CHAPTER 10

MICROBES IN HUMAN WELFARE

10.1 Microbes in Household

Products
10.2 Microbes in Industrial Besides macroscopic plants and animals, microbes are
Products the major components of biological systems on this earth.
10.3 Microbes in Sewage You have studied about the diversity of living organisms
Treatment in Class XI. Do you remember which Kingdoms among
the living organisms contain micro-organisms? Which are
10.4 Microbes in Production of
Biogas the ones that are only microscopic? Microbes are present
everywhere – in soil, water, air, inside our bodies and that
10.5 Microbes as Biocontrol
of other animals and plants. They are present even at sites
Agents
where no other life-form could possibly exist – sites such
10.6 Microbes as Biofertilisers as deep inside the geysers (thermal vents) where the
temperature may be as high as 1000C, deep in the soil,
under the layers of snow several metres thick, and in highly
acidic environments. Microbes are diverse– protozoa,
bacteria, fungi and microscopic animal and plant viruses,
viroids and also prions that are proteinacious infectious
agents. Some of the microbes are shown in Figures 10.1
and 10.2.
Microbes like bacteria and many fungi can be grown
on nutritive media to form colonies (Figure 10.3), that can
be seen with the naked eyes. Such cultures are useful in
studies on micro-organisms.

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(a)

(b)

(a) (b)

(c)
(c)
Figure10.1 Bacteria: (a) Rod-shaped,
magnified 1500X ; (b) Spherical Figure 10.2 Viruses: (a) A bacteriophage; (b)
shaped, magnified1500X; (c) A rod- Adenovirus which causes respiratory
shaped bacterium showing flagella, infections; (c) Rod-shaped Tobacco
magnified 50,000X Mosaic Virus (TMV). Magnified about
1,00,000–1,50,000X

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(a) (b)

Figure 10.3 (a) Colonies of bacteria growing in a petri dish;

(b) Fungal colony growing in a petri dish

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In chapter 8, you have read that microbes cause a large number of

diseases in human beings. They also cause diseases in animals and plants.
But this should not make you think that all microbes are harmful; several
microbes are useful to man in diverse ways. Some of the most important
contributions of microbes to human welfare are discussed in this chapter.

10.1 MICROBES IN HOUSEHOLD PRODUCTS

You would be surprised to know that we use microbes or products
derived from them everyday. A common example is the production of
curd from milk. Micro-organisms such as Lactobacillus and others
commonly called lactic acid bacteria (LAB) grow in milk and convert it
to curd. During growth, the LAB produce acids that coagulate and
partially digest the milk proteins. A small amount of curd added to the
fresh milk as inoculum or starter contain millions of LAB, which at
suitable temperatures multiply, thus converting milk to curd, which
also improves its nutritional quality by increasing vitamin B12. In our
stomach too, the LAB play very beneficial role in checking disease-
causing microbes.
The dough, which is used for making foods such as dosa and idli is
also fermented by bacteria. The puffed-up appearance of dough is due to
the production of CO2 gas. Can you tell which metabolic pathway is
taking place resulting in the formation of CO2? Where do you think the
bacteria for these fermentations come from? Similarly the dough, which
is used for making bread, is fermented using baker’s yeast
(Saccharomyces cerevisiae). A number of traditional drinks and foods
are also made by fermentation by the microbes. ‘Toddy’, a traditional
drink of some parts of southern India is made by fermenting sap from
palms. Microbes are also used to ferment fish, soyabean and bamboo-
shoots to make foods. Cheese, is one of the oldest food items in which
microbes were used. Different varieties of cheese are known by their
characteristic texture, flavour and taste, the specificity coming from the
microbes used. For example, the large holes in ‘Swiss cheese’ are due to
production of a large amount of CO 2 by a bacterium named
Propionibacterium sharmanii. The ‘Roquefort cheese’ are ripened by
growing a specific fungi on them, which gives them a particular flavour.

10.2 MICROBES IN INDUSTRIAL PRODUCTS 181

Even in industry, microbes are used to synthesise a number of products
valuable to human beings. Beverages and antibiotics are some examples.
Production on an industrial scale, requires growing microbes in very large
vessels called fermentors (Figure 10.4).

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10.2.1 Fermented Beverages

Microbes especially yeasts have been used from
time immemorial for the production of beverages
like wine, beer, whisky, brandy or rum. For this
purpose the same yeast Saccharomyces
cerevisiae used for bread-making and
commonly called brewer’s yeast, is used for
fermenting malted cereals and fruit juices, to
produce ethanol. Do you recollect the metabolic
reactions, which result in the production of
ethanol by yeast? Depending on the type of the
raw material used for fermentation and the type
Figure 10.4 Fermentors
of processing (with or without distillation)
different types of alcoholic drinks are obtained.
Wine and beer are produced without distillation
whereas whisky, brandy and rum are produced
by distillation of the fermented broth. The
photograph of a fermentation plant is shown in
Figure 10.5.

10.2.2 Antibiotics
Antibiotics produced by microbes are regarded
as one of the most significant discoveries of the
twentieth century and have greatly contributed
towards the welfare of the human society. Anti is
a Greek word that means ‘against’, and bio means
Figure 10.5 Fermentation Plant
‘life’, together they mean ‘against life’ (in the
context of disease causing organisms); whereas with reference to human
beings, they are ‘pro life’ and not against. Antibiotics are chemical
substances, which are produced by some microbes and can kill or retard
the growth of other (disease-causing) microbes.
You are familiar with the commonly used antibiotic Penicillin. Do you
know that Penicillin was the first antibiotic to be discovered, and it was a
chance discovery? Alexander Fleming while working on Staphylococci
bacteria, once observed a mould growing in one of his unwashed culture
plates around which Staphylococci could not grow. He found out that it
was due to a chemical produced by the mould and he named it Penicillin
182 after the mould Penicillium notatum. However, its full potential as an
effective antibiotic was established much later by Ernest Chain and
Howard Florey. This antibiotic was extensively used to treat American
soldiers wounded in World War II. Fleming, Chain and Florey were awarded
the Nobel Prize in 1945, for this discovery.

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After Penicillin, other antibiotics were also purified from other

microbes. Can you name some other antibiotics and find out their
sources? Antibiotics have greatly improved our capacity to treat deadly
diseases such as plague, whooping cough (kali khansi ), diphtheria (gal
ghotu) and leprosy (kusht rog), which used to kill millions all over the
globe. Today, we cannot imagine a world without antibiotics.

10.2.3 Chemicals, Enzymes and other Bioactive Molecules

Microbes are also used for commercial and industrial production of
certain chemicals like organic acids, alcohols and enzymes. Examples of
acid producers are Aspergillus niger (a fungus) of citric acid, Acetobacter
aceti (a bacterium) of acetic acid; Clostridium butylicum (a bacterium) of
butyric acid and Lactobacillus (a bacterium) of lactic acid.
Yeast (Saccharomyces cerevisiae) is used for commercial production
of ethanol. Microbes are also used for production of enzymes. Lipases are
used in detergent formulations and are helpful in removing oily stains
from the laundry. You must have noticed that bottled fruit juices bought
from the market are clearer as compared to those made at home. This is
because the bottled juices are clarified by the use of pectinases and
proteases. Streptokinase produced by the bacterium Streptococcus and
modified by genetic engineering is used as a ‘clot buster’ for removing
clots from the blood vessels of patients who have undergone myocardial
infarction leading to heart attack.
Another bioactive molecule, cyclosporin A, that is used as an
immunosuppressive agent in organ-transplant patients, is produced by
the fungus Trichoderma polysporum. Statins produced by the yeast
Monascus purpureus have been commercialised as blood-cholesterol
lowering agents. It acts by competitively inhibiting the enzyme responsible
for synthesis of cholesterol.

10.3 MICROBES IN SEWAGE TREATMENT

We know that large quantities of waste water are generated everyday in
cities and towns. A major component of this waste water is human excreta.
This municipal waste-water is also called sewage. It contains large
amounts of organic matter and microbes. Many of which are pathogenic. 183
Have you ever wondered where this huge quantity of sewage or urban
waste water is disposed off daily? This cannot be discharged into natural
water bodies like rivers and streams directly – you can understand why.
Before disposal, hence, sewage is treated in sewage treatment plants (STPs)
to make it less polluting. Treatment of waste water is done by the

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heterotrophic microbes naturally present in

the sewage. This treatment is carried out in
two stages:
Primary treatment : These treatment
steps basically involve physical removal of
particles – large and small – from the sewage
through filtration and sedimentation. These
are removed in stages; initially, floating debris
is removed by sequential filtration. Then the
grit (soil and small pebbles) are removed by
sedimentation. All solids that settle form the
primary sludge, and the supernatant forms
the effluent. The effluent from the primary
Figure 10.6 Secondary treatment
settling tank is taken for secondary treatment.
Secondary treatment or Biological treatment : The primary
effluent is passed into large aeration tanks (Figure 10.6) where it is
constantly agitated mechanically and air is pumped into it. This allows
vigorous growth of useful aerobic microbes into flocs (masses of
bacteria associated with fungal filaments to form mesh like
structures). While growing, these microbes consume the major part
of the organic matter in the effluent. This significantly reduces the
BOD (biochemical oxygen demand) of the effluent. BOD refers to
the amount of the oxygen that would be consumed if all the organic
matter in one liter of water were oxidised by bacteria. The sewage
water is treated till the BOD is reduced. The BOD test measures the
rate of uptake of oxygen by micro-organisms in a sample of water
and thus, indirectly, BOD is a measure of the organic matter present
in the water. The greater the BOD of waste water, more is its polluting
potential.
Once the BOD of sewage or waste water is reduced significantly, the
effluent is then passed into a settling tank where the bacterial ‘flocs’ are
allowed to sediment. This sediment is called activated sludge. A small
part of the activated sludge is pumped back into the aeration tank to
serve as the inoculum. The remaining major part of the sludge is pumped
into large tanks called anaerobic sludge digesters. Here, other kinds
of bacteria, which grow anaerobically, digest the bacteria and the fungi
184 in the sludge. During this digestion, bacteria produce a mixture of gases
such as methane, hydrogen sulphide and carbon dioxide. These gases
form biogas and can be used as source of energy as it is inflammable.
The effluent from the secondary treatment plant is generally released
into natural water bodies like rivers and streams. An aerial view of such
a plant is shown in Figure 10.7.

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You can appreciate how microbes play a major

role in treating millions of gallons of waste water
everyday across the globe. This methodology has
been practiced for more than a century now, in
almost all parts of the world. Till date, no man-
made technology has been able to rival the
microbial treatment of sewage.
You are aware that due to increasing
urbanisation, sewage is being produced in much
larger quantities than ever before. However the
number of sewage treatment plants has not Figure 10.7 An aerial view of a sewage plant
increased enough to treat such large quantities.
So the untreated sewage is often discharged directly into rivers leading to
their pollution and increase in water-borne diseases.
The Ministry of Environment and Forests has initiated Ganga Action
Plan and Yamuna Action Plan to save these major rivers of our country
from pollution. Under these plans, it is proposed to build a large number
of sewage treatment plants so that only treated sewage may be discharged
in the rivers. A visit to a sewage treatment plant situated in any place
near you would be a very interesting and educating experience.

10.4 MICROBES IN PRODUCTION OF BIOGAS

Biogas is a mixture of gases (containing predominantly methane) produced
by the microbial activity and which may be used as fuel. You have learnt
that microbes produce different types of gaseous end-products during
growth and metabolism. The type of the gas produced depends upon the
microbes and the organic substrates they utilise. In the examples cited in
relation to fermentation of dough, cheese making and production of
beverages, the main gas produced was CO2.. However, certain bacteria,
which grow anaerobically on cellulosic material, produce large amount
of methane along with CO2 and H2. These bacteria are collectively called
methanogens, and one such common bacterium is Methanobacterium.
These bacteria are commonly found in the anaerobic sludge during
sewage treatment. These bacteria are also present in the rumen (a part of
stomach) of cattle. A lot of cellulosic material present in the food of cattle
is also present in the rumen. In rumen, these bacteria help in the
breakdown of cellulose and play an important role in the nutrition of
cattle. Do you think we, human beings, are able to digest the celluose 185
present in our foods? Thus, the excreta (dung) of cattle, commonly called
gobar, is rich in these bacteria. Dung can be used for generation of biogas,
commonly called gobar gas.
The biogas plant consists of a concrete tank (10-15 feet deep) in which
bio-wastes are collected and a slurry of dung is fed. A floating cover is

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placed over the slurry, which

keeps on rising as the gas is
produced in the tank due to the
microbial activity. The biogas
plant has an outlet, which is
connected to a pipe to supply
biogas to nearby houses. The
spent slurry is removed through
another outlet and may be used
as fertiliser. Cattle dung is
available in large quantities in
rural areas where cattle are used
for a variety of purposes. So
biogas plants are more often
built in rural areas. The biogas
thus produced is used for
cooking and lighting. The
picture of a biogas plant is
shown in Figure 10.8. The
Figure 10.8 A typical biogas plant technology of biogas production
was developed in India mainly
due to the efforts of Indian Agricultural Research Institute (IARI) and
Khadi and Village Industries Commission (KVIC). If your school is situated
in a village or near a village, it would be very interesting to enquire if there
are any biogas plants nearby. Visit the biogas plant and learn more about
it from the people who are actually managing it.

10.5 MICROBES AS BIOCONTROL AGENTS

Biocontrol refers to the use of biological methods for controlling plant
diseases and pests. In modern society, these problems have been tackled
increasingly by the use of chemicals – by use of insecticides and pesticides.
These chemicals are toxic and extremely harmful, to human beings and
animals alike, and have been polluting our environment (soil, ground
water), fruits, vegetables and crop plants. Our soil is also polluted through
our use of weedicides to remove weeds.
Biological control of pests and diseases: In agriculture, there is a
method of controlling pests that relies on natural predation rather than
186 introduced chemicals. A key belief of the organic farmer is that biodiversity
furthers health. The more variety a landscape has, the more sustainable
it is. The organic farmer, therefore, works to create a system where the
insects that are sometimes called pests are not eradicated, but instead
are kept at manageable levels by a complex system of checks and balances
within a living and vibrant ecosystem. Contrary to the ‘conventional’
farming practices which often use chemical methods to kill both useful

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and harmful life forms indiscriminately, this is a holistic approach that

seeks to develop an understanding of the webs of interaction between the
myriad of organisms that constitute the field fauna and flora. The organic
farmer holds the view that the eradication of the creatures that are often
described as pests is not only possible, but also undesirable, for without
them the beneficial predatory and parasitic insects which depend upon
them as food or hosts would not be able to survive. Thus, the use of
biocontrol measures will greatly reduce our dependence on toxic chemicals
and pesticides. An important part of the biological farming approach is
to become familiar with the various life forms that inhabit the field,
predators as well as pests, and also their life cycles, patterns of feeding
and the habitats that they prefer. This will help develop appropriate means
of biocontrol.
The very familiar beetle with red and black markings – the Ladybird,
and Dragonflies are useful to get rid of aphids and mosquitoes,
respectively. An example of microbial biocontrol agents that can be
introduced in order to control butterfly caterpillars is the bacteria Bacillus
thuringiensis (often written as Bt ). These are available in sachets as dried
spores which are mixed with water and sprayed onto vulnerable plants
such as brassicas and fruit trees, where these are eaten by the insect
larvae. In the gut of the larvae, the toxin is released and the larvae get
killed. The bacterial disease will kill the caterpillars, but leave other insects
unharmed. Because of the development of methods of genetic engineering
in the last decade or so, the scientists have introduced B. thuringiensis
toxin genes into plants. Such plants are resistant to attack by insect pests.
Bt-cotton is one such example, which is being cultivated in some states
of our country. You will learn more about this in chapter 12.
A biological control being developed for use in the treatment of plant
disease is the fungus Trichoderma. Trichoderma species are free-living
fungi that are very common in the root ecosystems. They are effective
biocontrol agents of several plant pathogens.
Baculoviruses are pathogens that attack insects and other arthropods.
The majority of baculoviruses used as biological control agents are in the
genus Nucleopolyhedrovirus. These viruses are excellent candidates for
species-specific, narrow spectrum insecticidal applications. They have
been shown to have no negative impacts on plants, mammals, birds, fish
or even on non-target insects. This is especially desirable when beneficial
insects are being conserved to aid in an overall integrated pest
management (IPM) programme, or when an ecologically sensitive area is 187
being treated.

10.6 MICROBES AS BIOFERTILISERS

With our present day life styles environmental pollution is a major cause
of concern. The use of the chemical fertilisers to meet the ever-increasing

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demand of agricultural produce has contributed significantly to

this pollution. Of course, we have now realised that there are problems
associated with the overuse of chemical fertilisers and there is a
large pressure to switch to organic farming – the use of biofertilisers.
Biofertilisers are organisms that enrich the nutrient quality of the soil.
The main sources of biofertilisers are bacteria, fungi and cyanobacteria.
You have studied about the nodules on the roots of leguminous plants
formed by the symbiotic association of Rhizobium. These bacteria fix
atmospheric nitrogen into organic forms, which is used by the plant as
nutrient. Other bacteria can fix atmospheric nitrogen while free-living in
the soil (examples Azospirillum and Azotobacter), thus enriching the
nitrogen content of the soil.
Fungi are also known to form symbiotic associations with plants
(mycorrhiza). Many members of the genus Glomus form mycorrhiza.
The fungal symbiont in these associations absorbs phosphorus from
soil and passes it to the plant. Plants having such associations show
other benefits also, such as resistance to root-borne pathogens, tolerance
to salinity and drought, and an overall increase in plant growth and
development. Can you tell what advantage the fungus derives from
this association?
Cyanobacteria are autotrophic microbes widely distributed in aquatic
and terrestrial environments many of which can fix atmospheric nitrogen,
e.g. Anabaena, Nostoc, Oscillatoria, etc. In paddy fields, cyanobacteria
serve as an important biofertiliser. Blue green algae also add organic matter
to the soil and increase its fertility. Currently, in our country, a number
of biofertilisers are available commercially in the market and farmers use
these regularly in their fields to replenish soil nutrients and to reduce
dependence on chemical fertilisers.

SUMMARY
Microbes are a very important component of life on earth. Not all
microbes are pathogenic. Many microbes are very useful to human
beings. We use microbes and microbially derived products almost every
day. Bacteria called lactic acid bacteria (LAB) grow in milk to convert it
into curd. The dough, which is used to make bread, is fermented by
yeast called Saccharomyces cerevisiae. Certain dishes such as idli and
dosa, are made from dough fermented by microbes. Bacteria and fungi
188 are used to impart particular texture, taste and flavor to cheese. Microbes
are used to produce industrial products like lactic acid, acetic acid
and alcohol, which are used in a variety of processes in the industry.
Antibiotics like penicillins produced by useful microbes are used to kill
disease-causing harmful microbes. Antibiotics have played a major role
in controlling infectious diseases like diphtheria, whooping cough and

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pneumonia. For more than a hundred years, microbes are being used
to treat sewage (waste water) by the process of activated sludge formation
and this helps in recycling of water in nature. Methanogens produce
methane (biogas) while degrading plant waste. Biogas produced by
microbes is used as a source of energy in rural areas. Microbes can also
be used to kill harmful pests, a process called as biocontrol. The
biocontrol measures help us to avoid heavy use of toxic pesticides for
controlling pests. There is a need these days to push for use of
biofertilisers in place of chemical fertilisers. It is clear from the diverse
uses human beings have put microbes to that they play an important
role in the welfare of human society.

EXERCISES
1. Bacteria cannot be seen with the naked eyes, but these can be seen
with the help of a microscope. If you have to carry a sample from your
home to your biology laboratory to demonstrate the presence of microbes
with the help of a microscope, which sample would you carry and why?
2. Give examples to prove that microbes release gases during metabolism.
3. In which food would you find lactic acid bacteria? Mention some of
their useful applications.
4. Name some traditional Indian foods made of wheat, rice and Bengal
gram (or their products) which involve use of microbes.
5. In which way have microbes played a major role in controlling diseases
caused by harmful bacteria?
6. Name any two species of fungus, which are used in the production of
the antibiotics.
7. What is sewage? In which way can sewage be harmful to us?
8. What is the key difference between primary and secondary sewage
treatment?
9. Do you think microbes can also be used as source of energy? If yes, how?
10. Microbes can be used to decrease the use of chemical fertilisers and
pesticides. Explain how this can be accomplished.
11. Three water samples namely river water, untreated sewage water and
secondary effluent discharged from a sewage treatment plant were 189
subjected to BOD test. The samples were labelled A, B and C; but the
laboratory attendant did not note which was which. The BOD values
of the three samples A, B and C were recorded as 20mg/L, 8mg/L and
400mg/L, respectively. Which sample of the water is most polluted?
Can you assign the correct label to each assuming the river water is
relatively clean?

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12. Find out the name of the microbes from which Cyclosporin A (an
immunosuppressive drug) and Statins (blood cholesterol lowering
agents) are obtained.
13. Find out the role of microbes in the following and discuss it with your teacher.
(a) Single cell protein (SCP)
(b) Soil
14. Arrange the following in the decreasing order (most important first) of
their importance, for the welfare of human society. Give reasons for
your answer.
Biogas, Citric acid, Penicillin and Curd
15. How do biofertilisers enrich the fertility of the soil?

190

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 Ever since the days of Rene Descartes, the French philosopher,
Chapter 11 mathematician and biologist of seventeenth century, all human
Biotechnology : Principles and knowledge especially natural sciences were directed to develop
Processes technologies which add to the creature comforts of human
lives, as also value to human life. The whole approach to
Chapter 12 understanding natural phenomena became anthropocentric.
Biotechnology and Its Physics and chemistry gave rise to engineering, technologies
Applications and industries which all worked for human comfort and welfare.
The major utility of the biological world is as a source of food.
Biotechnology, the twentieth century off-shoot of modern
biology, changed our daily life as its products brought
qualitative improvement in health and food production. The
basic principles underlying biotechnological processes and some
applications are highlighted and discussed in this unit.

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 Herbert Boyer was born in 1936 and brought up in a corner of western
Pennsylvania where railroads and mines were the destiny of most young
men. He completed graduate work at the University of Pittsburgh, in
1963, followed by three years of post-graduate studies at Yale.
In 1966, Boyer took over assistant professorship at the University of
California at San Francisco. By 1969, he performed studies on a couple
of restriction enzymes of the E. coli bacterium with especially useful
properties. Boyer observed that these enzymes have the capability of
cutting DNA strands in a particular fashion, which left what has became
known as ‘sticky ends’ on the strands. These clipped ends made pasting
together pieces of DNA a precise exercise.
This discovery, in turn, led to a rich and rewarding conversation in
Hawaii with a Stanford scientist named Stanley Cohen. Cohen had
been studying small ringlets of DNA called plasmids and which float
about freely in the cytoplasm of certain bacterial cells and replicate
HERBERT BOYER independently from the coding strand of DNA. Cohen had developed
(1936 ) a method of removing these plasmids from the cell and then reinserting
them in other cells. Combining this process with that of DNA splicing
enabled Boyer and Cohen to recombine segments of DNA in desired
configurations and insert the DNA in bacterial cells, which could then
act as manufacturing plants for specific proteins. This breakthrough was
the basis upon which the discipline of biotechnology was founded.

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CHAPTER 11

BIOTECHNOLOGY : PRINCIPLES
AND PROCESSES

11.1 Principles of Biotechnology

11.2 Tools of Recombinant DNA Biotechnology deals with techniques of using live
Technology organisms or enzymes from organisms to produce products
and processes useful to humans. In this sense, making
11.3 Processes of Recombinant curd, bread or wine, which are all microbe-mediated
processes, could also be thought as a form of
DNA Technology
biotechnology. However, it is used in a restricted sense
today, to refer to such of those processes which use
genetically modified organisms to achieve the same on a
larger scale. Further, many other processes/techniques are
also included under biotechnology. For example, in vitro
fertilisation leading to a ‘test-tube’ baby, synthesising a
gene and using it, developing a DNA vaccine or correcting
a defective gene, are all part of biotechnology.
The European Federation of Biotechnology (EFB) has
given a definition of biotechnology that encompasses both
traditional view and modern molecular biotechnology.
The definition given by EFB is as follows:
‘The integration of natural science and organisms,
cells, parts thereof, and molecular analogues for products
and services’.

11.1 PRINCIPLES OF BIOTECHNOLOGY

Among many, the two core techniques that enabled birth
of modern biotechnology are :
(i) Genetic engineering : Techniques to alter the
chemistry of genetic material (DNA and RNA),

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to introduce these into host organisms and thus change the

phenotype of the host organism.
(ii) Bioprocess engineering : Maintenance of sterile (microbial
contamination-free) ambience in chemical engineering processes
to enable growth of only the desired microbe/eukaryotic cell in
large quantities for the manufacture of biotechnological products
like antibiotics, vaccines, enzymes, etc.
Let us now understand the conceptual development of the principles
of genetic engineering.
You probably appreciate the advantages of sexual reproduction over
asexual reproduction. The former provides opportunities for variations
and formulation of unique combinations of genetic setup, some of which
may be beneficial to the organism as well as the population. Asexual
reproduction preserves the genetic information, while sexual reproduction
permits variation. Traditional hybridisation procedures used in plant and
animal breeding, very often lead to inclusion and multiplication of
undesirable genes along with the desired genes. The techniques of genetic
engineering which include creation of recombinant DNA, use of
gene cloning and gene transfer, overcome this limitation and allows us
to isolate and introduce only one or a set of desirable genes without
introducing undesirable genes into the target organism.
Do you know the likely fate of a piece of DNA, which is somehow
transferred into an alien organism? Most likely, this piece of DNA would
not be able to multiply itself in the progeny cells of the organism. But,
when it gets integrated into the genome of the recipient, it may multiply
and be inherited along with the host DNA. This is because the alien piece
of DNA has become part of a chromosome, which has the ability to
replicate. In a chromosome there is a specific DNA sequence called the
origin of replication, which is responsible for initiating replication.
Therefore, for the multiplication of any alien piece of DNA in an organism
it needs to be a part of a chromosome(s) which has a specific sequence
known as ‘origin of replication’. Thus, an alien DNA is linked with the
origin of replication, so that, this alien piece of DNA can replicate and
multiply itself in the host organism. This can also be called as cloning or
making multiple identical copies of any template DNA.
Let us now focus on the first instance of the construction of an artificial
recombinant DNA molecule. The construction of the first recombinant
DNA emerged from the possibility of linking a gene encoding antibiotic
194 resistance with a native plasmid (autonomously replicating circular
extra-chromosomal DNA) of Salmonella typhimurium. Stanley Cohen and
Herbert Boyer accomplished this in 1972 by isolating the antibiotic
resistance gene by cutting out a piece of DNA from a plasmid which was
responsible for conferring antibiotic resistance. The cutting of DNA at
specific locations became possible with the discovery of the so-called

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‘molecular scissors’– restriction enzymes. The cut piece of DNA was

then linked with the plasmid DNA. These plasmid DNA act as vectors to
transfer the piece of DNA attached to it. You probably know that mosquito
acts as an insect vector to transfer the malarial parasite into human body.
In the same way, a plasmid can be used as vector to deliver an alien piece
of DNA into the host organism. The linking of antibiotic resistance gene
with the plasmid vector became possible with the enzyme DNA ligase,
which acts on cut DNA molecules and joins their ends. This makes a new
combination of circular autonomously replicating DNA created in vitro
and is known as recombinant DNA. When this DNA is transferred into
Escherichia coli, a bacterium closely related to Salmonella, it could
replicate using the new host’s DNA polymerase enzyme and make multiple
copies. The ability to multiply copies of antibiotic resistance gene in
E. coli was called cloning of antibiotic resistance gene in E. coli.
You can hence infer that there are three basic steps in genetically
modifying an organism —
(i) identification of DNA with desirable genes;
(ii) introduction of the identified DNA into the host;
(iii) maintenance of introduced DNA in the host and transfer of the DNA
to its progeny.

11.2 TOOLS OF RECOMBINANT DNA TECHNOLOGY

Now we know from the foregoing discussion that genetic engineering or
recombinant DNA technology can be accomplished only if we have the
key tools, i.e., restriction enzymes, polymerase enzymes, ligases, vectors
and the host organism. Let us try to understand some of these in detail.

11.2.1 Restriction Enzymes

In the year 1963, the two enzymes responsible for restricting the growth
of bacteriophage in Escherichia coli were isolated. One of these added
methyl groups to DNA, while the other cut DNA. The later was called
restriction endonuclease.
The first restriction endonuclease–Hind II, whose functioning
depended on a specific DNA nucleotide sequence was isolated and
characterised five years later. It was found that Hind II always cut DNA
molecules at a particular point by recognising a specific sequence of
six base pairs. This specific base sequence is known as the
recognition sequence for Hind II. Besides Hind II, today we know more
than 900 restriction enzymes that have been isolated from over 230 strains 195
of bacteria each of which recognise different recognition sequences.
The convention for naming these enzymes is the first letter of the name
comes from the genus and the second two letters come from the species of
the prokaryotic cell from which they were isolated, e.g., EcoRI comes from
Escherichia coli RY 13. In EcoRI, the letter ‘R’ is derived from the name of

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strain. Roman numbers following the names indicate the order in which
the enzymes were isolated from that strain of bacteria.
Restriction enzymes belong to a larger class of enzymes called
nucleases. These are of two kinds; exonucleases and endonucleases.
Exonucleases remove nucleotides from the ends of the DNA whereas,
endonucleases make cuts at specific positions within the DNA.
Each restriction endonuclease functions by ‘inspecting’ the length of
a DNA sequence. Once it finds its specific recognition sequence, it
will bind to the DNA and cut each of the two strands of the double
helix at specific points in their sugar -phosphate backbones
(Figure 11.1). Each restriction endonuclease recognises a specific
palindromic nucleotide sequences in the DNA.

Figure 11.1 Steps in formation of recombinant DNA by action of restriction endonuclease

enzyme - EcoRI

196
Do you know what palindromes are? These are groups of letters
that form the same words when read both forward and backward,
e.g., “MALAYALAM”. As against a word-palindrome where the same
word is read in both directions, the palindrome in DNA is a sequence
of base pairs that reads same on the two strands when orientation of

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reading is kept the same. For example, the following sequences reads
the same on the two strands in 5' à 3' direction. This is also true if
read in the 3' à 5' direction.
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'
Restriction enzymes cut the strand of DNA a little away from the centre
of the palindrome sites, but between the same two bases on the opposite
strands. This leaves single stranded portions at the ends. There are
overhanging stretches called sticky ends on each strand (Figure 11.1).
These are named so because they form hydrogen bonds with their
complementary cut counterparts. This stickiness of the ends facilitates
the action of the enzyme DNA ligase.
Restriction endonucleases are used in genetic engineering to form
‘recombinant’ molecules of DNA, which are composed of DNA from
different sources/genomes.
When cut by the same restriction enzyme, the resultant DNA fragments
have the same kind of ‘sticky-ends’ and, these can be joined together
(end-to-end) using DNA ligases (Figure 11.2).

Recombinant DNA
Molecule

197
(Cloning Host)

Figure 11.2 Diagrammatic representation of recombinant DNA technology

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You may have realised that normally, unless one cuts the vector and
the source DNA with the same restriction enzyme, the recombinant vector
molecule cannot be created.
Separation and isolation of DNA fragments : The cutting of DNA by
restriction endonucleases results in the fragments of DNA. These fragments
can be separated by a technique known as gel electrophoresis. Since
DNA fragments are negatively charged molecules they can be separated
by forcing them to move towards the anode under an electric field through
a medium/matrix. Nowadays the most commonly used matrix is agarose
which is a natural polymer extracted from sea weeds. The DNA fragments
separate (resolve) according to their size through sieving effect provided
by the agarose gel. Hence, the smaller the fragment size, the farther it
moves. Look at the Figure 11.3 and guess at which end of the gel the
sample was loaded.
The separated DNA fragments can be
visualised only after staining the DNA
with a compound known as ethidium
bromide followed by exposure to UV
radiation (you cannot see pure DNA
fragments in the visible light and
without staining). You can see bright
orange coloured bands of DNA in a
ethidium bromide stained gel
exposed to UV light (Figure 11.3). The
separated bands of DNA are cut out
Figure 11.3 A typical agarose gel from the agarose gel and extracted
electrophoresis showing from the gel piece. This step is known
migration of undigested as elution. The DNA fragments
(lane 1) and digested set of purified in this way are used in
DNA fragments (lane 2 to 4)
constructing recombinant DNA by
joining them with cloning vectors.

11.2.2 Cloning Vectors

You know that plasmids and bacteriophages have the ability to replicate
within bacterial cells independent of the control of chromosomal DNA.
Bacteriophages because of their high number per cell, have very high
copy numbers of their genome within the bacterial cells. Some plasmids
may have only one or two copies per cell whereas others may have
198 15-100 copies per cell. Their numbers can go even higher. If we are able
to link an alien piece of DNA with bacteriophage or plasmid DNA, we can
multiply its numbers equal to the copy number of the plasmid or
bacteriophage. Vectors used at present, are engineered in such a way
that they help easy linking of foreign DNA and selection of recombinants
from non-recombinants.

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The following are the features that are required to facilitate cloning
into a vector.
(i) Origin of replication (ori) : This is a sequence from where
replication starts and any piece of DNA when linked to this sequence
can be made to replicate within the host cells. This sequence is also
responsible for controlling the copy number of the linked DNA. So,
if one wants to recover many copies of the target DNA it should be
cloned in a vector whose origin support high copy number.
(ii) Selectable marker : In addition to ‘ori’, the vector requires a
selectable marker, which helps in identifying and eliminating non-
transformants and selectively permitting the growth of the
transformants. Transformation is a procedure through which a
piece of DNA is introduced in a host bacterium (you will study the
process in subsequent section). Normally, the genes encoding
resistance to antibiotics such as ampicillin, chloramphenicol,
tetracycline or kanamycin, etc., are considered useful selectable
markers for E. coli. The normal E. coli cells do not carry resistance
against any of these antibiotics.
(iii) Cloning sites: In order to link the
alien DNA, the vector needs to have
very few, preferably single,
recognition sites for the commonly
used restriction enzymes. Presence of
more than one recognition sites within
the vector will generate several
fragments, which will complicate the
gene cloning (Figure 11.4). The
ligation of alien DNA is carried out at
a restriction site present in one of the
two antibiotic resistance genes. For
example, you can ligate a foreign DNA
Figure 11.4 E. coli cloning vector pBR322
at the BamH I site of tetracycline showing restriction sites
resistance gene in the vector pBR322. (Hind III, EcoR I, BamH I, Sal I,
The recombinant plasmids will lose Pvu II, Pst I, Cla I), ori and
tetracycline resistance due to insertion antibiotic resistance genes
(ampR and tetR). rop codes for
of foreign DNA but can still be selected
the proteins involved in the
out from non-recombinant ones by replication of the plasmid.
plating the transformants on
tetracycline containing medium. The transformants growing on
199
ampicillin containing medium are then transferred on a medium
containing tetracycline. The recombinants will grow in ampicillin
containing medium but not on that containing tetracycline. But,
non- recombinants will grow on the medium containing both the
antibiotics. In this case, one antibiotic resistance gene helps in
selecting the transformants, whereas the other antibiotic resistance

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gene gets ‘inactivated due to insertion’ of alien DNA, and helps in

selection of recombinants.
Selection of recombinants due to inactivation of antibiotics is a
cumbersome procedure because it requires simultaneous plating
on two plates having different antibiotics. Therefore, alternative
selectable markers have been developed which differentiate
recombinants from non-recombinants on the basis of their ability
to produce colour in the presence of a chromogenic substrate. In
this, a recombinant DNA is inserted within the coding sequence of
an enzyme, β-galactosidase. This results into inactivation of the
gene for synthesis of this enzyme, which is referred to as insertional
inactivation. The presence of a chromogenic substrate gives blue
coloured colonies if the plasmid in the bacteria does not have an
insert. Presence of insert results into insertional inactivation of the
β-galactosidase gene and the colonies do not produce any colour,
these are identified as recombinant colonies.
(iv) Vectors for cloning genes in plants and animals : You may be
surprised to know that we have learnt the lesson of transferring genes
into plants and animals from bacteria and viruses which have known
this for ages – how to deliver genes to transform eukaryotic cells and
force them to do what the bacteria or viruses want. For example,
Agrobacterium tumifaciens, a pathogen of several dicot plants is able
to deliver a piece of DNA known as ‘ T-DNA’ to transform normal
plant cells into a tumor and direct these tumor cells to produce the
chemicals required by the pathogen. Similarly, retroviruses in animals
have the ability to transform normal cells into cancerous cells. A
better understanding of the art of delivering genes by pathogens in
their eukaryotic hosts has generated knowledge to transform these
tools of pathogens into useful vectors for delivering genes of interest
to humans. The tumor inducing (Ti) plasmid of Agrobacterium
tumifaciens has now been modified into a cloning vector which is no
more pathogenic to the plants but is still able to use the mechanisms
to deliver genes of our interest into a variety of plants. Similarly,
retroviruses have also been disarmed and are now used to deliver
desirable genes into animal cells. So, once a gene or a DNA fragment
has been ligated into a suitable vector it is transferred into a bacterial,
plant or animal host (where it multiplies).

11.2.3 Competent Host (For Transformation with

200 Recombinant DNA)
Since DNA is a hydrophilic molecule, it cannot pass through cell
membranes. Why? In order to force bacteria to take up the plasmid, the
bacterial cells must first be made ‘competent’ to take up DNA. This is
done by treating them with a specific concentration of a divalent cation,
such as calcium, which increases the efficiency with which DNA enters

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the bacterium through pores in its cell wall. Recombinant DNA can then
be forced into such cells by incubating the cells with recombinant DNA
on ice, followed by placing them briefly at 420C (heat shock), and then
putting them back on ice. This enables the bacteria to take up the
recombinant DNA.
This is not the only way to introduce alien DNA into host cells. In a
method known as micro-injection, recombinant DNA is directly injected
into the nucleus of an animal cell. In another method, suitable for plants,
cells are bombarded with high velocity micro-particles of gold or tungsten
coated with DNA in a method known as biolistics or gene gun. And the
last method uses ‘disarmed pathogen’ vectors, which when allowed to
infect the cell, transfer the recombinant DNA into the host.
Now that we have learnt about the tools for constructing recombinant
DNA, let us discuss the processes facilitating recombinant DNA technology.

11.3 PROCESSES OF RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology involves several steps in specific
sequence such as isolation of DNA, fragmentation of DNA by
restriction endonucleases, isolation of a desired DNA fragment,
ligation of the DNA fragment into a vector, transferring the
recombinant DNA into the host, culturing the host cells in a
medium at large scale and extraction of the desired product.
Let us examine each of these steps in some details.

11.3.1 Isolation of the Genetic Material (DNA)

Recall that nucleic acid is the genetic material of all organisms
without exception. In majority of organisms this is
deoxyribonucleic acid or DNA. In order to cut the DNA with
restriction enzymes, it needs to be in pure form, free from other
macro-molecules. Since the DNA is enclosed within the
membranes, we have to break the cell open to release DNA along
with other macromolecules such as RNA, proteins,
polysaccharides and also lipids. This can be achieved by treating
the bacterial cells/plant or animal tissue with enzymes such as Figure 11.5 DNA that
separates out can be
lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
removed by spooling
You know that genes are located on long molecules of DNA
interwined with proteins such as histones. The RNA can be removed by 201
treatment with ribonuclease whereas proteins can be removed by
treatment with protease. Other molecules can be removed by appropriate
treatments and purified DNA ultimately precipitates out after the addition
of chilled ethanol. This can be seen as collection of fine threads in the
suspension (Figure 11.5).

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11.3.2 Cutting of DNA at Specific Locations

Restriction enzyme digestions are performed by incubating purified DNA
molecules with the restriction enzyme, at the optimal conditions for that
specific enzyme. Agarose gel electrophoresis is employed to check the
progression of a restriction enzyme digestion. DNA is a negatively charged
molecule, hence it moves towards the positive electrode (anode)
(Figure 11.3). The process is repeated with the vector DNA also.
The joining of DNA involves several processes. After having cut the
source DNA as well as the vector DNA with a specific restriction enzyme,
the cut out ‘gene of interest’ from the source DNA and the cut vector with
space are mixed and ligase is added. This results in the preparation of
recombinant DNA.

11.3.3 Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction. In this reaction, multiple
copies of the gene (or DNA) of interest is synthesised in vitro using two

202

Figure 11.6 Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation;
(ii) Primer annealing; and (iii) Extension of primers

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sets of primers (small chemically synthesised oligonucleotides that are

complementary to the regions of DNA) and the enzyme DNA polymerase.
The enzyme extends the primers using the nucleotides provided in the
reaction and the genomic DNA as template. If the process of replication
of DNA is repeated many times, the segment of DNA can be amplified
to approximately billion times, i.e., 1 billion copies are made. Such
repeated amplification is achieved by the use of a thermostable DNA
polymerase (isolated from a bacterium, Thermus aquaticus), which
remain active during the high temperature induced denaturation of
double stranded DNA. The amplified fragment if desired can now be
used to ligate with a vector for further cloning (Figure11.6).

11.3.4 Insertion of Recombinant DNA into the Host

Cell/Organism
There are several methods of introducing the ligated DNA into recipient
cells. Recipient cells after making them ‘competent’ to receive, take up
DNA present in its surrounding. So, if a recombinant DNA bearing gene
for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli
cells, the host cells become transformed into ampicillin-resistant cells. If
we spread the transformed cells on agar plates containing ampicillin, only
transformants will grow, untransformed recipient cells will die. Since, due
to ampicillin resistance gene, one is able to select a transformed cell in the
presence of ampicillin. The ampicillin resistance gene in this case is called
a selectable marker.

11.3.5 Obtaining the Foreign Gene Product

When you insert a piece of alien DNA into a cloning vector and transfer it
into a bacterial, plant or animal cell, the alien DNA gets multiplied. In
almost all recombinant technologies, the ultimate aim is to produce a
desirable protein. Hence, there is a need for the recombinant DNA to be
expressed. The foreign gene gets expressed under appropriate conditions.
The expression of foreign genes in host cells involve understanding many
technical details.
After having cloned the gene of interest and having optimised the
conditions to induce the expression of the target protein, one has to
consider producing it on a large scale. Can you think of any reason
why there is a need for large-scale production? If any protein encoding
gene is expressed in a heterologous host, it is called a recombinant
protein. The cells harbouring cloned genes of interest may be grown
on a small scale in the laboratory. The cultures may be used for 203
extracting the desired protein and then purifying it by using different
separation techniques.
The cells can also be multiplied in a continuous culture system wherein
the used medium is drained out from one side while fresh medium is
added from the other to maintain the cells in their physiologically most

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active log/exponential phase. This type of culturing method produces a

larger biomass leading to higher yields of desired protein.
Small volume cultures cannot yield appreciable quantities of products.
To produce in large quantities, the development of bioreactors, where
large volumes (100-1000 litres) of culture can be processed, was required.
Thus, bioreactors can be thought of as vessels in which raw materials are
biologically converted into specific products, individual enzymes, etc.,
using microbial plant, animal or human cells. A bioreactor provides the
optimal conditions for achieving the desired product by providing
optimum growth conditions (temperature, pH, substrate, salts, vitamins,
oxygen).
The most commonly used bioreactors are of stirring type, which are
shown in Figure 11.7.

(a) (b)

Figure 11.7 (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which
sterile air bubbles are sparged

A stirred-tank reactor is usually cylindrical or with a curved base to

facilitate the mixing of the reactor contents. The stirrer facilitates even
mixing and oxygen availability throughout the bioreactor. Alternatively
air can be bubbled through the reactor. If you look at the figure closely
you will see that the bioreactor has an agitator system, an oxygen delivery
system and a foam control system, a temperature control system, pH
204 control system and sampling ports so that small volumes of the culture
can be withdrawn periodically.

11.3.6 Downstream Processing

After completion of the biosynthetic stage, the product has to be subjected
through a series of processes before it is ready for marketing as a finished

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product. The processes include separation and purification, which are

collectively referred to as downstream processing. The product has to be
formulated with suitable preservatives. Such formulation has to undergo
thorough clinical trials as in case of drugs. Strict quality control testing
for each product is also required. The downstream processing and quality
control testing vary from product to product.

SUMMARY
Biotechnology deals with large scale production and marketing of
products and processes using live organisms, cells or enzymes.
Modern biotechnology using genetically modified organisms was
made possible only when man learnt to alter the chemistry of DNA
and construct recombinant DNA. This key process is called
recombinant DNA technology or genetic engineering. This process
involves the use of restriction endonucleases, DNA ligase,
appropriate plasmid or viral vectors to isolate and ferry the foreign
DNA into host organisms, expression of the foreign gene, purification
of the gene product, i.e., the functional protein and finally making a
suitable formulation for marketing. Large scale production involves
use of bioreactors.

EXERCISES
1. Can you list 10 recombinant proteins which are used in medical
practice? Find out where they are used as therapeutics (use the internet).
2. Make a chart (with diagrammatic representation) showing a restriction
enzyme, the substrate DNA on which it acts, the site at which it cuts
DNA and the product it produces.
3. From what you have learnt, can you tell whether enzymes are bigger or
DNA is bigger in molecular size? How did you know?
4. What would be the molar concentration of human DNA in a human
cell? Consult your teacher.
5. Do eukaryotic cells have restriction endonucleases? Justify your answer.
6. Besides better aeration and mixing properties, what other advantages
do stirred tank bioreactors have over shake flasks?
7. Collect 5 examples of palindromic DNA sequences by consulting your teacher. 205
Better try to create a palindromic sequence by following base-pair rules.
8. Can you recall meiosis and indicate at what stage a recombinant DNA
is made?
9. Can you think and answer how a reporter enzyme can be used to monitor
transformation of host cells by foreign DNA in addition to a selectable
marker?

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10. Describe briefly the following:

(a) Origin of replication
(b) Bioreactors
(c) Downstream processing
11. Explain briefly
(a) PCR
(b) Restriction enzymes and DNA
(c) Chitinase
12. Discuss with your teacher and find out how to distinguish between
(a) Plasmid DNA and Chromosomal DNA
(b) RNA and DNA
(c) Exonuclease and Endonuclease

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