EcoProduction.

Environmental Issues in Logistics and Manufacturing

Arpita Saxena Editor

Biotechnology
Business
- Concept
to Delivery
EcoProduction

Environmental Issues in Logistics and Manufacturing

Series Editor
Paulina Golinska-Dawson, Poznań, Poland
The EcoProduction Series is a forum for presenting emerging environmental issues
in Logistics and Manufacturing. Its main objective is a multidisciplinary approach
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sustainability research. It encompasses topical monographs and selected conference
proceedings, authored or edited by leading experts as well as by promising young
scientists. The Series aims to provide the impulse for new ideas by reporting on the
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turing systems, environmentally conscious operations management and reverse or
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It aims to bring together academic, industry and government personnel from
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Arpita Saxena
Editor

Biotechnology Business -
Concept to Delivery

123
Editor
Arpita Saxena
SPAK megAcorp
Aurangabad, India

ISSN 2193-4614 ISSN 2193-4622 (electronic)

EcoProduction
ISBN 978-3-030-36129-7 ISBN 978-3-030-36130-3 (eBook)
https://doi.org/10.1007/978-3-030-36130-3
© Springer Nature Switzerland AG 2020
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This work is dedicated to selfless efforts of
Mentors, who, with their expertise and
experience nurture young start-ups and
entrepreneurs throughout the globe.
Foreword

It gives me pleasure to write a foreword for the book compiled with the basic
rationale of culturing the next generation with an enterprising attitude. As a leader
of synthetic biology in one of the leading business houses, I have addressed various
challenges to sustainable business solutions. In all this, I see the drive to bring about
the mental accommodation of an individual to channel towards a new idea. To
come out of one’s comfort zone and strive for excellence is desired by every fresh
mind but when it comes to practically doing it, things are pushed towards doubt and
drift. It needs a good mentor and a strong sense of belonging to the idea to actually
reach the zenith.
This book provides clear landmarks which any start-up would come across.
Right from the beginning of the book till the end, the process of conceiving a
business idea to delivering it in the form of a product is discussed. To give it more
appealing, the parts of an entrepreneur’s journey are projected in the form of
musical scales. To begin with, the first chapter details from the beginning of
biotechnology up to its contemporary extent. Research students’ favourite topic is
covered in the next part, where the molecular, genetic or nano targets, the ones used
to manipulate the working pathways at various levels, are discussed. The book in
the following chapters covers nutraceuticals and their importance in health care and
biofuels and their contribution to the environment.
The chapters in the following parts cover other pertinent aspects like regulations,
IP, commerce and management of a biotech start-up. These are the important
aspects which are to be precisely known to individuals when they start on their
own.
The knowledge and contribution of the authors of the book are commendable as
they have played the role of mentors for the readers. I would also congratulate the
editor of the book Dr. Arpita Saxena for the idea and effort put in the book. I hope

vii
viii Foreword

this book is used well by the young students to earn experience and know-how to
start-up and proceed with interesting and useful ideas. Wishing the best to the entire
team related to this book.

July 2019 Dr. Santanu Dasgupta

Senior Vice President
Synthetic Biology
Reliance Industries Limited
Mumbai, India
Preface

The first thing that comes to our minds when we read the title of this book is—what
is the business of biotechnology, or business of anything which is a discipline
itself? How can one approach towards a subject as a profit or loss statement? And
many more thoughts that cross our brains, which are by the way a symbol of our
affection, respect and sense of gratitude towards the subject…
So, how did I think about it as a title for my next book? This opens up the
Pandora’s box of my past experiences and story of how this idea looks genuinely
graceful and useful to me. Few days back, a self-proclaimed enterprising professor
in one of his addresses to the candidates who were attending an entrepreneurial skill
development program in one of the university incubation centres said, ‘if I give you
a thousand bucks to sell vegetables for one day, who amongst you would like to do
it?’ and before anybody could raise their hands, he himself answered his question as
‘obviously none of you… since you are all Ph.D.s, and the task will not give you
job satisfaction’.
This was the time when I had left my job from a start-up company to have my
own venture, and I was struggling to pay my bills. The first thought that crossed my
mind was—what actually gives job satisfaction? The ability to be on your own or
the perfection at whatever you do? Or maybe growth which gives you a boost at
work or an environment that keeps you charged and motivated? If all this counts,
what is wrong with selling vegetables without any monetary investment and
making thousand bucks a day? The answer may be different for different people and
that is what defines our approach to our subjects. To me, being able to tap the
smallest of opportunity and trying to solve the most complex problems in simplistic
ways are the attitude of an entrepreneur with fair chances of success.
The day left me reflecting on one more point and that was, if somehow the next
generation is told that there are no high and low profiles in work when you own
your business, and that challenges have to be met with wit and zeal, they might be
better prepared for things to come. Even better is to discuss the subject w.r.t.
application, discuss real challenges that confront them to realize their goals and tell
them real stories of success as well as failures of entrepreneurs. This book is an

ix
x Preface

attempt in the same direction although this also might have some imperfections, but
the sincerity in efforts is promised.
Biotechnology, as the name has it, is a beautiful orchestration of multiple
technologies working with living cells. These natural and human intervened
combinations of A-T, G-C bring about lot of products and alternatives to combat
the challenges in health care, agriculture, nutraceuticals as well as environmental
protection. This book is a next step towards strengthening the entrepreneurial
environment in the field of biotechnology specifically in India. Rhythm is the basis
of life. So whether it is the lub-dub rhythm of a living heart or the rhythm of the life
of an entrepreneur, efforts have to be continuous, dedicated and enriched with
knowledge and experience. Here, we bring every important aspect of a biotech
enterprise in the form of Musical scales. Step by step as the young biotechnologists
walk this road, they may be able to compose their own songs…
The first part of the book Do—‘Why biotechnology’ draws attention of people
about what to expect from biotechnology as a discipline. It flows from past of
human curiosities and discoveries to present scope of their applications. Here, an
insight into the scope and depth of the biotechnology as a subject is provided to the
readers.
The next is the most interesting part Re—‘Tiny targets big impact’. This part
will include the innovations through targeting at nano/genetic/molecular levels of
cells, to better the existing systems of health care, crop production/protection, etc.
The revolutionary CRISPR-Cas9 genome editing tool will be discussed along with
its applications. Another chapter in the same category discusses the role of nan-
otechnology in increasing the efficiency of delivering new forms of therapeutics to
the target tissue. This field is the newest and most exciting, hence an important
include in the book.
Mi—Food for all; Nutraceuticals ‘The intellectual quests only start when the
tummy is full’. Ensuring nutrition for all, this segment describes various approaches
in nutrition management and nutraceuticals production, scope which play a huge
role in disease prevention and general health promotion. As we talk about
entrepreneurship, it is important to understand the demand and supply chain. The
food and nutrition market especially those targeting specific groups like infants,
children, pregnant women and patients is immense, and a newcomer needs to
realize the need and choice of his/her product.
Fa—Biofuels—Recently, biofuels have surfaced as alternative source of energy
which are sources from plants or specifically algae. Considering the limitations of
first and second generation of biofuels, the third generation of biofuels has attracted
the research communities. Microalgae is striving to establish its place globally as an
alternative to conventional fuels. Necessity of fuels is obvious and with the con-
temporary fuels nearing extinction, the world looks forward to alternatives.
Biofuels, being one of those alternatives, makes an important placeholder in
research as well as this book.
So—Regulations—Every production is monitored with certain set of rules and
regulatory bodies to ensure safety and quality. This monitoring becomes a pre-
requisite when the modifications at DNA or molecular level are concerned and that
Preface xi

why regulations part will be discussed in this segment of book. Once an entre-
preneur is ready with an idea of product, it is indispensable to know the rules and
restrictions for its production.
La—Intellectual Rights. The production is not always in terms of materials but
also in terms of intellectual assets. Here, the author throws light on the intellectual
property rights as only material wealth is no more all that a person has, and it is
important for a start-up to understand the legalities of registration of assets as well
as ways to protect them.
Ti—Commerce and Management. Latest trends in biotechnology market and
various approaches to enter into it will be the focus of this segment. Once the
budding researcher turns a budding entrepreneur, he/she needs management skills
specific to a biotech industry. This segment gives insights into this aspect.
Products of modern biotechnology are discussed in the end to motivate the
readers. It also gives them an exposure about the line of products available already,
in the process of production or the ones which are not thought of still.
This book will serve the undergraduates and graduates with the guidance to
prepare themselves with entrepreneurial skills and mindsets, so that when they are
at the verge of completion of their courses, they are capable of joining big industries
and/or start their own start-ups.
This book is a guide through the various aspects of biotechnology as a subject
and the opportunities it offers to the coming scientific community. The authors
of the book belong to various specialized sectors from all around the globe. They
have a technique of changing their most significant research into simple, under-
standable and lucid English. Their contributions bring on the table keen research
ideas demanding a common man’s attention.
The impact of this book would be long imprinted in the minds of the readers.
Wishing you a happy read.

Aurangabad, India Arpita Saxena

Contents

Why Biotechnology?
Biotechnology: Discoveries and Their Applications
in Societal Welfare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Harsh Kumar

Tiny Targets, BIG Impact!

CRISPR: The Revolutionary Gene Editing Tool with Far-Reaching
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
Sohinee Bhattacharyya and Anindit Mukherjee
Nanotechnology in Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Anindit Mukherjee and Sohinee Bhattacharyya

Food for All; Nutraceuticals

The Rise of Nutraceuticals: Overview and Future . . . . . . . . . . . . . . . . . 67
Nitika Kapoor, Vijay Lakshmi Jamwal, Manish R. Shukla
and Sumit G. Gandhi

Biofuels
Algae Biodiesel: Fundamentals and Future Prospects . . . . . . . . . . . . . . 95
Ranjana Bhati
Biotechnology of Biofuels: Historical Overview, Business Outlook
and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Vijay Lakshmi Jamwal, Nitika Kapoor and Sumit G. Gandhi

xiii
xiv Contents

Regulations
Regulations for Health Care Biotechnology Products in Major
Markets of the World . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Durga Prasad Mindala, Yashbir S. Bedi, Satish Kumar Gupta,
Sumit G. Gandhi and Inshad Ali Khan

Intellectual Rights
Intellectual Property Rights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Tabassum Zafar

Commerce and Management

Evolution of Biotechnology as a Million Dollar Market:
The Management and Commerce of a Biotech Start-up . . . . . . . . . . . . . 161
Gaurav Verma and Srividhya Ravichandran

Some Success Stories and Products

Products of Biotechnology: The Out-Turn of Research
and Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Arpita Saxena
Editor and Contributors

About the Editor

Arpita Saxena is an entrepreneur, heading a start-up ‘SPAK megAcorp’ an ana-

lytical services and training provider based in Aurangabad, Maharashtra, India. She
is striving to better the education system in India with her concept of ‘AAPT’,
which is a social venture of ‘SPAK megAcorp’. Arpita received her Ph.D. in
biotechnology in 2012 off campus from Guru Nanak Dev University, Amritsar,
India, while working at the department of Cancer Pharmacology, Indian Institute of
Integrative Medicine (CSIR), Jammu, India. During Ph.D., her work was focused
on understanding the methods to evaluate cell death, apoptosis and the mechanisms
underlying apoptosis. While during screening a new synthetic, semisynthetic or
natural entity for its potential as an anticancer agent on in vitro, in vivo, mecha-
nistic, acute toxicity models, her specific topic was ‘Anticancer potential of Spiro
derivatives of Parthenin (a weed in Indian Subcontinent) against leukaemia cell
lines’.
Later, she was working with a start-up at Aurangabad, funded by BIRAC, India,
for 2 years where she was making targeted therapies for blood cancer. She also got
an exposure of material science practices during this therapeutic development
process. Arpita is associated with BYST (a not-for-profit organization to encourage
entrepreneurship amongst Indian youth). Teaching and writing continue to be her
motivation.

Contributors

Yashbir S. Bedi Indian Institute of Integrative Medicine, Jammu, India

Ranjana Bhati Department of Microbiology, Bundelkhand University, Jhansi,
Uttar Pradesh, India
Sohinee Bhattacharyya Oregon Health and Science University, Portland, USA

xv
xvi Editor and Contributors

Sumit G. Gandhi Plant Biotechnology Division, CSIR-Indian Institute of

Integrative Medicine, Jammu, India;
Indian Institute of Integrative Medicine, Canal Road, Jammu, India
Satish Kumar Gupta Indian Institute of Integrative Medicine, Jammu, India
Vijay Lakshmi Jamwal Plant Biotechnology Division, CSIR-Indian Institute of
Integrative Medicine, Jammu, India
Nitika Kapoor Plant Biotechnology Division, CSIR-Indian Institute of Integrative
Medicine, Jammu, India
Inshad Ali Khan Indian Institute of Integrative Medicine, Jammu, India
Harsh Kumar Regional Centre for Biotechnology, NCR Biotech Science Cluster,
Faridabad, Haryana, India;
Manipal Academy of Higher Education, Manipal, Karnataka, India
Durga Prasad Mindala Indian Institute of Integrative Medicine, Jammu, India
Anindit Mukherjee Oregon State University, Corvallis, USA
Srividhya Ravichandran Department of Biotechnology, Indian Institute of
Technology, Chennai, India
Arpita Saxena AAPT Foundation, SPAK megAcorp, Aurangabad, India
Manish R. Shukla Research and Development Center, Reliance Industries
Limited, Navi Mumbai, India
Gaurav Verma Clinical Research Center, Lunds University, Malmo, Sweden
Tabassum Zafar Department of Bioscience, Barkatullah University, Bhopal,
India
Why Biotechnology?
Biotechnology: Discoveries and Their
Applications in Societal Welfare

Harsh Kumar

Abstract The present chapter describes the field of biotechnology from earliest
known mention till modern-day developments. The introduction throws light on
understanding the practice of using microorganisms for making food since prehistoric
times and covers aspects on key discoveries in recent past like vaccines and drugs. The
section journeys through important seminal contributions that laid the foundation of
many branches of biotechnology which would eventually be applied for the benefit
of the population. The gradual advancement in the field of knowledge about life,
its complexities and processes governing these led to the generation of medicines,
diagnostic tests, industrially important materials and environmentally sustainable
products in future years. Biotechnology can be classified based on the broad spectrum
of deliverables it caters to the society. Starting with the healthcare, which constitutes
red biotechnology, the text details significant discoveries and inventions that have
greatly enhanced biomedical research. Green biotechnology, mentions important
methods which have been applied in the field of crop and livestock improvement to
ensure food security. Blue biotechnology, an emerging area enlists and highlights
important marine genetic resources which are being adapted for various demands
of global economy. Finally, the last section details the application of biotechnology
in the field of industry and environment for the generation of better raw materials
and its clean-up, respectively.

Keywords Biotechnology · Health care · Gene · Agriculture · GM · Marine ·

Drugs · Environment · Industry

H. Kumar (B)
Regional Centre for Biotechnology, NCR Biotech Science Cluster, Faridabad, Haryana 121001,
India
e-mail: [email protected]
Manipal Academy of Higher Education, Manipal, Karnataka 576104, India

© Springer Nature Switzerland AG 2020 3

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_1
4 H. Kumar

1 Introduction

Biotechnology is the integrated use of biochemistry, microbiology, and engineering sciences

in order to achieve technological (industrial) application of the capabilities of microor-
ganisms, cultured tissue cells and parts thereof. (European Federation of Biotechnology
1981)
Biotechnology means any technological application that uses biological systems, living
organisms, or derivatives thereof, to make or modify products or processes for specific use.
(Convention on Biological Diversity, United Nations 1992)

The term biotechnology has been defined in different ways by people and organiza-
tions. Scientific community by and large agrees that the science at its core involves
the usage of other life forms for human betterment. Since time immemorial, peo-
ple across various cultures have been using living organisms as food or medicine
either in their natural or processed forms. These included whole microbes or parts
of plants, animals or their derived products. Processing of raw food items required
more elaborate methods which employed whole organisms, e.g. yeast. The technique
of fermentation in preparation of edible items (e.g. bread, cheese, curd, wine, etc.)
was a traditional knowledge across various civilizations. Since 6000 B.C., herdsmen
of Central Asia knew the art of curd/yogurt making by carrying the milk in animal
stomachs. Around the same time, the people of Babylon and Sumer (present-day
Iraq) had the know-how of wine making by utilizing yeast. During 4000 B.C., peo-
ple of ancient Egypt employed yeast in bread making (Bud 1993). Chinese in 500
B.C. used poultices made up of curds having growth of moulds in it as a source of
antibiotics for curing boils, and farmers in Ukraine applied cheese moulds on wounds
to cure infection. At around 470 B.C., Greek philosopher Socrates expounded the
observation of shared features between the parents and their offspring. Another Greek
philosopher Aristotle in around 300 B.C. stated that children inherit traits from their
father (Daston and Lunbeck 2011).
Life in primitive times was principally dependent on agriculture and livestock.
Man began collecting wild plants for consumption since 7000 B.C. First organized
method of agriculture dates back to 3000 B.C. in China where crop rotation was prac-
ticed. Since then, man started observing plants closely. Theophrastus (322 B.C.) men-
tioned about the diseases affecting crop plants and pointed out bad air and nutrition as
their cause (Singh 2018). Domestication of crop plants was a significant achievement
as it ensured food security by preserving the seeds for next seasonal round of sowing.
Animals that were domesticated for the first time were mainly cattle, sheep and goats
as they helped in the cultivation of crops and also provided milk and meat. To carry
forward the generation of animal, people began paying attention to animal breeding
methods whereby they learned simple mating process that helped in propagation
of livestock. Although, people at that time were unaware of the causative agent(s)
behind these processes, yet they possessed experiential knowledge. As the thought
process evolved, people began to provide a more rational explanation for biologi-
cal phenomena they observed. Centuries later, from 1700 A.D. onwards numerous
milestone discoveries were made that led to the improvement in our understanding
Biotechnology: Discoveries and Their Applications in Societal … 5

of heredity, causes and cures of diseases, better agricultural practices, etc. Impor-
tant contributions in the field of basic biology expanded the horizon of knowledge
leading to identification of their translational potential (Fig. 1). Many of the older
practiced methods were better explained later when people began scientific quest to
Understand natural processes. For example, fermentation was explained by Louis
Pasteur who in multiple experiments demonstrated bacteria caused souring of milk
due to lactic acid formation (Pasteur 1879). Around the same time, Edward Jenner’s
discovery of smallpox vaccine was based on the observation of acquired immunity
and heralded a new era of immunization-based preventive medicine (Riedel 2005).
The curing of the wound infection was achieved through curd or cheese moulds in
prehistoric times without knowing that poultices made up of them contained antibi-
otics. Alexander Fleming (1928) purified penicillin antibiotic from the moulds and
demonstrated its curing abilities of bacterial infections. The discovery is considered
to be one of the greatest scientific achievements in post World War II era as it was
able to save countless lives (Aldridge 1999).

Fig. 1 Brief timeline of events in the development of biotechnology

6 H. Kumar

2 Historical Biotechnology—1700s Onwards

This section describes selected milestone inventions and discoveries that influenced
the society in the much needed way in those times. Hunger and disease have been
the major challenges before the society since ever. The population was experiencing
fatalities caused by several infectious diseases during the eighteenth and nineteenth
centuries, e.g. smallpox, tuberculosis, rabies, etc. For most of the diseases, their
causative agents were unknown at that time. Identification of disease pathogens and
preventive immunization were major scientific breakthroughs of those times which
greatly improved public health and increased life expectancy.

2.1 Smallpox Vaccine

Smallpox was the deadliest of all diseases of classical times that had the potential
to decimate vast majority of population as can be read in history. Edward Jenner, an
English physician, got the idea of developing a crude vaccine after observing that
milkmaids working on a cow farm were immune to smallpox virus. This made him
reason that may be the exposure of milkmaids to cowpox made them resistant to the
smallpox virus and therefore such a challenge to a healthy individual may lead to a
similar outcome. Jenner liked to experiment this idea for which he inoculated pus
taken from a milkmaid suffering from cowpox into the arm of a boy named James
Phipps who was found immune many days later. The observations were submitted to
the Royal Society in 1797 (Plotkin 2014). Jenner established the field of vaccinology
which has now become one of the exciting areas of biotechnology research with
great commercial potential.

Edward Jenner. Photo

Courtesy of the History
of Medicine Division at
the U.S. National Library
of Medicine
Biotechnology: Discoveries and Their Applications in Societal … 7

2.2 Rabies Vaccine and Germ Theory of Disease

In 1885, another vaccine, this time against rabies was developed by Louis Pasteur and
Emile Roux. Rabies developed as a result of bites from the dogs that were carrying
rabies virus in their saliva. The virus is known to attack central nervous system that
triggers encephalitis and kills the infected person. The vaccine was a great boon for
the society because until then all rabies infections led to death (Hicks et al. 2012). The
modern understanding of diseases came in the late nineteenth century from the studies
done by Louis Pasteur and Robert Koch. Pasteur contributed to the development
of “germ theory of diseases” which Koch later extended through his findings. Germ
includes any microscopic pathogen like bacteria, virus and protists. The theory states
that infectious diseases are caused by the growth and multiplication of germs after
they invade human or animal body. Pasteur discovered the theory while examining
a patient with puerperal fever whose blood was infected with pyogenic vibrio and
suggested the use of boric acid as a potent and safe antiseptic to prevent the growth
of germs (Ernst 2014). Germ theory of disease is still acceptable in medical sciences
and has greatly improved the understanding of disease aetiology which has led to
many preventive strategies and therapeutic interventions.

2.3 Koch’s Postulates

Dr. Robert Koch, a German physician, in 1882, put forth his famous theory of estab-
lishing a link between the disease and the pathogen. He synthesized four important
parameters from his studies on tuberculosis (TB) that became gold standard for judg-
ing a pathogen for its role in any disease. It must be noted that contemporary research
on tuberculosis was unable to identify pathogen causing the disease, and Koch’s pos-
tulates came out from the studies in which Robert Koch systematically showed the
presence of the stained bacillus in the tuberculous tissue. Following are the Koch’s
postulates:
(1) The microorganism must be found in abundance in all organisms suffering from
the disease, but should not be found in healthy organisms.
(2) The microorganism must be isolated from a diseased organism and grown in
pure culture.
(3) The cultured microorganism should cause disease when introduced into a
healthy organism.
(4) The microorganism must be reisolated from the inoculated, diseased experi-
mental host and identified as being identical to the original specific causative
agent.
Until now, TB was wreaking havoc leading to mass deaths due to its spread; there-
fore, in the light of Koch’s findings, public was provided better treatment and care.
TB sanatoria were built where the patients were kept in isolation to prevent further
8 H. Kumar

transmission (Walker et al. 2006). Koch was awarded Nobel Prize for Medicine and
Physiology in 1905 in recognition of his contribution.

2.4 Fermentation Theory

Besides working in the disease biology, Pasteur also demonstrated for the first time
the scientific basis of microbial fermentation process by propounding “fermenta-
tion theory” (1857). The theory rejected the idea of spontaneous creation and was
later itself replaced by the germ theory of disease. It was perhaps the only scien-
tific discoveries of its times that had the potential of industrial scalability. Through
systematic experiments, Pasteur showed the role of yeast in fermentation process
of ethanol production while also giving terms like “aerobic and anaerobic” (Barnett
2003). Fermentation till today continues to be one of the major areas of application of
biotechnology on which many food, chemical and drug industries thrive. There are
numerous health benefits of consuming fermented food products. Besides improv-
ing digestion, fermented edible items have also been shown to boost immunity and
relieve from stress. Fermentation is also used to manufacture important industrial
chemicals like ethanol, acetic acid; enzymes like cellulase for use in paper and pulp
industry; antibiotic like penicillin by culturing fungus Penicillium notatum on a large
scale (Parvez et al. 2006).

2.5 Penicillin Discovery

Professor Alexander Flemming (1928), a Scottish physician is credited with the dis-
covery of first true antibiotic penicillin having a broad spectrum bactericidal activity.
The antibiotic was discovered as an observation of inhibited bacterial growth on
a plate having mould (P. notatum) growing. The “mould juice” was the antibiotic
secreted out by the fungus which was effective in killing wide range of bacteria
like Staphylococcus (causes abscess), Meningococcus (causes meningitis and sep-
sis), Streptococcus (causes pneumonia) and diphtheria bacilli (causes diphtheria)
(Fig. 2). The antibiotic proved to be a massive life saver in post World War II era to
treat wounded soldiers and general public. An otherwise wound infection that led to
the development of abscess and subsequent gangrene could now be effectively treated
and cured (Gaynes 2017). Fleming eventually received Nobel Prize for Physiology
or Medicine in 1945.
Biotechnology: Discoveries and Their Applications in Societal … 9

Fig. 2 Schematic showing

inhibitory effect of
penicillium mould on
growing bacteria

Prof. Alexander Fleming. Photo

Courtesy of the History of
Medicine Division at the U.S.
National Library of Medicine

2.6 Cell Theory and Laws of Heredity

Observing cells for the first time under a microscope was a major breakthrough
that totally revolutionized biological research in a way that was going to impact
human life in years to come. Zacharias Jansen and his father Hans are credited with
inventing the first compound microscope in 1590 (Helden 2010). It was Antonie
Philips van Leeuwenhoek who for the first time saw living cells: bacteria, protists,
blood cells rotifers, etc. (Lane 2015). Thereafter, some of the most path-breaking
reports were published at successive intervals that included the discovery of nucleus
by Robert Brown (1831), formulation of “cell theory” by Matthias Schleiden and
Theodore Schwann (1839) and addition of new dimension to cell theory by stating
“Omnis cellula e cellula: all cells arise from pre-existing cells”; by Rudolf Ludwig
Carl Virchow (published in Cellular Pathology, 1858) that greatly enhanced our
understanding of basic functioning of cellular physiology (Kuiper 2010).
Contemporary discoveries of laws of heredity/inheritance by Gregor Johann
Mendel (1866; considered to be father of modern genetics) helped in understand-
ing the transmission of traits from one generation to next. The work of Mendel
was largely rediscovered by Hugo de Vries, Erich von Tschermak, Carl Correns and
William Jasper Spillman. Through simple crossing experiments performed in garden
10 H. Kumar

pea (Pisum sativum) plant, Mendel was able to conclude the presence of “dominant”
and “recessive” traits. What he observed was, when two pure-bred varieties of pea
plant (having tall and short traits) were crossed, then the second generation progeny
had a mix of population: two were tall and short and remaining two were hybrids
(Gros 1992). Initially, Mendel designated the traits as “factors”; however, it was
Wilhelm Johannsen (1909) who introduced the term “gene” and William Bateson,
the word “genetics” (1905) (Gerstein et al. 2007).

2.7 The Term “Biotechnology”

Karl Ereky, a Hungarian agriculture engineer coined the term biotechnologie in 1919
to explain the biological method of converting raw materials into useful products.
He wrote a book titled “Biotechnology of Meat, Fat and Milk Production in an
Agricultural Large-Scale Farm” in which he expressed his views on solving the
problem of food crisis (Fiechter 2000).

2.8 Identification of DNA as Genetic Material

Present-day biotechnology revolves around DNA/RNA or protein-based science and

its uses. However, to even make use of these molecules for societal application, a
detailed and thorough knowledge about them was lacking in the first half of the
twentieth century. Frederick Griffith, a British bacteriologist, in 1928 demonstrated
the importance of transforming principle or the genetic material for the process
of bacterial transformation (Lorenz and Wackernagel 1994). Later, in 1944, Avery,
McLeod and McCarty showed DNA to be the real transforming principle. In 1953,
James D. Watson and Francis H. C. Crick showed the double helix structure of
deoxyribonucleic acid (DNA) for which they received the Nobel Prize in Medicine
or Physiology (1962) (Crick and Watson 1954; Daston and Lunbeck 2011). With
the basic knowledge of DNA, further deep studies in molecular biology became
possible which led to the foundation of recombinant DNA technology and genetic
engineering.

3 Modern Biotechnology—1970s Onwards

Nirenberg and Matthaei (1961) are credited with the discovery of the genetic code
which helped in revealing the information contained in the genes. The triplet arrange-
ment of bases in the mRNA (codons) is matched with the respective anticodons
present on the tRNA which carries particular amino acid. During the process of
mRNA translation, the codons direct the amino acids they encode to be incorporated
Biotechnology: Discoveries and Their Applications in Societal … 11

in the growing nascent polypeptide chain. The genetic code was further completed
and presented as a table by the efforts of Har Gobind Khorana and Robert Holley.
The code was found to be degenerated (64 codons encoding 20 amino acids) which
meant simply that many codons encode the same amino acid. Nirenberg, Khorana
and Holley shared the Nobel Prize in Physiology or Medicine in 1968 (Landmarks
2009).
Werner Arber and Matthew Messelson (1962) discovered “restriction endonu-
cleases” or restriction enzymes that possessed the property of recognizing certain
inverted repeat sequences (palindromic sequences) in a plasmid and digest it specifi-
cally. The plasmid was a covalently closed circular form of DNA that contained sites
for many restriction enzymes called multiple cloning sites (MCS) which allowed
incorporation of any gene. Nathans (1971) demonstrated the digestion of the phage
DNA of simian virus 40 and resolving of the digested fragments by gel electrophore-
sis. These findings paved the new exciting area to work with DNA molecule and
tune it to do molecular cloning that involved creating an exact replica of a gene (gene
cloning). By this time, the central dogma of molecular biology was already in place
that dictated the synthesis of protein to be guided by the sequence contained in the
gene. People then began exploring the feasibility of cloning the genes that encoded
for necessary proteins and began expressing them in suitable host systems (Arber
and Linn 1969; Danna and Nathans 1971). The clone (plasmid carrying the gene
of interest) created in this manner was called a recombinant. The work extended
towards the production of recombinantly expressed proteins on an industrial scale,
and thus several agricultural, pharmaceutical and other companies jumped into the
fray of commercialization, patenting, licensing and mass production of them.

3.1 Hybridoma Technology

Cesar Milstein and Georges J. F. Kohler (1975) invented the hybridoma cells that were
capable of synthesizing monoclonal antibodies (mAbs). The purification of single
epitope recognizing antibody from the vast repertoire produced by the immune cells
had been a challenge before the investigators. Milstein and Kohler fused a plasma
B cell (secretes antibodies) with the myeloma cell (cancerous B cells) with the help
of sendai virus and created what is called a hybridoma that secreted antibody recog-
nizing single epitope. The antibody thus produced was monoclonal unlike the con-
ventional polyclonal ones that recognized several epitopes (Fig. 3). The hybridoma
was transplanted in mice peritoneum where it led to tumour growth and secretion of
vast amount of mAb in ascites fluid (Milstein 1999). Milstein and Kohler got Nobel
Prize for Medicine and Physiology in the year 1984 for the revolutionizing impact
their study had on immunology. With the availability of mAbs, it became possible to
selectively identify many tumour cells possessing unique antigens, their separation
and subsequent purification. Today, mAbs are used extensively in biomedical and
biotechnology research for immunodiagnostics of cancer and its immunotherapy;
12 H. Kumar

Fig. 3 Schematic illustrating the multiple versus single epitope recognition by a polyclonal antibody
and hybridoma-derived monoclonal antibody, respectively

identification of infectious pathogens; affinity purification of single protein from a

complex mixture, etc.

3.2 Insulin: The First Commercial Recombinant Product

Insulin is a hormone which is responsible for glucose metabolism in the body.

Impaired levels of insulin are a hallmark of type 1 diabetes (T1D). The beta cells of
islet of langerhans of pancreas produce insufficient or no insulin due to the autoim-
mune triggered death. Consequential to which the person affected manifests hyper-
glycaemia (increased blood sugar levels) symptoms. The patients of T1D are adminis-
tered insulin by subcutaneous injection (Chiang et al. 2014; CDC 2017). The number
of diabetics has increased from 108 million (in 1980) to 422 million (in 2014). It
is one of the major causes of renal failure, blindness, cardiac attacks, strokes, etc.
(WHO 2018). Herbert Boyer and Robert A. Swanson together founded Genentech,
Inc. (headquartered in California, USA; now a subsidiary of Roche) in 1976 which
became the first biotechnology-based company. Boyer and Swanson entered into the
contract agreement to manufacture recombinant human insulin that was until now
obtained from pigs. Boyer took the responsibility of scientific aspects of design, exe-
cution, purification, etc., while Swanson took care of the finances and legal aspects.
The company came out with recombinant human insulin (expressed in and purified
from Escherichia coli; Fig. 4) in 1978. It was licensed to Eli Lilly and Co. (to be sold
as Humulin) and was approved by Food and Drug Administration, USA, in 1982 (US
F&DA 1982; Hughes 2011).
Biotechnology: Discoveries and Their Applications in Societal … 13

Fig. 4 Schematic depicting the workflow of recombinant human insulin production

Since the original production by Eli Lilly and Co. (Indiana, US); Sanofi (Paris,
France) and NovoNordisk (Copenhagen, Denmark) have also come up with their
recombinant insulin products (named Insuman and Novolin, respectively) (Landgraf
and Sandow 2016). Today, insulin and its derivatives enjoy global sales of over $4.5
billion. The technology of insulin production has moved ahead and is now produced
in human cells besides yeast and other suitable expression systems for increased
yield and better efficacy (Walsh 2005). Arabidopsis thaliana, lettuce and tobacco
plants have been engineered for human insulin production and have been successful
(Nykiforuk et al. 2006; Boyhan and Daniell 2011).

3.3 Human Growth Hormone (hGH) by Genentech

The human growth hormone (hGH or somatotropin) is released by the anterior pitu-
itary gland. It is a protein composed of 191 amino acids and triggers cell reproduction,
metabolism and overall body growth. Choh Hao Li, a US biochemist of Chinese origin
synthesized and purified hGH for the first time (Cole 1996). Thereafter, Genentech
pioneered its commercial production using the recombinant DNA approach and sold
under the trade name Protropin (1985). The product got discontinued and is now sold
as Nutropin since 1993 (Genentech 1993). Besides Genentech, there are other phar-
maceutical companies selling recombinant hGH like Pfizer, Roche and NovoNordisk
under various trade names. The hormone is administered subcutaneously to children
and adults suffering with chronic kidney disease (CKD), Turner’s syndrome and
growth hormone deficiencies (GHD). The global market sales of hGH are estimated
to be generating revenues worth $5261 million by 2026 (TMR 2018).
14 H. Kumar

3.4 Polymerase Chain Reaction (PCR)

Kary Mullis (1983) developed a method for exponential amplification of a piece of

DNA using sequence-specific oligonucleotide primer and DNA polymerase (Fig. 5).
The process was carried out in a specialized machine (thermal cycler) capable of
rapid temperature switching (called thermal cycling). The method is now indispens-
ably used in every basic and medical research laboratory for specific amplification
or cloning of genes or any nucleotide sequence. The method proved to be a path-
breaking invention in the field of biotechnology which made exact identification of
any bacteria, virus, cell type or any gene possible. PCR had an immense impact on
state-of-the-art methodologies of molecular biology practised worldwide. Mullis was
awarded Nobel Prize in Chemistry in 1993. The PCR technique had direct application
in the field of gene cloning, site-directed mutagenesis, parentage identification, pre-
natal sex determination, pathogen identification, DNA matching from crime scene
sample (e.g. blood, semen, hair, skin, etc.), tumour cell testing, etc. For many infec-
tious diseases, cancer stage identification or legal dispute of biological parentage,
PCR has now become a standard method of diagnosis and investigation (Bartlett and
Stirling 2003).

3.5 The Era of Genomics and Proteomics

Modern times is witnessing tremendous ramification of biotechnology in health care,

agriculture and environment sectors. The wealth of data that has been generated by
aforementioned discoveries is stored, annotated, analysed and interpreted in vari-
ous ways. This enormous task has generated an entirely new arm of biotechnology
called bioniformatics. The in silico approach towards solving biotechnology-based
questions have been applied in the fields of genetics, molecular biology and protein
science. The classical genetics approach aimed at studying one or few “genes to
phenotype” on a case to case basis; whereas, genomics includes the entire genome
characterization in one go. Whole genome sequencing, single nucleotide polymor-
phism identification, screening of chemical library for fishing out the genomic tar-
gets, creation of genomic libraries, etc., all together constitute genomics. The similar
approach when applied to study the total RNA population dictated by the genomic

Fig. 5 Diagrammatic representation of the concept of polymerase chain reaction (PCR)

Biotechnology: Discoveries and Their Applications in Societal … 15

parent is known as transcriptomics which includes whole transcriptome profiling,

RNA sequencing, microarrays-based gene expression analysis and their validation.
The idea of sequencing the entire human genome was conceived at National Insti-
tutes of Health, USA, (1984) from where the original funding for implementation
of the idea came. The official launch commenced in 1990 and was termed complete
in 2003. A parallel effort was undertaken by a company known as Celera Genomics
which was spearheaded by J. Craig Venter. Currently, 92% of human genome stands
sequenced which corresponds to euchromatic area; whereas, remaining heterochro-
matic region still awaits the process. The sequence-related information is stored
in the publicly accessible databases like GenBank at NCBI (National Centre for
Biotechnology Information, NIH, USA), DDBJ (DNA Database of Japan, National
Institutes of Genetics, Shizuoka, Japan) and EMBL (European Molecular Biology
Laboratory, Heidelberg, Germany). The three databases frequently exchange infor-
mation with each other. The information generated by the human genome sequencing
was hailed as a historic milestone in the field of biology and medicine. It was envis-
aged as a tool to identify many oncogenes, developmental disorders-related genes,
identification of novel drug targets, throw light on understanding bacterial and viral
pathogenesis, etc. (Collins et al. 2007). The information contained in the genome is
eventually “executed” by the proteome (the sum total of all the proteins present in the
cell). The science aimed at studying and a characterization of the proteome is called
as proteomics. The term has virtually replaced the phrase protein science. Nowa-
days, any attempt either to study a single or complex of proteins is included in the
term proteomics. Present-day state-of-the-art proteomic methodologies use protein
purification strategies, 2-D gel electrophoresis, mass spectrometry analysis, peptide
microarrays, Fourier resonance energy transfer (FRET), etc., to identify and charac-
terize the function of proteins (Anderson and Anderson 1998). The protein-related
information has been stored in free databases like UniProt Consortium compris-
ing EBI (European Bioinformatics Institute, Hinxton, UK), SIB (Swiss Institute of
Bioinformatics, Lausanne, Switzerland) and PIR (Protein Information Resources,
Georgetown University, Washington, US) (Wu et al. 2002).

3.6 Metabolomics

The sum total of all the metabolites produced in the cell constitutes the metabolome
of the cell. The science involving the study of the metabolome profile of cell or
individual is known as metabolomics. The subject matter differs from genomics and
proteomics in a way that it studies the actual processes that proteins (which are in
turn dictated by the genome) are doing as end products. In this way, it rises above
the molecules and takes the investigation to the “systems biology” level. Metabolome
directly tells the fate of the cellular processes working in the cell. The study stems
from background literature where clinicians or investigators used to analyse the
presence of certain compounds in body fluids by the means available in those times.
For example, physicians in ancient China employed ants to check the presence of
16 H. Kumar

glucose in diabetes patients. Nowadays, modern techniques like gas chromatogra-

phy–mass spectrometry (GC–MS) and nuclear magnetic resonance (NMR) are used
for identification of secondary metabolites in the biological samples (Sussulini 2017).
METLIN (2005) was the first database of human metabolome that is maintained by
the Scripps Research Institute (La Jolla, USA). It contains the mass spectrometry data
(like molecular mass, structure and formulae) of the metabolites (Smith et al. 2005).
A human metabolome repository is maintained at University of Alberta, Canada,
named Human Metabolome Database (HMDB) that contains information of human
body metabolites derived from NMR and GC/LC-MS analyses. At present, it is the
most comprehensive and updated metabolomics database that is widely accessed
(Wishart et al. 2007).

4 Areas/Branches of Biotechnology

Biotechnology has been classified in multiple ways. Recently, there was a colour-
based rainbow-coding of various arms of biotechnology to provide a more holistic
view. These colours comprised red, green, blue and white. Each colour specified the
particular areas of application of biotechnology under its ambit (Fig. 6), e.g. red was

Fig. 6 Schematic description of the branches of biotechnology

Biotechnology: Discoveries and Their Applications in Societal … 17

constituted by biotech-based biomedical research; agriculture-oriented application

was included in green; biotechnological application in marine or aquatic life forms
and their usage comprised the blue and that pertaining to industry and environment
was termed as white biotechnology (Frazzetto 2003; DaSilva 2004; Kafarski 2012).
This section briefly discusses some of the recently emerging representative examples
from such categories and highlights their significance in the improvement of lives
on earth and its environment.

4.1 Red Biotechnology

The biotechnological inventions and discoveries pertaining to biomedical research

or animal health have been collectively included in the umbrella term red biotechnol-
ogy. It comprises significant developments in the field of stem cell biology, embryo
manipulation, transgenic animals (with better nutritive or commercial value; like
high milk-yielding cattle), biopharmaceuticals (recombinant vaccines, hormones and
other therapeutic proteins production), forensics (DNA fingerprinting), genetic inter-
ventions like gene editing and gene therapy, disease diagnostics (pathogen identi-
fication, oncogene examination) and personalized medicine (SNPs-related disease
predictions in cancers, infection and metabolic disorders).

4.1.1 Genetic Techniques

• Recombinant DNA technology for production of vaccines

“Prevention is better than cure”—Desiderius Erasmus

Perhaps, the aforementioned statement best describes the significance of vaccines

in saving lives from diseases. Ever since Jenner laid the foundation of the field,
vaccinology has come a long way from use of live-attenuated or heat-killed vaccines,
to recombinantly expressed or the plasmid-based ones. In classical times, infectious
virus was passaged for several generations in cell culture to get it mutated, and
upon introducing back into the original host it was unable to infect yet elicit an
immune response. In this way, it was said to have become “attenuated”. Examples
of live-attenuated vaccines include polio, rotavirus, measles and H1N1 flu vaccines.
Usage of chemicals like formaldehyde or heat to reduce their virulence resulted in
inactivated or killed vaccines, e.g. cholera, typhoid or influenza vaccines (Benn et al.
2013).
The recombinant approach applied to manufacture vaccines is twofold: one is
called the vectored approach whereby antigenic regions of the pathogen are deliv-
ered to patients via bacterial or viral vectors; second approach involves heterologous
expression of antigen encoding genes in yeast systems like Saccharomyces cerevisiae
or Pichia pastoris or mammalian and insect cell lines and large-scale purification of
18 H. Kumar

the recombinant protein thereafter. The first approach is easy to handle and employs
bacterial or viral vector for expressing genes encoding antigenic regions obtained
from a wide variety of pathogens. Intramuscularly injected antigens use several
viral or bacterial vectors like adenovirus (Ad5 serotype), adeno-associated viruses,
recombinant Mycobacterium bovis BCG (rBCG strain), Salmonella (Shata et al.
2000; Rollier et al. 2011) (Fig. 7b). For antigens requiring post-translational mod-
ifications, the first approach proves to be inadequate and therefore use of yeast or
mammalian and insect cell lines becomes indispensable. For this, the antigen encod-
ing genes (e.g. Hepatitis B surface antigen) is cloned and expressed in yeast (e.g.
S. cerevisiae) and cultured in fermenters on a large scale. The recombinant protein
assembles in virus-like particles (called VLPs) which are secreted by yeast in the
external medium from where it is purified (Fig. 7a). Glaxo Smithkline Plc., Lon-
don, manufactures hepatitis B vaccine via this approach which is directed against its
surface antigen under the brand name Engerix-B (Keating and Noble 2003).
A third category of recombinantly administered vaccines comprises DNA vac-
cines. The antigen encoding region is expressed in a vector that contains a bacterial
origin of replication, a strong viral promoter (e.g. cytomegalovirus, CMV), an MCS
region for cloning of the segment and an antibiotic selection marker. Modes of deliv-
ery of such vaccines have been intramuscular injection or via gene guns (Fig. 7c).
The DNA is adsorbed on gold particles and bombarded on the localized site of
action. The idea is to transfer the construct directly in the cells where it expresses
and mimics the condition of natural infection. The method has been used for vac-
cination against TB, leishmaniasis, influenza, HIV, etc. (Yang et al. 1990; Oliveira
et al. 1999). Improved methods now incorporate changes made in the construct so as
to prevent its degradation inside the cells upon entry or to simultaneously co-express
inflammatory cytokines to heighten the immune response (Belakova et al. 2007).
• Gene editing: CRISPR-Cas (clustered regularly interspaced short palindromic
repeats—CRISPR associated)
Originally discovered as an immune system of bacteria conferring resistance against
the invading viral DNA, the technology of CRISPR has been adapted into a toolkit for
genome editing. The acronym describes a particular stretch of repetitive nucleotide
sequences in the bacterial DNA that became part of the genome because of a previous
viral infection. The sequences are capable of degrading viral DNA in case of a future
infection by the same or similar DNA virus thus conferring immunity to the bacteria
against viral attack. The repeat sequences were discovered in 1987 in E. coli by group
of investigators in Osaka University, Japan. The group noticed the unusual feature
of the repeats which was the presence of interrupting nucleotides between them;
however, they could not assign any function to them (Ishino et al. 1987). The acronym
CRISPR was given in 2002 by a group that published bacterial genomic loci which
harboured interspaced repeat sequences (Jansen et al. 2002). Thereafter, a surge of
reports describing the phenomenon was observed in the scientific community which
not only established it as a novel form of natural bacterial defence mechanism but
also opened the possibility of applying the method for artificial genome editing. The
Biotechnology: Discoveries and Their Applications in Societal … 19

Fig. 7 Schematic describing types of recombinant vaccines. a An example of recombinant HbSAg

vaccine. Recombinant vaccine administration. b Adenovirus (Ad5) mediated recombinant vaccine
delivery. c Gene gun driven
20 H. Kumar

technology holds immense possibility to correct genetic mutations causing diseases,

genetically engineer crops or livestock of desired traits in a cheap and precise manner.
Simply speaking, whenever a DNA virus attacks a bacteria, then bacterial Cas
enzyme captures spacer sequences from the invading DNA and integrates into the
CRISPR loci in the bacterial genome in arrays. This array encodes an RNA transcript
which matures through different types of CRISPR pathways (I, II and III). In type
I pathway, the premature or precursor form of CRISPR-RNA or crRNA is cleaved
by CRISPR-associated RNAses leading to multiple crRNAs. The type III system
employs an unknown RNAse to generated final mature crRNA. The type II system
which has been adapted for artificial genome editing (with some modifications)
utilizes another trans-activating RNA or tracrRNA which hybridizes with the crRNA
and together with Cas endonucleases binds and cuts the target DNA by introducing
DSBs (Hsu et al. 2014). The three components, namely crRNA, tracrRNA and Cas9
endonuclease together constitute what is called as CRISPR system which is capable
of performing its function even in vitro (Gasiunas et al. 2012; Jinek et al. 2012). For
artificial purposes, the former two components have been combined, and a single
guide RNA or sgRNA has been used successfully (Jinek et al. 2012). These findings
have paved the way of translating the basic knowledge of this phenomenon into
useful biomedical and agricultural applications.
In health care, the method has been applied to correct the genetic mutations causing
Duchenne’s muscular dystrophy (DMD), cystic fibrosis, haematological malignan-
cies and HIV pathogenesis. Using the CRISPR technology in mouse model system,
DMD gene expression was rescued by delivering its functional copy through adeno-
associated virus vector. The muscular abilities of mice were found to be partially
restored as compared to the control group exhibiting degenerating muscle condi-
tions (Tabebordbar et al. 2016). The genome of HIV has been targeted to stop its
replication in the infected cells without any toxicity. The achievement could be
viewed as a significant advance over the currently practised method to contain or
stop viral replication in preclinical systems (Hu et al. 2014). In a parallel study, the
CXCR4 receptor of HIV was removed successfully from human T cells using the
CRISPR system; a feat which could be mobilized vertically for edited bone marrow
transplants from patients suffering from AIDS (Schumann et al. 2015).
There is growing concern about the potential misuse of CRISPR-Cas technol-
ogy, particularly for the purpose of editing the genome of human embryos. Human
embryo manipulation is legal in China and several states of USA. However, there
are clear ethical concerns regarding the reckless use of the technology towards giv-
ing birth to only the ones with modified features. Such changes when introduced
are heritable and are passed on for generations with unpredictable future outcomes.
There has been a case of human embryo editing which attracted worldwide shock and
condemnation. It called for an urgent action against the erring group simultaneous
with rounds of brainstorm sessions held globally to chart out regulation(s) to prevent
misuse of CRISPR technology (Cyranoski and Reardon 2015; Liang et al. 2015).
Nevertheless, the method has invited global interest towards harnessing its potential
in drug development by pharmaceutical companies, improvement of crop traits by
Biotechnology: Discoveries and Their Applications in Societal … 21

agriculture-based industries and general biomedical research towards understanding

of biological processes governing several less characterized disease conditions.
• Gene therapy
The introduction of DNA into cells or organisms for therapeutic benefits is known as
gene therapy. The first idea of gene therapy was proposed by Theodore Friedmann
and Richard Roblin in 1972 by emphasizing on the beneficial aspects of supplying
exogenous DNA to the organism for correcting the genetic defects (Friedmann and
Roblin 1972). There are two types of gene therapy strategies, namely somatic cell
gene therapy and germline gene therapy. Transfer of therapeutic DNA/gene into any
cell other than germ cells or progenitor stem cells is called as somatic cell gene
therapy. Such interventions do not affect the germ cells of the person and as such
results are not heritable (Mavilio and Ferrari 2008). Two modes of delivery of the
therapeutic DNA exists, namely in vivo and ex vivo. The delivery of the DNA directly
into the patient’s or model system’s body is known as in vivo approach; whereas,
transfer of gene into cells isolated from the body and then introducing them back
after ascertaining its stable expression is called as ex vivo mode of gene therapy.
The method of delivery of the DNA is divided into two categories: viral-dependent
vectors and viral-independent vectors. The former employs the use of viruses devoid
of their original genome and instead inserted with the functional copy of the gene
for its delivery either into the body directly or in cultured cells. The latter group
comprises liposome, gene gun, electroporation or dendrimers-based DNA transfer
into the organism or cultured cells. Examples of viruses utilized for gene therapy
include herpes simplex, human immunodeficiency, adeno-associated and vaccinia
viruses (Mavilio and Ferrari 2008).
Adenosine deaminase (ADA) deficiency caused severe combined immunodefi-
ciency (SCID); an autosomal recessive disorder (Hirschhorn et al. 1979) was the first
target of somatic gene therapy in a clinical trial performed by R. Michael Blaese, W.
French Anderson and Kenneth Culver in 1990. It was characterized by the lack of
ADA enzyme which was essential for DNA synthesis. Using viral vector, the gene
encoding the enzyme was delivered to the patient (Culver et al. 1990; Rosenberg
et al. 1990). Haematological disorder like beta-thalassaemia is caused by reduced or
no production of beta globin chain of haemoglobin. Using lentiviral vector-mediated
beta globin gene delivery led to successful production of correct haemoglobin (Sade-
lain 2006). Gendicine was the first gene therapy drug approved for the treatment of
head and neck squamous cell carcinoma in China. The drug enters the cells via
receptor-mediated endocytosis and overexpresses p53 levels (Pearson et al. 2004).
Despite endowed with enormous potential of treating diseases and disorders, gene
therapy has been marred by several challenges that has led to its decline in recent
years. The lack of persistence of effect is the first of all issues that has been slowing
down the pace of gene therapy. The functional copy of the administered gene is lost
with successive cell divisions which lead to reducing effect of the gene therapy. As
a result, the patient needs to get administered the therapy multiple times so as to
maintain the therapeutic effect. Secondly, problem of immune challenge caused by
viral vectors poses a serious impediment towards the usage of such vehicles for gene
22 H. Kumar

delivery. The surface composition and pattern of many viruses elicit major immune
response in the person administered with viral vector-based gene therapy. A second
approach of using non-viral vectors has not met with much success. The low rates
of gene delivery through such approaches are a major obstacle which has prevented
their large-scale testing (Goncalves and Paiva 2017).
• DNA fingerprinting or profiling
The use of unique DNA sequence properties to identify an individual is known
DNA profiling or fingerprinting. The method was originally discovered by Sir Prof.
Alec Jefferys in 1984 at the University of Leicester, UK. The genome between two
unrelated individuals match up to 99% yet the unmatched portion offers enough to
discriminate between them. Professor Jefferys also observed that the fingerprint in a
child comprised half from both the parents. The classical method of fingerprinting
relies on the selective detection of certain unique repetitive DNA sequences called
as variable number tandem repeats (VNTRs) which are present in the genome in the
form of minisatellites and microsatellites (Roewer 2013). The technique involved
extraction of DNA from samples (e.g. blood, hair follicle, semen, nails or other body
parts or secretions) and its restriction digestion and electrophoresis to generate band
fragments on the basis of restriction sites present. The sample was then blotted to
nitrocellulose membrane and repetitive sequences were detected using specific com-
plementary radiolabelled probes. The minisatellites are six to hundred nucleotides
long and repeat for hundreds of times in the genome (Tautz 1993). Microsatellites
(also known as short tandem repeats or STRs) on the other hand are shorter in length
(as the name suggests) ranging from one to five nucleotides repeating for some
hundred times (Koreth et al. 1996).
The aforementioned method has now been replaced by a PCR-based technique
which involves DNA extraction followed by amplification of microsatellite region.
This has greatly improved the workflow of fingerprinting and has added to its rise
in success. The method now is widely used for parentage identification, cell lines
authentication, to check cancer progression but perhaps its greatest application has
been in the field of forensics where it is used for identification of criminal. Owing to
this, now many countries maintain a DNA database of their population with which
the fingerprinting results are compared to find out the real culprit. In the USA,
Coding for DNA Identification System (CODIS) is maintained by Federal Bureau of
Investigation (FBI) for the identification of criminals by using DNA fingerprinting
(Saad 2005).
The method also revealed through arbitrarily primed-PCR (AP-PCR), the muta-
tions in the microsatellite regions of cancer tissues and established the process of a
new mechanism of carcinogenesis. Since then fingerprinting has been successfully
used for identification of cancer mutations in the microsatellites thereby allowing to
identify the stages of progression (Perucho 1996). In an earlier study, 46 different
cell lines (including cancer cells) were authenticated by using DNA fingerprinting
of minisatellites (Gilbert et al. 1990). The case of disputed paternity is now routinely
solved based on DNA profiling as mandated by the courts worldwide. As mentioned
Biotechnology: Discoveries and Their Applications in Societal … 23

earlier, based on the shared fingerprint of the VNTRs in the child and the suspected
person, it is possible to nail down the biological father.

4.1.2 Embryological/Organismal Manipulation

• Somatic cell nuclear transfer (SCNT)

The technique involves transfer of a nucleus from donor cell to enucleated egg cell
or ovum and allowing it to develop into an embryo. After many rounds of mitotic
divisions, the embryo reaches blastula stage where it develops inner cell mass (ICM)
containing embryonic stem cells (ESCs). From here, as the need demands the embryo
can be implanted into a female animal if reproductive cloning is the objective or
for therapeutic cloning, the ESCs can be extracted out and can be used for tissue
regeneration by harnessing their pluripotent nature (Fig. 8) (McLaren 2000). Sir
Hans Spemann (1928), a German embryologist, is credited with the discovery of
the then embryonic induction, an idea considered to be the predecessor of modern-
day SCNT. Through microsurgical needle, Spemann and his colleagues inserted a
particular region of an embryo into another embryo which led to development of a
new embryonic growth irrespective of the area where it was transplanted (Spemann
1938). Dolly (sheep) was the first organism born out of reproductive cloning through
SCNT method in 1997 at Roslin Institute, The University of Edinburgh, UK. The
investigators incorporated a nucleus from an adult cell into an enucleated ovum and
allowed to develop the zygote till blastula stage of embryogenesis. Later, it was
implanted into a female sheep that served as a surrogate mother for the developing
foetus and gave birth to Dolly (Edwards 1999). Very recently, macaque monkeys

Fig. 8 Schematic depicting somatic cell nuclear transfer

24 H. Kumar

were cloned in China which marked the first successful reproductive cloning using
SCNT in primate species (Liu et al. 2018).
Therapeutic cloning is perhaps the best legacy that SCNT has left behind for the
welfare of mankind in general. The power of generating blastocyst and extraction
of ICM allowed the investigators to generate multiple tissues and organs of human
body which has direct application in the treatment of several diseases. Researchers
have been successful in generating pancreatic endocrine cells by the application
of SCNT technique which were found to be capable of producing hormones like
insulin, glucagon and ghrelin. An advancement like this holds immense possibility
towards better treatment of type 1 diabetes in which insulin secretion is compro-
mised (D’Amour et al. 2006). In another study done in mouse, investigators were
able to regenerate spinal cord using motor neurons from ESCs, an achievement of
medical importance in cases of paralysis (Liang et al. 2006).
• Stem cells and their applications
Stem cells are capable of differentiating into almost any cell type of the body and can
also regenerate themselves. The property by which a stem cell can differentiate into
myriad cell types is known as pluripotency, and such cells are known as pluripotent.
As mentioned in the previous section, the inner cell mass (ICM) of a blastocyst
houses the embryonic stem cells (ESCs) capable of generating all three germ layers
and their cell types (Thomson et al. 1998). Besides the ESCs, there is another method
of generating stem cells which involves reprogramming back a differentiated cell into
a state of stemness. The stem cells thus created are known as induced pluripotent stem
cell (Aoi et al. 2008; Chagastelles and Nardi 2011). Apart from stem cells derived
from the embryo, the bone marrow also contains a population of haematopoietic stem
cells (HSCs) which give rise to all the blood cell types. HSCs have been clinically
used since 1960s (Good et al. 1969) and are now obtained from umbilical cord and
placenta (Kogler et al. 2004).
The clinical uses of stem cells have opened a new avenue of research which has
great potential for offering cure against several diseases. Cardiomyocytes generated
from human ESCs (hESCs) have been successfully transplanted into mouse model
system and were found to restore the beating function of heart (Laflamme et al. 2005).
hESCs incorporated with gene essential for generation of cone photoreceptor cells
have been successfully used for treating retinal pigment epithelial degeneration (Zhou
et al. 2015). Another class of stem cells, known as mesenchymal stem cells (MSCs;
capable of differentiating into cells of mesodermal origin) derived from bone marrow
has been used to generate bladder tissue in baboons thus establishing a non-human
primate model for potential human applications (Sharma et al. 2011). HSCs have been
used for transplantation in patients suffering from various lymphomas, myelomas
and immunodeficiency diseases (Eaves 2015). Prior to administering HSCs to the
patient, the bone marrow is destroyed by high doses of chemotherapeutic drugs with
or without radiotherapy (myeloablation). Thereafter, HSCs obtained from the patient
are injected in the bloodstream from where they are able to reach and replenish the
destroyed tissue. This mode of administering patient’s own HSCs to him or her is
known as autologous as compared to allogenic in which HSCs are obtained from
Biotechnology: Discoveries and Their Applications in Societal … 25

healthy donor with HLA match type (Russell et al. 2000). As pointed out earlier,
mother’s umbilical cord and placenta are two prime organs which are storehouses
of HSCs; therefore, people are now getting their cord blood cells stored in blood
banks. The stem cells derived from such banks can be very helpful in case of future
haematological malignancies, inherited disorders or other genetic diseases.

4.1.3 Molecular Diagnostics

The use of DNA/RNA or protein(s) for medical diagnosis or prognosis of diseases

or disorders is collectively referred as molecular diagnostics. The methods com-
monly utilize the detection of DNA or RNA converted to cDNA through PCR or
hybridization-based techniques. Proteins are generally detected by using antibod-
ies and antibody-dependent techniques like enzyme-linked immunosorbent assay
(ELISA) or flow cytometry in a method called as immunophenotyping (Poste 2001).
The methods are fast, precise and specific as compared to traditional diagnostic
approaches.
• PCR-based detection
Since the inception of PCR by Kary Mullis, the method has evolved into a more
advanced technique which has enabled the detection of DNA or RNA in real time
through a technique called Real-Time or Quantitative PCR (qPCR). qPCR works
on the principle of usual PCR except that the amplified product can be visualized
by using fluorescent dye (e.g. SYBR green) or fluorescent-labelled complementary
probes (TaqMan probes) whose signal rises as the amplified product accumulates
(Kubista et al. 2006). qPCR is of great relevance in the field of molecular and diag-
nostic virology where one can identify viral serotypes in the patient’s body sample.
Since clinical samples are often limited, a PCR-based method of detection proves
to be of immense advantage because the target gene can be amplified exponentially
and can be monitored live. The method has been successfully applied to detect and
quantify causative agents of several infectious diseases; viruses like HIV, hepati-
tis B virus (HBV), herpes simplex virus (HSV) 1 and 2, human papilloma virus
(HPV) in patient’s samples (Valones et al. 2009). For HIV, p24 gene is routinely
used for amplification for detecting the presence of HIV-1 in patient’s dried blood
spots (Lakshmi et al. 2011). All four serotypes of dengue virus (DENV 1–4) are
routinely detected in clinical laboratory using PCR methodology to amplify C and E
genes using serotype-specific primers (Das et al. 2008). HBV has been successfully
detected using TaqMan probe-based qPCR technique in clinical samples (Zhao et al.
2005). For bacterial strain identification, 16S rRNA gene (considered to be universal
reference) of each strain is amplified through PCR. Bacteria like Mycobacterium
tuberculosis and Helicobacter pylori are detected by their sequence-specific primers
against 16S rRNA (Barghouthi 2011). Another target is the outer membrane protein
(OMP) gene routinely used for detection of bacteria belonging to Chlamydia sp.
which primarily causes respiratory illnesses. Many species of Chlamydia have been
known to cause Alzheimer’s, arthritis, atherosclerosis, etc. (Yamamoto 2002).
26 H. Kumar

• FISH (fluorescence in situ hybridization)

The technique of FISH involves hybridization of fluorescently conjugated DNA/RNA
probes with its target genomic region or mRNA. Its analysis requires an imaging sys-
tem like a fluorescence microscope. The method can be performed on cells and fixed
tissues. The samples (cells or fixed tissues) are permeabilized and incubated with
the fluorescently conjugated DNA or RNA probes which then hybridize with their
target. FISH-based diagnostic tests are routinely used for detection of HER2 expres-
sion levels in various cancers (Hicks and Tubbs 2005). FISH probes are widely
used for detecting chromosomal translocations in the case of lymphomas (Tem-
pest et al. 2008). Using the probes, BCR-ABL translocation gene has been success-
fully detected in peripheral blood granulocytes of patients suffering from chronic
myelogenous leukaemia (CML) (Takahashi et al. 2005).
• Immunophenotyping
The method of detection of surface proteins through antibodies and flow cytometry
is known as immunophenotyping. The method involves labelling cells with their
respective cell surface markers, e.g. CD markers; CD8 in case of T cells or any other
cell surface-expressed protein used for identification of cell type and developmental
stage. Both high and low expression profiles of the markers are used as indica-
tor of particular cell type and its developmental stage. These two parameters help
in the diagnosis and prognosis of the disorder, respectively. The technique allows
to discriminate between various stages of cells based on their development stage
specific surface marker profile. This is helpful in deciding the prognostic strategy
when a patient is undergoing therapy. The technique also enables to identify the
lineage between cells of B or T lymphocyte origin. The method has been of great
use in haematological disorders including malignancies like chronic lymphocytic
leukaemia (CLL) (Ivancevic et al. 2014). Cancer cells tend to acquire drug resis-
tance over the period of therapy which can be ascertained by immunophenotyping
P-glycoprotein (MDR-1) or multidrug resistance-associated protein (MRP-1) (Jakab
et al. 2005). In the case of HIV infection, CD8+ T lymphocytes decrease in population
which can be accurately diagnosed by flow cytometry-based immunophenotyping.
B cell lymphomas are detected by low expression profile of CD20 marker. Using
the method, leucocyte adhesion deficiency disorders can be identified accurately by
looking at the decreased expression profile of the leucocyte β2 integrin receptor
complex which comprises CD11/CD18 complex (Brown and Wittwer 2000).

4.1.4 Personalized Medicine

The utilization and practice of the knowledge of genetic information of a patient to

evaluate disease risk assessment, drug response and deciding the therapy are known
as personalized medicine (Mancinelli et al. 2000). Through genetic analysis like
whole genome sequencing, it is possible to identify variations in the genes encoding
receptors or enzymes that may predict the outcome of a particular drug treatment.
Biotechnology: Discoveries and Their Applications in Societal … 27

Advancement in the field of genomics after the completion of Human Genome Project
has led to the generation of vast knowledge regarding underlying genetic differences
in a particular population. This knowledge when applied to study disease-related
predisposition markers or drug response of patients is known as pharmacogenomics
(Vogenberg et al. 2010).
• Genome-wide association studies (GWAS)
The comparison of genetic differences associated with any particular trait or disease
condition in a population between healthy and diseased subjects is known as genome-
wide association studies. The common genetic differences taken into account while
doing these analyses are single nucleotide polymorphisms (SNPs) which have been
shown to be present in varying frequencies in a population of healthy and diseased
individuals. Almost one million SNPs can be genotyped in one scan of a patient’s
DNA sample (Spencer et al. 2009). The first GWA study was reported in 2005 in
patients suffering from age-related macular degeneration (leads to blindness in elder
people). The study identified two SNPs present in complement factor H gene which
were linked with the increased risk of the disease (Haines et al. 2005).
In a separate study performed by the Wellcome Trust Case Control Consortium
(as reported in 2007), around 2000 patients for each of seven diseases, namely type
1 diabetes, type 2 diabetes, Crohn’s disease, rheumatoid arthritis, bipolar disorder,
coroner artery disease and hypertension were analysed for the variation in their SNPs
using Affymetrix SNP array. This comprehensive effort revealed several new genetic
loci associated with risk of diseases under study (Consortium 2007).

4.2 Green Biotechnology

Application of biotechnology in agriculture sector with the aim of increasing the

productivity and ensuring food security is called as green biotechnology (Kafarski
2012). The methodology adopted for realization of aforesaid goal is somewhat similar
to what is used in healthcare sector or red biotechnology due to the universality of
molecular biology principles. Broadly, the branch includes gene manipulations done
for crop or livestock improvement by increasing the crop or animal-based yield or
conferring resistance against biotic or abiotic stress factors.

4.2.1 Genetic Transformation in Plants

There are a number of methods for incorporating foreign genes in plant systems,
namely Agrobacterium tumefaciens-mediated gene transfer, electroporation, gene
gun or biolistics and microinjection (Lorence and Verpoorte 2004). Among all these,
Agrobacterium-dependent DNA transfer has been the most successful method, espe-
cially in dicotyledonous plants. The bacterium is a natural plant pathogen that upon
infection transfers a portion of tumour-inducing plasmid (Ti plasmid) DNA called
28 H. Kumar

T-DNA, which then triggers a tumour-like growth (called crown gall disease) in the
plant (Nester Gordon et al. 1984). The T-DNA is flanked by border repeat sequences,
which are retained while replacing the rest of T-DNA with the gene of interest for
subsequent infection and incorporation of the gene by A. tumefaciens in the cul-
tured plant cells (Quispe-Huamanquispe et al. 2017). Other technique like gene gun-
dependent DNA transfer involves coating gold or tungsten particle with the desired
plasmid DNA. The particles are then shot in the growing cultured cells for incorpo-
ration in the genome. Bt maize is an example of transgenic plant derived from gene
gun-mediated DNA transfer (Slater et al. 2008).
• Transgenic plants
Also called genetically modified or engineered crops (GM/GE crops) are created
by transferring genes to organisms from a different species which confers a trait not
found in the recipient’s species. The transgenic plants have been successfully created
and field tried for their property to yield more, resist herbicides or insects, be resistant
to drought or high soil salinity conditions, etc. (Banjara et al. 2012). The total produce
of GM crops has increased from 1.7 million in 1996 to more than 175 million by 2013.
The USA leads in the use of biotech crops (cotton, soybean, maize and canola) by
accounting for over 40% of global produce. India and China are the biggest growers of
Bt cotton (Clive 2015). Gossypium (cotton) species was genetically altered to express
Cry protein (endotoxin) of Bacillus thuringiensis which is highly effective in killing
lepidopteran insects. The crop was first commercially introduced by Monsanto, Inc.
in the USA, in 1996 and later in India in 2003 (Rocha-Munive et al. 2018).
Crop plants of nutritional importance like rice have been genetically modified to
produce golden rice variety. Oryza sativa (rice) was transformed into biosynthetic
gene encoding β carotene which led to its higher yield as compared to wild-type strain.
Two varieties were generated of different yield values of β carotene: golden rice-1
yielding 1.6 μg of vitamin A per gram of rice by transferring genes from daffodil and
golden rice-2 yielding 35 μg of vitamin A per gram of rice by transferring genes from
maize. The varieties are of considerable importance towards fulfilment of vitamin A
deficiency which causes night blindness and xerophthalmia (Tang et al. 2009).
Transgenic plants of ornamental importance are now being generated with
increased floral scent content or changed colours of flowers. The floral scents in the
flowers are due to the presence of volatile organic compounds, namely terpenoids,
benzenoid and aromatic amino acids (Piechulla and Effmert 2010). BEAT gene has
successfully transferred from Clarkia breweri into Eustoma grandiflorum which
induced fragrance in the petals of the recipient plant (Aranovich et al. 2007).

4.2.2 Genetic Modifications in Livestock

Microinjecting the embryos with foreign DNA or somatic cell nuclear transfer is the
most feasible methods for developing transgenic animals of agricultural importance.
The first method being the most widely practised in which embryos are microinjected
with the gene of interest cloned suitably in an expression cassette and implanted back
Biotechnology: Discoveries and Their Applications in Societal … 29

in the female (Kues and Niemann 2011). The progeny is screened for the presence
of the transgene by southern hybridization, pcr or DNA sequencing (Setlow and
Hollaender 2002). The main objectives behind such modifications are increased milk
and meat production, to render the livestock disease-free and utilize the animal for
production of therapeutic proteins through the process called biopharming.
• Recombinant human lactoferrin
Human lactoferrin (hLF) is an 80 kD iron-binding glycoprotein present in milk and
multiple bodily secretions. It is essential for iron uptake in body and kills several
harmful iron requiring bacteria by sequestrating iron away from them (Lonnerdal
and Iyer 1995). Using native gene (comprising all introns and exons) instead of
only cDNAs encoding the protein has been found to be yielding higher amounts
of therapeutic proteins in the milk of mammals (Choi et al. 1991; Whitelaw et al.
1991). With the former approach, whole genes (having introns and exons) encoding
human lactoferrin, transgenic cows have been produced, via both microinjection
and nuclear transfer methods yielding ~3–3.4 mg/ml of human lactoferrin in their
milk (van Berkel et al. 2002; Yang et al. 2008). The latter study utilized bacterial
artificial chromosome for accommodating the hLF construct and bovine fibroblast
cells for microinjecting the DNA. Somatic cell nuclear transfer led to the birth of
two transgenic cows secreting levels of hLF as mentioned earlier. Biochemical assays
revealed their iron-binding and release efficiency similar to the natural hLF.
• Recombinant antithrombin
In 2006, the European Medicines Agency and later in 2009, US FDA approved the
first transgenically obtained drug—a recombinant human antithrombin III protein
derived from the milk of goat, for surgical and childbirth applications. The trans-
genic goat was developed by the then GTC Biotherapeutics, Inc. at Massachusetts
(USA) in collaboration with Louisiana State University Agriculture Center (USA)
by injecting goat cells with human antithrombin gene (RD 2004). The protein is
purified from goat’s milk and is sold currently under the brand name ATryn by rEVO
Biologics, Southborough, MA (USA) (Erickson 2009). The drug is a lifesaver for
those who suffer from hereditary antithrombin deficiency which puts them at the
risk of developing deep vein thrombosis or pulmonary embolism (Maksimenko et al.
2013).
• Transgenic Salmon fish
AquAdvantage or transgenic salmon fish was developed at Aqua Bounty in Canada
in 2006. Wild-type Atlantic salmon (Salmo salar) eggs were injected with the growth
hormone encoding gene from Chinook salmon (Oncorhynchus tshawytscha) under
the regulatory elements of an antifreeze protein derived from an ocean pout (Zoarces
americanus). Whereas a wild-type salmon feeds only in spring or summer and takes
3 years to attain its full length, the transgenically created salmon could reach the
same length in less than two years. These fishes have triploid genomic content as
compared to their diploid wild-type counterparts and are sterile, thus preventing the
risk of interbreeding (Yaskowiak et al. 2006). The US FDA approved AquAdvantage
30 H. Kumar

for human consumption only in 2015 while Canada did the same six months later
(Waltz 2017).

4.2.3 Germplasm Conservation

For stable maintenance of the transgenic or wild-type traits of plants and animals, it
is essential to preserve the organisms’ genetic information so that the traits can pass
on from generation to generation without getting exhausted. The body parts contain-
ing such genetic information which are viable despite being preserved at ultra-low
temperature for long periods of time are collectively called as germplasm. For plants,
seeds, calli, pollen, excised embryos or root/shoot bud tips comprise the germplasm.
Animal germplasm includes semen, oocytes or embryos. With the advancement of
techniques like artificial insemination, the need of storage and preservation of male
and female gametes was realized. There are two principal modes of germplasm con-
servation of both plants and animals, namely in situ and ex situ. The preservation of
the germplasm in the natural habitat of the organism is called as in situ; whereas,
the artificial methods executed outside the boundary of natural habitat are known as
ex situ modes of conservation, respectively. The method of cryopreservation is an
example of the latter whereby the hereditary information containing components of
plant or animal bodies is mixed with a cryoprotectant chemical and frozen in liquid
nitrogen (Ruane et al. 2006).
Cryopreservation is achieved via two routes: slow freezing and vitrification (flash
freezing). In the former method, the rapid formation of ice crystal is avoided by using
chemicals like polyvinyl alcohol or synthetic biopolymers like alginates. The rate of
cooling is brought down to 1 °C per minute using a freezing box and cryoprotectants
like glycerol and dimethyl sulfoxide (Vutyavanich et al. 2010; Sambu 2015). Almost
majority of animal cells, tissues and embryos are frozen by this method. Vitrification,
also known as flash freezing, on the other hand bypasses the formation of ice crystals
at all. The sudden plunge in ultra-low temperature (megakelvins per second) in
cryoprotectant (e.g. ethylene glycol or sucrose) leads to amorphous ice formation
which is different from ice crystal sheet formation in which water molecules arrange
to create hexagonal lattice. The method was first used to freeze human oocytes which
were used to deliver a healthy baby girl through in vitro fertilization (Kuleshova et al.
1999).

4.3 Blue Biotechnology

Officially, the term blue biotechnology has not been defined yet by any
government/non-governmental organization. Although, the realm of blue biotechnol-
ogy encompasses marine bioresources and their applications, a more widely accept-
able definition of marine biotechnology is used alternatively for the purpose. The
European Marine Board defines the area of marine biotechnology as following:
Biotechnology: Discoveries and Their Applications in Societal … 31

The application of science and technology for the production of knowledge, goods and
services from (marine) biological resources. (Adapted from the Organization for Economic
Co-operation and Development general definition of biotechnology 2005)

The field is relatively nascent as compared to other areas of biotechnology and not
entirely independent from the rest. Blue biotechnology has clear overlaps with other
areas of biotechnology because of the technical know-how derived from them. The
marine realm of the biosphere comprises 70% of the total earth’s resources. Most of
the phyla known till date belong to this realm. A majority of them have been used for
societal applications. Broadly speaking, marine microorganisms, phytoplanktons,
red, green and brown algae, cnidarians, sponges, echinoderms and mangroves are a
few classes from which products of commercial value have been extracted (Blunt
et al. 2013). Figure 9 describes the phyla wise contribution of marine life forms for
biotechnological, industrial or domestic purposes (Arrieta et al. 2010).
The use of marine life forms on such an enormous scale has led to rise in marine
organism’s gene sequence-related patents being filed/granted at the International
Patents Office. These approvals have been granted in different sectors of biotechnol-
ogy affecting various aspects of human life (Arrieta et al. 2010). Most of these range
from the patents impacting human health to the field of biofuel generation as shown
in Fig. 10.
The field starts with the discovery and bioprospecting of marine bioresources with
potential biotechnological application. The commercial potential of these resources

Fig. 9 Schematic depicting marine life forms usage for biotechnology or other human benefits.
Each histogram indicates per cent organisms present in the respective taxon utilized for indicated
purposes shown in the upper legend (reproduced with kind permissions from the National Academy
of Sciences, USA and Dr. Jesus M Arrieta, Spain; (Arrieta et al. 2010)
32 H. Kumar

Fig. 10 Graphical representation of distribution of 460 patents deposited with International Patents
Office in the area of marine biotechnology. Since one patent may fall in more than one category,
sum total percentage exceeds 100% (reproduced with kind permissions from the National Academy
of Sciences, USA and Dr. Jesus M Arrieta, Spain; (Arrieta et al. 2010)

ranges from health care in the form of novel pharmacologically active substances to
the development of biofuels for providing clean energy. The European biotechnol-
ogy industry generates annual revenue of e 754 million from the blue biotech sector
(ECORYS 2014; ERA-NET 2017). Identification of key gene(s) and or other biolog-
ically active molecule and their characterization forms the next step. This involves
the screening, selecting and identifying vast number of marine organisms for desired
activity. Such organisms range from algal seaweeds to bacteria residing in deep sea
hydrothermal vents. Usually, such organisms are difficult to grow in laboratory due
to the requirement of pressure, temperature and light conditions in which they other-
wise grow. Upon the establishment of successful growth conditions, the organism’s
DNA is isolated for the purpose of identification of the potential gene encoding the
desired product. If the desired product is a biochemical, then its laboratory scale
purification is undertaken. The gene or chemical compound identified in such man-
ner is sequenced or assayed for activity in laboratories. Genes are matched to known
databases to find out the uniqueness which indicates their evolutionary difference
with other known organisms. An example is the famous GFP gene isolated from a
bioluminescent marine cnidarian (Aequorea victoria), encoding a green fluorescent
protein utilized in biomedical research.
After ascertaining the sequence, further research and development programmes
are undertaken to characterize the genomic sequence for its expression, purification
of the protein and standardization of biological/toxicological assays, and the efficacy
in the biological process is formally reported. Examination of commercial feasibility
is the deciding factor for the technology adaptation for upscaling to industries and
sometimes also the major bottleneck. However, there are several products of marine
origin which have seen the light of day, and these are being sold as food, medicine,
Biotechnology: Discoveries and Their Applications in Societal … 33

cosmetic ingredients, biofuels and bioremediation of the polluted environment. Both

marine flora and fauna have contributed to the expanding market of such commer-
cial products on which several industries falling in aforementioned sectors thrive.
Alginates are extracted from brown seaweeds (class: Phaeophyceae) like Laminaria,
Macrocystis and Ascophyllum and are used in food, textile and pharmaceutical indus-
tries. In food industries, it is used for increasing the viscosity like in making jellies.
Another brown sea algae, Fucus vesiculosus, is used in its extract form as a dietary
supplement. Algae-derived oils are good source of omega-3-fatty acids (monounsat-
urated and polyunsaturated fatty acids; EPA and DHA) that equates to oils obtained
from salmon fish (Doughman et al. 2007). Microalgae, Haematococcus pluvialis,
yields a red-coloured pigment called astaxanthin that is used in the manufacturing of
fish feed and is sold by Fuji Chemical Ltd., Japan. Calcium alginates have been used
in skin wound dressings for healing and micro-encapsulation of natural polyphenols
from wine wastes (Lansdown 2002; Aizpurua-Olaizola et al. 2016). Ecteinascidia
turbinata, a sea squirt, was identified by the National Cancer Institute (NIH, USA)
for producing an anticancer compound. The compound ecteinascidin 743 was puri-
fied many years later and is now commercially sold as Trabectedin by PharmaMar
S.A. (Spain) (Rinehart 2000). Ziconotide is a powerful analgesic drug which was first
isolated from cone snail (Conus magus) in early 1980s by scientist Michael McIntosh
at University of Utah (McIntosh et al. 1982). The compound which is a peptide got
an FDA approval in 2004 and was sold by the name Prialt by Elan Pharmaceuticals,
Ireland (Pope and Deer 2013).
Cosmetic industry has been deriving a considerable amount of raw material from
marine bioresources. Brown algae, Ascophyllum nodosum and Halopteris scoparia
(class: Phaeoophyceae), contains compounds that protect skin from harmful UV rays
of sun. The algal extract mix has been patented by Gelyma (France) and is sold by
the name Actiseane by Biosil Technologies Inc., NJ (USA) (Andre et al. 2002).
Alcanivorax borkumensis is a marine flagellar bacterium whose genome contains
sequences encoding petroleum degrading gene products. The bacterium uses alkanes
as its diet and therefore is employed for degradation of oil spills in oceans. It is
therefore also known as “oil-eating” bacterium (Schneiker et al. 2006). Mutant form
of Alcanivorax is used for production of polyhydroxy alkanoates (PHA) which are
used for manufacturing biodegradable bioplastics (Sabirova et al. 2006). Several
marine algae are currently explored and used as replacement of tradition fossil fuels
for powering engines. Botryococcus braunii (family: Trebouxiophyceae) a colonial
green microalga is known to secrete hydrocarbon rich oils in the exterior which has
been recently purified using electric currents to disrupt the bacterial colony (Banerjee
et al. 2002; Guionet et al. 2017).

4.4 White Biotechnology

The term white biotechnology is essentially European in origin. It is alternatively

called as industrial biotechnology which simply means application of living cells,
34 H. Kumar

microbes, yeast, fungi, plants and enzymes, etc., for industrial applications (Lee and
Jang 2006a, b; Ribeiro et al. 2016). Microbes and enzymes are collectively referred
to as biocatalysts which are used to manufacture paper, antibiotics, biopolymers, etc.
(Heux et al. 2015). Since long time, even before the coinage of the term white biotech-
nology, living organisms have been used to manufacture products on an industrial
scale by techniques like fermentation for making ethanol, acetic acid and various
secondary metabolites. Fermentation simply means the decomposition of complex
organic compounds to simpler products by enzymes secreted by bacteria and yeast.
Modern-day reaction takes place in a steel vessel called the fermenter. The medium
for fermentation is composed of carbohydrates, salts, nitrogen, trace elements, etc.
The selection of growth medium and the starter culture that includes the inoculum
of choice depends upon the product desired. Say, e.g. production of ethanol requires
carbohydrate-rich molasses in the growth medium and invertase secreting yeast as
inoculum. For production of cheese, lactic acid fermenting bacteria are added to the
curdling milk in the fermenters. Examples of fermenting bacteria are Lactobacillus,
Enterococcus, Streptococcus, etc., and are known for their beneficial effects on gut
microbiota. These are typically used in inoculum as starter culture and fed into the
fermenting food (e.g. milk for production of cheese). Characterization of the strain
of inoculum (here in this case lactic acid-producing bacteria) is a norm as per the
regulatory guidelines to ensure safety and maintain dietary consumption quality (Hill
et al. 2017). The method of culturing microbes and obtaining commercially important
products through the method of fermentation is briefly described by Fig. 11.
The major thrust areas at present are, namely the development of biodegrad-
able polymers and clean green energy by creating biofuels using the tools available
through white biotechnology. Since the use of plastics has greatly damaged the envi-
ronment that does not require further elaboration, a shift towards the alternatives
of synthetic non-biodegradable polymers has become imminent. Also, use of fos-
sil fuels like petroleum is slowly being reduced due to its limited reserves. Various
environmental protection agencies and government constituted panels have called
for research looking into alternative fuel sources which are environment compatible.

Fig. 11 Schematic describing the fermentation process and its products

Biotechnology: Discoveries and Their Applications in Societal … 35

Fig. 12 Switchgrass bunch grassland field (Panicum virgatum). Photo courtesy of Robert H.
Mohlenbrock, hosted by the USDA-NRCS PLANTS Database/USDA SCS. 1991. Southern wetland
flora: Field office guide to plant species. South National Technical Center, Fort Worth

Such fuels when derived from biomass meaning any plant or animal material are des-
ignated as biofuels as per US Environment Information Agency (EIA 2018). The most
commonly used biofuels are bioethanol and biodiesel. Bioethanol is conventionally
produced from carbohydrate-rich corn syrup or molasses. Recently, there has been a
tilt observed towards the production of ethanol from cellulosic biomass. Switchgrass
(Panicum virgatum; family: Poaceae) is a widely growing perennial bunch grass in
most of the USA as shown in Fig. 12. The US Department of Agriculture (USDA)
has selected the plant for production of bioethanol derived from the plant biomass
because of its ease of growing and high yield. The ethanol thus produced is currently
being mixed with gasoline at a final concentration of 10% (E10 fuel) and is used for
automobiles (EIA 2018).
Biodiesel is a liquid fuel which is currently promoted as an alternative to diesel for
its usage in public transport and commercial vehicles. It is manufactured by vegetable
oils and animal fats by esterification of fatty acids with an alcohol (Omidvarborna
et al. 2014). The National Biodiesel Board of America describes biodiesel as mono-
alkyl esters of fatty acids and designate as B100 in its purest form. There are other
parts mixed forms of biodiesel available, like B20 where it is 20% biodiesel of the
total fuel. Plants utilized for the purpose of producing biodiesel include soybean, jat-
ropha, rapeseed, etc. Salicornia bigelovii is a halophyte growing in coastal shorelines
of America that has been identified as biomass feedstock for generating biofuel. It
can survive well on salty water and is therefore suitable for large-scale cultivation by
irrigating with salt-rich water, a common industrial effluent (Bullis 2010). A similar
application of biomass lies in production of bioplastics which uses vegetable oils,
straw, woodchips and food waste (McMillan 2010). Starch-based thermoplastics are
used for making drug capsules by pharmaceutical companies. These plastics are actu-
ally a blend of several chemicals like polyhydroxy alkanoates and polylactic acid
which are endowed with better water resistance and improved mechanical properties.
Starch plastics are also used in the manufacturing of films used for packaging of food
items (Halley and Avérous 2014). Another biodegradable polymer polylactic acid
36 H. Kumar

Fig. 13 Chinese brake fern (Pteris vittata); a hyperaccumulator of arsenic. Photo courtesy of https://
suzandtell.wordpress.com/2015/02/18/the-chinese-ladder-fern/

is being produced by fermenting metabolically engineered strain E. coli for supply

to industries manufacturing it. Bacteria like Citrobacter, Klebsiella and Enterobac-
ter have been known to produce 1,3-propanediol which is used for manufacturing
adhesives, solvents and antifreeze agents (Lee and Jang 2006a, b).
Another area of growing importance is the field of phytoremediation that involves
the usage of plant for environmental clean-up. The industrial pollutants are dis-
charged into land, water and air which include heavy metals, organic compounds
and high salt-containing water. These have become a growing menace for the society
as they are severely damaging the environment and leading to rise in diseases and
deaths. Heavy metals like cadmium, mercury and lead have been found to perco-
late down the soil and reach groundwater table thus polluting it and making it unfit
for drinking or agricultural purpose. The plants that are able to extract these metals
are used for the purpose of phytoremediation of the polluted soil. Plants capable of
accumulating heavy metals more than 1000 mg/kg dry weight are called as hyper-
accumulators (Reeves 2003). Thlaspi caerulescens (family: Brassicaceae) is being
successfully used as hyperaccumulator for phytoremediation of cadmium and zinc.
Arsenic remains one of the biggest threats to groundwater that is used for direct
drinking purpose. A Chinese fern Pteris vittata of family—Pteridaceae (commonly
known as Chinese brake fern) was demonstrated to be a hyperaccumulator of arsenic
(Ma et al. 2001) (Fig. 13).

5 Summary

The field of biotechnology has evolved a lot since prehistoric times to what we see
it today and continues to evolve further. Improvization of pre-existing knowledge
and groundbreaking discoveries in the past centuries has led to significant over-
hauling of our understanding of biological processes. The causative agents of many
Biotechnology: Discoveries and Their Applications in Societal … 37

previously incurable diseases have now an identity, a method of diagnosis and a

possible treatment regime due to which life expectancy has increased considerably
from those times. Biotechnology has touched upon lives of people globally through
its various defined and undefined branches. Seminal discoveries governing the basic
process of life, their applications in the areas of health care, agriculture and environ-
ment have revolutionized the practice of science and adaptation of technology in an
unprecedented manner.
It is reasonable to conjecture that given the magnitude of applications in societal
welfare, the field of biotechnology will be going to be a major area of research and
investment. However, the endeavours pertaining to the investment are to be taken
up with certain precautions because many such areas need accumulation of enough
body of work before final commercialization can be realized. Specifically, areas
like blue biotechnology which holds immense possibility of discovery of several
crucial drugs, food supplements, industrially required materials are first needed to
be explored through deep research. Only thereafter, the true feasibility of the product
on a commercial scale can be adjudged. Areas like red biotechnology have many
promising inventions and discoveries in its basket. Gene editing tool like CRISPR
method is hailed across the globe as a major breakthrough in the sciences that can cure
several genetic diseases which are incurable till date. Application of the same in the
field of green biotechnology can lead to the generation of crops and livestock with
improved or superior traits for ensuring food and nutrition security of the public.
Increasing pollution of the ecosystem has prompted the scientists to adapt these
technologies for better environmental clean-up. The exploitation of phytoremediation
for absorbing heavy metals and degradation of oil spills in the oceans is an exciting
area of exploration. Increasing the industrial output by utilization of biologics or
living organisms helps in generation of several biodegradable materials which avoids
conventional hazardous chemical synthetic processes.
What remains to be done is an exhaustive list of tasks that needs to be taken up
seriously to ensure better practice of biotechnology throughout its various branches.
The formation of ethical guidelines for application of genetic techniques for embryo
manipulation needs to be carefully drafted. This is essential to prevent the abuse of
technology for personal fame and reputation at the cost of suffering of humanity.
Release of GM crops with proper evaluation of pros and cons must form an integral
part of crop improvement programs in poor and developing countries. The area of blue
biotechnology is still in its infancy, and therefore, more impetus is needed to attract
people towards exploring marine genetic resources. Marine resource exploitation
should be carried out with the sense of responsibility without damaging the ecosys-
tem. The concept of sustained development needs to be implemented in essence so
as to propel further research while ensuring judicious use of the natural resources.
38 H. Kumar

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Harsh Kumar did his doctorate from Regional Centre for Biotechnology, Faridabad (India) in
2019 and is a cell biology enthusiast. His doctoral work demonstrated novel biochemical and
functional crosstalk between early endocytosis and exocytosis-related proteins for facilitating the
abscission process in cytokinesis. He is currently working as a postdoctoral research associate
at the Genetics Division, Department of Pediatrics, All India Institute of Medical Sciences, New
Delhi (India).
Tiny Targets, BIG Impact!
CRISPR: The Revolutionary Gene
Editing Tool with Far-Reaching
Applications

Sohinee Bhattacharyya and Anindit Mukherjee

Abstract In the realm of scientific research, there is an immense value for biolog-
ical research tools, which can modify, insert, and delete DNA sequences of cells or
organisms in order to understand the function of specific genes. One of the most
revolutionary scientific revelations of the twenty-first century has been the devel-
opment of the programmable CRISPR-Cas9 genome engineering technology. The
groundbreaking CRISPR-Cas9 gene modulating system is derived from the original
type II CRISPR-Cas system, whose primary purpose was to endow bacteria with
adaptive immunity to viral infections. CRISPR-associated protein 9 or simply Cas9
is an enzyme that belongs to the class endonuclease by function. In brief, Cas9 func-
tions to introduce double-stranded breaks in the target DNA sequence by using a
guide RNA sequence to form base pairs with target DNA sequences. The elegance
and simplicity of the CRISPR-Cas9 genome editing system combined with its cost-
effectiveness and efficiency in precisely targeting and editing genomic sequences
have heralded a transformative phase in basic biological research. Scientists around
the globe are using this technology for novel groundbreaking applications that range
from improving human therapeutics, improving disease resistance of industrially
important crops, and for basic biology research. This chapter aims to present the key
milestones in the evolution of this revolutionary CRISPR-Cas9 genome editing tool
and will also discuss the far-reaching impacts of this transformative technology in
the domains of basic science, biotechnology, and medicine.

Keywords CRISPR · Cas9 · TALEN · Homologous recombination · Genome

engineering · Gene editing

S. Bhattacharyya (B)
Oregon Health and Science University, Portland, USA
e-mail: [email protected]
A. Mukherjee
Oregon State University, Corvallis, USA

© Springer Nature Switzerland AG 2020 47

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_2
48 S. Bhattacharyya and A. Mukherjee

1 Development of the CRISPR-Cas9 Genome Editing Tool

The human genome sets the stage for the blueprint of life. The accurate and precise
transfer of genetic information from gene to messenger RNA and ultimately to protein
(the central dogma of molecular biology) is indispensible in preventing the develop-
ment of genetic anomalies and diseases (Crick 1970). Rare events where fidelity of
genetic information transfer is compromised may lead to the development of seri-
ous genetic diseases. Some starring examples include genetically inherited BRCA1
mutations in breast cancer patients and in chronic myeloid leukemia patients, somatic
BCR-ABL1 fusions. These diseases are a couple of examples where mutations in
crucial genes have been directly linked to the development of human pathologies.
As a matter of fact, although strategies to target human diseases often are directed
toward inhibiting metabolic pathways or cellular signaling pathways, the most effica-
cious therapeutic regimens often target the underlying genetic anomalies. Scientists
have been developing technologies to edit underlying genomic abnormalities (such
as mutations), and technologies for manipulating genetic information (DNA) hold
tremendous potential for treating human genetic diseases, even certain cancers. In
order to correct underlying genetic abnormalities, scientists are now using harnessing
the power of genome engineering tools to introduce specifically targeted modifica-
tions in the gene pool of living cells. The path to the discovery of genetic tools that
could introduce site-specific modifications to the genetic code was not an easy path.
It took scientists around the world several years of hard work and research to dis-
cover the mechanism behind the revolutionary genetic engineering breakthrough, the
CRISPR/Cas9 system. Early developments in the field of genome engineering lead to
the discovery of site-directed “zinc finger nucleases (ZFNs), TAL effector nucleases
(TALENs),” which relied on the principles of “DNA–protein recognition” (Carroll
2011). However, due to the laborious efforts involved in protein engineering, both
these systems had severe drawbacks in large-scale studies (Carroll 2011). Finally,
after years of research, a breakthrough in the scientific field was the discovery of
the “CRISPR/Cas9 system.” Compared to the older gene editing technologies, the
CRISPR/Cas9 system offered the researchers site specificity, versatility, high effi-
ciency, and ease of use. By simply programming guide RNA sequences (guide RNAs
direct the Cas9 endonuclease), Cas9 endonuclease can be specifically directed to edit
defined DNA sequences.
The process of genome engineering involves the introduction of targeted modifi-
cations to the genome. There is a growing demand for technologies that can achieve
this easily and efficiently in mammalian cells as technologies that can easily modify,
delete, and insert DNA sequences hold immense potential to transform the realms
of biotechnology, basic science, and therapy. Genomes of eukaryotic organisms are
complex in nature and contain billions of DNA bases. This complex structural organi-
zation makes it immensely difficult to introduce new modifications into the existing
DNA sequence. One of the earliest methods used for genome targeting has been
the use of homologous recombination (Capecchi 1989). “Homologous recombina-
tion” is a biological process that is widely used by cells to repair double-stranded
CRISPR: The Revolutionary Gene Editing Tool with Far … 49

breaks, and it can also be used to integrate external repair sequences that are sequen-
tially homologous to the donor site. Homologous recombination-mediated genome
editing downstream of targeted DNA double-stranded breaks was demonstrated very
elegantly in a group of pioneering studies by Rudin et al. (1989). Following this, Car-
roll and Chandrasegaran developed zinc finger protein designer nucleases for precise,
sequence-specific homologous recombination (Bibikova et al. 2001, 2002, 2003).
However, one of the major drawbacks of this process is that homologous recombina-
tion occurs extremely rarely (1 in 106–109 cells) (Capecchi 1989). This presents an
enormous challenge to the adaptation of this method for large-scale applications. To
overcome this obstacle, scientists developed four principal classes of elegant genome
editing tools that could introduce locus-specific double-stranded breaks in the DNA:
(I)Meganucleases—originally coming from mobile genetic elements of microbes,
introns, or inteins (Smith et al. 2006). Meganucleases are functionally classified as
homing endonucleases which may be further broadly classified as intein endonucle-
ases and intron endonucleases. Mobile genetic elements such a sintrons propagate
by intervening at a specific locus in the genomic sequence, and the expression of
the meganuclease introduces a cleavage in the corresponding intron free allele. This
initial cleavage event leads to the duplication of the intron sequence at the cleavage
site by double-stranded DNA break repair (homologous recombination). (II) Zinc
finger (ZF) nucleases—this class of nucleases were developed by artificially fusing
a DNA cleavage functional domain to a “zinc finger DNA-binding domain.” This
class of nucleases can be engineered to modify specific genetic sequences and rely
on eukaryotic transcription factors (Urnov et al. 2005; Miller et al. 2007). (III) “Tran-
scription activator-like effectors (TALEs)”—TAL effectors are secreted proteins that
are derived from Xanthomonas bacteria (Miller et al. 2011; Christian et al. 2010;
Boch et al. 2009; Cong et al. 2013) when they infect plants. TALENs or “Transcrip-
tion Activator-Like Effector Nucleases” are a group of restriction enzymes that can
be modified to target specific genetic sequences. They are artificially synthesized
by structurally fusing a TAL effector DNA-binding domain to a functional DNA
cleavage domain. (IV) The “RNA-guided DNA endonuclease Cas9—derived from
bacterial adaptive immune system CRISPR” (Cong et al. 2013; Mali et al. 2013).
Meganuclease, ZF, and TALE proteins exploit the same principle; they bind to
specific DNA sequences through specific protein–DNA interactions. However, they
have severe drawbacks that make it challenging for use in genome engineering.
For example, meganucleases, ZF nucleases, and TALEs lack precise specificity to
their target DNA sequences and strategies to overcome this limitation can render
the method expensive and labor-intensive. Given these challenges, scientists worked
toward the development of a simpler and more precise nuclease. The CRISPR story
started gaining momentum 1987 (Ishino et al. 1987) when a set of 29 nucleotide
repeats downstream of the iap gene (the iap gene encodes for an enzyme which is
involved in isozyme conversion of alkaline phosphatase in E. coli) was reported.
As more microbial genomes were deciphered, it was discovered that unique sets of
clustered repeat elements are present in greater than 40% of all bacteria sequenced
and a vast majority of archaea (Mojica et al. 2005). Despite this compelling find-
ing, the precise function of these repeat elements was unknown until 2005, when
50 S. Bhattacharyya and A. Mukherjee

through investigations of the nonrepetitive spacer sequences separating the repeat

elements suggested that they were derived from bacteriophages (Pourcel et al. 2005;
Bolotin et al. 2005) These findings coupled to earlier studies that elucidated that
bacteriophages are unable to infect bacterial cells harboring spacer sequences that
correspond to their own genomic sequence, led to the possibility that “CRISPR
arrays” function as microbial immunity modules and act to mediate viral defense.
The first concrete proof that the CRISPR array is a mechanism of antiviral defense
came from the studies with the bacterial strain Streptococcus thermophilus, which is
routinely used in dairy cultures; they demonstrated a nucleic-acid-based functional
module in which “CRISPR spacers” regulate preciseness of sequence selectivity,
while Cas enzymes regulate acquisition of spacer sequences and develop immunity
to the phage (Barrangou et al. 2007).
Around this time, two revolutionary studies elucidated the key components that
are required for efficient genome editing by the “CRISPR system.” In the first study,
Moineau and colleagues (Barrangou et al. 2007) elucidated Cas9 as the only nuclease
that mediates sequence-specific DNA cleavage (Garneau et al. 2010). In the second
study, Deltcheva and colleagues revealed the existence of a crucial component of the
CRISPR module—a “trans-activating crRNA (tracrRNA)” that forms a hybrid com-
plex with crRNA to facilitate RNA-guided trafficking of Cas9 to selected genomic
sequences (Deltcheva et al. 2011).

2 Domain Organization of the “CRISPR/Cas9 System”

The “CRISPR-Cas9 technology” originally evolved from “type II CRISPR-Cas sys-

tems,” whose primary function was to bestow upon bacteria immunity from infecting
viruses (Carroll 2011; Cong et al. 2013; Mali et al. 2013; Jinek et al. 2012; Cho et al.
2013; Cox et al. 2015; Wright et al. 2016; Doudna and Charpentier 2014; Wieden-
heft et al. 2012; Fineran and Charpentier 2012; Horvath and Barrangou 2010). In
bacteria, CRISPR-Cas9 loci are composed of an array of identical CRISPR repeats
and an operon (a set of linked genes with the capacity to regulate other genes) that
encodes the Cas nuclease complex (Carroll 2011; Cong et al. 2013; Mali et al. 2013;
Jinek et al. 2012; Cho et al. 2013; Cox et al. 2015; Wright et al. 2016; Doudna and
Charpentier 2014; Wiedenheft et al. 2012; Fineran and Charpentier 2012; Horvath
and Barrangou 2010; Ran et al. 2013). The CRISPR nuclease Cas9 achieves target
specificity with the help of a short guide RNA that hybridizes to and cuts target DNA
via Watson–Crick base pairing. Typically, the CRISPR RNA guide sequences match
phage genomic sequences in order to render immunity to the bacteriophages. How-
ever, one huge advantage of the CRISPR system is that these targeting sequences can
be easily switched with a selected genomic sequence of interest to reassign the Cas9
nuclease. Upon invasion by viruses and plasmids, adaptive immunity in conferred in
three key steps: (i) A short sequence of the invading DNA is acquired and inserted
into the CRISPR loci, (ii) maturation of precursor crRNA to generate functional RNA
CRISPR: The Revolutionary Gene Editing Tool with Far … 51

duplex, tracrRNA:crRNA duplexes with invader targeting sequences, and (iii) site-
specific double-stranded break in the DNA by Cas proteins at sites corresponding to
the RNA duplex, tracrRNA:crRNA guide sequence (Carroll 2011; Cong et al. 2013;
Mali et al. 2013; Jinek et al. 2012; Wright et al. 2016; Doudna and Charpentier 2014;
Wiedenheft et al. 2012; Fineran and Charpentier 2012; Horvath and Barrangou 2010;
Ran et al. 2013). Very recently, scientists engineered the dual tracrRNA:crRNA to a
single-guide RNA. This novel and elegant engineering advance generated an easy-to-
use bipartite system (Doudna and Charpentier 2014). The easily programmable Cas9
targeting system, its high accuracy, and precise sequence specificity as a sequence-
specific nuclease have opened up a diverse range of biological applications that range
from basic research to biotechnology and medicine.

3 Revolutionary Applications of the CRISPR/Cas9 System:

Genome Engineering and Beyond

3.1 Generation of Knockout Cell Lines for Basic Science

Research

The easy-to-use “CRISPR-Cas9 genome editing system” has made the generation of
genetic knockout cells very feasible and efficient for a diverse array of cell systems
including cancer-derived patient organoids, primary immune cells as well as stem
cells. Generating such knockouts is invaluable to the advancement of scientific and
clinical research as it allows researchers to rapidly establish functional roles of their
genes of interest (Jinek et al. 2013; Grobarczyk et al. 2015; Matano et al. 2015; Hou
et al. 2013; Drost et al. 2015; Mandal et al. 2014) (Fig. 1).

4 Rapid Generation of Animal Models

Aside from being invaluable in the field of cell biology and basic science research,
CRISPR-Cas9 system has drastically enhanced the efficiency of generating animal
models of human pathologies (Shen et al. 2013; Li et al. 2013a, c; Wang et al. 2013).
CRISPR-Cas9 system can now be used to generate mice with genetic alterations in
the desired gene of interest in order to ultimately understand the functions of the
genes involved. This can be achieved by using electroporation or microinjection of
zygotes instead of using the traditional expensive and labor-intensive procedure of
embryonic stem cell (ESC) manipulation (Shen et al. 2013; Li et al. 2013a, c; Wang
et al. 2013).
52 S. Bhattacharyya and A. Mukherjee

Fig. 1 Mechanism of action of CRIPSR Cas9 module (i) a short sequence of the invading DNA
is incorporated into the CRISPR loci, (ii) maturation of precursor crRNA to generate functional
RNA duplex, tracrRNA:crRNA duplexes with invader targeting sequences, and (iii) site-specific
double-stranded break in the DNA by Cas proteins at sites complementary to the RNA duplex,
tracrRNA:crRNA guide sequence
CRISPR: The Revolutionary Gene Editing Tool with Far … 53

5 Using CRISPR-Cas to Make Novel Therapeutic

Approaches

Apart from its tremendous potential in enhancing the knowledge base of the genetic
basis of human malignancies, the power of the “CRISPR-Cas9 system” can be har-
nessed to develop novel therapeutic approaches. A starring example is the use of
CRISPR-Cas to engineer CAR T cells that have tremendous promise in the area
of cancer immunotherapy (Schumann et al. 2015). These genetically engineered T
cells, which express specific tumor-targeting receptors, have demonstrated tremen-
dous therapeutic potential for lymphoma and leukemia patients (Schumann et al.
2015).
Another excellent example to demonstrate the clinical efficacy of the “CRISPR-
Cas9 system” is the treatment of patients with haemoglobinopathies like sickle
cell disease (SCD). Sickle cell disease is caused by a mutation in the Hemoglobin
gene. Recently, SCD patients have been subjected to ex vivo gene correction using
CRISPR-Cas9 genome modification (Mandal et al. 2014; Hoban et al. 2015; DeWitt
et al. 2016). This mutational event causes a shift in the amino acids coded. More
specifically, substitution of a glutamate (Glu) to valine (Val) is the causal genetic
mutation that is found in all sickle cell disease patients. CRISPR-Cas9 has been able
to correct this mutation by using a single-stranded oligonucleotide introduction or
by using an integrase defective lentiviral vector (Mandal et al. 2014; Hoban et al.
2015; DeWitt et al. 2016).

6 “CRISPR/Cas9 System” in Genetically Modifying Plants

and Cattle Strains

Recently, scientists around the world have started using the “CRISPR-Cas system”
to genetically modify agricultural crops and cattle strains in order to produce more
economically beneficial, improved strains. For example, in economically important
agricultural crops like rice, wheat, sweet orange, and sorghum, target genes such
as genes that make the plants susceptible to microbial pathogenic infections have
been edited out using “CRISPR-Cas9 technology” (Li et al. 2013b; Nekrasov et al.
2013; Xie and Yang 2013; Jiang et al. 2013; Upadhyay et al. 2013). As an added
advantage, it was recently reported that these sites directed changes introduced by
“CRISPR-Cas9” did not have any detrimental off target effects and importantly;
these newly introduced genetic edits were inherited by the next generation of plants
without the development of new spontaneous and random mutational events. Thus,
“CRISPR-Cas9” genome editing tool is also set to revolutionize the agriculture and
dairy industry by creating new disease resistance strains that are genetically protected
from microbial diseases and pests (Li et al. 2013b; Nekrasov et al. 2013; Xie and
Yang 2013; Jiang et al. 2013; Upadhyay et al. 2013).
54 S. Bhattacharyya and A. Mukherjee

7 Conclusions

To summarize, the “CRISPR/Cas9 system” is poised to revolutionize the fields of

drug discovery and targeted therapy by enabling the precise and specific modula-
tion of genetic information in disease models. In the near future, the power of the
CRISPR/Cas9 system will enable scientists and clinicians to introduce corrective
mutations or modify gene regulatory elements or correct gene-splicing patterns.

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Sohinee Bhattacharyya In the Sherman Lab at OHSU, Sohinee is presently working on the gene
regulatory interaction networks that exist between pancreatic tumor and stroma. She joined the
University of Nebraska Medical Center in 2009 to pursue her Ph.D. in pathology and microbi-
ology where she investigated the novel role of the endocytic recycling protein EHD1 in mouse
early embryonic morphogenesis. After completing her Ph.D., she joined the University of Pitts-
burgh School of Medicine as a postdoctoral fellow. Here, under the guidance of Dr. Ora Weisz,
she investigated the cellular signaling pathways that regulate apical endocytosis in proximal tubule
cells in response to fluid shear stress.
56 S. Bhattacharyya and A. Mukherjee

Anindit Mukherjee received his Ph.D. degree in cellular and integrative physiology from the
University of Nebraska Medical Center in 2012. He is presently working at Oregon State Uni-
versity as an expert in epithelial biology and is currently focused on treatment of cystic fibrosis
with the help of nanoparticle-delivered therapeutics.
Nanotechnology in Medicine

Anindit Mukherjee and Sohinee Bhattacharyya

Abstract Nanotechnology is at the frontier of medicine. It plays a critical role in

expanding diversity and reach of the drug or drug-like molecules in patients. Nanopar-
ticles act as a protected transport vehicle for hydrophobic drugs or therapeutics such
as mRNA, siRNA, and DNA which are normally not bioavailable. In this chapter,
we review the different kinds of non-viral nanocarriers that are being employed
in modern-day medicine. We also discuss the challenges to effective delivery and
function of these particles as well as their application in various aspects of disease
treatment.

Keywords Nanotechnology · DNA · RNA · Liposome · Polyplex · Dendrimer ·

Quantum dot · Metal nanoparticle · Endocytosis · Macropinocytosis ·
Phagocytosis · Clathrin · Caveolin · Therapeutics · Niemann–Pick type C · Fabry ·
AIDS · HIV · Cancer

1 Introduction

Nanoparticles are engineered nanoscale structures that are increasingly being used in
the field of biomedical science for targeting and delivery of therapeutics. Most of these
particles are less than 100 nm in size. Nanomaterials have been used as a protective
vehicle to increase solubility and stability of therapeutics in the bloodstream. This
ability has helped expand the repertoire of cargo such as hydrophobic drugs, DNA,
RNA, peptides, and antibodies that are normally labile in the blood stream, to be
used as therapeutics. Additionally, particles have been engineered to take advantage
of biological and cellular processes for enhancing targeting and delivery of cargo
into the intended cells or tissues. However, the additional challenge for nanoparticle
engineering is to design the particles that are at once capable of protecting their cargo
in the blood stream but once inside the cells they will fall apart to release their cargo.

A. Mukherjee (B)
Oregon State University, Corvallis, USA
e-mail: [email protected]
S. Bhattacharyya
Oregon Health and Science University, Portland, USA
© Springer Nature Switzerland AG 2020 57
A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_3
58 A. Mukherjee and S. Bhattacharyya

2 Types of Nanoparticles and Their Application

in the Biomedical Field

A wide variety of materials have been engineered to be used as nanoparticles.

They include polymers, lipids, inorganic particles, viral vectors, cell ghosts, etc.
In this review, we will focus on the synthetic particles, namely liposomes, nanopar-
ticle albumin-bound (nab) technology, polymeric nanoparticles, dendrimers, metal
nanoparticles, and molecular-targeted nanoparticles.
Liposomes or lipid nanoparticles or lipoplexes are single-layered or multilay-
ered spherical lipid vesicles with an aqueous core. Liposomes are made with natural
or synthetic lipids and are capable of packaging both hydrophilic and hydrophobic
cargoes. Some of the earliest of attempts to introduce exogenous genes into cells
were performed using lipid-based particles. By the 1980s, various genetic materi-
als such as DNA and RNA were introduced into cells by utilizing the fusogenic
potential of liposomes. Once inside the endosome, the liposomes destabilize and
fuse with the endosomal membrane in an inside-out manner and release their cargo
into the cell cytoplasm. Several liposomal formulations are approved for clinical
applications. Cationic lipids such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethyl-
ammonium chloride (DOTMA) have been frequently used for formulating lipoplexes.
Variations of these formulations such as ones using ionizable lipids such as Dlin-
MC3-DMA (MC3) or novel ionizable lipid (lipid 5) have been successful in greatly
increasing intracellular nucleic acid delivery in cell and animal models. Liposomes
can be formulated with polymers such as polyethylene glycol (PEG) or with fuso-
genic lipids such as dioleoylphosphatidyl-ethanolamine (DOPE) and cholesterol for
increasing transfection efficacy. Cholesterol helps in cholesterol-dependent mech-
anisms for cellular entry of the nanoparticles, whereas adding polymers such as
PEG to the surface increases stability of the particles in serum. Nanoformulation of
doxorubicin, an anticancer drug, has resulted in markedly increased bioavailability
(Torchilin 2005).
Protein nanoparticles made with albumin-bound technology (nab) have made
use of capacity of albumin to carry and release hydrophobic cargo. Additionally,
albumin is transcytosed via glycoprotein 60 receptor which helps in intracellular
delivery of the cargo. Paclitaxel, a hydrophobic drug, is made soluble using which
nab technology is sold by the name Abraxane (Benson et al. 1978).
Polymeric nanoparticles are made with biocompatible block co-polymers con-
sisting of two or more block polymer chains with differing hydrophilicity. The
hydrophobic block forms the core, while the hydrophilic block forms the outer shell
that interacts with the aqueous phase. Various therapeutics can be packaged in the
core of these particles for delivery (Zhang et al. 2008).
Dendrimers are well-defined branched monodispersed polymers that can trap
molecules within their core. Dendrimers can also be conjugated with therapeutic or
diagnostic molecules. Most dendrimer formulation is 1–20 nm in size. These particles
are made with polymers such as polyamidoamine (PAMAM), melamine, poly l-
glutamic acid (PG), polyethyleneimine, or natural elements such as amino acids,
Nanotechnology in Medicine 59

sugars, and nucleotides. Polyamidoamine (PAMAM), melamine, poly l-glutamic

acid (PG), polyethyleneimine consist of a core, branched units emerging from the
core in layers, and an outer layer with repeat units of functional end groups. The
solvent-filled core forms the nanoscale cargo hold, while the defined exterior surface
allows it to interact with biological domains (Svenson and Tomalia 2005).
Metal nanoparticle are synthesized using biocompatible inert metals such as
gold, nickel, and silica. Different shapes such as nanoshell or quantum dots or
nanorods have been fashioned with metals. Unique optical and magnetic properties of
these nanoparticles have been variously used for diagnostic and imaging purposes,
e.g., MRI contrast imaging. Supermagnetic nanoparticles made with maghemite,
cobalt ferrite, etc., that oscillate between a strongly magnetic and a non-magnetic
state in the presence and absence of a magnetic field, respectively, are being studied
for in vivo applications. Oscillating magnetic field causes these particles to generate
heat, which is being used for intracellular hyperthermia-based applications.
Unique optical properties of semiconductor-based quantum dots are also being
extensively being used for biological applications, particularly in the field of imag-
ing. Several properties of these materials including their ability to absorb and emit
quantized packets of energy, the wide range of absorption spectrum, a large differ-
ence between the absorption and emission spectra and harnessing the discrete and
resistance to photobleaching have made them very attractive for biological imaging
application.
Metal nanoparticles have also been conjugated with hydrophobic drug for bet-
ter dispersal and delivery. For example, gold nanoparticles have been coated with
amphiphilic thiols, whose hydrophobic pockets’ package take up the hydropho-
bic therapeutics such as tamoxifen, and the hydrophilic group makes the particles
water-soluble (Minelli et al. 2010).
Molecular-targeted nanoparticles have their surface decorated with targeting
molecules such as monoclonal antibodies, aptamers, peptides, and small molecules
(such as folate) against cell-specific markers. These molecules bind to specific marker
proteins on cells and get internalized via receptor-mediated endocytosis. Targeted
nanoparticles are attracting a lot of attention. Formulations such as BIND-014, a
polymeric nanoparticle formulation of docetaxel, with controlled release property
went into clinical trial (Wang et al. 2008).

3 Barriers to Nanoparticle Delivery

Cellular uptake mechanisms at once present challenges and opportunities for

nanoparticle-based intracellular delivery mechanisms. Cells take up materials from
the extracellular milieu using a variety of mechanisms broadly known as endocyto-
sis (Sahay et al. 2010). Endocytosis can be subdivided into phagocytosis, a process
by which cells engulf large particles up to 20 μm in diameter and pinocytosis, a
more generalized form of endocytosis, involving uptake of solutes along with extra-
cellular fluids (Conner and Schmid 2003). Phagocytosis is primarily observed in
60 A. Mukherjee and S. Bhattacharyya

specialized phagocytic cells such as macrophages, neutrophils, monocytes, and den-

dritic cells, whereas pinocytosis is observed in all cells. Depending on the molecular
mechanism of uptake, pinocytosis can be primarily of four types: clathrin-mediated,
macropinocytosis, caveolin-mediated, and clathrin-caveolin-independent. Of which,
the last three together are known as clathrin-independent mechanisms. Clathrin-
independent uptake mechanisms involve receptors and membrane-associated pro-
teins other than clathrin, such as caveolin, ARf6, Flotillin, CDC42, and RhoA that
aid in forming outside-in membrane vesicles for uptake of extracellular materials.
Calthrin-mediated endocytosis is a hallmark of all cells, while clathrin-
independent mechanisms display some cell-type bias. For example, polarized epithe-
lial cells lack caveolae on their apical membrane (Lahtinen et al. 2003; Vogel
et al. 1998; De et al. 2014), whereas caveolae formation is prevalent in muscle,
endothelium, and fibroblasts (Doherty and McMahon 2009).
Depending on the cargo, cell type, and the vesicle type, majority of the endocy-
tosed materials are either degraded via lysosomal pathway, transcytosed out of the
cell or recycled back to the cells surface. Only a small percentage gets delivered into
the cytosol via a mysterious mechanism, broadly termed as endosomal escape.
Nanoparticle-based intracellular delivery strategies have tried to take advantage
of the heterogeneity of uptake mechanism by changing composition, size, shape, and
charge of the particles to target them to specific endocytic compartments in order to
enhance endosomal escape (Varkouhi et al. 2011). Additionally, clathrin-independent
mechanisms have been targeted to enhance particle delivery to non-epithelial cells
(Oh et al. 2007).

4 Application of Nanoparticles in Diseases

Lysosomal storage disorder: Lysosomal storage disorders (LSDs) are an attractive

nanoparticle-based therapeutic target, while small molecule screen in HeLa cells
identified several effectors such as V-ATPase, arachidonic acid metabolism, and
Cathepsin that affect delivery of LNP to lysosomes (Sahay et al. 2013). The inherent
defects in intracellular trafficking result in accumulation of material inside the late
endosomes and lysosomes. This type of accumulation has been utilized for enhancing
the effectiveness of nanoparticle delivery.
Niemann–Pick disease type C: Niemann–Pick Type C (NPC) is an inherited auto-
somal recessive lysosomal storage disorder that affects approximately 1:120,000
children globally and results in hepatomegaly, progressive neurodegeneration, and
finally death (Futerman and van Meer 2004; Vanier 2010). The disease is caused by
mutation in NPC1 and 2 that result in cholesterol accumulation in the endosomes and
blocks efflux out of the late endosomal compartments (Ikonen 2008). Superparam-
agnetic iron oxide nanoparticles (SPIONs)-based ferrofluid nanoparticles were used
to isolate late endosomes and study the difference in the composition of lysosomal
glycocalyx (Tharkeshwar et al. 2017; Kosicek et al. 2018). Increase in retention time
and defective degradation machinery of endosomes facilitated endosomal escape
Nanotechnology in Medicine 61

of siRNA into the cytosol (Sahay et al. 2013; Eltoukhy et al. 2014). Interestingly,
PEG-based lipid nanoparticles have shown to be a bioactive agent by facilitating
cholesterol solubilization and efflux from these compartments (Brown et al. 2016).
In Niemann–Pick type B, caused due to acid sphingomyelinase deficiency,
clathrin-mediated endocytosis is impaired (Rappaport et al. 2014). ICAM1-
conjugated nanocarriers were used to deliver acid sphingomyelinase to the lysosome
by clathrin-independent route by utilizing ICAM1-dependent route and decreased
sphingomyelin storage in mouse lung (Garnacho et al. 2008; Muro et al. 2003, 2005).
In Fabry disease caused due to α-galactosidase A (α-Gal) deficiency and in Pompe
disease caused due to acid α-glucosidase (GAA) deficiency, nanoparticle was used to
α-Gal and GAA, to reduce lysosomal accumulation of globotriaosylceramide (Gb3)
and to decrease excessive glycogen storage, respectively, in mice (Hsu et al. 2011,
2012).
AIDS: Nanoparticles have been used to tackle particularly challenging targets
such as HIV virus. HIV infects and hides inside patient’s immune cells such as
peripheral monocytes and macrophages. The virus thus uses the immune system as
a protective reservoir as well as a vehicle for transporting and spreading the virus
to different parts of the body (Amiji et al. 2009; Aquaro et al. 2002). Although
saquinavir is very effective in reducing virus load, it is highly hydrophobic which
makes for a key hurdle in using it for effective therapy. Poly(ethylene oxide)-modified
poly(epsilon-caprolactone) (PEO-PCL) and polyhexylcyanoacrylate-encapsulated
saquinavir nanoparticles were used to overcome the challenge of poor solubility and
increase intracellular delivery of the drug and effective reduction of HIV infection
(Bender et al. 1996; Shah and Amiji 2006).
Cancer: The use of nanotechnology for treatment of cancer is an active area
of biomedical research. Nanoparticles are particularly useful in solubilizing several
cancer drugs that are mostly hydrophobic molecules which short plasma half-life.
Moreover, solid tumors are characterized by leaky vasculature that arises due to
rapid and dysregulated proliferation of endothelial cells as well as due to decreased
abundance of pericytes. This unique leakiness allows nanocarriers along with their
cargo to be differentially enriched in the tumor microenvironment and not be cleared
through the lymphatic system as would be the case in normal tissue. This enrich-
ment is termed as enhanced permeability retention (EPR) effect. Polyethylene glycol
(PEG)-coated doxorubicin, a hydrophobic drug, has been coated to enhance solubil-
ity, increase circulation time, enhance EPR, and decrease non-specific toxicity of the
drug (Barenholz 2012; Unezaki et al. 1995). Doxorubicin nanoparticles have been
widely used to treat different types of cancer, including Kaposis sarcoma, ovarian
cancer lung carcinoma, prostate cancer, etc. (Sakakibara et al. 1996; Northfelt et al.
1996; Stürzl et al. 1994; Vaage et al. 1997; Muggia et al. 1997; Huang et al. 1992).
Albumin-bound nanoparticle (Nab) technology has been used to encapsulate the
hydrophobic antitumor agent, paclitaxel to enhance albumin driven endocytosis via
the cell surface receptor GP60 and SPARC (secreted protein, acidic, and rich in cys-
teine), a protein that is upregulated in multiple tumors (Tai and Tang 2008; Tiruppathi
et al. 1997; Minshall et al. 2000). This product is commercially available as Abrax-
ane. Docetaxel, another anticancer drug, was encapsulated in a controlled release
62 A. Mukherjee and S. Bhattacharyya

poly(lactide-co-glycolide)-poly(ethylene glycol) (PLGA-PEG) polymer that is dec-

orated with peptide ligands against prostate-specific membrane antigen (PSMA)
that is overexpressed in certain prostate cancers. The product named BIND-014 is
currently in phase 2 trial (Autio et al. 2018; Hoff et al. 2016). Drugs conjugated
with protein also form another important class of nanomedicine. Doxorubicin and
Camptothecin conjugated with albumin and a cathepsin cleavable linker. Enzyme-
catalyzed cleavage of the linker in the lysosomal compartment leads to release of
the drug (Akinc and Battaglia 2013; Schmid et al. 2007). Similarly, antitumor drugs
are conjugated to antibodies via low-pH hydrolysable or Cathepsin-like enzyme
cleavable linker. The antibodies bind specific proteins on the tumor cell surface and
are internalized along with the drug. The low endosomal pH or specific lysosomal
enzymes then cleave the linker and release the drug into the cancer cells (Diaman-
tis and Banerji 2016; Ducry and Stump 2010). Several nanodrugs, such as Ontak,
Mylotarg, Adcetris, Kadcyla, and Vintafolide, designed on the basis of aforemen-
tioned principle are either approved or in various phases of clinical trials (Nabhan
and Tallman 2002; Younes et al. 2010; Verma et al. 2012; Lorusso et al. 2012). Ontak,
for example, is an interleukin-2 (IL-2)–diphtheria toxin fusion protein that is used to
treat cutaneous T-cell lymphoma (CTCL). It is targeted against the CD25 subunit of
the IL-2 receptor CTCL cell surface (Olsen et al. 2001).
Nanoparticle-based gene therapy has also been studied in treating various cancer
types (Babu et al. 2016). Nanocarriers have been deployed to deliver tumor suppres-
sor genes such as p53 or siRNA against oncogenes such as c-Myc/MDM2/VEGF,
surviving, hypoxia-inducible factor 1α, and KSP (Ramesh et al. 2001, 2004). siRNA-
based nanoparticles have also being studied (Vaishnaw et al. 2010; Young et al. 2016;
Feng et al. 2017; Gillespie et al. 2015; Chen et al. 2010).

5 Conclusion

Nanotechnology has opened exciting frontiers in the treatment of intractable med-

ical problems. While current research is full of tremendous promise, challenges
remain in taking basic research from bench to bedside. However, newer engineering
and advances in materials and molecular techniques hold tremendous potential for
nanomedicine to truly take hold and deliver personalized medicine with lower side
effects.

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Food for All; Nutraceuticals
The Rise of Nutraceuticals: Overview
and Future
Nitika Kapoor, Vijay Lakshmi Jamwal, Manish R. Shukla
and Sumit G. Gandhi

Abstract Recent changes in lifestyle and dietary patterns, along with increased
industrialization, rising pollution and work pressure, have led to increased suscep-
tibility to a plethora of disorders such as hypertension, obesity, diabetes, inflamma-
tory and autoimmune conditions, and cancers. Recent advancement in the medical
sciences has considerably helped in treatment of these conditions; however, it has
been recently realized that nutrition management may play a huge role in preven-
tion as well as in treatment to some extent. The market is presently flooded with
hundreds of nutraceuticals/food supplements that not only help to prevent and tackle
such disease conditions but may also provide general health promotion. Nutraceuti-
cals are food substances intended to be supplemented to regular diet and may con-
tribute to general well-being, provide protection from diseases, or may ameliorate
disease conditions. Nutraceuticals may contain vitamins, lipids, proteins, carbohy-
drates, minerals, herbs or herbal extracts, or other necessary nutrients, depending
on their emphases and health claims. This chapter provides an overview of different
categories of nutraceuticals, their health applications, and business outlook.

Keywords Amino acids and proteins · Fatty acids · Herbs · Minerals ·

Microalgae · Nutraceuticals · Probiotics · Prebiotics · Vitamins

N. Kapoor · V. L. Jamwal · S. G. Gandhi (B)

Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu
180001, India
e-mail: [email protected]
M. R. Shukla
Research and Development Center, Reliance Industries Limited, CU-127, TC-30-C, 2nd Floor,
RCP Thane-Belapur Road, Ghansoli, Navi Mumbai 400701, India

© Springer Nature Switzerland AG 2020 67

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_4
68 N. Kapoor et al.

1 Introduction

1.1 Defining Nutraceuticals

The term Nutraceutical was coined in 1989 by Stephen De Felice. He joined the
terms nutrition and pharmaceuticals to describe how nutrition can be used for dis-
ease prevention (DeFelice 1995). Food or food additives that supplement the diet
and provide health benefits are popularly known as nutraceuticals, functional foods,
dietary supplements, etc. (Dureja et al. 2003). Nutraceutical products may have sub-
stances that are traditionally not consumed as food, but may have a positive phys-
iological benefit such as prevention or treatment of disease condition (Singh 2013;
Kalra 2003). Nutraceuticals thus lie somewhere between food and drugs, although
they are generally not as strictly regulated as the conventional modern drugs (Gulati
and Ottaway 2006; Pandey et al. 2010). As per various regulatory agencies, there
are minor differences between the definitions of nutraceuticals, functional foods, and
dietary supplements. For instance, functional foods are defined as foods cooked using
scientific intelligence and providing the body with the requisite quantity of vitamins,
proteins, carbohydrates, minerals, fats, etc., for its healthy survival (El Sohaimy
2012). However, if such a food helps in prevention or treatment of a disease, other
than anemia, then it may be called as a nutraceutical. Dietary supplement as defined
by The Dietary Supplement Health and Education Act of 1994 (DSHEA) of the USA
includes products other than tobacco that may contain mineral, vitamin, amino acids,
herbs, etc., in the form of a syrup, capsule, or pills (Zeisel 1999). Such supplements
are not projected to be used as sole food item or as conventional food and may
help to increase longevity, provide health benefits, or fulfill the special age-related
dietary requirements, etc. (Gupta et al. 2018). However, since we consider these as
minor regulatory variations, and this chapter intends to give the reader an overview
of nutraceuticals, we have clubbed them together in a single term nutraceutical and
may be used interchangeably in the text with other terms functional foods and dietary
supplements. As such in some countries, these terms are treated synonymously.

1.2 Need for Nutraceuticals

Sedentary lifestyle patterns and unhealthy eating habits, along with increased indus-
trialization, rising pollution, and work pressure, have led to increased susceptibility
to a plethora of disorders/diseases such as hypertension, obesity, diabetes, joint and
skeleton problems, inflammatory and autoimmune conditions, psycho-neurological
issues, and cancers (Prasad et al. 2012) (Fig. 1). Additionally, the average lifespan has
steadily increased over the past few decades, with a large subset of adult population
who suffer from such lifestyle-related disorders/diseases. Vegetarian diet and food
rich in carbohydrates and fats may lead to protein deficiency (Elorinne et al. 2016).
Protein deficiency is associated with fatty liver disease, loss of muscle mass, skin and
The Rise of Nutraceuticals: Overview and Future 69

HERBALS

FATTY-ACIDS

NUTRITION
PROBIOTICS AND
NUTRACEUTICAL PREBIOTICS

PHARMACEUTICAL VITAMINS AND

MINERALS

AMINO- ACIDS &

PROTEINS

Fig. 1 Nutraceuticals and its major classes

hair problems, greater risk of bone fractures, and increased severity of infections.
Increase in consumption of processed and fast food may also lead to high cholesterol
and high blood sugar, which may eventually lead to cardiovascular diseases, obesity,
and diabetes (Pereira et al. 2004; Swinburn et al. 2004; Bahadoran et al. 2015). Fur-
ther, such diets may also be deficient in fiber, leading to difficult bowel movement
and constipation (Cencic and Chingwaru 2010). Fiber-deficient diet has also been
linked to diverticular disease (Ünlü et al. 2012), heart disease (McRae 2017), dia-
betes (Schultz et al. 2004), and inflammation (Krishnamurthy et al. 2012). Low-grade
chronic inflammation, on the other hand, has been associated with heart disease, can-
cer, rheumatoid arthritis, diabetes, obesity, etc. (Coussens and Werb 2002). Obesity
and diabetes are on the rise even in younger population and children (Pozzilli et al.
2011). Further, recent trends show an increase in vitamin and mineral deficiency, not
only in economically weaker sections of the society, but also in affluent households.
Such deficiencies often lead to reduced vitality and fatigue and, if not tackled in time,
may also lead to neurological, skeletal, or metabolic disorders (Di Somma et al. 2017;
Christodoulou et al. 2013). This not only puts excess pressure on the state-funded
healthcare systems, but also negatively affects individual efficiency and productivity
as well as social structure. This problem is further accentuated by increasing medical
costs. Genetic predisposition is certainly a crucial factor, but other factors, such as
exposure to toxins, stress, and the food we eat, have significant impact on disease
incidence and progression. Since the kind of food that we eat has an important influ-
ence on our health, it is vital that our food contains agents that may be helpful in
preventing and/or treating diseases.
70 N. Kapoor et al.

1.3 Cues from Traditional Systems of Medicine

The use of plants or their parts/extracts/concoctions occupies center stage in almost all
the traditional systems of medicine (TSM), practiced worldwide. Fruits of the genus
Citrus are generally rich in Vitamin C, folic acid, pectin, and potassium. They have
been used in TSM to relieve palpitation, flatulence, as treatment for coughs and colds,
as antispasmodic, antiemetic, antihypertensive, antipyretic, antiseptic, antihyperlipi-
demic, etc. (Turner and Burri 2013). Nutraceutical products based on bioflavonoids
of Citrus are available for prevention as well as amelioration of cardiovascular prob-
lems. Ginkgo biloba has been traditionally used for treatment of memory impairment
(Mullaicharam 2013). Several nutraceutical products based on this plant are available
for treatment of dementia and improvement of memory (Chauhan et al. 2013). Sim-
ilarly, green tea, which is popularly used as a nutraceutical, consumed as decoction
or extract, finds its roots in traditional Chinese medicine. It was used for reduction
of weight, positive effect on metabolism, prevention of heart ailments, etc. (Ahmad
et al. 2014; Shinde et al. 2014). Garlic (Allium cepa) has been used in TSM world-
wide, for various ailments such as fevers, swellings, and antibacterial (Chauhan et al.
2013). Garlic contains sulfur compounds: agoene, allicin, and alliin, minerals such as
selenium, amino acids: cysteine, methionine, glutamine and isoleucine, flavonoids:
cyanidin, allistatin I & II, quercetin, vitamins: A, B, C and E (Ayaz and Alpsoy 2007).
Garlic finds use in prevention of cardiovascular diseases, regulation of blood pres-
sure as well as reduction of cholesterol and blood sugar (Bhagyalakshmi et al. 2005;
Wang et al. 2017). Garlic heart-care capsules are available as nutraceuticals. These
are few popular examples where cues from TSM have been used for preparation of
nutraceutical products. The use of herbs and phytochemicals in nutraceutical prod-
ucts will be revisited in the later section of this chapter, where it will be discussed in
detail.

1.4 Contrasting TSM with Modern Medicine

Preventive health care in modern medicine is practiced at primary level by popular-

izing healthy living through regular exercise and appropriate diet (Finckh and Deane
2014), immunizations, and use of specific aids to protect from workplace hazards,
such as use of earplugs in aerodromes. Secondary-level prevention is carried out by
risk profiling through family history and epidemiological data, regular health check-
ups to identify diseases at presymptomatic stage, and early treatment. Tertiary level
includes more elaborate procedures such as use of cardiac stents for prevention of
possible myocardial infarction, removal of tumor tissue to prevent metastasis, etc.
(Karunathilake and Ganegoda 2018). On the other hand, disease prevention through
dietary changes and/or inclusion of specific minerals, herbs, or herbal extracts in diet
formed a major pillar of TSM (Pandey et al. 2013). Further, use of allopathic or mod-
ern medicine is popularly believed to be associated with side effects (Bandaranayake
The Rise of Nutraceuticals: Overview and Future 71

2006), while the general public perception toward herbal drugs is positive and they
are thought to be free from toxic side effects. Nutraceuticals draw similarity and
strength from TSM, and the consumer perception toward them is quite positive, as
evident from the rising market share of nutraceutical products (El Sohaimy 2012;
Whitman 2001).

2 Major Classes of Nutraceutical Products

2.1 Herbal Extract/Plant-Based

Nutraceuticals hold an immense potential for health promotion and prevention of

diseases using botanical products (Nasri et al. 2014). Compounds such as polyphe-
nols, carotenoids, limonoids, flavonoids, and bioflavonoids obtained from different
herbs like green tea, pomegranate, ginger, turmeric, and citrus fruits exhibit phar-
maceutical properties and show promise as nutraceuticals (Shen et al. 2012). In the
market, various nutraceutical products containing herbs are available in the form of
capsules, powder, tablets, extract, syrup, etc., like chyawanprash, green tea, psyllium
husk, brahmi, mucuna, etc. Plantago ovata (Plantaginaceae) is a source of Psyllium
husk which is popularly used as a dietary supplement for its laxative properties. It is
available in the form of granules, capsules, powder, or flakes. It has been found to
be helpful in reducing cholesterol in addition to the treatment of constipation (Xing
et al. 2017). It has also been shown to relieve diarrhea, improve appetite, and aid
weight loss (Wärnberg et al. 2009; Mehmood et al. 2011; Verma and Mogra 2013).
Brahmi or Bacopa monnieri (Scrophulariaceae) has been used as a tonic for men-
tal health since ages (Gohil and Patel 2010). The key constituents of Brahmi are
triterpenoid saponins: asiaticoside, madecassoside, bacosides A and B (Russo and
Borrelli 2005; Kapoor 2017). Pharmacological studies have shown that Brahmi pos-
sesses activities such as memory enhancement (Kunte and Kuna 2013), antidepres-
sant (Kadali et al. 2014), anti-Alzheimer’s, neuroprotective, etc. (Rao et al. 2012).
Mucuna pruriens (Fabaceae), popularly known as velvet-bean or dopa-bean, due
to high content of L-dopa, is used for treatment of nervous disorders and Parkin-
son’s disease (Patil et al. 2015). Several nutraceutical products based on Mucuna are
available in the form of tablets, for treatment of stress, mood elevation, and mental
well-being. Tinospora cordifolia (Menispermaceae), commonly called as giloy or
guduchi, is used for treatment of fevers in traditional medicine (Kavya et al. 2015).
It possesses anti-inflammatory and antipyretic properties and is often used in condi-
tions like dengue, swine flu, malaria, and urinary tract infections (Saha and Ghosh
2012). Green tea is made from the leaves of Camellia sinensis (Theaceae) and is one
of the most commonly consumed beverages around the world. It is known to har-
bor several health benefits such as cardiovascular protection, weight maintenance,
skin care, allergy inhibition, and protection from osteoarthritis (Katiyar and Raman
2011; Wu et al. 2012; Zink and Traidl-Hoffmann 2015). Green tea has thermogenic
72 N. Kapoor et al.

and metabolic stimulant activity and is a rich source of antioxidants such as epi-
gallocatechin 3-gallate (Nagle et al. 2006), vitamin C and E. Curcumin, the main
bioactive compound present in the rhizome of Curcuma longa (Zingiberaceae), is
well known for its anti-inflammatory properties (Kohli et al. 2005). Being fat-soluble,
the bioavailability of curcumin is very low, and so in most ayurvedic formulations,
it is taken along with black pepper, which contains piperine that acts as a bioavail-
ability enhancer (Shoba et al. 1998). Curcumin finds use as a health supplement to
relieve inflammation and arthritis (Hewlings and Kalman 2017). Ginger, the rhizome
of Zingiber officinale (Zingiberaceae), is a widely used condiment in Asian cuisines
(Sharma 2017). Capsules/tablets made from ginger are used as nutraceutical for their
antiemetic, carminative, and anti-inflammatory properties (Malhotra and Singh 2003;
Palatty et al. 2013; Funk et al. 2016). Ashwagandha, the roots of Withania somnifera
(Solanaceae), are known in traditional medicine for their restorative and rejuvenating
benefits (Sharma et al. 2011). It is widely used as a nutraceutical in various forms
like powder, capsules, tablets, etc., for providing benefits such as vigor enhancement,
and stress management (Lopresti et al. 2019). Emblica officinalis (Phyllanthaceae),
known as Indian gooseberry or amla, is an ingredient of Triphala, one of the popu-
lar nutraceuticals consumed in India (Peterson et al. 2017). Triphala is used for its
properties in aiding digestion, relieving constipation and anti-inflammatory action
(Mukherjee et al. 2006; Kalaiselvan and Rasool 2015). Azadirachta indica (Meli-
aceae), popularly known as neem, is an important medicinal herb known to purify
blood, detoxify body, and neutralize free radicals. It also supports immune system,
provides radiant skin, supports healthy digestion, boosts liver function, etc., and is
available in the form of tablets and capsules (Mandal-Ghosh et al. 2007; Bhowmik
et al. 2010; Pingale Shirish 2010). Ginkgo biloba (Ginkgoaceae) has been used in
traditional Chinese medicine for the treatment of respiratory diseases, cardiovascu-
lar diseases, neurodegenerative diseases such as Alzheimer’s disease and fatigue for
several years (Ramamurthy et al. 2014; Mahadevan and Park 2008). Ministry of Food
and Drug Safety (MFDS) of Korea has accepted the use of Ginkgo biloba for memory
enhancement and improving blood circulation. EGb 761® extract obtained from the
leaves of Ginkgo biloba has displayed neuroprotective and cardiovascular protective
property (Maclennan et al. 2002; W˛asik and Antkiewicz-Michaluk 2017; McKeage
and Lyseng-Williamson 2018) and has been shown to ameliorate mild memory and
cognitive impairment.

2.2 Proteins and Amino Acids

Proteins are an important component of normal diet and are a must requirement for
healthy functioning of human body. They are important structural and functional units
in all life forms. A large population, especially in developing countries, is known
to consume diets deficient in proteins (Müller and Krawinkel 2005). Traditional
vegetarian diets, in general, are low in protein content. Further, protein-digestibility
corrected amino acid score (PDCAAS) of pulses is generally much lower compared
The Rise of Nutraceuticals: Overview and Future 73

to meat and egg. Milk and soy, though have a good PDCAAS (Schaafsma 2000),
may not suit the diets of all individuals due to issues such as lactose intolerance
and casein allergy (milk) and adverse effect on thyroid function (soy). However,
to address these issues, several nutraceutical products in the form of protein pow-
ders, cookies, shakes, etc., are available in the market. Many of these products may
contain total whey or soy protein, or their hydrolysates (Manninen 2009). Further,
obese people who intend to lose weight are advised low-carbohydrate–low-fat–high-
protein diets. Such individuals also consume protein shakes. Athletes, who require
higher muscle mass, often supplement their diets with protein-based nutraceuticals
(Phillips and Van Loon 2011). Many such products also contain higher amounts of
specific amino acids such as l-arginine. l-arginine is metabolized to nitric oxide,
which increases blood flow, thereby helping in better muscle development (Álvares
et al. 2012). l-arginine is also metabolized to creatinine that facilitates better muscle
mass and higher anaerobic work capacity. l-arginine also helps to increase the levels
of resting growth hormone, which in turns helps in gaining muscle mass (Kanaley
2008). l-Arginine soft-gel helps in reduction of menopause symptoms (Stanislavov
and Rohdewald 2014). Agmatine powder has been used to enhance rate of absorp-
tion and overall bioavailability (Schwedhelm et al. 2008). Some of these supple-
ments may also contain branched chain amino acids (BCAA) like leucine, valine,
and isoleucine which are metabolized into glutamine and alanine (Holeček 2018).
These help to increase immunity, boost muscle growth, provide neuro-protection, and
benefit people with liver disease (Shimomura et al. 2006; Fernstrom 2005; Tajiri and
Shimizu 2013). Aromatic acid tryptophan is also available as supplement for helping
thyroid hormone biosynthesis and maintaining metabolism. Similarly, phenylalanine
and tryptophan supplementation is known to provide relief from stress and anxiety
and improve mental health (Fernstrom and Fernstrom 2007; Jenkins et al. 2016).
Shark cartilage powder that mainly contains the collagenous protein which forms
the tough skeleton of shark is also available as a nutraceutical. It is thought to sup-
port and repair joint tissues as well as for the prevention and/or control of arthritis
(Merly and Smith 2015).

2.3 Fatty Acids

Polyunsaturated fatty acids (PUFAs) contain at least one double bond in their back-
bone and may be either omega-3 (n-3) or omega-6 (n-6) fatty acids. α-linolenic acid
(ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are examples
of omega-3 FA. They are crucial for normal functioning of the body. Various studies
suggest that omega-3-fatty acids have anti-arrhythmic (Leray et al. 2001; Stoll et al.
1999), hypolipidemic (Bucher et al. 2002; Nemets et al. 2002), and antithrombotic
(Stoll et al. 1999; Bucher et al. 2002; Albert et al. 2002) activities. Further, they are
important for brain development as well as its proper functioning. Supplementing
diet with omega-3 (EPA + DHA) fatty acids has also been shown to boost immunity,
cardiovascular health, and cognition ability and provide relief from joint pain. These
74 N. Kapoor et al.

have to be acquired through the diet. However, mostly vegetarian food is deficient
in both EPA and DHA (Escott-Stump and Mahan 2000); hence, dietary supplements
containing these are popular. Omega-3 (ALA, EPA, and DHA) are present in sev-
eral dietary supplement formulations, such as fish oil, krill oil, and cod liver oil
soft-gel capsules (Fialkow 2016). Omega-6 FA mainly includes linoleic acid (LA),
γ-linolenic acid (GLA), and arachidonic acid (ARA). The main source of LA is veg-
etable oils such as corn, safflower, soybean, and sunflower. ARA is mainly obtained
from animal products like meat, poultry, and eggs. Some observational studies pre-
dict that the alpha-linolenic acid is a promising nutraceutical for the prevention of
stroke (Blondeau et al. 2015).

2.4 Small Molecules, Vitamins, and Minerals

Glucosamine and chondroitin supplementation has been reported to improve joint

pain, swelling, and physical function of joints. Nutraceutical products comprising
both glucosamine and chondroitin have been found to be helpful to osteoarthritis
patients (Huskisson 2008; Vasiliadis and Tsikopoulos 2017). Similarly, Vitamin D
and calcium are important for maintenance of bone health. Due to changing lifestyle
and eating habits as well as relatively reduced exposure to sun, vitamin D deficiency is
becoming commonplace. Several supplements containing vitamin D, calcium along
with glucosamine and chondroitin are available to help skeletal function (Jerosch
2011; Abrams et al. 2018). Vitamin D deficiency has also been related to poor immune
response to infections and increased incidence of autoimmune disorders and has been
associated with neuropsychiatric disorders (Holick 2004; Sotodeh-Asl et al. 2014).
Similarly, deficiency of vitamin B12 is associated with tiredness, weakness, shortness
of breath, pale skin, as well as nerve problems like numbness or tingling. Vegetarian
diet is perpetrated to be deficient in vitamin B12 (Antony 2003). Several multivitamin
dietary supplements are available to address this need. Dietary supplements with
combinations of vitamins with whey protein lysate or with omega-3 fatty acids are
also available in the market (Andreeva et al. 2012; Kemse et al. 2014; Chanet et al.
2017). A large population in developing countries also suffers from anemia, due to
deficiency of vitamin B12 and iron. Several supplements are available which club
iron and multivitamin to address this need. Due to increased usage of reverse osmosis
purified and demineralized water, the incidence of mineral deficiency, such as those of
calcium, magnesium, potassium, zinc, selenium, and iron, is increasing. Deficiencies
of minerals are associated with various diseases and disorders (Kozisek 2005). Some
of these minerals are important for bone health, while others may be required for other
physiological functions (Campbell 1995; New 1999). Several protein supplements
available in the market include the recommended daily dose of the required vitamins
and minerals, to address these needs.
The Rise of Nutraceuticals: Overview and Future 75

2.5 Probiotics and Prebiotics

A probiotic comprises of alive microorganisms as dietary supplements, which when

taken in an adequate amount benefits the health by improving intestinal microbial
balance (Fuller 2012). It is mandatory that the microorganism to be used as a probi-
otics comes under the category of GRAS (generally recognized as safe) (Bouchard
et al. 2013). The most common sources of probiotics are yogurt, cultured buttermilk,
and cheese. Most probiotic preparations currently in the market consist of lactic acid
bacteria (like Lactobacilli, Streptococci, and Bifidobacteria), which are predomi-
nately present in healthy human gastrointestinal microbiota (Das et al. 2012). They
are readily available in the forms of powder, gel or paste or granule, capsule, etc.
(Suvarna and Boby 2005). Various nutraceutical capsules consisting of Lactobacil-
lus rhamnosus, Lactobacillus plantarum, Lactobacillus salivarius, etc., are known
to boost immunity and maintain healthy intestinal flora which are readily available
in the market. There are evidences that consuming probiotic supplements reduces
the risk of systemic conditions, like allergy, asthma, urinary tract infections, and
otitis in children (Lenoir-Wijnkoop et al. 2007). Many probiotics are mainly used
to treat various gastrointestinal (GI) problems such as lactose intolerance, diarrhea
and antibiotic-associated GI side effects (Doron et al. 2005), irritable bowel syn-
drome (IBS) (Lacy and De Lee 2005), etc. Helicobacter pylori is the causal agent
of peptic ulcer disease and is also associated with carcinogenesis. It has been found
that, in vitro, multiple strains of Lactobacilli and some flavonoids have the potential
to reduce the H. pylori growth. Probiotics have also been shown to inhibit gastric
mucosal adhesion of H. pylori (Ushiyama et al. 2003; Chatterjee et al. 2003). In
addition to this, Lactobacillus casei-supplemented milk product to a standard triple
therapy regimen (omeprazole, amoxicillin, and clarithromycin) in children resulted
in a remarkably higher eradication rate of H. pylori and conferred an enhanced ther-
apeutic benefit (Sýkora et al. 2005). Lactose intolerance is a genetically determined
beta-galactosidase deficiency which results in the inability to break down lactose
into the monosaccharides glucose and galactose. As a result, individuals suffering
from lactose intolerance develop diarrhea, bloating, abdominal pain, flatulence, etc.,
after consumption of dairy products. It has been suggested that Streptococcus ther-
mophilus and Lactobacillus delbrueckii subsp. bulgaricus have the ability to digest
milk products due to the presence of beta-galactosidase enzyme (Kechagia et al.
2013).
Prebiotics are dietary ingredients which modulate gut microbiota composition
and thereby may enhance metabolic rate, increase uptake of minerals, augment
immunity level, and improve role of intestinal mucosal barrier (Nagpal and Kaur
2011; Anadón et al. 2016). The most widely accepted prebiotics are fructo-oligo-
saccharides (fruits and vegetables), inulin (Chicory root, Agave tequilana), and
galacto-oligosaccharides (milk). Prebiotics may also include polydextrose, soybean
oligosaccharides, isomalto-oligosaccharides, xylo-oligosaccharides, palatinose, dex-
trins, gentio-oligosaccharides, chito-oligosaccharides, and sugar alcohols (Anadón
76 N. Kapoor et al.

et al. 2016). Most prebiotics are short-chain carbohydrates with a degree of poly-
merization of two or more and are mainly not susceptible to digestion by pancreatic
and intestinal brush border enzymes (Steed and Macfarlane 2009). Prebiotics can
also be used as a supplement that can be taken either directly in the form of capsules
and tablets or indirectly by sprinkling directly on food as well as by stirring into
beverages. Lactulose, a non-absorbably sugar, is available as a prebiotic for reliev-
ing constipation and symptoms of hepatic encephalopathy (Gibson 2004; Conway
2001; Delzenne 2003). Similarly, inulin is helpful in alleviating inflammatory bowel
disease by altering the intestinal microflora (Schultz et al. 2004; Cherbut et al. 2003;
Furrie et al. 2005; Kelly et al. 2005).

2.6 Microalgae as Nutraceuticals

Microalgae like Nostoc, Botryococcus, Anabaena, Chlamydomonas, Scenedesmus,

Chlorella, etc., contain minerals, vitamins, essential amino acids, fatty acids, and sec-
ondary metabolites, thus making it possible to exploit these as nutraceuticals with
some of them possessing antioxidant and anti-aging properties (Bishop and Zubeck
2012). Microalgae-based nutraceuticals are becoming popular and are acquiring a
large market share (Nicoletti 2016). Cell extracts or dried biomass produced from
Spirulina or Chlorella is among the most common blue-green algae-based nutraceu-
ticals (Suganya et al. 2016). They have been found to have positive effects on cardio-
vascular diseases, diabetes, hypertension, and blood pressure lowering and reduc-
ing cholesterol (Iwata et al. 1990). Spirulina is available in different forms such as
tablets, capsules, or powder, alone or in combination with other algae, plant extracts,
or vitamins, for both human and animal food. Iron-enriched Spirulina tablets are
available as a food supplement to compensate iron requirement, reduce fatigue, and
improve anemia. Chlorella contains high content of proteins, carotenoids, and vita-
mins and is used as a dietary protein supplement (Andrade et al. 2018). Dunaliella
(D. salina, D. bardawil) is a unicellular green alga, known for the production of
large amounts of carotenoid (β carotene), glycerine, and protein (Bental et al. 1988;
Murthy et al. 2005). The algal products in various forms like β-carotene capsules,
extracts, Dunaliella powder, and dried Dunaliella are accessible in the market for
human and animals. Haematococcus (H. pluvialis) is a unicellular green alga that is
used in several nutraceutical products mainly because of high content of astaxanthin
along with other vitamins and minerals (Lorenz and Cysewski 2000). Astaxanthin
possesses strong free-radical scavenging activity, which is 1000 times more that of
vitamin E (α-tocopherol) (Naguib 2000). Astaxanthin has been shown to exhibit pro-
tection against diseases such as inflammation, antibacterial, diabetes, diabetic nerve
damage, neurodegenerative, and eye diseases (Yuan et al. 2011). The annual world
market of astaxanthin is estimated to be about $200 million (Irianto and Austin 2002;
The Rise of Nutraceuticals: Overview and Future 77

Guerin et al. 2003). Aphanizomenon (blue-green alga) found in freshwater systems

throughout the world is also used as a nutraceutical products (Pulz and Gross 2004)
as it produces omega-3 and omega-6 polyunsaturated fatty acids in large quantity
(DeWille et al. 1979). Figure 1 summarizes the major classes, and Table 1 lists a few
examples of nutraceutical products available in the market.

3 Regulatory Aspects of Nutraceuticals

It is an important to regulate nutraceutical products to protect the public from spurious

and possibly toxic products (Bagchi et al. 2004; Halsted 2003; Hasler 2002). As
previously mentioned, a class of nutraceutical products includes natural products
directly or as a component. The natural products industry faces diverse challenges
starting from the collection of raw material to manufacturing. Collection of sample,
identification, quantification, and standardization methods are of critical importance
for maintenance of uniform quality. Parameters asserting product safety and efficacy,
such as acute and chronic and toxicity studies, genotoxicity, reproductive toxicology,
teratogenicity, etc., should be carried out. If long term consumption of nutraceutical
is intended, then supplementation studies in animals and limited clinical trials may
also be necessary (Bagchi 2006). Regulations should require that the nutraceuticals
be judged safe before they are marketed, especially considering that they may be
taken over long periods of time and would be available over-the-counter without the
need for prescription. Nutraceuticals are predominately considered as food product;
however, their classification and regulation may vary in different regions of the world.
So, they are regulated differently in different jurisdictions (Table 1).
UNFAO (United Nation food and Agricultural Organizations) and WHO (World
Health organization) have given specific guidelines for the production of and market-
ing foodstuffs and their derivatives. Different countries employ these guidelines for
the regulation of dietary supplements, which briefly explain health claims according
to the nutrient function; enhanced function; and reduction of probability of diseases.
The nutrient function claim can be defined as the correlation between the role of
nutrient and function of the human body. In the USA, Food and Drug Administra-
tion (USFDA) defines nutraceuticals or dietary supplements as products other than
tobacco that may be as tablet, capsule, soft-gel, or powder which is intended to
supplement the diet or enhance the health. Such a product may contain vitamins,
minerals, amino acids, botanicals, or other dietary substance (Hoadley and Row-
lands 2014). In USA, the FDA is also responsible for taking legal action against any
adulterated supplement product after it reaches into the market (Santini et al. 2018).
Under the Dietary Supplement Health and Education Act (DSHEA 1994), it is the
liability of manufacturers and distributors of dietary supplements for the evaluation
of the safety, efficacy, and labeling of all the items before marketing to ensure that the
safety of the nutraceutical. The USFDA Modernization Act (1997) mentions that at
least four months before a supplement is marketed, the FDA has to be notified about
the health claims and/or the nutrient content claims on the product label of a dietary
78 N. Kapoor et al.

Table 1 Nutraceuticals along with their source and applications

S. Category Species/product Application References
No
1 Herb Camellia sinensis Free radical Nagle et al. (2006)
(Epigallocatechin scavenger
gallate) Antioxidant
2 Herb Curcuma longa Antioxidant Hewlings and
(Curcuminoids) Anti-inflammatory Kalman (2017)
3 Herb Mucuna pruriens Enhance dopamine Patil et al. (2015)
(L-Dopa) levels and protects
neurons, promote
brain health
4 Plant Glycyrrhiza glabra Free radical Kalsi et al. (2016)
(liquorice) scavenger,
Hepato-protective
5 Herb Panax ginseng Anti-inflammatory Kim et al. (2017)
(Ginsenoside)
6 Herb Ocimum sanctum Anti-microbial Baliga et al. (2013),
(Eugenol) Anti-stress Prakash and Gupta
Anti-diabetic (2005)
Hepatoprotective
Anti-inflammatory
Neuroprotective
Cardioprotective
7 Plant Scutellaria baicalensis Antioxidant Shieh et al. (2000)
(Baicalein) Anti-inflammatory
8 Plant Azadirachta indica Immune system, Mandal-Ghosh et al.
radiant skin, support (2007), Bhowmik
healthy digestion, et al. (2010), Pingale
boosts liver function Shirish (2010)
9 Plant Coffea arabica Caffeine Regulate oxidative Martini et al. (2016)
stress
10 Herbal Ginkgo biloba extract Memory Maclennan et al.
extract (EGb 761) enhancement, (2002), W˛asik and
Improvement in Antkiewicz-Michaluk
blood circulation (2017), McKeage and
Lyseng-Williamson
(2018)
11 Mineral Magnesium Required for the Bhutto et al. (2005)
active transport of
ions like K and Ca
and provide strength
to the bone mass
12 Herb Larrea tridentata Antioxidant Rahman et al. (2011)
(Nordihydroguaiaretic Antiviral
acid) Anti-inflammatory
(continued)
The Rise of Nutraceuticals: Overview and Future 79

Table 1 (continued)
S. Category Species/product Application References
No
13 Mineral Vitamin B complex Neuro modulatory Kennedy (2016), Dai
function, bone and Koh (2015)
strength
14 Vitamins Vitamin C and E Free radical Grosso et al. (2013)
scavenger
15 Vitamin Vitamin D and Vitamin Maintain calcium Veldurthy et al.
C homoeostasis, aging (2016), Harrison and
Immuno-modulatory May (2009)
effects
Regulate dopamine
levels
16 Mineral Calcium supplement Strength to the bone Reid et al. (2015)
17 Amino-acids L-Arginine softgel Reduction of Stanislavov and
menopause Rohdewald (2014)
symptoms
18 Amino-acids Agmatine powder Enhance rate of Schwedhelm et al.
absorption (2008)
19 Amino acids BCAA (Branched Chain Increase immunity, Shimomura et al.
Amino-Acids) provide (2006), Fernstrom
neuro-protection as (2005), Tajiri and
well as benefit Shimizu (2013)
people with liver
disease
20 Amino-acids Aromatic amino acids Relief from stress, Fernstrom and
anxiety and improve Fernstrom (2007)
mental health
21 Herbs Centella Nerve tonic, Chauhan et al. (2013)
asiatica/Brahmi anti-anxiety,
spasmolytic
22 Herbs Aegle marmelos/Bael Digestive, appetizer, Neeraj and Johar
treatment of (2017)
diarrhoea and
dysentery
23 Plant Ferula assafoetida Stimulant, Mahendra and Bisht
(Asafoetida/ferulic and expectorant, (2012)
umbellic acid) carminative,
laxative, etc
24 Herb Panax ginseng Stimulate immune Kang and Min (2012)
(Ginsenosides and and nervous system
Panaxosides)
25 Herb Ginger (Zingiberene and Stimulant, chronic Sharma (2017)
gingerols) bronchitis,
hyperglycemia
(continued)
80 N. Kapoor et al.

Table 1 (continued)
S. Category Species/product Application References
No
26 Herb Echinacea purpurea Anti-inflammatory, Manayi et al. (2015)
(alkylamide and anti-viral and
echinacoside) immunomodulatory
27 Plant Allium sativum (Alliin Anti-inflammatory, Arreola et al. (2015)
and Allicin) immunomodulation,
nervine tonic
28 Herb Tinospora cordifolia Anti-inflammatory Saha and Ghosh
Giloy and anti-pyretic (2012)
activity
29 Herb Withania somnifera Aphrodisiac, liver Sharma et al. (2011)
(Ashwagandha) tonic,
anti-inflammatory
agent, astringent,
asthma
30 Herb Cassia angustifolia Purgative Ramchander and
(Sennosides) Middha (2017)
31 Herb Emblica officinalis Anti-inflammatory, Jain et al. (2015)
(Amla) fever, anaemia, etc
32 Herb Hydrastis Canadensis Anti-microbial, Asmi and Lakshmi
(Hydrastine and astringent, treatment (2013)
berberine) of mucosal
inflammation
33 Herb Valeriana officinalis Tranquillizer, Pilerood and Prakash
(valerenic acid and migraine, intestinal (2013)
valerate) cramps, bronchial
spasm
34 Minerals Glucosamine and Treatment of Huskisson (2008),
chondroitin osteoarthritis Vasiliadis and
Tsikopoulos (2017)
35 Herb Glycyrrhiza glabra Anti-inflammatory Shin et al. (2007)
Liquorice and anti-allergy
36 Prebiotic Lactulose Treatment of liver Gibson (2004),
diseases and relief Conway (2001),
from symptoms of Delzenne (2003),
constipation Marteau and
Boutron-Ruault
(2002)
37 Prebiotic Fructo-oligosaccharides Treatment of Cherbut et al. (2003),
inflammatory bowel Schultz et al. (2004),
disease and relief Furrie et al. (2005),
from constipation Kelly et al. (2005)
(continued)
The Rise of Nutraceuticals: Overview and Future 81

Table 1 (continued)
S. Category Species/product Application References
No
38 Prebiotic Raffinose, Prevention of Mitsuoka et al.
galacto-oligosaccharide, cholesterol (1987), Kohmoto
isomalto-oligosacharides gallstones et al. (1988)
39 Microalgae Arthrospira platensis Shows anti-oxidant Nuhu (2013)
and antibacterial
property
40 Microalgae Chlorella vulgaris Health food, food Batista et al. (2013),
Supplement, feeds Paniagua-Michel
(2015)
41 Microalgae Dunaliella salina Free radical Murthy et al. (2005),
(Lutein, betacarotene) scavenger and Hsu et al. (2008)
provide
hepato-protection
42 Microalgae Haematococcus Health food, feeds Paniagua-Michel
pluvialis (Astaxanthin) (2015)
43 Microalgae Schizochytrium Dietary, nutritional Chu (2012)
(Docohexaenoic acid) supplements
44 Micro algae Aphanizomenon UV-screening agent; Chu (2012)
flos-aquae sunscreen
Mycosporine-like amino
acids
45 Micro algae Crypthecodinium cohnii Oil for the infant Saha and Murray
formula as DHA (2018)
source

supplement which may be authorized by a statement of the Academy of Sciences or

another federal body. European Food Safety Authority (EFSA) approves the health
claim of a nutraceutical product before it can be put on the market. Over and above
the EFSA’s opinion on a product, the member states can individually set specific reg-
ulations for approval. However, unlike the USFDA, EFSA is not mandated to take
a legal action against an unsafe product (Hasler 2005). In Canada, the regulation of
nutraceuticals is more like a drug than food category (L’Abbé et al. 2008). In India,
these products are regulated by the Food Safety and Standards Regulations (2016).
These regulations cover nutraceuticals, food for special dietary requirement or for
special medical conditions. These products may include botanicals, prebiotics, pro-
biotics, etc. However, these regulations do not allow the use of hormones, steroids, or
psychotropic substances. FSSAI has published a list of dietary components and their
maximum daily doses. As such, there are no requirements for any non-clinical or
clinical data as long as the product contains a dietary component that is listed by the
FSSAI and follows other regulations regarding permitted dosage and labeling norms.
In case a new substance is to be added to a product, then safe history of human usage,
scientific statements by academia, acute or chronic safety and toxicology studies, and
82 N. Kapoor et al.

limited human trials may be required. Similarly, in Japan FoSHU (Food for Specific
Health Uses) system was introduced in 1991 by the Ministry of Health and Welfare,
now known as the Ministry of Health, Labor, and Welfare (MHLW), as a regulatory
system to approve statements concerning the effects of the food on the human body
(Shimizu 2003). So, for the approval of food supplements with health-promoting
activities, only requirements by FoSHU are safety of food, nutritional value of food
ingredients, etc., even if these perpetrated pharmacological activities are not vali-
dated with any scientific proof (Santini et al. 2018; Saito 2007). In any countries,
like in Australia or China, the nutraceuticals are regulated mainly as a category of
food (Tee et al. 2002; Tapsell 2008; Yang 2008). Detailed evaluation of nutraceutical
product with clinical trial as well as safety assessment study should be carried out
before it is marketed. A health claim substantiated with safety and efficacy data,
based on an understanding of the mode of action and the absence of any unwanted
side effects, further validated through clinical evidence should ideally be required,
in the interest of public safety, for a nutraceutical product to be approved.

4 Business Outlook

Due to the increasing prevalence of lifestyle diseases and rising awareness regarding
preventive healthcare measures, the demand of nutraceutical has soared worldwide,
during the past two decades. Initially, from 1999 to 2002 the nutraceutical industry
grew at 7% per year, the next few years up to 2010 saw almost twice that growth
at 14% per annum (Verma and Popli 2018). In 2017, the nutraceutical market is
estimated to be worth $379 billion. It has been predicted that globally the nutraceuti-
cal market was $230.9 billion in 2018 which is expected to reach $336.1 billion
by 2023 at a compound annual growth rate (CAGR) of 7.8%, according to the
BCC research report Nutraceuticals: Global Market to 2023 (www.bccresearch.com/
market-research/food…nutraceuticals-global-markets). Consumption of dietary sup-
plements is expected to rise at a CAGR of over 9.7% from 2017 to 2025 worldwide
due to the increasing awareness about preventive health care. These supplements are
available in various forms like herbal extracts, tablets, capsules, powders, etc. Their
low-cost, easy over-the-counter accessibility, and health-promoting properties are
the main driving factors anticipated to increase their demand over the next few years
(www.grandviewresearch.com/industry-analysis/nutraceuticals-market). In Europe
also the nutraceutical market is dominated by the dietary supplement with the mar-
ket share of 30.1% in 2016 and is predicted to grow at a CAGR of 6.4% from 2017 to
2025. The dietary supplements market, comprising vitamins, are the most common
supplement consumed by the people and account for 46.8% out of all total dietary
supplements followed by the herbal extract with an estimated growth at a CAGR of
9.8%. The nutraceuticals comprising proteins and amino acids may grow at a CAGR
9.4% from 2017 to 2025 (www.figlobal.com). In Africa, the infant nutrition industry
and the demand for nutritious food and beverages are the key drivers for the nutraceu-
tical market’s growth. Countries, like Egypt and South Africa, are trying to acquire
The Rise of Nutraceuticals: Overview and Future 83

more herb-based nutraceutical due to the larger acceptability. This may offer
good market opportunity in the Africa. However, the microalgae-based omega-3-
fatty acid is the new emerging field in the nutraceutical market of Africa (www.
mordorintelligence.com). Vitamins and minerals dominated the Chinese market fol-
lowed by the herbal supplements segment. According to the report Indian Nutraceu-
ticals Market Outlook: Vision 2022 published by ASSOCHAM and RNCOS, the
Indian dietary supplement market was valued at US$2.8 billion in 2015 which is
expected to reach a value of US$8.5 billion by 2022. The nutraceutical market for
herbal supplements in India was valued at approximately US$0.6 billion in 2015,
which is expected to reach a value of US$1.7 billion by 2022. The vitamin and min-
eral market in India is expected to grow in the coming years and estimated to reach at
a value of US$2.1 billion by 2022. Similarly, proteins and amino acids are an impor-
tant class of supplements that have gained a lot of popularity in the recent years. From
2015 to 2022, the market for these supplements is expected to rise from US$0.4 bil-
lion to US$1.09 billion by 2022. The market for the dietary supplements including
fatty acids and antioxidants in India was valued at approximately US$0.1 billion in
2015 and is estimated to reach a value of US$0.23 billion by 2022. In 2015, the
market for functional foods like oats, soy, probiotic yogurt, fortified baked goods,
and fortified edible oils was valued at US$0.7 billion and expected to rise at worth of
US$2 billion by 2022. Similarly, the market for functional beverages was US$0.3 bil-
lion in 2015 and predicted to reach an approximate value of US$1.1 billion by 2022
(www.assocham.org/newsdetail.php?id=6259).
Thus, in conclusion, it may be summarized that increasing awareness, posi-
tive public perception, and ever-increasing medical costs may push the market of
nutraceuticals which are perceived as one of the major drivers of preventive health
care. The market and hence business opportunities for nutraceutical products may
continuously show an upward trend for next few years. Indeed in countries such as
India, a lot of start-up countries are focusing on this segment. However, with such a
high growth, there is a need for strong regulatory setup so as to protect the interest
of consumers.

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Nitika Kapoor Masters in Human Genetics. Awardee of ICMR Junior and Senior research fellow-
ships. Presently pursuing Ph.D. at CSIR-Indian Institute of Integrative Medicine, Jammu. Work-
ing on regulatory aspects of secondary metabolism in medicinal plants, employing cutting edge
molecular tools, transcriptomics, metabolomics, etc.
92 N. Kapoor et al.

Vijay Lakshmi Jamwal Masters in Human Genetics. Presently pursuing Ph.D. at AcSIR.
Working at CSIR-Indian Institute of Integrative Medicine (Jammu), towards understanding the
physiological and ecological roles of secondary metabolites in high value medicinal plants.
Trained in molecular tools such as CRISPR-Cas9, transcriptomics, metagenomics, metabolomics,
transgenics, etc.

Manish R. Shukla With over seventeen years of experience in contributing and leading various
research and commercial areas of Biotechnology worked as a Senior Scientist in TIFR, Mumbai
establishing various protein analytics platforms and researched on various aspects of Algal stress
metabolism. During his PhD from MSU Vadodara and Postdoctoral studies at Louisiana State Uni-
versity, USA, under the aegis of the NASA NSF, added deeper understanding in the areas of bacte-
rial radiation resistance and their potential to Mars survival. In Reliance R&D, he has established
platforms to develop robust understandings on the metabolomics and proteomics of Algae, to har-
ness and optimize their productivity for commercial use. Further Dr Shukla is driving the potential
of Algal technology proficiency at Reliance R&D for various food and Ingredients segment.

Sumit G. Gandhi Working as senior faculty (Principal Scientist) at CSIR-IIIM (Jammu), on var-
ious aspects of secondary metabolism in microorganisms and medicinal & aromatic plants, since
more than a decade. PhD (JNU, Delhi) at CCMB (Hyderabad) focused on understanding molecu-
lar effectors and mechanisms of epigenetic regulation. Bachelors & Masters in Microbiology. Dr.
Gandhi has resourcefully collaborated with industries and academia, on various projects and has
been a recipient of grants from public and private sectors.
Biofuels
Algae Biodiesel: Fundamentals
and Future Prospects

Ranjana Bhati

Abstract Global economy has been benefited from fossil fuels in the past but at
the cost of increasing CO2 level and other environmental hazards. Fast-growing con-
cern about global warming, exhaustion of nonrenewable energy reserves and rising
cost of petroleum-derived fuels, led to quest for other sustainable renewable energy
alternatives. In current scenario, renewable and environment-friendly biofuel pro-
duction is the only sustainable alternative to shrinking petrodiesel reserves. Over
the years, biodiesel has grabbed the most focus as a potential liquid biofuel. First-
generation biodiesel was produced mainly from edible vegetable oils and provoke
large number of debate, primarily owing to competition with overall food produc-
tion. Subsequently, second-generation biodiesel was produced by using nonedible oil
sources like karanja, jatropha and mahua oils. First- and second-generation biodiesel
have demerits, mainly the expensive set up, land requirements for plants cultivation
and competition with net food production. Moreover, biodiesel derived from these
sources could not practically fulfill the small part of current transport fuels require-
ments. Thus, the focus of researchers has been shifted to the third-generation biofuel
from microalgae, which is highly promising. Utilization of microalgae for different
types of renewable biofuel production is having diverse benefits of overcoming the
energy crisis and environmental pollution control. Microalgae are photosynthetic
eukaryotes and capable to grow in different growth conditions with CO2 biofixation.
Microalgae are 10–50 times efficient in solar energy capture than plants and also
higher in biomass accumulation than energy crops. Algae can grow in diverse aquatic
habitat and on land that is agriculturally barren, therefore, no arable land competition
for food and feed. The key attraction of biodiesel production with microalgae lies in
their ability to tolerate high CO2 concentration. Moreover, wastewater (rich in nutri-
ents) can be effectively utilized for microalgae cultivations and subsequently results
in wastewater recycling. Biodiesel from algae appears to be the most economic and
sustainable energy source with the CO2 utilization and wastewater recycling.

Keywords Algae · Biofuel · Biodiesel · Lipid productivity · Photobioreactors

R. Bhati (B)
Department of Microbiology, Bundelkhand University, Jhansi, Uttar Pradesh 284128, India

© Springer Nature Switzerland AG 2020 95

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_5
96 R. Bhati

1 Introduction

Ever-increasing energy demand, concern about climate change and exhaustion of

fossil fuels has driven the interest to look for eco-friendly and sustainable energy
resources. Due to environmental hazards imposed by the elevated CO2 level in the
atmosphere, United Nations (1997) introduced the Kyoto Protocol to lower the col-
lective greenhouse gases emission by 5.2% as compared to the emission in 1990, and
this protocol was ratified by more than 170 countries (Gutiérrez et al. 2008; Wang
et al. 2008; Ho et al. 2011). In addition, ‘carbon credit’ system with an approximate
unit price of US $270/ton was proposed by United Nations in 2010 (Stewart and Hes-
sami 2005). Further at the climate change convention (Copenhagen 2010), nations
decided to grant about US $100 billion for greenhouse reduction by 2020 (Kintisch
2010; Ho et al. 2011).
Over the last few years, immense uses of fossil fuels to fulfill the energy demand
impose serious environmental hazards by rising CO2 concentration in the atmosphere.
In 2012, total CO2 emission was 36% of total global emissions generated by the
utilization of liquid fuels and predicted that by the year 2035, emission of CO2 would
be doubled (EIA 2016). Owning to this, the European Union Renewable Energy
Directive (RED) advocates that by 2020, 15% of the total energy demand supplied to
the UK should be derived from renewable resources (Gul 2016; Adeniyi et al. 2018).
Currently, 80% of energy requirement is derived from fossils fuels. According to
these energy requirements, it is projected that after 2050, conventional oil reserves
will be completely exhausted (Demirbas and Demirbas 2011; Ullah et al. 2014). So,
the development of carbon neural, renewable and eco-friendly energy resources to
overcome the energy crisis is the need of the hour. Research and development efforts
are now, therefore, directed to store, fix and utilize CO2 in order to minimize its
release into the atmosphere. The potential liquid biofuel that has attracted the most
attention recently is biodiesel (Chisti 2007).
This chapter gives a detailed insight on algae as a potent host for biodiesel produc-
tion. The chapter focuses on different microalgae biodiesel production aspects that
lead to sustainable and cost-effective production process. Various cultivation systems
of algae, harvesting and downstream processes for biomass recovery are discussed
in detail. Lipid content with biodiesel production and major biofuel manufacturers’
are described. Detailed insight on technical challenges and economic feasibility of
commercial algal biodiesel production process are discussed with cost comparison
to fossil fuels.

2 Biodiesel: Introduction

Biodiesel is chemically monoalkylester made by transesterification reaction of free

fatty acids with alcohols and obtained from renewable resources like vegetable oils
and animal fats. Biodiesel is ecologically less toxic than petroleum-based diesel and
Algae Biodiesel: Fundamentals and Future Prospects 97

Fig. 1 Algal biodiesel production by transesterification, R1-3 are hydrocarbon group (Chisti 2007)

has chemical and physical properties similar to petrodiesel (Chisti 2007; Demirbas
2011; Adeniyi et al. 2018) (Fig. 1).
Biodiesel has received immense interest in past few years as the sustainable choice
to petroleum diesel fuel due to its renewability, nontoxic and eco-friendly character-
istics (Krawczyk 1996). Biodiesel has comparable conventional diesel engine perfor-
mance to petrodiesel and reduces the emissions of pollutants (100% less sulfur diox-
ide, 84% less particulate matter, 46% less carbon monoxide and 37% less unburned
hydrocarbons emissions) as compared to conventional diesel fuel (McMillen et al.
2005).
The first-generation biodiesel is produced from feedstock of edible oils like rape-
seed and soybean, generated a lot of conflicts due to food and feed competitions.
The second-generation biodiesel is produced by using nonedible oil sources. The
first- and second-generation biodiesel based on terrestrial plants are potentially com-
peting with net food production (Chisti 2008). The focus of researchers has now
been shifted to the next-generation biodiesel. The third-generation biodiesel is both
promising and different, and it is based on simple microscopic organisms that live in
aquatic habitat, i.e., microalgae.

3 Microalgae as Host for Biodiesel Production

Microalgae are receiving much importance as a potential candidate for CO2 seques-
tration and alternative host for renewable energy production. Algae are photosyn-
thetic organisms that can be cultivated in various environmental conditions with
ability to fix CO2 . The benefits of using microalgae for biofuels production lie in
their capacity to tolerate high CO2 concentration, allowing efficient CO2 capturing
(CO2 content 5–15%) (Hsueh et al. 2007). On an estimate, 1.83 kg of CO2 is utilized
for the production of 1 kg of algal biomass (dry cell weight) (Chisti 2007). Algae have
high solar energy capture efficiency, faster growth and higher biomass production in
comparison with energy crops (Wang et al. 2008; Brennan and Owende 2010). Algae
can grow in diverse aquatic habitat and on land that is agriculturally barren. There-
fore, algae do not affect the crop cultivation system and no arable land competition
for feed and food (Brown and Zeiler 1993; Aresta et al. 2005). Moreover, algae can
successfully utilize and grow in high nutrient concentrations of wastewater and have
98 R. Bhati

dual potential for waste treatment as they can utilize waste nutrients for growth and
in turn eliminate N and P from the wastewaters (Mallick 2002). This indicates that
algae are noncompetitive with other users for freshwater, thus reducing the nutrients
costs and preserving the valuable freshwater resources (Campbell 2008) (Fig. 2).

Fig. 2 Overview of biodiesel production with algae

Algae Biodiesel: Fundamentals and Future Prospects 99

4 Microalgae Lipid Content

Different microalgae species accumulate distinct ratios of proteins, lipids and car-
bohydrates. Microalgae lipid accumulation ought to be high to attain the economic
performance (Xu et al. 2006). Most algal species can be triggered to synthesize size-
able amount of lipids, resulting in high oil yield. The typical reported lipid content
ranges from 1 and 70%; however, with specific growth conditions, lipid accumulation
reached up to 90% (dcw) (Chisti 2007; Li et al. 2011). Lipid productivity in many
microalgal species can be increased by physiological stresses such as nutrient defi-
ciencies/limitations, varying physical parameters (light intensity, pH temperature),
mode of nutrition (phototrophic, heterotrophic and photoheterotrophic mixotrophic)
and optimization of medium composition (carbon substrate, salts, phosphorus, nitro-
gen and vitamins). Lipids are then converted to biofuel through a transesterification
reaction (Chisti 2007). Brown et al. (1969) reported an oil yield of 86% (dcw) from a
bloom of Botryococcusbraunii in a freshwater lake. But a very slow growth rate is the
main hindrance in exploring B. braunii as the commercial organism for the produc-
tion of biodiesel (Dayananda et al. 2007). Scenedesmussp. strain JPCC GA0024 was
found to accumulate lipid up to 73% (dcw) lipid content when grown in artificial sea-
water (Matsunaga et al. 2009). Glucose supplementation in growth media resulted
in lipid accumulation up to 61% (dcw) by Chlamydomonas reinhardtii CC1010
(Karpagam et al. 2015). Extensive research has been done for enhancing the lipid
production by varying the cultural and physical conditions. Table 1 summarizes some
significant attempts to increase the lipid yield of microalgal species with different
growth conditions.

5 Microalgae Cultivation

Algae can grow anywhere with abundant sunlight, carbon source, water, micro and
macronutrients with optimum temperature. Algal cultivation for biomass has several
advantages like higher oil percentage than other sources. Second, production of
extremely large amount of biomass and this algal biomass production has limited
food and feed competition in market. Moreover, algae can grow with brackish water,
seawater and freshwater resources (Campbell 2008).
For enhanced production of biodiesel, type of cultivation system for the growth of
algae is a significant factor. Cultivation systems for algal growth are broadly divided
into types: open-pond system and closed controlled systems. Open-pond system is
the oldest, simplest and cost-effective method for mass cultivation of algae. Raceway
pond is defined as a closed oval loop, ranges in depth from 20 to 30 cm. They are
incorporated with paddle wheel to mix the water and avoid culture settling and min-
imizing the shading effect. These are generally kept shallow for sufficient sunlight
penetration. There are several disadvantages associated with raceway pond: little pro-
ductivity as compared to photo bioreactors (Brennan and Owende 2010; Rawat et al.
100 R. Bhati

Table 1 Increase lipid content of diverse microalgae species with different growth conditions
Microalgal species Lipid Growth conditions References
content (%
dry cell
weight)
Botryococcusbraunii 86 Nitrogen limitation Brown et al.
(1969)
Chlorella vulgaris 53 Nitrogen limitation Piorreck and
Pohl (1984)
Chlorella protothecoides 58 Heterotrophy (0.1% Miao and Wu
glucose with reduced (2004)
nitrogen)
Chlorella sp. 38 Heterotrophy (0.1% Feng et al.
glucose with sodium (2005)
thiosulphate
supplementations)
Chlorellaprotothecoides 55 Heterotrophy with Xu et al. (2006)
corn powder
hydrolysate +
nitrogen limitation
Scenedesmussp. strain JPCC 73 Phototrophy with Matsunaga et al.
GA0024 artificial seawater (2009)
Scenedesmusobliquus 39 Carbon dioxide Ho et al. (2010)
Chlorella zofingiensis 55 Nitrogen limitation Feng et al.
(2011)
Chlorella vulgaris ESP-31 56 Photoautotrophic Yeh and Chang
(2011)
Chlorella vulgaris 57 Nitrogen, phosphorus Mallick et al.
and iron limitations (2012)
Chlorella sorokiniana 32 Glucose Li et al. (2013)
Chlorella sp. HQ 63 Photoautotrophic Zhang et al.
(2014)
Chlorococcumlittorale 48 Carbon dioxide Ota et al. (2015)
ChlamydomonasreinhardtiiCC1010 61 Glucose Karpagam et al.
(2015)
Scenedemusobliquus (SAG 34 Carbon dioxide with Arbib et al.
276-10) wastewater (2017)
Dunaliellatertiolecta 21 Carbon dioxide with Kumar et al.
Nacl and NaOH (2018)
supplementation
Selenastrum sp. GA66 49 Nacl and nitrate Chakravarty and
supplementation Mallick (2019)
Algae Biodiesel: Fundamentals and Future Prospects 101

2013; Slade and Bauen 2013). Other demerits are poor light utilization, uncontrolled
temperature, contamination by predator and heterotrophs, evaporation loss and CO2
dispersion into the atmosphere. However, cultivation with open-pond system aids in
wastewater treatment and requires fewer amounts of energy and manpower (Ugwu
et al. 2008; Brennan and Owende 2010).
Closed controlled systems are mainly photobioreactors of diverse shapes as flat
plate, tubular and column. In PBRs, the culture medium and required nutrients for
algal growth are circulated from a central reservoir and light, and pH level is main-
tained in a controlled process. Though the PBR offers improved control of culture
conditions, they are much costlier than natural cultivation. Auxiliary energy demand
and capital cost are also higher (Chisti 2007; Brennan and Owende 2010; Slade and
Bauen 2013).
To overcome the disadvantages associated with these two cultivation systems,
hybrid system is introduced. The hybrid system is a two-stage cultivation method
coupling the different growth stages of photobioreactor with open ponds. Higher
biomass concentration and culture purity maintenances are achieved with photo-
bioreactor during first growth stage. The second growth stage is attempted with
raceway pond in which algal cells are subjected to nutrient limitations, which boost
up the desired lipid accumulation (Huntley and Redalje 2007; Rodolfi et al. 2008).
Raceway ponds are suitable as algal cells are subjected for natural environmental
stress to increase lipid yield.

6 Harvesting Methods of Microalgae Biomass

Removal of algae biomass from broth is known as biomass harvesting. It is a difficult

and problematic task due to microscopic size of algal cells (Grima et al. 2003). A
suitable harvesting method is required to remove large water content and process-
ing of high volumes of algal biomass since microalgae broth is relatively diluted
(0.5–5 kg m−3 dry weight). Choice of best harvesting method depends on microal-
gal characteristics, e.g., size, density of microalgae and significance of the desired
products (Grima et al. 2003; Rawat et al. 2013). 20–30% the total biomass produc-
tion cost is incurred solely by algal biomass recovery from broth (Mata et al. 2010).
Biomass processing for biodiesel production is a significant step which influences
the overall usefulness of product. For economical biodiesel production, suitable,
energy-efficient and cost-effective harvesting methods are required. Centrifugation,
filtration, ultrafiltration, sedimentation and flocculation are the most common har-
vesting methods. Belt filtering, microstraining, sedimentation and flotation with float
collection are the four harvesting methods studied by Weissman and Goebel (1987).
These methods principally work on difference on a size and density for biomass
separation.
Flocculations increase the particle size and accelerate the sedimentation process.
Sedimentation process can be fastened by the addition of chemical flocculants such
as ferric chloride, alum and lime, but they are costly for large-scale operations and
102 R. Bhati

not eco-friendly sustainable alternatives. Nontoxic and low-cost flocculants could

be the best choices. Due to these demerits, flocculation is not widely used method
for cost-effective and efficient production (Grima et al. 2003; Greenwell et al. 2010;
Rawat et al. 2013).
Centrifugation is the most frequently used method for harvesting of algal biomass
where quick algal biomass separation is performed by increase in gravitational accel-
eration, thereby resulting in harvesting biomass with >95% efficiency at 13000 g
(Greenwell et al. 2010). As a result, for 15% total suspended solids, 150 times
increase in slurry concentration is practically feasible (Mohn 1980). This technique
is easy, quick, energy-intense, efficient and nondisruptive for high-value intracellular
products. The disadvantages associated with the process are high power expenses
and maintenance requirements, consequently increasing the production cost.
Conventional filtration is the most appropriate harvesting process for big size algae
such as Spirulina. Principally, it works under pressure and filtration aided by cellulose
or diatomaceous earth to enhance the effectiveness (Mohn 1980). Membrane micro-
filtration and ultrafiltration (a type of membrane filtration with hydrostatic pressure)
are technically feasible alternatives to conventional filtration and used for smaller size
algae suspension (<30 mm) (Petrusevski et al. 1995; Brennan and Owende 2010).
Microfiltration is more competent and appropriate method for fragile microalgal cells
harvesting (Grima et al. 2003). The key limitations of membrane filtration processes
are membrane replacement cost and pumping in larger production scales results in
high cost of process. Membrane filtration’ operational costs are low as compared
to centrifugation technique, thus making the technology more lucrative (Greenwell
et al. 2010).
Gravity sedimentation is the most frequently used and simplest harvesting tech-
nique for algae biomass treatment in large volumes of water and wastewater cultures.
However, process is accepted as suitable method for large microalgae such as Spir-
ulina. The process is cost effective, simple, and only a settling tank is required for
large-scale biomass harvesting. The major disadvantages associated with this process
are time consumption, ineffective for small size algae and separation required rela-
tively longer settling time (Grima et al. 2003; Chen et al. 2011). Due to high energy
requirement and operation cost, filtration and centrifugation appear to be very far
from commercial applications. However, mass harvesting methods like flocculation
offer a promising alternative with lower energy input and low cost.

7 Biodiesel Production

For biodiesel production, microalgal biomass needs to be processed for lipids and
fatty acids extraction. Several methods are used by researchers for extraction of lipids.
Extraction of lipid is expensive and one of the extensively discussed processes for
biodiesel production. Large number of microalgae grown in diverse aquatic habitat
produced neutral lipids due to their lower degree of unsaturation. These lipids are
ideal candidate for conversion to biodiesel (Viswanath et al. 2010).
Algae Biodiesel: Fundamentals and Future Prospects 103

Choice of the suitable lipid extraction methods depends on accuracy, efficiency,

cost effectiveness, ease to carry out, reproducibility and precision of the method
employed. First algal cells must be disrupted chemically or mechanically for lipid
extraction. Bligh and Dyer method, Folch method and gravimetric method are widely
reported for lipid extraction. A number of effective chemical solvent extraction meth-
ods such as ethanol, methanol, n-hexane and mixed polar/nonpolar chemical solvents
(e.g., chloroform/methanol) are also available, and efficiency of extraction is reliant
on algal species (Halim et al. 2012).
A novel method for lipid extraction from diluted algal cultures employing switch-
able polarity solvents (SPS) was developed by Samorì et al. (2013). Addition of CO2
into the extracting SPS results in 70–80% efficient lipid recovery. Use of wet algal
biomass for lipid extraction reduces the power consumption required for dewater-
ing process of biomass. Ideal lipid extraction method should be highly specific and
selective for algal lipids and should circumvent the co-extraction of other different
compounds like carbohydrates and protein (Halim et al. 2012). The Bligh and Dyer’s
(1959) lipid extraction method is the best and extensively used method for total lipid
extraction from microalgae where precipitation of proteins takes place in the mid-
dle of the two distinct liquid phases. This is widely accepted method and used for
large-scale lipid extraction (Rawat et al. 2013).
After lipid extraction processes, algal oil is converted into biodiesel through trans-
esterification. Biodiesel is mono-esters that are produced by transesterification, a
chemical reaction between triglycerides and alcohol in the presence of a catalyst
(Chisti 2007; Demirbas 2011; Adeniyi et al. 2018). Transesterification is commonly
used to reduce the viscosity of bio-oils and converting them to biodiesel. Since crude
algal oil is highly viscous, so their conversion to fatty acid alkyl esters (low in molec-
ular weight) is required (Demirbas 2008). Due to gravity separation, two layers are
formed at the end of the end of the reaction, biodiesel is separated at upper layer
(the main product), while glycerol is at bottom layer (by-product). This process was
followed by several purification steps like washing with water and evaporation to
obtain high purity and quality biodiesel (Demirbas 2008).
Culture conditions play a significant role in determining the lipid and fatty acid
contents of microalgae. Algae contain both saturated and monounsaturated fatty
acids, and high proportion of these fatty acids is desirable for good quality of fuel
(Sheehan et al. 1998; Demirbas 2011). Finally, a number of companies are investing
in algae biofuel research, funding on the research and development in order to make
biofuel from algae—a commercial reality. Table 2 presents a list of algae biofuel
manufacturers with location.

8 Techno-Economic Analysis

Economic concern and achieving the desired revenue are the important factors for
any production plant. Algae biodiesel presents a promising environment-friendly
production system; however, a number of barriers should be taken into account. These
104 R. Bhati

Table 2 Major algae biofuel producing companies (Chisti and Yan 2011)
Manufacturers Biofuel types Locations Website
AFS BiooilTM Biodiesel South San Francisco, www.afsbiooil.com
USA
Algenol Biofuels Biodiesel, ethanol, Florida, USA www.algenol.com
gasoline, jet fuel
Bionavitas, Inc. Biofuel Redmond, www.bionavitas.com
Washington USA
Petroalgae Inc. Algae oil New York, USA www.petroalgae.com
Poet LLC Bioethanol South Dakota, USA www.poet.com
Sapphire Energy Biofuel, green crude San Diego, sapphireenergy.com
Californian USA
Solix Biofuels Biofuel Fort Collins, USA www.solixbiofuels.
com
Seambiotic Ltd. Biodiesel, ethanol Israel www.seambiotic.com
Synthetic genomics Biodiesel La Jolla, CA, USA www.
incorporation syntheticgenomics.
com
Terra Via Holdings, Algae oil South San Francisco, http://terravia.com
Inc USA
(formlySolazyme)

barriers include high energy demand (to supply water and CO2 and for mixing), heat
requirement (for biomass drying) and nutrient requirements (carbon, phosphorus and
nitrogen) (Beal et al. 2015). Biodiesel production process needs to be efficient by
reducing the huge cost incurred by biomass production and downstream processing.
Further, biodiesel cost depends on choice of algal strain, oil content, microalgae
yield, over head cost, plant capacity, plant location and design. Biodiesel production
with soya oil or rapeseed enhances the cost of production since feedstock cost is
the largest expense. Further, considerable reduction in biodiesel cost is obtained by
exploitation of feedstock that is nonedible like jatropha oil. By financial analysis of
biodiesel production cost, it has been found that ethyl esters production costs from
jatropha is to be around 0.40 e per litre (Nevase et al. 2012), however ethyl esters
production costs from palm oil is 0.57 e per litre (Chisti 2007)
It has been found that the biomass production costs is the only relevant factor
after comparative evaluation of microalgae biodiesel production from photobiore-
actors and raceways. Capital investments for open-pond systems are ten times less
than photobioreactors (Chisti 2007). Biomass recovery cost for broth produced in
raceways is much higher than the cost of biomass recovery for broth produced by pho-
tobioreactors. The concentration of biomass produced in raceways is approx 30 times
less than concentration of biomass produced in photobioreactors (Chisti 2007). In
open ponds, typical microalgae productivity is 30–50 t/ha/y (Benemann and Oswald
1996; Sheehan et al. 1998). For open-pond systems and photobioreactors, estimated
cost of production from algal biomass is ($10/kg) and ($30–$70/kg), respectively.
Algae Biodiesel: Fundamentals and Future Prospects 105

When compared with conventional agricultural biomass, this cost is higher in two
and more than three order of magnitude, respectively (Carlsson et al. 2007). Pre-
sume that 30% of biomass weight is oil, free of cost CO2 is available (flue gas), and
estimated production cost for one liter of oil from raceway ponds and photobiore-
actors is $1.81 and $1.40, respectively (Chisti 2007). Moreover, for biodiesel to be
cost competitive with petrodiesel, algal oil cost should be less than $0.48/L (Chisti
2007; Demirbas and Demirbas 2011). Despite the high biomass productivity, algae
biodiesel production is still not economically competitive with petrodiesel, and these
costs are too high to address the current energy market.

9 Conclusion and Future Perspectives

To fulfill the growing energy requirement of mankind, algae biodiesel is cost effec-
tive, biodegradable and renewable alternative while sustaining growth. Algae are the
fastest-growing eukaryotes containing 50% of oil by weight. It is noteworthy here
that a number of reports for biodiesel production at laboratory scale or pilot scale
are available, but reports for large-scale studies are scarce. Though closed systems
are quite successful for biodiesel production, large-scale production and operation
of closed systems are the need of the hour.
A multidisciplinary approach involving mass algal cultivation with the utiliza-
tion of inexpensive sources like wastewaters, and CO2 from flue gas, coupled with
the extraction of value-added products is required make the process cost effective
and more economical. Most importantly, lucrative and power-competent harvesting
methods are also necessary to make the entire biodiesel production process more
efficient. Furthermore, algal biofuel production systems are in an early development
stage, and for approaching commercial profitability, extensive research is required to
explore the new approaches that could increase the productivity and lower the pro-
duction cost. Nevertheless, biodiesel forms algae coupled with CO2 sequestration,
and wastewater recycling is the best alternative that will contribute to environment
sustainability and future energy security.

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719

Ranjana Bhati is working as Assistant Professor at Department of microbiology, Bundelkhand

University, Jhansi. She has completed her doctorate degree from Indian Institute of Technology,
Kharagpur, in Algal Biotechnology. She has been awarded Dr. D. S. Kothari Postdoctoral Fellow-
ship (UGC) at Jawaharlal Nehru University, New Delhi. Her area of research is Algal Biotechnol-
ogy, Biofuels, Biopolymers, Applied and Environmental Microbiology. She has published several
book chapters and research papers in referred to national and international journals.
Biotechnology of Biofuels: Historical
Overview, Business Outlook and Future
Perspectives

Vijay Lakshmi Jamwal, Nitika Kapoor and Sumit G. Gandhi

Abstract Continued reliance on fossil fuels is a major risk to energy security, and
its extraction from natural reserves poses serious environmental threats. Exploration
of new and renewable sources of energy is important to mitigate these concerns. In
recent times, biofuels have come up as an inexhaustible and alternative source of
energy which may be produced from plants or algae. Past 3–4 decades have seen a
serious effort in this area and broadly can be classified in three generations of bio-
fuels. Commonly cultivated crop plants served as an energy base for the production
of the first generation of biofuel. However, this led to increase in food prices which
had a serious negative impact on the third world countries. The second generation
of biofuels was produced from non-crop high energy plants. However, both of these
were dependent on agriculture, which requires high cultivable land reserves, con-
siderable human resource involvement, irrigation facilities, is severely impacted by
changes in rainfall patterns and weather conditions as well as biotic factors such
as pathogen infections. In contrast, the third-generation biofuels are produced from
microalgae which overcome the disadvantages of first- and second-generation biofu-
els. Microalgae are regarded to be an attractive source for energy due to its biomass
productivity (dry weight per unit time per unit area) that is much higher than those of
higher plants. Globally, several private industries and government organizations are
involved in research and development of biofuels as alternative sources of energy.
Governments around the world have provided subsidies in different forms for the
production of biofuels. Brazil has come up as a world leader in sustainable biofuel
production, with most automobiles running on bioethanol or bioethanol blends. Over
the years, the use of biofuel has continuously increased in most countries. Recently,
the increased pumping out of fossil fuels from Middle Eastern countries has led to a
global decrease in crude oil putting pressure on alternative sources of fuels. However,
this may not be sustainable in long term, and continued research on biofuels is nec-
essary for future energy security. Present research trends include the isolation of new
algae, with higher biomass production and oil accumulation. Further, transgenesis
in algae has made it possible to increase photosynthetic rate, re-route metabolism

V. L. Jamwal · N. Kapoor · S. G. Gandhi (B)

Plant Biotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu
180001, India
e-mail: [email protected]

© Springer Nature Switzerland AG 2020 109

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_6
110 V. L. Jamwal et al.

toward higher oil production, make the algal membranes and cell walls withstand
high oil, etc.

Keywords Biofuels · Fermentation · Fossil fuels · Metabolic engineering ·

Microalgae · Transesterification · Plant oil

1 Introduction

1.1 World Energy Requirement

Fossil fuels (coal, gas and oil) contribute more than 80% of the world’s primary
energy supply (Council WE 2016). These sources of energy are used for generat-
ing power, electricity, energy and heat for different purposes including industrial,
domestic, commercial and transportation. Developing countries require to expand
their economies with increase in population which results in voracious demand for
fossil fuels. Greenhouse gases (CO2 ) are released from the combustion of fossil
fuels that cause global warming. According to International Energy Agency (IEA),
there is a need to change the world’s existing energy economy. It has been estimated
that world’s CO2 emission will increase by 50% up to the year 2030. Nations are
attempting to modify their existing energy mix and reduce reliance on fossil fuels
so as to reduce the CO2 emissions. International agreements by governments are
turning to new forms of renewable energy, such as biomass, biofuels, geothermal,
hydro, solar and wind, in order to reduce greenhouse gas emissions (Fig. 1). The IEA
predicted in 2017 that the global capacity of renewable electricity will expand by
over 920 GW between 2017 and 2022, with solar power comprising the largest com-
ponent of the increase (Wold Energy Outlook 2017). The consumption of renewable
energy sources for transportation is expected to elevate from over 4% in 2016 to 5%
in 2022. Biofuels are expected to stand for over 90% of total renewable energy for
transportation in 2022.

1.2 Conventional Sources of Energy

Conventional or non-renewable resources are not naturally replenished once they

have been used. Non-renewable resources, such as fossil fuels, become economically
inaccessible when they are excessively consumed (Khan 2006). The residues of
living organisms were subject to high temperature and pressure over millions of
years, which lead to the formation of fossil fuels. There are three types of fossil fuels
used as source of energy: coal, oil and natural gas (Bertine and Goldberg 1971).
Coal is a solid fossil fuel formed by the decomposition of plant remains over several
thousands of years. Comparing the two types of fossil fuels, coal is relatively profuse.
It is expected that the current coal supplies could last for more than 200 years. The
Biotechnology of Biofuels: Historical Overview … 111

DomesƟc Industries
Fossil fuel
Coal, Petroleum and
Natural gas
Transport
Uses
Non
renewable

Electricity

Renewable
Sources of
Biomass, Hydro, Solar
Bio-fuels
Energy
(Plant oil, Bioethanol,
Biodiesel, Biogas)

Fig. 1 Renewable and non-renewable sources of energy

consumption of coal has increased to double since the middle of 20th century. Many
developing countries including China and India depend on coal for their energy
supply (Bhattacharya and Jana 2009).
Petroleum oil is liquid fossil fuel that is formed from the decomposition of remains
of animals and microorganisms under high pressure and high temperature. Oil is
trapped in small spaces between the rocks and sediments. It is extracted by large
drilling platforms (Vassoyevich 1967). Petroleum consists of mixture of various con-
stituents such as petroleum gas, diesel, petrol and lubricating oil, which are separated
by refining it (Meinschein 1959). Natural gas is a fossil fuel that is adaptable and
comparatively cleaner than the other two forms of fossil fuel. In developed countries,
use of natural gas has overtaken coal. Natural gas mainly consists of methane (CH4 ).
The reserves of natural gas are distributed equally over the globe as compared to the
oil.

1.3 Depletion of Fossil Fuel Reserves and Environmental

Impact

Growth in world’s population and demand for urbanization, especially in underde-

veloped and developing countries, has resulted in an extensive use of fossil fuels.
The reckless wastage or suboptimal use of energy adds to the growing worry of fossil
112 V. L. Jamwal et al.

fuel extinction. With the rising fear of fossil fuels extinction, their prices have risen
inexplicably. Several countries depend on importing fossil fuels, with some of them
giving subsidies for public use. This puts excessive pressure on the nation’s economy
(Shafiee and Topal 2010). Excessive use of fossil fuels results in release of green-
house gases in environment which cause global warming. The major greenhouse gas
is carbon dioxide (CO2 ), and 70–75% is emitted out from fossil fuel combustion
(Davis and Caldeira 2010). These atmospheric changes in turn generate extensive
climate changes globally. Melting of the world’s glaciers and ice sheets due to rising
temperatures is a major concern. This could lead to rise in sea levels, which could
trigger significant population and infrastructure dislocations in the coming century.
Further, use of fossil fuels is associated with increasing air pollution, which neg-
atively affects the health of individuals (Hoel and Kverndokk 1996). This in turn
adds further pressure on nation’s health economy. Energy sources which can substi-
tute fossil fuels and do not affect the environment negatively are renewable energy
sources including solar energy, hydro energy, etc. Further, biofuels have come up
as a relatively cleaner and renewable energy source, which will be the focus of this
chapter.

1.4 Biofuels

Biofuels are fuels derived from biomass which comprises wood, agricultural crops
and products, forest products, wastes and residues. Biofuels may be solid, liquid
or gaseous. Solid biofuels include wood, charcoal, bagasse, etc. (Obernberger et al.
2006). Gaseous biofuels include methane gas which is produced from anaerobic
fermentation of domestic waste, animal waste, wastewater treatment sludge, etc.
(Stafford et al. 1980). Liquid biofuels include bioethanol, plant oils, vegetable oils and
the methyl esters produced after transesterification of these oils which is commonly
known as biodiesel (Granda et al. 2007).

2 Generation of Biofuels

Biofuels are classified in three different generations based on the source of feedstock
(Fig. 2). Biofuels of first generation were primarily generated from food crops like
grain, cane and plant oils, whereas biofuels of second generation were generated
from energy crops other than food crops such as miscanthus, forest residues and
woody biomass. In the third-generation biofuels, microalgae are used as source of
biomass for biofuel production (Dutta et al. 2014).
Biotechnology of Biofuels: Historical Overview … 113

• Derived from
sources like starch, SECOND
sugar, animal fats GENERATION • Derived from algae
and vegetable oil. mostly marine
• Examples: Biodiesel, • Derived from non-food algae
Vegetable oil, Bio crops, such as • Examples: Algal
ethers, Biogas, etc cellulosic bio fuels and bioethanol and
waste biomass algal biodiesel
FIRST • Examples: Advanced bio-
GENERATION fuels like bio-hydrogen, THIRD
bio-methanol GENERATION

Fig. 2 Generations of biofuels

2.1 First Generation

2.1.1 Historical Overview

First-generation biofuels, also refer to conventional biofuels, are produced from

sugar and starch from food crops or edible oil (Ho et al. 2014). First-generation
biofuels are produced using different processes such as fermentation, distillation
and transesterification (Ma and Hanna 1999). Sugars and starch are fermented to
produce primarily ethanol, and in smaller quantities, butanol and propanol (Sarkar
et al. 2012). Energy density of ethanol is less than gasoline, but it is presently used in
various countries, including the USA, as an additive to gasoline (Hansen et al. 2005).
Combustion of ethanol is comparatively cleaner than gasoline, thus emits out less
greenhouse gases. Biodiesel is another first-generation biofuel which is produced
through transesterification of plant or vegetable oils (Yusuf et al. 2011). Biodiesel
can be used as alternative of petroleum diesel in diesel engines or by blending the
two. These biofuels also supported agricultural industries and rural communities
through increased demand for crops.

2.1.2 Sources

Plant oils as biodiesels: Rapeseed oil, sunflower oil, soya, castor, coconut oil, etc.,
are used as biodiesels. Transesterification of plant oils with alcohol results in fatty
acid esters called fatty acid methyl ester (FAME) or fatty acid ethyl ester (FAEE),
depending on the kind of alcohol used (Martín and Grossmann 2014). There are
various physical and chemical properties of plant oils or vegetable oils that affect
114 V. L. Jamwal et al.

their suitability as fuels such as iodine value, ash content, cetane number, sulfur
content and heat value. (Demirbas 2008). The ash, sulfur and potassium contents
of the plant oil are some of the important characteristics. Plant oils or vegetable
oils with low iodine value are more combustible and comparatively more efficient
as fuel than oils with high iodine value. For example, coconut oil has low iodine
value, whereas linseed oil has very high iodine value. Oils with low iodine value
have high melting point and are solid at room temperature. Oils with low cetane
numbers produce noise, cause trouble in starting and turn out thick exhaust smoke
(e.g., linseed oil and rapeseed oil) (Schwab et al. 1987; Goering et al. 1982). The ash
content of fuels is inversely proportional to the heating value. Biofuels have lower
ash content and sulfur content as compared to the fossil fuels (Bozbas 2008).
Sugar-rich food crop for bioethanol: It is produced from the fermentation of dif-
ferent type of feedstocks that contain sugars or carbohydrates (Cardona et al. 2010).
These feedstocks mainly consist of edible food crops such as barley, potato, rice,
corn, sugarcane, wheat and vegetable oil such as mustard oil, olive oil, rapeseed oil,
soybean oil, coconut oil, sunflower oil and canola oil. (Rulli et al. 2016). Bioethanol
does not produce SO2 or NOx .
Biomass for biogas: It is formed by anaerobic decomposition of organic mate-
rial such as biodegradable waste materials, including animal sewage and household
waste. Under oxygen deprivation, anaerobic bacteria break down the organic matter
to produce methane (Kigozi et al. 2013). Biogas consists of carbon dioxide, sulfur
dioxide, methane and other gases in minute quantity. This method is used for sludge
stability in wastewater treatment system. Small-scale and low-cost units for degra-
dation of domestic waste are common in developing countries and production of gas
for cooking (Zupančič and Grilc 2012).

2.1.3 Ecological and Environmental Impact

Biofuels emit less greenhouse gases as compared to the fossil fuels, thus positively
affect the quality of air and water. First-generation biofuels included mainly ethanol
(bioethanol) and biodiesel. Production of feedstocks for first-generation biofuels
requires an agricultural bio-resource, considerable amount of water, fertilizers as well
as pesticides (Escobar et al. 2009). Most of the environmental effects are related to the
agricultural practices of the bio-energy crop cultivation. The net benefit of a biofuel
is evaluated by analyzing its effect on air, water, soil, food and on net energy gain
(Tilman et al. 2009). Biofuels derived from feedstocks produced from the plants
grown on degraded non-cultivable lands, residues of crops, sustainably harvested
forest remains as well as domestic and industrial waste (Escobar et al. 2009), will
reduce their advantages though not competing with food crops, reducing effects on
land clearing and providing real greenhouse gas reductions.
Biotechnology of Biofuels: Historical Overview … 115

2.1.4 Economic Impact

A number of countries have experienced rapid boost in supply as well as demand of

biofuels, but the US ethanol area distinctly stands out on the rate of growth experi-
enced in the last ten years, which has made the USA the leading producer of biofuels
worldwide (Gasparatos et al. 2013). In the USA, corn has been used for the production
of ethanol for more than three decades. In 2011, the level of ethanol output increased
by 80 folds relative to the levels in 1980. Methyl tertiary butyl ether (MTBE) was
popularly used as oxygenate gasoline additive (Belpoggi et al. 1995). Ethanol has
become known as the most feasible oxygenate alternative for MTBE, which also pro-
motes a valuable market position for ethanol as a key additive of gasoline (Belpoggi
et al. 1995).

2.1.5 Drawbacks and Need for Next Generation

First-generation biofuels have had severe limitations. The first key barrier is that the
feedstocks they require are used for food. Across the world, a number of countries
have cited biofuels as the reason for increased food prices, as supplies of crops for
food have had to compete against crops used for fuel (Gabrielle 2008). Following,
China’s decision that food has priority (Duggan and Naarajärvi 2015), the USA also
announced that it will be moving away from its corn ethanol fuel policy and into a
new direction for biofuels.
The second key barrier is that certain first-generation biofuels have been estimated
to have equal or higher CO2 emissions than petrol when indirect land-use effects were
taken into account. First-generation biofuels provide a small benefit over fossil fuels
in regards to greenhouse gases which cause global warming, but cultivation and
processing of feedstocks require very high amount of energy as compared to the
energy supplied by them (Gabrielle 2008). Moreover, the agricultural intensification
increases the adverse environmental effects including acidification, nutrient pollu-
tion, eco-toxicity and weakening of ozone cover (Doornbosch and Steenblik 2008;
Quadrelli and Peterson 2007; Tomei and Upham 2009).
Mass production of feedstock for first-generation biofuels needs more cultivable
lands which result in reduction of land for food crop production (Gabrielle 2008).
Furthermore, the method for the production of feedstocks used for making first-
generation biofuels is responsible for environmental deprivation. This decreased
the enthusiasm about first-generation biofuels, thus potentiating the need for newer
discoveries to mitigate the issues (Janda et al. 2012; Naqvi and Yan 2015).
116 V. L. Jamwal et al.

2.2 Second Generation

2.2.1 Historical Overview

Biofuels of second-generation were designed to address the negative issues of first-

generation biofuels that endangered food supply through the resources used. Pro-
duction of second-generation biofuels comprises a range of non-food crops which
includes energy crops, stalks of wheat and corn, woody trees, etc. (Eisentraut 2010).
Second-generation biofuels mainly focused on two things, i.e., these biofuels utilize
the non-food parts of food crops that are left over after the extraction of the food
part. In these cases, the relevant crop parts are the stems, leaves and husks. Another
focus has been to utilize non-food such as jatropha, palm and switchgrass. (Pimentel
and Patzek 2005).
Second-generation biofuels also generate higher energy yields than first-
generation fuels. The technology is fairly immature, so it still has potential of cost
reductions and increased production efficiency as scientific advances occur (Antizar-
Ladislao and Turrion-Gomez 2008). However, some biomasses used for the produc-
tion of second-generation biofuels still compete with cultivable land used for food
crops. In addition, the process to produce second-generation fuels is more elab-
orate, requires more energy and material than first-generation biofuels because it
requires pre-treating the biomass to release the trapped sugars (Antizar-Ladislao and
Turrion-Gomez 2008).

2.2.2 Sources

Different types of feedstocks are used for the production of the second generation of
biofuels which includes energy crops such as amaranth, bamboo, eucalyptus grass,
miscanthus, oilseed rape, poplar, salix, sugarbeet and sweet sorghum; agriculture and
wood residues such as barn, citrus waste, corn stover, sugarcane bagasse, sawdust,
wheat straw, waste rice-straw and wood; organic waste; vegetable oils such as jat-
ropha oil and palm oil (Antizar-Ladislao and Turrion-Gomez 2008; Sanderson and
Adler 2008). Cellulosic ethanol production requires freeing of sugar molecules from
woody lignocellulosic material using enzymes, steam heating or other pre-treatments,
followed by the fermentation of these sugars to produce bioethanol (Somerville
2007). Lignin is the resulting by-product which can be used as a fuel. The second-
generation biofuel industry has had difficulty in producing ethanol from cellulose
(Sims et al. 2010).

2.2.3 Ecological and Environmental Impact

Land usage: Feedstock in the second generation consists of switchgrass and mis-
canthus, which have relatively lower water and nutritional requirement and may
Biotechnology of Biofuels: Historical Overview … 117

positively affect the environment as these crops can grow on wastelands (Keshwani
and Cheng 2009). Woody crops are also used as high energy feedstock. These have
relatively high yield potential, are widely distributed geographically and require low
levels of input in comparison with the annual crops (Smeets et al. 2007). In case
of woody energy crops, use of sustainable forest practices can help in controlling
deforestation (Ciccarese et al. 2012).
Air pollution: Second-generation biofuels emit relatively lesser amount of green-
house gases as compared to fossil fuels, and they are in general more eco-friendly
and socio-friendly as compared to first-generation biofuels (Dias et al. 2011). Woody
energy crop obtained from forests stimulates indirect land-use change (iLUC) effects,
which affect the balance of greenhouse gases (Witzke et al. 2010). Second-generation
biofuels production by sustainably sourced wood (rather than fossil fuels) may reduce
overall emissions.
Water usage: The characteristic feature of second-generation feedstocks (woody
energy crop) includes higher transpiration rate due to their large root system, high
leaf area index, long growing season and height (Versfeld and Van Wilgen 1986).
High water use efficiency is important especially in the areas with limited water
availability (Oliver et al. 2009).
Soil quality regulation: Second-generation feedstock (woody energy crop) can
control the erosion of soil by wind and water also regulates the risk of flood. Trees
require no annual tillage, provide soil cover throughout the year and exert a bene-
ficial impact on soil properties including the enhancement of water fluxes leading
to reduction in surface runoff, changes in size and stability of soil aggregates and a
decrease in wind erosion (Kittredge 1948).

2.2.4 Economic Impact

First-generation biofuels contributed minimally to the mitigation of carbon emis-

sion and had upward effect on food prices as compared to the second generation
(Mohr and Raman 2013). In addition to short-time gasolines pricing damping, trade
improvements and wealth transfer, increased energy production in second-generation
biofuels offered significant advantages. The use of cellulosic biomass for energy
production resulted in significantly higher energy return on investment (EROI) and
carbon sequestration in comparison with biofuels produced from starch and sugar
(Tilman et al. 2006; Sheehan et al. 2003; Farrell et al. 2006).
The prices of second-generation biofuels depend significantly on the source of
the feedstock, production process as well as its transport.

2.2.5 Drawbacks and Need for Next Generation

Second-generation biofuels continue harboring a number of limitations present in

first-generation biofuels such as environmental pollution because of its combustion,
water pollution because of the waste material from the processing and production
118 V. L. Jamwal et al.

of biofuels, and high land use. Further, unmanaged woody energy crops that are
harvested unsustainably may significantly damage the environment and ecology due
to deforestation.
The biofuels so far used have recorded limitations in speed of growth (in case
of woody energy crop) (Kozlowski 1999) and yield compared (low yield in case of
inedible crops) to the required volume of fuel, within a set timeframe. The production
process for producing biofuels may be complex, multi-layered and time consuming.
The land that can be used for the cultivation of food crops is limited, and harsher
unused lands may sometimes not be utilized. Using land designated for food crops
for the production of fuel would further reduce the land available for the cultivation
of foods and put excessive pressure on food prices. All these factors make the pro-
duction of second-generation biofuel unprofitable and commercially unsustainable.
Microalgae are reasonable renewable source of energy as substitutes for first- and
second-generation biofuels (Saladini et al. 2016). Microalgae can avail various types
of renewable biofuels, such as biodiesel, bioethanol, methane and bio-hydrogen.
Microalgae are capable of producing 15–300 times more biodiesel as compared to
the traditional crop used for biofuel production (Lee and Lavoie 2013). The life cycle
of microalgae is very short, and growth rate is very high. Additionally, the production
of microalgae does not require high-quality cultivable land (Dragone et al. 2010).

2.3 Third Generation

2.3.1 Historical Overview

Microalgae are single-celled microorganisms which occur in freshwater and marine

environment. Microalgae convert solar energy more efficiently as compared to higher
plants. Due to high photosynthetic rate, microalgae are excellent candidates for fast
and increased biomass production, lipid fabrication and biofuel production. In third-
generation biofuels, microalgae biomass is used as feedstock for the production of
biofuels. Microalgae are capable of growing throughout the year, and thus, oil yield
per area of microalgae cultures could significantly enhance the yield of best oilseed
crops. Cultivation of microalgae requires less water as compared to the food crops as
well as energy crops and does not require herbicide or pesticide (Brennan and Owende
2010; Mata et al. 2010; Um and Kim 2009). Further, microalgae feedstock can grow
on non-cultivable land, can use salty-marine water and thus does not compete with
first- and second-generation feedstocks.

2.3.2 Sources

Microalgae are used as the major source for the production of third-generation bio-
fuels. Microalgae are grown in a variety of aquatic environments, such as fresh or
Biotechnology of Biofuels: Historical Overview … 119

seawater, domestic or industrial wastewaters. Microalgae required sufficient quan-

tity of carbon, nitrogen and phosphorus for their growth (Chen et al. 2011). Biofuel
production by using microalgal biomass first includes the selection of microalgal
strain, cultivation of microalgae, harvesting, drying of biomass and extraction of oil
for production of bioethanol and biodiesel.
(a) Cultivation of microalgae
Cultivation of microalgae can be done by using two different cultivation systems:
one is suspended cultures which include open ponds and closed-loop system (not
exposed to air) or more complex systems such as photo-bioreactors (Chen et al.
2011).
(b) Harvesting of microalgae
Harvesting of microalgae has been thought-out as main blockage on the way to the
commercial-scale processing of microalgae biomass for biofuel production. The cost
of recovering the microalgal biomass from the medium is 20–30% of total biomass
production cost (Grima et al. 2003). Harvesting of microalgal biomass can be attained
using various chemical, physical and biological ways such as filtration, auto-flotation,
air-flotation and flocculation. (Salim et al. 2011; Vandamme et al. 2013).
(c) Extraction of oil algal biomass
Drying of algae is required prior to lipid extraction. Sun drying the biomass is
probably the cheapest method of drying, followed by cell disruption that includes
high-pressure homogenisers, autoclaving and addition of hydrochloric acid, sodium
hydroxide or alkaline lysis (Brennan and Owende 2010; Schenk et al. 2008; Mendes-
Pinto et al. 2001). Several methods are used for extraction of lipids from microal-
gal biomass. Most commonly used methods are oil pressing, solvent extraction,
supercritical fluid extraction (SFE) (Clifford and Williams 2000), etc.
After the extraction of microalgal oil, it is processed to convert into biodiesel
through transesterification (Vasudevan and Briggs 2008). The transesterification
reaction consists of transforming triglycerides into fatty acid alkyl esters, in the
presence of an alcohol, such as methanol or ethanol, and a catalyst. Production of
bioethanol from microalgae biomass is carried out through starch hydrolysis fol-
lowed by fermentation and then distillation. It requires less energy and simplified
processes as compared to the production of biodiesel (Harun et al. 2010).

2.3.3 Ecological and Environmental

Large-scale production of microalgae also has variety of environmental impacts.

These environmental impacts include
120 V. L. Jamwal et al.

(a) Land use

Production of microalgae can be done on marginalized land, which is unusable for
the cultivation of crops; thereby, it minimizes the competition with food production
(Borowitzka 1999). It requires considerably less land compared to that required
for the cultivation of plants used for the production of first-generation or second-
generation biofuels. However, topography and soil type may be a factor affecting
the land availability for raceway pond systems as the installation of large shallow
ponds requires relatively flat land. This may especially be a concern in hilly areas.
Depending on the permeability of soil, there may be requirement of lining and sealing
of pond for the cultivation of microalgae (Lundquist et al. 2010).
(b) Nutrient pollution
Leaching of residual nutrients culture medium into local aquatic systems causes neg-
ative as well as positive impact. Negative impact includes nutrient pollution (eutroph-
ication), whereas a positive impact includes use of algae production in the treatment
of water bodies which are suffering from excess nutrient supply (Graneli et al. 2008;
AquaFUELs 2011).
(c) Toxic effect of algae
Algal species can produce toxins at certain stages of their life cycle. The effect of
these toxins ranges from acute to chronic (Collins 1978). The production of toxins
may depend on environmental conditions and the species and strain or algae that
were used. Due to leaching of culture or the discarded wastewater from the pond,
these toxins may be released in the water bodies and affect the environment (Collins
1978; Rellán et al. 2009).
(d) Carbon fixation
Carbon dioxide is required for the cultivation of microalgae, and theoretically, CO2
fixation efficiency of 20–90% can be achieved. In open ponds, the efficiency of
CO2 fixation is less than 10%, whereas for thin layer cultivation, the CO2 fixation
efficiency is approximately 35%. CO2 fixation efficiency of about 75% has been
reported for closed photo-bioreactors (Weissman et al. 1988; Acién et al. 2012).
Carbon neutral alternative renewable fuels are sought after for improvement of air
quality and reducing the adverse effects on environment. Microalgae cultivation
practices may be optimized to attain a high rate of carbon sequestration so that the
amount of CO2 released from the biofuel may almost equal the amount fixed during
photosynthesis (Pokoo-Aikins et al. 2010).

2.3.4 Economic Impact

Japan pioneered large-scale commercial production of microalgae as an alternative

food. (Krauss 1962). Several other countries followed the lead during 1970s and
1980s. By the year 2004, the industrial production of microalgae reached 7000 tonnes
of dry mass per annum (Werner et al. 2004).
Biotechnology of Biofuels: Historical Overview … 121

Economy has significant role in the commercial feasibility of production of bio-

fuels using microalgae. Cost of microalgal oil production relies on different factors,
such as biomass yield, oil content, scale of production systems and cost of recov-
ering oil (Rodolfi et al. 2009). The factors that can sway the cost of production of
microalgae include illumination, stirring, photosynthetic efficiency, medium for cul-
tivation and CO2 . The production costs may further reduce, through optimization
of these factors, making algal biofuel more luring and a viable alternative to fossil
fuels, especially as the production of fossil fuels would decline in coming years due
to the depletion of reserves. More than one type of biofuels can be extracted from
algal biomass, which increases the value of biomass.

3 Business Outlook

3.1 Production

About 85% of world’s ethanol production occurs in the USA and Brazil, followed by
EU and China. According to Renewable Fuels Association (RFA), world’s ethanol
production improved from 2007 levels and reached their highest in 2017 after plung-
ing briefly during the 2011 and 2012. In 2017, the USA attained a record production
of 16 billion gallons of bioethanol (Association RF 2010).
According to OECD-FAO Agricultural Outlook 2017–2026, bioethanol and
biodiesel production in developed countries will increase between 2017 and 2023,
and then, it will slightly decrease after 2023. But in developing countries, the pro-
duction of bioethanol is projected to keep increasing from 2017 to 2026. The trends
of bioethanol production in world are predicted to increase by 11% and biodiesel by
8% between 2017 and 2026 (OECD-FAO 2017).

3.2 Consumption

According to IEA, globaly, the rate of production of biofuel is not able to keep up
with increasing demand. In 2018, consumption of biofuels increased by 7% and
reach to 152 billion liters, but average production increased only 3% per year. This
will result in falling short sustained annual growth by 10% through to 2030.
Ethanol consumption worldwide is predicted to increase by about 12 billion liters
and by the year 2026. Developing countries like Brazil, India, China and Thailand
will account for 80% of the increased consumption. Use of ethanol may increase by
5.4 billion liters in Brazil alone. Consumption of ethanol is expected to increase by
a billion liters in China as well (OECD-FAO 2018).
Over the next few years, biodiesel use will peak out in developed countries, while
it may continue to increase firmly in developing countries. By 2027, Indonesia may
122 V. L. Jamwal et al.

annually consume 4.1 billion liters of biodiesel, whereas in Argentina and Brazil,
its demand may grow up to 1.9 billion liters and 5.6 billion liters, respectively.
Requirement of biodiesel will also increase in Colombia, Malaysia, Paraguay, India,
Philippines and Thailand because of blending regulations. In most countries, cur-
rently, only 1–3% of total diesel requirement is fulfilled with biodiesel (OECD-FAO
2018).

3.3 Trade

Factors such as increasing domestic consumption within the countries that are
presently exporting bioethanol, decreased demand from the countries that presently
import bioethanol due to a boost in domestic production, as well as increasing reliance
on alternate energy sources for transportation, such as electricity, will impact the
global trade volumes of bioethanol, which is expected to marginally reduce by about
1% between 2017 and 2027. The USA may remain a net exporter of bioethanol pro-
duced from maize feedstock. Export of bioethanol from Brazil may slightly reduce
due to increasing domestic demand. Import of bioethanol by countries like Japan and
Canada is also expected to reduce (OECD-FAO 2018).
For similar reasons, as mentioned for biofuels, the trade volumes of biodiesel are
also expected to decline, worldwide. Argentina is expected to remain as topmost
exporter of biodiesel, followed by other countries such as Malaysia, Indonesia and
Canada (OECD-FAO 2018).

4 Future Perspectives

First-generation biofuels have been phased out from most countries except in some
developed nations, where corn starch is being used for production of bioethanol
(Mohanty and Swain 2019). Second-generation biofuel, due to higher costs, is no
longer a viable alternative. Third-generation biofuels are the major thrust area (Neto
et al. 2019). Recently, there has been a decrease in crude oil (fossil fuel) prices
internationally due to political issues and over-pumping in the Middle East nations
(Bantacut et al. 2019). This has put excessive pressure on the biofuel industry, which
was even earlier under economic stress. Moreover, the subsidies for biofuel industry
have also been continuously declining, at least in the developed nations. For the third
generation, microalgal biofuel to become economically viable and sustainable, its
production capacity needs a major boost (Maity et al. 2014). There is a need for much
higher biomass accumulation with significantly higher oil content, preferably in open
ponds, throughout the year (Park et al. 2011). This presents major challenges because
open ponds are subject to contamination from the environment (Day et al. 2012).
Seasonal variations can have devastating effects on algal biomass (Olofsson et al.
2012). Water requirements are also an important issue which needs to be addressed
Biotechnology of Biofuels: Historical Overview … 123

as this also adds to the cost (Farooq et al. 2015). In addition, harvesting of small-sized
microalgae from high volume liquid culture is energy and cost intensive. Compared
to the conventional agriculture practice, cultivation of microalgae is more expensive,
complex and requires specialized expertise, adding to the costs incurred on trained
manpower (Vandamme et al. 2013).
Some of these difficulties may be overcome through biotechnological interven-
tion. This may involve technological strategies such as: (a) development of biorefin-
ery (Galkin and Ananikov 2019), (b) development of cost-effective technologies for
biofuel production (He et al. 2018; Correa et al. 2019), (c) strain selection, such that
it may be cultivated throughout the year without much impact of seasonal variation
(Gnouma et al. 2018), (d) selected strains may be cultivable in saline seawater, with
plants developed along the coast, to reduce the requirement of freshwater (Sydney
et al. 2019) and (e) development of genetic tools and genome editing methodologies
to engineer metabolism for higher biomass and lipid production (Naghshbandi et al.
2019; Jagadevan et al. 2018).

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Vijay Lakshmi Jamwal Masters in Human Genetics. Presently pursuing Ph.D. at AcSIR. Work-
ing at CSIR-Indian Institute of Integrative Medicine (Jammu), toward understanding the physio-
logical and ecological roles of secondary metabolites in high value medicinal plants. Trained in
molecular tools such as CRISPR-Cas9, transcriptomics, metagenomics, metabolomics and trans-
genics.

Nitika Kapoor Masters in Human Genetics. Awardee of ICMR Junior and Senior research fellow-
ships. Presently, pursuing Ph.D. at CSIR-Indian Institute of Integrative Medicine, Jammu. Work-
ing on regulatory aspects of secondary metabolism in medicinal plants, employing cutting edge
molecular tools, transcriptomics, metabolomics, etc.

Sumit G. Gandhi Working as senior faculty (Principal Scientist) at CSIR-IIIM (Jammu), on var-
ious aspects of secondary metabolism in microorganisms and medicinal & aromatic plants, since
more than a decade. Ph.D. (JNU, Delhi) at CCMB (Hyderabad) focused on understanding molec-
ular effectors and mechanisms of epigenetic regulation. Bachelors & Masters in Microbiology. Dr.
Gandhi has resourcefully collaborated with industries and academia, on various projects and has
been a recipient of grants from public and private sectors.
Regulations
Regulations for Health Care
Biotechnology Products in Major
Markets of the World
Durga Prasad Mindala, Yashbir S. Bedi, Satish Kumar Gupta,
Sumit G. Gandhi and Inshad Ali Khan

Abstract Biotechnology products have applications in many areas such as medicine,

agriculture, energy, and environmental protection. Drugs and food supplements are
amongst the major biotechnology-based products that cater to the health care sector.
The products that are intended for use in diagnosing, curing, mitigating, or treat-
ing disease fall in the category of a drug and regulations are applicable to it. As a
general rule of thumb, such products must subscribe to the best available science
at the time. Regulatory authorities/systems are entrusted with the task of secur-
ing health of common people, as well as safety of subjects and environment while
accelerating economic growth, innovation, competitiveness, and job creation. The
regulatory agency has the responsibility to review laboratory and clinical research
before approval of such products for commercial use. Similarly, products such as
food supplements that are intended to prevent a disease or help in ameliorating a
disease condition also require appropriate regulations. Although the general princi-
ples on which the regulatory guidelines are based remain almost the same, minor
procedural and nomenclature differences exist in the regulatory agencies in various
countries. In the present article, we provide specific and compiled knowledge of
current regulatory procedures in the European Union (EU), USA, and India.

Keywords Drug · Regulatory authorities · Biotechnology · European Union ·

USA and India

Abbreviations

ADME Absorption, Distribution, Metabolism, and Excretion

BLA Biologics License Application
CBER Center for Biologics Evaluation and Research

D. P. Mindala · Y. S. Bedi · S. K. Gupta · S. G. Gandhi (B) · I. A. Khan (B)

Indian Institute of Integrative Medicine, Canal Road, Jammu 180001, India
e-mail: [email protected]
I. A. Khan
e-mail: [email protected]
© Springer Nature Switzerland AG 2020 131
A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_7
132 D. P. Mindala et al.

CDER Center for Drug evaluation and Research

CNS Central Nervous System
CVS Cardiovascular System
DBT Department of Biotechnology
DLC District-Level Committees
eCTD Electronic Common Technical Document
ETIPC Emerging Technologies Interagency Policy Coordination Committee
EU European Union
GEAC Genetic Engineering and Approval Committee
GLP Good Laboratory Practice
IAEC Institutional Animal Ethics Committee
IB Investigator’s Brochure
ICH International Conference on Harmonization
IDE Investigational Device Exemption
INAD Investigational New Animal Drug
IND Investigational New Drug
IRB Institutional Review Board
MoEF Ministry of Environment and Forests
MS Multiple Sclerosis
NDA New Drug Application
NIH National Institutes of Health
OECD Organization for Economic Cooperation and Development
PD Pharmacodynamics
PK Pharmacokinetics
r-DNA Recombinant Deoxy Ribonucleic Acid
RS Respiratory System
SBCC State Biotechnology Coordination Committees
SmPC Summary of Product Characteristics
USDA US Department of Agriculture
USEPA US Environmental Protection Agency
USFDA US Food and Drug Administration

1 Introduction

Biotechnology advancements have revolutionized the health care sector through

biotechnology-derived pharmaceuticals (biological). Initial development of such
products was reported in early 1980s; however, marketing permissions of these prod-
ucts took another 10 years. Regarding safety assessment of the biologics, several
guidelines and documents were published from by various regulatory agencies at
various time points. This information available with the regulatory authorities was
of utmost use for the review/development of such products and their background in
Regulations for Health Care Biotechnology Products … 133

developing new biopharmaceuticals/biologics. A thorough analysis of this informa-

tion is mandatory to develop scientifically acceptable preclinical safety evaluation
programs.
“The Coordinated Framework for Regulation of Biotechnology” ref was finalized
in 1986, after successful use of r-DNA techniques, and some of the formulations
from such techniques were put to use commercially. “The Office of Science and
Technology Policy”, USA, was of the opinion that “existing statutes provide a basic
network of agency jurisdiction over both research and products; this network forms
the basis of this coordinated framework and helps assure reasonable safeguards for
the public (OSTP 1986)”. The existing laws passed by US legislative bodies on
the subject were regulated by “US Environmental Protection Agency (EPA), the
US, Food and Drug Administration (FDA), and the US Department of Agriculture
(USDA)”.
The stimulus for regulations on research and development of the r-DNA technol-
ogy was provided in an International Conference held at Asilomar, California, in
1975. With passage of time, the onus for these regulations shifted from national to
international regulatory authorities, resulting in the publication of the “blue book”
titled, “Recombinant DNA Safety Considerations”, by the Organization for Eco-
nomic Cooperation and Development (OECD) (Development 1986). This blue book
describes three general approaches to be considered while r-DNA organisms and
r-DNA techniques. First, the risks rose by r-DNA organisms and conventional organ-
isms are similar in nature. Second, the traits exhibited by genetically modified organ-
isms through r-DNA techniques are more predictable in nature than regular methods
of living cell manipulation. Third, legislation cannot be justified on scientific basis.
In addition, OECD and the National Institutes of Health (NIH) have made many
other recommendations. As on day, countries at large observed the guidelines with
different approaches, i.e. no regulation (south pacific countries), appropriate reg-
ulation (USA and some European countries), or stringent biotechnology specific
regulation (German) (Brown 1991). Two distinct but closely interrelated regulatory
agencies carry out regulatory functions to protect public health and the safety of the
public at large regulated by the government of the said country. These are consumer
and occupational safety to consume biotechnology products and to enter a workplace
where biotechnology products or biotechnological means of production are in use.

2 Classifications

Biotechnology and life sciences contribute to the modernization of industry and shall
be classified into three broad groups:
a. Health care applications:
It includes the development of advanced medicines, therapies, diagnostics, and
vaccines. The generation of novel drugs for diseases, that did not exist earlier
and upgradation of the existing therapeutics.
134 D. P. Mindala et al.

b. Agriculture, livestock, veterinary products, and aquaculture:

In this sector, applications include the production of vaccines, improvement of
crop production and animal feed, bettering the quality of livestock, focussed
breeding, efficient food processing, and improved diagnostics for detecting a
spectrum of diseases.
c. Industrial processes and manufacturing:
Industrial applications include the use of enzymes in the production of various
products of commercial use such as pulp and paper, detergents, textiles, and
biomass. Most contemporary processes include fermentation and enzyme bio-
catalysis instead of traditional chemical synthesis.

3 Regulations

Regulatory authorities in the world aims providing safe items for its consumption
and reliable regulatory systems. However, regulatory approach differs country wise
with respect to manufacturing and commercialization of the biologics. Taking into
consideration the set up in USA, the new Biotechnology Working Group under
the “Emerging Technologies Interagency Policy Coordination Committee (ETIPC)”
from the Executive Office of the President is governed as detailed at introduction
section (Gottlieb 2018). The working group shall harmonize with other federal agen-
cies and offices where ever needed. In Europe, national law implements legal mea-
sures through regulations that are immediately applicable. The legal approach pro-
vides necessary framework for the processes and/or products, including technical
specifications enforced by the “European Committee for Standardization (CEN and
CENELEC)”, with the help of the national entities (90/220/EEC, E.C. 1990). These
standards are adopted on a voluntary basis without any regulatory requirements. In
India, “Department of Biotechnology (DBT)” constituted under the Ministry of Sci-
ence and Technology is the nodal agency for policy, promotion of R&D, international
cooperation, manufacturing activities, etc. With coordination of Genetic Engineer-
ing and Approval Committee (GEAC) constituted under “Ministry of Environment
and Forests (MoEF)”, CDSCO and DBT are the regulatory bodies in the area of
biotechnology in India (Rao 2018).

3.1 In USA

FDA approves any new drug for its manufacture as well as commercialization only
if its safety and effectiveness are proven through trials. The financial burden of these
testing and trials is borne by the manufacturer/sponsor. The safety studies shall be
conducted step wise as per the regulatory guidelines.
Regulations for Health Care Biotechnology Products … 135

3.1.1 Pre-market Approval of the Biological Product

In the USA, “biological products” are licensed by Center for Biologics Evaluation
and Research (CBER) thorough Biologics License application (BLA) process ensur-
ing its safety, purity, and potency regulated by the laws in force in the said country.
The nonbiological drug requires laboratory studies and animal testing to define their
pharmacologic and toxicological effects before they can be studied in humans similar
to that of the biological products under “Good Laboratory Practice (GLP) regula-
tions”. However, biologics require a “flexible, case-by-case, science-based approach”
to preclinical testing.
Preclinical studies are undertaken to establish initial safe dose in human, potential
target organs for toxicity, and safety parameters to be considered during clinical
monitoring.
For biotechnology-derived pharmaceuticals, the FDA follows the “International
Conference on Harmonization of Technical Requirements for the Registration of
Pharmaceuticals for Human Use (ICH) S6 guidelines”.
Sponsor must perform in vitro and in vivo assay studies to determine the pharma-
cological activity, i.e. pharamacokinetic (PK), pharmacodynamics (PD), and mech-
anism of action of the investigative agent (Group IEW 2011). Evaluate effective-
ness/safety of the agent on specific organs and systems on these systems sepa-
rately, like CNS, CVS, RS, etc., (Guideline IHT 2011). Sponsor shall also con-
duct dose-dependent biopharmaceutical studies like PK and toxicokinetic studies
to measure absorption, disposition, and metabolism (ADME) with reference to
antibody-mediated clearance and investigate dose-response relationships (Guideline
IHT 2011). This information helps to determine safe dose for human to conduct the
clinical studies. Immunogenicity and carcinogenicity testing might be included in
the preclinical studies of the agent though animals are not always a good predictor
of human immunogenecity (Guideline IHT 2011). Carcinogenicity studies may be
done based on the “duration of clinical dosing, population, and biological activity
(Guideline IHT 2011)”. These studies are done as per ICH S6 guidelines. Similar to
carcinogenicity studies, reproductive and developmental toxicity studies may or may
not be required but based on “the product, clinical indication, and intended patient
population”.

3.1.2 Investigational New Drug Application (IND)

According to current regulations, new R&D products in the USA, which are
not approved for marketing, need to be submitted as an IND to the office of
Food and Drugs Administration (FDA). In USA, different sections/offices for the
approval/licensing of different products are an official process, for example, non-
biological application shall be submitted to the “Center for Drug Evaluation and
Research (CDER) and for biological Center for Biologics Evaluation and Research
(CBER)” (Table 1). As per US regulations, 21 CFR 312.22 and 312.23 contains the
136 D. P. Mindala et al.

Table 1 Summary of IND application submission in regulatory market

Contents United States Europe India
1. IND application Division of Heads of medical Central drugs
submission/departments communication and agency (HMA), standard control
consumer affairs, office European medicine organization
of communication, agency (EMA), (CDSCO)
outreach and Spark building Directorate general
development, center Orlyplein 24 of health services
for biologics 1043 DP Ministry of health
evaluation and research Amsterdam and family welfare
(CBER), food and drug The Netherlands Government of
administration India
(FDA)—10903
2. Mode of submission Electronic common Electronic Online submission
technical document common technical through SUGUM
(eCTD) document eCTD online portal
through EudraLink
3. Application forms Cover sheet (Form Annex-1: clinical Form CT-04
FDA-1571) trial application
form
4. Application fee $2,588, 477 EUR 87600 for INR 3,00,000
initial request
5. Guidelines ICH S6, OECD and ICH S6, OECD OECD/ICH and
USFDA and EMA schedule Y of
guidelines drugs and cosmetic
act, 1940
6. Directives 21 CFR 312.22, 312.23 Article 9(8) of Schedule Y of
and 312.30 directive drugs and cosmetic
2001/20/EC act, 1940

basics and the general requirements for an IND Submission (Regulations, C. o. F.,
21 2018). An enumerated list of studies and documents are mentioned below.

3.1.3 Cover Sheet (Form FDA-1571) (CFR 2019)

Form FDA-1571 is an application form to seek the permission to conduct the clinical
trials on the human subject. The application form contains the general information of
the sponsor and investigational agent (Drug) (Table 1). Sponsor shall mention clearly
in the application, the phase to be conducted. An “Institutional Review Board” (IRB)
is constituted in every organization committed to comply with the requirements
described in part 56 and is responsible for the approval and review of each of the
studies in the proposed clinical investigation and report to the IRB (CFR, Part 56,
Institutional Review Board 2018). Complete details of the persons responsible for
monitoring the progress of the investigations as well as reviewing and evaluation of
information relevant to the safety of the drug, has to be provided in the application.
Regulations for Health Care Biotechnology Products … 137

3.1.4 Introductory Statement and General Investigational Plan

The Introductory statement should contain detailed information of the Active Phar-
maceutical Ingredients (API) and the structural elucidation of the molecule, infor-
mation on the formulation, its dosage form, route of administration, and the broad
objectives and duration of the proposed clinical investigation(s). In addition to that,
previous human experience with the drug, marketing experience in other countries
that may be relevant to the safety of the drug (Regulations, C. o. F., 21 2018).
The investigational plan requires additional information such as
(a) The rationality for the drug, the research, and development study;
(b) The therapeutic indications;
(c) The basics to be followed in evaluating the drug;
(d) The kind of clinical trials to be conducted to the estimated number of patients;
and
(e) Any risks of particular severity or seriousness anticipated on the basis of the
preclinical studies or previous studies in humans with the drug or related drugs.

3.1.5 Investigator’s Brochure (IB)

Investigator’s brochure, containing the following information: An introduction on

the drug substance and the formulation, along with the structural formula (structural
elucidation of the drug molecule). The data on summary of the preclinical studies
like toxicity, efficacy, and ADME of the drug in animals.

3.1.6 Investigational Protocols

Investigational protocols should be submitted in accordance with 312.30(a) (Reg-

ulations, C. o. F., 21 2018). The Phase 1 studies are primarily done to access the
safety of the new product in humans. Phase 1 protocols give an outline of the number
of human subjects to be enrolled, a description of safety and dosing plan including
duration, dose, or method be used in quantify dose. The details of the parameters
critical for the safety are given in detail. Phase 2 and 3 studies are conducted after
the completion of Phase 1 studies when the safety of the product of humans is estab-
lished. Phase 2 and 3 are efficacy trials. All detailed protocols describing aspects of
the efficacy of the study should be submitted.
A protocol should include the following, with the specific requirements:
(a) “A statement depicting the purpose of the study.
(b) The curriculum vitae or other statement of qualifications of each investigator
and the name of each sub-investigator (e.g. research fellow, resident) working
under the supervision of the investigator; the name and address of the research
facilities to be used; and the name and address of each reviewing Institutional
Review Board.
138 D. P. Mindala et al.

(c) Criteria for inclusion and exclusion of the patient and an estimate of the number
of patients to be studied.
(d) Design of the study and a description of methods to be used by investigators
and analysts.
(e) The method for determining the dose(s) to be administered.
(f) A description of clinical procedures, laboratory tests to monitor the effects of
the drug in human subjects and to minimize risk”.

3.1.7 Chemistry, Manufacturing, and Control Information (CMC)

In the IND application CMC should provide sufficient information to assure quali-
tative and quantitative, and strength of the investigational agent. The strength of the
desired dosage form shall vary from phase 1 to 2. Final release specifications for the
drug substance and drug product are not expected until the end of the investigational
study. The stability data is also required in addition to the CMC information.
Drug substance: A description of the drug substance pertaining to its physical,
chemical, or biological characteristics is required for the planned clinical studies.
Drug product: A list of all components, including the inactive and active compounds
during manufacture of the investigational agent as well as quantitative composition
of the investigational drug product required.

3.1.8 Pharmacology and Toxicology Information

Information pertaining to the pharmacology and toxicology reports from in vitro

animal studies is required for deciding whether to go ahead with human clinical
trials, as well as the dosing that may be required. This information along with other
mechanistic data is also useful as well as mandatory, in deciding the scope, duration,
and the end points for a clinical investigation (USFDA 2015).

3.1.9 Toxicology

Depending on the type of the drug and duration of treatment required for a disease
condition, acute, sub-acute, and chronic toxicity data needs to be generated (USFDA
2015). Toxicity profile would also include tests for mutagenicity, genotoxicity, affects
on developing foetus, as well as special toxicity tests related to route of administration
(e.g. inhalation, dermal, or ocular toxicology). A full tabulation of data has to be
compiled in the form of dossier.
Regulations for Health Care Biotechnology Products … 139

4 In Europe

In EU, biologics cover a broad spectrum of medicinal products that are biological
in origin and are more complex than a synthetic one. Such a biological substance is
produced by or extracted from a biological source and requires its characterization
by physico-chemical-biological testing for its quality, including production process
and its control. Annex II to the EU GMP guidelines are followed for the above studies
(Commission E 2018). ICH S6 marketing guideline is followed like USA.

4.1 Clinical Studies

Directive 2001/20/EC, Article 9(8) of the EU, mandates that Good Clinical Practices
(GCP) are to be followed in a clinical trials. The clinical trials may be conducted at
a single location or at multiple locations spanning a single or more member states
of the EU. The applicant has to register at the EudraCT Community Clinical Trial
System and obtain a unique identification number. A covering letter quoting the
EudraCT number along with a dossier containing complete documentation detailing
the CMC of the product, pharmacological activities, mechanism of action, toxico-
logical reports, clinical trial protocols, and any other information about the investi-
gational drug, as may be required in specific cases are to be furnished to appropriate
authorities in the member states of the EU, for obtaining an approval to conduct a
clinical trial. Further trial needs to be permitted by the ethics committee (Council
E 2001). Moreover, such an approval must not be construed as scientific advice on
investigational medicinal product (IMP) (Commission E 2010).

4.2 Clinical Protocol

According to Article 2(h) of mentioned directive, the objectives, design, method-

ology, statistical consideration, and organization of a trial shall be decided by the
sponsor’s protocol and be identified by the title of the study. The protocol will refer
section 6 of the community guidelines on Good Clinical Practice (ICH, ICH E6 (R1)
2002) (CPMP/ICH/135/95) containing relevant information for ethics committee’s
assessment.
The conditions on all the articles, i.e. Article 6(3)(a), Article 6(3)(b), Article
6(3)(g), and Article 6(3)(k) of Directive 2001/20/EC are followed in letter and spirit
for designing a investigational protocol.
140 D. P. Mindala et al.

4.3 Investigator’s Brochure

Investigator’s brochure (IB) containing clinical and non-clinical data also needs to be
submitted to the trial authorization agency, along with other documents as mentioned
above. It also contains the clinical trial protocol, including dose, frequency/interval,
methods of administration, and safety monitoring procedures covered by Article 2(g)
of Directive 2001/20/EC.
The IB if requires to be updated must comply to Article 8(1), Directive
2005/28/EC, and “Requirements for authorization for the manufacturing or importa-
tion of such products and with the community guideline on Good Clinical Practice”
(CPMP/ICH/135/95) prepared from all available information on clinical trials. After
approval of the drug, the IB may be revised, if required, and such a document is then
called “Summary of Product Characteristics” (SmPC). The SmPC should contain
the safety information regarding any adverse reaction that may have been recorded
in the clinical trial. If the IMP is approved only in one member state and the active
substance contained in it is to be tested in a multilocational trial for approval in other
countries, then the SmPC document may be used as IB in other countries where trial
is to be conducted.

5 In India

In India, the regulatory authority that is designated to evaluate the safety, efficacy,
and quality of drugs is the Central Drug standard Control Organization (CDSCO).
Further, Review Committee on Genetic Manipulation (RCGM) of the DBT (Depart-
ment of Biotechnology) has been entrusted the responsibility to oversee the devel-
opment and preclinical evaluation of recombinant biologics. Recombinant biolog-
ics are regulated under the provisions of Drugs and Cosmetics Act, 1940, and the
rules framed there under for the manufacture, use, import, export, and storage of
hazardous microorganisms/genetically engineered organisms or cells, 1989 (Rules,
1989) notified under the Environment (Protection) Act, 1986.

5.1 Pharmaceutical Biotechnology

Along with other statutory approvals, approval on Research and Development,

manufacturing import is equitably important.
Regulations for Health Care Biotechnology Products … 141

5.2 Research & Development

The applicant shall submit proposal (r-DNA) to Institutional Bio-safety Committee

(IBSC) with details about the organisms and vectors to be used, protocols, risks
involved as well as risk mitigation strategies. If the experiments involve production
in larger quantities (up to 20 l) or animal studies, then the proposal is further taken up
with RCGM seeking its permission to conduct the experiment (Limited Capacity).
RCGM if approves the experiments, then it is supposed to intimate its decision to
State Biotechnology Coordination Committee (SBCC) and Drug Controller General
of India (DCGI).
A prior approval is also required from the Institutional Animal Ethics Committee
(IAEC) to conduct animal trials. Where the safety pharmacology studies are part
of toxicology studies, these studies should be conducted in an accredited laboratory
(GLP). These studies are to be conducted as per the OECD/ICH guidelines. After
obtaining such permissions, proposal is submitted to RCGM for animal study.

5.3 Permission to Manufacture and Import of New Drugs

An application form (Form CT-12/13 (API/formulation) and Form CT-16 (Import))

shall be submitted to the CDSCO through SUGUM online portal to seek permission to
manufacture new drugs for testing or analysis or clinical trials or BA/BE studies. The
Central Licensing Authority may, after scrutiny of the CMC information, rationality,
and documents furnished with the application, grant the permission to manufacture
unapproved active pharmaceutical ingredient in Form CT-15, to manufacture its
formulation in Form CT-14 and for import of new drug in Form 17 under relevant
rules of Drugs and Cosmetics Act, 1940, and the permission is valid up to three years
unless suspended/cancelled by the competent authority.

5.4 Clinical Trials

As mentioned in the USA and EU, detailed information compiled in IB is required

in India as well, for conducting a clinical trial. Such a document contains infor-
mation comprising chemical, pharmaceutical information including stability data,
animal pharmacology, and toxicology data which prove the safe dose for the human
subject/clinical investigations along with investigational protocol containing infor-
mation about study centre, dose, frequency/interval, methods of administration, and
safety monitoring procedures. The sponsor shall submit the application form (Form
CT-04) (Table 1) through online portal to CDSCO, and same shall be submitted to
the RCGM to seek the permission to conduct the clinical trials (Phase 1 or II or III)
as per the protocol.
142 D. P. Mindala et al.

After successful clinical trials (Phase III), an application is submitted to the

Genetic Engineering Appraisal Committee (GEAC) for environmental clearance,
and thereafter, CDSCO will grant the permission to the state licensing authorities to
manufacture, market/import the drug as per the Drugs and Cosmetics Act, 1940.
The summary of IND application submission in Regulatory market presented in
Table 1.

6 Conclusion

It may be observed from the brief overview of the regulatory procedures presented
here that in various countries around the world, including India, the main stress
is of the regulatory agencies is towards safety of human subjects. The procedures
undergo amendment from time to time, to keep up with the new data from R&D
as well as with the technological development. The regulatory agencies also keep
a constant oversight on the approved drugs available in the market. Despite the
stringent regulations, in case a safety issue is not noticed during the trials, and the
drug is approved and following its marketing and public use, a significant adverse
reaction is noticed, it may be banned, or its use may be restricted to only certain
settings. With new innovations, such as regenerative medicine, stem cell therapy,
artificial organs, the regulatory procedures will continue to evolve, to allow such
innovations to be used safely and in the public interest.

References

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trials on medicinal products for human use. In: DIRECTIVE 2001/20/EC, Council, E. P. a. o. t.,
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Yashbir S. Bedi Professor at Academy of Scientific and Innovative Research and Scientist and
Head, at CSIR-Indian Institute of Integrative Medicine, Jammu. Dr. Bedi has a long experience
and strong networking skills resulting in over 100 publications and numerous patents.

Sumit G. Gandhi Senior scientist and faculty at CSIR-Indian Institute of Integrative Medicine
(Jammu) and AcSIR—Academy of Scientific and Innovative Research. Working on various
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Inshad Ali Khan Principal Scientist at CSIR-Indian Institute of Integrative Medicine, Jammu.
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Intellectual Rights
Intellectual Property Rights

Tabassum Zafar

Abstract Intelligence has no limit, and innovations have no boundaries. In this era
of fast pace globalization, any information could be stolen and modified very easily.
Creations of mind, whether it is an idea, thought, plan, gizmo, gadget, technology or
fiction, they all covered under the legal term called intellectual property. To prevent
the misuse of intellectual contents and ideas, intellectual property rights are very
essential. Intellectual property rights are the legal rights, which secure the write-ups,
inventions designs, methods, artistic works, innovations and intellectual ideas from
being stolen by anyone else than the real owner. The registered trademark, copyright
or patented idea, technology or invention remains secure for theft or being stolen.
For every budding researcher and entrepreneurs, it is very important to know the
scientific rights covered under the shelter of intellectual property rights. The present
chapter elaborates various aspects of intellectual property rights in an interesting and
concise way.

Keywords Intellectual property rights · Patent · Copyright · Scientific ethics ·

Trademarks · Trade secrets

1 Introduction

Intellectual property (IP) is a intangible property that is the result of human creativity
and intellect. IP could be an idea, a product, a formula, a creation, a machine or
service. IP covers the legal rights of any of them to the actual concept originator
and protects them from being stolen or misused. Intellectual property rights (IPRs)
are the rights that protect the intellectual idea or products and services based on that
idea. It seems relatively novel legal branch, but it actually exists in society since long
ago.
IPRs are the statuary rights that are territorial in nature for limited period protection
of intellectual content for management of monopoly of commercial or noncommer-
cial inventions and artistic works to avoid any exploitation of that intellectual idea

T. Zafar (B)
Department of Bioscience, Barkatullah University, Bhopal, India
e-mail: [email protected]
© Springer Nature Switzerland AG 2020 147
A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_8
148 T. Zafar

or invention in public domain. IPRs are the basic bottom line of commercial sci-
ence as it provides legal certification of scientific ideas and concepts, which is very
important in commercialization of a scientific idea or discovery. IPRs are relatively
recent but an emerging topic of interest. It is still not taught under many scientific
branches during the routine studies, but it is an essential requirement for researchers,
businessmen and entrepreneurs in the 21st century. The present chapter is an effort
to introduce and elaborate the IPRs in an interesting and understanding way (Jolly
et al. 2012).

2 Historic Preview of IPR

Intellectual property rights have an interesting history in spite of their recent revival.
Even in the ancient and old times, the threat for the misuse and theft of the original
ideas persisted. Due to this, ancient intellectuals had developed a policy very similar
to modern IPR system. In 500 B.C.E., the few popular pieces of evidence of protection
of intellectual concept existed. According to this policy, only the Sybaris’s Greek
colony chefs retained the right to cook specific culinary cuisines. IPR in ancient times
was discussed in Bruce Bugbee’s formidable work. ‘The Genesis of American Patent
and Copyright Law,’ where the incidence of Vitruvius, was discussed. Vitruvius
(257–180 B.C.E.) was a poet, who recited the words, poems and verses written by
any other author in a literary contest and got exposed in Alexandria. In consideration
to IPR, different ownership interests were also visible among Roman (first century
C.E.) painters and poets. Apart from such type of rights protection, still the ancient
Greece or Rome world lacks any typical written law or institution for the convention
of intellectual property protection. The intellectual ideas were however saved from
being stolen by the involvement of franchises, privileges and royal favors granted.
Bugbee was the person who primarily gets involved in the distinguishing between
franchises or royal favors. It was distinguished that franchises and royal favors restrict
the access to intellectual idea preexisting in the public domain (Bugbee 1967; Adam
and Ken 2018).
The English system of American institutions of intellectual property protection
initiated when the Statute of Monopolies in 1624 and the Statute of Anne in 1710
come in light. In the Statute of Monopolies, a fourteen-year monopoly was provided
to the authors and inventors. However, literary works remained largely unprotected
until the Gutenberg’s printing press arrived during the fifteenth century. The Statute
of Anne in 1710 is the foundation of the first statute of modern copyright. The law
in Statute of Anne covers the protection to the author by granting fourteen-year
copyrights, along with a fourteen-year renewal, if the author was alive (Farnley et al.
2004).
European countries such as Belgium, Holland, Italy and Switzerland started fol-
lowing the unsaid law generated by England (Bugbee 1967). The expansion of the
geographic scope of intellectual property protection was spread over the globe when
Intellectual Property Rights 149

various international treaties like the Berne Convention treaty and the trade-related
aspects of intellectual property (TRIPS 1994) come in picture.

3 Various Forms of IPR

In the 21st century, intellectual property rights can be easily classified into four major
divisions named trade secret, trademark, copyright and patent.

3.1 Trade Secret

Trade secret is the confidential information related to market capitalization by which,

an entrepreneur can cover the intellectual rights related to the particular secret infor-
mation or industrial formula. Trade secret covers the formula process, design, prac-
tice, pattern, commercial method, instruments, marketing, export or sales strategies.
A trade secret is widely used in industry and manufacturing business plans, where the
rights related to the secret formula of manufactured product retained confidentially
by the manufacturers using the trade secret. Trade secrets provide the economic ben-
efit to the trade secret holders usually by commercialization (Patino 2009). One of
the best example of trade secret keeping is associated with the name of a popular cold
during manufacturer named “Coca Cola”, This company is successfully keeping the
formula as a trade secret without keeping any patent for many years. It is possible
to patent someone’s trade secret if found by the legitimate means. Legal circum-
stances related to trade secrets are less complicated in comparison with patents. A
trade secret is a weak protection against the misuse, but it works with an immediate
effect. Keeping the trade secret is easy and does not require any legal disclosure or
registration cost but if; the trade secret is stolen, no legal action could be taken in
that case. Anything that could be easily disclosed by the keen inspection of a product
cannot be kept as a trade secret (Rockman 2004; Becker and Susan 2005). The secret
formula of Coca-Cola is one of the best examples of trade secrets.

3.2 Trade Mark

Trademark is another category of intellectual property rights that distinguish the

product, design, expression or service from a registered provider from other sources
or competitors. Any phrase, logo, symbol, design, image, color, smell, or sound
(like jingles) or service could be protected under the trademark policy. It is legally
possible to purchase, sell or license the trademark. Trademark actually used only for
the management of the goodwill of the product or service it symbolizes. There are
various types of trademark that are denoted by various means. ™ is the symbol for
150 T. Zafar

an unregistered trademark, which is used to product or brand. ™ is an unregistered

service mark, which is used to promote services provided by any particular brand.
is the symbol for a registered trademark (Tiwari et al. 2011). All the symbols used
for trademarks and service marks are written as a superscript over the brand names
during commercial popularization. However, unregistered trademarks provide less
protection against trademark infringement in comparison with registered trademark
(Anonymous 2004).

3.3 Copyright

Copyrights are a form of IPR that protects the creative work such as books, articles,
movie, short films, report, diary, songs, poems, literature, music, drama, artistic
works, paintings and cinematography. Copyright could be applied through proper
channel with various benefits. Copyright is mentioned by using the symbol of circled
letter c ( or ). Sound recording copyrights are mentioned by capital letter P
enclosed by a circle . A registered trademark used to symbolize by the capital
letter R enclosed by a ( ), which denotes that product is registered in the trademark
office. For mask work protection of the content, a non-obligatory symbol capital
letter M enclosed by a circle ( ) is used. The infringement of the copyright allows
the copyright holders to receive the penalty, if the copyright was earlier registered
with the original creator. The copyright is provided for a limited period of time for
software. The length of copyright varies over the world. From Yemen to Jamaica,
the copyright lasts minimum life plus 30 years to life plus 95 years. Copyright
protection is a safe way to cover the license related to the work for the author’s entire
life plus seventy years after the author’s death. Copyright provides various rights to
the owner including, reproduction of work, distribution of work, cinematographic
rights, share in economic benefits generated from the copyrighted work. However,
to avoid the restricted publicity of copyrighted work in the public domain, some
exceptions were considered under IPR. In order to protect the interests of users,
exceptional use by non-copyright holders is considered as fair use. For educational,
research, religious, criticism, performance, a limited user of the copyrighted material
is considered (Alastair et al. 2010).

3.4 Patent

The patent is the form of IPRs that cover new patentable invention according to their
novelty and utility under the strict monitoring and examination. According to the
national law, patents’ law changes for each location worldwide with some common
points of consideration. A patent should be novel and useful enough for the consid-
eration as a patent. An invention, which is frivolous or contrary to law or morality
or injurious to public health, cannot be a patent. A discovery, mathematical formula,
Intellectual Property Rights 151

calculation or scientific theory cannot be patentable. An invention relating to atomic

energy, an invention, which is in effect, is traditional knowledge cannot be patented.
A method of horticulture, cultivation or agriculture cannot be patented. Apart from
these, there are various such things that are not allowed to be a patent; however, some
of them could be secured using other types of IPRs (Saha and Bhattacharya 2011).
Patented material or process provides the inventor a kind of monopoly right gain
over the invention that anyone else could not use it without asking for permission.
There could be two different classes of patents termed as process patent and product
patent. It could also be stated that utility patents and design patents are the subclasses
available in patenting systems.
A design is defined as the ‘surface ornamentation’ including the configuration or
shape of an object. A design patent protects only the object’s appearance. It could be
obtained only when the design must be inseparable from the object. A utility patent is
the patent type that provides protection for the functional or structural features of the
patented object. It is the most common and most useful type of patent, which could
be obtained for new machines, devices, manufacturings, processes, and method. If,
an inventor wishes to get patent for both the functional features and the design of the
object, they should apply for utility and design patent both (Tulasi and Rao 2008).

4 IPR in Biodiversity and Bio-resources

Biological diversity includes the diverse forms of a variety of organisms, including

their genetic diversity in the world. This natural biological wealth and its interrela-
tionship of genes, species and ecosystems are precious for human society. Millions
of plants, animals, fungi and other organisms are the glamorous bio-resource that
we owe. Biological resources include organism’s populations, genetic resources and
another biotic component of ecosystems, which have significant potential use or
value for humanity (Kannaiyan 1992).
It was a matter of debate that whether biodiversity and bio-resource should be
covered under the terms and laws of IPR or not. The Traditional Knowledge of Biodi-
versity was earlier untouched by the contemporary debates related to the inclusion of
these properties under the margins of intellectual property rights. The concerns about
the inclusion of indigenous resources and genetic resources and bio-resources rose
enormously when the use and demands for the bio-products raised with each passing
year. The coverage of bio-resource under the IPR becomes a global need indeed. In
the recent decades, international agreements such as the convention on biological
diversity (CBD) and the trade-related aspects of intellectual property rights (TRIPS)
help in the commercialization of the traditional knowledge associated with the bio-
diversity all over the world. Bio-rich countries, such as Third World and India, cover
the countless varieties of animals, genetic resources, plants, trees, microbes which
have potential applications of commercial and economic importance. To avoid the
serious threat to the biodiversity and bio-prospecting, it becomes really an important
aspect to cover the use of bio-resources under IPR (Utkarsh et al. 1999).
152 T. Zafar

The global biodiversity pool has 1.75 million identified species among which 2.7
lakh belongs to plant kingdom. Such a huge pool of animal and plant bio-resource
is not easy to handle with equal distribution and prevented misuse.
The distribution of plants and animal species is diverse and unequal across the
globe. It is really very difficult to equally provide the resources among biodiversity-
rich countries and areas with moderate and little biodiversity. These life forms,
which are important for medicine, trade, food, fashion, tourism, energy, recreation,
are nowadays covered under the boundaries of IPR. Various acts and laws under
the edge of IPR in bio-resource sector cover the law related to the common her-
itage of mankind, biological diversity, patentable, microorganisms varieties, protec-
tion of plant varieties and farmers’ rights, sovereign right of nations: conservation,
sustainable utilization, equitable benefit sharing, etc.

5 IPR in the Pharmaceutical Industry

Pharma industry is a fast-growing business relative to other biotechnology and med-

ical science-based commercial benefits. It is very competitive to maintain the quality
of the drug while introducing new drugs for emerging diseases. Pharma industry
has nowadays shifted its focus to long-term consumable medicines rather than acute
medication. It is really costly for the company to introduce the new drug to the
market, while there is a huge market full of piracy. In this fast-globalized market,
it becomes essential to get a patent or IP coverage for any new or generic formula
(Angell 2000; Lexchin 2005).
Over the period, the applications to cover the IPRs related to drug and pharma
industry almost tripled in the last decade. This makes the competent authorities
to be a little choosy for patent granting and IPR protection for new drugs. These
are the reasons that drive the consequent reduction in patent protection coverage in
years. The drug industry also faces many others conflicting requirements such as
keeping the cost low to meet public demands, which is not an easy task because of
biotechnological research and development, production, management and marketing
chain (Mrudula et al. 2009). Pharma industry has a tough competition worldwide, and
thus, they usually do not disclose the invention or formula by publication until they get
it patented by proper channel. Patenting protects the drug formula from being stolen
or copied and retains the commercial rights within the inventors and real owners.
The procedure of IP protection is a bit complicated for biotechnologically developed
biopharmaceuticals as compared to simple conventional drugs. Each microbial strain
that is used in development should be identified before the IP protection, and if it
is an existing one, then the database was considered, but if it is novel, it should
be deposited and registered in the appropriate repository before patenting the drug
(Glasgow 2001).
There are many limitations and hurdles in pharma industry patents as IPRs’
conservation in the pharma industry is a multidimensional task. Writing patents
in the pharma industry requires special expertise in professional, scientific, legal
Intellectual Property Rights 153

and technological expertise. The inventor could also patent new inventions, as well
as new combinations, which have not used earlier. Combination of already existing
substances into something useful could also be patented.

6 IPR in Bioinformatics and Computational Biology

Bioinformatics is the branch of science that deals with the computer or computer-
based information technology to create the interdisciplinary and unambiguous
approach to manage biological data and information. From the discovery of DNA
and RNA structure, the need for bioinformatics in biological science study increased
exponentially. In a modern world, the generation and management of various
software-based scientific information are the biggest outcome of bioinformatics
(Harrison 2003). The genesis of bioinformatics becomes more and more signifi-
cant each day by development, proliferation and management of molecular science
data consisting of the gene, DNA, RNA, protein and functional sequence-based data.
Understanding of the biological approaches, the discovery of new drugs, detection
and identification of various gene-based medicines contains lots of efforts time and
funding, and thus, it becomes very essential to cover each novel and existing finding
within some legal framework (Fernandez and Chaw 2005; Fernandez et al. 2013).
IPR coverage of bioinformatics outcomes ensures the benefits and economic advan-
tages should reach to the real inventors for the rest of life. The patenting system
is one of the prompt tools to ensure the protection of IPRs within the bioinformat-
ics domain. In this way, the patent containing authorities without any risk of theft,
copy or misuse retains the authority to marketing and commercializing the prod-
uct. Security of fundamental rights related to economic arguments is also covered by
copyright protection and trademark development (Konig et al. 2015). However, there
are limitations also in the case of the genome and gene-based personalized studies.
Any database could be a subject matter of patenting or not depending upon the data
presentation and information it consists. However, software is more likely to be a
patentable intellectual property for the developers, which allows them to raise money
by the commercialization. The useful, tangible and concrete information that is pro-
vided by any particular software can be covered by IPRs regime by manufacturers
(McBride 2002).
Synthetic biology has grown enormously within the last few years with the devel-
opment of various computational aids in research. Life constructing pathways are
studied under these types of studies at the genetic level. These efforts are ahead of the
scale and extent of traditional recombinant DNA technology and similar approaches.
Biological engineering is a nonconventional branch that faces tremendous flaw under
IPR protection. While techniques related to biotechnology have mainly difficul-
ties for patent law, computers and computational methods both face copyright and
patent problems. Copyright is considerable for original works of expression, explic-
itly excluding works that are functional. However, patent law considers functional-
ity and traditionally excludes formulas and algorithms. Thus, many computerized
154 T. Zafar

approaches do not fulfill the considerations of neither the copyright nor the patent
box. It was too functional for copyright, too close to a collection of algorithms and
ideas for a patent (Gopalan 2009). Some popular foundation Biotechnological out-
comes such as monoclonal antibodies and recombinant techniques either were not
patented or patented. If patented, the owners of the patents were generally made the
techniques and benefits available widely at a reasonable cost. But one thing is sure
the law and IPRs are still struggling in between hardware and software inventions
(Rai and Boyle 2007).

7 IPR in Biotechnology and Corporate Biotechnology

Transfer

Biotechnology is a sister branch of biology with enormous possibilities of growth and

development. Since ancient era, human beings were using applications of biotech-
nology in a silent manner, but within a recent decade, the development of noncon-
ventional methods, the introduction of genetic modification in plants, animals and
microbial system help this branch to bloom as a lovely flower. Because of new
inventions, day by day, it is really essential for the inventors to introduce their inven-
tions with IPRs. There are various factors that justify the essentiality of IPRs in
biotechnology corporate arena.
All the common IPRs forms (trademark, trade secret, copyright, patent) are equally
applicable in the biotechnology industry depending upon the subject matter. In India,
IPRs in biotechnology field also face a variety of limitations. In India, it is not possible
to patent any plant species. Similarly, it is not possible to patent any developed
software in India, but still, the software can be secured under the functionality of
copyright protection. Plant breeder’s rights are the protection that covers the legal
benefits of plant species, which are not possible to get patent. There are many other
limitations and overcomings available in corporate biotechnology market with their
existing alternative support systems (Saukshmya and Chugh 2010).
Biotechnology sector requires an updated and relevant IPRs system because of its
own limitation of generating new inventions day by day. IPRs not only protect the
material, invention, process, information, technology secure from unfair use but it
helps significantly in revenue generation too. When a license is awarded for patented
material or technology, it generates royalty for the owner in two forms. Granting
permission for fair use allows the owner to receive royalty due to license sharing, but
if the use is unfair, it generates compensation from the illegal users. Revenues are
the biggest source of income for organizations scientists, researchers and inventors.
This constant influx of funds remains in the rotation. Research and development
branch requires lots of time, effort and money investment, which will recover from
the patented or copyrighted product revenue. By this process, the consistent funds
remain available for further survival of the industry (Giugni and Giugni 2010).
Intellectual Property Rights 155

Apart from this, IPRs not only provide the financial and legal cover but it also feeds
the moral and psychological reward system of the inventors. It is really worthless if
the inventors have no name, fame and money associated with the hard work done
earlier. Due to IPR patenting and copyright system, the individual and organization
retain the rights of name, fame and revenue with them. This helps the inventors to
become psychologically carefree and pleasant that they have sufficient platform to
portray their skills for their own as well as social benefit simultaneously.
In biotechnology, it is possible to get patents for nucleotide sequences for a limited
period of years, which then release in public domain; with a little controversy, gene
patenting systems also exist in few countries. However, in general, animals, human,
their genes are cells are covered by non-patentability approach. But exceptions are
available. Microorganisms obtained by genetic engineering are patentable. Genet-
ically modified crops, such as ‘tryptophan over producing maize,’ were patented
in the USA in 1985. Similarly, the ‘oncomouse’ was patented in 1988, the USA,
for being genetically modifiable. A plant patent is another type of nonconventional
patent, which is a little different from a conventional utility patent. A plant patent
is available for a plant that is obtained from asexual reproduction but not covers the
tuber propagated the plant. Trade secrets are also popular IPR form in biotechnology,
which is quite popular for hybridization techniques, cell line processing, customer
consumer lists and corporate merchandising plans. Indian Copyright Act, 1957, has
a later amendment in 1999 that reflects the Berne Convention on copyright. India is a
member party of many conventions and organizations including Berne Convention,
Geneva Convention, World Intellectual Property Organization (WIPO), Geneva and
UNESCO.

8 Biological Database and IPR

Biological databases are the libraries like information source about existing bio-
logical data. The biological database could be classified under various sub-divisions
including sequence, structural and functional database on the basis of the information.
All primary (Genbank, EMBL, DDBJ, SWISS-PROT, PIR, PDB), secondary (Uni-
Gene, PRINTS, BLOCKS) and composite databases (NCBI) also have significant
roles in IPR management. Primary databases are usually collected by government
or social funding resources; hence, they usually open to the public without any IPR
protection, while secondary databases are usually involved, capital, labor, time, etc.,
and thus, they are considered to be covered under IP protection (Groombridge 1992).

9 Conclusions

IPRs are very useful but only for those, who intellectually understand their right and
take actions to cover it. It is very beneficial to decide early, whether any information
156 T. Zafar

or idea is essential to protect under the legal line of defense using IPRs. As early as
the professional step taken to cover the idea, service or product, the rights become
more secured in professional front from the theft or competitors encroachment. It
should be chosen very carefully that what types of IPRs are useful for a particular
case. Understanding the IPR is a basic prerequisite for better management and com-
mercialization of biotechnological applications. Identification of the rights, proper
planning to secure the intellectual property helps to globalize the commercialization
of technology, creativity or invention without any fear of theft or misuse. IPR plays a
vital role in the management of the protection of inventions and creativity. The need
of the hour is to evolve IPR policies and incorporation of these into management
systems as a foundation strategy. A wise decision, in the beginning, is the protector
of safe and sound commercial future.

Acknowledgements Author is thankful to Prof. Vinoy K. Shrivastava, Head, Department of Bio-

sciences, Barkatullah University and Dr. Bashirulla Shaik, Assistant Professor, National Institute of
Technical Teachers Training, Bhopal, for their valuable guidance, consistent support and blessings.

Conflict of Interests Author declares that there is no conflict of interest.

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Dr. Tabassum Zafar is a guest faculty and researcher currently associated with Department of
Biosciences, Barkatullah University Bhopal, Madhya Pradesh, India. She is the recipient of vari-
ous fellowships including CSIR research internship award by Council of Scientific and Industrial
Research, India, DST-Inspire fellowship by Department of Science and Technology, Government
of India, FTYS—fellowship for training of young scientist by Madhya Pradesh Council of Sci-
ence and Technology. She has also received many Young Scientist Awards and felicitation from
Madhya Pradesh Council of Science and Technology, Royal chemical society of London, (North
India Chapter) and society of Lifesciences.
Commerce and Management
Evolution of Biotechnology as a Million
Dollar Market: The Management
and Commerce of a Biotech Start-up

Gaurav Verma and Srividhya Ravichandran

Abstract This chapter focuses on the commercial and management aspect of

biotechnology’s business in today’s scenario. The recent trends and the develop-
ments in the business models of the biotech sector would be dealt in detail. The recent
economy predicts the pharmaceutical sector as the most capital intensive market area
but the challenges in taking up a career in biotechnology and ethical principles that
govern the setting up of an industry in core areas of biotechnology are huge. The
role of biotech industry in terms of commercial, industrial and economical grounds
gains importance since it requires a lot of manpower in the intellectual and in the
commercial front. A success story of a biotech sector involves a lot of challenges
to be faced. Hence, the chapter would highlight the challenges that equip oneself to
be an entrepreneur amidst the growing demands of the biotech sector. The chapter
also throws light on the core and sub-domains of life science and biotech companies
which have grown enormously in the past two decades.

Keywords Biotechnology · Health care · Industrial biotechnology · Gene editing

tools · Plant breeding techniques · Bioprocess engineering · Entrepreneurship

1 Current Perspectives in the Biotech Sector

Biotechnology is recognized as one of the emerging areas in science and technology

having significant potential with a wide array of applications in health care, agricul-
ture, process industry and service sectors. Biotechnology is the future of the country,
and the private sector is the driving force of biotechnology supporting industrial
R&D and production capabilities which in turn greatly influence the human resource
development (Ninawe 2019). The ultimate aim of biotechnology is to deliver materi-
als, molecules or processes of interest for the medical, agricultural and environmental

G. Verma (B)
Clinical Research Center, Lunds University, Malmo, Sweden
e-mail: [email protected]
S. Ravichandran
Department of Biotechnology, Indian Institute of Technology, Chennai, India

© Springer Nature Switzerland AG 2020 161

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_9
162 G. Verma and S. Ravichandran

market. The past role of a biotech company has been more or less like an intermediary
between science and commercial applications (Joshi 2018).
The biotech industry, just like any other industry, is dedicated towards product
development but the difference is that there are stringent rules monitoring the produc-
tion (Kaitin 2010; Khilji et al. 2006; Maak and Wylie 2016). The amount of money
invested in the industry is very high while an uncertainty always remains about the
end profits (Chok and Sun 2007). With passing time, this industry continued growing
by academic collaborations and in-licensing of valuable research outcomes (Stuart
et al. 2007) accounting for the biotech industry which seems to be an important
part of modern economy. There have been intensive investments in this sector glob-
ally, especially in 2015 where this sector received the second highest funding and
hence rapid generation of business models (Segers 2015). Learning is important in
industries such as biotechnology in which knowledge is developing rapidly and the
focus is on new innovations and is indeed often a primary motive for entering into
partnerships.

2 Fields of Biotechnology

The different fields of biotechnology can be classified by colours, as a “rainbow”

methodology (Kafarski 2012) (Fig. 1)
In this sense, the several areas of biotechnology identified and classsified include:
• white—industrial biotechnology
• violet—law, ethical and philosophic issues
• blue—marine (aquatic)
• green—agriculture
• yellow—nutritional biotechnology
• red—medicine and human health
• grey—environmental protection
• brown—desert and dry regions
• gold—bioinformatics, computer science and chip technology
• dark biotechnology—bioterrorism and biological weapons.
There are some areas like nanotechnology which are grooming vigorously which
are not coded by the above classification; yet, they stand out vibrantly in the arena
of biotechnology. The industry could be broadly divided into pharmaceutical and
non-pharmaceutical sectors (Horvath et al. 2019). The pharmaceutical companies
have been on the top list in terms of the investments made since the evolution of a
new drug takes its time and money. Hence, the non-pharmaceutical companies find
a better position in the industry itself.
Evolution of Biotechnology as a Million Dollar Market … 163

Fig. 1 Areas of biotechnology. The figure represents various streams of biotechnology as given by
their color codes; green-agriculture; yellow-nutrition; red-health and medicine; white—industrial;
purple—ethics and law; gold—bioinformatics; blue—marine; grey—environment; brown—arid
and black—bioweapons

3 Commerce and Management of the Agricultural

Biotechnology

Genetic engineering has revolutionized food and agriculture to an enormous level.

Gene manipulation techniques have rendered many useful products like nutritional
supplements, food flavouring substances and food additives which are useful to
enhance the standard of the food industry to greater extents. Metabolic engineering
plays a progressive role in the production of different types of genetically modified
foods in the food industry. The development of functional foods plays an important
role in the area of food biotechnology. The presence of secondary metabolites and
also the genetic engineering of the microorganisms to incur the production of use-
ful products have been under research for the past three decades. The development
of metabolomics and metabolic engineering is some niche areas where the green
revolution has really paid off in the recent past.
The field of plant biotechnology is being transformed by new plant breeding
techniques, which is an orchestration of novel genetic techniques. There are tools
available to manipulate DNA at targeted sites to achieve specific traits. Of these tools,
clustered regularly interspaced short palindromic repeats (CRISPR) —CRISPR-
associated protein (Cas9), transcription activator-like effector nucleases (TALENs)
164 G. Verma and S. Ravichandran

and zinc-finger nucleases (ZFNs) have already found widespread use in research and
development (Kleter et al. 2019).
Metabolomics, used for crop improvement, is one of the most emerging tech-
nologies these days. It facilitates study of complex networks of biological processes,
aiming to accelerate the sustainable crop production. A complete understanding of
the enzymes present in traditional food and their nature of presence is extremely
important for the food manufacturing industries which are involved in their produc-
tion and application. It is time to find out novel approaches and easier methods that
mitigate with the genetic modification techniques and are also expected to contribute
to the value addition in terms of nutritive value of plant-based food and also the qual-
ity of enzymes used in the food industry (Meshram et al. 2019). The principle point
of GM crops is to accomplish food security or to improve nutrition, enhance suste-
nance and advance manageable farming (Chaudhary and Singh 2019). The greatest
benefit of the agricultural sector is the technology development and genetic engi-
neering techniques to bring in herbicide-tolerant and sustainable crops. However, at
times, the social and ethical dilemmas drive the industry very hard but GM crops are
already into human consumption after successful field trials.
The products produced by the genetically modified microorganisms are in use for
the promotion of health benefits and prevention of malnutrition problems. For exam-
ple, it is clear from a report that there are about 3000 different species of microalgae
which have the potential to produce triacylglycerol which are known precursors for
biofuels. Hence, focus would be laid on tapping the potential of biofuel generating
microalgae to capture the current market. Inventions are in progress to generate and
modify better molecular tools and technologies to instigate the process of synthe-
sizing triglycerides in lipid-accumulating microalgae. The quality and quantity of
agricultural products obtained using biotechnology are huge. The products in com-
mercial biotechnology are also on the rise every year. In other words, biotechnology
has yielded unique solutions for sustainable environment-friendly products through
industrial development. As a benchmark, biotechnological innovations must owe
to provide sustainable solutions for environmental, economical and cultural prob-
lems apart from providing nutritive food. Figure 2 illustrates the happenings in the
agricultural and nutritional biotechnology.
Continuous demand for agricultural growth in developing countries and the lack
of supply owing to less productivity have affected agricultural market trends in a large
scale. The contributory factors for loss of balance in supply-demand cycle are the
dwindling natural resources and the change in the global climatic conditions. Small-
scale farming had been the highest per cent of agricultural suppliers in developing
countries where agriculture becomes the major occupation till date. Agricultural
biotechnology has really contributed by contributing new ideas, techniques and pro-
cesses to keep up their roles and also by yielding sustainable agriculture solutions to
small farmers. Nevertheless, GM crops have been proven to be disease and herbicide
resistant and also earned a label for global identification for the economic growth of
the agricultural biotechnology sector. Science and technology remains the sole prob-
lem solver amidst adverse climatic, social, economical and environmental problems
being imposed on the farmers. Research and development at the field level along with
Evolution of Biotechnology as a Million Dollar Market … 165

Fig. 2 Status of biotechnology in agriculture. Biotechnology in agriculture encompasses gene

editing and manipulating tools which results in the rise of the GM crops but the success of the same
is not likely to address its problems of worldwide acceptance owing to the regulatory compliance
of the industries and processes involved

information practices should reach the farmers for them to get hands-on training and
solutions to develop their capacity. Public governance and innovation governance
play an important role in the commercialization and globalization of the GM prod-
ucts. There must be some decisive changes at the policy and operational level to bring
out new financial policies which strengthens public–private partnerships that could
initiate a global cascade of changes and a positive transformation of research and
development, product development and technology transfer from small- to medium-
scale farmers which would improve the economy of the agricultural biotechnology
sector and render a greener solution for the existing hurdles which might turn out to
be big barriers of growth of the sector in the long run.
166 G. Verma and S. Ravichandran

4 Commerce and Management of the Industrial

Biotechnology Sector

Industrial biotechnology involves the use of microorganisms to synthesize a number

of products impacting our lives and life spans and is valuable to human beings (Vijay-
Paul et al. 2019). Products used in daily routine are the major focus of the industrial
biotechnology sector (Pessoa et al. 2019). FMCG industry has its development on
a daily basis. For instance, food, cosmetic, pharma manufacturing and related fields
are looking for alternative approaches to sustain their market in a rather sustainable
way. The supply-demand cycle is the main criterion which decides the market trend.
We must also realize the fact that there is an increasing demand from consumer mar-
ket for bio-degradable and natural products instead of synthetic derivatives. Today’s
industrial biotechnology is ready to face the real challenges imposed by the consumer
end and also enter into the business in a full swing to meddle with the trend and also
take care of the supply-demand cycle.
Recent advancements in the genetic engineering techniques have paved way for
commercial production of food products which are consumed widely across the
world. Promoter engineering is an example of such technique to regulate gene expres-
sion and optimize metabolite biosynthesis in metabolic engineering and synthetic
biology. The gene expression techniques which involve direct or cascading effects
on the multi-gene pathways could significantly contribute to the metabolic flux distri-
bution and also to the maximization of the production of certain metabolites which
are real need of the hour (Xu et al. 2019). The end product of gene modification
could be of direct utilization in food, pharma and other industries which retain the
consumer market in an equipped manner.
Metabolic engineering, enzyme engineering, genomics, proteomics,
metabolomics, etc., play an important role in the process of intriguing the
genetic potential of the microorganisms to increase the yield of useful products like
secondary metabolites, nutraceuticals and essential phytonutrients, biopolymers,
pharmaceuticals and clinically useful enzymes. Several advancements in the indus-
trial biotechnology corridor have opened up new vistas for budding biotechnologists
who would get an opportunity to work under any of those branches. Right from the
internship level, opportunities have been huge for the future biotechnologists who
aspire to take up any research and developmental activities in any of the industrial
areas related to biotechnology. Figure 3 clearly depicts the prospects of the industrial
biotechnology with reference to the industrial demand of the novel products in the
market.
Industrial biotechnology has a lot of scope, and its potential contribution towards
economic growth is very huge and is supposed to pave way for a sustainable global
economy in the future. As discussed in the previous segment, academia-industry part-
nership can strengthen the industrial biotech sector in a very good manner. Consul-
tancy, contract research, public–private partnerships and bilateral partnership firms
are at their good deals to open up their economy stride into the industrial sector. In
the industrial biotech sector, few small and medium sized enterprises (SME) do exist
Evolution of Biotechnology as a Million Dollar Market … 167

Fig. 3 Industrial biotechnology—the past, present and the future. Industrial biotechnology has
always been an indispensable tool in meeting the needs of the consumers. The products rendered
by industrial biotechnology have added value, improved sustainability and kept the supply-demand
cycle at a threshold

when compared to the biopharmaceutical sector. These SMEs are in partnerships

with multinational companies. Biotech start-ups are a welcome note to the indus-
trial biotechnologists where the academia/universities are in a collaborative mode of
working. The technical know-how is transferable, and the ideas could be incubated
into these start-ups which would then develop into an independent firm. To create
an ideal business model is very difficult with the start-ups but the academia-industry
partnerships have definitely improved the success of the industrial biotechnology
sector to the next grade. A huge cost of commercialization and a lack of marketing
knowledge are barriers to technical work up involved in the technology transfer pro-
cess happening with biotech start-ups. A completely niche product would definitely
cross all the barriers and be successful in the industry. Hence, it is mandatory that
the start-ups must think in the ways of the consumer as well as the producer of the
same.
168 G. Verma and S. Ravichandran

5 Healthcare Industry a Major Investment Corridor

of Biotechnology in Future

Innovation has always been the backbone and underlying strength of the pharma-
ceutical industry. During decades, the industry has delivered multiple life-saving
medicines contributing to new treatment options for several medical needs (Khanna
2012). The accomplishments of the biotechnology sector have been formidable in
the improvement of health care. These include 16 biotechnological platforms used
in more than 200 novel products, more than 250 indications representing expanded
and improved patient care, more than $160 billion in global sales, more than 4,000
biotechnology companies worldwide and 170 biotechnology company acquisitions
by pharma firms represented a value of $185 billion (Evens and Kaitin 2018).
The pharmaceutical sector bound by the healthcare industry has grown enor-
mously in recent years. The developments in the pharmaceutical sector are vividly
seen by the drug expenditure of the total world’s population which is on the high
rise and expected to touch 1.5 trillion $ by the end of 2021, and also the revenues
gained by the sector is touching their peak levels when compared to the previous
decade. There is a prospect of growth in drug spending of around 6% by 2021, the
global biotechnology market will surpass USD 775 billion by 2024, and the areas
earmarked for the increased drug expenditures would be in oncology, diabetes and
autoimmune disorders. In order to meet the demand, a lot of inventions would be
expected in these areas in the future also.
The major forecasts for the healthcare sector are on the rise since the healthcare
costs for chronic ailments like cancer and neurodegenerative disorders are already
peaking. The demand for effective drugs and vaccines is the need of the hour. These
trends would be accommodating the drug market which is driven by pharmaceutical
biotechnology sector. Currently, nearly two-thirds of the funding for biotech research
and development comes from the pharmaceutical industry, while over one-quarter
comes from government sources, and less than four per cent comes from universities
(National Science Foundation).
The global recognition of the pharmaceutical industry is incomparable, and it has
reached its peak in the past decade and continues to be a powerful industry all the
more. Nevertheless, the European and American countries remain to be the major
producer of products formed from the entrepreneurial biotechnology sector. This
includes biotech-based products in health care whose value would be around 70 bil-
lion US dollars by 2020 which is a huge sum and reveals a dependency of the global
market on these service providers like US, UK and Germany. To mention, a notewor-
thy contribution towards health care is explicit when the utilization of recombinant
proteins expressed in plants has been a novel approach in biopharmaceutical indus-
try that renders practical as well as safety advantages over traditional approaches
(Moustafa et al. 2016). During decades, there have been several players (4300) in
pharma sector but only a small subset (261) seems to have tasted success with at
least one NME approved (Munos 2009).
Evolution of Biotechnology as a Million Dollar Market … 169

6 Commerce and Management of the Pharmaceutical

Biotechnology Sector

Healthcare industry is facing major challenges owing to the dynamicity of the market
trends in this sector. It has become a capital intensive market, and major hurdles faced
by the pharma industry are huge cost of investment towards research and development
right from the start-up stage to fully developed MNCs. Even in developed countries,
venture capitals face a setback owing to the high investment barrier in this industry.
In order to keep up with the market trends and stay profitable, companies are trying to
build up their economic scales. The scenario is really worsening in a long timescale.
Much attention is needed to keep their heads up in the gearing situation and to take
bold initiatives to be in the game. Drug discovery costs are huge, and big pharma
companies are not able to cope up with the investment challenges of the same.
Neurodegenerative diseases and rare genetic disorders are imposing challenges
to the pharmaceutical leaders to invest huge sum of money towards R&D in the drug
discovery market, and the economy seems to be much profitable when compared
to the past. Amidst the problem of expiring patents, pharma leaders are trying to
strike a balance between the achieved profits and new investments by carrying out
basic research and focus on generics diversification and collaborative research. The
drug development costs are at their skyrocketing levels in the past two decades. In
order to comply with the rules and regulations of a pharma manufacturing practices,
increased cost of production, scale-up and regulatory approval processes of new
drugs, pharma companies are facing a lot of challenges. Many large biopharma cor-
porations are trying to operate in unison with the local biopharmaceutical industry
situated at their offsites in any developing economic countries to perform manufac-
turing in these countries, with local contract manufacturing organizations (CMOs)
and trying to be a solution provider for huge investment and long duration process of
biopharmaceuticals. Merger of small pharma and medical biotech companies into one
and acquisition of pharma companies by profited multinationals are small stop-gap
solutions foreseen.

7 Nano Biotechnology, Promising Era of Biotechnology

Nanotechnology is established on the design, development and application of mate-

rials and devices possessing at least one dimension sized in a nanometre scale (Assa
et al. 2016). Nanotechnology became one of the leading sectors of biotechnology
that contributes to the global rise in the market and has risen to become a trillion
dollar industry in the recent past. The predominant areas of use of nanotechnology
in medicine, classified as nanomedicine, are in drug delivery and as diagnostic imag-
ing and biosensors. Nanotechnology is also emerging as an indispensable tool in
various parts of the biotechnology industries. The nanomaterials which are devel-
oped using nanotechnology could be used instead of chemical pesticides, fertilizers
170 G. Verma and S. Ravichandran

Fig. 4 Products of nanotechnology. Nanotechnology has grown as the biggest field of biotechnol-
ogy and the products given by nanotechnology include nanoparticle based biosensors, nanotubes,
diagnostic nanomaterials, nanocapsules and targeted drug delivery systems

and herbicides by promoting green and sustainable agriculture through the use of
nanofertilizers, nano pesticides and detection and control of plant diseases by using
nanoparticles; development of diagnostic tools for detection and control of human
diseases (Rohela et al. 2019). Figure 4 represents the products that are obtained from
the development in nanotechnology.
These diagnostic tools could have implications in molecular diagnostics, drug dis-
covery processes, targeted drug delivery, lab-on-chip, tissue engineering and regen-
eration. High-throughput microfluidics has revolutionized biotechnology assays,
enabling intriguing new approaches often at the single-cell level (Nagendran et al.
2018). Targeted delivery of drug using nanoparticles has always been a theme of
interest in nanoscience and nanotechnology. Scientists have been trying hard to use
these nanodelivery systems to target cancer cells and tumour tissues which otherwise
would proliferate to a greater extent even with conventional protocols for treatment. A
complete utilization of the nanoparticle- and nanomaterial-based approaches would
enhance the future technology and determine the fate of the market in agriculture.
Production of products based on nanotechnology has also faced the hurdles in their
initial phases, and currently, there is a welcome note added to the inventions from
the nanotechnology corridor. Futuristic innovations might include nanorobots for
performing surgery or as artificial organ substitutes and a lot more inventions to
come.
Evolution of Biotechnology as a Million Dollar Market … 171

8 Career Paths for Biotechnology Students

Biotechnology has proved to be a viable vehicle for the development and utiliza-
tion of technologies, which has brought not only advances to society, but also career
opportunities to nation-states that have enabling conditions (Dettenhofer et al. 2018).
Because biotechnology has applications in many industries, professionals can choose
to work for a variety of organizations, including government agencies, private com-
panies, regulatory bodies or clinical laboratories. Biotechnology employers range in
size and type from small start-ups to global pharmaceutical leaders. Employed by
medical communications agencies, contract research organizations and pharmaceu-
tical companies, medical writers distil and translate complex clinical and scientific
data to develop documentation spanning the entire pharmaceutical product life cycle,
from clinical development to registration and marketing (Hager 2019).
Though the biotechnology industry is a major economic driver, generating approx-
imately $140 billion in revenue and also the demand for skilled professionals will
continue to rise, to open up a career immediately after biotechnology in the core
field is yet another challenging task for biotech/life science students. As the indus-
try is still not self-sufficient, the students must be more dynamic and open towards
the opportunities which are coming to them soon after their degree. A postgraduate
diploma course in applied fields/information technology would help them mend their
candidature towards a good opportunity in biotechnology. Figure 5 shows the various
career paths of a biotech student in a pictorial form.
Flexibility and adaptability are two important mantras that every biotech student
has to keep in mind. If they choose to work in the core area of biotechnology, there
will be a limited number of options available. Henceforth, the students must be smart
enough to make themselves strong to choose their career option in biotechnology
and its allied fields.

9 Entrepreneurs in Biotechnology—Current Scenario

and Future Prospects

According to many experts, over the next few years, an innovative-driven or

knowledge-based economy, in which the production and the use of knowledge and
innovation as a source of gaining wealth and competitive advantage, will replace
the current traditional economics (Tohidyan Far and Rezaei-Moghaddam 2019).
Researchers have shown that entrepreneurship-related education could not lever-
age the process of transformation of additional products into growth, and founders
who start their businesses immediately after completing their studies might struggle
when they seek growth by adding more products.
The entrepreneurial experience would be adopted from their co-workers or a
known guide who could instigate the entrepreneurial ways to them. The ecosys-
tem for successful start-ups typically starts with proper mentorship with a mix of
172 G. Verma and S. Ravichandran

Fig. 5 Career prospects of a biotech student. A biotechnology student can acquire several career
prospects depending upon his/her area of expertise. Some of the jobs include microbiologist, med-
ical writer/knowledge management expert, pharmacovigilance/regulatory affairs expert, product
development scientist, a bioprocess engineer or a data scientist

technological and directorial support focusing on the career trajectory of biotech-

nology (Munshi 2019). Scientists from US universities find that the number of job
changes is positively related to the likelihood to become an entrepreneur, as indi-
viduals with many job experiences have had frequent employment offers and are
therefore assumed to possess valuable skills (Stuart and Ding 2006).
The hallucinations and the imaginative thought processes about the pros and
cons of the biotechnology industry obtained in the past would be deceiving to the
forthcoming and budding entrepreneurs, as the industry itself is dynamic and unpre-
dictable. The commercial part of biotechnology lies in the core domains of the sub-
ject itself while entrepreneurship and business environment differs across countries
(Cardon and Kirk 2015) and contextual factors such as cultural, social and economic
environments highly influence entrepreneurship research (Zahra 2007).
Some small towns and semi-cities have started to witness a revolution in the
growth of entrepreneurship, and there are people who are taking very traditional
businesses with a new dimension. One such example is biotech entrepreneur Sarah
D’Souza, Founder and Chairman of Biosyl Technologies, a biotech-based company,
headquartered in Hubballi, which is a small town in India which deals with both man-
ufacturing and services. Entrepreneurial activity involves a lot of newer technologies
Evolution of Biotechnology as a Million Dollar Market … 173

flowing in, which are often found as complex process formed by the co-evolution
of technology commercialization and entrepreneurship (Giones and Brem 2017). A
smaller technology might get a warmer note when it begins its real commercialization
into the market. There are times when a product developed by a huge venture capital
has failed to survive in this big arena, owing to the technology commercialization
and increased changes in the market. Hence, a vibrant situation and a trendsetting
attitude towards the entrepreneurial approach are the deepest truth of the market that
every entrepreneur has to keep in his mind. For an easy understanding, readers could
refer to Fig. 6.
This era is marked as a knowledge era as the intellectual capital plays an indis-
pensable role in the economy and growth of any market in a global pattern. Surplus
economic development and technological advances are seen in the areas such as
pharmaceuticals, aerospace and telecommunications. Though these areas are widely
separated in their distribution and consumerism, knowledge becomes a key factor
in controlling their growth levels (Lastres and Sarita 1999). Without an intellectual
capital driven by highly motivated entrepreneurial skills, a start-up would not exist
in the present economic situation.

Fig. 6 Qualities of a budding biotechnologist. A biotechnologist who wishes to become an

entrepreneur has to imbibe certain qualities like a good thinker and a decision maker, an expert
in analytical skills and a strong team player
174 G. Verma and S. Ravichandran

Support from educational and research institutions would also lead to the birth of
an entrepreneur from any moderate student who wants to survive in the core industry.
The shortcomings in entrepreneurial development are addressed to the issues owing
to the lack of conceptualism, understanding of how a few core institutions influence
entrepreneurship and the negligence of the scholars towards incrementally innovative
ventures (Dilli et al. 2018) which constitute a distinct (and under-researched) type
of entrepreneurship next to the (over-researched) form of radically innovative, high-
growth or high-tech entrepreneurship.
In order to find a business environment and capital friendly, a funding source
becomes indispensable. We could see that the Indian industry has faced a lot of
challenges in terms of funding. The government has also been a crucial factor in
such states where there is a real lack of other sources like venture capitalists who
face the real challenge of the market risks all the time. Today’s complex problems
require multidisciplinary approaches and team science, with investigators who are
equipped with sophisticated data analysis skills, expertise in ethical research and
other advanced capabilities (Katz and Glass 2019).
Biomedical research faces a scarcity of scientists who are able to work effectively
across diverse scientific disciplines (Lamb and Curtin 2019). The opportunities that
develop in these research areas are huge which need a strong interaction between
the academic and industrial researches, where each side is open to collaborate and
learn from the other and also focuses on those research fields where the individual
partners can provide real excellence.

10 Sustainability Development—A Better Option

for Entrepreneurial Biotechnology

Life science sector shows rise and fall depending on the industry. Certain areas of
development include entrepreneurship in sustainability development and environ-
mental management. The society and industrial sectors are facing important chal-
lenges regarding the production of bioproducts influenced by social responsibility
and environmental consequences (González-García et al. 2019). It would be nice to
recall the story of a life sciences entrepreneur Edward Robinson and his colleagues
who were seeking financial and technical support to develop a radical approach to
fish farming. The main aim of their farming strategy was to create a zero-waste
sustainable facility that would be friendly with the ecosystem. Their magnum opus
fetched them the state-of-the-art scientific facilities and also some guidance from
faculty experts and researchers. Their success story continued to secure $660,000
from a national funding agency that supported more growth.
One of the primary goals of bioeconomy is the development of new bioproduction
systems to produce fuels, chemicals and other materials at large scale from renewable
resources or various feedstocks including industrial waste streams using bio-based
conversion technologies (Zeng 2019). Converting generated biomass (which has
Evolution of Biotechnology as a Million Dollar Market … 175

the capacity to grow on wastewaters) from microalgae into the product such as
pharmaceuticals, biofuels and food supplements could also bring in greener solutions
for the major challenges faced today. Also these products would be sustainable and
environment-friendly and also cope up with the increasing demand of the food and
energy needs of mankind.
There is an increasing demand of oil products, and substitution of the same with
bio-based products would pave way for the emergence of a new bioeconomy and
new industrial processes paying tributes to the sustainability development concept.
Industrial biorefinery can be developed on the principle that any residues of one
can then be exploited as raw material by others in an industrial metabolism (Octave
and Thomas 2009). Post-bioremediation yields the chance of marketing high value-
added products obtained from the agricultural waste. Such type of sustainability
ventures would face a welcome note by the people per se. Process biotechnology
industries which are involved in bioremediation could also offer employment to self-
help groups and create an opportunity to the people around the place. Some start-up
ventures involved in the management of agricultural waste using microalgal sources
stand as a good example of this.
Methods of genetic engineering which include genome editing or gene transfer
would easily overcome the adverse environmental conditions by increasing the pho-
tosynthetic efficiency and production of biomass. It becomes mandatory to invent
techniques for optimizing biofuel production and also to provide environmentally
friendly fuels which are now possible with the utilization of biotechnology tech-
niques. These methods also counteract with the global warming and efficiently fight
with the problem of environmental pollution implicated by the greenhouse gases
(Delangiz et al. 2019). Thus, biofuel industries are a very important model of sus-
tainability development in biotechnology. As they have conceptualized to be the alter-
native for the fossil fuels, scientists and researchers have to overcome the barriers in
setting up one such industry or be a part of it.
Starting from the process optimization to the fine-tuning of the various processes
available to get the product out of the industry is really cumbersome. To be successful,
the organizations must continuously be responsive to the changing forces of the
business environment (Downes and Mui 2000) and strive for distinctively excellent
quality through their effective and efficient business processes. To strike a balance
between the realistic and the imaginative ideas put forth in any research-based idea
is also challenging while setting up a biotech industry, especially those which work
on the sustainability sector. Business creation from biotechnology could be done
through curriculum setting, financial and other policy incentives, and using their
convening power to start-ups and shorten the learning curve (Hohnen 2017).

11 Summary

The process of training young researchers is a key to ensure the feasibility of these
start-ups in the long term; however, many universities have curricula that are far
176 G. Verma and S. Ravichandran

from generating conditions that favour the development of new businesses. In one
of the recent articles in the European journal of innovation management, there was
a statement which indicates that the driven passion within oneself and the ability
to collaborate with peers and participate in the creation of new ventures in life
science start-ups have always overshadowed the downside risks and the lower level
of economic compensation. Passion-driven and motivation-filled work atmosphere
would also be a fun-filled one. The overwhelming incentives and lucrative earnings
of the other industries would pose a threat to biotechnology entrepreneurs at the first,
but the passion within themselves would definitely allow them to choose a career in
a start-up and to make a mark rather than earning high-rise figures from a venture
capital industry.

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178 G. Verma and S. Ravichandran

Gaurav Verma works at Clinical Research Center, Lunds University, Malmo, Sweden, to advance
the knowledge of diabetes and to explore the mechanisms to treat the diabetic individuals. He is
involved to identify a novel or unknown process through which T2D progresses and emphasizes
on the development of novel therapies to reverse diabetes.

Srividhya Ravichandran did her research work on the effect of polyphenols on the oxidative
stress mediated neuronal alterations in ageing rat brain. Currently, she is working as a technical
staff at IIT Madras. Her passion towards science education drives her to be a scientific writer as
well.
Some Success Stories and Products
Products of Biotechnology: The
Out-Turn of Research and Production

Arpita Saxena

Abstract This chapter covers information about various products of biotechnology

in the market already, or about to hit the market. The prehistoric, historic and modern
findings in biological and biotechnological research have led to harnessing the capa-
bilities of basic unit of life, i.e., the cell and its internal machinery. This is closely
responsible for the advancements in the fields of microbiology, physiology, medicine,
biochemistry and environmental studies; thereby, leading to a rapid amplification of
tools and techniques of recombinant DNA technology, cell fusion techniques, tissue
culture methods, genetic probes and bio-markers. All this has emerged as modern
biotechnology, which has closely bound research with industry and business. These
fundamental discoveries need to be translated into applications within a fixed or pre-
dictable time frame. Although conversion of scientific concepts into ideas, ideas into
proof of concept (POC) and POC into finished products is not an easy task and it
takes a long time, immense efforts and lot of financial investments to complete this
journey, still a lot of entrepreneurs have been able to accomplish this and have made
a big difference in the socio-economic lives of commoners.

Keywords Products · POC · Business · Bioprocessing · Enzymes

1 Introduction

All research should ideally lead to business and all business leads to products. These
products can be in the form of goods or services. Some products are designed for
consumption by common people directly, known as consumer products and some for
serving various business levels like producers, who further produce other products
out of these primary products or retailers, etc. Biotechnology industry is generally
characterized by frequently changing business models pertaining to the innovations
that keep the sector enormously diverse and flexible. Although this flexibility also
brings along uncertainty which is an indispensable part of biotechnology business

A. Saxena (B)
AAPT Foundation, SPAK megAcorp, S-9, Opp. DKMM Homeopathic Medical College,
Aurangabad 431004, India
e-mail: [email protected]

© Springer Nature Switzerland AG 2020 181

A. Saxena (ed.), Biotechnology Business - Concept to Delivery, EcoProduction,
https://doi.org/10.1007/978-3-030-36130-3_10
182 A. Saxena

(Konde 2009). Market economy is one of the fundamental driving forces that drive
this industry; other forces being social, political, environmental demands of the world
population, to continue driving this industry further (Soetaert and Vandamme 2006).
This industrial revolution in the field of biotechnology has led to product-oriented
research. This is a modern wave which has surpassed the earlier two waves, namely
medical or red biotechnology and agricultural or green biotechnology (Singh 2014).
In the Indian scenario, the government has decided to invest US$5 billion to develop
infrastructure and research laboratories so as to achieve a growth up to US$100
billion by 2025. Presently, the global share of Indian biotech industry is about 2%
growing at a compound annual growth rate (CAGR) of 20% (Next what business,
Accessed 2019).

2 General Products of Biotechnology

The traditional methods of food preservation have a long history dating back to the
discovery of fermentation, brewing, pickling and so on. These included microbes and
their use which was well explored since a long time. These methods led to processing
of certain range and variety of products. Followed by this, events like bioprocess-
ing and enzymatic process yielding, led to production of many useful products with
large market such as amino acids, organic solvents, acids, vaccines, etc. (Demain
2007). Today these methods have been industrially exploited and have been devel-
oped rapidly from the business point of view. There are specific broad sectors of
biotechnology industry like food, pharmaceuticals, medical, agriculture and envi-
ronment. I shall try to address some of them separately in this chapter. There are also
some general consumer products besides the above-mentioned categories, which are
manufactured using biotechnological processes such as the following (Biotechnology
Industry organization 2019).
• Cosmetics and personal care items—biotechnologically textured products are
highly pure, non-irritating, smoother, less greasy, enhanced hair-care properties
and environmentally sustainable.
• Bread—genetically modified microorganisms with enzymes to ensure better
quality bread having longer shelf life and eliminates the carcinogen potassium
bromate.
• Vitamin B2—genetically enhanced microbes produce this in one step fermentation
reducing 33% CO2 emission and use of energy.
• Diapers—using bacillus a biodegradable polymer is produced which is ecofriendly
and skin friendly as well.
• Detergent—biotech enzymes like proteases, amylases and lipases help cleaning
clothes brighter and save energy.
• Tissue paper—using wood bleaching enzymes the process of pulping is faster,
more environmentally friendly and cost effective.
Products of Biotechnology: The Out-Turn of Research … 183

• Textiles and stonewashed jeans—use of biotech cellulose creates fabrics that dye
better and are soft.
• Foam and nylon—The biotech processed foam is used to make furniture and nylon
to make carpets of softer better quality fiber. This is more durable, stain and UV
resistant than traditionally manufactured nylon.
• Plastics—biodegradable polymer, manufactured using bacillus microbe are very
useful in packaging of food and beverages, in making food service wares and
containers and are ecofriendly.
• Synthetic rubber—natural substances are polymerized to synthetic rubber and
other elastomers. These are high in purity and low in cost.
Apart from these, there are some more products (Table 1) that are either about to hit
the market floor or are in the market already. These products have started making
their customer base and their specific niche in the market is rapidly expanding.

3 Pharmaceutical Products

A usual product of ‘drug like’ capabilities on an average, takes ten years to find its
place in the market. It starts from virtual screening of millions of molecules through
tools of bio-informatics. This in silico study not only facilitates screening, refinement,
characterization of side effects and resistance of drug candidate, but also profiles a
high throughput data related to its genome, epigenetics, proteomics and ribosome-
related information (Xia 2017). The in vitro study follows this virtual screening
process and on a daily basis, and it is capable of generating data on hundreds and
thousands of compounds (Blass 2015). This is followed by mechanistic study along
with the clinical studies to examine the dosage regimen, efficacy, effects and side
effects, pharmacokinetics and quality control for the drug entity (Aronson et al.
2018). The road to make a biotech drug is filled with a lot of hurdles and U-turns
and the probability of failure haunts the scientists on every step. As per Professor
Gary P. Pisano, a renowned business analyst, it is critically important to get the right
people to work as he says, “Once you attract bright, young scientists, you need to get
them to realize this is not a postdoc lab, this is a business. It’s not enough just to do
the experiment; somebody’s got to take the ball and run with it” (Hanna 2000). Most
biotech drugs can be tailored and the patients can be treated more effectively and even
the genetic makeup of the patient can be changed depending upon the case to case
basis. Pisano further adds that “The $1 and $2 billion drugs will give way to $200–
$300 million drugs. That will be a very different world for big drug companies, with
different cost structures and resource-allocation processes” (Hanna 2000). Experts
say that the process of drug discovery is a complex process and the genome revelation
will facilitate the identification of molecular interactions for a drug. Listed in Table 2
are some biopharmaceuticals in global market and their manufacturers from around
the world along with their uses and applications.
184 A. Saxena

Table 1 Some general biotech products (Sotiriadis 2017)

S. no. Product name Manufacturing Advantages
company/laboratory
1. Drinks made with algae LifeDNA Vegetarian DHA and
(Grebow 2017) TerraVia vegetarian Omega-3
Algal protein and lipid
powder
2. Aquamin Stauber Aquamin derived from
Lithothamnion red algae
and is suited for
everything from bakery
products to confectionery,
snacks, smoothies, fruit
juices, and vegetable
drinks and is rich in
calcium and trace minerals
3. Synthetic spider silk University of Cambridge It is found to be stronger
(Matchar 2017) than steel and tougher than
Kevlar making it one of
the sturdiest materials
found in nature. It can be
stretched several times its
length before it breaks
4. Mushtari (Oh 2015) Mediated matter group It is a 3D-printed wearable
and stratasys that can convert sunlight
into usable products like
sucrose, desired pigments,
drugs, food, fuel or scents
5. Cow-free cow milk Perfect day Contains same proteins
like an actual cow minus
lactose
Can be produced more
locally and sustainably
Shelf life of six months so
storage and logistics
becomes easy
6. Lab-grown meat Memphis Meats Cheap, scalable, no animal
suffering, low pressure on
environment,
programmable food and
meat produced molecule
by molecule
(continued)
Products of Biotechnology: The Out-Turn of Research … 185

Table 1 (continued)
S. no. Product name Manufacturing Advantages
company/laboratory
7. Cell free Cell-free Tech User-friendly easy to use
kits containing plasmids
carrying the genetic
software instructions like
an app. This eliminates the
need for microbial
culturing, training and
equipment and customized
products can be obtained
8. MicrobeMiner Prospective research It mimics the stimuli
present in soil to activate
the silent operons of
Streptomyces bacteria
thereby producing variety
of antibiotics
9. Mushroom lamp Ecovative Eco-lamps, biodegradable,
sustainable, decorative and
turns down plastic
pollution

4 Food Products from Industrial Biotechnology

Food is the basis of sustenance and food processing has made food a way of recre-
ational activity as well. Socialization over food is a common thing since oldest times
and these days, it has become just more significant. Keeping this in mind, the food
sector has rapidly evolved and significant business associations have formed all over
the world liaisoning modern biotechnology research skills with large-scale manu-
facturers and marketers of food products (Lawrence 1988). Biotechnology, which
was earlier only being looked upon as medical and pharmaceutical revolution, now
is making a billion-dollar market in food as well. Some of the food and beverages
products are discussed (Maud Kordylas 1992) in Table 3.

5 Products of Agricultural Biotechnology

For a very long time research in agricultural biotechnology has been focussed on
improving crops yield, crop nutritional value and protection of crops against dis-
eases, pests and natural calamities. This has led to a variety of products with higher
yield, insect resistance, disease resistance, altered nutritional profile, enhanced shelf
life, draught tolerance, herbicide tolerance and many more specific traits desired by
agriculturists. There have been debates around the world about safety of biotech
crops, still biotech crops have been cultivated for nearly two decades and consumed
186 A. Saxena

Table 2 Biopharmaceutical products (Konde 2009; Demain 2007; Singh 2014; Biotechnology
Industry Organization (BIO), 2003)
S. no. Biopharmaceuticals Applications Manufacturing
company/companies
1. Plasma derivatives, Medical and clinical Bharat Serums and
monoclonals, hormones, applications Vaccines, Mumbai
antifungals, anesthetics,
diagnostic products
2. AranespTM (darbepoietin Anemia induced due to Amgen
alfa) Chemotherapy in patients
with non-myeloid
malignancies
3. Human growth hormone Growth deficiency Genetech, Pharmacia
4. Interferon-α cancer, hepatitis Schering-Plough, Roche,
Serono, Schering AG
5. Interferon-β Multiple sclerosis, Biogen, Serono, Schering
hepatitis AG, Chiron
6. Recombinant drugs, Heart diseases and Bharat Biotech
cardiovascular diseases, vaccines for various International, Hyderabad
vaccines diseases
7. Human insulin Diabetes Novo Nordisk, Eli Lilly
8. Argatroban Anticoagulant for use in Texas Biotechnology
patients with or at risk of Corp. and
heparin-induced GlaxoSmithKline
thrombocytopenia
undergoing percutaneous
coronary interventions
9. Tissue plasminogen Blood clot Genentech
activator
10. BOTOX ® COSMETIC Temporary improvement Allergan Inc.
(botulinum toxin type A) in appearance of moderate
to severe frown lines in
adults 65 or younger
11. Pediatric and childhood Animal- and human-health Indian Immunologicals,
vaccines, DNA-based products Hyderabad
vaccines
12. FOLLISTIMTM Treatment of primary and Organon (unit of Akzo
(follitropin-beta) secondary Nobel)
hypo-gonadotropic
hypogonadism in men
13. Polyvalent vaccines, Human indications Panacea Biotec, New
drugs/formulations including pain Delhi
management, diabetes and
renal disease
14. Granulocyte-colony Neutropenia/White blood Amgen, Roche, Schering
stimulating factor (G-CSF) cell
(continued)
Products of Biotechnology: The Out-Turn of Research … 187

Table 2 (continued)
S. no. Biopharmaceuticals Applications Manufacturing
company/companies
15. Factor VIII Hemophilia Bayer
16. Rituximab Non-Hodgkin’s lymphoma Genentech/Idec
17. INTEGRA ® dermal Repair of scar contractures Integra Life Sciences
regeneration template Holding Corp and Ethicon
Inc.
18. Glucocerebroidase Gaucher’s disease Genzyme
19. OLUX ® Foam Short-term topical Connetics Corp.
(clobetasol proprionate) treatment of mild to
moderate plaque-type
psoriasis of non-scalp
regions excluding the face
and intertriginous areas
20. Blood plasma proteins, Reliance Life Sciences,
recombinant proteins Mumbai
21. Erythropoietin Anemia Amgen, Johnson &
Johnson, Roche, Kirin,
Sankyo
22. Therapeutic antibodies Cancer Glaxo, Amgen, Genentech
23. SecreFloTM (synthetic Aid in location and Repligen Corp.
porcine secretin) cannulation of pancreatic
ducts in patients
undergoing endoscopic
retrograde
cholangiopancreatography
24. Etanercept Rheumatoid arthritis Amgen, Wyeth
25. Xyrem ® (sodium Cataplexy associated with Orphan Medical Inc.
oxybate) narcolepsy
26 Infliximab Crohn’s disease Johnson & Johnson
27. Orfadin ® (nitisinone) Hereditary tyrosinemia Swedish Orphan
type 1 International AB and Rare
Disease Therapeutics Inc.
28. Trastuzumab Breast cancer Roche
29. GleevecTM (imatinib Gastrointestinal tumors Novartis AG
mesylate) and Philadelphia
chromosome-positive
chronic myeloid leukemia
30 Palivizumab Prevention against Medimmune
respiratory syncytial virus
188 A. Saxena

Table 3 Food products obtained by using biotechnological processing (FAO International

Technical Conference 2010)
S. no. Products Country/Company Benefits Process of
production
1. Rob Sudan Butter extraction Fermentation
(Dirar 1992)
2. Merissa, Opaque Sudan Local beer 5% sorghum malt
beer is used along with
a caramelized
sorghum product
called surji (Dirar
1976, 1978)
3. Fermented soy Thailand, Japan, Economic gain for Using starter
sauce China the soy sauce culture technology
industry and and the use of
greater value added improved
to the product in bioreactor
terms of quality technology.
and safety Aspergillus oryzae
and
Saccharomyces
rouxii are used as
microbes for
culturing and
fermentation
(Valyasevi and
Rolle 2002)
4. Nham (fermented Thailand, Viet Traditionally Acidity of the
pork sausage) Nam, Lao and produced nham is culture was rapidly
Combodia considered high increased that
risk by Thai health prevented the
authorities, while growth of bacterial
biotechnologically pathogens. Nitrite
processed Nham was checked and
was endorsed for monitored. An
its safety and innovative pH
quality indicator was used
to mark end point
of fermentation.
RAPD markers
were used for the
molecular typing
of approximately
100 bacterial
strains (Paukatong
and Kunawasen
2001)
(continued)
Products of Biotechnology: The Out-Turn of Research … 189

Table 3 (continued)
S. no. Products Country/Company Benefits Process of
production
5. Som Fug South east Asian High Lactobacillus
(Fermented fish countries discriminatory pentosus and Lact.
paste) power of Plantarum are the
biotechnology in garlic fermenting
differentiating lactic acid bacteria
lactic acid bacteria associated with
at the strain level Som Fug
fermentations
(Paludan-Muller
et al. 2002)
6. Flavor production African Savannah High-value Alkaline
from region compounds such as fermentation of the
alkaline-fermented flavor compounds African locust
beans are derived from bean, dawadawa
low investment (Steinkraus 1995)
fermentation
7. Vanillin, Monascin Flavoring and
coloring pigments

by billions of people around the world. Scientists believe that to improvise the crops,
traditional breeding methods have the same consequences as using biotechnological
techniques. An interesting application of selective alteration of specific genes is that
specific allergens in particular crops can be checked and a non-allergenic variety
of the same crop can be developed targeting the consumer group those are allergic
to specific type of foods. Generally speaking, the scientists aim to check hunger
and malnutrition through gene alterations and specifically bringing up varieties of
foods with particular desired traits. Some biotechnologically grown crops have been
enlisted in Table 4 along with their advantages over the previous traditional crops.

6 Summary

Apart from the crops that are genetically modified for achieving specific traits, there
are other applications of agricultural biotechnology research that has led to products
of high commercial value like vaccines, antibiotics and nutritional supplements.
Genetically modified foods may be prepared with specific antigenic proteins that
trigger immune response in consumers and/or boost their resistance to pathogens.
The antibiotics obtained through biotech applications in plants reduce the cost of
production, risk of contamination and production time. This book includes a chapter
on nutraceuticals as they are a major player in economic growth and commercial
value addition of food and pharma products. The beta carotene-rich golden rice is
a transgenic grain rich in Vitamin A and certain other nutrients, which is valuable
190 A. Saxena

Table 4 GMO crops and their advantages (Young 2017; ISAAA 2019)
S. no. Crops/GMOs Plantation areas Advantages
1. Maize USA, South Africa, Argentina, 85% maize grown in the USA is
Brazil, Canada, Paraguay, genetically modified.
Colombia, EU, Philippines, Herbicide-tolerant and
Vietnam, Uruguay, Cuba, insect-resistant varieties available
Honduras
2. Soy USA, South Africa, Argentina, Most economically relevant crop.
Bolivia, Brazil, Canada, Herbicide-tolerant and
Paraguay, Mexico, Chile, Costa insect-resistant varieties
Rica available, another variety
available is with high oleic acid
content
3. Canola Canada, USA Herbicide-tolerant gene
containing variety is available
along with a high Laureate
variety, which is used for
chocolate candy coatings, icings,
toppings, coffee whiteners and
few cosmetics
4. Tomato China and USA These tomatoes contain genes for
delayed ripening that provide
longer shelf life, higher
commercial value and better taste
5. Alfalfa Canada, Mexico, USA Herbicide-tolerant variety contain
genes that provide resistance to
broad-spectrum herbicides thus
reducing crop injury
6. Potato USA and Canada Insect-resistant variety is
resistant to pests and insects. The
virus-resistant and low
acrylamide varieties are also
available
7. Rice USA Herbicide-tolerant variety
provides protection against broad
spectrum and environmentally
benign herbicides
8. Cotton USA, South Africa, Australia, Herbicide-tolerant and
Argentina, Brazil, Costa Rica, insect-resistant varieties available
Colombia, Mexico, Paraguay
9. Papaya China and USA There is a virus-resistant variety
that provides in-built protection
against Papaya Ringspot virus
(PRSV)
10. Sugar beet USA and Canada A herbicide-tolerant gene
facilitates the growers with
reduced number of cultivations
Products of Biotechnology: The Out-Turn of Research … 191

to check dietary deficiencies and resist diseases. Applications of agro-biotech in

producing specific nutrient-rich crops are classic example of success of scientific
intervention over traditional methods.
There are success stories of start-ups that transformed into Pvt. Ltd companies like
Advanced Biotech Products (http://www.advanced-biotech.com/), Chennai. They are
affiliated to Encoll Corp., a USA-based company, and have patented a technology
to manufacture highly purified collagen. This collagen is surface modified using
collagen-phosphorylation technology to make tissue regenerative and remodeling
products for clinical and surgical purposes. They provide products for dermal sur-
gical, general surgical, dental surgical and skincare cosmetics. They are working to
achieve gene therapy, stem-cell delivery and target-based cure for diseases, using
their technology base.
There are many companies that manufacture self-test kits to monitor health and
diagnose diseases/pregnancy cases. Just like the popular pregnancy testing kit, there
are ovulation detector kits, HIV and STD test kits and many more options for con-
sumers to personally diagnose these conditions. Forensic DNA analysis tools, lie-
detecting equipment and biometric capturing centers are another category of prod-
ucts/services those have a demand in the market. There is a fairly big market for such
products.
Energy sector remains a major investment platform as society is transforming
from oil economy to bioeconomy. Bio-diesel, bio-ethanol and bio-oils are being
produced from feedstocks of good quality. Microbial strains are being engineered
for large spectrum applications. There are many companies that are into culture,
genetic modification and production of such microbes like Aequor Inc. that engi-
neers marine microbe that kills foul smell and biofilm over the water body; or Chain
Biotechnology Ltd. that makes microbial hosts for engineering anaerobic bacte-
ria. Similarly, Greenlight Biosciences facilitates cell-free bioprocessing technology,
while Synthetic Genomics with its advanced genetic engineering techniques can
produce microbial cell lines, DHA Omega-3 and Astaxanthin.
Indian biotechnology industry was primarily into manufacturing and production
of biopharmaceuticals with a USA-based company as a technology partner. Grad-
ually the scene has changed and there are many biotech start-ups mushrooming
up with proprietary technology and innovation. The government support is also
very favorable for start-up culture with many policies and funding support schemes
for new entrepreneurial ideas. Education system itself has started to accommodate
entrepreneurial skills in the curriculum which represents a positive change.

7 Conclusion

Research has moved from microbial fermentation related enzymes to DNA and RNA
modifications and customized production of desired entities. With this transforma-
tion, a horizon has opened up for stakeholders of new biotech products and services.
There are scientific and academic communities collaborating with each other to
192 A. Saxena

match up with the market demand and speed. Specific categories of products from
pharmaceutical, food and agricultural sectors are always flowing into market dynam-
ically while offbeat general products are matching up with these categories building
up a niche. As the education and awareness in the society increases, acceptance of
these products will increase resulting in the vertical mobility of biotech products on
the ladder of new innovations and inventions.

Acknowledgements I thank my mentor Dr. A. K. Saxena.

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Arpita Saxena is presently self employed at SPAK megAcorp, her proprietary venture. Arpita was
awarded her PhD in Biotechnology in 2012 off campus from Guru Nanak Dev University, Amrit-
sar, India while working at the department of Cancer Pharmacology, Indian Institute of Integrative
Medicine (CSIR), Jammu, India. During PhD her work was focused on understanding the methods
to evaluate cell death, apoptosis and the mechanisms underlying apoptosis.