jangoux,**

_tific Fisheries_ **52(9), 1681–1685.**

**M. (1997).** Heavy metals in _Di-_

**Kessing, J. (2001).** The ecology of _Heli-_

_adema setosum_ (Echinodermata

_ocidaris erythrogramma_. _In_ : Law-

Echinoidea) from Singapore coral

rence, J.M. (ed.). Edible Sea Ur-

reefs. _Journal of Sea Research_ **38,**

chins: Biology and Ecology, Else-

**35–45.**

vier, Amsterdam, The Netherlands.

**George, S. B., Lawrence, J. M. and**

**pp. 261–270.**

**Lawrence, A. L. (2004).** Com-

**Keesing, J. K. and Hall, K. C. (1998).**

plete larval development of the

Review of the status of world sea

sea urchin _Lytechinus variegatus_

urchin fisheries points to opportu-

fed an artificial feed. **** _Aquaculture_ ****

nities for aquaculture. _Journal of_

**242(1-4), 217–228.**

_Shellfish Research_ **17(5),1597–**

**Ghirardini, V. A, Chiaraa, L., Alessan-**

**1604.**

**draa, A. N., Alvisea, B., Hisb, E.**

**Kelly, M. S. (2000).** The reproductive

**and Francesco, G. (2005). __**_Myti-_

cycle

of

the

sea

urchin

_lus galloprovincialis_ as bioindica-

_Psammechinus_ _miliaris_ (Gmelin)

tor in embryotoxicity testing to

(Echinodermata: Echinoidea) in a

evaluate sediment quality in the

Scottish sea loch. _Journal of the_

lagoon of Venice (Italy). _Chemis-_

_Marine Biological Association of_

_try and Ecology_ **21(6), 455–463.**

_the United Kingdom_ **80, 909–919.**

**Grignard, J. C., Flammang, P., Lane, D.**

**Kelly. M. S.** **(2002).** Survivorship and

**J. W. and Jangoux, M. (1996).**

growth rates of hatchery-reared

Distribution and abundance of the

sea urchins. _Aquaculture Interna-_

echinoid _Diadema setosum_ (Echi-

_tional_ **10, 309–316.**

nodermata) on sediment-stressed

**Kelly, M. S. (2005).** Echinoderms: their

coral reefs in Singapore. **** _Asian_

culture and bioactive compounds.

_Marine Biology_ **13, 123–132.**

_In_ : Matranga, V. (ed.). Echino-

**Grosjean, P., Spirlet, C. and Gosselin,**

dermata: Progress in Molecular

**P. (1998).** Land-based, closed-

and Subcellular Biology, Subse-

cycle echinoculture of _Paracen-_

ries: Marine Molecular Biotech-

_trotus lividus_ (Lamarck) (Echinoi-

nology, Springer-Verlag, Berlin

dea: Echinodermata): a long-term

Heidelberg, **pp. 139–165.**

experiment at a __ pilot scale. **** _Jour-_

**Kelly, M. S., Brodie, C. C. and McKen-**

_nal of Shellfish Research_ **17(5),**

**zie, J. D. (1998).** Somatic and

**1523–1531.**

gonadal growth of the sea urchin

**Hagen, N. T. (1996).** Echinoculture: from

_Psammechinus miliaris_ (Gmelin)

fishery enhancement to closed-

maintained in polyculture with the

cycle cultivation. **** _World Aquacul-_

Atlantic salmon. _Journal of Shell-_

_ture_ **27, 6–19.**

_fish Research_ **17(5), 1557–1562.**

**Hagen, N. T. (2000).** Echinoderm culture. ****

**Kobayashi, N. (1971).** Fertilized sea ur-

_In_ **:** Stickney, R. R. (ed.), Encyclo-

chin eggs as indicatory materials

pedia of aquaculture. Wiley-

for marine pollution bioassay, pre-

Interscience, New York, **pp. 247–**

liminary experiment. _Publications_

**253.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 438

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

of the _Seto Marine Biological La-_

**Lessios, H. A., Kessing, B. D. and**

_boratory_ **18, 379–406.**

**Pearse, J. S. (2001).** __ Population

**Kobayashi, N. (1973).** Studies on the ef-

structure and speciation in tropical

fects of some agents on fertilized

seas. Global phylogeography of

sea urchin eggs, as a part of the

the sea urchin _Diadema. Evolution_

bases for marine pollution bioas-

**55(5), 955–975.**

say. _Publications_ of the _Seto Ma-_

**Marimuthu, K., Gunaselvam, P., Rah-**

_rine Biological Laboratory_ **21(2),**

**man, M. A., Xavier, R., Arock-**

**109–114.**

**iaraj,**

**J.,**

**Subramanian,**

**S.,**

**Kobayashi, N. (1980).** Comparative sen-

**Yusoff, F. M. and Arshad, A.**

sitivity of various developmental

**(2015).** Antibacterial activity of

stages of sea urchins to some

ovary extract from sea urchin _Di-_

chemicals. _Marine Biology_ **58,**

_adema setosum_. _European Review_

**163–171.**

_for Medical and Pharmacological_

**Kobayashi, N. and Fujinaga, K. (1976).**

_Sciences_ **19, 1895–1899.**

Synergism of inhibiting actions of

**Masuda, R. and Tsukamoto, K. (1998).**

heavy metals upon the fertilization

Stock enhancement in Japan: Re-

and development of sea urchin

view and perspective. **** _Bulletin of_

eggs. _Science_ and Engineering

_Marine Science_ **62(2), 337–358.**

Review of _Doshisha University_

**McClanahan, T. R. and Muthiga, N. A.**

**17(1), 54–69.**

**(2001).** The ecology of _Echinometra_.

**Lawrence, J. M. (2007).** Edible Sea Ur-

_In_ : Lawrence, J. M. (ed.). Edible Sea

chins: Biology and ecology. Else-

Urchins: Biology and Ecology. Else-

vier, Boston, USA. **p. 380.** ****

vier, Amsterdam, the Netherlands, **pp.**

**Lawrence, J. M., Olave, S., Otaiza, R.,**

**225–243.**

**Lawrence, A. L. and Bustos, E.**

**Miskelly, A. (2002).** Sea Urchins of and

**(1997).** Enhancement of gonad

Indo-Pacific, Capricornica Publica-

production in the Sea Urchin _Lox-_

tions, Sydney, Australia.

_echinus albus_ in Chile fed extrud-

**Moylan, E. (1997).** Gonad conditioning

ed feeds. _Journal of the World_

and wild stock enhancement of the

_Aquaculture Society_ **28(1), 91–96.**

purple sea urchin _Paracentrotus_

**Lawrence, J. M., Lawrence, A. L.,**

_lividus_ on the west coasts of Ire-

**McBride, S. C., George, S. B.,**

land. _Bulletin of the Aquaculture_

**Watts, S. A. and Plank, L. R.**

_Association of Canada_ **97(1), 38–**

**(2001).** Developments in the use

**45.**

of prepared feeds in sea-urchin

**Nacci, D., Jackim, E. and Walsh, R.**

aquaculture. _World Aquaculture_

**(1986).** Comparative evaluation of

**32(3), 34–39.**

three rapid marine toxicity tests:

**Lebedev, A.V., Levitskaya, E. L.,**

sea urchin early embryo growth

**Tikhonova, E. V. and Ivanova,**

test, sea urchin sperm cell toxicity

**M. V. (2001).** Antioxidant proper-

test and microtox. _Environmental_

ties, autoxidation and mutagenic

_Toxicology and Chemistry_ **5, 521–**

activity of echinochrome A com-

**525.**

pared with its etherified derivative.

**Orensanz, J. M. and Jamieson, G. S.**

_Biochemistry_ **66, 885–893.**

**(1998).** The assessment and man-

**Lessios, H. A. (1981).** __ Reproductive peri-

agement of spatially structured

odicity of the echinoids Diadema

stocks: an overview of the North

and Echinometra on the two

Pacific Symposium on Inverte-

coasts of Panama. _Journal of Ex-_

brate Stock Assessment and Man-

_perimental Marine Biology and_

agement _. In_ : Jamieson, G. S. and

_Ecology_ **50(1), 47–61.**

Campbell, A. (eds.). Proceedings

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 439

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

of the North Pacific Symposium

ceedings of the 2nd International

on Invertebrate Stock Assessment

Coral Reef Symposium. Brisbane,

and management. _Canadian Spe-_

ICRS, Australia, **1, 235–240.**

_cial Publication of Fisheries and_

**Pena-Cabrera, E. and Liebeskind, L. S.**

_Aquatic Sciences 125_ **pp. 441–445.**

**(2002).** Squaric acid ester-based

**Oshima, T., Wada, S. and Koizumi, C.**

total synthesis of Echinochrome

**(1986).** Lipid deterioration of salt-

A. _Journal of Organic Chemistry_

ed gonads of sea urchin during

**67, 1689–1691.**

storage at 5oC. _Bulletin of the_

**Phillips, D. J. H. (1990).** Use of

_Japanese Society of Scientific_

macroalgae and invertebrates as

_Fisheries_ **52(3), 511–517.**

monitors of metal levels in estuar-

**Pagano, G., Cipollaro, M., Esposito, A.,**

ies and coastal waters. _In_ : Furness,

**Ragucci, E., Giordano, G. G. and**

R. W. and Rainbow, P. S. (eds.).

**Trieff, N. M. (1986).** The sea ur-

Heavy Metals in the Marine Envi-

chin: Bioassay for the assessment

ronment, CRC Press, Boca Raton,

of damage from environmental

FL, **pp. 81–99.**

contaminants. _In_ : Cairns, J. Jr.

**Phillips, B. M., Nicely, P. A., Hunt, J.**

(ed.). Community Toxicity Testing,

**W., Anderson, B. S., Tjeerdema,**

Association for Standard Testing

**R. S., Palmer, S. E., Palmer, F.**

and Materials, Philadelphia, **pp.**

**H. and Puckett, H. M. (2003).**

**67–92.**

Toxicity of cadmium–copper–

**Parvez, M. S., Rahman, M. A. and**

nickel–zinc mixtures to larval

**Yusoff, F. M. (2016a).** Sea urchin

purple sea urchins. _Bulletin of En-_

fisheries in Malaysia: status, po-

_vironmental Contamination and_

tentials and benefits. _In_ : Rahman,

_Toxicology_ **70, 592–599.**

M. A. and Monticolo, D. (eds.).

**Proksch, P. and Muller, W. E. G.**

Proceedings of the 5th Interna-

**(2006)**. Frontiers in Marine Bio-

tional Conference on Chemical

technology. Horizon Bioscience,

Engineering and Biological Sci-

Norfolk, UK.

ences (ICCBS-16), International

**Pulz, O. and Gross, W. (2004).** Valuable

Scientific Academy of Engineer-

products from biotechnology of

ing and Technology, Kuala Lum-

microalgae. _Applied Microbiology_

pur, Malaysia, **pp. 14–16.**

_and Biotechnology_ **65(6), 635–648.**

**Parvez, M. S., Rahman, M. A., Yusoff,**

**Rahim. S. A. K. A. and Nurhasan, R.**

**F. M. and Arshad, A. (2016b).**

**(2012).** Edible sea urchin species

Status, prospects and potentials of

in Sabah waters. Research Bulle-

the commercially important spe-

tin, Faculty of Resource Science

cies of sea urchin, _Tripneustes_

and Technology (FRST), Univer-

_gratilla_ (Linnaeus 1758) in Ma-

siti Malaysia Sarawak, **1, 2–3.**

laysia. _International Journal of_

**Rahman, M. A. and Yusoff, F. M.**

_Biological, Ecological and Envi-_

**(2010).** Sea urchins in Malaysian

_ronmental Sciences_ **5(1), 50–54.**

coastal waters. The Oceanog-

**Pearse, J. S. (1970).** Reproductive perio-

rapher, **4(1), 20–21.**

dicities of Indo-Pacific inverte-

**Rahman, M. A., Uehara, T. and Aslan,**

brates in the Gulf of Suez III. The

**L. M. (2000).** Comparative viabil-

echinoid _Diadema_ _setosum_ (Les-

ity and growth of hybrids between

ke). _Bulletin of Marine Science_ **20,**

two sympatric species of sea ur-

**697–720.**

chins (genus _Echinometra_ ) in

**Pearse, J. S. (1974).** __Reproductive pat-

Okinawa. _Aquaculture_ **183, 45–56.**

terns of tropical reef animals:

**Rahman, M. A., Uehara, T. and Pearse,**

three species of sea urchins.  __ Pro-

**J. S. (2001).** Hybrids of two close-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 440

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

ly related tropical sea urchins (ge-

from the coastal waters of Johor,

nus

_Echinometra_ ):

evidence

Malaysia. _Asian Journal of Ani-_

against

postzygotic

isolating

_mal and Veterinary Advances_ **8(2),**

mechanisms. _Biological Bulletin_

**301–308.**

**200, 97–106.**

**Rahman, M. A., Yusoff, F. M. and Ar-**

**Rahman, M. A., Uehara, T. and Pearse,**

**shad, A. (2014a).** Potential and

**J. S. (2004).** Experimental hybrid-

prospect for sea urchin resource

ization between two recently di-

development in Malaysia. _Fish-_

verged species of tropical sea ur-

_mail_ **21, 16–18.**

chins, _Echinometra mathaei_ and

**Rahman, M. A., Arshad, A. and Yusoff,**

_Echinometra oblonga_. _Inverte-_

**F. M. (2014b).** Sea urchins (Echi-

_brate Reproduction and Develop-_

nodermata: Echinoidea): Their bi-

_ment_ **45, 1– 14.**

ology, culture and bioactive com-

**Rahman, M. A., Uehara, T. and Law-**

pounds. _In_ : Kao, J. C. M. and

**rence, J. M. (2005).** Growth and

Rahman, M. A. (eds.). Proceed-

heterosis of hybrids of two closely

ings of the International Confer-

related species of Pacific sea ur-

ence on Advances in Environment,

chins (genus _Echinometra)_ in

Agriculture and Medical Sciences

Okinawa. _Aquaculture_ **245, 121–**

(ICAEAM"14),

International

**133.**

Academy of Arts, Science &

**Rahman, M. A., Amin, S. M. N., Yusoff,**

Technology, Kuala Lumpur, Ma-

**F. M., Arshad, A., Kuppan, P.**

laysia, **pp. 23–27.**

**and Shamsudin, M. N. (2012a).**

**Rahman M. A., Yusoff, F. M. and Ar-**

Length weight relationships and

**shad, A. (2015).** Embryonic, lar-

fecundity estimates of long-spined

val and juvenile development of

sea urchin, Diadema setosum,

tropical sea urchin, _Diadema se-_

from the Pulau Pangkor, Peninsu-

_tosum_. _Iranian Journal of Fisher-_

lar Malaysia. _Aquatic Ecosystem_

_ies Sciences_ **14(2), 409–424.**

_Health and Management_ **15, 311–**

**Rahman, M. A. Yusoff, F. M., Arshad,**

**315.**

**A. and Ara, R. (2016).** Growth

**Rahman, M. A., Yusoff, F. M., Arshad,**

and survival of the tropical sea ur-

**A. Shamsudin, M .N. and Amin,**

chin, _Salmacis sphaeroides_ fed

**S. M. N. (2012b).** Embryonic, lar-

with different macroalgae in cap-

val, and early juvenile develop-

tive rearing condition. _Journal of_

ment of the tropical sea urchin,

_Environmental Biology_ **37, 855–**

_Salmacis sphaeroides_ (Echino-

**862.**

dermata: Echinoidea). _The Scien-_

**Richard, M. (2004).** The little urchins

_tific World Journal_ **2012, 1–9.**

that can command a princely price.

**Rahman, M. A., Arshad, A., Yusoff, F.**

The Sydney Morning Herald.

**M. and Amin, S. M. N. (2013a).**

**Robinson, S. M. (2004).** The evolving

Hybridization and growth of trop-

role of aquaculture in the global

ical sea urchins. _Asian Journal of_

production of sea urchins. _In_ : Sea

_Animal and Veterinary Advances_

Urchins: Fisheries and Ecology

**8(2), 177–193.**

proceedings of the international

**Rahman, M. A., Yusoff, F. M., Arshad,**

Conference on Sea-Urchin Fisher-

**A., Amin, S. M. N. and**

ies and Aquaculture. Puerto Varas,

**Shamsudin, M. N. (2013b).** Pop-

Chile, March 25-27, 2003. DES-

ulation characteristics and fecun-

tech Publications. Lancaster, PA.

dity estimates of short-spined

**Saito, K. (1992).** Sea urchin fishery of

white

sea

urchin,

_Salmacis_

Japan. _In_ : Anonymous, The man-

_sphaeroides_

(Linnaeus,

1758)

agement and enhancement of sea

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 441

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

urchins and other kelp bed re-

**Service, M. and Wardlaw, A. C. (1984).**

sources: a Pacific rim perspective.

Echinochrome-A as a bactericidal

California Sea Grant College, La

substance in the coelomic fluid of

Jolla, Californoa, Report No. T-

_Echinus esculentus_. _Comparative_

CSGCP-028.

_Biochemistry and Physiology Part_

**Sakai, Y., Tajima, K. I. and Agatsuma,**

_B_ **79, 161–165.**

**Y. (2004).** Mass production of

**Shpigel, M., McBride, S. C., Marciano,**

seed of the Japanese edible sea ur-

**S. and Lupatsch, I. (2004).** Prop-

chins _Strongylocentrotus droe-_

agation of the European sea urchin

_bachiensis_ and _Strongylocentrotus_

_Paracentrotus lividus_ in Israel. _In_ :

_nudus_. _In_ : Lawrence, J. M. and

Lawrence, J. M. and Guzman O.

Guzman, O. (eds.). Sea urchins:

(eds.). Sea urchins: fisheries and

fisheries and aquaculture, DES-

aquaculture, DEStech, Publica-

tech

Publications,

Lancaster,

tions, Lancaster, Pennsylvania, **p.**

Pennsylvania, **pp 287–298.**

**85.**

**Sanderson, C. J. (1994).** Interim Sea Ur-

**Shimabukuro, S. (1991).** _Tripneustes_

chin Report June 1994. Depart-

_gratilla_ (sea urchin). _In_ : Shokita,

ment of Primary Industry and

S., Kakazu, K., Tomomi, A., To-

Fisheries Marine Research Labor-

ma, T., Yamaguchi, M. (eds.).

atory International Report 8

Aquaculture in Tropical Areas.

**Sanderson, C. J. (1995).** Interim Sea Ur-

Midori Shobo Co. Ltd. Tokyo, **pp.**

chin Report February 1995. De-

**313–328.**

partment of Primary Industry and

**Sivertsen, K. (2004).** Harvestable sea

Fisheries Marine Research Labor-

urchin _Strongylocentrotus_ _droe-_

atory International Report 21.

_bachiensis_ resources along the

**Sanderson, C. J. Le Rossignol, M. and**

Norwegian coast. _In_ : Lawrence,

**James, W. (1996).** A pilot pro-

J.M. and Guzman, O. (eds.). Sea

gram to maximize Tasmania"s sea

Urchins: Fisheries and Aquacul-

urchin

( _Heliocidaris_

ture, DEStech Publications, Lan-

_erythrogramma_ ) resource. Fisher-

caster, Pennsylvania, **p. 85.**

ies Research and Development

**Sloan, N. A. (1985).** Echinoderm fisher-

Corporation Final Report 93/221.

ies of the world: a review. _In_ :

**Scheibling, R. E. and Hatcher, B. G.**

Keegan, B. F. and O' Connor, B.

**(2001).** The ecology of _Strongylo-_

D. S. (eds.). Echinoderms, A.A.

_centrotus_

_droebachiensis_.

_In_ :

Bolkema, Rotterdam, **pp. 109–**

Lawrence, J. M. (ed.). Edible Sea

**124.**

Urchins: Biology and Ecology.

**Strathmann, R. R., Fenaux, L. and**

Elsevier, Amsterdam, The Nether-

**Strathmann, M. F. (1992).** Het-

lands, **pp. 271–297.**

erochronic developmental plastici-

**Schoppe, S. (2000).** A guide to common

ty in larval sea urchins and its im-

shallow water sea stars, sea ur-

plications for evolution of non-

chins, sea cucumbers and feather

feeding larvae. _Evolution_ **46, 972–**

stars (Echinoderms) of the Philip-

**986.**

pines. Times Editions, Singapore,

**Tan, L. W. H. and Ng, P. K. L. (1988).**

**p. 144.**

A Guide to Seashore Life. Singa-

**Seifullah, R. D., Ankudinova, I. A. and**

pore Science Centre, Singapore, **p.**

**Kim, E. K. (1995).** Seksual"noe

**160 _._** ****

provedenie muzhchin (sexual be-

**Taylor, P.H. (2004).** Green Gold: Scien-

havior of men). Izd. Yaguar, Mos-

tific findings for management of

cow.

Maine"s Green Sea Urchin fish-

ery. Maine Department of Marine

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 442

_Biotech Sustainability (2017)_

_Sea Urchin - A New Potential Bio-resource for Human Health Rahman et al._

Resources, Boothbay Harbour,

(ed.). Edible Sea Urchins: Biology

Maine, USA.

and Ecology, Elsevier, Amster-

**Tegner, M. (2001).** The ecology of

dam, The Netherlands, **pp. 161–**

_Strongylocentrotus_

_fransiscanus_

**175.**

and _Strongylocentrotus purpu-_

**Watts, S. A., McClintock, J. B. and**

_ratus_. _In_ : Lawrence, J. M. (ed.).

**Lawrence, J. M. (2001).** The

Edible Sea Urchins: Biology and

ecology of _Lytechinus variegatus_.

Ecology. Elsevier, Amsterdam,

_In_ : Lawrence, J. M. (ed.). Edible

The Netherlands, **pp. 307–331.**

Sea Urchins: Biology and Ecolo-

**Tuason, A. Y. and Gomez, E. D. (1979).**

gy. Elsevier, Amsterdam, The

The reproductive biology of _Trip-_

Netherlands, **pp. 375–394.**

_neustes gratilla_ Linnaeus (Echi-

**Warnau, M., Temara, A., Jangoux, M.,**

nodermata:

Echinoidea),

with

**Dubois, P., Iaccarino, M., De Bi-**

some notes on _Diadema setosum_

**ase, A. and Pagano, G. (1996).**

Leske. Proceedings of the Interna-

Spermiotoxicity and embryotoxici-

tional Symposium of Marine Bio-

ty of heavy metals in the echinoid

geography and Evolution in the

_Paracentrotus lividus_. _Environ-_

Southern Hemisphere. Auckland,

_mental Toxicology and Chemistry_

New Zealand **. 2, 707–716.**

**15, 1931e1936.**

**USEPA. (1994).** Methods for Derivation

**Yur'eva, M. I., Lisakovskaya, O. V.,**

of Inhalation Reference Concen-

**Akulin, V. N. and Kropotov, A.**

trations and Application of Inhala-

**V. (2003).** Gonads of sea urchins

tion

Dosimetry

EPA/600/8-

as the source of medication stimu-

90/066F. United States Environ-

lating sexual behavior. _Russian_

mental

Protection

Agency

_Journal of Marine Biolog_ y **29,**

(USEPA), Washington DC, USA.

**189–193.**

**Vasquez, J. (2001).** The ecology of _Lox-_

_echinus albus_. _In_ : Lawrence, J. M.

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 443

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P444-455_

**Marine Pollution and Its Impacts on Living Organisms**

****

**Thavasimuthu Citarasu* and Mariavincent Michael Babu**

_Centre for Marine Science and technology, Manonmaniam Sundaranar University,_

_Rajakkamangalam- 629 502, Tamilnadu, India; michaelmsu@live.com (MMB);_

_*Correspondence: citarasu@gmail.com_

**Abstract:** Marine environments are seriously affected by different pollutants created by

human activities. The main entry of pollutants to marine environment is through atmos-

phere, water bodies, ships and other human activities. The pollutants seriously affect the

marine flora, fauna and disturb the food supply chains. Several synthetic chemical residues

including carbon tetrachloride, polychlorinated biphenyls, trichloroethylene and vinyl chlo-

ride are found in the marine sediments and flora and fauna. This type of marine pollution

does have direct or indirect effect on human health. The biotechnological approaches such

as bioremediation, probiotics, waste treatments by micro algae and seaweeds are useful to

restrict and reduce the pollutants in the effluents before they reach the marine water bodies.

This article highlights various aspects of marine pollution and its impacts on living organ-

isms.Several pollution preventive measures and awareness programs are also discussed in

this chapter. ****

_**Keywords**_ **:** Marine biotechnology; marine ecosystem; marine pollution; microplastics; pol-

lution awareness

**1. Marine ecosystem and its resources**

goons, salty marshes, mangroves, deep

sea and sea floor ecosystems which are

The ocean occupy 71 percentages

important for marine (and terrestrial) liv-

of earth"s surface; they are interconnected

ing organism (Barange _et al_., 2010). They

and traditionally divided into four large

provide goods and various services to the

basins including North and South Atlan-

human society such as good climate, vital

tic, North and South Pacific, Arctic and

foods, medicines, bio processing and em-

Indian oceans. The average depths are

ployments including fishing, process in-

13,216, 10,932, 12,786 and 3,665 feet for

dustries, aquaculture and coastal tourism

Pacific, Atlantic, Indian and Arctic

etc.

oceans, respectively. Marine environment

****

is most important for life on earth, the

**2. Marine pollution**

living organisms originated in marine and

they emigrated to terrestrial and freshwa-

The oceans are susceptible for

ter bodies. Oceans are the main regulators

pollution ever by human activities by pol-

of climate and temperature to the terres-

luting with different agents and physical

trial ecosystem. Phytoplanktons are im-

destruction ocean environments. The def-

portant for oxygen production; they yield

inition of Marine Pollution as " _Introduc-_

around eighty percentages of oxygen

_tion by man, directly, or indirectly, of_

which is used by the animals and plants

_substances or energy to the marine envi-_

for breathing in terrestrial and aquatic

_ronment resulting in deleterious effects_

ecosystems (Bigg _et al_., 2003). Marine

_such as: hazards to human health, hin-_

ecosystem is the largest ecosystem with

_drance to marine activities, impairment of_

intertidal zones, coral reefs, estuaries, la-

_the quality of seawater for various uses_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 444

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ _and reduction of amenities_ ". Marine pol-their feathers (Kachel, 2008). The crude

lution is mainly classified to coastal

oil is highly toxic to the marine organisms

sources by riverine inputs, deposition

because of it contains toluene, xylene,

from atmosphere and offshore inputs etc.

benzene and polycyclic aromatic hydro-

Coastal diffuse sources are contaminants

carbons and these chemicals bioaccumu-

via coastal industry, sewages and devel-

lated to planktons, fishes, shellfishes, sed-

opment sites (Clark, 2001). Alternative

iments constitute a long lasting threaten-

inputs are site-specific discharges, agri-

ing to the benthic animals. Polycyclic Ar-

culture lands and forests, mainly for nu-

omatic Hydrocarbons (PAHs) is having

trients leakage to groundwater bodies,

many pathetic effects to the living organ-

further they transported into the marine

isms including mutagenic, carcinogenic

ecosystem by backwater bodied and final-

and act as a teratogens (Kachel, 2008).

ly huge quantities of contaminants depos-

ited marine environments (GESAMP _,_

_3.2. Persistent toxic substances (PTS)_

1993). Municipal wastes and sewage wa-

Persistent

Toxic

Substances"

ter effluents are not properly treated and

(PTS) also called Persistent Organic Pol-

discharged in to sea, these problems

lutants (POPs) are noxious, long-lived

mainly happened in developing countries.

and less persistent. The prolonged usage

Emissions through air craft"s are main

and dispersion of PTS may cause serious

sources of atmospheric pollution to sea

problems and responsible for chemical

and the pollutants are dispersed a vast ar-

and biological degradation. Their physical

eas by the wind flow and weather chang-

characteristics are chlorinated or halogen-

es. Marine pollution in offshore caused by

ated affected to water solubility levels and

discharges of pollutants from vessel based

high lipid solubility, no degradability

at least 10 % of the total marine pollution

leading to fatty tissues bioaccumulationsn

and they contribute by different ways

(El-Shahawi _et al_., 2010). POPs constitute

(Anon, 2005). The other sources are in-

remarkable societal advantages, or not

cluding crude oil extraction and of miner-

planned by-products of burning process-

al extraction etc.

es, such as dioxin. The halogenated hy-

drocarbons derivatives of tributyl tin

**3. Pollutant Sources**

(TBT), dibutyl tin and monobutyltin that

****

are the disruptors of endocrine organs

_3.1. Oil pollutants_

(Kachel, 2008).

Hydrocarbons are classified into

alkanes, naphthenes and aromatics. The

_3.3. Heavy metal pollution_

crude oil also contains nitrogen, oxygen

Through riverine input, heavy

and vanadium compounds. The spillage

metals are entering the sea and accumu-

of oils from the ships/ cargo, platforms of

late in marine sediments as well as flora

offshore oil and on-shore refineries, it

and fauna. The important heavy metals

may be produced serious effects to the

are mercury (Hg), cadmium (Cd) and lead

marine environments in multiple ways.

(Pb), accumulations are important for tox-

The causes of oil pollution producealtera-

icity (Burger and Gochfeld, 2002). They

tions in physic chemical levels, also in-

generally share the features of PTS, be-

volved toxication of marine habitats and

cause they are bioaccumulate, non-

the flora and fauna seriously affected af-

degradable and generate stringent or long

termath of large spills. The turbidity of oil

standing toxic effects. The toxicity effect

prevents the light penetrations and lead to

are vary based on the heavy metal types,

photosynthesis processes for phytoplank-

for example if absorption of mercury as in

tons. Lose of waterproofing qualities

very little doses, cause severe harmto

faced by the larger animals, aquatic birds

brain and the central nervous system. The

getting lost of waterproofing by losing

main sources of oceanic and atmospheric

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 445

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ contamination heavy metal contamina-ing the toxin secretion from the microbes

tions provided by electric utilities, fuel

and harmful algal blooms in the marine

combustion, iron manufacturing, incinera-

water create other environmental prob-

tion of urban refuse and fuel additives.

lems. Environmental changes causespar-

Marine anti-fouling paints are very dan-

ticular animals can change their living

gerous to marine organisms because it

place to alternate place due to warmer

contains. Lead is another big poison cre-

waters and reproduction also affected by

ates the problems like neurotoxicity, men-

increasing temperature. Marine organisms

tal health problems, lead is mainly re-

lay of undeveloped eggs or the tempera-

leased from batteries, sewage and fuel

ture prevents the normal development of

combustions. Electroplating factories,

particular eggs due to rise of temperature

batteries and sewages are also responsible

level in marine waters. Enzymatic activity

for cadmium toxicity and it affects bones

and metabolic rate also raised and in-

by deformities and kidney dysfunction.

creased food consumption due to increase

Nickel poisoning is also a potent carcino-

temperature. This is seriously affecting

gen that is toxic at relatively low concen-

the stability of food chain and modifies

tration. Crude oil contains selenite with

the balance of species composition (Brett,

sulfur, reported as a toxic metal found out

1970).

in marine environment at a huge level.

The other dangerous heavy metals con-

_3.5. Nuclear radiation_

taminations released through various

In marine environments, radia-

routes including rain of pollutants from

tions may classified in to natural and hu-

the atmospheric air, fallout from ship de-

man activities, natural radiations are

struction and contaminated land runoff

emission of cosmic rays, potassium-40

creating dangerous environmental prob-

by earth's crust and decay products of

lems (Kennish, 1998).

uranium etc. Human activities including

__

nuclear power plants and reprocessing,

_3.4. Thermal pollution_

nuclear weapons testing and accidents,

Nuclear reactors release huge

fallouts of nuclear wastes, radiation using

amount of heat to the marine environ-

food conservation, medical diagnosis

ments leading to decreased level of oxy-

combustion, land-based mining, phos-

gen can disastrous effects on ecosystems

phate production, and oil exploration etc

and its communities.The decreased level

(Shinde and Gawande, 2015). High level

of oxygen in marine water, degrade the

radioactive waste dumping is not permit-

quality of wildlife animals that lives un-

ted in the ocean, even low level wastes is

derwater. The increased temperature

still permitted to the marine trenches, be-

holds less oxygen and creates suffocation

cause low level wastes containing low

to marine fauna including copepods and

radioactivity than high level waste. High

amphibians (Gautam _et al.,_ 2016). Indus-

level nuclear waste had half-life for

tries are responsible for decrease the qual-

24,100 years whereas the short-lived ra-

ity marine life and destroy habitats by al-

dioactive elements had the half life of 30

tering the natural environments. Natural

years. The radioactivity may absorbed by

destruction including geothermal activity

micro algae, zooplankton, and other small

and volcanoes under the marine can in-

marine organisms and then this will be

duce warm lava to increase the tempera-

transmitted or biomagnified to the food

ture of marine waters. Thunders and

chain, to fish, marine mammals, and hu-

lightening is also responsible for tremen-

mans. In human concerns, the radiations

douslevel of heat into the marine that

cause cancer, changes in "DNA" that en-

leads to raise the temperature of marine

sures cell repair (Kachel, 2008).

water. The high temperature discharge

from industries is responsible for induc-

_3.6. Excess nutrients_

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 446

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ Emissions and discharges of agri-get the diseases such cholera, typhoid etc

culture sources are a significant pollu-

(Yazhini _et al_., 2015).

tions to

the coastal

zone particularly affects biogeochemical

_3.8. Noise pollution_

cycles and primary producers. Eutrophi-

Life in marine can be susceptible

cation is created by the pollutants includ-

for noises from different noise pollutants

ing discharge of ammonia and methane,

including oil exploration, seismic surveys,

use of insecticides and other pesticides

passing ships, and naval low-frequency

affecting marine flora and fauna. Excess

sonar waves. Sound is very fast in water

nutrients discharges including phosphorus

than in the atmosphere and the noise pol-

and compounds are responsible to eu-

lution in marine are increased at ten folds

trophication. High concentrations of nu-

during the period of 1950 to 1975. Marine

trients, based on the physico-chemical

mammals including dolphins, porpoises

properties of the affected marine envi-

and whales are using sound as communi-

ronments, may lead to increased growth

cation and sensation because sound is not

of phytoplanktons (Sarkar and Malchow,

or less effective for their communications.

2005). Hydrogen sulphides also induce

Also the particulates scattering the light

the negative impacts, if the oxygen level

and it interferes the communications in

decreases, hydrogen sulphides levels also

marine mammals. Also it is impossible to

increased. Hydrogen sulphides induced

using smell as communications, because

low resistance to the aquatic living organ-

the chemical molecules in water diffuse

isms leading to die off. The dead micro

more slowly than in air are not possible

algae due to hydrogen sulphide toxicity

(Payne, 1983). Sound speed is four times

floats on the sea surface leading to inter-

higher in water than atmosphere so it is

fere the penetration of sunlight. Fertilizer

opt to communicate the marine mammals.

runoffduring heavy rainthe organic ferti-

Sonar waves is very dangerous to marine

lizers run off from the agricultural field

mammals it interfere the sound wave

and it affects the marine environment and

communications and causes severe inju-

back water bodies.

ries including lose of sense organs leading

to death. Also the sounds emanated from

_3.7. Microbial contamination_

the passing ships other instruments for

Generally microorganisms are re-

marine mining activities also disturbed

leased from waste water, waste products

the dolphin and whale populations.

and sometimes human and animal wastes

into the environment seriously spoiled the

_3.9. Ship based threatening_

marine ecosystem. The viruses do not

Ship based threatening mainly

replicate without the help of host animals.

caused by operationally or accidentally

First it may infect some other host cells

and releases the pollutants to the marine

and multiply. Seafood contaminations are

ecosystem and these pollutants mainly

the important pollution. The effluents

damage the flora and fauna. The pollu-

from fish processing units, shrimp farms,

tants also somewhat released more in ship

hatcheries are creating big problems. The

are on voyage than accidental ships. The

effluents contain serious bacterial patho-

pollutants released including tank resi-

gens such as salmonella, pathogenic Vib-

dues, bunker oils, chronic discharge of

rio, WSSV and the fungal pathogen

sewage, garbage, exchange of ballast wa-

Fusarium. Due to the broader host range

ter, other emissions from vessel and anti-

of these pathogens it may infect very easi-

fouling paints etc. The sewage discharges

ly in the marine animals and affect the

causes severe problems including micro-

food chain. This also affects the consumer

bial pollution especially bacterial infec-

levels very easily and there is an easy to

tions it will be seriously damage the bio-

diversity, fisheries and food chains etc.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 447

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ The excess nutrient discharges also affect

and deposited the sediments may add

the primary production etc (Katchel,

dead organic matter load. Further dead

2008). Pollutants are released by acci-

organic matter may cause bad effects to

dental pollution including contacts with

the normal microbial flora and depleted

external objects, due to collisions, cargo-

the oxygen and this will affect the de-

transfer failures, sinking or loss of cargo,

composition process. Shrimp farm efflu-

explosions and groundings etc. It is also

ents including algal bloom die off, large

very hazardous, the toxic materials spill-

amount of dead organic load accumulate,

ing from cargoes which carry large quan-

affect filter feeding animals, shrimps and

tities. The oil tankers happened in acci-

lobsters.

dent may spill larger quantities of crude

__

oil to the marine environments. The oil

_3.11. Ocean mining_

may drift to the sea shore and seriously

Deep sea mining is the mineral re-

affected the ecosystem particularly the

trieval process from the ocean floor by

rocky animals including oyster and mus-

disturbing the marine organism from ben-

sel beds, backwater etc. The places which

thic regions. Ocean mining takes place

affected by oil spills may suffer a long

about from 1,400 to 3,700 meters below

times, until the oil pollution completely

the oceans" surface for extinct or active

degraded or solved (Katchel, 2008). The

hydrothermal vents (Ahnert and Bor-

hydrocarbons form the oil spills pene-

owski, 2000). The mine deposits are drill

trates the marine and related ecosystems

out from the ocean deeps by hydraulic

sediments alter the community structure

pumps or buckets that take ore to the sur-

especially the phytoplanktons and worms.

face. Removal of marine floors disturbs

Ship can also harm to the marine habitats

the benthic habitats. Removing parts of

and wildlife by damaging the marine an-

the seafloor disturbs the habitat of benthic

imals by physical destruction by anchor-

organisms depending upon the places and

ing. Anchoring may seriously affected by

type of mining; sometimes it may disturb-

the coral and sponge beds. Another phys-

ances to the marine benthos permanently.

ical hindrance including collisions by

Leakage, corrosion and spilling might be

ships, ship"s propellers and ship strikes

altering the area of mining bycontamina-

are cause the damages and death to the

tionof chemicals. Surface plumes may

marine mammals including whales etc.

cause more serious effects and based on

__

the particles size and water currents by

_3.10. Harmful algal blooms (HABs)_

the plumes could spread over vast areas

Harmful

algal

bloom (HABs)

of the floor.Sand mining is a prac-

causes pathetic effects to other marine

tice mined from beaches, inland dunes

animals by mechanical damage through

and dredged from ocean beds and river

the secretion of toxins.HABs are involved

beds. Sand mining also emits radiation

in large-scale marine mortality of marine

problems and pathetic effects to the living

animals and they secreted with different

organisms in sea shore peoples (Pitchaiah,

toxins responsible for shellfish poisonings

2017).

(Nancy, 2012). Red tides caused by

__

HABs that can affect estuarine and salt

_3.12. Plastics and polythenes_

lakhs areas by depletion of oxygen, in-

Plastic pollution is one of the seri-

creasing the carbon dioxide and secrete

ous concerns in recent years because the

toxins. Thousands of harmful algal spe-

plastic pollutions in ocean become signif-

cies were identified among that one hun-

icant environmental issues related to gov-

dred species ware produce toxins that will

ernmental and nongovernmental organi-

seriously affect the shellfish species by

zations, scientists and members of the

ingested the HABs by filter feeding. The

public worldwide. The main risk to ma-

excess amount algal bloom may dies off

rine biota posed by different activities in-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 448

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ cluding marine vessels, commercial fish-head sea turtles, _Caretta caretta_ from

ing, marine-industries and recreational

Central Mediterranean sea (Gramentz,

coastal tourism. These are the major

1988). Polythenes is the another danger-

sources for generating the plastic and pol-

ous pollutants Generated from household,

ythene wastes hat can directly enter the

industrial wastes and Recreational beach-

marine environment. During World War

es. Sea turtles mistakenly easting the pol-

II end, approximately eighty percent of

ythene bags. During degradation of poly-

the marine debris is considered as plastic

thenes, it releases phenol, bisphenol,

wastes.

phathalate and gases it causing cancer,

Marine vessels have been reported as a

heart failures in humans.

major contributor of marine litter and it is

estimated that the commercial fishing

_3.13. Microplastics_

fleet dumped more than 23,000 tons of

Microplastic

contamination

in

plastic packaging materials during 1970s

ocean has been a serious issue nowadays

(Pruter, 1987). The waste materials in-

with harmful effects to marine biota.

cluding nylon netting, plastic monofila-

They are very tiny plastic granules de-

ment line and discarded or waste fishing

rived from the breakdown of macroplas-

gears are neutrally buoyant can therefore

tics and the plastic particles used in cos-

drift at variable depths within the oceans.

metics and detergents. Primary microplas-

The waste plastic materials are problem-

tics in personal care and cosmetic prod-

atic to marine animals because they cause

ucts are a minor source of releases of mi-

entanglement of marine biota, known as

croplastics to the environment. Micro-

""ghost fishing"" (Lozano and Mouat,

plastics which used in detergents, cosmet-

2009).

ics and also in air-blasting media are en-

Waste six pack rings, plastic bags

tering the freshwater bodies and reached

and other forms of plastic waste cause

to domestic or industrial drainage systems

dangerous effects to wildlife and fisher-

(Derraik, 2002). Most of the microplastic

ies. Plastic fishing nets can be lost or left

granules are trapped the waste-water

by fishermen to the ocean are dangerous

treatment plants unfortunately the small

and create so many problems including

microplastic granules from cosmetics and

entangle to fishes, sea turtles, sharks, dol-

detergents are passing through such filtra-

phins, crocodiles, crabs, seabirds, and

tion systems and finally reached the ma-

other creatures, restricting movement,

rine environments (Browne _et al_., 2007).

causing starvation and infection etc. Plas-

The small size nature, microplastics are

tic waste debris during bulky or tangled, it

considered bio-available to marine organ-

is very difficult to move, and may become

isms throughout the food-web. Microplas-

permanently trapped in the digestive

tics in intertidal sediments have been

tracts of marine animals finally leading to

shown to reduce the thermal conductive

blocking the food passage and causing

properties and alter drainage. Due to the

death through infection or starvation.

tinny size and availability of microplas-

Moser and Lee (1992) conducted a study

tics in pelagic and benthic ecosystems,

for plastic pollution in sea birds, they col-

they have easily ingested by the marine

lected 1033 birds and among the birds,

biota including microalgae, crustaceans,

fifty five percentages of the birds guts

fishes and sea birds etc (Blight and Burg-

containing plastic particles. Carpenter _et_

er, 1997). Devriese _et al._ (2015) studied

_al_. (1972) observed different species of

the microplastic contamination in the

fishes had plastic wastes in their guts es-

brown shrimp, _Crangon crangon_ sampled

pecially white plastic spherules had been

Europe and the study revealed that, the

ingested, indicating that they feed selec-

shrimps are heavily contaminated with

tively. The same pattern of white plastic

microplastics. Ingestion of microplastics

debris ingestion also observed in logger-

may release toxins to the food chain and

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 449

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ lead to the bioaccumulation and biomagnifications. Additives used in microplas-

_3.15. Coastal tourism_

tics

such

aspolybrominateddiphenyl

Coastal tourism creates so many

ethers, phenol and bisphenol-A are inhibit

harmful effects to the marine environ-

the synthesis of endogenous hormones

ments adding pollution by waste disposal,

leading to affect the reproduction.

input of local waste structures and habi-

Phthalates are associated with broad

tats under enormous pressure. For new

range of genotoxic damage including mi-

tourists, over developments including

cronuclei and apoptosis in mussel haemo-

construction of resorts, marinas, golf

cytes, suppressed the locomotion in inver-

courses and airports etc are not advisable

tebrates and intersex conditions in fishes

to clean environments. For the new de-

(Oehlmann _et al_., 2009). Consumption of

velopments peoples removed the man-

microplastics and nano-plastics by hu-

grove forests and sea grass meadows.

mans through marine foods including

Piers and other related structures are built

shrimps, shellfish and small fishes may

directly from the top of coral reefs related

cause health problems. Ingestion of mi-

to the tourist developments is not advisa-

croplastics by young fishes causing

ble and spoil the marine environments.

deaths, stunted growth, altering behavior,

The overcrowding of tourists in the

sometimes killed and prevented from

beaches may affect the nesting sites of the

reaching maturity.

marine endangered turtles by destruction.

__

Resorts from beaches may discharges

_3.14. Ocean acidification_

their sewage effluents directly to the ma-

The oceans are act as a carbon

rine waters which is seriously affect the

sink, because they absorbing more carbon

coral reefs and other sensitive marine

dioxide from atmosphere. If the carbon

habitats. Coral reefs also damaged by dif-

dioxide levels increased in the oceans the

ferent tourist"s activities including fish-

Ocean become more acidic. The acidic

ing, snorkeling, diving and careless boat-

nature is seriously affected by the for-

ing etc. Boating and other human activi-

mation of calcium carbonate in corals,

ties also disturbed the marine animals in-

shellfishes including shrimps, oysters,

cluding seals, dugongs, whales, whale

mussels, clams and other molluscs etc

sharks, dolphins and marine birds. Over-

shells (Caldeira and Wickett, 2003). The

fishing in a particular area for seafood

higher acidity in the seawater also creates

consumption affected local fish popula-

more pathetic effects to the marine organ-

tions(Sunulu, 2003).Tourists also dis-

isms in combinations with environmental

charges the polythene bags, bottles and

stressors including higher ocean tempera-

other plastic materials to the beach by

ture and other pollutants etc. Marine or-

improper disposal and it reaches to the

ganisms like algae and zooplanktons us-

sea create so many environmental prob-

ing carbonate ions to produce calcium

lems. Tourism can cause pollutions in-

carbonate shells and skeletons. Unfortu-

cluding solid waste and littering, releases

nately, the acidification leads the less

of sewage, oil and chemicals, air emis-

availability of carbonate ions in marine

sions, noise and even architectural activi-

waters and it interfere the formation of

ties. Tourism development of marinas and

shells. Also the ocean acidification inter-

breakwaters also can changes in currents

fere the iron, phosphorous, nitrogen and

and coastlines. ****

other elements absorption from marine

__

waters by the marine organisms for their

_3.16. Ballast water_

vital growth. More acidity interfere the

Ships need ballast to maintain the

attachment of iron to other organic com-

balance successfully and safely. Dis-

pounds may hindrance to the normal ma-

charges of ballast waters from ships to

rine life.

marine environment have negative im-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 450

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ pacts to various ways. Huge quantities of

living organisms and passes through the

waters are loaded by cruise ships, cargos

food chain.

and large tankers from one place for bal-

__

ancing and discharge to some other plac-

_3.18. Marine littering_

es. Ballast water have various biological

Littering to marine environment is

materials such as seaweeds, sea grass,

a global concern, they affect ecosystem

phytoplanktons, zooplanktons, small fish-

very seriously. Several million tons of

es, invertebrates, microbes including bac-

litters are discharged to the worldwide

teria, fungi and virus etc. The biological

every year creating economic, environ-

materials are nuisance, non-native and

mental, health and aesthetic issues

exotic species can create extensive eco-

(Strieker, 1998).The important land based

logical and economic problems to the

disposals including industrial outfalls,

aquatic ecosystem with human health is-

rivers and floodwaters, discharge from

sues etc. The important preventive

storm water drains, untreated municipal

measures for treating the ballast water

sewerage, land-fills and littering from

bytreating with ultraviolet irradiation, fil-

coastal tourism. The marine based litters

tration, heat, gas super saturation and

including shipping transport, offshore

electrical fields etc (Silva _et al_., 2004),

mining and extraction, fishing industry,

chemical treatments byiodides, borates,

illegal dumping at sea and discarded fish-

chlorates, ozone, ionization, copper ions,

ing gear etc. The vast majority of marine

hydrogen

peroxide,

adjustment

of

litter is plastic, which never truly breaks

pH,changes in salinity for eradication of

down. Another serious problem is nuclear

organisms, ozonization and deoxigeniza-

waste disposal to the marine trenches,

tion etc (Wright _et al_., 2003).

Mariana

trench

is

the

main

site

__

for nuclear waste disposal because of its

_3.17. Natural calamities_

huge depth 36, 201 feet which is situated

Hurricanes and floods can induce

in the western Pacific Ocean, to the east

waste transportation from land to the ma-

of the Mariana Islands (Sheavly and Reg-

rine environment. Earthquakes and tsu-

ister, 2007). Ocean is used as a dumping

nami can cause ground, air, and water

ground for disposing the nuclear materials

pollution (Shaw, 2006). The pollutants

for many decades after Second World

including detergents, chemicals from pro-

War II, and then it was banned interna-

cessing plants, farm wastes and fertilizers

tionally. Thirteen nuclear capable coun-

from crops are swept downstream and

tries are dumped their nuclear/radioactive

deposited to marine through large river

waste including started from 1946 to

floods. Volcanic eruptions have also been

1993. The important nuclear waste mate-

creating fluorine-containing compounds

rials are medical products, industrial

deposition to sea and also harm to the ma-

products, weapons, house hold containers,

rine flora and fauna. High amount of min-

reactor vessels, with and without spent or

erals are accumulated to the marine envi-

damaged nuclear fuel etc. The discarded

ronments from demolishing of the build-

wastes emit nuclear radiation leading to

ings, bridges during natural disasters and

health issues to the marine living organ-

small amount of hazardous materials

isms.

reached to marine environments that

threatening to marine living organisms.

**4. Impact of pollutants to marine life**

Heavy floods in rivers also washed out

****

the sewages to the accumulated in the ma-

The pollutants which are affect a

rine environments that contains pathogen-

broad range to the environments as well

ic bacteria fungi and virus which causes

as the living organism in the entire world

serious pathological effects to the marine

directly or indirectly. Climate changes

are spoiled by extreme weather conditions

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 451

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ as well as rising of sea level, sea surface

pesticides may change hormonal system,

warming by temperatures and ocean acid-

reduce fertility, weaken immune system

ification. Corals reefs are important for

and create cancer. The degraded deriva-

preventing acidification etc, loss by an-

tives like phenol, bisphenols and phath-

thropogenic stress activities, collection

alates also creating heart diseases and

and recreational activities and coral beds

cancers to humans by consuming sea

may be affected. Mangrove forest is im-

foods.

portant for preventing barrier for the natu-

ral calamities like tsunami. Degradation

**5. Remedies to solve the pollutants by**

of mangroves by over exploitation may

**biotechnological approach**

affect the marine environments. Illegal

and over exploitation of capture fisheries

Bioremediation is defined as _"The_

by trawlers may spoil the fishery re-

_act of adding materials to contaminated_

sources leading to decline the particular

_environments such as oil spill sites, to_

fish species. The pollution in beaches by

_cause an acceleration of the natural bio-_

coastal tourism causes several environ-

_degradation process"._ The microbes uti-

mental impacts and the oil pollution caus-

lize the nutrients from the polluted water

es reduction in benthic organisms. Biodi-

bodies and reduce or neutralize the pollu-

versity is important for produce organic

tants. The extremophilic microbes includ-

material, decompose organic material,

ing halophilic, thermophilic, acidophilic

capture and store energy, cycling water

and alkaliphilic are useful to bioremedia-

and nutrients and helps to regulate climate

tion purposes for to neutralize the pollu-

and atmospheric gases. The pollutants

tants, because they withstand high tem-

seriously interrupt the food chains leading

perature, low and high pH and higher sa-

to several problems. In concern with the

linity. The polluted effluents/ water bod-

human health point of view, the chemical

ies, fish farm effluents, effluents from any

poisoning in food chain creates the bioac-

waste sources may remediate with mi-

cumulation and biomagnifications prob-

crobes before reaching the water bodies

lems (Table 1). The harmful chemical

like river, back water may help to reduce

contamination in seafood crate several

or remove the pollutants and solve the

problems to humans. The pathogenic bac-

pollution problems. Genetically modified

teria including pathogenic vibrios, salmo-

microbes like super bug also helps de-

nella contaminations from waste water

grade the oil from oil contaminated water

disposal from fish processing industry

bodies.

may cause the disease problems in the

The water probiotic microbes like

fishes/ shrimps from the sea. The con-

_Lactobacillus_ sp and _Bifidobacterium_ sp

sumption of infected fish/ shrimp may

also clean the aquaculture ponds by re-

cause diseases to humans. The residue of

ducing or removal pathogenic microbes

****

**Table 1:** Impact on human heath by some synthetic chemicals detected in ocean

No

Chemical residues

Human health impacts

1.

Benzene

Anemia, blood disorders and chromosomal damage

2.

Carbon tetrachloride

Cancer; central nervous system, liver, kidney and

lung damages

3.

Dioxin

Cancer and skin disorders

4.

Ethylene dibromide

Sterility and Cancer

5.

Polychlorinated biphenyls Kidney, lung and liver damages

6.

Trichloroethylene

CNS, cancers, liver and kidney damage and skin

problems

7.

Vinyl chloride

Cardiovascular problems, liver, kidney, and lung

damages and gastrointestinal problems

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 452

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ and unwanted physicochemical parame-dress the pollution problems, especially

ters which solve the effluent contamina-

for school students and create awareness.

tion to the marine environments. The mi-

Because school students and youngsters

cro algae like _Chlorella_ sp., _An-_

take their responsibility and awareness to

_kistrodesmus_ sp. are also useful to remov-

the public peoples, families and related

al of excess nutrients and Co2 in waste

communities related to the marine pollu-

water systems, Solve BOD problems by

tions and the future conservations etc.

releasing oxygen which will help to efflu-

Coastal cleanup is the major awareness

ent treatment. The seaweeds like _Sargas-_

programme,

the

International Coastal

_sum_ sp also helpful to chelate the heavy

Cleanup is one of the largest volunteer

metal contaminates in the water bodies

program in worldfor cleaning the coastal

especially the effluents. These types of

areas. Volunteers are clean or remove

treatments which restrict the polluted ef-

trash from the coastal areas especially

fluent water to the marine environments. ****

beaches the entire world. Waste manage-

****

ment is also an important strategy for re-

**6. Suggestions to protecting marine en-**

strict the entry of pollutants through ef-

**vironment**

fluents to the clean water bodies. Indus-

****

tries are advised to set up waste proper

Several measures for protecting

treatment plants, minimize waste for

the marine environments from the pollu-

adopting suitable measures, waste recy-

tants including legislation for plastics and

cling and reuse and recovery of waste wa-

polythenes, set standards protocols for

ter effluents.

sewage and other effluent discharges, low

level use of pesticides, coastal cleaning

**7. Concluding remarks**

programmes,

public

awareness,

re-

strictions of polluter pays principal and

The entry of different pollutants

implementation of laws pertaining to pre-

from atmosphere, through water bodies,

vention by strictly and coastal zone man-

by ships, from beaches and other human

agement activities before constructing

activities are very harmful to the marine

new industries on the coast. Also strictly

environments and affect all marine flora

advice to the peoples for following the

and fauna. The pollutants also directly or

marine act s including National Marine

indirectly affect the human community

sanctuaries Act of 1972, Fisheries Man-

through food chain. Among the pollutants

agement and Conservation Act – 1976,

plastics and micro plastics are the serious

Clean Water Act of 1977, Endangered

concerns to the marine environments and

Species Act, Oceans Act of 2000, Estuar-

affect lower to higher organisms and indi-

ies and Clean Waters Act of 2000 and Es-

rectly affect human"s health. Restriction

tuaries and Clean Waters Act of 2000. It

or reducing the pollution is in our hand,

is also important to conserve the marine

we can take care of the environments by

ecosystem and marine animals we must

avoiding pollution to save our environ-

follow the international marine legislation

ments for future. Several pollution pre-

such as Convention on the Prevention of

ventive measures can be adopted includ-

Marine Pollution by Dumping wastes and

ing picking up and disposing various

Other Matter. One of the important legis-

types of litters at proper place; by reduc-

lation, Protocol to the International Con-

ing, reusing and recycling the waste mate-

vention for the Prevention of Pollution

rials; bring reusable, biodegradable bags

from Ships (MARPOL) address the ma-

to the grocery stores; disposing properly

rine pollution problem caused by marine

the trash; promoting awareness among

vessels to the marine environment in 1978

your friends and family; restrict the usage

(Lentz, 1987). Education is also a very

of plastic bags; awareness programs to the

important and powerful criterion to ad-

public especially the school children, un-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 453

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ educated publics to restrict / stop the pol-Caldeira, K. and Wickett, M. E. (2003).

lution will definitely help in minimizing

Anthropogenic carbon and ocean

the pollution.

pH". _Nature_ **425 (6956), 365–365.**

**Carpenter, E.J., Anderson, S.J., Har-**

**References**

**vey, G.R., Miklas, H.P. and Peck,**

****

**B.B. (1972).** Polystyrene spherules in

**Ahnert, A. and Borowski, C. (2000).**

coastal waters. _Science_ **178, 749–750**

Environmental risk assessment of an-

**Clark, R.B. (2001).** Marine Pollution.

thropogenic activity in the deep-

GESAMP, Protecting the Oceans

sea". _Journal of Aquatic Ecosystem_

from

Land-Based

Activities,

_Stress and Recovery_ **7 (4), 299–315**.

_GESAMP Report and Studies_ No. 71

**Anonn (2005).** Land-based activities ac-

. **p. 17.**

count for roughly 80 per cent of re-

**Derraik, J.G.B. (2002).** The pollution of

leased pollutants; cf. UN Doc.

the marine environment by plastic

A/60/63, Oceans and the Law of the

debris: a review. _Marine Pollution_

Sea – Report to the 60th session of

_Bulletin_ **44, 842–852**

the General Assembly, 4 March

**Devriese, L.I. , van der Meulen M.D, ,**

2005, **p. 104.**

**Maes , T. , Bekaert, K.B , Pont,**

**Barange, M., Cheung, W. W. L., Meri-**

**I.P. , Frère, L., Robbens, J. and**

**no, G. and Perry, R. I. (2010).**

**Vethaak, A.D. (2015).** Microplastic

Modelling the potential impacts of

contamination in brown shrimp

climate change and human activities

( _Crangon crangon_ , Linnaeus 1758)

on the sustainability of marine re-

from coastal waters of the Southern

sources. _Current Opinion in Envi-_

North Sea and Channel area. _Marine_

_ronmental_

_Sustainability_

**2(5-6),**

_Pollution Bulletin_ **1(2), 179-87**

**326–333.**

**El-Shahawi, M.S., Hamza, A., Ba-**

**Bigg, G.R., Jickells, T.D., Liss , P.S.**

**shammakhb, A.S. and Al-Saggaf,**

**and Osborn, T.J. (2003).** The role

**W.T. (2010).** An overview on the

of the oceans in climate. _Int. J. Cli-_

accumulation, distribution, transfor-

_matol_. **23, 1127–1159**

mations, toxicity and analytical

**Blight, L.K. and Burger, A.E. (1997).**

methods for the monitoring of persis-

Occurrence of plastic particles in

tent organic pollutants. _Talanta_ **80,**

seabirds from the eastern North Pa-

**1587–1597.**

cific. _Marine Pollution Bulletin_ **34,**

**Gautam, N., Rai, P. and Chandra, H.**

**323–325**

**(2016).** A case study on environmen-

**Brett,**

**J.R.**

**(1970).**

Temperature-

tal air pollution and control methods,

Animals-Fishes. Marine Ecology,

3rd International conference on sci-

Vol.1. Environmental Factors, Part 1.

ence, technology and management,

**pp. 515-560**.

India international centre , New Del-

**Browne, M.A., Galloway, T. and**

hi.

**Thompson, R. (2007).** Microplastic

**GESAMP (1993).** _Supra note_ 106, **p.50.**

– an emerging contaminant of poten-

**Gramentz, D. (1988).** Involvement of

tial concern?. _Integrated Environmen-_

loggerhead turtle with the plastic,

_tal Assessment and Management_ 3,

metal, and hydrocarbon pollution in

**559–561**

the Central Mediterranean. _Marine_

**Burger, J. and Gochfeld, M. (2001).**

_Pollution Bulletin_ **19, 11–13.**

Heavy metals in commercial fish in

**Kachel, M.J. (2008).** Particularly sensi-

New Jersey. _Environ. Res_. **99, 403-**

tive Sea Areas, the IMO"s Role in

**412**

protecting vulnerable marine areas,

International Max Plank research

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 454

_Biotech Sustainability (2017)_

_Marine Pollution and Its Impacts on Living Organisms Citarasu and Babu_ school of marine affairs at the uni-plankton control algal blooms – a

versity of Hamburg.

spatio-temporal study in a noisy en-

**Kennish M.J. (1998).** Pollution impacts

vironment. _J. Biosci._ **30(5), 749–760**

on marine biotic environments; CRC

**Shaw, R. (2006).** Indian Ocean tsunami

Press. **pp. 310.**

and after-math: need for environ-

**Lentz, S.A. (1987).** Plastics in the marine

ment-disaster synergy in the recon-

environment: legal approaches for in-

struction process _. Disaster Preven-_

ternational action. _Marine_ _Pollution_

_tion and Management_ **7(1), 5-20.**

_Bulletin_ **18, 361–365**

**Sheavly, S. B. and Register, K. M.**

**Lozano, R.L. and Mouat, J. (2009).** Ma-

**(2007).** Marine Debris & Plastics:

rine litter in the North-East Atlantic

Environmental Concerns, Sources,

Region: Assessment and priorities

Impacts and Solutions. _Journal of_

for response. KIMO International.

_Polymers_

_and_

_the_

_Environ-_

**Moser, M.L. and Lee, D.S. (1992).** A

_ment_. **15 (4), 301–305.**

fourteen-year survey of plastic inges-

**Shinde,**

**R.**

**and**

**Gawande,**

**S.**

tion by western North Atlantic sea-

**(2015).** Ocean pollution and its per-

birds _. Colonial Water birds_ **15, 83–94**

spective. _International journal of en-_

**Nancy,**

**D.**

**(2012).**

Phytoplankton

_gineering and general science_ , **3,4**.

Blooms:

The

Basics.

NOAA

**Silva J.S.V., Fernandes, F.C., Souza,**

FKNMS.

**R.C.C.L., Larsen, K.T.S., Danelon,**

**Oehlmann, J., Schulte-Oehlmann, U.,**

**O.M. (2004).** Ballast Water and

**Kloas, W., Jagnytsch, O., Lutz,I.,**

Bioinvasion.

Rio

de

Janeiro:

**Kusk, K.O, Wollenberger, L, San-**

Interciencias, **pp.1-10.**

**tos, E.M., Paull, G.C., Van Look,**

**Strieker , G. (1998).** __ Pollution invades

**K.J.W. and Tyler, C.R. (2009).** A

small Pacific island. CNN. __

critical analysis of the biological im-

**Sunlu U. (2003).** Environmental impacts

pacts of plasticizers on wildlife. _Phil._

of tourism.CIHEAM, **pp. 263-270**

_Trans. R. Soc. B._ **364, 2047–2062**

**Wright, D.A., Dawson, R., Mackey,**

**Payne, R. (1983).** Communication and

**T.P., Cutler, H.G., Cutler, S.J.**

behaviour of whales. Westview

**(2003).** Some shipboard trials of bal-

Press.

last water treatment systems in the

**Pitchaiah, P.S. (2017).** Impacts of Sand

United States. International Ballast

Mining on Environment – A Review.

Water

Treatment

Symposium,

_SSRG International Journal of Geo_

Globallast Monograph Series, Lon-

_informatics and Geological Science_.

don, **pp. 15.**

**4(1): 1.**

**Yazhini , R., Pavithra , M., Suganthy ,**

**Pruter, A.T. (1987).** Sources, quantities

**C., Elumalai, E.K. and Ganesh-**

and distribution of persistent plastics

**kumar, G. (2015).** Microbial con-

in the marine environment. _Marine_

tamination of sea water at Puducher-

_Pollution Bulletin_ **18, 305–310**

ry sea shore. _Malaya Journal of Bio-_

**Sarkar, R.R and Malchow, H. (2005).**

_sciences_ ,

**2(2),**

**115-118.**

Nutrients and toxin producing phyto-

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 455

**Biotechnology for Sustainability**

__

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P456-474_

**Ecology, Distribution and Diversity of Bioluminescent**

**Bacteria in Palk Strait, Southeast Coast of India**

****

**Srinivasan Rajendran1, Ganapathy selvam Govindarasu2 and Govindasamy**

**Chinnavenkataraman1,***

_1Department of Oceanography and Coastal Area Studies, School of Marine Sciences, Ala-_

_gappa University, Thondi Campus, Thondi – 623 409, Ramanathapuram District, Tamil_

_Nadu, India; 2Division of Algal Biotechnology, Department of Botany, Annamalai Universi-_

_ty, Annamalainagar, Chidambaram, Tamil Nadu, India;_

_*Correspondence: sandalsrini@gmail.com; Tel.: +91 9788780266_

**Abstract:** The present study was carried out to determine the influence of ecological char-

acteristics and bioluminescent bacterial distribution in the seawater and sediment of Palk

Strait, Southeast coast of India during July 2010 - June 2012. The physico-chemical param-

eters such as., atmospheric temperature range was varied from 24.3 - 35.3°C, surface wa-

ter temperature 23.2 - 33.5°C, pH (6.2 - 8.91), dissolved oxygen concentration ranged from

3.84 - 7.73ml l-1, salinity fluctuated between 23.12 and 39 ppt, POC concentration ranged

from 0.78 - 2.56mg dry wt.l-1. The seawater was analysed and results suggest that it con-

tains _,_ inorganic phosphate (4.12 - 21.6μM), reactive silicate (4.3 - 19.26μM), inorganic ni-

trate (1.95 - 12.25μM), inorganic nitrite (1.1 - 5.5μM), total nitrogen (1.5 - 10.39μM), cal-

cium (120 - 990 mg l-1) and magnesium (730 - 2460mg l-1). Bottom water temperature

ranged from 20.4 to 27.1ºC, sediment temperature (17 to 25ºC), sediment pH (6.2 to 9.1),

concentration of sediment nutrients and heavy metals phosphorus concentration ranged

from 0.065 to 0.315%, potassium (3.18 to 8.57%), calcium (4.08 to 25.44%), magnesium

(0.29 to 1.7%), silicon (33.2 to 56.53%), sodium (1.56 to 3.35%), sulphur (0.32 to 2.06%),

chloride (1.3 to 5.71%), aluminium (4.63 to 11.87%), titanium (1.55 to 9.15%), manganese

(0.08 to 0.26% ), iron (3.2 to 11.13%), cobalt (3 to 11ppm), copper (4 to 29ppm), chromium

(26 - 85ppm), nickel (5 to 18ppm) and lead (11 to 27ppm). Seawater colony forming unit

(CFU) for the bioluminescent bacterial population density was varied from 1.06 x 104 to

9.44 x 104 CFU/ml and sediment (2.6 x104 to 23.2 x104 CFU/g). Statistical analysis sea-

water colony forming unit (CFU) of bioluminescent bacteria showed a positive correlation

with salinity and water pH. Sediment colony forming unit of bioluminescent bacteria

showed a positive correlation sediment temperature, sediment pH, sediment silicon, sodium

and chloride. **** It can be concluded that the ecological parameters were observed in water and

sediment which highly influence the bioluminescent bacterial populations and their diversi-

ty. ****

_**Keyword**_ **:** Bioluminescent bacteria; colony formation Palk Strait; diversity; physico-

chemical parameters

**1. Introduction**

present in water, sediment and also har-

bored in the light organs of some fish and

Bioluminescence is a ubiquitous

gut of many marine organisms (Hastings,

feature of the world oceans and originates

1986; Govindasamy and Srinivasan,

from organisms representing all tropic

2012). Bioluminescence is also subject to

levels. They are ecologically versatile,

control by a number of environmental

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_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

factors. Low oxygen tension may increase

vironmental parameters in Devipattinam,

luminescence and luciferase levels (Ruby

Thondi and Manamelkudi region of Palk

and Nealson, 1978; Nealson and Hasting,

Strait, Southeast coast of India.

1979). Although on large scales biolumi-

nescence may be correlated with hydro-

**2. Materials and methods**

graphic features (Swift _et al.,_ 1995). The

marine environment contains an immense

A total of 144 water and sediment

diversity of microbes that have evolved to

samples were collected from three differ-

perform equally diverse functions in their

ent places of Palk Strait _viz.,_ Devipat-

respective environments and ecological

tinam **** (Station I; Lat. 9˚28N; Long.

niches. Particularly when their popula-

78˚54'E), Thondi (Station II; Lat. 9˚45'N;

tions are dense, disturbance of the water

Long. 79˚3'E) and Manamelkudi (Station

during the night causes bright blue biolu-

III; Lat. 10˚3'N; Long. 79˚13'60'E) dur-

minescent display that have been reported

ing July 2010 - June 2012. The surface

(Harvey, 1952) and are now known to

water sample was collected with sterile

occur globally (Lynch, 1981). Its wide-

polypropylene bottle and sediment sample

spread distribution, bioluminescence is

were also collected using alcohol-rinsed,

clearly a predominant form of communi-

air dried, Peterson grab sampler. The cen-

cation in the sea, with important effects

tral portion of the top 2cm sediment sam-

on the immense daily vertical migration,

ples was taken out with the help of a ster-

predator-prey interactions and the flow of

ile spatula and the samples were then

material through the food web. Biolumi-

transferred into a sterile polythene bag.

nescent bacteria emit light continuously,

All samples were transported to the labor-

whereas higher organisms usually emit

atory within 1-3 hours of collection under

light in pulses, accomplished by localiz-

ice cold condition.

ing the systems into organelles that are

**** Atmospheric, surface water, bottom water

regulated by pH change, calcium flux and

and sediment temperatures were meas-

oxygen (Nelson and Hastings, 1979).

ured using standard mercury filled centi-

These are among the most extensively

grade thermometer. Salinity was estimat-

studied group of marine bacteria with re-

ed with the help of hand refractometer

gard to ecology, taxonomy and phyloge-

(Model 2491 Master-S/Milla). Seawater

ny. Many studies have suggested that the

pH was measured using high configura-

distribution and species composition of

tion digital pH pen. Soil pH was meas-

luminous bacteria are influenced by sea-

ured at a soil/water ratio of 1:2.5 (w/w).

son, temperature, nutrient concentration,

Air-dried soil (10g, 2.8 mm) and 25 ml

depth and geographical location (Nair _et_

distilled water were shaken together for 2

_al._ 1979; Ramaiah and Chandramohan,

min and left to settle for 30 min, this pro-

1988). Furthermore, the chemical reac-

cedure was repeated once and then the pH

tion, which produces light in biolumines-

was determined using high configuration

cent bacteria, is essentially the same for

digital pH pen (Model No: Reed 8690;

all species (Nealson and Hastings, 1979).

±0.001) (Rousk _et al_., 2009). Dissolved

In spite of the fact that a wide

Oxygen, Particulate Organic Carbon

range of habitats are occupied by lumi-

(POC), inorganic Phosphate (PO4), reac-

nous bacteria, very little information con-

tive Silicate (SiO3), inorganic Nitrate

cerning their distribution of luminous bac-

(NO3), inorganic Nitrite (NO2), Total Ni-

teria in Palk Strait, coastal region espe-

trogen (TN), and Calcium (Ca) and Mag-

cially in seagrass meadows sediments.

nesium (Mg) were analyzed as described

Hence, the present study was undertaken

by Parsons _et al._ (1984). All sediment

to understand the ecology, distribution

samples were air-dried at 25°C in the la-

and diversity of bioluminescent bacteria

boratory of Department of Oceanography

in this seagrass ecosystem along with en-

and Coastal Area Studies, Alagappa Uni-

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_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

versity, India. The soil particles were dis-

5g, Agar 15g, Seawater 1000ml, pH

aggregated, crushed and sieved through

7.2±01). The plates were incubated at

10mm nylon sieve and then stored in pol-

32°C for 24 hrs. After incubation, the

ythene bags and labelled at 4°C until the

colonies were counted in the dark room

analysis. All the elemental present in the

and expressed in CFU ml/g and identified

sediments were analyzed by using

by the standard procedure (Nealson,

Bruker™ S4-Pioneer model wavelength

1978). The isolated bioluminescent colo-

dispersive X-ray fluorescence spectropho-

nies were purified and stored at 4°C with

tometer (WD-XRF). The samples were

40% glycerol until analysis (Kita-

ground to particle size well below 100µm

Tsukamoto _et al_., 2006). All the data were

using ball mill in order to minimize the

computed using SPSS v 16.0 statistical

grain

size

interference

on

XRF-

software to obtain Pearson's correlation

measurement. The major elements _viz.,_

co-efficient (r) for the statistical interpre-

Phosphorus (P), Potassium (K), Calcium

tation. The correlation coefficient and

(Ca), Magnesium (Mg), Silicon (Si), So-

standard deviation (SD ±) were calculated

dium (Na), Sulphur (S), Chloride (Cl),

between the physico-chemical variables

Aluminium (Al), Titanium (Ti), Manga-

and CFU of bioluminescent bacterial

nese (Mn), Iron (Fe), Cobalt (Co), Copper

population in all samples collected from

(Cu), Chromium (Cr), Nickel (Ni) and

all the stations from June 2010 to May

Lead (Pb) were analyzed.

2012.

Then, 1ml seawater/ 1g sediments

of samples were mixed with 99/100ml of

**3. Results __**

50% aged sterile seawater mixed vigor-

ously and used for serial dilution in test

Atmospheric temperature was var-

tubes upto 10-2 dilutions up to 10-7. About

ied from 24.3 to 35.3°C (Figure 1), the

0.1ml from the each dilution was spread

surface water temperature was varied

in luminescent agar (LA) medium (Dehy-

from 23.2 to 33.5°C (Figure 2). The pH

drated Nutrient broth 8g, Sodium Chlo-

range was from 6.2 to 8.91 (Figure 3), the

ride 30g, Glycerol 3g, Calcium Carbonate

**Figure 1:** Monthly variations of atmospheric temperature (°C) recorded at stations I, II and

III from July 2010 to June 2012.

****

**Figure 2:** Monthly variations of surface water temperature (°C) recorded at stations I, II

and III from July 2010 to June 2012.

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**Figure 3:** Monthly variations of hydrogen ion concentration (pH) in seawater recorded at

stations I, II and III from July 2010 to June 2012.

****

**Figure 4:** Monthly variations of dissolved oxygen (ml l-1) recorded at stations I, II and III

from July2010 to June 2012.

**Figure 5:** Monthly variations of salinity (S%o) in seawater recorded at stations I, II and III

from July 2010 to June 2012.

**Figure 6:** Monthly variations of particulate organic carbon (mg dry wt.l-1) recorded at sta-

tions I, II and III from July 2010 to June 2012.

dissolved oxygen concentration ranged

showed that seawater contains nutrients

from 3.84 to 7.73ml l-1 (Figure 4) and the

_viz.,_

inorganic

phosphate

(4.12

to

salinity was fluctuated between 23.12 and

21.6µM; Figure 7), reactive silicate (4.3

39 ppt (Figure 5). Particulate organic car-

to 19.26µM; Figure 8), inorganic nitrate

bon concentration ranged from 0.78 to

(1.95 to 12.25µM; Figure 9), inorganic

2.56mg dry wt.l-1 during the study period

nitrite (1.1 to 5.5µM; Figure 10), total ni-

at all the stations (Figure 6). The results

trogen (1.5 to 10.39µM; Figure 11),

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**Figure 7:** Monthly variations of inorganic phosphate (µM) concentration in seawater rec-

orded at stations I, II and III from July 2010 to June 2012.

**Figure** **8:** Monthly variations of reactive silicate (µM) concentration in seawater recorded

at stations I, II and III from July 2010 to June 2012.

**Figure 9:** Monthly variations of inorganic nitrate (µM) concentration in seawater recorded

at stations I, II and III from July 2010 to June 2012.

__
__

**Figure** **10:** Monthly variations of inorganic nitrite (µM) concentration in seawater recorded

at stations I, II and III from July 2010 to June 2012.

**Figure** **11:** Monthly variations of total nitrogen (µM) concentration in seawater recorded at

stations I, II and III from July 2010 to June 2012.

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calcium (120 to 990 mg l-1; Figure 12)

25.44%; Figure 20), magnesium (0.29 to

and magnesium (730 to 2460mg l-1; Fig-

1.7%; Figure21), silicon (33.2 to 56.53%;

ure13). The bacterial diversity revealed

Figure 22), sodium (1.56 to 3.35%; Fig-

that, the bioluminescent bacterial popula-

ure 23), sulphur (0.32 to 2.06%; Figure

tion density was varied from 1.06 x 104 to

24), chloride (1.3 to 5.71%; Figure 25),

9.44 x 104 CFU/ml in seawater (Figure

aluminium (4.63% to 11.87%; Figure 26),

14).

titanium (1.55 to 9.15%; Figure 27), man-

Bottom water temperature was in

ganese (0.08 to 0.26%; Figure28), iron

the range of 20.4 to 27.1ºC during the

(3.2 to 11.13%; Figure 29), cobalt (3 to

study period at all three stations (Figure

11ppm; Figure 30), copper (4 to 29ppm

15). Sediment temperature varied from 17

Figure 31), chromium (26 to 85ppm; Fig-

to 25ºC (Figure 16). Sediment pH varied

ure 32), nickel (5 to 18ppm; Figure 33)

from 6.2 and 9.1 at all stations (Figure

and lead (11 to 27ppm; Figure 34). In sed-

17). The results for sediment nutrients

iment, the bioluminescent bacterial popu-

showed that __ phosphorus (0.065 to

lation density varied from 2.6 x104 to 23.2

0.315%; Figure 18), potassium (3.18 to

x104 CFU/g (Figure 35).

8.57%; Figure 19), calcium (4.08 to

**Figure 12:** Monthly variations of calcium (mg l-1) concentration in seawater recorded at

stations I, II and III from July 2010 to June 2012.

**Figure** **13:** Monthly variations of magnesium (mg l-1) concentration in seawater recorded at

stations I, II and III from July 2010 to June 2012.

**Figure** **14:** Monthly variations of bioluminescent bacteria (CFU/ml x 104) populations in

seawater recorded at stations I, II and III from July 2010 to June 2012.

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**Figure** **15:** Monthly variations in bottom water temperature (ºC) recorded at stations I, II

and III from July 2010 to June 2012.

**Figure** **16:** Monthly variations in sediment temperature (ºC) recorded at stations I, II and III

from July 2010 to June 2012.

**Figure** **17:** Monthly variations of hydrogen ion concentration (pH) in sediment recorded at

stations I, II and III from July 2010 to June 2012.

**Figure** **18:** Monthly percentage variations of phosphorus (%) concentration in sediment

recorded at stations I, II and III from July 2010 to June 2012.

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**Figure** **19:** Monthly percentage variations of potassium (%) concentration in sediment rec-

orded at stations I, II and III from July 2010 to June 2012.

**Figure** **20:** Monthly percentage variations of calcium (%) concentration in sediment rec-

orded at stations I, II and III from July 2010 to June 2012.

**Figure** **21:** Monthly percentage variations of magnesium (%) concentration in sediment

recorded at stations I, II and III from July 2010 to June 2012.

**Figure** **22:** Monthly percentage variations of silicon (%) concentration in sediment record-

ed at stations I, II and III from July 2010 to June 2012.

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**Figure** **23:** Monthly percentage variations of sodium (%) concentration in sediment record-

ed at stations I, II and III from July 2010 to June 2012.

**Figure** **24:** Monthly percentage variations of sulphur (%) concentration in sediment record-

ed at stations I, II and III from July 2010 to June 2012.

**Figure** **25:** Monthly percentage variations of chloride (%) concentration in sediment rec-

orded at stations I, II and III from July 2010 to June 2012.

**Figure** **26:** Monthly percentage variations of aluminum (%) concentration in sediment rec-

orded at stations I, II and III from July 2010 to June 2012.

**Figure 27:** Monthly percentage variations of titanium (%) concentration in sediment rec-

orded at stations I, II and III from July 2010 to June 2012.

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**Figure** **28:** Monthly percentage variations of manganese (%) concentration in sediment

recorded at stations I, II and III from July 2010 to June 2012.

**Figure** **29:** Monthly percentage variations of iron (%) concentration in sediment recorded at

stations I, II and III from July 2010 to June 2012.

**Figure** **30:** Monthly variations of cobalt (ppm) concentration in sediment recorded at sta-

tions I, II and III from July 2010 to June 2012.

**Figure** **31:** Monthly variations of copper (ppm) concentration in sediment recorded at sta-

tions I, II and III from July 2010 to June 2012.

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**Figure** **32:** Monthly variations of chromium (ppm) concentration in sediment recorded at

stations I, II and III from July 2010 to June 2012.

**Figure** **33:** Monthly variations of nickel (ppm) concentration in sediment recorded at sta-

tions I, II and III from July 2010 to June 2012.

**Figure** **34:** Monthly variations of lead (ppm) concentration in sediment recorded at stations

I, II and III from July 2010 to June 2012.

**Figure** **35:** Monthly variations of bioluminescent bacteria (CFU/g x 104) populations in

sediment recorded at stations I, II and III from July 2010 to June 2012.

**4. Discussion**

tool for the assessment and monitoring of

coastal ecosystems. The similar results

Based on the results, the physico-

and trend was observed in Muthupattai

chemical parameters, heavy metals in wa-

mangroves, Southeast coast of India

ter and sediments would form a useful

(Ashokkumar _et al.,_ 2009; Senthilnathan

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and Balasubramanian, 1999). The higher

luminescent bacteria showed a positive

concentration of metals were recorded

correlation with salinity at station-I (r=

during monsoon season, which could be

0.773; P< 0.001), station-II (r= 0.903; P<

mainly due to land runoff and influx of

0.001) and station-III (r=0.837; P< 0.001)

metal rich freshwater that in turn reflects

and water pH at station-I (r=0.726; P<

in the metal concentration in sediment

0.01), station-II (r= 0.631; P< 0.01) and

(Athalye and Gokhale, 1989). The as-

station-III (r=0.874; P< 0.001) The counts

sessment of trace metal concentrations

were at low levels during the active mon-

and distribution in marine water and sed-

soon period because of high rainfall. The

iment leads to an understanding of their

monsoonal flood water may have altered

behaviour and detects the pollution source

the luminous bacterial population from

in marine environment (Forstner and

sediment as the Vellar estuary is shallow

Wittman, 1979). Besides substantiating

(Ramesh _et al.,_ 1989). Further, the maxi-

higher biological productivity, higher

mum atmospheric, surface water and sed-

densities of luminous bacteria in this bay

iment temperature were recorded during

also signify pollution free environment in

summer at station I and minimum was

this region.

recorded during monsoon at station III.

Bioluminescent bacteria are being

Maximum bottom water temperature was

found in marine environment. Microbial

recorded during pre-monsoon at station I

activities play an important role in marine

and minimum was recorded during mon-

food webs, nutrient mineralization and

soon at station III. Surface water tempera-

recycling. The ecological importance of

ture was slightly higher than the bottom

bioluminescence in the ocean is evident in

water at all the stations. Surface and bot-

the dominance of light emitters in open

tom water temperature of all stations are

waters. Ecology of bioluminescent bacte-

slightly varied monthly. Temperature is

ria has focused primarily on distribution

an important environmental factor, can

of these organisms in marine environment

influence the diversity of luminous bacte-

(Nealson and Hastings, 1979); Atalntic

ria (Ruby and Nealson, 1978; Yetinson

Ocean (Ramaiah and Chandramohan,

and Shilo, 1979; Ruby _et al_., 1980). Dun-

1988), Indian Ocean (Lynch, 1981) and

lap (2009) reported that the temperature

near shore water Porto Nova (Ramesh _et_

relationships of luminous bacteria appear

_al.,_ 1989).

to be a specific to _Vibrio_ species _._ Accord-

The present study was carried out

ing to Govindasamy _et al._ (2000), the sur-

to understand the role of ecological pa-

face water temperature could be changed

rameters of the bioluminescent bacteria

by the intensity of solar radiation; evapo-

(during July 2010 - June 2012) at differ-

ration, freshwater influx, cooling and it

ent stations of Palk Strait region, India.

might mix up with ebb and flow form ad-

The maximum counts of bioluminescent

joining neritic water. It is further evident

bacteria in seawater/ sediments samples

that the atmospheric temperature showed

was recorded during summer season (May

positive correlation at station-II (r= 0.663;

2011) at station I; whereas, minimum

P< 0.01) and at station - III (r= 0.685; P<

counts of bioluminescent bacteria sea-

0.01) to seawater with CFU microbial

water/ sediment samples was recorded at

counting at all the stations. Surface water

monsoon season (2010) at station III. The

temperature was found low during mon-

CFU values suggested that the higher

soon because of rainfall but the maximum

population counts were recorded during

temperature was observed during summer

the summer months during the study peri-

(Kannapiran _et al.,_ 2008). This could be

ods at all the stations. This variation

attributed due to high solar radiation as

might be due to the high-saline relativity.

reported by Ashok Prabu _et al._ (2008).

The statistical analysis revealed that sea-

Lower temperature was observed due to

water colony forming unit (CFU) of bio-

cloudy sky and rainfall that brought down

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temperature to minimum (Kannan and

fall and the resultant freshwater mixing.

Kannan, 1996). In addition, the surface

The minimum dissolved oxygen was

water temperature showed positive corre-

found during summer months, which

lation with colony forming unit (CFU) at

could be mainly due to reduced agitation

station - II (r= 0.635; P< 0.02) and at sta-

in the coastal and estuarine water (Ruby

tion - III (r= 0.627; P< 0.02). Further, the

and Nealson, 1978; Nealson and Hasting,

statistical analysis revealed that the sedi-

1979). Further, this is evidenced by the

ment colony forming unit of biolumines-

negative correlation between the dis-

cent bacteria showed a positive correla-

solved oxygen and seawater CFU at sta-

tion with sediments temperature at station

tion-III (r= -0.823; P< 0.001).

\- I (r= 0.806; P< 0.001), station-II (r=

Maximum particular organic car-

0.842; P< 0.001) and station-III (r= 0.784;

bon (POC) was recorded during the

P< 0.001).

month of November (2010) at station-II

Surface water salinity was reduced

and minimum POC was observed during

greatly during the monsoon and it was

the month of May (2012) at station-I. The

gradually increased from postmonsoon to

maximum POC content in water was

summer at all stations. The maximum sa-

mainly due to the organic matter brought

linity could be due to low amount of rain-

in from the land through run-off. Further,

fall and higher rate of evaporation in the

it could be also due to the presence of

shallow coastal area owing to high at-

plant (seagrass and seaweeds) and animal

mospheric temperature (Govindasamy

organic matter within the seagrass ecosys-

and Kannan, 1991). Significantly positive

tem and exported from the adjacent eco-

correlation was observed between sea-

system by wind and wave action. Further,

water salinity and CFU of bioluminescent

the seasonal variation in POC content in

bacteria at station-I (r= 0.773; P< 0.001),

the water could be related to the plankton

station-II (r= 0.903; P< 0.001) and sta-

productivity (Kannapiran _et al.,_ 2008).

tion-III (r= 0.837; P< 0.001). The present

This is further evidenced by the negative

study results are in line with the research

correlation between POC and seawater

findings of Abraham _et al._ (2003).

CFU at station-I (r= -0.786; P< 0.001),

The high pH values recorded dur-

station-II (r= -0.841; P< 0.001) and sta-

ing summer and this might be due to the

tion-III (r= -0.832; P< 0.001).

influence of seawater penetration and

Maximum inorganic phosphate

high biological activity. These findings

was observed during the monsoon season

are in accordance with the previous report

(December 2010) at station-III and mini-

(Smith and Key, 1975). The statistical

mum was recorded during the summer

analysis shows the positive correlation

season (May 2011) at station-I. Possibly,

between pH and CFU station - I (r=0.726;

the maximum concentration of phosphate

P< 0.01) and station - III (r= 0.874; P<

was due to invasion of upwelling of wa-

0.001) to seawater colony forming unit.

ter, which increased the level of phos-

The statistical analysis shows the positive

phate. Low values of phosphate observed

correlation with sediments pH station-I

to utilization by phytoplankton, seagrass-

(r= 0.692; P< 0.001), station-II (r= 0.641;

es and other primary producers (Rajaseg-

P< 0.001) and station-III (r= 0.813; P<

ar, 2003). The statistical analysis shows

0.001) to sediment colony forming unit.

the negative correlation to seawater colo-

Dissolved oxygen is one of the most im-

ny forming unit with inorganic phosphate

portant abiotic parameters influencing the

at station - I (r= - 0.734; P< 0.01), station-

life in the coastal environment. In the pre-

II (r= -0.832; P< 0.001) and station-III (r=

sent study, the maximum dissolved oxy-

-0.828; P< 0.001).

gen was recorded during monsoon and

Minimum concentration of silicate

this might be due to the cumulative effect

was observed during the summer season

of higher wind velocity coupled with rain-

(May 2012) at station II and maximum

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during the monsoon season (November

was recorded during the summer (May

2010) at station III. It might be due to the

2011) at station I. It might be due to

heavy rain, land runoff water mixing was

freshwater inflow was high in the mon-

high level. It has been reported that the

soon season so high level of nitrogen was

silicate from the bottom sediment might

recorded. The statistical analysis showed

have been exchanged with overlaying wa-

total nitrogen a negative correlation with

ter in this mangrove environment (Go-

seawater CFU of bioluminescent bacteria

vindasamy and Kannan, 1996). The low

at station-I (r= -0.829; P< 0.001), station-

value observed in summer and post-

II (r= -0.929; P< 0.001) and station-III (r=

monsoon season could be attributed to

-0 .896; P< 0.001).

uptake of silicates (Saravanakumar _et al.,_

Maximum and minimum level of

2008). The statistical analysis of silicate

seawater calcium was observed during the

showed negative correlation with sea-

monsoon (December 2010) and summer

water colony forming unit at station-II (r=

seasons (May 2011) at station-III. Maxi-

-0.786; P< 0.001) and station-III (r= -

mum level of sediment calcium was ob-

0.841; P< 0.001).

served during the monsoon season (De-

Nitrate concentration was found

cember 2010) at station-II and minimum

lower than that of nitrate and same trend

level of calcium was observed during the

of fluctuation was reported in Muthpettai

summer (May 2011) at station-II. Maxi-

mangrove (Ashokkumar _et al.,_ 2011).

mum level of seawater magnesium was

The statistical analysis showed nitrate a

recorded during the monsoon season De-

negative correlation with seawater colony

cember (2010) at station I and minimum

forming unit at station-I (r= -0.868; P<

was recorded during the summer season

0.001), station-II (r= -0.888; P< 0.001)

May (2011). Maximum level of sediment

and station-III (r= -0.926; P< 0.001).

magnesium was observed during the post

Maximum level of nitrite was rec-

monsoon season (January 2011) at sta-

orded during December 2011 at station-II

tion-I and minimum level of magnesium

and minimum was observed during the

was recorded during the summer season

May 2011 at station-I. It might be due to

(May 2011) at station-II. The statistical

the minimum nitrite were recorded during

analysis showed seawater calcium a nega-

the summer may be due to high salinity.

tive correlation to seawater colony form-

The higher nitrate value in monsoon sea-

ing unit at station-II (r= -0.816; P< 0.001)

son could be due to the increased phyto-

and station-III (r= -0.762; P< 0.001). In

plankton excretion, oxidation of ammo-

addition, to that the seawater magnesium

nia, reduction of nitrate, the recycling of

a negative correlation with seawater colo-

nitrogen and bacterial decomposition of

ny forming unit at station-II (r= -0.829;

planktonic detritus (Govindasamy _et al_.,

P< 0.001) and station-III (r= -0.840; P<

2000; Asha and Diwakar, 2007) and also

0.001). Sediment calcium a negative cor-

due to denitrification and air-sea interac-

relation with sediment colony forming

tion exchange of chemicals were also re-

unit at station-I (r= -0.843; P< 0.001), sta-

sponsiple for this increased values (Raja-

tion-II (r= -0.832; P< 0.001) and station-

segar, 2003; Ashok Prabu _et al.,_ 2008).

III (r= -0 .909; P< 0.001); Sediment mag-

The statistical analysis which showed ni-

nesium showed a negative correlation

trite a negative correlation with seawater

with sediment colony forming unit at sta-

colony forming unit at station-I (r= -

tion-I (r= -0.710; P< 0.001), station-II (r=

0.767; P< 0.001), station-II (r= -0.834; P<

-0.675; P< 0.001) and station-III (r= -

0.001) and station-III (r= -0.882; P<

0.721; P< 0.001). It might be due to the

0.001).

calcium and magnesium higher level was

Maximum level of total nitrogen

recorded in monsoon and post monsoon

was recorded during the monsoon (De-

seasons. Calcium and magnesium concen-

cember 2010) at station III and minimum

trations could be increased due to the in-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 469

_Biotech Sustainability (2017)_

_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

put of freshwater (Sundararajan and Nate-

ment colony forming unit at station-I (r= -

san, 2010).

0.661; P< 0.001), station-II (r= -0.856; P<

Maximum sediment total phos-

0.001) and station-III (r= -0.882; P<

phorus was observed during the month of

0.001).

December-2010 at station-I and minimum

Maximum concentration of sedi-

was recorded during the month of May-

ment sodium was observed during the

2011 at station-III. and this might be due

month of 2011 at station-III and minimum

to the domestic, municipal and agricultur-

was recorded at station-II and this might

al waste (non-pointed sources). The re-

be due to the high counts of microbial

generation and releases of total phospho-

populations depending on the sodium

rus from bottom mud into the water col-

concentration and it was varied spatially.

umn by turbulence and mixing was also

The statistical analysis which showed sed-

attributed to recorded higher values

iment sodium a positive correlation with

(Chandran and Ramamoorthy, 1984). The

sediment colony forming unit at station-I

statistical analysis showed sediment total

(r= 0.834; P< 0.001), station-II (r= 0.824;

phosphorus a negative correlation with

P< 0.001) and station-III (r= 0.845; P<

sediments colony forming unit at station-I

0.001). Maximum and minimum level of

(r= -0.848; P< 0.001), station-II (r= -

chloride was recorded during the summer

0.843; P< 0.001) and station-III (r= -

and post monsoon seasons at station III

0.861; P< 0.001). Maximum concentra-

and station I respectively. The statistical

tion of sediment potassium was observed

analysis of sediments chloride shows a

during the monsoon season (December

positive correlation with sediment colony

2010) at station-I and minimum concen-

forming unit station-I (r= 0.665; P<

tration of potassium was recorded during

0.001), station-II (r= 0.840; P< 0.001) and

the summer (June 2010) at station-II. It

station-III (r= 0.811; P< 0.001).

might be due to high concentration of po-

The maximum sediment alumini-

tassium are inflected by heavy rainfall

um was observed recorded during the post

inflow on in peak values was recorded

monsoon month of January 2012 at sta-

during the monsoon seasons. Further, evi-

tion-I and minimum was observed during

denced by the statistical analysis showed

the month of April 2011 at station-III. It

potassium a significant negative correla-

might be due to contribution from detrital

tion with sediments colony forming unit

mineral grains supplied from through the

at station-II (r= -0.656; P< 0.001) and sta-

rivers in addition to the precipitation of

tion-III (r= -0.751; P< 0.001).

their dissolved species under prevailing

Maximum and minimum sediment

estuarine condition. The statistical analy-

silicon was observed during the May -

sis which showed a negative correlation

2012 at station II and November -2011 at

to sediment colony forming unit with al-

station-III. This might be due to its bio-

uminium at station-II (r= -0.706; P<

logical significance; the flux of particulate

0.001). Removal of dissolved river-borne

silica from surface waters plays an im-

aluminum by coagulation in the coastal

portant role in the cycling of other ele-

areas is common (Holliday and Liss,

ments in the marine environment such as

1976).

radium, barium and germanium (Li _et al.,_

In sediment, the maximum and

1973; Froelich and Andreae, 1980). The

minimum titanium level was recorded

statistical analysis showed silicon a sig-

during the monsoon 2010 and summer

nificant positive correlation with sediment

April 2011 at stations III and I respective-

colony forming unit at station-I (r= 0.745;

ly. Moreover, the statistical analysis re-

P< 0.001), station-II (r= 0.824; P< 0.001)

vealed that the sediment titanium shows a

and station-III (r= 0.836; P< 0.001). The

negative correlation to sediment colony

statistical

analysis

showes

sulphur

forming unit at station-I (r= -0.732; P<

showed a negative correlation with sedi-

0.001), station-II (r= -0.792; P< 0.001)

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_Biotech Sustainability (2017)_

_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

and station-III (r= -0.888; P< 0.01). Max-

_al._ (2004) has reported that effluent of

imum and minimum level of sediment

metal finishing industry and corrosion of

manganese was recorded during the mon-

building materials play the main role to

soon (2010) at station III and summer

increase chromium level in marine envi-

(2012) seasons at station I. The statistical

ronment. The statistical analysis showed

analysis showed sediment manganese a

sediment chromium a negative correlation

negative correlation with sediment colony

with sediment microbial count at station-I

forming unit at station-I (r= -0.732; P<

(r= -0.760; P< 0.001), station-II (r= -

0.001), station-II (r= -0.819; P< 0.001)

0.768; P< 0.001) and station-III (r= -

and station-III (r= -0.800; P< 0.001).

0.896; P< 0.001).

Maximum and minimum concen-

Maximum sediment nickel con-

tration of sediment iron was recorded dur-

centration was recorded during the mon-

ing the post monsoon (2011) and summer

soon (2010) at station I and minimum was

(2012) seasons at station I and III. In gen-

recorded during the summer seasons

eral, marine environment and discharge of

(2012) at station II. The statistical analy-

aquatic ponds, domestic wastes, land and

sis sediment nickel showed a negative

agricultural drainage, boating activities

correlation with sediment colony forming

such as loading and unloading of materi-

unit at station-I (r= -0.801; P< 0.001), sta-

als, antifouling paints from boating activi-

tion-II (r= -0.778; P< 0.001) and station-

ties contribute to enhance the metal level

III (r= -0.684; P< 0.001). Maximum and

in marine environment (Govindasamy and

minimum concentration of sediment lead

Azariah, 1999; Ashokkumar _et al.,_ 2009).

was recorded during the monsoon (2010)

The statistical analysis showed sediment

and summer (June 2010) seasons at sta-

iron a negative correlation with sediment

tion-III. It might be due to the heavy rain

colony forming unit at station-I (r= -

river runoff and sewage discharges in the

0.643; P< 0.01), station-II (r= -0.695; P<

coastal region. Chatterjee _et al._ (2006)

0.001) and station-III (r= -0.758; P<

reported that 25.3 to 33.4mg/kg were ob-

0.001).

served in Hugi (Ganges) estuary, north-

Maximum and minimum level of

east coast of Bay of Bengal. The statisti-

copper was observed during the month of

cal analysis sediment lead showed a nega-

January 2012 at station III and summer

tive correlation with sediment colony

month of May (2012) at station I. It might

forming unit station-I (r= -0.778; P<

be due to the discharge of maximum level

0.001), station-II (r= -0.835; P< 0.001)

of freshwater in the central west coast of

and station-III (r= -0.801; P< 0.001).

India (Sankaranarayanan and Reddy,

1973). The statistical analysis showed

**5. Conclusion**

sediment copper a negative correlation

with sediment colony forming unit sta-

The marine environment supports

tion-I (r= -0.675; P< 0.001), station-II (r=

the survival, reproduction and the meta-

-0.699; P< 0.001) and station-III (r= -

bolic reactions of the flora and fauna.

0.770; P<; 0.001).

Among them, the microbial community

Maximum sediment chromium

plays a vital role in the food webs, bioge-

was recorded monsoon season (2010) at

ochemical cycles and the energy flow

station III and minimum was recorded

mechanisms in the marine ecosystem. But

during summer season (2011) at station-

the microbial diversity in seagrass ecosys-

II. It might be due to huge amount of

tem is structured and determined by the

freshwater river flow carry agricultural

prevailing temporal and spatial variability

water, domestic sewage, animal and

of ecological parameters in the native en-

chromium in river-borne solids transport-

vironment. The physico-chemical pa-

ed in estuaries is relatively higher than

rameters of seawater were highly influ-

that of near shore sediments. Mannan _et_

encing the bioluminescent bacterial popu-

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_Biotech Sustainability (2017)_

_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

lation in the Palk Strait region. Most of

behavior of zinc and copper. _Ma-_

the parameters such as the atmospheric

_hasagar Bulltion National Institute_

temperature, salinity, pH were significant-

_of Ocanography_ **22, 185-191.**

ly correlated with the bioluminescent bac-

**Chandran, R and K. Ramamoorthi.**

terial population in all the three stations

**(1984)**. Hydrobiological studies in

studied.

the gradient zone of the Vellar es-

tuary. I. Physicochemical parame-

**References**

ters. _Mahasagar Bulletin_. National

****

Institute of Oceanography **17, 69-**

**Abraham, T.J., S.A. Shanmugam, R.**

**78.**

**Palaniappan and K. Dheven-**

**Chatterjee, M., E.V. Silva Filho, S.K.**

**daran. (2003)**. Distribution

and

**Sarkar,**

**S.M.**

**Sella,**

**A.**

abundance of luminous bacteria

**Bhattacharya, K.K. Satapathy,**

with special reference to shrimp

**M.V.R. Prasad, S. Chakraborthy**

farming activities. _Indian Journal_

**and B.D. Bhattacharya. (2006)**.

_of Marine Sciences_ **32(3), 208 -**

Distribution and possible source of

**213.**

trace elements in the sediment

**Asha, P.S and K. Diwakaran. (2007)**.

cores of tropical macrotital estuary

Hydrobiology of the inshore waters

and ecotoxicological significance

off Tuticorin Bay, Gulf of Mannar.

**33(3), 346-356.**

_Journal of Marine Biologycal As-_

**Dunlap, P.V. (2009)**. Bioluminescence.

_sociation of India_ **49(1), 07-11.**

_Microbial_ _Physiology._ 48-61. ****

**Ashok Prabu, V., M. Rajkumar and**

**Forstner, V and G.T.W. Wittmann.**

**P. Perumal. (2008** ). Seasonal vari-

**(1979)**. Metal pollution in the

ation in physico- chemical charac-

aquatic environment. Springer-

teristics of Picavaram mangroves,

Verlag, Berlin, **486.**

southeast coast of India. _Journal of_

**Froelich, P.N and M.O. Andreae.**

_Environmental Biology_ **29, 945-**

**(1980)**. Germanium in the oceans:

**950.**

eka-silicon. _Trans Am_

_Geophys._

**Ashokkumar, A., V.S. Reddy Belum,**

_Union_ **61, 987.**

**B. Ramaiah, P. Sanjana Reddy,**

**Govindasamy, C and R. Srinivasan.**

**K.L. Sahrawat and H.D. Upadh-**

**(2012)**. Association of biolumines-

**yaya. (2009)**. Genetic variability

cent bacteria from blue swimmer

and plant character association of

crab _Portunus pelagicus_ (Linneaus,

grain Fe and Zn in selected core

1758). _Asian Pacific Journal of_

collections of _Sorghum_ germplasm

_Tropical Disease_ **S699-S702.**

and breeding lines. _Journal of SAT_

**Govindasamy, C and J. Azariah.**

_Agricultural Research_ **7, 1-4.**

**(1999)**. Seasonal Variation of Dis-

**Ashokkumar, S., G. Rajaram, P.**

solved Metals in Coastal Water of

**Manivasagam, S. Ramesh, P.**

the Coromandel Coast, India. _Indi-_

**Sampathkumar and P. Mayavu.**

_an Journal of Marine Sciences_ **28,**

**(2011)**. Studies on hydrographical

**249- 256.**

parameters, nutrients and microbial

**Govindasamy, C and Kannan, L.**

population of Mullipallam Creek in

**(1991)**. Rotifers of the Pitchavaram

Muthupettai Mangrove (Southeast

Mangroves (Southeast Coast of In-

coast of India). _Research Journal_

dia): A Hydrographical Approach.

_of Microbiology_ **6(1), 71-86.**

_Mahasagar- Bulletin National In-_

**Athalye, R.P and K.S. Gokhale.**

_stitute of Oceanography_ **24(1), 39-**

**(1989)**. Study of selected trace

**45.**

metals in the sedimends of

Thane

**Govindasamy, C and L. Kannan.**

creek near Thane city- Antagonistic

**(1996)**. Ecology of Rotifers of

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 472

_Biotech Sustainability (2017)_

_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

Pichavaram mangroves, southeast

regarding the marine geochemistry

coast of India. _Indian Hydrobiolo-_

of Ba and Ra. _Earth Planet Sci Let._

_gy_ **1, 69-76.**

**19, 352-361.**

**Govindasamy, C., L. Kannan and Ja-**

**Lynch, R.V. (1981)**. Distribution of

**yapaul Azariah. (2001)**. Seasonal

luminous marine organisms: a lit-

variation in physico-chemical

erature review, In Biolumines-

properties and primary production

cence: current perspectives, (Eds)

in the coastal water biotopes of

K.H. Nealson, Burgess Publising

Coromandal coast. _Indian Journal_

Co, _Minnesota,_ **153-159.**

_of Environmental Biology._ **21, 1-7.**

**Mannan, M., B. Zourarah, C. Car-**

**Harvey, E. N. (1952)**. Biolumines-

**ruesco, A. Aajjane and J. Nadu.**

cence. Academic Press Inc., New

**(2004)**. Distribution of heavy met-

York, **p. 649**

als in Sidi Moussa lagoon sediment

**Hastings, J. W. (1986)**. Biolumines-

(Atlantic Moroccan Coast). _Journal_

cence. _Annual Review of Biochem-_

_of American Earth Sciences_ **39,**

_istry_ **37, 597-630.**

**473-484.**

**Holliday, L.M and P.S. Liss. (1976).**

**Nair, G.B., M. Abraham and R. Na-**

The behaviour of dissolved iron,

**tarajan. (1979).** Isolation and iden-

manganese and zinc in the Beau-

tification of luminous bacteria from

lieu estuary. _Estuarine Coastal_

Porto Novo estuarine environs. _In-_

_Marine Sciences_ **4, 349-353.**

_dian Journal of Marine Sciences_ **8,**

**Kannan, R and L. Kannan (1996)**.

**46-48.**

Physico-chemical characteristics of

**Nealson, K.H and J.W. Hastings.**

seaweed beds of the

Palk

Bay,

**(1979).** Bacterial bioluminescence:

Southeast coast of India. _Indian_

its control and ecological

signifi-

_Journal of Marine Sciences_ **25,**

cance. _Microbiological_ _Reviews_ **43** ,

**358-362**.

496-518.

**Kannapiran, E., L. Kannan, A.**

**Nealson, K.H. (1978).** Isolation, identi-

**Purushothaman and T. Thanga-**

fication and manipulation of lumi-

**rajdou. (2008)**. Physico-chemical

nous bacteria. _Methodes in Enzy-_

and microbial characteristics of the

_mology_ **57,153-166.**

coral reef environment of the Gulf

**Parsons, T.R., Y. Maita and C.M.**

of Mannar marine biosphere re-

**Lalli, (1984).** A Manual of Chemi-

serve, India. _Journal of Environ-_

cal and Biological Methods

Sea-

_mental Biology_ **29(2), 215- 222.**

water Analysis. Pergamon Press,

**Karuppasamy, P.K and P. Perumal.**

Oxford, New York, Toronto, Syd-

**(2000)**. Biodiversity of zooplank-

ney, Frankfurt, **1-173.**

ton at Pichavaram mangroves,

**Rajasegar,**

**M.**

**(2003).**

Physico-

South India. _Advanced in Biologi-_

chemical characteristics of the Vel-

_cal Sciences_ **19, 23-32**.

lar estuary in relation to shrimp

**Kita-Tsukamoto, K., K. Yao, A. Ka-**

forming. _Journal of Environmental_

**miya, S. Yoshizawa, N. Uchiya-**

_Biology_ **24** , 95-101.

**ma, K. Kogure and M.**

**Wada.**

**Ramachandran, S., S. Anitha, V. Bal-**

**(2006)**. Rapid identification of ma-

**amurugan, K. Dharaanirajan,**

rine bioluminescent bacteria by

**K.E. Venthan and M.I.P. Divien.**

amplified 16S ribosomal

RNA

**(2005).** Ecological impact of tsu-

gene restriction analysis. _FEMS_

nami on Nicobar Islands (Camrta,

_Microbiology Letters_ **258(2), 320.**

Katchal, Nancowry and Trinkat).

**Li, Y., T.L. Ku, G.G. Mathieu and K.**

_Current Sciences_ **89(1), 195-200.**

**Wolgemuth. 1973**. Barium in the

**Ramaiah, N and D. Chandramohan.**

Antartic Ocean and

implications

**(1988).** Distribution of luminous

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 473

_Biotech Sustainability (2017)_

_Ecology, Distribution and Diversity of Bioluminescent Bacteria Srinivasan et al._

bacteria and bacterial luminescence

**Thivakaran. (2008).** Seasonal var-

in the equation region of the Indian

iations in physicochemical charac-

Ocean. _Microbiologica_ **11, 243-**

teristics of water, sediment and soil

**254.**

texture in arid zone mangrove of

**Ramesh, A., B.G. Loganathan and K.**

Kachchh Gujarat. _Journal of Envi-_

**Venugopalan. (1989).** Seasonal

_ronmental Biology_ **29, 725-732.**

distribution of luminous bacteria in

**Senthilnathan. S and T. Balasubra-**

the sediments of a tropical estuary.

**manian. (1999).** Heavy metal dis-

_Journal of Genetics and Applied_

tribution in Pondicherry harbour,

_Microbiology_ **35, 363-368.**

southeast coast of India. _Indian_

**Rousk, J., P.C. Brookes and E. Baath.**

_Joumal of Marine Sciences_ **28,**

**(2009).** Contrasting Soil pH Effects

**380- 382.**

on Fungal and Bacterial

Growth

**Sundararajan, M and U. Natesan.**

Suggest Functional Redundancy in

**(2010).** Environmental significance

Carbon Mineralization. _Applied_

in recent sediments along Bay of

_and Environmental_

_Microbiology_

Bengal and Palk Strait, East Coast

**75(6), 1589-1596.**

of India: A geochemical approach.

**Ruby, E.G and K.H. Nealson (1978).**

_International Journal of Environ-_

Seasonal changes in the species

_mental Research_ **4(1), 99-120.**

composition of luminous

bacteria

**Swift,**

**E.,**

**J.M.**

**Sullivan,**

**H.P.**

in near shore seawater. _Luminology_

**Batchelder, J. Van Keuren, R.D.**

_and Oceanography_ **23, 530-533.**

**Vaillancourt and R.R. Bidigare.**

**Ruby, E.G., E.P. Greenberg, and**

**(1995).** Bioluminescent organisms

**J.W. Hastings. (1980).** Planktonic

and

bioluminescence

marine luminous bacteria: species

measurements in the North Atlantic

distribution in the water column.

Ocean

near

latitude

59.5ºN,

_Applied_ _Environmental_ _Microbiol-_

longitude 21º. _**** World Journal of _

_ogy_ **39,302-306.**

_Geophysis and Reserach_

**100,**

**Sankaranarayanan, V.N. and C.V.G.**

**6527-6647.**

**Reddy. (1973).** Copper content in

**Yetinson, T and M. Shilo. (1979).** Sea-

the inshore and estuarine

waters

sonal and geographic distribution

along the central west coast of In-

of luminous bacteria in the eastern

dia. _Current Sciences_ **4, 223-224.**

Mediterranean and the Gulf of Elat.

**Saravanakumar, A., M. Rajkumar, J.**

_Applied_ _Environmental_ _Microbiol-_

**Sesh**

**Serebiah**

**and**

**G.A.**

_ogy_ **37, 1230-1238.**

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 474

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P475-485_

**Synthesis of Biocompatible Silver Nanoparticles Using**

**Green Alga ( _Ulva reticulata_** **) Extract**

****

**Ganapathy Selvam Govindarasu1, Srinivasan Rajendran2 and Sivakumar**

**Kathiresan1,***

****

_1Division of Algal Biotechnology, Department of Botany, Annamalai University,_

_Annamalainagar-608 002, Tamil Nadu, India; 2Department of Oceanography and Coastal_

_Area Studies, School of Marine Sciences Alagappa University, Thondi Campus, Thondi –_

_623 409, Ramanathapuram District, Tamil Nadu, India; *Correspondence:_

 _vgs.biot@gmail.com_ _/ kshivam69@gmail.com; Tel: +91 9786330511_

**Abstract:** The synthesis of nanoparticles has become a matter of great interest in recent

times due to their various advantageous properties and applications in a variety of fields.

We have reported biological synthesis of nano-sized silver particles. The nanoparticles of

silver were formed by the reduction of silver nitrate to aqueous silver metal ions while ef-

fect of marine seaweed _U_. _reticulata_ extract was investigated; silver nanoparticles were

characterized using UV-visible absorption and room temperature photoluminescence. The

X-ray diffraction results revealed that the synthesized silver nanoparticles were in the cubic

phase. The existence of functional groups was identified using Fourier transform infrared

spectroscopy. The morphology and size of the synthesized particles were studied with

atomic force microscope. Further, the photocatalytic degradation of methyl orange was

measured spectrophotometrically by using silver as nanocatalyst under visible light illumi-

nation. Silver nanoparticles synthesized from _U. reticulata_ by facile method from can able

to degrade dyes in the presence of visible light and paves way for ecological health and en-

vironmental bioremediation. Despite numerous studies conducted over the last decade,

there are still considerable gaps in our knowledge about the biotechnological potential of

green-synthesized nanoparticles. Furthermore, the precise basis of their antibiotic activity

has yet to be defined. The biological methods are generally cost effective, nontoxic, and

ecofriendly. This chapter focuses on the methods involved in algal-synthesized nanoparti-

cles and its applications.

_**Keywords**_ **:** AFM; Ag nanoparticles; antibacterial activity; __ cubic phase; methyl orange; pho-

tocatalytic degradation; _Ulva reticulata_

**1. Introduction**

methods for the synthesis of nanomateri-

****

als that do not use toxic chemicals in the

Nanotechnology deals with the

synthesis protocols so as to avoid adverse

synthesis of nanoparticles that exhibit

effects in medical applications. Among

completely new or improved properties

the nanoparticles, silver nanoparticles

based on specific characteristics such as

(AgNPs) have received considerable at-

size, distribution and morphology. New

tention due to their attractive physico-

applications of nanoparticles and nano-

chemical properties (Elechiguerra _et al_.,

materials are emerging rapidly (Jahn

2005). The metallic nanoparticles are

1999;

Naiwa

2000;

Murphy

most promising and considered as re-

2008).Currently, there is a growing need

markable biomedical agents. Due to their

to develop eco- friendly and sustainable

large surface volume ratio, they govern

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 475

_Biotech Sustainability (2017)_

_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

interest of researchers on microbial re-

pieces and boiled in 100 ml of sterile dis-

sistance. Among the developed nanopar-

tilled water for 5 min. The crude extract

ticles, silver (Ag) nanoparticles are per-

was passed through Whatman No.1 filter

taining to have a wide range of applica-

paper and the filtrate was stored at 4°C

tion in the fields of physical, chemical

for further use the methods suggested by

and biological sciences. In the past dec-

Jha _et al_. (2009).

ade, several kinds of the biological organ-

isms (microbes, plants and seaweeds)

_2.3. Synthesis of silver nanoparticles_

have been employed and well-studied for

Analytical grade (AR) Silver ni-

the ability of silver nanoparticles (Ag-

trate (AgNO3) was purchased from E.

NPs) synthesis (Ramanathan _et al_., 2011;

Merck (India). In the typical synthesis of

Ahmad _et al.,_ 2003; Shankar _et al.,_ 2003;

silver nanoparticles, 10 ml of the aqueous

Mohanpuria _et al.,_ 2008; Kumar _et al.,_

extract of _Ulva reticulata_ was added to 90

2012a). Application of green chemistry in

ml of 1 mM aqueous AgNO3 solution in

synthesizing nanomaterials has vital role

250 ml conical flask and incubated at

in medicinal and all technological aspects

room temperature for 72 h by agitating at

(Mondal _et al.,_ 2011, Begum _et al.,_ 2009).

120 rpm. Suitable controls were main-

Biologically synthesized Ag-NPs have

tained throughout the experiments (Para-

wide range of applications because of

shar _et al._ , 2009). The bio-reduction of

their remarkable physical and chemical

AgNO3 into AgNPs can be confirmed

properties. The literature on the extra cel-

visually by the change in colour from

lular biosynthesis of Ag-NPs using plants

light yellow to brown indicating the for-

and pure compounds from plants are in-

mation of silver nanoparticles (Figure 1).

significant (Kattumuri _et al.,_ 2007; Song

and Kim 2008; Gilaki 2010).

_2.4. Characterization techniques_

In this article, we describe a sim-

The colour change in reaction

ple one step method for the synthesis of

mixture (metal ion solution + seaweed

Ag-NPs by the reduction of aqueous Ag-

extract) was recorded through visual ob-

ions using extracts of green seaweed, at

servation. The bio reduction of silver ions

direct sunlight conditions.

in aqueous solution was monitored by pe-

riodic sampling of aliquots (0.5 ml) and

**2. Materials and methods**

subsequently measuring UV-Vis spectra

****

of the solution. UV-vis spectra of these

_2.1. Screening and selection of sample_

aliquots were monitored as a function of

Fresh sample of _Ulva reticulata_

time of reaction on UV-Vis spectropho-

green seaweed was collected in the month

tometer UV-2450 (Shimadzu).

of January 2013 from Pudumadam coastal

The Ag-NPs solution thus ob-

region (78.99°'E, 9.27°'N), in Gulf of

tained was purified by repeated centrifu-

Mannar, Tamil Nadu, India. Sample was

gation at 5000 rpm for 20 min followed

immediately brought to the laboratory in

by resuspention of the pellet of Ag-NPs in

polythene bags and cleaned thoroughly

10 ml of deionized water. After freeze

with fresh water to remove adhering de-

drying of the purified Ag-NPs, its struc-

bris and associated biota. The alga sample

ture and composition was analyzed by

was cleaned with distilled water using

XRD. The dried mixture of Ag-NPs was

brush for the removal of the epiphytes.

collected for the determination of the

__

formation of Ag-NPs by an X'Pert Pro x-

_2.2. Preparation of aqueous extract_

ray diffractometer (PAN analytical BV,

The whole _U_. _reticulata_ samples

The Netherlands) operated at a voltage of

were initially rinsed thrice in distilled wa-

40 _kV_ and a current of 30 _mA_ with Cu Kα

ter and dried on paper toweling. Twenty

radiation in θ- 2 θ configurations. The

five (25) gram sample was cut into fine

crystallite domain size was calculated

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_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

**Figure 1:** Silver nitrate (AgNO3) solution and others colour changes during the reduction of

AgNO3 into AgNPs by the extract of _U_. _reticulata_ after 20 min of incubation.

from the width of the XRD peaks, assum-

thesized _Ag-NPs_ (Rashed and El-Amin

ing that they are free from non-uniform

2007). All the experiments were per-

strains, using the Scherrer's formula.

formed outdoor with sun as the main

D= 0.94 λ / β Cos θ

source of light (Wang _et al.,_ 2000). Prior

where D is the average crystallite domain

to the experiment, a suspension was pre-

size perpendicular to the reflecting planes,

pared by adding 20 mg of _Ag-NPs_ to 50

λ is the X-ray wavelength, β is the full

ml of methyl orange solution (Fisher Sci-

width at half maximum (FWHM), and θ

entific). Later, the mixture was allowed to

is the diffraction angle. To eliminate addi-

stir constantly for about 30 min in dark-

tional

instrumental

broadening

the

ness to ensure constant equilibrium of _Ag-_

FWHM was corrected, using the FWHM

_NPs_ in the organic solution. During the

from a large grained Si sample.

reaction, the mixture was kept under sun-

β corrected = (FWHM2 sample-

light within a Pyrex glass beaker and

FWHM2si) 1/2

stirred constantly. The mean temperature

This modified formula is valid only when

was found to be 29˚C with 10 h mean

the crystallite size is smaller than 100 _nm_.

shine duration. The absorption spectrum

of the suspension mixture was measured

The silver nanoparticles were ob-

periodically using a UV–visible spectro-

served using SEM. Sample was prepared

photometer (Shimadzu, UV-2450, Japan)

by placing a drop of AgNPs on carbon

after centrifugation to ensure the degrada-

coated copper stuff and subsequently dry-

tion of methyl orange solution.

ing air, before transferring it to the micro-

scope operated at an accelerated and volt-

_2.6. Antibacterial activities_

age of 120KV (JOEL Model JSM-5010

Experimental pathogens namely,

LV with INSA EDS) and followed for

Gram positive bacterium _Staphylococcus_

Energy Dispersive Spectrophotometer

_aureus_ , Gram negative bacteria _Pseudo-_

analysis.

_monas aeruginosa_ , _Escherchia coli_ , _Pro-_

The morphology of the product

_teus mirabilis_ and _Proteus vulgaris_ were

was observed by Nano Surf Easy Scan 2

obtained from Raja Sir Muthaiya Medical

Atomic Force Microscope (AFM) meas-

College, Annamalai University. Patho-

urement to study the morphology and size

gens were loaded in inoculated in medium

of the _Ag-NPs_ (Al-Warthan _et al.,_ 2010).

(Muller Hinton Agar medium) [100

µg/ml, 50 µg/ml, 25 µg/ml ( _Ag-NPs_ )] and

_2.5. Photocatalytic degradation_

AgNO3 was used as negative control.

The photocatalytic degradation of

Each culture was spread on to Muller

methyl orange was evaluated by biosyn-

Hinton Agar plates. Sterile paper discs of

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6mm diameter along with streptomycin

(Positive control) antibiotic containing

_3.2. FTIR spectrum_

discs were placed in each plate. Bacterial

FTIR analysis was used for the

growth inhibition was determined as the

characterization of the extract and the re-

diameter of the inhibition zones around

sulting nanoparticles. FT-IR measure-

the discs. All tests were performed in trip-

ments were carried out to identify the

licate. Then, Petri dishes were incubated

possible biomolecules responsible for the

at 37°C for 18–24 h aerobically, inhibi-

reduction of Ag+ ions and the capping of

tion zone were measured and data was

the bioreduced AgNPs synthesized. FT-

recorded (Bauer _et al._ , 1966).

IR spectrum (Figure 3) showed different

major peaks positioned at 3405.37,

**3. Results and discussion**

2955.75, 2922.87, 2851.82, 1654.15,

****

1637.15, 1512.61, 1457.45, 1418.38,

_3.1. UV–Visible spectroscopy_

132.90, 1250.54, 1175.74, 1109.93,

UV–visible absorption is one

812.05 and 699.73 cm-1. The presence of

among the most important techniques to

peak at 3405.37 cm-1 could be due to O-H

identify the formation of metal nanoparti-

group in alcohols and phenols. A small

cles, provided surface plasmon resonance

peak observed at 2955.75, 2922.87, and

exists for metal (Binupriya _et al.,_ 2010). It

2851.82 cm-1 is due to C-H stretching of

is well known that silver nanoparticles

alkanes. Sharp and intense bands ob-

exhibit yellowish brown color in aqueous

served at 1654.15, 1637.15, 1560.38,

solution due to excitation of surface

1512.61, and 1490.30 cm-1 are due to –

plasmon vibrations in silver nanoparticles

C=C- stretch, N-H bend, NO2 asymmet-

(Shankar _et al.,_ 2004). As the extract was

rical stretch and nitro compounds, respec-

mixed in the aqueous solution of the sil-

tively. Another bands were positioned at

ver ion complex, it started to change the

1457.45 (C-H bend alkanes), 1418.32 (C-

color from watery to yellowish brown due

C stretch (in-ring) aromatics), 1362.90

to reduction of silver ion which indicated

(C-H rock alkanes) and 1250.74 (C-N

formation of silver nanoparticles. It is

stretch aromatic compounds). The ob-

generally recognized that UV–Vis spec-

served bands ranging between 1109.93

troscopy could be used to examine size

and1032.31 cm-1 are due to C-N stretch

and shape controlled nanoparticles in

band of aliphatic amines. Bands observed

aqueous suspensions (Wiley _et al.,_ 2006).

at 812.05 and 743. 34 cm-1 are due to N-H

The UV-Vis spectra recorded from the

wag band of primary and secondary

reaction medium after 4 hours is shown in

amines. A band positioned at 699.73 cm-1

Figure 2. A strong silver plasmon absorp-

is due to –C (triple bond) C-H; C-H bond

tion maximum was recorded at 410-420

alkynes. After bio-reduction, there is a

nm in UV-Vis spectroscopy. The ob-

shift in the absorption and band at

served band in this range has been associ-

2955.75, 1457.45, 1362.90, 812.05 and

ated with Ag-NPs confirming the synthe-

743.34 cm-1 may be due to the binding of

sis of spherical _Ag-NPs_ with narrow size

(NH) C=H and N-H wag group with the

distribution has been revealed (Henglein

nanoparticles. The (NH) C=H groups

1993 and similar observation were also

within the cage of cyclic peptides are in-

made by Kumar _et al.,_ 2012b). Elevation

volved in stabilizing the nanoparticles.

in temperature results in formation of

Thus, the peptides may play an important

spherical and octahedral shaped nanopar-

role in the reduction of _Ag-NPs_. This

ticles of size 5-100 nm (Lengke _et al.,_

could be due to the ability of reducing and

2007). Similarly, shape-controlled _Ag-_

capping agents present in _U. reticulata_

_NPs_ can be also synthesized using biolog-

which were revealed by FT-IR studies.

ical system under varying temperature

Fourier Transform Infra-Red (FT-IR)

(Bansal _et al.,_ 2012).

spectroscopy analysis showed that the

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_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

**Figure 2:** UV–visible absorption spectra of silver nanoparticles after 30 min of incubation.

**Figure 3:** FT-IR spectra of AgNPs synthesized by _U_. _reticulata_.

synthesized nano-Ag was capped with

tern revealed that the sample contains a

bimolecular compounds which are re-

mixed phase (cubic and hexagonal) struc-

sponsible for reduction of silver ions

tures of silver nanoparticles. The average

(Jegadeeswaran _et al.,_ 2012). The above-

estimated particle size of this sample was

mentioned shift was also observed in

10 nm derived from the FWHM of peak

_Codium capitatum_ (Kannan _et al.,_ 2013).

corresponding to 90 plane (Figure 4). X-

ray diffraction showed the average parti-

_3.3. Crystal structures analysis and de-_

cle size of 15 nm as well as revealed their

_termination of crystallite size_

cubic structure (Geethalakshmi and Sara-

The XRD pattern showed three

da 2010).

intense peaks in the whole spectrum of 2θ

value ranging from 10 to 80. Average size

_3.4. Particles morphology (SEM and_

of the synthesized particles was 10 nm

_AFM measurements)_

with size range 10 - 50nm with cubic and

The SEM image (Figure 5 a and b)

hexagonal shape. The typical XRD pat-

depicts the high density Ag-NPs synt -

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_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

**Figure 4:** XRD patterns of silver nanoparticles synthesized after 120 h of incubation. ****

****

**Figure 5:** (a) SEM micrograph of Silver nanoparticles synthesized from the extracts of _U_. __

_reticulata_ ; (b) energy dispersive spectrometer analysis

_-_ hesized by the _U. reticulata_ and confirms

are spherical shaped and well distributed

the development of silver nanostructures

without aggregation in solution. Ag-NPs

with energy dispersive spectrometer. The

predominantly spherical well distributed

SEM micrographs of nanoparticle ob-

with an average size 15 nm (Saraniya De-

tained in the filtrate showed that Ag-NPs

vi _et al.,_ 2013). It is known that the shape

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_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

of metal

nanoparticles

considerably

within 10 h of incubation time. There is

changes their optical electronics proper-

no considerable shift in peak position for

ties (Xu and Kall, 2002). Similar phe-

methyl orange solution without exposure

nomenon was reported by Chandran _et al._

to Ag-NPs. Kansal _et al_. (2006) have re-

(2006).

ported that compared to other irradiation

The surface morphology and size

techniques, solar light was found to be

of the Ag-NPs harvested after 120 h of

faster in decolorizing methyl orange in

incubation were studied by AFM. The

the presence of metal catalyst. The ad-

two- and three-dimensional images of the

sorption of Ag-NPs on to the methyl or-

nanoparticles are shown in Figure 6a and

ange solution was initially low and further

6b. From the 2D view, well-separated

increased with constant increase in time.

spherical particles are seen. The sizes of

Altogether, the photocatalytic properties

the particles are in the range of 1–40 nm.

of Ag-NPs in visible light may be well

However, most of the particles are in the

due to excitation of SPR, which is nothing

range of 10 nm. The 3D view revealed

but oscillation of charge density that can

that the growth direction of all the parti-

propagate at the interface between metal

cles was almost same confirming the sin-

and dielectric medium (Garcia, 2012).

gle crystalline nature of the cubic phase

Ag-NPs are potential, highly efficient and

of _Ag-NPs_. Williams, (2008) reported that

stable photocatalysts under ambient tem-

nanoparticles are clusters of atoms in the

perature with visible light illumination for

size range of 1–100 nm. Morphology and

degrading organic compounds and dyes

size of the synthesized particles were

(Wang _et al.,_ 2008).

studied with atomic force microscope

(shanmugam _et al.,_ 2014).

_3.6. Antibacterial activities_

Highest inhibition zone (10mm) in

_3.5. Photocatalytic degradation_

_Proteus mirabilis_ was observed in _U. re-_

Photocatalytic degradation of me-

_ticulata_ at 100 µg/ml, lowest inhibition

thyl orange dye was investigated using

zone (7 mm) was observed in _U. reticula-_

biometrically synthesized silver nanocata-

_ta_ 25 µg/ml. _U. reticulata_ at 100 µg/ml

lysts by solar irradiation technique at dif-

exhibits high inhibition zone of 8 mm in

ferent time intervals as shown in Figure 7.

_Escherichia coli_ and lowest inhibition

The characteristic absorption peak of me-

zone (7 mm) was present in _U. reticulata_

thyl orange solution was found to be 420

at 25 µg/ml. Ag-NPs from _U. reticulata_

nm. Degradation of methyl orange was

was compared effectively with silver ni-

visualized by decrease in peak intensity

trate solution and standard antibiotic

**Figure 6:** (a). AFM images of synthesized silver nanoparticles using extract of _U_. _reticula-_

_ta_ ; (b) corresponding 3D view.

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_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

__

**Figure 7:** Photocatalytic degradation of methyl orange using silver nanoparticles synthe-

sized from _Ulva reticulata._

__

**Figure 8:** Antibac-

terial activity of sil-

ver nanoparticles

synthesized from _U._

_reticulata_ against

human pathogens.

Con, Control; A, 100µ g/ml; B, 50µg/ml;

C, 25 µg/ml; S,

Ampicillin; (1) _Staphylo-_

_coccus aureus_ ; (2) _Pseu_ _domonas aerugino-_

_sa_ ; (3) _Escherichia coli_ ; (4) _Proteus mira-_

_bilis,_ and (5) _Proteus vu_ _lgaris_.

streptomycin, Ag-NPs exhibited more

In this present investigation, the

activity than silver nitrate solution. Max-

environmental friendly synthesis of Ag-

imum inhibitory activity was observed in

NPs using fresh extract of the green sea-

Ag-NPs from _U. reticulata_ , when com-

weed _U. reticulata_ is described. Despite

pared to control. Raimondi _et al._ (2005)

numerous studies conducted over the last

and Morones _et al._ (2005) corroborated

decade, there are still considerable gaps in

that the bactericidal effect of silver nano-

our knowledge about the biotechnological

particles is size dependent, the antimicro-

potential of green-synthesized nanoparti-

bial efficacy of the nanoparticle depend

cles. Furthermore, the precise basis of

on the shapes of the nanoparticles also,

their antibiotic activity has yet to be de-

this can be confirmed by studying the in-

fined. In addition, improvements in the

hibition of bacterial growth by differen-

way that green-synthesized nanoparticles

tially shaped nanoparticles.

are incorporated into medical devices

****

could increase their efficacy and diminish

**4. Conclusion**

any side effects; but, further research is

****

required to perfect this technology. The

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_Biotech Sustainability (2017)_

_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

morphology of silver nanoparticles was

**Begum, N.A., Mondal, S., Basu,S., Las-**

characterized by SEM and AM. The na-

**kar, R.A. and Mandal, D. (2009).**

noparticles were found to be active in de-

Biogenic synthesis of Au and Ag

grading methyl orange solution with visi-

nanoparticles using aqueous solu-

ble light illumination. The antimicrobial

tions of Black Tea leaf extracts.

activity of synthesized Ag-NPs is promis-

_Colloids Surf B Biointerfaces_. **71,**

ing. In a nutshell, synthesis and character-

**113–118.**

ization of Ag-NPs with regard to novel

**Binupriya AR, Sathishkumar M, Vi-**

morphology are of great interest in the

**jayaraghavan K, Yun SI. (2010)**.

fabrication of antibacterial materials.

Bioreduction of trivalent aurum to

nano-crystalline gold particles by

**Acknowledgments** __

active and inactive cells and cell-

free extract of _Aspergillus oryzae_

The authors wish to thank Profes-

var. viridis. _Journal of Hazardous_

sor and Head, Department of Botany, An-

_Materials_. **177, 539-545.**

namalai University. We also thank Dr. S.

**Chandran SP, Chaudhary M, Rasricha**

Barathan, Professor and Head, Depart-

**R, Ahmad M, and Sastry. (2006).**

ment of Physics. Authors cordially thank

Synthesis of gold nanotriangales

Dr. B.Shanthi and Dr. G. Sivakumar,

and silver nanoparticles using _Al-_

CISL Lab, Department of Physics, An-

_oevera_ plant extract. _Biotechnology_

namalai University, for their help in

_Progress._ **22, 577.**

providing access to SEM and AFM and

**Elechiguerra JL, Burt JL, Morones JR,**

for his suggestions while performing the

**Bragado AC, Gao X, Lara HH,**

research work.

**Yocaman M. (2005)**. Interaction of

silver nanoparticles with HIV- 1.

**References**

_Journal of Nanobiotechnology._ **3, 6.**

****

**Garcia M.A. (2012).** Surface plasmons in

**Ahmad A, Mukherjee P, Senapati S,**

metallic nanoparticles: fundamen-

**Mandal D, Khan MI, Kumar R,**

tals and applications. _Journal of_

**Sastry M. (2003)**. Extracellular bio-

_Physics D: Applied Physics_. **44(28),**

synthesis of silver nanoparticles us-

**389501.**

ing the fungus _Fusarium oxyspori-_

**Geethalakshmi R, Sarada DVL. (2010).**

_um_. _Colloids Surf B Interface_. **28,**

Synthesis of plant-mediated silver

**313–318.**

nanoparticles

using

_Trianthema_

**Al-Warthan A, Kholoud MM, El-Nour**

_decandra_ extract and evaluation of

**A, Eftaiha A, Ammar RAA.**

their antimicrobial activities. _Inter-_

**(2010).** Synthesis and applications

_national Journal of Engineering_

of silver nanoparticles. _Arabian_

_Science and Technology_. **2(5), 970-**

_Journal of Chemistry_. **3, 135–140**.

**975.**

**Bansal V, Bharde A, Ramanathan R,**

**Gilaki, M. (2010)**. Biosynthesis of silver

**Bhargava SK. (2012).** Inorganic

nanoparticles using plant extracts.

materials using 'unusual' microor-

_Journal of Biological Sciences_.

ganisms. _Advances in Colloid and_

**10(5), 465-467.**

_Interface Science_. **179–182 (1),**

**Henglein A. (1993).** Physicochemical

**150–168.**

properties of small metal particles in

**Bauer AW, Kirby WMM, Sherris JC**

solution: ''microelectrode'' reac-

**and Turck M. (1966)**. Antibiotic

tions, chemisorptions, composite

susceptibility testing by a standard-

metal particles, and the atom-to-

ized single disk method. _Journal of_

metal transition. _The Journal of_

_Clinical Pathology_. **45(4), 493-496.**

_Physical Chemistry_. **97, 5457–5471.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 483

_Biotech Sustainability (2017)_

_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

**Jahn, W. (1999).** Chemical aspects of the

cals for its anti-bacterial activity.

use of gold clusters in structural bi-

_Nano-Biomedical Engineering_. **4(4),**

ology. _Journal of_

_Structural_

**12-16.**

_Biology_. **127, 106-112.**

**Lengke MF, Fleet ME, Southam G.**

**Jegadeeswaran P, Rajeshwari Shivaraj,**

**(2007)**. Biosynthesis of silver nano-

**Venckatesha R. (2012).** Green syn-

particles by filamentous cynaobacte-

thesis of silver nanoparticles from

ria from a silver (I) nitrate complex.

extract of _Padina tetrastromatica_

_Langmuir_. **23, 2694-2699.**

leaf. _Digest Journal of Nanomateri-_

**Mohanpuria P, Rana NK, Yadav SK.**

_als and Biostructures._ **7(3), 991 –**

**(2008).** Biosynthesis of nanoparti-

**998.**

cles: technological concepts and fu-

**Jha B, Reddy CR, Thakur MC, Rao**

ture applications. _Journal of Nano-_

**MU. (2009).** Dordrecht, Nether-

_particle Research_. **10, 507–517.**

lands: Springer; Seaweeds of India:

**Mondal S, Roy N, Laskar RA, Sk I,**

The diversity and distribution of

**Basu S, Mandal D. (2011)**. Biogen-

seaweeds of Gujarat coast; **89.**

ic synthesis of Ag, Au and bimetal-

**Kannan RRR, Stirk WA, Van Staden**

lic Au/Ag alloy nanoparticles using

**J. (2013).** Synthesis of silver nano-

aqueous

extract

of

mahogany

particles using the seaweed _Codium_

(Swietenia

mahogani

JACQ.)

_capitatum_

P.C.

Silva

(Chloro-

leaves. _Colloids interfaces B. Bioin-_

phyceae). _South African Journal of_

_terfaces_. **82, 497-504.**

_Botany._ **86, 1–4.**

**Morones**

**JR,**

**Elechiguerra**

**JL,**

**Kansal SK, Singh M, Sudo D. (2006).**

**Camacho A and Ramirez JT.**

Studies on TiO2/ZnO photocata-

**(2005).** The bactericidal effect of

lysed degradation of lignin. _Journal_

silver nanoparticles. _Nanotechnolo-_

_of Hazardous Materials_. **153, 412-**

_gy_. **16, 2346-2353.**

**417.**

**Murphy C.J. (2008).** Sustainability as a

**Kattumuri V, Katti K, Bhaskaran S,**

design criterion in nanoparticle syn-

**Boote EJ, Casteel SW and Fent**

thesis and applications. _Journal of_

**GM. (2007)**. Gum Arabic as a phy-

_Materials Chemistry._ **18, 2173–**

tochemical construct for the stabili-

**2176.**

zation of gold nanoparticles. _In Vi-_

**Naiwa H.S.** ( **2000).** Ed., Hand Book of

_vo_

_Pharmacokinetics_

_and_

_X-_

Nanostructural Materials and Nano-

_RayContrast-Imaging Studies_. **3(2),**

technology Academic Press New

**333-341.**

York. **1-5.**

**Kumar P, Senthamilselvi S, Laksh-**

**Parashar, U.K., Saxenaa, P.S. and Sri-**

**mipraba A, Premkumar K, Mu-**

**vastava, A.** **(2009).** Bioinspired

**thukumaran R, Visvanathan P,**

synthesis of silver nanoparticles.

**Ganeshkumar RS, Govindaraju**

_Digest Journal of Nanomaterial and_

**M. (2012a)**. Efficacy of biosynthe-

_Biostructure_ , **4, 159** - **166.**

sized silver nanoparticles using _Ac-_

**Raimondi F, Scherer G.G, Kotz R and**

_anthophora spicifera_ to encumber

**Wokaun A. (2005).** Nanoparticles

biofilm formation. _Digital Journal_

in energy technology: examples

_of Nanomaterial Bioscience_. **7, 511–**

from electrochemistry and catalysis.

**522.**

_Angewandte Chemie International_

**Kumar P, Senthamilselvi S, Laksh-**

_Edition in English_. **44, 2190-2209.**

**miprabha A, Premkumar K,**

**Ramanathan R, O'Mullane AP, Parikh**

**Ganeshkumar RS, Govindaraju**

**RY, Smooker PM, Bhargava SK,**

**M. (2012b).** Synthesis of silver na-

**Bansal V. (2011)**. Bacterial kinet-

noparticles from _Sargassum ten-_

ics-controlled shape-directed bio-

_errimum_ and screening phytochemi-

synthesis of silver nanoplates using

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 484

_Biotech Sustainability (2017)_

_Synthesis of Biocompatible Silver Nanoparticles... Ganapathy Selvam et al._

_Morganella psychrotolerans_. _Lang-_

**Song JY, Kim BS. (2008).** Biological

_muir_. **27(2), 714–719.**

synthesis of bimetallic Au/Ag nano-

**Rashed MN, El-Amin AA. (2007).** Pho-

particles using Persimmon ( _Diopy-_

tocatalytic degradation of methyl

_ros kaki_ ) leaf extract. _Korean Jour-_

orange in aqueous TiO2 under dif-

_nal of Chemical Engineering_. **25(4),**

ferent solar irradiation sources. _In-_

**808-811.**

_ternational Journal of Physical Sci-_

**Wang G, Liao C, Wu F. (2000).** Photo-

_ences._ **2, 73-81.**

degradation of humic acids in the

**Saraniya Devi J, Valentin Bhimba B,**

presence of hydrogen peroxide.

**Magesh peter. (2013)**. Production

_Chemosphere_. **42, 379-387.**

of biogenic Silver nanoparticles us-

**Wang P, Huang B, Qin X, Zhang X,**

ing _Sargassum longifolium_ and its

**Dai Y, Wei J, Whangbo MH.**

application. _Indian Journal of Geo-_

**(2008).** Efficient and stable photo-

_marine Science._ **42(1), 125-130.**

catalyst under visible light. _Chemi-_

**Shankar SS, Ahmad A, Sastry M.**

_cal Engineering Journal_. **14, 10543-**

**(2003)**. Geranium leaf assisted bio-

**10546.**

synthesis of silver nanoparticles. _Bi-_

**Wiley BJ, Im SH, McLellan J, Siek-**

_otechnological_ _Progress_. **19, 1627–**

**kinen A. Xia Y. (2006).** Maneuver-

**1631.**

ing the surface plasmon resonance

**Shankar SS, Rai A, Ankamwar B,**

of silver nanostructures through

**Singh A, Ahmad A, Sastry M.**

shape-controlled

synthesis.

The

**(2004).** Biological synthesis of tri-

_Journal of Physical Chemistry_.

angular gold nanoprisms. _Nature_

**110(32), 15666- 15675.**

_materials_. **3(7), 482-488.**

**Williams D. (2008).** The relationship be-

**Shanmugam N, Rajkamal P, Cholan**

tween biomaterials and nanotech-

**S, Kannadasan N, Sathishkumar**

nology. _Biomaterials_. **29(12), 1737-**

**K, Viruthagiri G, Sundaraman-**

**1738.**

**ickam A. (2014).** Biosynthesis of

**Xu H, and Kall M. (2002).** Morphology

silver nanoparticles from the marine

effects on the optical properties of

seaweed _Sargassum wightii_ and

nanoparticles. _Journal of Nanosci-_

their antibacterial activity against

_ence and Nanotechnology_. **4, 254-**

some human pathogens. _Applied_

**259.**

_Nanoscience_. **4(7), 881–888.**

****

****

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

****

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 485

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P486-511_

**Diversity and Ethno-Botanical Potential of Tree Plants of**

**Katarniaghat Wildlife Sanctuary, Bahraich (UP) India:**

**An Overview**

**Tej Pratap Mall***

****

_Postgraduate Department of Botany, Kisan PG College, Bahraich-271 801, Uttar Pradesh,_

_India; *Correspondence: drtejpratapmall@gmail.com; Te: +91 945 042 5622_

**Abstract:** Plants are of variable purpose having efficiency as ethno-botanical, ethno-

medicinal, ethno-veterinary and even in agro-forestry wherein they act as shelter, source of

habitat for several organisms, _etc_. They even have a role in improving the soil conditions.

Many useful products such as fruits, timber, fire wood and variety of metabolic chemicals

are also obtained from plants. In _Katarniaghat_ Wildlife Sanctuary (KWS), there are fifty

five tree plant species representing forty five genera belonging to thirty one families. _Mora-_

_ceae_ was found to be the largest family with seven plant species; whereas _Euphorbiaceae_

and _Mimosaceae_ with five; _Anacardiaceae, Myrtacea_ and _Rubiaceae_ with three; _Caesal-_

_piniaceae, Ehretiaceae, Papilionaceae_ and Louraceae with two and rest twenty one fami-

lies, _viz_., _Rutaceae, Apocyanaceae, Baringtoniaceae, Bombocaceae, Dilleniaceae, Ebena-_

_ceae, Tiliaceae, Ulmaceae, Malvaceae, Lythraceae, Sapotaceae, Annonaceae, Rutaceae,_

_Sapindaceae, Dipterocarpaceae,_ _Sterculeaceae, Bignoniaceae, Verbinaceae, Combreta-_

_ceae, Meliaceae_ and _Rhamnaceae_ with single plant species only. This chapter is an attempt

to summarise the information available on plant species found in KWS which are yet not

popular due to limited research.

****

_**Keywords**_ **:** Ethnobotanical; ethnomedicinal; ethnoveterinary; Katarniaghat wildlife sanctu-

ary; nutrimental tree

**1. Introduction**

cities and towns, trees provide shade and

shelter, and their flowers brighten the

We cannot survive without plants.

surroundings, Plants in parks and gar-

We depend on plants for food: directly in

dens contribute to the serene and

the form of grains, roots and tubers, fruits,

peaceful environment, making such plac-

vegetables, spices, oil and beverages.

es favourite retreats (Chin, 2005).

Much of our food also comes indirectly

The knowledge of utilizing wild

form plants. We get our meat and milk

plants was painstakingly passed on

from animals that are dependent on plants

from generation to generation database

for food. Plants provide fuel, either as

of valuable information of the plants

firewood or in the form of fossil fuel, to

around him. It is natural to assume that

cook our food, keep us warm, run our

certain members of the tribe were gradu-

machinery and light up our homes and

ally entrusted with such knowledge. The-

cities. We also depend on trees for con-

se individuals were known as shamans,

struction materials to build our houses

bomohs, healers or witchdoctors. As

and to craft our furniture. From cotton

communications between settlements was

and flax we get fibres for our clothes.

then poor, it is likely that such knowledge

Plant dyes colour our clothes, at least be-

was developed independently in different

fore synthetic dyes were developed. In

locations (Chin, 2005). The primitive

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 486

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ man, through his trial and error, has

tries, but also in developed countries, where

selected many wild fruits which are

modern medicines are predominantly used.

edible and subsequently domesticated

While the traditional medicines are derived

them which played a very vital part

from medicinal plants, minerals, and organic

in supplementary diet knowingly or

matter, the herbal drugs are prepared from

medicinal plants only. Use of plants as a

unknowingly. Although due to the igno-

source of medicines has been inherited and is

rance of modern generation the im-

an important component of the health care

portance of wild plants were recently

system in India. There are about 45,000 plant

have been decreasing yet many people

species in India, with high concentration in

especially in rural areas still use them

the region of Eastern Himalayas, Western

extensively as a supplementary to their

Ghats and Andman and Nicobar Island. The

basic food requirement. A scientific study

officially documented plants with medicinal

of wild fruits is important for the po-

potential are 3,000 but traditional practioners

tential sources which are protective

use more than 6,000. India is the largest pro-

foods. The nutrients/pigments present

ducer of medicinal herbs and is appropriately

in the fruits prevent different degrada-

called the botanical garden of the world. In

rural India, 70 percent of the population is

tive/ageing process in our body and thus

dependent on the traditional system of medi-

via restoring health offer longevity

cine, the Ayurveda, which is the ancient Indi-

(Singh, 2011). These wild fruits would be

an therapeutic measure renowned as one of

utilized at the time of scarcity or culti-

the major systems of the alternative and com-

vated as a source of food material for

plementary medicine (Bhatia, _et al_., 2013).

ever increasing population (Rashid _et_

In _Katarniaghat_ Wildlife Sanctuary

_al_., 2008).

there are fifty five tree plant species repre-

India has been considered as one of

senting forty five genera belonging to thirty

the 17 mega-diversity centers of the word

one families. _Moraceae_ was found to be the

with a wide range of phyto-geographical var-

largest family with seven plant species

iations. It consists of about 64 million hec-

whereas _Euphorbiaceae_ and _Mimosaceae_

tares forest covers out of which 86% is tropi-

with five; _Anacardiaceae_ , _Myrtacea_ and _Ru-_

cal forest comprising 54% dry deciduous,

_biaceae_ with three; _Caesalpiniaceae, Eh-_

37% moist deciduous and 9% wet evergreen

_retiaceae, Papilionaceae_ and _Louraceae_ with

& semi-evergreen (Kaul and Sharma, 1971).

two and rest twenty one families, _viz_., _Ru-_

As a characteristic feature, the tropical forest

_taceae,_

_Apocyanaceae,_

_Baringtoniaceae,_

shows a huge variation in tree species diversi-

_Bombocaceae, Dilleniaceae, Ebenaceae, Til-_

ty place to place (Pitman _et al_., 2002).

_iaceae, Ulmaceae, Malvaceae, Lythraceae,_

Among the different phyto diverse regions

_Sapotaceae, Annonaceae, Rutaceae, Sapin-_

found in the country, the Terai region is one

_daceae, Dipterocarpaceae, Sterculeaceae,_

of them existing from Uttarakhand to West

_Bignoniaceae, Verbinaceae,_ _Combretaceae,_

Bengal. It is the transition zone between two

_Meliaceae_ and _Rhamnaceae_ with single plant

eco-climatic zones, the Gangetic plain to-

species only. The available literature reveals

wards south and Bhabhar towards north,

that most of the tree plants found in _Katar-_

along with the sub- Himalayan tracts (Tripa-

_niaghat_ Wildlife Sanctuary (KWS) are multi-

thi and Singh, 2009). The region has lost ma-

purpose, ethno-botanical, nutrimental, ethno-

jority of its natural forest due to deforestation

medicinal, ethno-veterinary and of environ-

chiefly for agriculture and lack of sustainable

mental use in agro-forestry which provide

forest management in last many centuries

shade, habitat for organisms, soil improve-

(Bajpai _et al_., 2012a, b). Now the natural for-

ment, _etc_., many useful products are also ob-

ests of the region have been restricted to the

tained such as fruits, timber, fire wood and

wildlife protected areas only. _Katerniaghat_

variety of metabolic chemicals which may be

Wildlife Sanctuary (KWS) is also one of

used in the form of home remedies and for

them.

traditional medicine. Considering the multi-

Traditional medicines are used by

purpose importance of these trees of KWS,

about 60 percent of the world's population.

the present overview is an attempt to summa-

These are not only used for primary health

rize the information's available on these

care just in rural areas, in developing coun-

plants which are yet not popular due to one

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 487

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ reason or the other despite providing an array

while description of the plant. Since the

of benefits.

list of the trees is big, the description of

all is beyond the scope of this manuscript

**2. Study area**

so we have taken thirteen plants, _viz_.,

****

_Acacia catechu, Acacia concinna, Aegle_

The study area _Katerniaghat_ Wild-

_marmelos,_

_Albizia_

_lebbeck,_

_Albizia_

life Sanctuary (KWS) is situated in Bah-

_procera, Alstonia scholaris, Bombax cei-_

raich district of Uttar Pradesh in India. It

_ba, Diospyros cordifolia, Ficus racemosa,_

lies along Indo-Nepal international board-

_Madhuca latifolia, Shleichera oleosa,_

er and is situated between 27° 41' – 27°

_Syzygium cumuni_ and _Ziziphus mauritia-_

56' N and 81° 48' – 81° 56' E covering

_na_ for detail description.

an area of 440 km2 with 116 to 165 m el-

evation. The sanctuary comes under the

_4.1. Acacia catechu_ Willd.Khair, Catechu

tropical moist deciduous forest of the

(Mimosaceae)

Himalayan Terai-Bhabar region (Cham-

It is a moderate size tree. Leaves

pion and Seth, 1968; Rodgers and

are pinnate. Flowers yellow in globose,

Panwar, 1988). The forest of the sanctu-

peduncled axillary heads. Pods strap

ary area has been classified into two ma-

shaped, dark brown. Phenology - August

jor forest types (i) The Sal forest and (ii)

to February. In _Katarniaghat_ Wildlife

The miscellaneous forest (Champion and

Sanctuary it is found only in miscellane-

Seth, 1968). Pedagogically the study area

ous forest with IVI value 18.3.

is made up of the alluvial soil of the

****

_Kaudiyala_ and _Saryu_ rivers and its tribu-

_Ethnobotanical potential_

taries flowing adjoining to it. Geological-

 Catechu, a multipurpose tree species

ly the sanctuary area has been divided

is widely used by the inhabitants for

into high and low land areas.

fodder, fuel, building material and in

health care.

**3. Climate**

 The heartwood of the tree is mainly

**__**

used for extracting _Katha_ and Cutch

A typical tropical monsoonal cli-

(decoction obtained after filtration)

mate with three distinct seasons, _i.e_.,

which are sold in the market.

summer (April to June), winter (Novem-

 _Katha_ is commonly used in ayurve-

ber to February) and warm-rainy (July to

dic preparations.

September) prevails in the study area.

 _Katha_ serves as one of the major

March and October are considered as

components in masticatory, _i.e._ ,

transition months between the seasons.

chewing of betel leaf (pan) in India.

The mean maximum temperature ranges

 _catechu_ is a valuable bio-resources

from 22°C in January to 40°C in May and

and has been exploited commercially

the mean minimum temperature ranges

in tannin and _Katha_ industry for dec-

from 8°C in January to 27°C in June. The

ades.

annual rainfall ranges from 36 to 142 cm

 Besides its commercial importance,

in winter, 34 to 662 cm in summer and

it is equally significant for the people

1294 to 1689 cm in warm-rainy seasons

particularly rural communities living

(Bajpai _et al_., 2012).

in the vicinity of catechu forests as it

is a subsidiary source of income to

**4. Observations**

them. To a certain extent, these peo-

ple are dependent on this plant to ful-

At present the KWS has been di-

fill their day to day need of fuel, fod-

vided in to three types of forests- miscel-

der, building material, _etc_.

laneous forest, sal forest and teak planta-

tion forest. The IVI of the plants in all the

_Ethno-medicinal potential_

three forests are presented respectively

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 488

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_

 The decoction of bark mixed with

handles of axe, saw, sickle, hammer,

milk is taken to cure cold and cough.

spade and combs.

 The bark decoction is either alone or

used in combination with opium to

_Socio-religious beliefs_

cure severe diarrhea.

 Khair is considered one of the sa-

 _Katha_ after drying is applied on lem-

cred trees by the natives and wood

on slice and taken regularly with

is used in the religious ceremonies

empty stomach to cure piles.

at the time of havans (yagya).

 Heartwood of _khair_ is boiled with

 Wood is considered sacred and used

other ingredients to prepare the de-

as one of the religious plants along

coction. It is taken as tea by the

with bhoj patra **(** _Betula utilis_ **)** at the

pregnant ladies to keep warm their

funeral ceremony. It is believed to

body. It is also given to cure fever

provide _mukti_ or _moksha_ (peace to

due to cold during the pregnancy. ****

the heavenly soul).

 A decoction is served to women after

2-3 days of child delivery, prepared

_Fencing_

by boiling _Katha_ along with carda-

 Cut branches are extensively used

mom. It is believed that it provides

for fencing purpose by the farmers

strength to the body and also helps in

to protect agricultural fields and lo-

secretion of milk.

cal grasslands from domestic live-

 The water boiled with the heartwood

stock and wild animals.

chips of Khair _,_ is used to take bath

by women after delivery. It is con-

_Tanning_

sidered beneficial to cure the body

 The cutch is used locally for tanning

pains.

leather and as dye to a great extent.

 _Katha_ or decoction of heartwood is

__

applied in mouth and on tongue to

_Economic Importance_

cure mouth ulcer. It is also applied

 Besides traditional utility, _A. cate-_

externally on ulcers, boils, skin erup-

_chu_ is widely utilized commercially

tions and on gums **as** disinfectant.

for extracting Katha from the heart

wood which costs around US $ 4-6

_Fuel_

per kilogram in Indian markets.

 The dried logs, twigs and branches

 Cutch is used as adhesive in ply-

are largely used as fuel.

wood industry and it is also used in

preparing polishes and paints (Singh

_Fodder_

and Lal, 2006).

 The trees are lopped heavily for

their leaves used as **** fodder particu-

From the present study, it is en-

larly for sheep and goats.

visaged that _A. catechu_ has a great socio-

economic importance as it is widely used

_Building and furniture material_

for different purposes by the natives. Be-

 The wood is considered durable and

sides, traditional and commercial im-

widely used by the inhabitants for

portance, it has tremendous ecological

house building material as pole and

significance. Because of its leguminous

to prepare furniture like bed poles,

nature and soil binding abilities, it is a

tables etc.

suitable species for wasteland develop-

ment **.**

_House hold articles_

****

 Wood of khair is preferred to pre-

__
__

_4.2. Acacia concinna_ (Willd.) DC __

pare various parts of local plough,

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_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ Synonyms: _Acacia sinuata_ (Lour.)

mouth ulcers, gumboils and pain in

Merrill, _Acacia rugata_ Lamk., _Mimosa_

the throat.

_sinuate_ Lour.

 It aids in the prevention of tooth

_Acacia concinna_ is a prickly bush or

degradation

and

formation

of

climbing shrub and grows in tropical for-

plaque.

ests of India, common in the warm plains

 It is also helpful in lowering the

leaflets membranous. The leaves are bi-

chances of encountering diabetes **.**

pinnate and the thorns are soft and small.

 _Acacia concinna_ is a good herbal

Flowers are white and pink in copiously

treatment for lowering the body

panicled globose heads in the axils of

cholesterol.

leaf. Pods strap shaped, straight, thick,

 It is a fruitful remedy in curing di-

succulent when dry shriveled and rough

gestive disorders and relieving con-

with straight waved sutures. There are

stipation. It facilitates proper bowl

about 6-10 seeds in a pod. Phenology is

movement and improves the flow of

January-March & November to February.

urine.

The tree is food for the larvae of the but-

 It also possesses the attribute of be-

terfly  _Pantoporia hordonia_. **** Alkaloids are ing a contraceptive which helps in

found in the tree's fruit. In _Katarniaghat_

birth control.

Wildlife Sanctuary it is found only in

 It is utilized in the preparation of

miscellaneous forest with IVI value of 1.0

savoury jams (chutneys). It adds to

****

the flavor.

_Ethno-botanical potential_





_Acacia concinna_ has been used tra-

The leaves, pods has astringent ac-

ditionally for hair care in the Indian

tion and useful in treating cuts,

Subcontinent __since ancient times.

wounds and oral problems. The de-

 It is one of the Ayurvedic medicinal

coction of pods is prepared and used

plants.

for washing and cleaning of wound

 Fruit for hair are being used as a

for quick healing. _Acacia concinna_

traditional shampoo. In order to

is a good herbal remedy for hair. It

prepare it the fruit pods, leaves and

is an advantageous herb for bald-

bark of the plant are dried, ground

ness. Its usage helps in preserving

into a powder then made into a

the natural oil of our hair and nur-

paste. While this traditional sham-

tures the scalp. It encourages the

poo does not produce the normal

growth of hair and strengthens

amount of lather which are found in

them. It is an effective hair cleanser.

sulfate-containing shampoo, it is

It prevents dandruff and lice.



considered a good cleanser. It is

It is used in the manufacture of

mild, having a naturally low pH,

shampoos, soaps and hair packs.



and doesn't strip hair of natural oils.

It is a good herbal treatment for

Usually no conditioner is needed,

black fever (Visceral Leishmaniasis)

for shikakai which also acts as a de-

and fever due to Malaria.

tangler.

 _Acacia concinna_ is a herbal treat-

 An infusion of the leaves has been

ment for skin ailments. It is advan-

used in anti-dandruff preparations.

tageous in curing psoriasis (genetic

 Since _A. concinna_ extracts are used

disease) and the spreadable diseases

in natural shampoos or hair powders

like eczema. It provides a relief in

so the tree is now grown commer-

scabies, rashes, cuts, bruises and

cially in India.

cures them.





The plant parts used for the dry

It is effectual in curing oral ail-

powder or the extract are the bark,

ments. It helps in suppressing bad

leaves or pods. The bark contains

breath. It is helpful in treating

high levels of saponins,  which are-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 490

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ foaming agents found in several

_Acacia concinna_ for 15 minutes.

other plant species used as sham-

Wash the area with hot water and

poos or soaps. Saponin-containing

wipe it.

plants have a long history of use as

mild cleaning agents.

_Acacia concinna for constipation:_

 Saponins from the plant's pods have

 Discard the _Acacia concinna_

been traditionally used as a deter-

seeds after crushing the fruit.

gent, and in Bengal for poisoning Soak it in 1 glass of water for

fish; they are documented to be po-

one hour. Take the infusion.

tent marine toxins.

 The leaves have an acidic taste and

_Acacia concinna (sikakai) for jaundice_

are used in chutneys.

 Take out the _Acacia_ seeds after

crushing the fruit. Soak it in a

_Chemical constituents_

glass of water for an hour. Take

 In commercial extracts, when the

one fourth of the glass infusion. ****

plant is hydrolyzed it yields lupeol,

****

spinasterol, acacic acid, lactone, and _Acacia concinna (shikakai) for dandruff_

the natural sugars glucose, arabinose

 _Acacia concinna_ (shikakai) is a

and rhamnose. It also contains hex-

boon for getting rid of dandruff.

acosanol, spinasterone, oxalic acid,

Boil a handful of coarsely crushed

tartaric acid, citric acid, succinic ac-

_Acacia concinna_ (Shikakai) in a

id, ascorbic acid, and the alkaloid

litre of water for 10 minutes. Cool

scalyctomine and nicotine.

it and use the filtered decoction to

 _Acacia concinna_ is a thorny medici-

wash hair. Do it daily for week

nal plant, native to south Asia,

and then twice a week or make a

widely known for the organic sham-

paste of powdered _Acacia concin-_

poo derived from its fruit, shikakai.

_na_ (Shikakai) by adding water in

 The pods (shikakai), have medicinal

it. Apply it over scalp and hair.

properties and are used for hair

Leave it for an hour. Wash hair

cleansing and enhancement. Shika-

with normal water. It also cleans

kai is traditionally preferred over

your hair from roots to tips. ****

commercially available shampoo,

__

across the Indian Subcontinent.

_Acacia concinna for age spots_

 Shikakai is also used for manufac-

 Make a fine paste of _Acacia con-_

turing body and facial care creams,

_cinna_ fruit. Apply every day for

across the personal hygiene indus-

15 minutes. Wash three times a

try.

day.

 A particular extract from _Acacia_

_concinna_ leaves has shown to be ef-

_Acacia concinna for gum diseases_

fective in treatment of malarial fe-

 Take half table spoon _Acacia con-_

ver.

_cinna_ and boil it in two cups of

water. Gargle with this lukewarm

_Ethno-medicinal potentialty_

water three times a day.

 Shikakai is also used in traditional

medicine to treat jaundice, constipa-

_Acacia concinna for skin diseases_

tion and skin problems.

 Boil one table spoon _Acacia con-_

_cinna_ powder in one cup water.

_Acacia concinna for pain_

Cool it and apply on the skin.

 Apply some castor oil on the af-

fective area. Heat with a hot water

_Acacia concinna (shikakai) pods for lep-_

bag. Now massage with powdered

_rosy_

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_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_

 Squeeze the juice of _Acacia con-_

dandruff. Take 3 to 4 tablespoon

_cinna_ (shikakai) pods. Apply it on

of _Acacia concinna_ (shikakai)

effected parts twice a day.

powder. Make a paste by adding

lemon (nimbu) juice. Apply it

_Acacia concinna for psoriasis:_

over scalp and leave it for an hour

 Take few pods of _Acacia concin-_

or two. Wash with water. Do it for

_na_. Boil them. Apply them over

a week every day and you may see

affected areas.

half your dandruff has gone. ****

****

_Herbal treatment for constipation_

_Herbal treatment for head lice_

 Make a paste of three leaf buds of

 Grind ten dried fruits of _Acacia_

_Acacia concinna_ , two cloves of

_concinna_ after discarding the

garlic and some black salt. Take it

seeds, half cup each of fenugreek

with cooked rice. ****

seeds, wild turmeric roots, roots of

****

Indian Sarsaparilla and Sandal-

_Herbal treatment for fever_

wood chips. Massage the head

 Make a paste of three leaf buds of

with coconut oil and apply this

_Acacia concinna_ , two cloves of

powder. Rinse off after thirty

garlic and some common salt.

minutes. ****

Take it with cooked rice.

****

_Herbal treatment for jaundice_

_Herbal treatment for dandruff_

 Make a paste of one tea spoon

 Grind three to four dried fruits of

tender _Acacia concinna_ leaves,

_Acacia concinna_ (shikakai) with-

three pepper corns, one tea spoon

out seeds. Add one to two tea-

tamarind pulp, half red chilli and

spoons of fenugreek (methi in In-

some salt to add taste. Eat it with

dia) seeds, one teaspoon full wild

cooked rice. ****

turmeric (aamahaldi in India) root

****

powder, one teaspoon of Indian

_Herbal treatment for stomach ache_

Sarsaparilla (anantmool in India)

 Boil one cup _Acacia concinna_ tree

root powder and one teaspoon of

bark in one litre water and sieve it.

sandalwood powder. Mix well.

Add powdered Indian Pennywort

Add water to form a thick paste.

in it. Also add the powder of four

Massage the scalp with coconut

black peppercorns, two carda-

oil, and then apply this paste.

mom, cinnamon, two cloves, one

Leave it for half an hour. Wash off

fourth nutmeg and half tablespoon

and shampoo. Do this once a

long pepper. Mix it and leave it

week. ****

for a month before it is taken for

 Massage your scalp with luke-

medicinal use. Take one table-

warm sesame oil. Boil four to five

spoon twice a day. ****

dried _Acacia concinna_ (Shikakai

****

in India) pods in two glass of wa-

_Herbal treatment for grey hair_

ter. Strain well. Let it cool. Use its

 Soak Indian gooseberry, soap nut

water to rinse your scalp after an

seeds (Ritha) and pots of _Acacia_

hour. It gives relief from dandruff

_concinna_ (Shikakai) in three cups

and promotes hair growth. ****

of water for a night. Grind it. Use

 _Acacia concinna_ (shikakai) and

it as shampoo.

lemon both are beneficial herbs in

__

treating dandruff. The mixture

_Herbal treatment for frizzy hair_

made up of these two, works

 Take following herbs in men-

wonder to make your hair free of

tioned quantity, two hundred g In-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 492

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ dian gooseberry (Amia), two hun-

& heart tonic, ulcer, antiviral, intesti-

dred g _Acacia concinna_ (Shika-

nal parasites, gonorrhoea, epilepsy.

kai), twenty g _Bacopa monnieri_

 _Root_ : Dog bite, gastric troubles, heart

(Bhrahmi), one g Asphaltum

disorders, intermittent fevers, ant-

(Shilajit), forty g dried pomegran-

amoebic, hypoglycemic, rheumatism.

ate peel (Anar ka chilka), twenty

 _Bark:_ Stomach disorders, intermittent

g _Eclipta alba_ (Bhringraaj), Soak

fevers, heart disorders.

them overnight in any iron bowl.

 _Seed:_ Febrifuge.

Grind to make paste. Apply it on

 _Flower_ : Expectorant, epilepsy.

your hair and Scalp. Wash after 3

 _Whole Plant:_ Abdominal pain, ab-

hours with lukewarm water. Do

scess, astringent back ache, dog bite,

not use shampoo. Shampoo may

breast pain, cholera constipation, con-

be used on next day.

vulsions, cramp, diabetes, diarrhoea,

dysentery, fevers, eye complaints,

_4.3. Aegle marmelos_ (L.) Corr __

gastric trouble, abdominal disorders,

It is a medium-sized, deciduous,

jaundice, laxative, nausea night fever,

armed tree. Leaves are tri-

heart disorders, snakebite, stomach

foliolate. Flowers are yellowish.

disorder, vomiting tonic, cut &

Fruits are large, globose. Phenolo-

wounds.

gy is April-May & March-July. In

 _Root bark_ : Fish poison.

_Katerniaghat_ Wildlife Sanctuary

 _Seed mucilage_ : Plaster for walls.

_Aeglemarmelos_ is found only in

 _Seed oil_ : Laxative.

teak plantation forest.

 _Wood_ : Beads worn by low caste, spe-

cial couches for rheumatic patients.

_Ethnobotanical potentiality_



Bael is one of the most important

_Gum around seed_ : To improves adhe-

tree species used in various indige-

sive strength of water paints.



nous system of medicine in India,

_Unripe fruit rind, Bark_ : Yellow dye.

China, Burma, and Sri Lanka. Bael is

 _Stem_ : Pestles of oil and sugar mills.

used in all _tridosa_ \- vista (air), _Pitta_

The medicine is prepared in the form

(phlegm) and _kapha_ (cough). Out of

of pills, powder and paste. Ayurvedic

more than 66 ethno-botanical uses of

practitioners commonly use the roots

bael, 48 are exclusively for medicinal

of bael as an ingredient of dasmula

purposes. Almost all parts of bael are

(ten roots), which is useful in recover-

used in preparing medicine (Kala,

ing the loss of appetite and use fruits

2006). ****

in the preparation of chawanprash.





_Leaf:_ Abscess, backache, eye com-

Bael fruits regarded as an astringent

plaints, abdominal disorders, vomit-

are frequently used by various ethnic

ing, cut & wounds, ulcer, destroy, ber-

communties for the treatment of diar-

iberi, weakness of heart, cholera, diar-

rhoea, dysentery, constipation, stom-

rhoea, cardio tonic, blood sugar, inju-

ach ache, intestinal ulcer, diabetes,

ries caused by animals, nervous disor-

dyspepsia, heart diseases and cholera

ders, hair tonic, acute bronchitis, child

due to its digestive and carminative

birth, veterinary medicine for wounds,

properties. ****

killing worms, fodder for sheep, goat

 Bael is highly valued in Ayurvedic

and cattle, stimulation of respiration

medicine for the treatment of chronic

and contraction of de-nerved nictitat-

diarrhoea and dysentery and as brain

ing membrane in anaesthetized cats. ****

tonic. Bael possesses antiviral, anti-

 _Fruit:_ Astringent, diarrhoea, gastric

helminthic anti-inflammatory, anti-

troubles, constipation, laxative, tonic,

bilious, anti-parasitical, anti-pyretic,

digestive, stomachic, dysentery, brain

anti-scorbutic, aromatic, astringent, di-

gestive, febrifuge, haemostatic, anti-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 493

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ diarrheal, laxative and nutritive proper-

 For Hindus, the Bael is sacred tree,

ties. ****

which they dedicate to the lord Shiva ****

Ripe bael fruit is sweat, aromatic

by offering of Bael leaves. Its three

and nutritive, whereas fresh fruit is

leaflets are assumed by the symbols of

stringent and has laxative properties.

three gunas or attributes (e.g., satva _,_

Bael fruit powder exhibits anti-

rajas and tamas _,_ literally meaning mo-

cancerous and anti-proliferative activi-

rality, superiority and immorality, re-

ties. The combinations of five parts of

spectively); three Gods (Brahma,

bael, such as, fruit, leaf, bark, root and

Vishnu and Mahesh); and three lives

flower is assumed to be effective for

(past, present and future). Bael is con-

certain mental disorders.

sidered to be extremely auspicious and

 Unripe fruits pulp mixed with boiled

cultivated around most of the Hindu

rice water is taken twice a day to cure

temples.

vomiting in pregnancy. Unripe fruits

__

pulp mixed with sugar is taken with

_Phytochemicals of Aegle marmelos_

milk twice daily for curing urinogenital

_A. marmelos_ has been reported to con-

disorders.

tain several phyto-constituents mainly

 Half roasted unripe fruit pulp mixed

marmenol, marmin, marmelosin, mar-

with equal quantity of sugar is taken

melide, psoralen, alloimperatorin, ruta-

twice a day to cure dysentery. Unripe

retin, scopoletin, aegelin, marmelin, fa-

fruit pulp powder is taken twice daily

garine, anhydromarmelin, limonene, a-

to cure abscess.

phellandrene, betulinic acid, marmesin,

 Bael leaf extract is taken twice a day to

impertorin, marmelosin, luvangentin and

remove the intestinal worms. Leaf

auropetene Rahman and Parvin .

poultice is used as remedy in ophthal-

 Due to the presence of various

mic problems and ulcer.

phyto-constituents the plant has an-

 Leaf juice is reported to have multiple

ti-diarrhoeal, anti-microbial, anti-

medicinal uses, including controls of

cancerous, anti-pyretic, anti-

diabetes. Cooling delicious drink pre-

genotoxic,

anti-fertility,

anti-

pared from fruit pulp along with sugar

inflammatory anti-diabetic and diuret-

and tamarind diluted with water is use-

ic activities.

ful for health.

 The essential oil isolated from the

 Baelroot decoction is given twice daily

leaves of _A. marmelos_ tree has proved

to cure fever and cold. Extract of bael

to have antifungal activity against an-

root, pyaz __ ( _Allium cepa_ Linn _._ ) _,_ and

imal and human fungi like _Tri-_

haldi ( _Curcuma domestica_ Valeton)

_chophyton mentagrophytes_ , _Tri-_

mixed in equal proportion is put in the

_chophyton rubrum_ , _Microsporum_

ears to relive earache and secretion

_gypseum_ , _Microsporum audounii_ ,

from ears. Root decoction is used in

_Microsporumcookie_ , _Pidermophyton_

the treatment of intermittent fevers and

_floccosum, Aspergillus niger, Asper-_

heart palpitation.

_gillus flavus_ and _Histoplasma capsu-_

 Root and stem bark decoction is used

_latum._

in the treatment for fever and various

 The leaf extracts and fractions have

types of heart disorders. Bael root is

fungicidal activity against various

used in the treatment of abdominal

clinical isolates of dermatophytic fun-

pain, heart palpitation and urinary

gi.

troubles.

 Various extracts of _A. marmelos_

 Bael tea is good for health and is used

leaves, roots ad fruits have been re-

for flatulence, gastrointestinal prob-

ported to be active against many bac-

lems, cough and chronic intestinal dis-

terial strains.

eases in children.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 494

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ _Ethno-botanical potential_

_back_ has a density of 0.55-0.66g/cm3

 Bael fruits are edible, contain high

or higher (Babu _et al_., 2009).

protein and are used in making tasty

 Even a where it is not native, some

aromatic cold drinks and jam. Its fresh

indigenous herbivores are liable to

juice is better and pungent. In Myan-

utilize lebbeck as food resource. For

mar, Bael fruits are used in making

example, the greater rhea ( _Rhea_

paints. Fruits are also used as a substi-

_Americana)_ has been observed feed-

tute for soap, as source of essential

ing on it in the cerrado of Brazil.

oils and perfumes. The mucilage of

__

bael seed is good cementing material.

_Ethno-madicinal potential_

Bael wood is used in building houses,

 Lebbeck is an astringent, also used

making carts, agricultural implements,

by some cultures to treat boils,

pestles, handles of tools and combs. A

cough, to treat the eue, flu, gingivitis,

yellow dye is obtained from the rind

lung problems, pectoral problems, is

of unripe fruits and is used in calico

used as a tonic, and is used to treat

printing. An essential oil is also dis-

abdominal tumors.

tilled from the rind. Dried fruit after

 The bark is used medicinally to treat

removing the pulp are used as pill

inflammation.

boxes for keeping valuable medicines

 In Sidha system of medicine the bark

and sacred ashes. Bael stem yields

and flowers of this plant are used to

gum, which is used for improving the

treat arthritis (Mudaliar, 1936).

adhesive potency of water paints. Its

 The tribal people in Himachal Pra-

wood is suitable for making charcoal.

desh and Kashmir use this plant to

treat inflammation (Srivastava _et al_.,

_4.4. Albizzia lebbeck_ **** (Linn.) Benth. ****

1986; Jain, 1991; Kapur, 1993).

Synonym: _Mimosa lebbeck_ Linn.

 Balasubramaniam (1992) reported

 It is a tall, unarmed, and deciduous

that the tribals point Calimere Wild-

tree distributed throughout India from

life Sanctuary, Tamilnadu use this

the plains up to 900m in the Himala-

plant to treat fractures.

yas. It is tree growing to height of 18-

 In Ayurvedic system of medicine, the

30 m tall with a trunk 50 cm to 1 m in

stem bark of this plant is used to treat

diameter. The leaves are bipinnate,

diarrhoea (Nadkarni, 1954), edema,

7.5-15 cm long, with one to four pairs

poisoning, asthma and bronchitis

of pinnae, each pinna with 6-18 leaf-

(Gupta, 2004).

lets. The flowers are white, with nu-

 Inflammation is complex patho-

merous 2.5-3.8 cm long stamens, and

physiological process medicated by a

very fragrant. The fruit is a pod 15-30

variety of signalling molecules pro-

cm long and 2.5-5.0 cm broad, con-

duced by leucocytes, macrophages

taining six to twelve seeds. In Katar-

and mast cells as well as by the acti-

niaghat Wildlife Sanctuary it is found

vation of complement factors that

only in miscellaneous forest with IVI

bring about edema formation as a re-

0.9.

sult of extravasation of fluid and pro-

__

teins and accumulation of leucocytes

_Ethnobotanical potential_

at the inflammatory site (White,

 It uses included in environmental

1999). All the steroidal and non-

management, forage, medicine and

steroidal anti-inflammatory drugs

wood. It is cultivated as a shade tree

(NSAID's), despite their great num-

in North and South America. In India

ber, cause undesired and serious side

and Pakistan, the tree is used to pro-

effects. Therefore, development of

duce timber. Wood from _Albizia leb-_

new and more powerful drugs is still

needed.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 495

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_

 Research in plants with medicinal

gland near the base of the petiole, leaflets

properties and identification of the

rigidly sub coriaceous, grey beneth, gla-

chemical components responsible for

brous obliquely truncate at the base.

their activities have corroborated the

Flowers whitish in copiously panicled

traditional uses of ancient healing

heads. Pods thin, brown, glabrous. White

wisdom and lore and have proven the

siris is a large, fast growing tree with an

enduring healing potential of many

open canopy that is almost evergreen but

plant medicines even in today's hi-

becomes leafless for a short time in the

tech community.

dry season. It grows up to 30 meters tall.

 It is previously reported that the al-

The bole can be straight or cooked; it can

coholic extract of _Albizia lebback_

be branchless for up to nine meters and up

protects the guinea pig against the an-

to sixty cm in diameter. An ornamental

tigen induced challenge (Tripathi _et_

tree, it is often planted along avenues and

_al_., 1977; Barua _et al_., 1997).

in gardens to beautify them. Phenology is

 Further there it also reduced the level

in May-June and September-March. In

of histamine and raised the plasma

Katarniaghat Wildlife Sanctuary it is

cortisol in antigen challenged guinea

found both in miscellaneous forest and

pigs (Tripahti and Shukla, 1979) as

Sal forest with IVI of 2.8 and 0.5, respec-

well as in bronchial asthma patients

tively.

(Tripathi _et al_., 1978). Das _et al_.,

(2003) and Pramanik _et al_., (2005)

_Ethno-botanical potential_

previously

reported

the

anti-

 The tree is extensively harvested

inflammatory activity of the metha-

from the wild for its timber, many

nol extract of _Albizia Lebback_ bark.

natural forests being managed on a

Many saponins, such as lebbekanin

forty year rotation. ****

A-H (Varshney and Khan, 1961;

 The tree is also grown as a plantation

Varshney and Sharma, 1969; Varsh-

crop in Asia, Africa and the Ameri-

ney _et al_., 1973, 1976) and Albizzi-

cans. ****

asaponin A-C (Pal _et al_., 1995),

 Fuel wood plantations are managed

which contain oleanolic acid, echino-

on a 20-year rotation. ****

cystic acid or acacic acid as sapogen-

 The cooked leaves are eaten as a

ins were reported from various parts

vegetable. In times of scarcity the

of this plant. Further, melanoxetin

bark can be ground into a powder,

okenin-3-one, (+) pinitol, (-) leuco-

mixed with flour and eaten. ****

pelargonidin (Gupta _et al_., 1966).

 The tree is widely planted for its

 Alternative medicine for the treat-

good soil binding capacity. ****

ment of various diseases is getting

 It is occasionally cultivated as shade

more popular. Many medicinal plants

tree for tea and coffee plantations,

provide relief of symptoms compara-

where it also acts as a wind and fire-

ble to that of conventional medicinal

break. ****

agents (Verpoorte, 1999).

 It is popular for the rehabilitation for

seasonally dry, eroded and degraded

_4.5. Albizia procera_ (Roxb.) Benth. __

soils. Its ability to grow on dry,

_Synonym: Mimosa procera_ Benth __

sandy, stony and shallow soils makes

The habitat ranges from monsoon

it a useful species for reforestation

forest, mixed deciduous forest, savannah

for difficult sites. ****

woodlands, pyrogenic grasslands, road-

 Good survival and rapid early growth

sides and dry gullies, to stunted, seasonal

have been reported in reforestation

swamp forest. It is commonly found in

trials on both saline and alkaline

open secondary forest. It is a large decid-

soils, which are widely cultivated in

uous tree. Leaves bi-pinnate with a large

agro-forestry system. ****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 496

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_

 The bark can provide tanning materi-

bre length is 0.9 mm, mean fibre di-

al. It is used in India for tanning and

ameter is 0.021mm). ****

dying. However, its low tanning con-

 The calorific value of dried sapwood

tent (12-17%), considerable weight

is 4870 kcl/kg, and that of heartwood

loss in dying and difficult harvesting

4865 kcl/kg. An excellent charcoal

has limited its importance. ****

(39.6%) can be prepared from the

 When injured, the stem exudes large

wood, and it is widely used as a fuel. ****

amounts of a reddish-brown gum that

****

is chemically similar to, and used as a

_Ethno-medicinal potential_

substitute for, gum arabic (obtained

 White siris is commonly used in tra-

from _Acacia senegal_ and other spe-

ditional medicines. Some research

cies). ****

has been carried out into the medici-

 The leaves are known to have insec-

nal activities of the plant and a num-

ticidal and pesticidal properties. ****

ber of active compounds have been

 The branches (twigs) are used by tea

recorded.

planters as stakes for lying out tea

 All parts of the plant are anti-

gardens. These are found to split

cancerous.

well. The species is popular along

 The roots contain alpha-spinasterol

field borders. ****

and a saponin that possess spermi-

 Pods and fallen leaves should be con-

cidal activity at a dilution of 0.008%.

sidered not as undesirable litter but as

 A decoction of the bark is given for

potential energy sources. It seems

the treatment of rheumatism and

probable that if the pods of the relat-

haemorrhage.

ed species _A. lebbeck_ can yield ten

 It is also considered useful in treating

barrels of ethanol per hectare, then

problems of pregnancy and for stom-

this species could as well. ****

ach-ache. The leaves are poultice on

 The timber has large amount of non-

to ulcers.

durable, yellowish-white sapwood. ****

 The heartwood large and heavy, light

_4.6. Alstonia scholaris_ (Linn.) R.Br. __

or dark brown with light and dark

_Synonym: Echites scholaris_ Linn. __

bands. Due to the broadly interlocked

_Alstonia scholaris_ found in India,

nature of the grain, it is more suitable

Sri Lanka, Pakistan, Nepal, Thailand,

for use in large section where a bold-

Burma, South East Asia, Africa, Northern

er effect is desired, such as in large-

Australia, Solomon Islands, and Southern

sized panels and tabletops. ****

China. _Alstonia scholaris_ is an evergreen

 It seasons and polishes well. The

tropical tree up to 80 ft in height, having

wood is used chiefly for construction,

greyish rough bark with lenticels, secret-

furniture, veneer, cabinet work, floor-

ing white milky latex-rich in poisonous

ing, agricultural implements, mould-

alkaloid, lateciferous which is bitter in

ing, carts, carriages, cane crushers,

taste. Leaves grow in clusters of seven,

carvings, boats, oars, oil presses and

coriaceous, elliptic-oblong, 10 to 20 cm

rice pounders. It is resistant to several

long, 3 to 4.5 cm wide, pointed at the

species for termites. ****

base, rounded at the apex, glossy green on

 The chemical analysis of the wood

the upper surface, white or greyish on the

indicates that it is a suitable material

underside. The tip of the leaf is rounded

for paper pulp. Bleached pulp in sat-

or shortly pointed, tapering towards the

isfactory yields (50.3%) can be pre-

base. The blokes of larger trees are

pared from _A. procera_ wood by the

strongly fluted to 10 m. the outer blaze is

sulphate process. It is suitable for

cream to yellowish in colour with abun-

writing and printing paper (mean fi-

dant, milky latex that flows rapidly when

cut. The inflorescence is a much-branched

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 497

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ terminal panicle, up to 120 cm long;

 The bark and roots are boiled with

flowers 7-10 mm long white, cream or

rice and eaten by girls daily for sever-

green, the tube hairy, lobes sparsely or

al weeks to treat excessive vaginal

densely pubescent, 1.5-4 mm long, the

discharge.

left margins overlapping, strongly per-

 The roots and bark are used in tradi-

fumed. The fruits are thin pods that can

tional medicine as an anthelmintic, as-

grow up to 20 inches long. Fruit is made

tringent tonic, alternative antidiar-

up two slender follicles which are pendu-

rhoeaticum, antiperiodicum, _etc_.

lous and cylindrical follicles, 20 to 40 cm

 The latex is used to clean wounds and

long, 4-5 mm in diameter. Seeds are 3 to

can we used for chewing gum.

4 mm long, with brown ciliate hairs on

the ends. Phenology- December-June. In

_4.7. Bombax ceiba ****_ DC. __

Katarnia Ghat Wildlife Sanctuary it is

It is a large deciduous tree. Leaves are

found only in miscellaneous forest with

digitate, leaflets 5-7, flowers red or yel-

IVI 0.4.

lowish, capsules ovoid. Phenology is

March-April. In Katarniaghat Wildlife

_Ethno-botanical potential_

Sanctuary is being found only in miscel-

The wood is too soft for making

laneous forest with IVI 11.00.

anything- so it is usually used in making

packing boxes, blackboards, _etc_. _Alstonia_

_Ethno-botanical potential_

_scholaris_ tree has been used to make pa-

 The silk cotton tree is often re-

per.

ferred to as the silent doctor for the

host of medicinal benefits that is of-

_Ethno-madicinal potential_

fers almost each part of the tree, in-

 _Alstonia scholaris_ has many medici-

cluding the bark, flowers, fruits, seed

nal properties like antimicrobial, anti-

and leaves, gums, thorns have

amoebic, anti-diarrheal, antihyperten-

therapeutic potential **.**

sive, anti-malarial, febrifuge, stimu-

 A herbal composition made from the

lant,

hepoprotective,

immune-

bark of the tree, for example is admin-

modulatory,

anti-cancer,

anti-

istered for the treatment of male sexu-

asthmatic, antioxidant, analgesic, anti-

al and gastro-intestinal disorders like

inflammatory,

anti-fertility,

anti-

dysentery and diarrhoea. The pharma-

diabetic, _etc_.

cological benefits are basically due to

 _Alstonia scholaris_ used in the treat-

the presence of Glycosides and tan-

ment of fevers, chronic diarrhea, dys-

nins in the root and stem. ****

entery, ulcers, rheumatic pains, can-

 It has haemostatic properties and is

cer, malarial fever etc.

administered during menorrhagia. ****

 The ripe fruit of the plant are used in

 Silk cotton extracts are used in eve

syphilis and epilepsy.

care, tentax forte, acne pimple

 The milky juice of _Alstonia Scholaris_

cream. The plant is also being used

has been applied to treat ulcers.

for general debility, diabetes, impo-

 The bark of the _Alstonia scholaris_ is

tence, spermatorrhoea, urinary stones

used in Ayurvedic medicine to treat

and liver disorders. ****

fever, malaria, troubles in digestion,

 Some of the diseases for example di-

tumors, ulcers, asthma and so forth.

arrhoea, dysentery, asthma, rheuma-

 The leaves and the latex are applied

tism, leprosy, leucorrhoea, body

externally to treat tumors.

pain, wounds are included in anti-

 The dried leaves of the _Alstonia_

inflammatory, analgesic, anti-

_scholaris_ are used as an expectorant.

microbial and oxytocic activities

 The leaves can be used to treat skin

of plant as indirect evidence of

diseases.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 498

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ scientific validation (Jain and Vertings of stem and root shakers or by seeds

ma, 2014). ****

also. The flowers are pollinated by very

****

small wasps. Phenology: April – July. In

_4.8. Diospyros cordifolia_ Linn. __

Katarniaghat Wildlife Sanctuary is being

It is a large shrub or small tree.

found both in miscellaneous forest and

Leaves are ovate-oblong, ovate lanceo-

Teak Plantation forest with IVI of 9.7 and

late, acute, base cordate or rounded and

1.9, respectively.

hirsute on both surface. Flowers pale

****

white in axillary cymes. Fruits are glo-

_Ethnobotanical potentiality_

bose yellow at maturity. Phenology-

 Traditionally it is used in Indian

March-June and June-September, In

medicinal practice as astringent,

Katarniaghat Wildlife Sanctuary is being

carminative, stomachic, vermicide

found both in Sal forest and Teak Planta-

etc (Mall and Tripathi, 2017).

tion forest with IVI of 3.2 and 3.0 respec-

 The extract of fruit is used in leprosy,

tively.

diarrhoea, menorrhagia. It is useful in

****

the treatment of leucorrhoea, blood

_Ethnobotanical potentiality_

disorder, burning sensation, fatigue,

 It is of great use for human being.

urinary discharge, intestinal worms

 It has commercial value being used in

and as carminative.

Bidi industry as a raw material.

 Leaves are astringent to bowels and

 Leaves are being used in stupefying

good in case of bronchitis; leaves are

fishes.

used in dysentery young tender leaves

 It is being used in several ailments

are used for fair complexion. The de-

either as a cure or for the well-being,

coction of leaves is used to wash the

_viz_., used for lever disorders, whoop-

wounds and ulcers.

ing cough, leprosy, ulcers, gonor-

 Bark is useful in asthma and piles.

rhoea, fever as emetic and anti-

The latex or milky juice is adminis-

helminthic. Alcoholic extract are an-

tered in chronic infected wounds,

ti-inflammatory, antipyretic and an-

haemorrhoids, boils, traumatic swell-

algesic. It is depressant, spasmolytic

ing, toothache, vaginal disorder,

producing bradycardia and hypoten-

wounds it promote healing very soon.

sion. Aqueous extract is being used

 The root sap is used for treating dia-

in critical jaundicised condition.

betes.

 The fruits are consumed because of its

 Phytochemical properties: The leaf of

juicy and sweet nature by local inhab-

this plant contains sterols, triterpe-

itants (Mall, 2016).

noides (lanosterol) and alkaloids, tan-

_****_

nins and flavonoids. Stem bark gives

_4.9. Ficus racemosa_ Linn __

gluanol acetate, β-sterol, lupenol,

__

stigmasterol. Fruit contains gluanol

Synonym: _Ficus glomerata ****_ Roxb.

acetate, glucose, tiglic acid, esters of

A large deciduous tree, buttressed

taraxasterol, lupeol acetate and other

at the base. Bark smooth reddish brown;

phytosterols.

blaze pink, fibrous with white latex turn-

ing yellow on exposing. Leaves are 5-15

_4.10. Madhuca latifolia_ Roxb. __

x 2.5-6.5 cm, alternate, ovate or elliptic-

Madhuca is a large deciduous tree

lanceolate, entire, sub-acute, base round-

reaching a height up to 20m. Leaves are

ed or acute, glabrous above, minutely dot-

large and broadly elliptic 12-20cm long.

ted beneath. Receptacles 2-3.2 x 2-3.5

The bark is 1.2 cm thick. Flowers white to

cm, clustered on leafless branches,

cream colour with tubular, fleshy and

smooth or pubescent, red or pink at ma-

juicy corolla. Fruit berry, ovoid, green

turity. Plant is propagated by using cut-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 499

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ at maturity and turn pinkish yellow

osa is derived from the Latin word 'ole-

when ripe.

osa' meaning oil, as the seed kernels are

rich in oil. Synonymously the tree is also

_Ethnobotanical potentiality_

reffered as _Schleichera trijuga_ Willd.,the

 The madhuca contains protein, carbo-

word trijuga stands for 'three pairs',based

hydrate, fat, minerals, calcium, phos-

on thepresence of three pair of leaflets in

phorus, iron, carotenes, sugar. Vita-

a leaf. Kusum is a large forest tree with

mins and many other nutrimental

dense green foliage. Leaves pinnate with

chemical constituents.

three pairs of leaf lets .Inflorescence ra-

 The bark of Madhuca is used to

ceme. Flowers are white and fruits small.

cure leprosy and to heal wound.

The fruits are berry shaped, globose or

 The flower decoction is used for

ovoid with a hard skin. The seeds are

headache due to cough and cold.

brown, irregular elliptic, slightly com-

 Paste of fresh bark is useful on joint

pressed oily and enclosed in a succulent

and muscles pain.

aril. The oil content of the seed is around

 Whole plant decoction is taken orally

59-72% with yellowish green color. Phe-

which is useful in joint and muscles

nology is in October-November.

pain (Mall and Tripathi, 2017a).

It is locally known as kusum. The

other common names are kusum, kusumb,

_4.11. Syzygium cumini_ Skeels. __

kosumb, koshamara, Celon oak kosamara,

It is a large evergreen tree with whit-

lac tree, honey tree, gum lac tree, macas-

ish brown bark. Every year the bark is

sar oil tree, sukoshka, skrataka, jatud-

shed off. Its leaves are simple pointed at

ruma,koshamra, jantu vriksha and kshu-

the tip, somewhat leathery, oval to rec-

dra maukkuli, etc. It occurs in the Indian

tangular and somewhat shiny. Flowers are

sub-continent and south East Asia. There

mostly white and appear in cluster from

are many trees that are grown for multiple

axil to leaves.The fruit is berry.

products. They are known as multipur-

pose trees (MPTS), a term widely used in

_Ethnobotanical potentiality:_

agro-forestry. Kusum is also one among

 The fruit contains 88% moisture,

the multipurpose trees which has been

0.7% protein, 0.1% fat, 19.7% car-

proved to be useful in numerous ways

bohydrate and 0.4% minerals. Fresh

from times immemorial.

fruit

had

the

antioxidants

708

In Katarniaghat Wildlife Sanctu-

mg/100g AEAC units. The ripe fruit

ary (KWS) it occurs in all the three types

contains anthocyanin pigment (Rao _et_

of forests with different IVI values. In

_al.,_ 2006). _Syzygium cumini_ is a well-

miscllaceous forest, Sal forest and Teak

known anti-diabetic herb.

plantation the IVI value of the kusum



plant is 1.5, 4.3 and 4.4, respectively.

It is a good immune modulator. It is

The available literature reveals

also used in blood pressure, dysen-

that this multipurpose ethno-botanical,

tery, diarrhoea and gingivitis.

nutrimental,

ethno-medicinal,

ethno-

veterinary and plant of environmental use

_4.12. Schleichera oleosa_ (Lour.) Oken. __

in agro-forestry which provide shade,

Synonims: _Pistacia oleosa_ Lour.

habitat for organisms, soil improvement,

_Schleichera trijuga_ Willd., _Cussambium_

_etc._ , many useful products are also ob-

_oleosum_ Kuntze., _Melicocca trijuga_ Juss.

tained such as fruits, timber, fire wood

It is a monotypic genus belonging

and variety of metabolic chemicals which

to the same family to which the popular

fruit 'Litchi' belongs

may be used in the form of home reme-

**.** The generic name

dies and for traditional medicine. Con-

of kusum, Schleichera is derived after the

sidering the multipurpose importance of

Swiss botanist J. C. Schleicher who first

the tree, which is yet not popularise due

descrived the tree. The species name ole-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 500

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ to one reason or the other despite provid-duced in winter and summer season.

ing an array of benefits (Mall and Tripa-

About 34-38% of the total lac pro-

thi, 2017b).

duction of India is shared by the

Kusum tree as lac host. There are al-

_Ethno-botanical potential_

so other lac hosts but the quality of

 The leaves, twigs and the seed- cake

kusum lac is far superior. The dence

are used as fodder to feed cattle.

foliage of the mature kusum tree pro-

 The wood is suitable as firewood and

vides an additional advantage of sup-

makes excellent charcoal.

porting brood lac (inoculums stick

 Pressed oil cakes from kusum tree are

lac with emerging larvae from the

rich source of crude protein, carbo-

female resin cells) viability even dur-

hydrate, fibre and other minerals

ing the very hot summer season, oth-

and serves as nutritive cattle feed.

erwise summer mortality of lac in-

 The oil extracted from the seed,

sects is a common problem with oth-

called as kusum oil is used for culi-

er host plants like _Butea monosperma_

nary and lighting purposes.

(palas) and _Ziziphus mauritiana_

 The kusum oil is being used to cure

(ber).

itching, acne, burn and other skin

 The seeds of kusum are a very rich

problems.

source of oil (60-72%) for industrial

 The oil is used in rheumatism by ex-

implications. The seed oil called

ternal massage.

kusum oil is an important component

 Kusum oil is used in hair dressing as

of the Makassar oil used for hair

well as for promoting hair growth.

dressing and cooling bath oil.





The pinkish-brown heart wood is

Kusum oil is used in textile industry

very hard, durable and excellent to

for batik applications and also for

make pestles, cartwheels, axles,

making soap.



plows, tool handles, and rollers of

The bark of kusum tree produces tan-

sugar mills and oil presses.

nins and dyes that are occasionally

 Kusum plant is known for lac cultiva-

used in small-scale industries like

tion. It is one of the major host plant

tanning in leather industry.



commercially exploited for cultiva-

Young leaves and shoots-raw cooked

tion of the Indian lac insect ( _Kerria_

in soups or steamed and served with

_lacca_ ). It supports the kusmi strain of

rice.

lac insect, which produces good qual-

 The ripe fruit is eaten raw which has

ity, natural, biodegradable and com-

a pleasant acid flavor.

mercially important, light colored lac

 The unripe fruits are pickeled.

resin of demand by lac industry, thus

 Oil obtained from the seed called

fetching high remunerative prices to

macassar oil, is sometimes used for

lac growers. The lac resins serves as

culinary purposes. It contains cyano-

a livelihood support to millions of

genic compounds, which may cause

poor farmers in states like Jharkhand,

giddiness and should be removed if

Chattisgarh, Orissa, Andhra Pradesh

the oil is used for human consump-

and West Bengal. Immature lac in-

tion.

sects prefer semi-tender twig of

 The kusum tree is also grown as an

kusum tree for sap sucking and start

avenue tree or wayside tree.

secreting resins surrounding their

 The tree is utilized for multifarious

body. The resinous coatings of close-

purposes and is a boon for a subsist-

ly settled sessile insects eventually

ence farmer.

coalesce together to form an encrus-

 The extended foliage and canopy of

tation in five to six months. On

the kusum tree provides good shade

kusum tree, two lac crops are pro-

and is therefore, suitable for mixed

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 501

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ farming with other heat susceptible

and Gandhi _et al_., 2011). Therefore, it is

economic plants.

used in production of biodiesel. In a re-

port by Gandhi _et al.,_ 2011 methyl ester

_Importance of S. oleosa as a biodiesel_

was produced using _S. oleosa_ seeds.

_fuel_

__

The depletion of the conventional

_Phytoremediation properties_

petroleum resources has become a prob-

 _Callophyllum inophyllum_ L. and _Bixa_

lem of major concern in recent years. Ex-

_orellana_ L. (Chaturvedi _et al_., 2012).

tensive research is going on to find an al-

 Mining, smelting of metalliferrous,

ternative fuel. Since vegetable oils have

dumping of waste, chemicals used in

properties similar with that of diesel, they

agriculture etc. Are the different

are replacing diesel in the field of com-

source of soil pollution, but the waste

mercial transportation and agricultural

rocks generated by mining is the main

machinery. But the direct use of vegetable

source of the metal pollution of soil.

oil is having adverse effects on the com-

The direct consequences of the depo-

bustion engine. Therefore, these vegetable

sition of waste rocks on the surface

oils are converted to biodiesel. Blending,

are the loss of cultivatable lands, for-

emulsification, thermal cracking, and

est and grazing land (Clemente _et al_.,

trans-esterification are the few techniques

2007, Rio _et al_., 2006 and Freitas _et_

used for the conversion of crude vegeta-

_al_., 2004). Activity such as grinding,

ble oil into biodiesel. At present, biodiesel

crushing, washing and smelting, used

is produced by sunflower oil, palm oil and

to extract and concentrate metals,

soybean oil by trans-estrification process.

generate waste rocks and tailings.

These oil due to their non-toxic, biode-

Most of the tailings exhibit acidic pH

gradable and renewable nature, have

due to which the microbial activity

gained a lot of alteration by the research-

decreases which in turn leads to the

ers. Cetane number for biodiesel is higher

death of plants. Tailings fo not con-

than that of petroleum. Moreover, bio-

tain organic matter and are character-

diesel does not contain aromatic compo-

ized by the high concentration of ar-

nents. The emission of carbon monoxide,

senic, cadmium, copper, manganese,

hydrocarbon and particulate matter is also

lead, zinc and other heavy metals

less as compared to that of diesel fuel.

(Mukhopadhyay and Maiti, 2010).

High cast of the above mentioned oil is

However some plants can exist in the

the basic disadvantage associated with

region of high concentration of metals

them. Hence, the non-edible type of oils

(Das and Maiti, 2008). Such plants

yielded from tress such a mahua, sal, in-

can be used to restore the contaminat-

seed, caster, karanji neem, rubber,

ed sites by the process of phytoreme-

jatropha, kusum, cashew, restaurants

diation. Phytoremediation is an envi-

waste oils and greases slong with animal

ronmental friendly and cost efficient

fats are best suited for the production of

technique used to treat the contami-

biodieses, for instance, _S. oleosa_ seed oil,

nated soil, air or water through the use

one of the many non-edible seed oil is

of plant without employing any soil

found to have many cyanogenitic materi-

excavation or mechanical clean up

als and free fatty acides (FFA) such as

method. Although many physic-

myristic acid, palmitic acid, palmitoleic

chemical techniques are also available

acid, cis oleic acid, trans linolelaidic acid,

to extract metals such as acid leaching

cis linoleic acid, alpha linolenic acid,

and electro-osmosis, but these tech-

eicosadienoic, heneicosanoic, behenic

niques are quite costly and can decon-

acid, erucic acid, lingoceric acid, do-

taminate only small portions of land

cosahexaenoic acid (Mikolajczek and

(Ramadhas and Murleedharan, 2005).

Smith, 1971, Canacki and Gerpen, 2001

Moreover, these techniques also dete-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 502

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ riorate biological activity of the soil

 The bark is known to contain medical-

and adversely affect its physical struc-

ly important compounds like lupeol

ture. Therefore, the phyto-remediation

used in preparing analgesic and anti-

is the preferred technique to decon-

tumerous agents like betulin and betu-

taminate the soil. This approach to

lic acids.

remove the metal is called green min-

 It balances kaph, useful in productive

ing because further extraction of met-

cough and asthema.

als can be done from the plant tissue

 It cleanses intestine.

(Clemente _et al_., 2007).

 It is used in bleeding disorders like

nasal bleeding and heavy periods.

_Use as livestock feed_

 The unripe fruits are absortant, useful

 A few non-conventional agro-

in diarrhea, neuralgia, paralysis, con-

industrial by-products including _S._

stipation and bloating.

_oleosa_ cake were checked for their ef-

 The fruit pulp improves hair strength

fectiveness a livestock feed (Punj,

and promotes hair growth.

1988). The presence of tannins ad-

 The ripe fruit improves digestion

versely effects the utilization of vari-

strength, improve taste and relieves

ous nutrients (Mc Leod, 1974). In ad-

anorexia.

dition, tannins are believed to create

 The leaf, seed, oil, and bark are used

toxic effects by breaking down the al-

for treating rheumatoid arthritis,

imentary canal tissues and the hydro-

headache, myalgia,,skin disease, ma-

lysable tannins make pathological

larial fever and prophylactic against

changes in liver, kidney, heart, etc.

cholera.

When their concentration in blood in-

 The bark is astringent and is used in

creases further than the competence of

fever as antipyratic, useful in pruritus.

the liver to deify them. The levels of

 The kusum oil is bitter, sour, sweet

tannins were determined using various

which improves strength and immuni-

chemical and biological methods. It

ty and can be taken regularly. It im-

was observed that in _S. Oleosa_ , tannin

proves taste and relieves anorexia.

levels in terms of total phenols (TP)



and condensed phenols (CP) were

The oil is digestive, induce mobility,

low, and protein-precipitation capaci-

causes diarrhea, purgative and re-

ty (PPC) could not be detected be-

lieves constipation.



cause of its very low level. Hence, it

The fine paste of the bark which is

can be considered safe for incorpora-

astringent is mixed with oil is applied

tion in livestock feed since the harm-

to cure itch and acne and other skin

ful factors are absent (Makkar, 1990). ****

eruptions.



****

The oil is useful in worm infection,

_Ethno-medicinal potential_

skin diseases, in toxic conditions, poi-

 Different plant parts (stem bark, seed,

soning, ulcer and wounds.

fruit and seed oil) of kusum are used

 The seed oil is also used for the cure

in traditional medicines.

of itch and acne.





The seed oil is used by the local vaids

The seed oil is stimulating and has

for curing skin diseases like scabies,

cleansing applications.

itching, and acne.

 The ripe fruit is often served with salt

 The bark decoction is also used

which improves digestion, useful in

against skin inflammation and ulcers.

anorexia and nourishing.

 The bark decoction is also infused for

curing malaria.

_Ethno-veterinary potential_





The fine paste of the bark of Kusum is

The seed is grinded so as to make

often used to control tissue swelling.

fine powder. It is mixed with wa-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 503

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ ter and given to cattle for remov-tion of an extract prepared from the bark

ing worms from the stomach.

and stem of Sri lankan tree _S. oleosa_ re-

 The fine powder of the seeds is

sults in the isolation of seven sterols,

applied to wounds and ulcers of

Scheicherastins (1-7) and two related

cattles to remove maggots.

sterols 8 and 9 designed as Schleicheols 1

and 2. The isolated Scheicherastins exhib-

_Phyto-chemical constituents_ ****

ited cancer cell growth inhibitory proper-

Phytochemical

studies

have

ties (Pettit _et al_., 2000).

shown that its bark contains lupeol, lupeol

acetate, betulin, betulinic acid, beta-

_Antioxidant activity_

sitosterol, and scopoletin (Dan and Dan,

Oxygen is used for generating

1986). A very recent report have also

metabolic energy in our body but it also

shown the existence of taraxerone and

produces reactive oxygen as by products

tricadenic acid A in the outer bark of the

during its various reactions in the body.

above plant (Ghosh _et al_., 2011). The

Reactive oxygen species are usually at-

bark also contains about 10% tannin and

oms or a group of atoms having odd (un-

antitumor agents such as betulin and betu-

paired) electrons, in aerobic cells these

linic acid have also been isolated from it. ****

are produced during mitochondrial elec-

****

tron transport and several oxidation reac-

_Anticancer activity_

tions (Forman and Torres, 2002). These

Cancer is a term used for a disease

reactive species acan, react with DNA

in which abnormal cells trend to prolifer-

and several other bio-molecules causing

ate in an uncontrolled way and, in some

what is called 'oxidative damage to DNA'

cases metastasize. Extensive research has

this damage causes changes in DNA such

been done in order to find therapeutic

as stand breaks; changes at cross links

drug for the treatment of cancer. Plant

between DNA and protein; changes as

based products have been frequently ex-

base tree sites among other changes (Diz-

amined as potential anticancer agents.

daroglu _et al_., 2002). Several medicinal

The screening of various medicinal plants

plants, fruits, vegetable can decrease the

results in the isolation of bioactive com-

risk of oxidative damage as they comprise

pounds which have been reported as ef-

of vitamins, carotenes, phenolic com-

fective chemopreventive as well as chemo

pounds, flavanoids, alkanoids, tannins,

therapeutic agents (Kawamori _et al_.,

_etc_. which act as chemo-preventive agents

1999, Choi _et al_., 2001, Kirana _et al_.,

(Dhir _et al_., 1993, Cozzi _et al_., 1997 and

2003 and Sandhya _et al_., 2006). The phy-

Thind _et al_., 2012). These phyto-

tochemical screening of _S. oleosa_ re-

chemicals can prevent damage by their

vealed the presence of lupeol and butilinic

radical scavenging ability. Thind _et al._

acid type triterpene which have antineo-

evaluated the hydroxyl radical scavenging

plastic activity (Bhatia _et al_., 2013). This

potential of _S_. _oleosa_. Extracts of roots of

study provides a step toward the explora-

_S_. _oleosa_ with different solvents were

tion of _S. oleosa_ as a chemo preventive

tested for their anti-proliferative activity.

agent against cancer. A bulk of research

Antioxidants are molecules which

revealed that the phyto-chemicals exhibit

can safely interact with free radicals and

their anticancer properties either by sup-

terminate the chain reaction before vital

pressing the proliferation of tumor cells

molecules get damaged. The free radical

_via_ suppression of various cell signalling

damage can be prevented by several en-

pathways or by induction of apoptotic

zymes and the principle antioxidants such

death in tumor cells by generation of free

as vitamin E, beta-carotene, and vitamin

radical, such as reactive oxygen/nitrogen

C, present in the defence system of our

species (Bharti _et al_., 2003 and Pettit _et_

body. Several studies have shown that

_al._ , 2000). A report involving the separa-

plant phenolics also have antioxidant

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 504

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ properties

(Velioglu

_et_

_al_.,

1998,

tivity against various microbes. It was

Maisuthisakul _et al_., 2007 and Li _et al_.,

also found that this plant has various en-

2008). Natural polyphenols can have sim-

vironment aspects to it as well. The bio-

ple structure for example phenolic acid,

diesel produced from it. Is found to have

phenylpropanoids, flavonoids can have

many properties similar to that of diesel

simple structures like polymers, _e.g_., lig-

e.g. viscosity and volatility. Also its ce-

nis, melanins, tannins (Bravo, 1998). Free

tane number is higher than that of petro-

radical scavenging property, metal chelat-

leum; therefore it can replace diesel for

ing property, effects on cell signalling

the combustion engine. On the basis of

pathways and on gene expression con-

physic-chemical,

growth

and

bio-

tributes to the potential of phenolics as

chemical parameters _C. inophyllum_ and

antioxidant therapeutic agents (Soobrattee

_B. orellana_ were found to be more capa-

_et al_., 2003). _S.oleosa_ has been found as

ble for phyto-remediation of the con-

potent antioxidant due to the presence of

tained soil compared to _S. oleosa_. Fur-

phenolic compounds (Thind _et al_., 2011).

thermore, it was observed that it con-

tained low tannin levels, thus it can be

_Antimicrobial activities_

considered safe to be used as a livestock

In recent study two (Ghosh _et al_.,

feed.

2011) triterprnoids, namely taraxerone

The major use of kusum tree is for

and tricadenic acid A were isolated from

cultivation of lac. However, other uses of

the outer bark and preliminary study on

kusum tree are currently underexploited

their antimicrobial activities were done

but hold promise to benefit human life in

against five different fungal pathogens

many spheres. The plantation of kusum in

namely

_Colletotrichum_

_camelliae,_

suitable areas needs to be promoted in

_Fusarium equisti, Altermaria alterata,_

view of its advantages as MPT. There is a

_Curvularia eragrostidis, Colletotrichum_

tendency to prefer quick growing trees in

_gloeosporioides_ by in vitro antifungal as-

the forestation programmes, which may

say (Suleman _et al._ , 2002 and Saha _et al_.,

not always be advantageous or even eco-

2005) and against four bacterial patho-

friendly in long run.

gens namely. _Escherichia coli, Bacillus_

The role of kusum plantation can

_subtilis, S aureus_ and _Enterobacter_ by

mainly be envisaged in terms of economic

antibacterial assay. It was found that both

benefits to the resource-constrained farm-

taraxerone and tricardenic acid A had

ers dwelling around forest areas.

prominent activities against the fungal

and bacterial pathogens.

_4.13. Ziziphus mauritiana_ Lam. __

The

enumerations

collectively

__

_Ziziphus mauritiana_ is an ex-

show the various pharmacological activi-

tremely drought hardy and native fruit of

ties of _S. oleosa_. It has potential of anti-

India, found wild and cultivated.

cancer, antioxidant and antimicrobial ac-

tivities. It contains various poly phenolic

_Ethnobotanical potentiality_

compounds. The poly phenols scavenge

It is useful as food, fodder, nutrient,

free radicals and do not allow them to

medicinal, construction material and fuel.

damage the cell. Due to its free radicals

_Z. mauritiana_ is having tremendous

scavenging activity, _S_. _oleosa_ is a potent

medicinal properties, attributed by di-

antioxidant. Free radical scavenging ac-

verse group of secondary metabolites

tivity can also be correlated to cyto-

such as alkaloids, flavonoids terpenoids,

toxicity. It exhibits toxicity against vari-

saponin, pectin, triterpenoic acids and

ous cell lines and was found to be as ef-

lipids. Jujubosides (saponin) isolated

fective anticancer agent. It, moreover, has

from Ziziphus reported to have haemolyt-

a great scope of being an effective anti-

ic, sedative, anaxiolytic, and weetness

microbial agent since it showed good ac-

inhibiting properties. Whereas, cyclopep-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 505

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ tide alkaloids, found to have sedative,

**Bajpai, O., Kumar, A., Mishra, A. K.,**

antimicrobial, hypoglycaemic, anti-

**Sahu, N., Pandey, J., Behera, S.**

plasmodial, anti-infectious, anti-diabetic,

**K. and Chaudhary, L. B. (2012b).**

diuretic, analgesic, anticonvulsant and

Recongregation of tree species of

anti-inflammatory activities (Goyal _et_

Katarniaghat Wildlife Sanctuary,

_al.,_ 2012).

Uttar Pradesh, India. _Journal of Bi-_

In spite of the fact that _Ziziphus mau-_

_odiversity and Environmental Sci-_

_ritiana_ having medicinal properties it is

_ences_ **2, 24-40.**

neither considered as important medicinal

**Babu, N. P., Pandikumar, P. and Igna-**

plant nor utilized for medicinal use in

**cimuthu, S. W. (2009).** Anti-

main stream therapeutic.

inflammatory activity of Albizia

From the present study, it is envisaged

lebbeck Benth., an ethnomedicinal

that the trees of _Katarniaghat_ Wildlife

plant, in acute and chronicanimal

Sanctuary has great socio-economic im-

models of inflammation **.** _Journal of_

portance as they are being widely used for

_Ethnopharmacology_ **. 125, 356-360.**

different purposes by the natives. Nature

**Balasubramaniam, P. (1992)**. Observa-

has provided a complete store house of

tion on the utilization of forest

remedies to cure ailments of mankind.

plants by the tribal's of point Cali-

Besides, traditional and commercial im-

mere wild life sanctuary, Tamil Na-

portance, they have tremendous ecologi-

du. _Bulletin of Botanical survey of_

cal significance. Trees which are of le-

_India_ **34, 100-111**.

guminous nature and soil binding abili-

**Barua, C. C., Gupta, P. P., Patnaik, G.**

ties, they all are suitable species for

**K., Kulsherestha, D. K. and Dha-**

wasteland development **.** However, other

**wan, B. N. (1997).** Studies on the

uses of many trees are currently underex-

antianaphylactic

activity

of

ploited but hold promise to benefit human

franctions of Albizzia lebbeck. _Cur-_

life in many spheres. The plantation of

_rent science_ **25, 397-399.**

plants in suitable areas needs to be pro-

**Bharti, A. C., Donato, N., Singh, S. and**

moted in view of their advantages as

**Aggrawal, B. B. (2003)**. Curucu-

malty purpose trees (MPT). There is a

min down regulates the constitutive

tendency to prefer quick growing trees in

activation of nuclear factor-kappa B

the forestation programmes, which may

and Ikappa B alpha kinase in human

not always be advantageous or even eco-

multiple myeloma cells, leading to

friendly in long run. The trees must be

suppression of proliferation and in-

conserved and more plantations should be

duction of apotosis. _Blood_ **101,**

done either by utilisation of Biotechnolo-

**1053-1062.**

gy or through traditional methods. People

**Bhaumik, S., Aanjum, R., Rangara, N.,**

conserve what they love. They love what

**Pardhasaradhi, B. V. V. and**

they understand and they understand

**Khar, A. (1999)**. Curicumin medi-

what they are taught.

ated apotosis in AK-5 tumor cells

involves the production of reactive

**References**

oxygen intermediates. _FEBS Lett._

****

**456, 311-314**.

**Bajpai, O., Kumar, A., Mishra, A. K,**

**Bhatia, H., Kaur, J., Nandis, S., Gur-**

**Sahu, N., Behera, S. K. and**

**nam, V., Chowdhary, A., Reddy,**

**Chaudhary, L. B. (2012a).** Pheno-

**P. H., Vashishtha, A. and Rathi,**

logical study of two dominant tree

**B. (2013).** A review on _Schleichera_

species in tropical moist deciduous

_oleosa_ : Pharmacological and Envi-

forest from the Northern India. _In-_

ronmental

aspects.

_Journal_

_of_

_ternational Journal of Botany_ **8, 66-**

_Pharmacy Research_ **6(1),224-229.**

**72**.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 506

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ **Bravo, L. (1998).** Polyphenols: chemis-dealing of na abandoned copper-

try, dietery sources, metabolism and

tailing pond. _Environ Monit Assess_.

nutritional significance. _Nutr Rev._

**137, 343-350.**

**56, 317-333.**

**Das, A.K., Ahmad, F., Bachar, S.C.,**

**Celemente, R., Paredes, C. and Bernal,**

**Kundu, J., Dev, S., 2003.** Anti-

**M. P. (2007).** A field experiment

inflammatory effect of Albezia

investigating the effects of olive

lebbeck (Benth.) Bark. _Online_

husk and cow manure of heavy

_Journal of Biological Science_ **3,**

metal availability in a contaminated

**685-687.**

calcareous

soil

from

Murcia

**Dhir, H., Kumar, A. and Sharma, A.**

(Spain). _Agric Ecosyst Environ_.

**(1993).** Relative efficiency of Phyl-

**118, 319-326.**

lanthus emblica fruit extract and

**Champion, H. G. and Seth, S. K.**

ascorbic acid in modifying lead and

**(1968).** A Revised Survey of the

aluminium-induced sister-chromatic

Forest Types of India. _Publication_

exchanges in mouse bone marrow.

_Division, Govt. of India, New Delhi_.

_Environ Mol Mutagen_. **21, 229-236.**

**Chanacki, M. and Van Gerpen, J.**

**Dizdarogula, M., Jaruga, P., Bi-**

**(2001).** Biodiesel production from

**rincioglu, M. and Rodrigeuez, H.**

oils and fats with high free fatty ac-

**(2002).** Free radical induced damge

ids. _Trans Am Soc Agric Engineers._

to DNA: mechanisms and meas-

**44, 1429-1436.**

urement. _Free Radicals Biol Med._

**Chaturvedi, N., Dhal, N. K. and**

**166, 1102-111.**

**Ramareddy, P. S. (2012).** Compar-

**Forman HJ and Torres M. (2002).** Re-

ative phytoremediation potential of

active oxygen species and cell sig-

Callophyllum inophyllum L., Bixa

naling:

burst

in

orellana L. and Scheichera oleosa

macrophage signaling. _Am J. Respir_

(Lour.) Oken on iron ore trailings.

_Crit Care Med._ **166, S4-S8.**

_Int J Min Reclam Environ._ **26,104-**

**Freiteas, H., Prasad, M. N. V. and Pra-**

**108**.

**tas, J. (2004).** Plant community tol-

**Chin, W.Y. (2005).** Plants That Heal,

erant to trace elements growing on

Thrill and Kill SNP International,

the degraded soils of Sao Domingos

Singapore **pp.172.**

mine in the south east of Portugal:

**Choi, J. A., Kim, J.Y. and Lee, J. Y.**

enhvironmental implications. _Envi-_

**(2001).** Induction of cell cycle ar-

_ron Int._ **30, 65-72.**

rest and apoptosis inhuman breast

**Gandhi, M., Ramu, N. and Raj, S. B.**

cancer cells by quercetin _. Int J. On-_

**(2011).** Methyl ester production

_col_. **19, 837-844.**

from Schleichera oleosa. _Int J_

**Cozzi, R., Ricordy, R., Aglitti, T., Gat-**

_Pharma Sci Res_. **2, 1244-1250.**

**ta, V., Perticone, P. and De salvia,**

**Ghosh, P., Chakraborty, P., Mandal,**

**R. (1997).** Ascorbic acid and β-

**A., Rasul, M. G., Chakraborty,**

carotene as modulators off oxida-

**M. and Saha, A. (2011).** Triterpe-

tive damage. _Carcinogenesis._ **18,**

noids from Schleichera oleosa of

**223-236.**

Darjeeling foothills and their anti-

**Dan,**

**S.**

**and**

**Dan,**

**S.**

microbial ativity. _Indian J. Pharm_

**(1986).** Phytochemical

study

of

_Sci._ **73, 231-233.**

Adensonia digitata, Psychotria ad-

**Goyal, M., Sasmal, D. and Nagori, B. P.**

enophylla and S. oleosa. _Fitoterapia_

**(2012).** Review on ethnomedicinal

**57, 445-446.**

uses, pharmacological activity and

**Das, M. and Maiti, S. K. (2008).** Com-

phytochemical constituents of

parison between availability of

Ziziphus mauritiana ( _Z. jujuba_

heavy metals in dry and wetland

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 507

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ Lam., nonMill). _Spatula DD_ **2, 107-Kowamori, T., Lubet, R. and Steele, V.**

**116.**

**E. (1999).** Chemopreventive effect

**Gupta, A. K. (2004).** Reviews on Indian

of curcumin, a naturally accuring

Medicinal Plants.Vol

I

Indian

anti-inflammatory agent, during the

Council of Medical Research, New

promotion/progression stages of co-

Delhi, **pp. 445-480**.

lon cancer. _Cancer Res_ **59, 597-**

**Gupta, A.K., Varalakshmi, P. (1998).**

**601.**

Anitinflammatory activity of Lu-

**Lee, K. W., Hur, J. H. and Lee, C. Y.**

peol linoleate in adjuvant induced

**(2005).** Antiproliferative effects of

arthritis _. Fitoterapia_ **69, 13-19.**

dietary phenolic substances and hy-

**Gupta, S. R., Malik, K. K. and Sesha-**

drogen peroxide. _J. Agric food_

**dri, T. R. (1966).** Chemical compo-

_Chem._ **54. pp. 7036-7040.**

nents of Albizzia lebbek heart

**Li, H. B., Wong, C., Cheng, K.W. and**

wood. _Indian Journal of Chemistry_

**Chen, F. (2008).** Antioxidant prop-

**4, 139-141.**

erties in vitro and total phenolkic

**Iwasa S. (1997).** Schleichera oleosa

contents in methanol extracts from

(Lour.) Oken. I. faridah Hanum.

medicinal plants. _Lwt-Food Sci_

L.J.G. van der Maesen (Eds.). plant

_Technol._ **41, 385-390.**

resources of South-east Asia No.

**Mahaptma, S. P.and Sahoo, H. P.**

11. Auxilary Plants, Prosea Founda-

**(2008).** An ethno-medico botanical

tion, Bogor Indonesia. **pp. 227-229.**

study of bolangi, Orissa, India: na-

**Jhansi, P., Kalpana, P. and Ramanu-**

tive plant remedies against gynae-

**jan, G. K. (1994).** _Apidologie._ **25,**

cological diseases. _Ethanobot Leafl_.

**289-296.**

**12, 846**

**Jain, S.K., (1991).** Dictionary of Indian

**Maisuthisakul, P., Suttajit, M. and**

folk medicine and Ethnobotany.

**Pongsawatmanit, R. (2007).** As-

Deep Publications, Lucknow, **pp.**

sessment of phenolic content and

**17.**

free radical-scavenging capacity of

**Jain, V. and Verma, S. K. (2014).** As-

some Thai indigenous plants. _Food_

sessment of credibility of some folk

_Chem_. **100, 1409-1418.**

medicinal claims on Bombax ceiba

**Makkar, H. P. S. (1990).** Tannin levels

Linn. _Ind. J. Trad. Knowl_. **13, 87-**

and their degree of polymerization

**94.**

and specific activity in soma agro-

**Kala, C. P. (2006).** Etnhobotany and eth-

industrial by products. _Biol Wastes_

noconservation of Aegle marmelos

**31, 137-144.**

(L.)Correa. _Indian Journal of Tradi-_

**Mall, T.P. (2016).** Diospyros cordifoia

_tional Knowledge_ **5, 537-540.**

Roxb.- An under exploited potent

**Kapur, S.K., 1993.** Ethno-medico plants

ethnomedicinal feed- A review

of Kangra valley (Himachal Pra-

_World J. Pharmaceut. Res_. **5, 172-**

desh). _Journal of Economic and_

**177.**

_Taxonomic Botany_ **17, 395-408**.

**Mall, T. P. and Tripathi, S. C. (2017).**

**Kaul, O. N. and Sharma, D. C. (1971).**

Diversity of Wild Nutrimental

Forest type statistics. _Indian Forest-_

Fruits of District Bahraich, Uttar

_er_ **97, 432-436.**

Pradesh, India. _Int. J. Curr. Biosci._

**Kirana, C., Mcintosh, G. H., Record, I.**

_Plant Biol._ **4, 65-77.**

**R. and Jones, G. P. (2003).** Anti-

**Mall, T. P. and Tripathi, S. C. (2017).**

tumor activity of extract of _Zingiber_

Kusam-A Multipurpose Plant from

_aromaticum_ and its bioactive ses-

Katarniaghat Wildlife Sanctury of

quiterpenoid

Zerumbone.

_Nutr_

Bahraich

(UP)

India-A

Re-

_Cancer_. **45, 218-225.**

view. _World Journal of Pharmaceu-_

_tical Research_ **. 6, 463-477.**

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 508

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ **McLeod, M. N. (1974).** Plant tannis-their

mination of accurate extinction co-

role in forage quality. _Nutr Abstr_

efficients and simultaneous equa-

_Rev._ **44, 803-815.**

tions for assaying chlorophylls a

**Mikolajczak, K. L. and Smith, C. R.**

and b extracted with four different

**(1971).** Cyanolipids kusum (Schlei-

solvents: verification of the concen-

chera trijuga (seed oil). Northern

tration of cjlorophyll standards by

Regional

research

Laboratory

atomic absorption spectroscopy _. Bi-_

ARS/USDA _Lipids_. **6, 349-350.**

_ochem Biophys Acta._ **975, 384-394**.

**Moon, A., Khan, A. and Wadher, B.**

**Pramanik, K. C., Bhattacharya, P.,**

**(2009).** Biotherapeutic antibacterial

**Chatterjee, T. K. and Mandal, S.**

of potential Schleichera oleosa

**C. (2005).** Anti-inflammatory activ-

against drug resident isolates. _J_

ity of methanol extracts of Albizzia

_Pure Apl Mirobiol._ **3, 181-186.**

lebbeck (Mimosaceae) bark. _Euro-_

**Mukhopadhyay, S. and Maiti, S. K.**

_pian Bulletin of Drug Research_ **13,**

**(2010).** Phytoremediation of metal

**71-75.**

mine waste. _Appl Ecol Environ Res_. ****

**Punj, M. L. (1988).** Agricultural by-

**8, 207-222.**

products and industrial wastes for

**Mudaliar, K. S. M., (1936).** Siddha Ma-

Livestock and poultry feeding. All

teria. Department of Indian medi-

India Co-ordinated research project

cine and Homeopathy. Chennai, **pp.**

India council of Agriculture Re-

**799-800.**

search, New Delhi.

**Nadkarni, A. K. (1954).** Indian Materia

**Ramadhas, S. and Murleedharan, C. J.**

Medica, Vol. 1. Popular Book De-

**S. (2005).** Performance and emis-

parment, Bombay. ****

sion evaluation of a diesel engine

**Nemeikaite, C. A., Imbrasaite, A.,**

fueled with methyl esters of rubber

**Sergediene, E. and Cenes, N.**

seed oil.

**(2005).**

Quantitative

structure-

**Rao, V. K., Shivshankara, S. and**

activity relationships in pro-oxidant

**Prakas, G. S. (2006).** Antioxidant

cytotoxicity of polyphenols: role of

properties of some underutilized

potential of phenoxyl radical/phenol

fruits.National Symposium on Un-

redox couple. _Arch Biochem Bio-_

derutilized

Horticultural

Crops,

_phys_. **441, 182-190.**

IIHR, Bangalore. **pp54.**

**Palanuvej, C. and Vipunngeun, N.**

**Rashid, A., Anand, V. K., Serwar, J.**

**(2008).** Fatty acid constituents of

**(2008).** Less known wild edible

Schleichera oleosaa (Lour) Oken

plants used by Gujjar tribes of

seed oil. _J. Health Res._ **22, 203-212.**

Dirtrict Ralouri, Jammu aand

**Pettit, G. R., Numata, A. and Cragg, G.**

Kashmir State, India. _Int. J._

**M. (2000).** Isolation and structures

_Bot._ **4, 219-224.**

of

Scheicherastins

(1-7)

and

**Rio, M. D., Font, R., Moreno-Rojas, R.**

Schleicheols 1 and 2 from the teak

**and De Haro-Bailon, A. (2006).**

forest medicinal tree Schleichera

Uptake of lead and Zinc of wild

oleosa _. J. Nat Prod._ **63, 72-78.**

plants growing on contaminated

**Pitman, N. C. A., Terborgh, J. W., Sil-**

soils _. Ind Crop Prod._ **24, 230-237.**

**man, M. R., Percy, N. V., Neill, D.**

**Rodgers, W. A. and Panwar, H. S.**

**A., Ceron, C. E., Palacios, W. A.**

**(1988).** Planning a Protected Area

**and Aulestia, M. (2002).** A com-

Network in India. Vol I & II. The

parison of tree species diversity in

Report of Wildlife Institute of India,

two upper _Amazonian Forests._

Dehradun, India.

_Ecology_ **83, 3210-3224.**

**Rout SD, Panda T and Mishra N.**

**Porra, R. J., Thompson, W. A. and**

**(2009).** Ethno-medical plants used

**Kriedmann, P. E. (1989).** Deter-

to cure different diseases by tribal

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 509

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ of Mayuribhanj district of North

agent of black scorch of date palm.

Orissa _. Ethno-med._ **3, 27-36.**

_Biocontrol._ **47, 207-216.**

**Sandhya, T., Lathika, K. M, Panday, B.**

**Srivastava, T.N., Rajesekaran, S.,**

**N. and Mishra, K. P. (2006).** Po-

**Badola, D.P. and Shah, D.C.**

tential of traditional ayurvedic for-

**(1986).** An indedx of the available

mulation, Triphala, as a novel anti-

medicinal plants used in Indian sys-

cancer drug. _Cancer Lett._ **231, 206-**

tem of medicine from Jammu and

**214.**

Kashmir State. _Ancient Science of_

**Saha, D., Dasgupta, S. and Saha, A.**

_Life_ **6, 49-63.**

**(2005).** Antifungal activity of some

**Thind, T. S., Rampal, G., Agrawwal, S.**

plant extracts against fungal patho-

**K., Saxena, A. K. and Arora, S.**

gen of tea (Camellia sinensis).

**(2010).** Diminution of free raaadical

_Pharm Biol._ **43, 87-95.**

induced DNA damage by ex-

**Sakagami, H., Jiang, Y. and Kusama,**

tracts/fractions from bark of Schlei-

**K. (2000).** Cytotoxic of hydrolysa-

chera oleosa (Lour.) Oken. _Drug_

ble against human tumor cell lines-a

_Chem Toxicol_. **33, 329-336.**

possible mechanism. _Phytomedi-_

**Thind, T. S., Rampal, G., Agrawwal, S.**

_cine._ **1, 39-47.**

**K., Saxena, A. K. and Arora, S.**

**Singh, J. (2011).** Sehat ke liya phal

**(2011).** In vitro antiradical proper-

khayein, Phal-phool, _Jan-Feb, IC-_

ties and total phenolic contents in

_AR, New Delhi_. **pp.17-20.**

meth extracts/fractions from bark of

**Singh, K. N. and Lal, B. (2006).** Notes

Schleicheraoleosa (Lour.) Oken.

on Traditional Uses of Khair ( _Aca-_

_Med Chem Res_. **20, 254-260.**

_cia catechu_ Willd.) by Inhabitants

**Thind, T. S., Rampal, G., Agrawwal, S.**

of Shivalik Range in Western

**K., Saxena, A. K. and Arora, S.**

Himalaya. _Ethnobotanical Leaflets_

**(2012).** Evaluation of cytotoxic and

**10, 109-112.**

radical scavenging activities of root

**Singleton, V. N. (1981)**. Naturally occur-

extracts of Schleichera oleosa

ring food toxicants; phenolic sub-

(Lour.) Oken. _Nat Prod Res._ **26,**

stances of plant origin common in

**1728-1731.**

food. _Adv Food_. **27, 149-242.**

**Tripathi, K. P. and Singh, B. (2009).**

**Sinha, A. K., Sharma, U. K., Abhisekh,**

Species diversity and vegetation

**S. and Nandini, S. (2010).** A pro-

structure across various strata in

cess for the preparation of crystal-

natural and plantation forests in

line and non-hygroscopic phenolic

Katerniaghat Wildlife Sanctuary,

rich colored fractions from plants.

North India. _Tropical Ecology_ **50,**

Int

Appl.

WO

Patent

**191-200.**

(2010)2010109286A1.

**Tripathi, S. N. and Shukla, P. (1979).**

**Soobrattee, M. A., Neergheen, V. S.,**

Effect of Histamine and Albizzia

**Luximon-Ramma, A., Aruoma,**

lebbeck Benth, on guinea pig ad-

**O. L. and Bahorun, T. (2005).**

renal glands. _Indian journal of ex-_

Phenolics as potential antioxidant

_perimental Biology_ **17, 915-917.**

therapeutic agents: Mechanism and

**Tripathi, S. N., Shukla, P., Mishra, A.**

Actions.

_Mutation_

_Re-_

**K, and Udupa, K. N. (1978).** Ex-

_search/Fundamental and Molecular_

perimental and clinical studies on

_Mechanism of Mutagenesis_ **579,**

adrenal function in bronchial asth-

**200-213.**

ma with special reference to the

**Suleman, P., Al-Musallam, A. and**

treatment with _Albizzia lebbeck_.

**Menezes, C. A. (2002)**. The effect

_Quarterly Journal of Surgical Sci-_

of

biofungicide

Mycostop

on

_ence_ **14, 169-176.**

Ceratosystis, redicicola, the casual

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 510

_Biotech Sustainability (2017)_

_Diversity and Ethno-Botanical Potential of Tree Plants... Mall_ **Varshney, I. P. and Khan, M. S. Y.**

_lebbek_ Benth. _Journal of Indian_

**(1961).** Saponins and Sapogenins

_Chemical Society_ **LIII, 859-860.**

XI. On the presence of echinocystic

**Velioglu, Y. S., Mazza, G., Gao, L. and**

acid and quercetin in flowers of the

**Oomah, B. D. (1998).** Antioxidant

_Albizzia_

_lebbek_

Benth. _Canidian_

and total phenolics om selected

_Journal of Chemistry_ **39, 1721-**

fruits, vagetables and grain prod-

**1723.**

ucts. _J Agric Food Chem_. **46, 4113-**

**Varshney, I. P. and Sharma, S. P.**

**4117.**

**(1969).** Chemical Investigation of

**Verpoorte, R. (1999).** Exploration of na-

the _Albizzia lebbek_ leaves and _Al-_

tures chemodiversity: the rolr of

_bizzia lucida_ seeds and bark. _Indian_

secondary metabolites as leads in

_Journal of Applied Chemistry_ **32,**

drug development. _Drug Discovery_

**10-11.**

_Today_ **3, 232-238.**

**Varshney, I. P., Geeta, H., Srivastava,**

**White, M. J. (1999).** Mediators of in-

**H. C. and Krishnamurthy, T. N.**

flammation of inflammatory pro-

**(1973).**

Partial

structure

of

cess. _Journal of Allergy and Clini-_

Lebbekanin A, a new saponin from

_cal Immunology_ **103, S378-S381.**

the seeds of Albizzia lebbek

**Yu, L., Haley, S., Perret, J., Harris, M.,**

Benth. _Indian Journal of Chemistry_ ****

**Wilson, J. and Qian, M. (2002).**

**11, 1094-1096.**

Free radical scavenging properties

**Varshney, I. P., Pal, R., and Vyas, P.**

of wheat extracts. _J Agric Food_

**(1976).** Studies on the Lebbekanin

_Chem.._ **50, 1619-1624.**

E, a new saponin from _Albizzia_

****

© 2017 by the author. Licensee, Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 511

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P512-524_

**Free Radical Scavenging Potential and Anticancer**

**Activity of _Primula denticulata_** **Sm. from North-Western**

**Himalayas**

**Bilal Ahmad Wani1, *, Mohammed Latif Khan1 and Bashir Ahmad Ganai2**

_**1** Department of Botany, Dr. Hari Singh Gour Central University, Sagar, India- 470003_ ****

_**2** Centre of Research for Development, University of Kashmir, Srinagar, 190006, India; ****_

_*Correspondence: bilalenvsci@gmail.com; Tel: +91-9407579802_

****

****

****

**Abstract** : The present research work was aimed to evaluate the free radical scavenging po-

tential and anticancer property of _Primula denticulata_ ethanol extract (PDEE) against dif-

ferent human cancer cell lines. The antioxidant potential was determined by total phenolic

and flavonoid content, DPPH free radical scavenging assay, hydroxyl radical scavenging

assay and DNA damage assay. The anticancer activity was determined by SRB assay.

Apoptotic induction in MiaPaca-2 cells was analysed by propidium iodide staining cell-cycle

and DNA content analysis, and mitochondrial membrane potential loss was measured using

rhodomine-123 as fluorescent dye through flow cytometry. The total phenolic and flavo-

noid content in PDEE was found to be 12.24±2.11 (mg GAE/g dry extract) and 7.06 ±1.31

(mg catechin/g dry extract) respectively. The extract at 600 µg/ml concentration induces

69.35% DPPH free radical inhibition. In hydroxyl radical scavenging, the extract showed

54.51% inhibition at 120 μg/ml concentration. PDEE prevents DNA damage against oxida-

tive stress in concentration dependent manner. Anticancer activity was evaluated against six

human cancer cell lines (MiaPaca-2, A-549, PC-3, THP-1, HCT-116 and HOP-620). The

extract showed significant anticancer activity in concentration dependent pattern. The high-

est activity was shown against MiaPaca-2 cell line. PDEE induces significant apoptotic in-

duction in cells. Exposure of MiaPaca-2 cells to PDEE (0-100 μg/ml) caused dose dependent

cell cycle arrest at G0/G1 phase and induced apoptosis by increasing accumulation of cells at

__
__

G0/G1 phase. PDEE increased the apoptotic cell population from 11.4% in case of control to

49.6% at 100 μg/ml. Further, PDEE induces loss of mitochondrial membrane potential (∆Ψm)

to 99.5% at 100 μg/ml from 24.4% in control cells. These primary results depict the free radical

scavenging potential and anticancer activity of _P. denticulata_ extracts. These findings may

serve as foundation to develop an anticancer drug from medically important _P. denticulata._

_**Keywords**_ **:** Apoptosis; DNA damage; DPPH; MiaPaCa-2 cells; _Primula denticulata_ ****

****

****

**1. Introduction**

cases and 13.5 million deaths by 2030.

Pancreatic cancer is the fourth most

Cancer after cardiovascular dis-

common cause of cancer-related deaths

eases is the second leading mortality

across the world with incidence equalling

cause and is rapidly becoming a global

mortality and continues to pose an enor-

pandemic. The worldwide incidence and

mous challenge to clinicians and cancer

mortality of cancer in 2008 were 12.66

scientists (Hariharan _et al.,_ 2008). Among

and 7.56 million cases respectively. Ac-

all pancreatic cancers, pancreatic ductal

cording to (World Health Organization,

adenocarcinoma (PDAC) is the most

2010) report, the global cancer burden is

common epithelial, exocrine pancreatic

expected to nearly double to 21.4 million

malignancy, representing more than 80%

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_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

of the malignant neoplasms of the pancre-

tine,

podophyllotoxin,

camptothecin,

as (Alexakis _et al.,_ 2004).

combretastatins, flavopiridol, bruceatin

Consumption of fruits and vegeta-

etc, with diverse chemical structures have

bles is known to impart reduction in the

been isolated from plants. Several biolog-

incidence of ischemic heart disease and

ically active analogues such as taxotere,

some types of cancer, particularly stom-

isotaxel (taxol analogues) topotecan, iri-

ach, oesophagus, lung, oral cavity and

notecan, rubitecan, lurtotecan, 9-Amino

pharynx, endometrial, pancreas and colon

CPT (camptothecin analogues), etoposide,

cancers (Mathew _et al.,_ 2004). Similarly

teniposide (podophyllotoxin analogues),

natural antioxidant supplements (ascorbic

vinorelbine, hydravin (Vinca alkaloid de-

acid, tocopherols, anthocyanin, β-carotene

rivatives) have been synthesised from the-

and other polyphenols have been associ-

se front line anticancer lead molecules

ated with lower incidences of cancers and

(Vandana _et al.,_ 2005). As a result, em-

cancer related diseases (Fleischauer _et al.,_

phasis has now been shifted towards the

2003). During some pathophysiological

screening of apoptotic inducers from nat-

conditions, excess amount of reactive ox-

ural sources particularly from plants in

ygen species (ROS) is being generated by

the form of extracts or as isolated com-

certain external agents such as UV-

pounds that specifically increase apoptot-

radiations, drugs, pollution, other xenobi-

ic cell death in cancerous cells.

otics and as well as by endogenous chem-

_Primula denticulata_ Sm. (Primu-

icals, especially stress hormones (adrena-

laceae) is an important member of genus

lin and noradrenalin). The superoxide

primula, which represent more than 400

dismutase (SOD) and other defence

species (Richards, 1993). _P. denticulata_ is

mechanisms in living organisms are una-

commonly known as __ drumstick primula

ble to scavenge excess of ROS complete-

or tooth-leaved primula. _P. denticulata_ is

ly, which causes damage to cellular mole-

20-30 cm tall perennial rarely annual, de-

cules such as DNA, RNA, enzymes, lipids

ciduous, clump-forming plant with com-

etc. that results in fluidity of bio-

pact heads of many flowers. The plant is

membranes (Dean and David, 1993) and

widely distributed from eastern Afghani-

development of degenerative diseases in-

stan and northern Pakistan, across the

cluding caners, cardiovascular, neuro-

Himalaya to Yunnan, Sichuan and Gui-

degenerative, Alzheimer's and inflamma-

zhou in China. In Kashmir Himalaya, the

tory diseases (Shahidi _et al.,_ 1992; Gerber

species is widely distributed (Map-1). The

_et al.,_ 2002; Di Matteo and Esposito,

species thrives best in moist, shady

2003; Sreejayan and Rao, 1996). Hence

slopes, mostly near melting glaciers and

there is growing interest in natural poly-

moist meadows, ranging in altitude from

phenolic compounds, present in medicinal

2100 – 4050 meters.

and dietary plants that might help attenu-

ate oxidative damage (Silva _et al.,_ 2005).

**2. Materials and methods**

The increased incidence of differ-

****

ent types of cancers during the last few

_2.1. Chemicals_

decades and the modern techniques for

Sulphorhodamine-B

(SRB),

separation, structure elucidation, screen-

RPMI-1640 medium, fetal bovine serum

ing and combitorinial synthesis have led

(FBS), streptomycin, sodium bicarbonate,

to the development of new anticancer

5-Fluorouracil, paclitaxel, gentamycin

drugs, drug combinations and chemother-

sulphate,

trypsin,

1,1-diphenyl-2-

apy strategies by exploration of enormous

picrylhydrazyl (DPPH), folin–Ciocalteu

pool of biological, synthetic and natural

reagent, catechin, gallic acid were pro-

products (Mukherjee _et al.,_ 2001). So far

cured from Sigma-Aldrich. Trichloroace-

several potential anticancer lead mole-

tic acid (TCA), butylated hydroxytoluene

cules such as taxol, vincristine, vinblas-

(BHT), thiobarbituric acid (TBA), hydro-

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**N**

****

****

**Map 1:** Distribution of _Primulla denticulata_ Sm. __ in Kashmir Himalaya, J&K- India.

gen peroxide (H2O2), ferric chloride, di-

tude of 2650 m. The healthy plant species

methyl sulfoxide (DMSO), potassium fer-

were randomly collected by hand-picking

ricyanide were purchased from Merck.

and later identified by Dr. Anzar A.

The other reagents used were all of ana-

Khuroo at department of Botany, Univer-

lytical grade.

sity of Kashmir. A specimen under

voucher number KASH-1743 was pre-

_2.2. Collection and identification of plant_

served for future reference.

_Primula denticulata_ Sm. at flow-

ering stage was collected from Gulmarg

_2.3. Extract preparation_

region of Kashmir Himalaya (latitude

Fresh and healthy leaves of _P._

34°3'27" N; longitude 74°23'9" E) at alti-

_denticulata_ (1Kg) were cleaned with dou-

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_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

ble distilled water, dried under shade (25±

pable of donating hydrogen or electron.

2°C) for 5-6 days. The dried plant materi-

100 μl of different concentrations (100-

al was ground to powder form. The plant

600 μg/ml) of plant extract or standard

powder was extracted in soxhelt apparatus

antioxidant was added to 1 ml DPPH so-

using ethanol as solvent at desirable tem-

lution (0.5 mM). The solution was slight-

perature. The filtered extract was concen-

ly shaken and kept stand for 30 min at

trated using Buchi rotavapour and stored

room temperature under dark conditions.

in glass vials at 4oC until used.

The yellow colour solution was read at

517 nm against ethanol (Brand-Williams

_2.4. Estimation of total phenolics_

_et al.,_ 1995). The free radical inhibition

The total phenolic content in leaf

was calculated as:

extract of _P. denticulata_ was determined

Percentage inhibition = [(Ac-As)/Ac] x

by Folin–Ciocalteu method as adopted by

100

Slinkard and Singleton (1977) with slight

Where, Ac and As are the absorbance of

modifications. To 0.2 ml of plant extract

control and sample respectively

(1mg/ml) was added to 2.5 ml of 10% di-

Butylated hydroxytoluene and α- tocoph-

luted Folin–Ciocalteu reagent and 2 ml of

erol were used as positive control.

2.5% aqueous Na2CO3. The reaction mix-

__

_●_

ture was incubated at room temperature

_2.7. Hydroxyl radical (HO ) scavenging_

with intermittent shaking. The blue colour

_assay_

solution was read at 765 nm on UV–

Deoxyribose assay was used to

visible spectrophotometer. The absorb-

evaluate the hydroxyl radical scavenging

ance of solution was compared against

potential of _P. denticulata_ leaf extract

●

standard Gallic acid (50 mg %) calibra-

(Halliwell _et al.,_ 1987). The HO generat-

tion curve.

ed in Fenton reaction attack deoxyribose

to form products that upon heating with

_2.5. Estimation of total flavonoids_

thiobarbituric acid at low pH yield a pink

The aluminium chloride colori-

chromogen (TBARS). A reaction mixture

metric method as described by Mcdonald

containing deoxyribose (25 mM), FeCl3

_et al.,_ (2001) was used to determine the

(10 mM), ascorbic acid (100 mM), H2O2

total flavonoid content of leaf extract. The

(2.8 mM) in 10 mM KH2PO4 (pH 7.4)

principle of this method is based on fla-

with or without plant extract at various

vonoid–aluminium complex formation,

concentrations (20-120 µg/ml) and incu-

which shows absorbance maximum at 430

bated at 37 0C for 1h. Then 1 ml of TBA

nm. Briefly 0.5 ml (1mg/ml) of extract

(1% w/v) and 1 ml of TCA (3% w/v)

was mixed with 1.5 ml of ethanol, 0.1 ml

were added and heated at 100 0C for 20

of 10% AlCl3, 0.1 ml of 1M potassium

min. Absorbance of TBARS was read at

acetate and 2.8 ml of distilled water. After

532 nm. Deoxyribose oxidation inhibition

5 min of incubation, the absorbance was

was calculated as:

read at 430 nm. Flavonoid concentration

Percentage inhibition = [(A-B)/A] ×100

was expressed as milligrams of catechin

Where, A is malonaldehyde produced

equivalents per gram dry weight.

when treated with extract and B is malo-

naldehyde produced without extract. Bu-

_2.6. DPPH assay_

tylated hydroxytoluene and α- tocopherol

DPPH assay is one of the most ex-

were taken as the positive control.

tensively used method for determining the

antioxidant potential of any biological

_2.8. DNA damage assay_

sample. DPPH is a purple stable free radi-

The Prevention of oxidative DNA

cal which is reduced to yellow colour

damage by PDEE was determined by

complex 1,1-diphenyl-2-picrylhydrazine

method as previously described by Ghan-

(DPPH-H) by compounds which are ca-

ta _et al.,_ (2007). Calf thymus DNA

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_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

(0.37μg) with and without plant extract

at room temperature. The optical density

(10, 30, 50, 80 and 100 μg) was incubated

was read at 570 nm using ELISA reader.

with 20 mM ferric nitrate 30 mM H2O2 in

The experiments were done in triplicates.

20.0 mM phosphate buffer (pH 7.4) in a

Percentage cell growth was calculated as:

final reaction mixture volume of 20 μl for

Percentage cell viability = [At/Ac] ×100

1h at 37 oC. Oxidative DNA damage was

Percentage cell growth inhibition = (100-

induced by hydroxyl radicals generated in

percentage cell viability)

Fenton reaction (Ani _et al.,_ 2006). Bro-

Where At and Ac are absorbance of treat-

mophenol blue (0.25%) and glycerol

ed and control cells, respectively.

(30%) were added to terminate reaction

mixture, followed by gel electrophoresis

_2.11. DNA content and cell cycle phase_

in 0.7% agarose. The gel was then visual-

_distribution_

ized and photographed on gel doc.

Human pancreatic (MiaPaca-2)

cells were seeded in 6-well culture plates

_2.9. Cells culture_

5

with cell density 2x10 cells/ml/well and

Pancreatic cell line (MiaPaca-2),

incubated for 24 hrs. After incubation, the

Lung cell line (A-549), Prostate cell line

cells were treated with PDEE (0, 30, 50

(PC-3), Leukaemia cell line (THP-1), Co-

and 100 mg/ml) and again incubated for

lon cell line (HCT-116) and Lung cell line

48 hrs. After 48 hrs treatment cells were

(HOP-62) were purchased from National

collected by 5 min centrifugation at 1000

Cancer Institute, U.S.A and European

rpm. The harvested cells were washed

collection of cell culture, UK. Cells were

twice with phosphate buffer solution and

cultured in RPMI-1640 and MEM medi-

fixed with 70 % ethanol at -20 ºC for 1h.

um supplemented with nutrients and anti-

The cells were then stained with DNA

biotics. Cells were grown at 37 oC and 5%

staining solution containing propidium

CO2 level with relative humidity of 98%.

iodide (20 mg/ml) and triton X-100 (1%)

in PBS for 30 min in dark. FACScan was

_2.10. Anticancer activity_

used to measure DNA content. For each

The anticancer activity of PDEE

data file, data was collected from 10,000

against different human cancer cell lines

cells. Cell Quest (Becton, USA) was used

was evaluated by SRB assay as described

for analysis of histograms.

by Monks _et al.,_ 1991. Briefly 100µl of

cell suspension (1x105 cells/well) were

_2.12. Loss of Mitochondrial Membrane_

cultured in 96-well plates and incubated

_Potential (ΛΨm)_

overnight at 37 0C and 5% CO2 level. 20

Flow cytometry was used to meas-

µl test material at various final concentra-

ure the mitochondrial membrane potential

tions (10-100 µg/ml) was added. Paclitax-

loss (ΛΨm). Human pancreatic (MiaPaca-

el (1 µM) and 5-fluorouracil (20 µM)

2) cells were plated in 6-well cultural

were used as standard anticancer drugs.

6

plates with cell density of 1x10

Cell growth was stopped after 48 hrs of

cells/ml/well and incubated for 24 hrs at 5

incubation by adding 50 μl of 50% TCA

% CO2 level. The cells were then treated

in each well and incubated further for 1h

with PDEE (0, 30, 50 and 100 mg/ml) and

at 4 0C. The plates were then washed, air

again incubated for 48 hrs. Rhodamine-

dried and stained with 50 μl of 0.4% SRB

123, a cell permeable cationic dye was

dye in 1% acetic acid, followed by incu-

added one hour before termination of ex-

bation for 30 min at room temperature.

periment and again incubated for 30 min.

The unbound dye was then removed by

The cells were washed with PBS and pel-

washing with 1% acetic acid and kept

lets were collected by centrifugation. The

overnight for drying. In each well, 100 μl

collected pellets were re-suspended in 300

of 10 mM tris-base was added to solubil-

ml of PBS. Florescence of Rh-123 in cells

ise the dye followed by stirring for 5 min

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_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

were analysed at 485 nm by flow cytome-

them to act as strong reducing agents, hy-

ter (Jung _et al.,_ 2006).

drogen donor's metal chelaters and singlet

oxygen quenchers (Miguel, 2010). The

_2.13. Statistical analysis_

total flavonoid content in DPEE was

All of the experiments were done in

found to be 7.06 ± 1.31 (mg catechin/g

triplicate. The data were recorded as

dry extract). Flavonoids are known exhib-

means ± standard deviations and were

it many biological activities like antioxi-

analysed with SPSS software.

dant, anticancer, antimicrobial and anti-

****

inflammatory properties (Hodek _et al.,_

**3. Results and discussion**

2002).

__

_3.1. Total phenolic and flavonoid content_

_3.2. DPPH free radical scavenging activi-_

Polyphenolic compounds are very

_ty_

important plant bioactive constituents be-

DPPH is a purple stable free radi-

cause of their scavenging potential due to

cal at room temperature with characteris-

the presence of hydroxyl groups (Hatano

tic absorbance at 517 nm. The nitrogen

_et al.,_ 1989). Folin-Ciocalteu method is

free radical of DPPH is easily quenched

most widely used to measure the poly-

by an antioxidant to yellow coloured

phenol contents, with the basic mecha-

complex

(1,1-diphenyl-2-picrylhydra-

nism of electron transfer and reducing

zine). The decolourization of purple col-

ability (Prior and Schaich, 2005). Using

our is stoichiometric depending on the

this quantitative assay, we found that the

number of electrons gained (Soares _et al.,_

total phenolic content (TPC) of ethanolic

1997; Mokbel _et al.,_ 2006; Singh _et al.,_

leaf extract of _P. denticulate_ was found

2002). DPPH radical scavenging potential

to be 12.24 ± 2.11 (mg GAE/g dry ex-

of PDEE at different concentrations in-

tract) as depicted in Figure 1. Considera-

vestigated in the present study was deter-

ble attention has been received by poly-

mined together with standard antioxidants

phenols for their physiological role as an-

(BHT and α-tocopherol) at the same con-

tioxidant and anticancer agents (Othman

centrations (Figure 2). PDEE showed sig-

_et al.,_ 2007). Polyphenolic compounds h-

nificant scavenging effect on DPPH free

**Figure 1:** Represents the total phenolic

and flavonoid content of PDEE. Each

value represents the mean ± SD (n = 3).

**Figure 2:** DPPH radical scavenging activ-

-ave been reported to possess strong anti-

ity of PDEE and known antioxidant BHT

oxidant potential their by scavenging free

and α-tocopherol. Values are means of

radicals and protect cells against such ox-

triplicate experiments (n = 3) ± standard

idative damages (Kahkonen _et al.,_ 1999).

deviation.

The antioxidant potential of medicinal

plants is due to the redox properties of

radical in concentration dependent man-

polyphenolic compounds, which enables

ner. When compared with standard anti-

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_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

oxidants used in the experiment, the ex-

hydroxyl-2-deoxyguanosine and thymine

tract showed relatively lower DPPH free

glycol which leads to mutagenesis and

radical scavenging potential. The extract

carcinogenesis (Ames _et al.,_ 1993). PDEE

at 600 µg/ml produced 69.35% inhibition,

prevents calf thymus DNA from oxidative

while as BHT and α-tocopherol produced

damage due to hydroxyl radicals generat-

85.95% and 89.65% inhibition at the

ed by FeSO4 and H2O2 in Fenton reaction

same concentration. The DPPH radical

using agrose gel electrophoresis. Figure 4

scavenging activity of PDEE as such

shows the protective effect of PDEE on

might prevent reactive radical species

calf thymus DNA. The Hydroxyl radicals

from damaging biomolecules such as

induce DNA strand breaks and causes

DNA, protein, polyunsaturated fatty acids

complete DNA damage ( _Lane_ 2). PDEE

(PUFA) and sugars in susceptible biologi-

at different concentration (10–100 μg/ml)

cal and food systems.

offered concentration dependent protec-

tion to DNA damage (Lane 3-7). Catechin

_•_

_3.3. Hydroxyl (HO ) radical scavenging_

(10μg/ml) was used as standard antioxi-

_activity_

dant ( _Lane_ 8). Thus, the results indicate

Among different free radicals

that PDEE prevents DNA damage against

generated in biological systems, hydroxyl

oxidative stress. The hydroxyl radical

**•**

radical (HO ) is one of the most reactive

quenching ability of polyphenolic com-

species, which is capable to damage al-

pounds of _P. denticulate_ could be respon-

most all the molecule in living cells,

sible for the protection against oxidative

which ultimately leads to carcinogenesis

damage.

and mutagenesis (Manian _et al.,_ 2008;

Hochestein and Atallah, 1988). This radi-

_3.5. Anticancer activity_

cal is considered to be one of the im-

The anticancer activity of PDEE was de-

portant initiators in the process of lipid

termined by SRB assay against six human

peroxidation, abstracting hydrogen atoms

cancer cell lines. The assay is based on

from unsaturated fatty acids (Kappus _et_

measuring the content of cellular protein

_al.,_ 1991). In the present study, hydroxyl

using SRB dye (Vanicha and Kanyawim,

radical scavenging ability was estimated

2006). Treatment of cells with PDEE (10-

by generating hydroxyl radicals using

100 µg/ml) exhibited concentration de-

ascorbic acid–iron-H2O2 (Fenton reac-

pendent anti-proliferative effect (Table 1).

tion). Antioxidant efficiency of PDEE

The results of the present study reveal that

was determined as the ability to scavenge

the plant extract is very active against all

the free radicals generated. The extract

the cell lines used. The highest percentage

exhibited a concentration dependent scav-

cell growth inhibition was observed in

enging of hydroxyl radicals which was

MiaPaca-2, HCT-116 and THP-1 with

comparable to the reference standards

mean percentage value of 99.06 ± 0.30,

(BHT & α-tocopherol) at the same con-

98.45 ± 0.65 and 98.33 ± 1.25 respective-

centration. The percentage inhibition of

ly. The susceptibility of cells to the drug

hydroxyl radical scavenging is shown in

exposure was characterized by its IC50

Figure 3. The extract showed antioxidant

values. Lower IC50 value indicates the

activity in concentration dependent man-

higher anticancer potential of plant ex-

ner. A 120 μg/ml of PDEE, BHT and α-

tract. The results of our study reveal that

tocopherol exhibited 54.51%, 81.20% and

PDEE inhibits proliferation of cancer cell

88.25% inhibition, respectively.

lines. The cytotoxic activity may be due

to individual polyphenolic phytochemi-

_3.4. Prevention of oxidative DNA damage_

cals that act synergistically with other

Oxidative damage by hydroxyl

compounds to display the anticancer ac-

radicals make DNA susceptible by oxida-

tivity, as has been suggested by Yang _et_

tion of guanosine or thymine to 8-

_al._ (2009).

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_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

****

**Figure 3:** Effect of PDEE and known antioxidant BHT and α-tocopherol on hydroxyl radi-

cal scavenging potential. Values are means of triplicate experiments (n = 3) ± standard de-

viation.

****

**Figure 4:** PDEE Protects calf thymus DNA f oxidative damage. _Lane_ 1: Native calf thymus

DNA; _Lane_ 2: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2; _Lane_

3: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2 + 10µg of extract;

_Lane_ 4: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2 + 30µg of

extract; _Lane_ 5: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM H2O2 +

50µg of extract; _Lane_ 6: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid + 30mM

H2O2 + 80µg of extract; _Lane_ 7: DNA + 20mM Ferric Nitrate + 100mM Ascorbic Acid +

30mM H2O2 + 100µg of extract; _Lane_ 8: DNA + 20mM Ferric Nitrate + 100mM Ascorbic

Acid + 30mM H2O2 + 10µg of catechin.

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_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

**Table 1:** Anticancer activity of ethanolic leaf extract of _Primula denticulate#_ ****

**Percentage cell growth inhibition**

HCT-

HOP-

Conc.

MiaPaca-2

A-549

PC-3

THP-1

**Sample**

116

62

(µg/ml) (Pancreatic)

(Lung)

(Prostate) (Leukaemia) (Colon) (Lung)

**PDEE**

100

99.06 ± 0.30 95.67 ±

97.09 ±

98.33 ± 1.25 98.45 ±

93.68

1.85

2.09

0.65

± 3.20

50

73.15 ± 2.76 39.18 ±

40.23 ±

93.27 ± 2.10 83.21 ±

47.08

1.90

3.45

1.67

± 2.34

10

44.36 ± 2.09 17.05 ±

15.68 ±

38.35 ± 2.43 28.80 ±

10.96

1.16

2.53

1.56

± 3.05

**5-FU**

20µM

\-

\-

\-

67.87 ± 1.56 67.00 ±

\-

1.87

**Paclitaxel**

1µM

\-

70.78 ±

\-

\-

\-

72.43

2.45

± 2.78

_**#**_ The results represent mean ± S.D of three experiments.

_3.6. Flow cytometric analysis_

al membrane potential loss using Rhoda-

To evaluate the action mechanism

mine-123 staining flow cytometry. Mi-

of cell growth inhibition by PDEE, further

aPaca-2 cells stained with PI were treated

experiments were performed on human

with PDEE (0, 30, 50 and 100 µg/ml) for

pancreatic (MiaPaca-2) cell line. In the

48 hrs showed that the percentage of

present study, apoptotic induction was

apoptotic nuclei increased to 57.00% at

assessed by two assays; Cell-cycle phase

100 µg/ml PDEE from 11.4% in control

distribution via propidium iodide (PI)

(Figure 5).

staining flow cytometry and mitochondri-

**Figure 5:** PDEE induces apoptosis of human pancreatic cancer (MiaPaca-2) cells via cell

cycle arrest. Flow cytometric analysis of MiaPaca-2 cells after propidium iodide staining.

Cells were incubated for 48 h in presence of PDEE (0, 30, 50 and 100 µg/ml). Figures show

the representative staining profile of one of two similar experiments. P1 is the population of

apoptotic cells, which increases from 11.4% in case of control to 57.00% in case of 100

µg/ml of PDEE.

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_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

**Figure 6:** __ PDEE induced loss of mitochondrial membrane potential (∆Ψm) in human pan-

creatic cancer cell line (MiaPaca-2) incubated with extract at different concentrations (0, 30

50 and 100 µg/ml) in 6 well plate for 48 h treatment. __

Apoptotic cells were characterised by de-

**4. Conclusion**

graded chromatin with high side-scatter

(SSC) and low forward-scatter (FSC)

The _P. denticulata_ ethanol extract

properties (Bachir _et al.,_ 2012). The re-

exhibit potent antioxidant property as re-

sults indicate that PDEE blocks cell cycle

vealed by different antioxidant assays.

progression, resulted in significant in-

PDEE showed strong anticancer activity

crease in population of cells in sub G0/G1

and induces apoptosis in MiaPaca-2 cell

phase, which may be due to fragmentation

line by arresting cells at G0/G1 phase and

of DNA, resulted in apoptotic cell death.

inducing loss of MMP (∆Ψm). Further re-

Disruption of MMP (∆Ψm) is one of the

search is required to explore the potential

earliest events that occur following apop-

of _P. denticulata_ in developing an anti-

tosis induction (Qi _et al.,_ 2010). MiaPaca-

cancer drug.

2 cells after treated with PDEE for 48 hrs

resulted in loss of (∆Ψm) from 24.4% in

**Acknowledgement**

control cells to a low of 99.5% at 100

µg/ml of PDEE (Figure 6). MMP loss is

Authors are thankful to UGC,

mainly due to mitochondrial permeability

New Delhi for providing financial support

transition pore, which causes Cyto-

to Bilal Ahmad Wani in the form of Dr.

chrome-C release from mitochondria and

D.S. Kothari Postdoc fellowship.

ultimately triggers other apoptotic factors

(Kroemer _et al.,_ 1997). The increase in

**References**

inner mitochondrial membrane permeabil-

****

ity is due to interaction of anticancer

**Alexakis, N., Halloran, C., Raraty, M.,**

agents with mitochondria (Fulda _et al.,_

**Ghaneh, P., Sutton, R., Ne-**

1998).

**optolemos, J.P. (2004).** Current

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 521

_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

standards of surgery for pancreatic

**Halliwell, B., Gutteridge, J.M., Aru-**

cancer. _British Journal of Surgery_

**oma, O.I. (1987).** The deoxyri-

**91, 1410-27.**

bose method: a simple "test tube"

**Ames, B.N., Shigenaga, M.K., Hagen,**

assay for determination of rate

**T.M. (1993).** Oxidants, antioxi-

constants for reactions of hydroxyl

dants, and the degenerative dis-

radicals. _Analytical Biochemistry_

eases of aging. _Proceedings of the_

**165, 215-219.**

_National Academy of Sciences_ **90,**

**Hariharan, D., Saied, A., Kocher, H.M.**

**7915–7922.**

**(2008).** Analysis of mortality rates

**Dean, R.T., David, M.J. (1993).** Reac-

for pancreatic cancer across the

tive species and their accumula-

world.

_International_

_Hepato-_

tion on radical damaged proteins.

_Pancreato-Biliary Association_ **10,**

_Trends in Biochemical Sciences_

**58-62.**

**18, 437-441.**

**Hochestein, P., Atallah, A.S. (1988).**

**Di Matteo, V., Esposito, E. (2003).** Bio-

The nature of oxidant and antioxi-

chemical and therapeutic effects

dant systems in the inhibition of

of antioxidants in the treatment of

mutation and cancer. _Mutation Re-_

Alzheimer's disease, Parkinson's

_search_ **202, 363-375.**

disease and amyotrophic lateral

**Hodek, P., Trefil, P., Stiborova, M.**

sclerosis. _Current Drug target_

**(2002).** Flavonoids- Potent and

_CNS neurological Disorders_ **2,**

versatile biologically active com-

**95-107.**

pounds interacting with cyto-

**Fleischauer, A.T., Simonsen, N., Arab,**

chrome P450. _Chemico-Biological_

**L. (2003).** Antioxidant supple-

_Interactions_ **139, 1-21.**

ments and risk of breast cancer re-

**Jung, G.R., Kim, K.J., Choi, C.H., Lee,**

currence and breast cancer-related

**T.B., Han, S.I., Han, H.K.**

mortality among postmenopausal

**(2007).** Effect of betulinic acid on

women. _Nutrition and Cancer_ **46,**

anticancer drug-resistant colon

**15–22.**

cancer cells. _Basic and Clinical_

**Fulda, S., Susin, S.A, Kroemer G, De-**

_Pharmacology & Toxicology_ **101,**

**batin KM. (1998).** Molecular or-

**277–85.**

dering of apoptosis induced by an-

**Kahkonen, M.P., Hopia, A., Vuorela,**

ticancer drugs in neuroblastoma

**H.J., Rauha, J.P., Pihlaja, K.,**

cells. _Cancer Research_ **58, 4453–**

**Kujala, T.S., Heinonen, M.**

**4460.**

**(1999).** Antioxidant activity of

**Gerber, M., Boutron-Ruault, M.C.,**

plant extracts containing phenolic

**Hercberg, S., Riboli, E., Scal-**

compounds. _Journal of Agricul-_

**bert, A., Siess, M.H. (2002).**

_ture and Food Chemistry_ **48,**

Food and cancer: state of the art

**1485–1490.**

about the protective effect of fruits

**Kappus, H., (1991).** Lipid peroxidation–

and vegetables. _Bulletin du Can-_

Mechanism and biological rele-

_cer_ **89, 293-312.**

vance. In Aruoma, O.I., Halliwell,

**Ghanta, S., Banerjee, A., Poddar, A.,**

B., (Eds.), _Free radicals and food_

**Chattopadhyay, S. (2007).** Oxi-

_additives_ (pp. **59-75** ).London, UK:

dative DNA damage preventive

Taylor and Francis.

activity and antioxidant potential

**Kroemer, G., Zamzami, N., Susin, S.A.**

of _Stevia rebaudiana_ (Bertoni)

**(1997).** Mitochondrial control of

Bertoni, a natural sweetener.

apoptosis, _Immunology Today_ **18,**

_Journal of Agricultural and Food_

**44–51.**

_Chemistry_ **55, 10962–10967.**

**Manian, R., Anusuya, N., Siddhuraju,**

**P., Manian, S., (2008).** The anti-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 522

_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

oxidant activity and free radical

**Qi, F., Li, A., Zhao, L., Xu, H., Inagaki,**

scavenging potential of two dif-

**H., Wang, D., Cui, X., Gao, B.,**

ferent solvent extracts of _Camellia_

**Kokudo, N., Nakata, M., Tang,**

_sinensis_ (L.) O. Kuntz, _Ficus ben-_

**W. (2010).** Cinobufacini, an aque-

_galensis_ L. and _Ficus racemosa_ L.

ous extract from Bufo bufo Gar-

_Food Chememisry_ **107, 1000–**

garizans cantor, induces apoptosis

**1007.**

through a mitochondria-mediated

**Mathew, A., Peters, U., Chatterjee, N.,**

pathway in human hepatocellular

**Kulldroff, M., Sinha, R. (2004).**

carcinoma cells. _Journal of Eth-_

Fat, fiber, fruits, vegetables, and

_nopharmacology._ **128, 654–661.**

risk of colorectal adenomas. _Inter-_

**Richards, J., Richards, A.J., Edwards,**

_national Journal of Clinical Nutri-_

**B. (1993).** Primula timber (Botan-

_tion_ **78, 517-520.**

ical illustrations). _Timber Press,_

**Mcdonald, S., Prenzler, P.D., Autolo-**

_Portland_. **Silva, B.A., Ferreres,**

**vich, M., Robards, K. (2001).**

**F., Malva, J.O., Dias, A.C.P.**

Phenolic content and antioxidant

**(2005).** Phytochemical and antiox-

activity of olive extracts. _Food_

idant characterization of _Hyperi-_

_Chemistry_ **73, 73–84.**

_cum perforatum_ alcoholic extracts.

**Miguel, M.G. (2010).** Antioxidant activi-

_Food Chemistry_. **90, 157–167.**

ty of medicinal and aromatic

**Singh, R.P., Murthy, K.N.C., Jayapra-**

plants. A review. _Flavour Fra-_

**kasha, G.K. (2002).** Studies on

_grance Journal_ **25, 291- 312.**

the antioxidant activity of prome-

**Mokbel, M.S., Hashinaga, F. (2006).**

granate ( _Punica granatum_ ) peel

Evaluation of the antioxidant ac-

and seed extracts using in vitro

tivity of extracts from buntan ( _Cit-_

models. _Journal of Agricultural_

_rus grandis_ Osbeck) fruit tissues.

_and Food Chemistry._ **50, 81−6.**

_Food Chemistry_ **; 94,529−34.**

**Slinkard, K., Singleton, V.L. (1977).**

**Monks, A., Scudiero, D., Skehan, P.,**

Total phenol analyses: automation

**Shoemaker, R., Paull, K., Visti-**

and comparison with manual

**ca, D., Hose, C., Langley, J.,**

methods. _American Journal of_

**Cronise, P., Vaigro-Wolff, A.,**

_Enology and Viticulture._ **28, 49–**

**Gray-Goodrich, M., Cambell,**

**55.**

**H., Mayo, J., Boyd, M. (1991).**

**Soares, J.R., Dinis, T.C.P., Cunha,**

Feasibility of a high-flux anti-

**A.P., Almeida, L.M., (1997).** An-

cancer drug screen using a diverse

tioxidant activities of some ex-

panel of cultured human tumour

tracts of _Thymus zygi_. _Free Radi-_

cell lines. _Journal of the National_

_cal Research_. **26, 469–478.**

_Cancer Institute_ **83, 757.**

**Sofowora, A. (1993).** Medicinal Plants

**Othman, A., Ismail, A., Ghani, N.A.,**

and Traditional Medicine in Afri-

**Adenan, I. (2007).** Antioxidant

ca. 2nd Edn. _Spectrum Books Lim-_

capacity and phenolic content of

_ited_ , Ibadan, Nigeria **1-153.**

cocoa beans. _Food Chemistry_.

**Sreejayan, N., Rao, M.N.A. (1996).** Free

**100,1523–1530.**

radical scavenging activity of cur-

**Prior, R.L., Wu, X., Schaich, K. (2005).**

cuminoids. _Arzneimittel forschung_

Standardized methods for the de-

**46, 169-171.**

termination of antioxidant capaci-

**Vandana, S., Arvind, S.N., Kumar,**

ty and phenolics in foods and die-

**J.K., Gupta, M.M., Suman,**

tary supplements. _Journal of Agri-_

**P.S.K. (2005).** Plant-based anti-

_cultural and Food Chemistry._ **53,**

cancer molecules: A chemical and

**4290–4303.**

biological profile of some im-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 523

_Biotech Sustainability (2017)_

_Free Radical Scavenging Potential and Anticancer Activity of Primula sp. Wani et al._

portant leads. _Bioorganic and Me-_

**Yang, J., Liu, R.H., Halim, L. (2009).**

_dicinal Chemistry_ **13, 5892–5908.**

Antioxidant and antiproliferative

**Vanicha, V., Kanyawim, K. (2006).** Sul-

activities of common edible nut

forhodamine B colorimetric assay

seeds. _LWT– Food Science and_

for cytotoxicity screening. _Nature_

_Technology_ **42, 1–8.**

_protocols_ **3, 1112-1116.**

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

****

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 524

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P525-533_

**Panchakavya: Organic Fertilizer and Its Stimulatory**

**Effect on the Seed Germination of _Abelmoschus_**

_**esculentus**_ **and _Solanum melongena_** ****

****

**V. Ramya and S. Karpagam***

_Department of Botany, Queen Mary's College, Chennai - 4, Tamil Nadu, India;_

_*Correspondence: s.karpagam98@gmail.com; Tel.: +91 9444944835_

**Abstract:** Panchakavya is an incredible source of growth promoting substances. From an-

cient period cow"s urine has been used as a medicine. In India, drinking of cow urine has

been practiced for thousands of years. Panchakavya is a term used in Ayurveda to describe

five important major substances, obtained from cow, which include cow"s urine, milk,

ghee, curd and dung all the five products posses medicinal properties against many disor-

ders and are used for the medicinal purpose singly or in combination with some other

herbs. This kind of treatment is called "panchakavya therapy" or "cowpathy". The indiscrim-

inate use of chemical pesticides resulted in environmental problems. An alternative to the

chemicals is the natural products, one such as the panchakavya. Panchakavya is the single

organic input that acts as a fertilizer, pesticide, growth promoter and immunity booster.

The effect of panchakavya (cow urine) on the seed germination was studied in two plants

namely _Abelmoschus esculentus L.Moench_ (Lady"s finger) and _Solanum melongena L._

(brinjal). The germination percentage was calculated. After germination the shoot and root

length was measured and seedling vigour index was calculated. Cytotoxic effect of pan-

chakavya on cell growth and cell division was studied in onion bulbs. The panchakavya at

1% concentration favoured the production of larger number of roots. This article highlights

that panchakavya is more effective, easy to prepare, environmental friendly and could be

used as a good fertilizer to boost the growth and productivity of agricultural crops. ****

_**Keywords**_ : Abelmoschus: mitotic index; __ panchakavya: _Solanum melongena_ : seed germina-

tion

****

**1. Introduction**

dation of soil; water resources and quality

****

of the food. At this juncture, a keen

Agriculture is means of livelihood

awareness has sprung on the adoption of

for millions of people in India and

organic farming as a remedy to cure the

worldwide with crops chiefly dependent

ills of modern chemical agricultural prac-

on rainfall and fertilizers. India is not on-

tice. It is very much essential to develop a

ly self-sufficient in food production but

strong workable and compatible package

also has a substantial reserve (Gupta and

of nutrient management through organic

Gopal, 2001). The latest trend is turned to

resources for various crops, based on sci-

organic farming, since the side effects of

entific facts, local conditions and eco-

chemical fertilizers and pesticides have

nomic viability (Nene, 1994; 1999). Cow

been established. India is the third largest

is described as "Kamdhenu" (one which

producer and consumer of chemical ferti-

fulfills all the wishes) since vedic times in

lizers in the world. Heavy use of chemi-

Indian civilization. According to ayurve-

cals in agriculture has weakened the eco-

da cow products are used to treat various

logical balance, in addition to the degra-

disease conditions in human beings. Five

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 525

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ products of cow called as panchakavya is

health and environmental problems due to

an important component of many rituals

the continuous use of pesticides resulted

and pooja in Hindus. (Gosavi and Jhon,

in the development of integrated pest

2012; Sathasivam _et al.,_ 2010). In San-

management and organic farming.

skrit, panchakavya means the blend of

Organic manure replaced chemi-

five products obtained from cow (all these

cal fertilizers, herbal extracts replaced

five products are individually called

pesticides and fungicides, but nothing was

"Gavya" and collectively termed as "Pan-

available to replace growth promoting

chakavya). When suitably mixed and

hormones and immunity for plants. The

used, has positive influence on living or-

organic system was imperfect and contin-

ganisms. Panchakavya had got reverence

ued to be incomplete for want of an input

in the scripts of Vedas (divine scripts of

to replace growth promoting hormones

Indian wisdom), and Vrkshayurveda (vrk-

and immunity boosters, to maximize the

sha means plant and ayurveda means

efficiency of cultivated crops and coordi-

health system). The texts on Vrkshayur-

nate the process leading to sustained

veda are systematization of the practices

higher productivity.

that the farmers followed at field level,

Many alternatives are available to

placed in a theoretical framework and it

reduce the effects pesticides have on the

defined certain plant growth stimulants;

environment. Alternatives include manual

among them panchakavya was an im-

removal, applying heat, covering weeds

portant one that enhanced the biological

with plastic, placing traps and lures, re-

efficiency of crop plants and the quality

moving pest breeding sites, maintaining

of fruits and vegetables (Natarajan, 2002).

healthy soils that breed healthy, more re-

Green revolution lead to intensi-

sistant plants, cropping native species that

fied agriculture to meet the ever increas-

are naturally more resistant to native pests

ing demand for food and fiber, which is a

and supporting biocontrol agents such as

practice at great cost to the environment

birds and other pest predators.

resulting in continuous loss of natural

Biological controls such as re-

ecosystems, ground water depletion, pol-

sistant plant varieties and the use

lution and other environmental degrada-

of pheromones, have been successful and

tion (Gupta and Gopal, 2001). Alterna-

at times permanently resolve a pest prob-

tive approaches to pest control is the con-

lem.  Integrated Pest Management (IPM)

cept of integrated pest management,

employs chemical use only when other

where synthetic pesticides are only ap-

alternatives are ineffective. IPM causes

plied as a last resort and is now consid-

less harm to humans and the environment.

ered common practice in professional ag-

The focus is broader than on a specific

riculture. The non-chemical alternatives

pest, considering a range of pest control

include cultural practices, use of resistant

alternatives. The use of phosphorous and

varieties, creation of an environment fa-

nitrogen fertilizer in the global level has

vourable for natural enemies of pests and

increased manifold, which effects the en-

use of biological products and agents, in-

vironment as run off into water bodies.

cluding beneficial insects.

The indiscriminate use of pesticide and

The indiscriminate use of chemi-

fungicide has increased at global level,

cal pesticides in modern agriculture re-

which is biomagnified at the tertiary level

sulted in the development of several prob-

consumers.

lems such as pesticide resistant insects,

Panchagavya or panchakavyam is

resurgences of target and non-target pest,

a concoction prepared by mixing five

destruction of beneficial organism like

products of cow and used in traditional

honey bee, pollinaters, parasites and

Indian rituals. The three direct constitu-

predators and pesticide residues in food

ents are cow dung,  urine, and milk; the and fodder. The awareness about the

two derived products are curd and ghee.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 526

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ **Figure 1:** Global map of phosphorus fertilizer application rates (kg per ha of grid cell area)

[source: https://ourworldindata.org/fertilizer-and-pesticides/#note-6].

**Figure 2:** Global map of nitrogen fertilizer application rates (kg per ha of grid cell area)

[Source: https://ourworldindata.org/fertilizer-and-pesticides/#note-7].

These are mixed in proper ratio and then

tender coconut, is believed to be a po-

allowed to ferment.  Panchamrita is a

tent organic pesticide and growth promot-similar mixture that replaces dung and

er

\-

this

is

considered

to

urine with honey and sugar. The mixture

be pseudoscience.

which is made using yeast as a fermenter,

The Sanskrit word Panchagavya m

bananas, groundnut cake, and the water of

eans "mixture of five cow products". It is

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 527

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ also called cowpathy treatment based on

products

obtained

from cows used

_2.1. Preparation of panchakavya_

in Ayurvedic medicine and of religious

The ingredients for panchakavya

significance for Hindus. Panchgavya is

sample was collected from cow farm

also used as fertilizers and pesticides in (Thiruvallur DT)) using sterile container.

agricultural operations, but has no scien-

Based on the detailed review of literature

tific evidence to back its claims.

panchakavya stock solution was prepared

Cow dung contained undigested

by using cow dung (2.5 Kg), cow"s urine

fibre, epithelial cells, pigments and salts,

(1.5 L), cow"s milk (1L), cow"s curd (1

rich in nitrogen, phosphorous, potassium,

L) and cow"s ghee (0.5 kg). In addition,

sulphur, micronutrients, intestinal bacteria

jaggery (1.5 Kg), tender coconut water

and mucous, cow dung is also rich in bac-

(1.5 L) and ripe banana (6 Nos.) were al-

teria, fungi and other microbial organisms

so added as modification. All the materi-

(Nene, 1999). Singh (1996) recorded that

als were placed in a wide mouthed mud

cow dung had water 82% and solid matter

pot and kept open under shade. The con-

18%. Reddy (1998) reported that cows

tents were stirred twice a day for about 20

urine is rich in urea and acted both as nu-

mintues, both in the morning and evening

trient as well as hormone. Cow milk was

to facilitate aerobic microbial activity.

used by farmers in ancient times and re-

The panchakavya stock solution will be

ported to be an excellent sticker, spreader,

ready after 30 d (care should be taken not

a good medium for saprophytic bacteria

to mix buffalo products, local breeds of

and a virus inhibitor (Nene, 1999).

cow is said to have potency then exotic

They are used as a Prasad in tem-

breeds) and is covered with a plastic

ples. A common usage is as a fertilizer

mosquito net to prevent houseflies from

and pesticide. Seeds can be treated with

laying eggs and the formation of maggots

panchagavya. This was found useful in

in the solution. Jaggery is dissolved in

rhizome of turmeric, ginger and sugar-

water and used while sugarcane juice is

cane and they yielded more, helps in plant

more suitable.

growth and immunity. The medicinal us-

age of panchagavya, particularly cow

_2.2. Seed collection_

urine, is practiced in Ayurveda. Propo-

The seeds of _Abelmoschus escu-_

nents claim that cow urine therapy is ca-

_lentus_ (ladies finger) and S _olanum_

pable of curing several diseases, including

_melongina_ (Brinjal) were bought from

certain types of cancer, although these

nursery Balaji traders, Chennai 601203,

claims have no scientific backing. In fact,

Tamil Nadu. Healthy Seeds of uniform

studies concerning ingesting individual

size and shape were used for sowing.

components of Panchagavya, such as cow

__

urine, have shown no positive benefit, and

_2.3. Treatments_

significant side effects, including convul-

Treatments were given as: 1. Con-

sion,

depressed

respiration,

and

trol (water); 2. Chemical (Cartap hydro-

death. Cow's urine can also be a source of

chloride 50% radon sp + tata tafgor dime-

harmful bacteria and infectious diseases,

thoate 30% ec = 2:1 ratio and dissolved

including leptospirosis. Proponents claim

in 10 ml of water); 3. Panchakavya; 4.

it is an antibiotic growth promoter in the

Panchakavya +Neem cake; 5. vermicom-

broiler diet, capable of increasing the

post were used.

growth of plankton for fish feed, the pro-

duction of milk in cows, the weight of

_2.4. Germination of seeds_

pigs, and the egg laying capacity of poul-

The seeds (40 numbers) were

try chicken.

soaked in sterile water in Petri plates with

filter paper. After 24 hours, the seeds

**2. Materials and methods**

were observed for germination. The ger-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 528

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ minated seeds were counted, the radical

_melongena_ was studied in sterile Petri

and plumule length were measured (cm)

plates. Seeds of 40 numbers were soaked

and tabulated.

in 2ml of water (Control), in 2ml of

Chemical control, (Cartap hydrochloride

_2.5. Seedling length_

50% radon sp + tata tafgor dimethoate

Two days after seed germination,

30% ec = 2:1 ratio and dissolved in 10

ten normal seedlings were taken out care-

ml of water), and 2 ml of panchakavya;

fully at random from each treatment and

2ml of Panchakavya + neem cake (2

seedling length was measured from the tip

gm), 2ml of vermicompost as treatment,

of primary root to the tip of apical shoot.

separately and was observed for 5 days.

The average length (cm) of ten seedlings

The germinated seeds were counted and

was calculated and expressed as mean

the germination percentage was calculat-

seedling length.

ed in each of the treatment (Table 1 and

__

2). The germination frequency was 100%

_2.6. Seedling vigour index_

in panchakavya and the adjuvant treat-

The seedling vigour index was

ment in _A. esculentus_ while it was only

calculated adopting the method suggested

90% in _S. melangena_. The germination

by Abdul-Baki and Anderson (1973) and

frequency was lesser in all the other

expressed whole number treatment wise

treatments and it was only 50% in control

for _S. melangena_. Among the pan-

_Vigour index = Germination percentage_

chakavya treated seeds, maximum germi-

_× Seedling length_

nation was found in both _A. esculentus_

_****_

and _S. melongena_.

_2.7. Effect of panchakavya on cell divi-_

After germination the seedling

_sion and cell growth_

vigour index was calculated by measur-

Cytological studies of root tip of

ing the length of shoot and root. The data

A _llium cepa_ was studied for 7 days. On-

on shoot and root length of different

ion bulbs were treated with diluted pan-

treatment were measured by cm scale.

chakavya (50%, 25%, 10% and 1%).and

The plants treated with 2ml pan-

tap water as control. A _llium cepa_ din

chakavya produced the longest shoot

(2n=16) (Rank and Nielsen 1994) were

legnth (2.3) and root length (2) in _Abel-_

used as test system. The root tip from

_mochus esculentus_. While in _Solanum_

control and experimental setup were thor-

_melongena_ longest shoot (1.2), root

oughly washed in distilled water and

length (1.5) was in panchakavya treat-

fixed in Carnoy"s fixative (ethanol, chlo-

ment which was comparatively higher

roform glacial acetic acid 6:3:1 v/v/v) and

than control. The seedling vigour index

chromosome studies were done by aceto-

was higher in panchakavya treated seed-

carmine staining technique. The fixed

lings in both the plants which was 430 for

root tip were washed in distilled water

_A. esculentus_ and 243 for _S. melangena_

and hydrolyzed in IN HCL for 10 min

which was higher than control and all

then treated in 45% acetic acid for 5 min

other treatments (Table 1 and 2).

and stained in acetocarmarine stain for

Among the panchakavya and pan-

10-15 minutes. After staining the root tips

chakavya + neem cake treated seeds,

were squashed and observed under phase

maximum germination frequency was

contrast microscope.

observed in _Abelmoschus esculents_ and

_Solanum melongena_. The shoot length

**3. Results and discussion**

was 2.5 and 1.2 in _A. esculentus_ and _S._

_melangena_ and root length was 2 and

The effect of panchakavya on

1.1in _A. esculentus_ and _S. melangena_.

seed germination of two plants namely

There was a slight difference in 2ml of

_Abelmoschus esculentus_ and _Solanum_

panchakavya + neem cake. The pan-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 529

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ chakavya and panchakavya + neem cake,

was measured in (cm) scale (Table 3 and

is more effective, when compared to con-

4).

trol as well as other treatments. The 100

The onion bulb developed more

%

germination

was

observed

in

number of roots in 1% panchakavya

_A.esculentus_ in panchakavya and pan-

which was 48, and the root length was

chakavya + neem cake and while only 90

7.5, and the shoot length was 7 which

% germination was seen in _Solanum_

was higher when compared to other con-

_melongena_ (Table 1 and 2).

centrations. At 1% concentration of pan-

The growth of onion roots was ob-

chakavya the number of cells in the mi-

served on panchakavya at the concentra-

croscopical field was 250 cells, of which

tions of, (50%, 25%, 10%, and 1%). The

48 of them was in metaphase stage.

highest number of onion roots, shoots

Whereas, in control it was lesser, 22 cells

length and root length was observed in 1

in metaphase stage out of the 250 cells in

% concentration of panchakavya, and it

the microscopical field. The 1% concen-

tration of panchakavya increased the

****

**Table 1:** Pesticide consumption (in lakh tones) during, 1994-1997 (Heisey and Norton,

2007)

**Country/Region**

**Herbicides**

**Insecticides**

**Fungicides**

China

NA

NA

NA

India

6.8

37.2

9.4

Other Asia

24.4

41

19.2

Middle East/North Africa

9.7

19.5

14.1

Sub-Saharan Africa

11.7

9.7

9

Latin America/Caribbean

85.8

39.8

31.8

All developing

138.4

147.2

83.6

Transitional

35.6

7.9

23.2

Industrialized

337.8

163.4

190.4

World

511.8

318.4

297.2

****

**Table 2:** Rate of seed germination in _Abelmoschus esculentus_

**No Treatment**

**Time**

**No of**

**No of seed**

**Germination**

**Vigour index**

**Duration Seeds germination**

**%**

**Seedling**

**(Hours)**

**Length**

1.

Control

48

40

28

70

98

2.

Chemical

48

40

32

80

184

3.

Panchakavya

48

40

40

100

430

4.

Panchakavya

48

40

40

100

450

\+ Neem cake

5.

vermicompost

48

40

40

100

410

****

**Table 3:** Rate of seed germination in _Solanum melongena_

**Time**

**Germination**

**Vigour index**

**No of**

**No of seed**

**No**

**Treatment**

**Duration**

**%**

**Seedling**

**Seeds**

**germination**

**(Hours)**

****

**Length**

1.

Control

48

40

20

50

70

2.

Chemical

48

40

28

70

133

3.

Panchakavya

48

40

36

90

243

Panchakavya +

4.

48

40

36

90

207

Neem cake

5.

vermicompost

48

40

32

80

168

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 530

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ **Table 4:** Effect of Panchakavya on growth of onion roots ****

**No. of roots**

**Concentration**

**Root length**

**Shoot length**

**S.No**

**After four**

**[%]**

**[cm]**

**[cm]**

**days**

1.

Control

26

5

3

2.

50

37

5.5

5

3.

25

37

6

6

4.

10

37

7

6.5

5.

1

48

7.5

7

number of onion roots, when compared

panchakavya, adjuvated with neem cake,

with control as well as other different

and vermicompost.

concentration of panchakavya.

The effect of adjuvants namely

The greatest challenge of the na-

vermicompost and neem cake showed a

tion in the coming years is to provide safe

slight difference, whereas the pan-

food for the growing population in the

chakavya alone was sufficient to enhance

country without degrading the environ-

the seed germination. The panchakavaya

ment. In this regard organic farming

favors cell growth, cell division (Table 5)

which is a holistic production manage-

which is known from the mitotic index.

ment system for promoting and enhancing

The onion bulbs produced longer roots

health of agro ecosystem, has gained wide

and shoots when compare to control. The

recognition as a valid alternative to con-

panchakavya at 1% concentration fa-

ventional food production and ensure

voured the production of larger number of

safer food supply for human consump-

roots. The presence of growth promoting

tion. This farming system avoid large use

substance present in the panchakavya fa-

of synthetic fertilizer, pesticides, growth

vored rooting. The present study reveals

regulators and livestock feed additives

that panchakavya preparation is easy and

and relies on green manures, crop rota-

it is cost effective and could be prepared

tion, crop residues, animal manures, bio

easily by a farmer with his household ex-

fertilizers, bio pesticides, different kinds

penses and availability. Rahul kumar _et_

of cow based liquid organic manures such

_al_. (2014) studied the effect of pan-

as panchakavya, sanjibani, kunapajala,

chakavya fortified with _Bauhinia_ plant

amrit pani etc.

extract which showed a positive response

Many advanced countries mainly

as an anthelminthic preparation.

depend upon the dairy products because

of their commercial, agricultural and nu-

**Table 5:** Effect of Panchakavya on cell

tritive properties. The dairy industries

division [onion root tips]

play a vital role in the development of the

**No. of cells**

**No.**

**Concentration**

country. When a new house or building or

**No**

**in**

**of**

**[%]**

even a temple constructed in India, the

**Metaphase cells**

first to enter the premises would be the

1

Control

22

250

cow because this is considered to be aus-

2

50

46

250

picious. Cow"s urine (Comiyam) is used

3

25

44

250

in almost all the Hindu rituals. The poten-

4

10

45

250

tial of using panchakavya as growth pro-

5

1

48

250

motor and biofertilizer is revealed in this

work. The present study revealed the

Sangeetha

and

Thevanathan

germination frequency of _Abelmoschus_

(2010) studied the potential of panchaga-

_esculentes_ , and _Solanum melongena_

vya as biofertilizer against certain pulses

grown under the different treatments

by growing in soil amended with seaweed

namely, Control, Chemical + fertilizer,

extract and panchagavya. Experimental

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 531

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ seedling recorded higher rates of linear

only enhances the plant yield and growth

growth of both shoots and roots as com-

rate, it also reduces the insect invasion

pared to controls. These seedlings pro-

and fungal attack. The results of the pre-

duced 264 to 390% more lateral roots

sent study clearly show that panchakavya

than the control and maximum lateral root

is an organic fertilizer and growth pro-

production was always observed in seed-

moter. It also increases the soil fertility.

lings grown in soil amended with sea-

It is very much essential to develop a

weed based panchagavya at low concen-

strong workable and compatible package

trations

of nutrient management through organic

In recent years the people have

resources for various crops, based on sci-

recognized a number of commercial, me-

entific facts, local conditions, and eco-

dicinal and agricultural values from the

nomic viability for the sustainability.

various products of dairy forms. Tharun

_et al_., (1983) carried out extensive works

**Acknowledgement**

in this aspects and the environmental

management in developing countries.

The authors would like to thank

The number of new methods of

Dr. S.Karpagam, Head of the department

recycling and controlling measures of or-

of Botany, Queen Mary"s College for

ganic waste in urban and rural habits was

providing the laboratory facilities.

proposed by (Furedy _et al.,_ 1989). The

****

current global scenario firmly emphasizes

**References**

the need to adopt eco-friendly agricultural

practices for sustainable agriculture.

**Abdul-Barki, A.A, and Anderson, J.D,**

Chemical input had an adverse impact on

**(1973).** Vigour determination in soy-

the health-care of not only soil but also

bean seed by multiple criteria. _Crop_

the beneficial soil microbial communities

_Science_ **13, 630 – 63.** __

and the crops. This eventually led to a

**Furedy,C, Bluemental,u.J, Strauss.M,**

high demand for organic products. Farm-

**Carnicross, L. (1980).** Model for the

ers all over the world realize the need to

effect of different control measures

detoxify the land by switching over to

in reducing health risks from waste

organic farming dispensing with chemical

water. _Sci.Tec.,_ **21, 567 – 577.** __

fertilizers, pesticides, fungicides and

**Gomathi, R., Isaivani Indrakumar and**

herbicides. In India, organic farming was

**S. Karpagam (2014).** Larvicidal ac-

a well developed and systematized agri-

tivity of _Monstera_ _adansonii_ plant

cultural practice during the past and this

extracts against _Culex quinequefacia-_

ancient wisdom obtained through Indian

_tus_. Journal of Pharmacognosy and

knowledge is the use of "panchagavya" in

Phytochemistry, **3(3), 160-162.**

agriculture for the health of soil, plants

**Gosavi D.D. and Jhon, P.S.** **(2012).** Ef-

and humans.

fect of panchakavya Ghritra on some

neurological parameters in albino

**4. Conclusion**

rats. Asian journal of pharma ceutical

science clinical Research, **5, 154 –**

Panchakavya is used in different

**156.**

forms. Such as foliar spray, soil applica-

**Gupta**

**and**

**Gopal**

**(2001).**

tion along with irrigation, seed treatment

http:/www.epa.gov/oecaagct/ag101/c

or seedling treatment etc. For foliar spray

roppesticidesuse

3% concentration is being used by organ-

**Natarajan, K. (2002).** Panchakavya a

ic farmers. Panchakavya was an important

manual. _Other India press, Mapusa,_

one that enhanced the biological efficien-

_Goa, India, pp, 33_.

cy of crop and the quality of fruits and

**Nene,Y.L. (1999).** Seed health ancient

vegetable production. Panchakavya not

and medicinal history and its rele-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 532

_Biotech Sustainability (2017)_

_Use Organic Fertilizers for Sustainability Ramya and Karpagam_ vance to present day agriculture _In_ :

**Sangeetha, V. and Thevanathan, R.**

Anicent and medical history of Indi-

**(2010).** Biofertilizer Potential of

an agric S.L.Choudhary, G.S,Sharma

Traditional

and

Panchagavya

and X.L,

Amended with Seaweed Extract. The

**Nene (ed) (1911).** _Proc. of the summer_

Journal

of

American

Science,

_school held from 28th may, college of_

6(2):61-67.

_Agric, Jaipur, Rajasthan._

**Sathasivam, A., Muthuselvam, M., Ra-**

**Rank, J. and Nielsen, M.H. (1994).** A

**jendran, R. (2010).** Antimicrobial

modified Allium test as a tool in the

Activities of Cow Urine Distillate

screening of the genotoxicity of

against Some Clinical Pathogens.

complex mixtures. _Hereditas_ **118,**

Global Journal of Pharmacology, **4,**

**49-53.**

**41-44.**

**Rahul Kumar, Amit Kumar, Kuldip**

**Tharun,G., N.,C., and Bidwelll, R.**

**Kumar,**

**Vaishnavee**

**Gupta,**

**(1983)**. Environmental Management

**Triveni Shrivas, Kishu Tripathi.**

for Developing countries, vol 1:

**(2014).** Synergistic anthelmintic ac-

Waste and Water Pollution control –

tivity of different compositions of

Review of Technical solutions. _Asian_

panchagavya and _Bauhinia_ _variegata_

_institute of Technology, continuing_

linn. International Journal of Phyto-

_Education centre, Bangokok, **pp.48 –**_

pharmacology, **5(2), 120-122.**

_**54.**_

© 2017 by the authors. Licensee, Editors and AIMST University, Ma-

laysia. This article is an open access article distributed under the terms

and conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 533

**Biotechnology for Sustainability**

__

_Achievements, Challenges and Perspectives_

_Biotech Sustainability (2017), P534-538_

****

_**Short Communication**_

****

**Increasing Human Interference in Katarniaghat**

**Wildlife Sanctuary**

****

**Shiv Pratap Singh***

_Department of Geography, Kisan P.G. College Bahraich, Affiliated to Dr. R.M.L. Awadh_

_University, Faizabad, Uttar Pradesh, India;*Correspondence: principalkpgc@yahoo.co.in;_

_Tel.: +919984146483_

**Abstract:** Katarniaghat Wildlife Sanctuary (KWS) is a part of Dudhwa Tiger Reserve. It is

managed with the Dudhwa National Park and Kishanpur Wildlife Sanctuary. It is located

near to the Indo-Nepal boarder. It is in the Bahraich district in Uttar Pradesh. The sanctuary

comes under the tropical moist deciduous forest of the Himalayan Tarai-Bhabhar region. It

is under laid on 31 may, 1976. It is situated between 27°41 – 27°56 N and 81°48 – 81°56 E

covering an area of 551 km. The KWS is divided into six divisions. Four division (Katrniya

,Nishangada, Murtiha, Dharampur) of it lies under the core zone and remaining two divi-

sions are in buffer zone. _Tharu_ is the main tribe of this area. Increasing human interference

has been studied in the related research paper that outsteps the concept of Biosphere Re-

serve. . Due to growing tourism and farming in core zone, and human activities and habitats

in buffer zone, it has be-come a critical situation to the bio-diversity of this wildlife sanctu-

ary. In this article, the challenges of the Katarniyaghat Wildlife Sanctuary are highlighted.

The highlighted challenges need to be addressed for the sustainable growth and develop-

ment of the region and the associated communities.

_**Keywords:**_ Biodiversity; conservation; environment; human interference; sustainable de-

velopment; wildlife

****

**1. Introduction**

environmental activities but availability

of fuel, food items, fodders and income

In the 21st century, due to growth in

also get affected. In this article, the ex-

materialistic view of life and population

ample of Himalayan Tarai Ecosystem of

explosion, the competition to make the

Uttar Pradesh and the problems pervaded

life more and more comfortable by utiliz-

in biodiversity of Katarniyaghat Wildlife

ing industrial products has become no-

Division are highlighted.

ticeable. To meet the consumers demand,

industrial production is increased drasti-

**2. Introduction of the study field**

cally; but unplanned and indiscriminate

industrialization elevated the level of en-

Katarniyaghat Wildlife division is

vironment pollution and ecological im-

situated in Nanpara Tahsil in Bahraich

balance. The growing number of animals

district of Uttar Pradesh in India. It lies

and human, and pressure of agriculture

along Indo-Nepal international border. It

and economic development destroyed the

is situated between 27°41 - 27°56 N and

forests. Deforestation is sensitive in India;

81°48 - 81°56 E. This division is extend-

because not only it has a bad effect on the

ed in about 551 km area; which is an im-

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_ 534

_Biotech Sustainability (2017)_

_Increasing Human Interference in Wildlife Sanctuary Singh_ portant part of Himalayan Tarai - Bhabhar

Tiger, Cheetal, Stages, Rhinocerous, Ele-

region. This wildlife sanctuary is included

phant, Leopard, Boar, Padha **** etc. Under

in the Project Tiger in 2003 for the con-

the endangered aquatic birds we find **** Lal-

servation of tigers, Saryu (Kaudiyala and

sar, Surkhab, Nilsar, Gugral, Kaj kurchhia

Girwa) river Dalphins, massive Croco-

and little Mew. Some other birds like

diles, Alligator and Turtles biodiversity.

Peacock, Moorcock, Pheasant, Dhanesh,

The forest of the sanctuary area has

Log log, Hairns, Submarine bird, Crane,

been classified into two major types: (1)

Wilture, Hawak, Kite, Owl, Caprimulgid,

The Sal forest and (2) the miscellaneous

Magpia, Woodpecker, Khanjan, Mynah

forest. This area is rich in biodiversity by

bird, Crow, Nightangle, Satbahin **** are also

including vast grasslands, dense forests of

observed.

Sal, Sakhu and Teak trees and Aquatic

Endangered reptiles namely, Iguanas,

areas.

Pythans, Black **** Cobras and Cobras are

Pedagogically, the study area is made

found here. For conserving crocodiles **** and

up of the alluvial soil of the Saryu River

alligator a Crocodile Project has been es-

and its tributaries flowing adjoining to it.

tablished in the Katarniyaghat. Currently,

The study area has a tropical mon-

this wild life division is being developed

soon type climate with three distinct sea-

as a tourist place of Bahraich district.

sons i.e. summer (April to June) winter

Tourists come here to see and watch wild

(November to February) and warm-rainy

animals and birds natural beauty as well

(July to September). March and October

from for-off places. The main tribe living

are considered as transition months be-

here is ' _**Tharu** '_ which lives in the buffer

tween the seasons. The mean maximum

zone of the forest area. They are able to

temperature ranges from 22 degree Celsi-

earn their livelihood by working in agri-

us in January to 40 degree Celsius in May

cultural or non-agricultural sectors.

and the mean minimum temperature

ranges from 8 degree Celsius in January

**3. Katarniaghat wildlife sanctuary and**

to 27 degree Celsius in June. The annual

**the concept of biosphere reserve**

rainfall ranges from 36 to 142 cm in win-

ter, 34 to 662 cm in summer and 1294 to

Under the concept of bio-sphere re-

1689 cm in warm-rainy seasons.

serve the conservation and enhancement

It is a natural aesthesis to see roaring

of endangered environmental condition is

Tigers, Leopard resting on tree, leaping

implied. The entire zone is divided into

Cheetal, Padha, Stage, Kakad and Boars

three zones. First zone is the specific zone

digging the forest land with their long

related to the internal part; where human

snout.

entries are restricted. Second zone is

To conserve the abundance of wild lives,

known as transitional zone or mid-zone

U.P. government has divided it in 6 sub

surrounded to the core zone; this zone is

divisions. Four sub divisions (Katarnia,

related to research centers, tourist places

Nishangara, Murtiha, Bharthapur) are de-

and utilization of fuels and non-living re-

clared as a core zone and remaining two

sources. Third zone which is situated

(Motipur, Kakraha) are declared as a

round the mid zone is related to the habi-

buffer zone. North-east railway line pass-

tats, agriculture, tourist places and utiliza-

es through this division connecting the

tion of non-living resources. But in the

Bichhia-Katarniya tourist place. This line

Bio-sphere reserve (Katarniyaghat Wild-

is parallel to the roadway that divides the

life Sanctuary), the core zone (Katarnia,

zone into two parts (Figure 1).

Nishangara, Murtiha, Bharthapur ) is be-

There is greatest biodiversity under

ing developed as Guest House (GH) and

the Katarniya zone. If we have a glimpse

Tourist Centre (TC). Meanwhile, in the

on biodiversity of biosphere reserve then

buffer zone, the activities of forestry and

we find endangered Ganga Dolphin,

agriculture are growing for the earning of

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 535

_Biotech Sustainability (2017)_

_Increasing Human Interference in Wildlife Sanctuary Singh_ livelihood for growing population densi-there is possibility of bio-diversity loss

ty. Thus, the area of this zone is decreas-

and imbalance of ecosystem.

ing day by day. If these trends persist then

**Figure 1:** A diagrammatic sketch (map) showing the Katarniaghat Wildlife Sanctuary loca-

tion in India. Katarniaghat Wildlife Sanctuary map shows the roads, railway lines and

guest houses located within the sanctuary.

**4. Human activities in Katarniaghat**

by _Lantana sp_. Over grazing and human

**wildlife sanctuary**

activities including fire are disturbing.

The Protected area also experiences pres-

Approximately 20% of the area con-

sure for fuel wood and fodder from the

sisting mostly of the grassland is infested

adjoining 25 villages and also from popu-

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 536

_Biotech Sustainability (2017)_

_Increasing Human Interference in Wildlife Sanctuary Singh_ lation on the Nepal side. There are some

responsible for the fragmentation are; as

forest villages (erstwhile Taungya villag-

follows:

es) and a Central State Seed Research

 Burgeoning human population

Farm near Girijapuri, situated inside the

(and therefore the escalating pres-

wildlife sanctuary covering 38.42 sq Km

sure for resources)

area that causes considerable disturbance

 Changed socio-economic scenario

to the wildlife, creating a break in the for-

(e.g. changed lifestyle of the Guj-

est connectivity.

jars and Khatta holders, therefore

Generally, we know that the har-

overgrazing over cutting of fire-

nessing of timber and fuel is right and to

wood timber and fodder species)

cut the forest for trade is illegal. It cannot

 Encroachment of forest lands ( by

be restricted without giving the employ-

agriculture monoculture plantation

ment to the effected people and maintain-

and other land use)

ing facultative measures. In the studied

 Infrastructure development (like

area, the most of the human activities are

rail, road, hydroelectric and irriga-

done by rural people; because, the activi-

tion projects)

ties of cattle rearing and forestry are done

 Various illegal/ legal (timber har-

directly. Along with these activities, ille-

vesting, boulder mining in river

gal wood cutting and hunting is found in

beds etc.)

abundant measure. In the buffer zone of

 approach of various departments

this sanctuary, the rural population and

 Little/ no community participation

unplanned habitats are growing continu-

 Obsolete policies less amenable to

ously. So the local people are overleaping

adapt to changes, and

the forest land and converting it into agri-

 Lack of adequate scientific

cultural land by cutting the forest. In fact

knowledge and proper monitoring

is a challenge of present time for the sanc-

plants.

tuary. In addition to this, the fast running

The magnitude of the above stated prob-

vehicles and north-east railway line cause

lems leading to habitat fragmentation and

the accident of wild lives of the forest ar-

biological invading varies. There are dif-

ea. The ecological system is widely dis-

ferent responses from various stakehold-

turbed by the noise pollution and savage

ers. The government forest department,

pollution caused by the tourists. As the

local communities, and the main stake-

Sanctuary is situated at the border of Ne-

holders that largely manage and use the

pal; Nepalies from the border region are

natural resources of the landscape. There

involved in lot of illegal activities related

are some broad responses, which could be

to forestry; which is also disturbing to the

generalized across the sites for the bet-

wildlife and forest of the Sanctuary.

terment of the sanctuary.

**5. Response to cope with the changing**

**6. Perspectives**

**theme (human activity)**

This paper highlighted the human ac-

Though the management plant of the

tivities in which are disturbing wildlife

Protect Area Sanctuary (PAS) and work-

sanctuary. To protect the biosphere re-

ing plant of Reserve Forest Sanctuary

serve people should be educated to incul-

(RFS) have not been specifically oriented

cate the importance of conserving wildlife

to cope with the identified Global Change

for the sustainability. The human interfer-

Factors per se yet, Many initiatives are

ence in the buffer and core zone generat-

taken up to minimize the factors that con-

ed the danger to the bio-diversity. Forest

tribute to habitat fragmentation and infes-

department has recently started the plans

tation of invasive species. Major factors

to supply income to the local people but it

is well know that only forest department

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 537

_Biotech Sustainability (2017)_

_Increasing Human Interference in Wildlife Sanctuary Singh_ cannot do this. Other institution will have

**McComb, C. Benda (2007).** Book: _Wild-_

to take responsibility to make more

_life Habitat Management Concepts_

chances to generate employments for the

_and Application in Forestry_ , Secand

people in the vicinity of the sanctuary.

Edition **, pp-162-168**.

_Jawahar Rojgar Yojna, Samekit Gramin_

**Verma, S. (2010).** _The Eco-Friendly_

_Vikas Karyakram_ and _MGNAREGA_ will

_Tharu Tribe: A Study in Socio- Cul-_

be helpful in providing proper jobs to lo-

_tural Dynamics;_ Joural Of Asia Pa-

cals to establish the right situation to con-

cific Studies **1, 177-187.**

serve the wild lives. Task is challenging;

**Pandy,R.K. (IFS 2005-2008).** _Tiger Pro-_

but, it is essential for the sustainable

_tection and Conservation in Indian_

growth and development of the people

_Tarai: Project Work:_ New Delhi.

and the region.

**Praveer, R. (2001).** Book: _Tharu Tribe_ ;

Bihar Hindi Granth Akadme, Patna.

**References**

**Sharma, P.B. (2007).** Book: _Ecology and_

****

_Environment_ ; Rastoge Publication,

**Annual Report (2007).** _Government of_

Meerut **; pp 168-171.**

_India ministry of Environment and_

**Semwal, R. L. (2005).** Book: _The Tarai_

_Forest_.

_Arc Landscap In India;_ Forests &

**Census of India (2001).** Table-D1005,

Biodiversity

Conservation

Pro-

_Ministry of Home Affair_ , Office of

gramme _,_ New Delhi.

the Registrar General Delhi; Kal-

**Prakash, S. and Prasad, S. L. (2009).** __

yani Pub. **, 240-250**.

_Some Aspects of Socio- Economic_

**Forest Department Gajetiar (2009).** Ut-

_Study of the Tharu in Uttar Pra-_

tar Prdesh.

_desh: An Asian Tribes Studies;_

**Gosal, G.S. (1961).** Internal Migration in

Jounral Annals **28(1).**

India; A Reginal Analysis; _Indian_

**Singh, Dr. savindra (2002).** Book- _The_

_Geographical Joural_ **36(2), 106-**

_Environment Geography_ , Indian

**121**.

Press Publication, pp. **484-490**

© 2017 by the author. Licensee, Editors and AIMST University, Malay-

sia. This article is an open access article distributed under the terms and

conditions of the Creative Commons Attribution (CC BY) license

(http://creativecommons.org/licenses/by/4.0/).

ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9 538

"Earth provides enough to satisfy every

man's needs, but not every man's greed" ****

**\--- Mahatma Gandhi**

**About Editors**

**Subhash Bhore, _PhD_** **:** Subhash completed his BSc (Botany) and MSc (Botany) degrees

education at University of Pune, India. Immediately after completing his

MSc (1996), he got an opportunity to work at 'Biochemical Engineering

Department' and 'Plant Tissue Culture Pilot Plant' of the CSIR-National

Chemical Laboratory, Pune, India. In June 2000, he received a Doctoral

Fellowship (GRA) to pursue a PhD Degree in Molecular Genetics at the

National University of Malaysia (UKM). In 2004, he was appointed as

Senior Research Officer at Melaka Institute of Biotechnology (MIB), a

research wing of Melaka Biotechnology Corporation, Malaysia. Based

on his performance, in April 2005, he was promoted as 'Principal Investigator & Head of

R&D Department' at MIB, Malaysia. In 2008, he was invited by the AIMST University as

a 'Visiting Faculty' for their Department of Biotechnology and now serving as a Senior

Associate Professor. In 2009, he was nominated for the AASIO (Association of

Agricultural Scientists of Indian Origin) Young Scientist Award. He has published more

than 50 peer-reviewed articles, 6 books and submitted more than 11,900 DNA sequences in

Gene Bank; filed one patent, and received more than 10 awards/fellowships. As of June

2017, he has supervised more than 72 students including postgraduates, undergraduates and

industrial trainees. He is actively involved in research as well as teaching and advising of

postgraduate and undergraduate students. You may contact him using email,

subhash@aimst.edu.my or subhashbhore@gmail.com

_________________________________________________________________________

**Kasi Marimuthu, _PhD_** **:** Marimuthu accomplished his BSc (Zoology); MSc

(Environmental Biotechnology); PhD (Environmental Biotechnology/

Zoology Interdisciplinary) degree education at Manonmaniam

Sundaranar University, Tamilnadu, India. In 2003 he joined as a Post-

Doctoral Fellow at School of Biological Sciences, University Science

Malaysia, Penang for 2 years. At present, he is working as a Professor

in the Department of Biotechnology AIMST University, Malaysia for

the last 12 years. He teaches Aquaculture, Biostatistics, Research

Methodology, Biology of Invertebrates and Vertebrates courses for undergraduate

biotechnology programme. His research interests include fish reproduction and breeding,

larval rearing, hatchery management, fish immunology and aquatic toxicology related

research. He has published 95 research papers in fisheries and aquaculture in various

reputed and indexed journals. He has participated in more than 35 local and international

conferences, seminars, and workshops. He has been appointed as an external examiner for

six Indian Universities (Manonmaniam Sundaranar University, Annamalai University,

Bharathiyar University, Bharathidasan University, Madras University, and Priest

University) Tamilnadu, India. He is a life member in National Academy of Biological

Sciences [NABS], India and Asian Fisheries Society. He is an editorial member in Indian

Journal of Natural Products and Resources and Acta Ichthyologica Et Piscatoria. **** He is also

presently serving as a Deputy Vice chancellor for Academic and International Affairs,

AIMST University, Kedah Darul Aman, Malaysia. You may contact him at

marimuthu@aimst.edu.my / aquamuthu2k@gmail.com.

**Manickam Ravichandran, _PhD:_** Prof M. Ravichandran obtained his

M.Sc Medical Microbiology from Christian Medical College, Vellore in

1991 and gained PhD degree from Anna University, Chennai, India in

1997. Currently his research interests include cholera vaccine, molecular

diagnostics and phage therapy. He has constructed several cholera

vaccine candidates for O139 Vibrio cholerae and developed several

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_

simple cold chain free molecular diagnostic kits. He has published 64 papers in

international journals (Cumulative citations 584), supervised 40 postgraduate students, filed

8 patents and commercialized 3 products on diagnostics. He has been awarded with 44

national and international awards for the academic and research excellence including Ideas

Inventions New Products (IENA), Nuremberg Germany; International Exhibition of

Inventions: New Technologies and Products, Geneva and Anugerah Inovasi Negara. He is

an associate member of Academy of Sciences Malaysia (ASM) and Expert panel member

for R,D&C grants, Sciencefund, Technofund, Community Innovation Fund (CIF),

Enterprise Innovation Fund (EIF), and Innovation (MOSTI); He was the Cluster Working

Group (CWG) Committee member on Human Capital Development, Malaysian

Biotechnology Corporation and the Committee member on 'Top Research Scientists

Malaysia'(TRSM) of Academy of Sciences Malaysia (ASM), Biotechnology Road map of

the Kedah state was formulated under his supervision. He is currently the Chief Executive

and Vice Chancellor of AIMST University, Kedah Darul Aman, Malaysia. He can be

contacted at ravichandran@aimst.edu.my.

_ISBN: 978-967-14475-3-6; eISBN: 978-967-14475-2-9_

**Biotechnology for Sustainability**

_**Achievements, Challenges and Perspectives**_

**Published by AIMST University**

__
