1 INTRODUCTION
1.1 WHAT IS BIOTECHNOLOGY?
Karl Ereky first coined the term “biotechnology” in 1917. At that time it referred to technology
related to large scale production of pigs using sugar beets as food source and production of products
from raw materials with the aid of living organisms. The term was redefined in 1961 to include
industrial production of goods and services by processes using biological organisms, systems and
processes. Biotechnology is in essence the deciphering and use of biological knowledge (see Table
1.1 for other definitions). It is highly multidisciplinary since it has its foundations in many disciplines
including biology, microbiology, biochemistry, molecular biology, genetics, chemistry and chemical
and process engineering (Fig. 1.1). It may also be viewed as a series of enabling technologies that
involve the practical application of organisms (particularly microorganisms) or their cellular
components to manufacturing and service industries and environment management.
Historically, biotechnology was an artisanal skill rather than a science, exemplified by the
manufacture of wines, beers, cheese, etc. where the techniques of manufacture were well worked
out and reproducible, while the biological mechanisms were not understood. As the scientific basis
of these biotechnology processes has developed, this has led to more efficient manufacturing of
traditional processes that still represent the major financial rewards of biotechnology.
Table 1.1: Some selected definitions of biotechnology
The application of biological organisms, systems or processes to manufacturing and service
industries.
The integrated use of biochemistry, microbiology and engineering sciences in order to achieve
technological (industrial) application capabilities of microorganisms, cultured tissue cells and
parts thereof.
A technology using biological phenomena for copying and manufacturing various kinds of useful
substances.
The application of scientific and engineering principles to the processing of materials by
biological agents to provide goods and services.
The science of production processes based on the action of microorganisms and their active
components and of the production processes involving the use of cells and tissues from higher
organisms. Medical technology, agriculture and traditional crop breeding are not generally
regarded as biotechnology.
The use of living organisms and their components in agriculture, food production and other
industrial processes.
The deciphering and use of biological knowledge.
The application of our knowledge and understanding of biology to meet practical needs.
Modern biotechnological processes have generated a range of new and novel products, including
antibiotics, recombinant proteins and vaccines, monoclonal antibodies, the production of which has
been optimized by improved fermentation practices. Biotechnology has been further
revolutionized by a range of new molecular innovations, allowing unprecedented molecular
changes to be made to living organisms. Genomics and proteomics are now heralding a new age of
biotechnology, especially in the areas of human health and food production. In the environment,
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biotechnology innovations are creating major advances in water and land management and also
remediating the pollution guaranteed by over-industrialization. New aspects of biotechnology,
such as genetic engineering, have aroused certain social sensitivities of an ethical, moral and
political character. Regulatory authorities throughout the world are now examining the
implications of these new and revolutionary techniques.
Fig 1.1: Biotechnology is the fusion of many areas of biology which has impacted several fields such as agriculture,
medicine and environmental pollution (Rastogi, 2007).
1.1.1
THE BEGININGS OF BIOTECHNOLOGY
The ancient Sumerians and Babylonians knew to make fermented liquors by using yeast, a practice
that continues today but with more refined technology (Table 1.2). Fermented foods such as bread
and cheese have been produced since olden times and may be cited as the crude beginnings of
biotechnology. It was not until the 14th century AD that fermentation techniques were employed
to produce alcoholic beverages by fermenting grains. The Chinese were perhaps the first people
who processed cheese, wines and other fermented food products. Indians since ancient times
produced vinegar and curd as food items. However, biotechnological practices got a boost in the
19th century due to scientific developments and the discovery of the role played by microorganisms.
Due to this, the term biotechnology is sometimes applied to processes in which microorganisms are
grown under strictly controlled conditions and thus brewing is sometimes called the oldest form of
biotechnology.
1.1.2
RISE OF THE FERMENTATION INDUSTRY
Another important milestone was the development of the food industry based on microbial enzyme
activity. While many beverages and cheeses are produced by the fermentation industry, microbial
spoilage of food is one of the problems faced by the industry. This led to technological progress in
processing and quality management (development of HACCP and ISO standards).
This milestone was due to the pioneering work of Pasteur who demonstrated in 1857 that the
souring of milk was caused by microorganisms. During the same time Koch (1843-1910), a German
physician, showed that specific microorganisms are the causative agents of diseases in humans and
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animals. Microorganisms have since been used for the synthesis of many drugs, steroids, vaccines
and intermediary products commonly used for therapeutic purposes.
Table 1.2: Historical Development of Biotechnology
Biotechnological
Sumerians and Babylonians were drinking beer by 6000BC; Egyptians
production of foods were baking leavened bread by 4000BC; wine was known in the Near
and beverages
East by the time of the book of Genesis. Microorganisms were first seen
in the seventeenth century by Anton van Leeuwenhoek, who developed
the simple microscope and the fermentative ability of microorganisms
was demonstrated between 1857 and 1876 by Pasteur – the father of
biotechnology. Cheese production has ancient origins, as does
mushroom cultivation.
Biotechnological
Ethanol, acetic acid, butanol and acetone were produced by the end of
processes
initially the nineteenth century by open microbial fermentation processes.
developed under non- Waste-water treatment and municipal composting of solid wastes
sterile conditions
created the largest fermentation capacity practised throughout the
world.
Introduction of sterility In the 1940s, complicated engineering techniques were applied to the
to
biotechnological mass cultivation of microorganisms to exclude contaminating
processes
microorganisms, e.g., in the cultivation of antibiotics, amino acids,
organic acids, enzymes, steroids, polysaccharides, vaccines and
monoclonal antibodies.
Applied genetics and Traditional strain improvement of important industrial organisms has
recombinant
DNA long been practised. Recombinant DNA techniques, together with
technology
protoplast fusion, allow new programming of the biological properties
of organisms.
Modern fermentation industry owes its existence to the First World War (1914-1918). Glycerol was
an important item in times of war for the manufacture of explosives. Usually glycerol is a by-product
of soap manufacture from fats, which are always in short supply during war time. During the First
World War, Germany faced acute shortages of glycerol due to the British Naval Blockade. They
made up this shortfall by using yeast to produce glycerol by fermentation of sugar by Neuberg’s
Method which also had the advantage of simultaneous production of alcoholic substances for
industrial use.
Industrial microbiology, the major foundation of biotechnology, arose out of empirical
developments in the production of wine, vinegar, beer and sake, and with the traditional fungal
fermentations used in Asia and Africa for the production of food. An experimental approach to the
production of microbial metabolites only began at the beginning of the 20th century. Up until the
time of World War II (1939-1945), the main microbial products that had developed from this
experimental approach were enzymes such as proteases, amylases and invertase.
A major breakthrough in biochemical and microbial engineering occurred after World War II as a
result of the large-scale production of the first antibiotic, penicillin. In order to produce this
antibiotic economically, important engineering developments had to be made, including the
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development of techniques for large-scale sterilization, aeration, and growth of microorganisms.
In addition, genetic methods for microbial strain improvement were perfected.
From World War II up until about 1960, the major new biotechnology products were antibiotics.
Through the intense efforts of the pharmaceutical industry, numerous new antibiotics were
discovered and of these around 20 were put into commercial production. In addition, in this early
post-World War II period, processes were developed for the chemical transformation of steroids.
Processes for the culture of animal cells for the production of virus vaccines were also perfected. In
the period from 1960 through 1975, new microbial processes for the production of amino acids and
5’-nucleosides as flavour enhancers were developed, primarily in Japan. Numerous processes for
enzyme production for industrial, analytic and medical purposes were also perfected. During this
same period, successful techniques for the immobilization of enzymes and cells were developed.
A further development was the use of continuous fermentation for the production of Single-Cell
Protein (SCP) from yeast and bacteria for use as human and animal food. SCP processes were
developed using microorganisms capable of using petroleum-based starting materials such as gas
oil, alkanes, and methanol. In this same period, microbial biopolymers such as xanthan and dextran,
used as food additives, were also developed into commercial processes. Somewhat distinctive
processes that were advanced during this period were the use of microorganisms for tertiary oil
recovery (an aspect of geo-microbiology) and the perfection of techniques for anaerobic cultivation
of microorganisms, derived out of studies on the sewage treatment process.
Since 1975 biotechnology has entered some important new phases. First was the development of
the hybridoma technique, for the production of monoclonal antibodies, of interest primarily in the
medical diagnosis field. Soon after was the production of human proteins using genetically
engineered Escherichia coli. The first product, human insulin, was introduced in 1982, followed
soon by Factor VIII, human growth hormone, interferons and urokinase. At present a vast array of
human proteins are in the development stage.
Although the production of human proteins by engineered bacteria is generally recognized as the
major “highlight” of the period since 1975, in actuality other products are economically more
important. For instance, the production of ethanol by immobilized cells has become a major
process. The enzyme glucose isomerase has become a 27 million dollar industry and is used to
produce high-fructose syrup which itself has a value of 2.5 billion dollars. Aspartame, a major
artificial sweetener, is produced microbially. Many new antibiotics have been introduced. Cheap
fats are being increased in value by enzymatic esterification, the enzymes being microbial products.
The biodegradation of persistent chemicals using specially developed microbial strains as starter
cultures is being field-tested.
1.2 BIOTECHNOLOGY – A FUSION OF SEVERAL FIELDS
Biotechnology is knowledge intensive and involves development of skills and manpower drawn
from various fields (Fig. 1.1).
1.2.1
EMERGENCE OF MOLECULAR BIOLOGY
The discipline of Molecular Biology that merged the structural, biochemical and informational
approaches with classic genetics, emerged in the 1950s. Watson and Crick proposed a model for
DNA – made up of genes encoding information determining the processes of replication and protein
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synthesis. This led to the deciphering of the genetic code, elucidating the machinery involved in
replication and protein synthesis, and opening the scope for DNA manipulation. Advances in
molecular biology saw the emergence of recombinant DNA technology or genetic engineering
which led to the potential to carry out deliberate and controlled modification of the genetic makeup
of living beings to solve problems.
1.2.2
EMERGENCE OF GENETIC ENGINEERING
The term genetic engineering was coined by A Jost. However, the Nobel laureate Joshua Lederberg
envisaged the dramatic applications of genetic engineering techniques to plants, animals and
microorganisms by the use of viruses and plasmids to transfer new genes into humans and plants
for treating diseases and crop improvement. By 1970 the techniques of genetic engineering with
bacteria were perfected. In 1972, Boyer and Helling successfully performed gene splicing
experiments and transferred foreign DNA via phage lambda into E. coli. It soon became reality that
by manipulating bacterial genomes, foreign DNA could be replicated and made to express genes
from higher organisms to synthesize foreign proteins. Thus, commercially important substances
such as insulin, growth hormone, interferon, interleukins, etc. could be produced, making bacteria
chemical factories.
Some important developments:
1 Construction of new vectors capable of replicating more efficiently in a bacterial cell.
2 Development of DNA cloning techniques: protocols to synthesize genes for small proteins;
Craig Venter’s shotgun technique that enabled unknown sequences of DNA to be cut into small
pieces by restriction enzymes for cloning purposes.
3 Methods to screen bacteria containing foreign DNA inserts so that desired clones could be
obtained.
4 DNA sequencing technique developed by Maxam and Gilbert (1975-76). Sequencing made it
possible to synthesize sticky ends of DNA fragments useful in gene splicing and cloning.
1.2.3
BIOTECHNOLOGY TECHNIQUES
Fermentation: Fermentation is a technique that requires bioreactors which are used to allow a
biological process to take place under optimum conditions, producing a useful compound in large
amounts. Simple bioreactors have been used to produce beer and wine. Because of advances in
genetic engineering, bioreactors can now manufacture complex biological substances. Recently
scientists have developed ways of culturing mammalian cells in bioreactors to produce antibodies
and other useful proteins. Some bioreactors carry out a chemical process without using living cells
– instead enzymes are used to trigger conversion of one chemical into another by a process called
biotransformation. Important substances like corn syrup and vitamin C are produced in this way.
Cell Fusion: Cell fusion involves combining two cells to make a single cell that contains the genetic
material of the original cells. This technique is being used to create new plants by fusing cells from
species that do not naturally hybridise, i.e. cross breed, and then generating new plants from the
fused cells. The most famous example is pomato which is a cross between potato and tomato. Cell
fusion is also used to make fused cells known as monoclonal antibodies, which are protective
proteins produced by a clone of a single cell. There are several ways of doing cell fusion.
Liposomes: Liposomes are very important in biotechnology as they are used as vehicles for
delivering certain drugs to the target tissues of the body. Drugs are encapsulated in liposomes
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which offer protection against digestive enzymes in the stomach. Liposomes are microscopic
spherical particles that are formed when lipids form a suspension in the water. The lipid molecules
in a liposome are arranged in a manner so as to carry a drug or any other material.
Cell and Tissue Culture: Tissue culture is the growth of living cells or organisms outside the body in a
suitable culture medium which provides nutrients to the growing cells. Individual cells grow and
divide in a sterile medium. The technique is extensively used in laboratories, e.g., cancer research,
plant breeding and for routine analysis of chromosome karyotypes.
Genetic Engineering: This is a technology requiring recombinant DNA technology and gene transfer
techniques in order to bring about a change in the genetic constitution of the organism to express
desired traits. Before the advent of this technology, animal and plant breeders used to employ
classical breeding (guided mating) methods to improve breeds, ensuring combination of genes. In
this way the breeders were able to produce economically important plants, cows, horses, dogs, etc.
Techniques for isolating and altering genes were developed by geneticists in the US in the 1970s by
manipulating the genome of bacteria.
They successfully produced insulin, interferon and
somatostatin.
1.3 MAIN AREAS OF APPLICATION OF BIOTECHNOLOGY
Biotechnology (Table 1.3) has unlimited potential and has proved immensely useful in wide ranging
areas in life (Table 1.4).
 Bioprocess technology for the production of High Quality Foods: High nutritional quality foods
have been produced by using modified and high performing strains of microorganisms. Bread,
cheese, wine, yogurt, beer and vinegar are made by culturing microorganisms. Food
biotechnology has also developed sensitive tests to detect toxins and other harmful
contaminants generated by microorganisms in foods. Cultivation of GM plants has gained
approval in many countries such as Japan, US, Canada and a number of European nations.
 Medicine: Bioreactors were first used for growing cultures of moulds to produce antibiotics. A
number of antimicrobial drugs such as ampicillin, amoxicillin, tetracyclines, erythromycin and
ciprofloxacin were produced by this method. Some hormones that are proteinaceous in nature
were also produced from genetically modified microbes. In Britain, genetically engineered
sheep were produced whose milk contains Factor VIII, a protein used as anticoagulant to treat
patients infected with haemophilia. Factor VIII obtained in this way can be produced on mass
scale in a cost efficient manner. Genetic engineering techniques have yielded monoclonal
antibodies to combat and inactivate foreign proteins or other antigens that may invade the
body. Several precious drugs such as interferons, interleukins, hormones with regulatory
functions, bacteria and viral vaccines are manufactured by DNA technology.
 Agriculture: Agriculture has been the greatest beneficiary of biotechnology. Several genetically
engineered crop plants, transgenic plants, pest and stress resistant plants, etc., have been
produced commercially to enhance agriculture productivity. These plants have improved
nutrition, disease resistance, keeping quality, improved yields and stress tolerance. With the
help of tissue culture technique, virtually unlimited plants can be propagated from a single plant
possessing desired traits. Genetic improvements in crop plants have been brought about by
inducing chromosome variations. Other applications include hybrid seeds, development of
artificial and synthetic seeds and production of photosynthesis improvers. By gene transfer
technique, nitrogen fixation ability of plants has been achieved. Biotechnology has offered
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farmers the means to adopt alternate strategies towards pest control – including the use of
biological control agents, attractants and growth affecting agents.
Fuel and Fodder: Biotechnology has been able to produce an alternative to traditional fossil
fuels. The tissue culture technique for mass multiplication offers not only rapid and mass
production of existing stock of germ plasm for woody and biomass energy production, but also
for rapid afforestation of depleted forests and regeneration of green cover. Biomass is the
renewable source of carbon produced in forests, grasslands and cultivated areas. Biotechnology
seeks to improve the conversion of biomass into fuel as energy source, yielding gaseous
methane and ethanol as by-products.
Waste technology: Although of long historical importance, more emphasis is now being made
to couple these processes with the conservation and recycling of resources; foods and fertilizers,
biological fuels.
Environmental technology: Great scope exists for the application of biotechnological concepts
for solving many environmental problems - pollution control, removing toxic wastes, recovery
of metals from mining wastes and low-grade ores.
Animal agriculture: The application of biotechnology in the area of livestock improvement has
augmented milk and meat production. Embryo transplantation used with cattle, goats, sheep
and pigs aims to increase the number of offspring from a quality female. In this procedure,
highly rated females are given hormone treatments to cause the hyper-production of eggs,
which are then transferred to another healthy female after fertilisation. Improved productivity,
etc., for animal farming. Improved food quality, flavour, taste and microbial safety. It is believed
that genetically engineered pigs may one day be able to provide compatible organs for
transplantation into humans – a technique known as xenotransplantation.
Biosensors: Biotechnology has impacted the electronic industry in the development of
biosensors. These make use of an enzyme combined with electronic components to trigger a
circuit when a particular type of chemical or metabolite is detected in extremely low levels.
Biosensors are available which can detect hormones, proteins, gases, acids, alcohols or
pollutants. Geologists also make use of biosensors in the mining industry to detect small
quantities of minerals in samples of ores.
Health and Molecular Medicine: Biotechnology has impacted the area of human health.
Genome research is expected to revolutionise the practice of medicine in both diagnosis and
treatment in the following ways:
o Diagnosis of a disease
o Rational drug design
o Detection of genetic predisposition to genetic disorders
o Gene therapy
o Control systems for drugs
o Pharmacogenomics in designing custom drugs
o Assessment of health damage caused by radiation hazards
o Detection of heritable mutations in early stages to reduce their likelihood
Many biotechnological processes may be considered as having a three component central core, in
which one part is concerned with obtaining the best biological catalyst for a specific function or
process, the second part creates (by construction and technical operation) the best possible
environment for the catalyst to perform, and the third part (downstream processing) is concerned
with the separation and purification of an essential product from a fermentation process.
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Table 1.3: Types of Companies Involved With Biotechnology
CATEGORY
Therapeutics
Diagnostics
Agriculture/forestry/horticulture
Food
Environment
Chemical Intermediates
Equipment
BIOTECHNOLOGY INVOLVEMENT
Pharmaceutical products for the cure or control of human diseases, including
antibiotics, vaccines and gene therapy
Clinical testing and diagnosis, food, environment, agriculture
Novel crops or animal varieties, pesticides
Wide range of food products, fertilizers, beverages, ingredients
Waste treatment, bioremediation, energy production
Reagents including enzymes, DNA/RNA and speciality chemicals
Hardware, bioreactors, software and consumables supporting biotechnology
Table 1.4: Major Products of Biological Processing
FERMENTATION PRODUCT
Bulk organics
Ethanol (non beverage)
Acetone/butanol
Biomass
Starter cultures and yeasts
for food and agriculture
Single cell protein
TYPICAL ORGANISM USED
Saccharomyces cerevisiae
Clostridium acetobutylicum
Lactic acid bacteria or baker’s yeast
Aspergillus niger
Aspergillus niger
Lactobacillus delbruckii
Aspergillus itaconicus
Vitamins
B12
Riboflavin
Propionibacterium shermanii
Pseudomonas denitrificans
Eremothecium ashbyii
Therapeutic proteins
Insulin
Growth Hormone
Erythropoietin
Factor VIII-C
Tissue
plasminogen
activator
TYPICAL ORGANISM USED
Amino acids
L-glutamic acid
L-lysine
L-phenylalanine
L-arginine
Corynebacterium glutamicum
Brevibacterium flavum
Corynebacterium glutamicum
Brevibacterium flavum
Corynebacterium spp.
Monoclonal antibodies
Hybridoma cells
Ergot alkaloids
Claviceps paspali
Xanthomonas campestris
Leuconostoc mesenteroides
Brevibacterium
ammoniagenes
Pseudomonas methylotrophus or
Candida utilis
Organic acids
Citric acid
Gluconic acid
Lactic acid
Italconic acid
Microbial transformations
Steroids
D-sorbital to L-sorbose (in
vitamin C production)
Antibiotics
Penicillins
Cephalosporins
Tetracyclins
Macrolide antibiotics
(e.g. erythromycin)
Polypeptide
antibiotics
(e.g. gramicidin)
Aminoglycoside antibiotics
(e.g. streptomycin)
Aromatic antibiotics
(e.g. griseofulvin)
Pigments
Shikonin
ß-carotene
FERMENTATION PRODUCT
Extracellular
polysaccharides
Xanthum gum
Dextran
Nucleotides
5’-guanosine
monophosphate
or
Rhizopus arrhizus
Acetobacter suboxydans
Vaccines
Diptheria
Tetanus
Pertussis (whooping
cough)
Poiliomyelitis
Penicillium chrysogenum
Cephalosporium acremonium
Streptomyces aureofaciens
Streptomyces erythreus
Bacillus brevis
Streptomyces griseus
Penicillium griseofulvum
Rubella
Hepatitis B
Lithospermum eryhrorhizon (plant
cell culture)
Blakeslea trispora
Recombinant Escherichia coli
Recombinant Escherichia coli
Or recombinant mammalian cells
Recombinant mammalian cells
Recombinant mammalian cells
Recombinant mammalian cells
Recombinant Escherichia coli
Interferon-∝2
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Insecticides
Bacterial spores
Fungal spores
Enzymes
Proteases
∝-amylase
Glucoamylase
Glucose isomerase
Pectinase
Rennin
Corynebacterium diptherium
Clostridium tetani
Bordetella pertussis
Live attenuated virus grown in
monkey kidney or human
diploid cells
Live attenuated virus grown in
baby hamster kidney cells
Surface antigen expressed in
recombinant yeast
Bacillus thuringiensis
Hirsutella thompsonii
Bacillus spp.
Bacillus amyloliquefaciens
Aspergillus niger
Bacillus coagulans
Aspergillus niger
Mucor miehei or recombinant
yeast
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