2020 Book BiotechnologyBusiness-ConceptT
2020 Book BiotechnologyBusiness-ConceptT
Biotechnology
Business
- Concept
to Delivery
EcoProduction
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
to link the scientific activities in various manufacturing and logistics fields with the
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
state-of-the-art and motivating for the future development of sustainable manufac-
turing systems, environmentally conscious operations management and reverse or
closed loop logistics.
It aims to bring together academic, industry and government personnel from
various countries to present and discuss the challenges for implementation of
sustainable policy in the field of production and logistics.
Biotechnology Business -
Concept to Delivery
123
Editor
Arpita Saxena
SPAK megAcorp
Aurangabad, India
This Springer imprint is published by the registered company Springer Nature Switzerland AG
The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland
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.
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.
Why Biotechnology?
Biotechnology: Discoveries and Their Applications
in Societal Welfare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Harsh Kumar
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
Contributors
xv
xvi Editor and Contributors
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.
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
1 Introduction
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).
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.
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.
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.
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.
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).
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
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).
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).
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.
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
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).
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
3.6 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
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
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
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.
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
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.
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).
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).
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).
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).
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
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. 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/
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
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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
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.
S. Bhattacharyya (B)
Oregon Health and Science University, Portland, USA
e-mail: [email protected]
A. Mukherjee
Oregon State University, Corvallis, USA
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
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.
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).
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
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).
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
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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
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
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
5 Conclusion
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64 A. Mukherjee and S. Bhattacharyya
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.
1 Introduction
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.
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
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.
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.
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).
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.
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).
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).
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).
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
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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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.
R. Bhati (B)
Department of Microbiology, Bundelkhand University, Jhansi, Uttar Pradesh 284128, India
1 Introduction
2 Biodiesel: Introduction
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.
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).
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.
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
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.
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
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
toward higher oil production, make the algal membranes and cell walls withstand
high oil, etc.
1 Introduction
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.
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)
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.
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
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).
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
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.
2.2.2 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).
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).
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).
2.3.2 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
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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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
Abbreviations
1 Introduction
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.
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
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”.
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.
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.
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
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.
(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”.
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.
3.1.9 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.
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).
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.
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.
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biotechnology. Executive Office of the President. Federal Register 51:23302. Policy, O. o. S. a.
T., Ed. Executive Office of the President: NEOB-Room 5005, Washington, DC 20506
Rao SN (2018) Regulations and guidelines on biosafety of recombinant DNA research and biocon-
tainment. In: Chapter 1, Department of Biotechnology, Ed. Ministry of Science and Technology,
India, p 148
USFDA, Safety Assessment for IND Safety Reporting, Guidance for Industry (2015) 10985dft
12-14-2015, (CBER), C. f. B. E. a. R., Ed. US, p 16
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
aspects of secondary metabolism in microorganisms and medicinal and aromatic plants since ten
years. Dr. Gandhi has resourcefully collaborated with industries on various projects and is asso-
ciated with many academic organizations. He did his Ph.D. at Center for Cellular and Molecular
Biology (Hyderabad) focused on understanding molecular effectors and mechanisms of epigenetic
regulation. His bachelors and masters were in Microbiology from Jawaharlal Nehru University,
Delhi.
Inshad Ali Khan Principal Scientist at CSIR-Indian Institute of Integrative Medicine, Jammu.
NASI-Reliance Platinum Jublee Award—2017 for application-oriented research in the area of bio-
logical sciences. Awarded DHR fellowship by Indian Council of Medical Research 2012 to work
in NIAID, NIH, Maryland, USA. He is a member of various biological societies and academies.
His areas of research have been anti-infective research with reference to identification of novel
scaffolds and antimicrobial resistance and microbial efflux pumps.
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.
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).
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.
In the 21st century, intellectual property rights can be easily classified into four major
divisions named trade secret, trademark, copyright and patent.
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
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.
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.
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).
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.
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.
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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
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
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
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
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
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
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
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
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.
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
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.
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
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.
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.
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.
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well.
Some Success Stories and Products
Products of Biotechnology: The
Out-Turn of Research and Production
Arpita Saxena
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]
(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).
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 (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
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.
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 (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
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.
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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.