TCH-465 Bio-System Process

Module-1
Principles of Microbiology
Micro-organisms and their activities are vitally important to virtually all processes on Earth.
Micro-organisms matter because they affect every aspect of our lives – they are in us, on us
and around us.
Microbiology is the study of all living organisms that are too small to be visible with the naked
eye. This includes bacteria, archaea, viruses, fungi, prions, protozoa and algae, collectively
known as 'microbes'. These microbes play key roles in nutrient cycling,
biodegradation/biodeterioration, climate change, food spoilage, the cause and control of
disease, and biotechnology. Thanks to their versatility, microbes can be put to work in many
ways: making life-saving drugs, the manufacture of biofuels, cleaning up pollution, and
producing/processing food and drink.
You have seen several kinds of plants and animals. However, there are other living organisms
around us which we normally cannot see. These are called microorganisms or microbes. For
example, you might have observed that during the rainy season moist bread gets spoilt and its
surface gets covered with greyish white patches. Observe these patches through a magnifying
glass. You will see tiny, black rounded structures.
These observations show that water and soil are full of tiny organisms, though not all
of them fall into the category of microbes. These microorganisms or microbes are so small in
size that they cannot be seen with the unaided eye. Some of these, such as the fungus that grows
on bread, can be seen with a magnifying glass. Others cannot be seen without the help of a
microscope. That is why these are called microorganisms or microbes. Microorganisms are
classified into four major groups. These groups are bacteria, fungi, protozoa and some algae.
Microorganisms may be single-celled like bacteria, some algae and protozoa, or multicellular,
such as many algae and fungi. They live in all types of environments, ranging from ice cold
climate to hot springs; and deserts to marshy lands. They are also found inside the bodies of
animals including humans. Some microorganisms grow on other organisms while others exist
freely.
Microbiology is the branch of biology that examines very small organisms and viruses. There
are many types of microorganisms and we will concentrate on bacteria and viruses. In this
course, we will use microorganisms to look at some of the basic processes of life shared by all
living organisms: growth, reproduction, cell structure and function, and the basics of genetics.
Measures used to control microbial growth will also be discussed, including antibiotics,
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disinfection, and sterilization. We will also be looking at the basics of how we interact with
microorganisms with an introduction to immunology.
Commercial Use of Microorganisms:
Microorganisms are used for the large-scale production of alcohol, wine and acetic acid
(vinegar). Yeast is used for commercial production of alcohol and wine. For this purpose, yeast
is grown on natural sugars present in grains like barley, wheat, rice, crushed fruit juices, etc.
Harmful Microorganisms: Microorganisms are harmful in many ways. Some of the
microorganisms cause diseases in human beings, plants and animals. Such disease-causing
microorganisms are called pathogens. Some microorganisms spoil food, clothing and leather.
Let us study more about their harmful activities.
Cell:
Unicellular organisms are capable of (i) independent existence and (ii) performing the essential
functions of life. Anything less than a complete structure of a cell does not ensure independent
living. Hence, cell is the fundamental structural and functional unit of all living organisms.
Anton Von Leeuwenhoek first saw and described a live cell. Robert Brown later discovered
the nucleus. The invention of the microscope and its improvement leading to the electron
microscope revealed all the structural details of the cell.
A cell is the smallest unit that is capable of performing life functions.
• All living things are made up of cells.
• Cells are the smallest working units of all living things.
• All cells come from preexisting cells through cell division
A description of cell structure and cell growth by division is given in the chapters comprising
this unit. Cell theory also created a sense of mystery around living phenomena, i.e.,
physiological and behavioural processes. This mystery was the requirement of integrity of
cellular organisation for living phenomena to be demonstrated or observed. In studying and
understanding the physiological and behavioural processes, one can take a physico-chemical
approach and use cell-free systems to investigate.
Cell Wall
As you may recall, a non-living rigid structure called the cell wall forms an outer covering for
the plasma membrane of fungi and plants. Cell wall not only gives shape to the cell and protects
the cell from mechanical damage and infection, it also helps in cell-to-cell interaction and
provides barrier to undesirable macromolecules. Algae have cell wall, made of cellulose,
galactans, mannans and minerals like calcium carbonate, while in other plants it consists of
cellulose, hemicellulose, pectins and proteins. The cell wall of a young plant cell, the primary
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wall is capable of growth, which gradually diminishes as the cell matures and the secondary
wall is formed on the inner (towards membrane) side of the cell. The middle lamella is a layer
mainly of calcium pectate which holds or glues the different neighbouring cells together. The
cell wall and middle lamellae may be traversed by plasmodesmata which connect the cytoplasm
of neighbouring cells.
FUNGI: These are heterotrophic eukaryotic organisms. Some of them use decaying organic
material as food and are therefore called saprotrophs. Others require a living protoplasm of a
host organism for food. They are called parasites. Many of them have the capacity to become
multicellular organisms at certain stages in their lives. They have cell-walls made of a tough
complex sugar called chitin. Examples are yeasts, molds and mushrooms.
Some fungal species live in permanent mutually dependent relationships with blue green algae
(or cyanobacteria). Such relationships are called symbiotic. These symbiotic life forms are
called lichens. We have all seen lichens as the slow-growing large coloured patches on the bark
of trees.

Fig: Fungi

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Fig: Five Kingdom Classification
Viruses are also microscopic but are different from other microorganisms. They, however,
reproduce only inside the cells of the host organism, which may be a bacterium, plant or animal.
Common ailments like cold, influenza (flu) and most coughs are caused by viruses. Seriou’s
diseases like polio and chicken pox are also caused by viruses. Diseases like dysentery and
malaria are caused by protozoa(protozoans) whereas typhoid and tuberculosis (TB) are
bacterial diseases.
VIRUSES:
Viruses may be generalized to define as 'very small sized etiological agents of disease that are
capable of passing through filters that retain even bacteria, increase only in the presence of
living cells, and give rise to new strains by mutation'.
Some define viruses as 'infectious nucleoproteins'. The word virus is derived from the latin
language meaning 'poisonous liquid or 'poison'. In 1935 Stanley crystallized the virus causing
tobacco mosaic disease, and demonstrated that the crystals retained their infectivity when
inoculated into healthy plants Hershey and Chase (1952) studied the T2 bacteriophage and
demonstrated that (1) the genetic information is carried in the phage DNA and (2) that infection
is the result of penetration of viral DNA into cells. The nucleic acid fraction of the virus is the
actual infectious agent was first shown by Gierrer and Schramm (1956). Phycophages were

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first isolated by Schafferman and Morris (1963) from blue green alga LyngbtJa. The phage
isolated by them was found to infect Plectono1lema and Phormidiunl also (hence named LPP-
1), these are cyanophages.
GENERAL Characters OF VIRUSES
(1) They do not occur free in nature but act as obligate intracellular parasite.
(2) They are extreme microscopic structure which can only be seen by electron
microscope.
(3) Mainly the size ranges from 100-2000 millimicron.
(4) They cannot be filtered by bacterial filters.
(5) The genetic material is either DNA or RNA which occurs in the form of single molecule
and can be single or double stranded.
(6) A single virus particle is known as virion which lacks functional autonomy.
(7) They lack their own enzyme system but interact with the host enzyme system and
synthesize new virus particles. Thus, they have a master and slave relationship.
(8) Outer capsid of virus is proteinaceous and harmless and provide cellular specificity to
the virus.
(9) They are intracellular obligate parasite and can't be cultured on artificial culture media.
(10) All animal and plant viruses have a narrow host range while others show a
broad host range.
(11) They show replication.
(12) They are highly infectious and spread disease very quickly.
(13) They show special kind of pathogenicity i.e., they cause disease at particular
temperature. Most of virus become inert at 56-69°C (for 30 minutes)
(14) They are haploid.
(15) They are un-effected by antibiotics.
(16) They show life between 5-9 pH.
(17) They remain active for a long time when kept in 50% glycerol solution.
(18) The extract of virus become inert at high pressure and high sound frequency.
(19) They get precipitated with ethyl alcohol and acetone.
(20) They can be inerted by treatment with ultraviolet rays, pyridine, urea and
hydrogen peroxide.
(21) They can be crystallized.
(22) They show response toward temperature, radiation and chemical substances.
(23) They lack cell wall, nucleus, protoplasm and cell organelles
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How do Virus differ from Bacteria and Mycoplasmas?
Viruses differ from bacteria and mycoplasmas in:
(i) not possessing any cellular organization. (ii) Not growing on inanimate media. (iii) not
multiply by binary fission. (iv) Not possessing both DNA and RNA together. (v) Not
possessing ribosome. (vi) Not showing any sensitivity to antibiotics. (vii) Showing sensitivity
to interferon.
NATURE OF VIRUSES
The nature of viruses is still not clear, because it is not easy to define them within the accepted
framework of living or non-living organisms. Some virologist regard viruses as animate object
(when present inside the host cell) whereas other consider them inanimate (when present
outside the host cell). Viruses are living because:
(i) They show growth and multiplication (only inside the host cell). (ii) They have genetic
material i.e., DNA/RNA. (iii) They can direct protein synthesis (though they use host
machinery for it). (iv) They show mutation. (v) They can be transmitted from the diseased host
to the healthy ones or possess the ability to infect. (vi) They react to heat, chemicals and
radiation and also shows irritability, a character of only living organisms. (vii) They possess
genetic continuity and have definite races/strains. (viii) Similarity between nucleoproteins of
viruses with the protein and nucleic acid of living organisms.
Viruses are non-living because:
(i) They can be crystallized (Stanley, 1935) (ii) They behave as inert chemicals outside the
host cell. (iii) A cell wall or cell membrane of any type is absent in viruses. (iv) They do not
show functional autonomy. (v) They do not respire or excrete or they do not show any sign of
metabolism except reproduction. (vi) They lack any energy producing enzyme system.
Occurrence: The occurrence of viruses in the cells of bacteria and higher plants and animals
is well established.
Plant viruses: Most plant viruses have been found in angiosperm (flowering plants). Relatively
few viruses are known in gymnosperm, ferns, fungi or algae. Plant viruses are of great
economic importance, since they cause plant diseases in a variety of crops.
Animal viruses: Virus diseases are known in a variety of vertebrates including fish,
amphibian, birds and mammals. Important virus diseases of humans include poliomyelitis,
small pox, rabies, mumps, measles, yellow fever, influenza and encephalitis.
Bacteriophages: Viruses have been found in practically all groups of bacteria. The host range
is confined within bacterial groups. A bacteriophage may multiply only in certain strains of E.
coli.
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Viruses in Eukaryotic Microorganism Virus like particles have been observed in species of
Protozoa, algae and fungi.
Protozoa: Viruses or virus like particles have been observed in several protozoa viz.,
leishmania, Entamoeba histolytica, Plasmodium vivax, P. berghei, Paramecium aurelia,
Carclzesill polypinum and Acanthamoeba sp. A virus like particle has been reported in
Plasmodium berghei. Its structure is like that of a cytoplasmic Polyhedrosis virus, a dsRNA
virus of insects.
Algae: Virus like particles have been reported in Aulacomonas subnzarina, Chara, Corralina,
Oedogonium spp., Uronema gigas. Bacteriophage like virus particles have been found in
Chiorella and have been called chlorellophages.
Fungi: Killer phenotypes associated with the strains of Saccharomyces cerervisiae and
Ustilago maydis have been shown to be virus related. dsDNA viruses have been detected in
cells of Penicillium moulds. dsRNA viruses are widely present in the higher fungi. The bacilli
form particles of Agaricus bisporous are the only fungal virus reported that contains ssRNA.
Size And Structure of Viruses: The size of viruses is variable. Most viruses are much smaller
than bacteria. Their size ranges from 10 nm - 250 nm. The size of viruses is determined by
electron microscopy, ultra-centrifugation and by filtration through colloid ion membrane of
known pore diameter.
The smallest virus is coliphage F2 measuring about 2 nm.
The smallest plant virus is satellite tobacco necrosis virus measuring 17nm.
The longest known plant virus is citrus tristeza virus-rod shaped measuring 2000 x 12 nm.
Foot and mouth virus of cattle is the smallest animal virus measuring about 10 nm.
Pox viruses are the largest and most complex animal viruses.
Parrot fever virus measuring 400 nm.
Structure of Viruses
The intact virus unit or infectious particle is called the virion. Each virion consists of a nucleic
acid core surrounded by a protein coat (capsid) to form the nucleocapsid. The nucleocapsid
may be naked or may be surrounded by a loose membranous envelope. It is composed of a
number of subunits called capsomeres. The capsid protects the nucleic acid core against the
action of nucleases. Structurally viruses occur in three main shapes viz. spherical or polyhedral,
cylindrical or helical and the complex type.
Bacteria are the sole members of the Kingdom Monera. They are the most abundant micro-
organisms. Bacteria occur almost everywhere. Hundreds of bacteria are present in a handful of

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soil. They also live in extreme habitats such as hot springs, deserts, snow and deep oceans
where very few other life forms can survive. Many of them live in or on other organisms as
parasites. Bacteria are grouped under four categories based on their shape: the spherical Coccus
(pl.: cocci), the rod-shaped Bacillus (pl.: bacilli), the comma-shaped Vibrium (pl.: vibrio) and
the spiral Spirillum.

Fig: Bacteria of different shapes

Though the bacterial structure is very simple, they are very complex in behaviour. Compared
to many other organisms, bacteria as a group show the most extensive metabolic diversity.
Some of the bacteria are autotrophic, i.e., they synthesise their own food from inorganic
substrates. They may be photosynthetic autotrophic or chemosynthetic autotrophic. The vast
majority of bacteria are heterotrophs, i.e., they depend on other organisms or on dead organic
matter for food.
Archaebacteria
These bacteria are special since they live in some of the most harsh habitats such as
extreme salty areas (halophiles), hot springs (thermoacidophiles) and marshy areas
(methanogens). Archaebacteria differ from other bacteria in having a different cell wall
structure and this feature is responsible for their survival in extreme conditions. Methanogens
are present in the gut of several ruminant animals such as cows and buffaloes and they are
responsible for the production of methane (biogas) from the dung of these animals.
Eubacteria
There are thousands of different eubacteria or ‘true bacteria’. They are characterised by
the presence of a rigid cell wall, and if motile, a flagellum. The cyanobacteria (also referred to
as blue-green algae) have chlorophyll a similar to green plants and are photosynthetic

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autotrophs (Figure). The cyanobacteria are unicellular, colonial or filamentous,
freshwater/marine or terrestrial algae. The colonies are generally surrounded by gelatinous
sheath. They often form blooms in polluted water bodies. Some of these organisms can fix
atmospheric nitrogen in specialised cells called heterocysts, e.g., Nostoc and Anabaena.
Chemosynthetic autotrophic bacteria oxidise various inorganic substances such as nitrates,
nitrites and ammonia and use the released energy for their ATP production. They play a great
role in recycling nutrients like nitrogen, phosphorous, iron and sulphur. Heterotrophic bacteria
are most abundant in nature.

A filamentous blue-green algae – Nostoc

The majority are important decomposers. Many of them have a significant impact on human
affairs. They are helpful in making curd from milk, production of antibiotics, fixing nitrogen
in legume roots, etc. Some are pathogens causing damage to human beings, crops, farm animals
and pets. Cholera, typhoid, tetanus, citrus canker are well known diseases caused by different
bacteria. Bacteria reproduce mainly by fission (Figure). Sometimes, under unfavourable
conditions, they produce spores. They also reproduce by a sort of sexual reproduction by
adopting a primitive type of DNA transfer from one bacterium to the other.
The Mycoplasma are organisms that completely lack a cell wall. They are the smallest living
cells known and can survive without oxygen. Many mycoplasma are pathogenic in animals and
plants.

Fig: A dividing bacterium

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In addition to proteins, viruses also contain genetic material, that could be either RNA or DNA.
No virus contains both RNA and DNA. A virus is a nucleoprotein and the genetic material is
infectious. In general, viruses that infect plants have single stranded RNA and viruses that
infect animals have either single or double stranded RNA or double stranded DNA. Bacterial
viruses or bacteriophages (viruses that infect the bacteria) are usually double stranded DNA
viruses (Figure 2.6b). The protein coat called capsid made of small subunits called capsomeres,
protects the nucleic acid. These capsomeres are arranged in helical or polyhedral geometric
forms. Viruses cause diseases like mumps, small pox, herpes and influenza. AIDS in humans
is also caused by a virus. In plants, the symptoms can be mosaic formation, leaf rolling and
curling, yellowing and vein clearing, dwarfing and stunted growth.
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. 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 LABs, 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.
Microbes In Industrial Products
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 fermenters.
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
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of the raw material used for fermentation and the type 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.
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 ‘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 after the mould Penicillium
notatum. However, its full potential as an effective antibiotic was established much later by
Ernest Chain and Howard Florey.
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.
Microbes In Sewage Treatment

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 We know that large quantities of waste water are generated every day 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. 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
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) is removed by sedimentation. All solids that settle form the primary sludge, and the
supernatant forms the effluent. The effluent from the primary settling tank is taken for
secondary treatment.
Secondary treatment or biological treatment: The primary effluent is passed into large
aeration tanks 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 litre 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 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.
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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 cellulose 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.
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 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.
Microbes As Biofertilizers
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 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
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switch to organic farming – the use of biofertilizers. Biofertilizers are organisms that enrich
the nutrient quality of the soil. The main sources of biofertilizers 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 biofertilizer. Blue green
algae also add organic matter to the soil and increase its fertility. Currently, in our country, a
number of biofertilizers 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.
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),
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
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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 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 ‘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;
15
(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.
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.
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 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 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). Each
restriction endonuclease recognises a specific palindromic nucleotide sequence in the DNA.

16
 Steps in formation of recombinant DNA by action of restriction endonuclease
enzyme – EcoRI
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 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).
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).

17
 Diagrammatic representation of recombinant DNA technology
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.

A typical agarose gel electrophoresis showing migration of undigested (lane 1) and digested
set of DNA fragments (lane 2 to 4)
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

18
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). The separated bands
of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known
as elution. The DNA fragments purified in this way are used in constructing recombinant DNA
by joining them with cloning vectors.
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 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. 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). 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 at the
BamH I site of tetracycline resistance gene in the vector pBR322. The recombinant plasmids
will lose tetracycline resistance due to insertion of foreign DNA but can still be selected out
19
from non-recombinant ones by plating the transformants on tetracycline containing medium.
The transformants growing on 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 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, b-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 b-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).

20
 E. coli cloning vector pBR322 showing restriction sites (Hind III, EcoR I, BamH I, Sal I,
Pvu II, Pst I, Cla I), ori and antibiotic resistance genes (ampR and tetR). rop codes for
the proteins involved in the replication of the plasmid.
Competent Host (For Transformation with 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 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.
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.

21
a. 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 lysozyme
(bacteria), cellulase (plant cells), chitinase (fungus).
You know that genes are located on long molecules of DNA interwined with proteins
such as histones. The RNA can be removed by 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).
b. 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 is mixed and ligase is added. This results in the preparation of recombinant DNA.
c. 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 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 (Figure).

22
 Polymerase chain reaction (PCR): Each cycle has three steps: (i) Denaturation;
(ii) Primer annealing; and (iii) Extension of primers
d. 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.
e. 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

23
cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be
used for 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 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.

(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 control system and sampling ports so
that small volumes of the culture can be withdrawn periodically.
f. 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 product. The processes include

24
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.
Nucleotide
A nucleotide is the basic building block of nucleic acids (RNA and DNA). A nucleotide
consists of a sugar molecule (either ribose in RNA or deoxyribose in DNA) attached to a
phosphate group and a nitrogen-containing base. The bases used in DNA are adenine (A),
cytosine (C), guanine (G) and thymine (T).
A nucleotide is an organic molecule with a basic composition of a nitrogenous base, pentose
sugar and phosphate.
DNA and RNA are polynucleotides, which contain a chain of nucleotides monomers with
different nitrogenous bases. Nucleotides are essential for carrying out metabolic and
physiological activities.
ATP (Adenosine triphosphate) acts as the energy currency of cells. Nucleotides form various
coenzymes and cofactors, such as NAD, NADP, FAD, coenzyme A, etc. and are essential for
many metabolic processes.
Table of Content
• Nucleotide Structure
• Nucleotides vs Nucleosides
• Nitrogenous Bases
• Nucleotide Function
Nucleotide Structure – What does a nucleotide look like?
A nucleotide consists of three units, which are covalently linked. They are:
1. Nitrogenous bases – Purine and Pyrimidine
2. Pentose Sugar – Ribose and Deoxyribose
3. Phosphate – monophosphate, diphosphate, triphosphate
1. Nitrogenous Base: They comprise pyrimidine or purine base. DNA contains adenine
(A), guanine (G), cytosine (C) and thymine (T) whereas RNA contains adenine,
guanine, cytosine and uracil (U).
2. 2. Sugar: A nucleotide comprises a pentose sugar. DNA (Deoxyribonucleic acid)
contains deoxyribose sugar and RNA (Ribonucleic acid) contains a ribose sugar.
3. A Nitrogenous base attached with the sugar is called “Nucleoside”.

25
 4. 3. Phosphate: Phosphate is associated with the sugar of nucleoside by an ester bond
with the 5thC hydroxyl group. Nucleotides at least contain one phosphate group.
5. Phosphate of one nucleotide attaches to the 3rd C-OH group of the sugar of the 2nd
nucleotide, thereby forming 5’ → 3’ linkage.
6. In DNA (double helix) there are two antiparallel strands of polynucleotides that are
linked together by hydrogen bonds between nitrogenous bases. Purine pairs with
pyrimidine base, A pairs with T and G pairs with C by two and three hydrogen bonds
respectively.
7. In RNA instead of thymine (T), A pairs with U.
8. Phosphate group interlinks the sugar molecules of two nucleotides forming a chain.
DNA and RNA are polynucleotides. Sugar phosphate chain forms the backbone of a
polynucleotide chain.
9. When the phosphate group attaches to the hydroxyl group of the same sugar, it forms
cyclic nucleotide, they are present as a single monomer, e.g. cAMP, cGMP used in
intracellular signal transduction processes.
RNA
Ribonucleic acid (abbreviated RNA) is a nucleic acid present in all living cells that has
structural similarities to DNA. Unlike DNA, however, RNA is most often single-
stranded. An RNA molecule has a backbone made of alternating phosphate groups and
the sugar ribose, rather than the deoxyribose found in DNA.
Nucleotides are organic molecules consisting of a nucleoside and a phosphate. They serve as
monomeric units of the nucleic acid polymers – deoxyribonucleic acid (DNA) and ribonucleic
acid (RNA), both of which are essential biomolecules within all life-forms on Earth.
Nucleotides are obtained in the diet and are also synthesized from common nutrients by the
liver.
Nucleotides are composed of three subunit molecules: a nucleobase, a five-carbon sugar (ribose
or deoxyribose), and a phosphate group consisting of one to three phosphates. The four
nucleobases in DNA are guanine, adenine, cytosine and thymine; in RNA, uracil is used in
place of thymine.
Nucleotides also play a central role in metabolism at a fundamental, cellular level. They
provide chemical energy in the form of the nucleoside triphosphates, adenosine triphosphate
(ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP) and uridine triphosphate
(UTP) throughout the cell for the many cellular functions that demand energy, including: amino
acid, protein and cell membrane synthesis, moving the cell and cell parts (both internally and
26
intercellularly), cell division, etc.[2] In addition, nucleotides participate in cell signalling (cyclic
guanosine monophosphate or cGMP and cyclic adenosine monophosphate or cAMP), and are
incorporated into important cofactors of enzymatic reactions (e.g. coenzyme A, FAD, FMN,
NAD, and NADP+).
RNA structure
RNA typically is a single-stranded biopolymer. However, the presence of self-complementary
sequences in the RNA strand leads to intrachain base-pairing and folding of the ribonucleotide
chain into complex structural forms consisting of bulges and helices. The three-dimensional
structure of RNA is critical to its stability and function, allowing the ribose sugar and the
nitrogenous bases to be modified in numerous different ways by cellular enzymes that attach
chemical groups (e.g., methyl groups) to the chain. Such modifications enable the formation of
chemical bonds between distant regions in the RNA strand, leading to complex contortions in
the RNA chain, which further stabilizes the RNA structure. Molecules with weak structural
modifications and stabilization may be readily destroyed. As an example, in an initiator transfer
RNA (tRNA) molecule that lacks a methyl group (tRNAiMet), modification at position 58 of the
tRNA chain renders the molecule unstable and hence non-functional; the non-functional chain
is destroyed by cellular tRNA quality control mechanisms.
Deoxyribonucleic acid (DNA): Deoxyribonucleic acid (DNA) is a polymer composed of two
polynucleotide chains that coil around each other to form a double helix carrying genetic
instructions for the development, functioning, growth and reproduction of all known organisms
and many viruses. DNA and ribonucleic acid (RNA) are nucleic acids. Alongside proteins,
lipids and complex carbohydrates (polysaccharides), nucleic acids are one of the four major
types of macromolecules that are essential for all known forms of life.
The two DNA strands are known as polynucleotides as they are composed of simpler
monomeric units called nucleotides. Each nucleotide is composed of one of four nitrogen-
containing nucleobases (cytosine [C], guanine [G], adenine [A] or thymine [T]), a sugar called
deoxyribose, and a phosphate group. The nucleotides are joined to one another in a chain by
covalent bonds (known as the phospho-diester linkage) between the sugar of one nucleotide
and the phosphate of the next, resulting in an alternating sugar-phosphate backbone. The
nitrogenous bases of the two separate polynucleotide strands are bound together, according to
base pairing rules (A with T and C with G), with hydrogen bonds to make double-stranded
DNA. The complementary nitrogenous bases are divided into two groups, pyrimidines and
purines. In DNA, the pyrimidines are thymine and cytosine; the purines are adenine and
guanine.
27
Lipids:
Lipids are fatty, waxy, or oily compounds that are soluble in organic solvents and insoluble in
polar solvents such as water. Lipids include: Fats and oils (triglycerides) Phospholipids.
A lipid is an organic substance found in living systems that is insoluble in water but is soluble
in organic solvents. Lipids vary widely in their structures. They have mostly C, H and some
have a few polar atoms/ functional groups.
Lipids include: fats and oils, steroids, waxes
Fats & oils make up 95% of the nutritional lipids, the other 5% are steroids. Waxes are
functional only.
Fats are solid triglycerides
Oils are liquid triglycerides
In biology and biochemistry, a lipid is a biomolecule that is only soluble in nonpolar solvents.[3]
Non-polar solvents are hydrocarbons used to dissolve other hydrocarbon lipid molecules that
do not dissolve in water, including fatty acids, waxes, sterols, fat-soluble vitamins (A, D, E,
and K), monoglycerides, diglycerides, triglycerides, and phospholipids.
The functions of lipids include storing energy, signalling, and acting as structural components
of cell membranes. Lipids have applications in the cosmetic and food industries as well as in
nanotechnology.
Scientists sometimes define lipids as hydrophobic or amphiphilic small molecules; the
amphiphilic nature of some lipids allows them to form structures such as vesicles,
multilamellar/unilamellar liposomes, or membranes in an aqueous environment.
Fats and oils are esters made up of glycerol (a 3-carbon sugar alcohol/polyol) and 3 fatty acids.
Fatty acids are hydrocarbon chains of differing lengths with various degrees of saturation that
end with carboxylic acid groups. Additionally, fatty acid double bonds can either
be cis or trans, creating many different types of fatty acids. Fatty acids in biological systems
usually contain an even number of carbon atoms and are typically 14 carbons to 24 carbons
long. Triglycerides store energy, provide insulation to cells, and aid in the absorption of fat-
soluble vitamins. Fats are normally solid at room temperature, while oils are generally liquid.
Lipids are an essential component of the cell membrane. The structure is typically made of a
glycerol backbone, 2 fatty acid tails (hydrophobic), and a phosphate group (hydrophilic). As
such, phospholipids are amphipathic. In the cell membrane, phospholipids are arranged in a
bilayer manner, providing cell protection and serving as a barrier to certain molecules. The
hydrophilic part faces outward and the hydrophobic part faces inward. This arrangement helps
monitor which molecules can enter and exit the cell. For example, nonpolar molecules and
28
small polar molecules, such as oxygen and water, can easily diffuse in and out of the cell.
Large, polar molecules, for example, glucose, cannot pass freely so they need the help of
transport proteins.
Another type of lipid is wax. Waxes are esters made of long-chain alcohol and a fatty acid.
They provide protection, especially to plants in which wax covers the leaves of plants. In
humans, cerumen, also known as earwax, helps protect the skin of the ear canal.
Polysaccharides
Polysaccharides are major classes of biomolecules. They are long chains of carbohydrate
molecules, composed of several smaller monosaccharides. These complex bio-
macromolecules functions as an important source of energy in animal cell and form a structural
component of a plant cell. It can be a homopolysaccharide or a heteropolysaccharide depending
upon the type of the monosaccharides.
The acid insoluble pellet also has polysaccharides (carbohydrates) as another class of
macromolecules. Polysaccharides are long chains of sugars. They are threads (literally a cotton
thread) containing different monosaccharides as building blocks. For example, cellulose is a
polymeric polysaccharide consisting of only one type of monosaccharide i.e., glucose.
Cellulose is a homopolymer. Starch is a variant of this but present as a store house of energy
in plant tissues. Animals have another variant called glycogen. Inulin is a polymer of fructose.
In a polysaccharide chain (say glycogen), the right end is called the reducing end and the left
end is called the non-reducing end.
Polysaccharides can be a straight chain of monosaccharides known as linear polysaccharides,
or it can be branched known as a branched polysaccharide.
Characteristics of Polysaccharides
Polysaccharides have the following properties:
1. They are not sweet in taste.
2. Many are insoluble in water.
3. They are hydrophobic in nature.
4. They do not form crystals on desiccation.
5. Can be extracted to form a white powder.
6. They are high molecular weight carbohydrates.
7. Inside the cells, they are compact and osmotically inactive.
8. They consist of hydrogen, carbon, and oxygen. The hydrogen to oxygen ratio being 2:1.
Types of Polysaccharides
Polysaccharides are categorized into two types:
29
 • Homopolysaccharides.
• Heteropolysaccharides.
Homopolysaccharides
A polysaccharide that contains the same type of monosaccharides is known as a
homopolysaccharide. Some of the important homopolysaccharides are:
1. Glycogen: It is made up of a large chain of molecules. It is found in animals and fungi.
2. Cellulose: The cell wall of the plants is made up of cellulose. It comprises long chains
of ꞵ-glycosides.
3. Starch: It is formed by the condensation of amylose and amylopectin. It is found largely
in plants, fruits, seeds, etc.
4. Inulin: It is made up of a number of fructofuranose molecules linked together in chains.
It is found in the tubers of dahlia, artichoke, etc.
Heteropolysaccharides
A polysaccharide that contains different types of monosaccharides is known as a
heteropolysaccharide. Some of the important heteropolysaccharides are:
1. Hyaluronic Acid: It is made up of D-glucuronic acid and N-acetyl-glucosamine. It is
found in connective tissues and skin.
2. Heparin: It is made up of D-glucuronic acid, L-iduronic acid, N-sulfo-D-glucosamine
and is largely distributed in mast cells and blood.
3. Chondroitin-4-sulfate: Its component sugars are D-glucuronic acid and N-acetyl-D-
galactosamine-4-O-sulfate. It is present in the cartilages.
4. Gamma globulin: N-acetyl-hexosamine, D-mannose, D-galactose are the component
sugars of this polysaccharide. It is found in the blood.
Functions of Polysaccharides
The polysaccharides serve as a structural organization in animals and plants. Other functions
of polysaccharides include:
1. They store energy in organisms.
2. Due to the presence of multiple hydrogen bonds, the water cannot invade the molecules
making them hydrophobic.
3. They allow for changes in the concentration gradient which influences the uptake of
nutrients and water by the cells.
4. Many polysaccharides become covalently bonded with lipids and proteins to form
glycolipids and glycoproteins. These glycolipids and glycoproteins are used to send
messages or signals between and within the cells.
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 5. They provide support to the cells. The cell wall of plants is made up of polysaccharide
cellulose, which provides support to the cell wall of the plant. In insects and fungi,
chitin plays an important role in providing support to the extracellular matrix around
the cells.
Sugar:
Sugar is the generic name for sweet-tasting, soluble carbohydrates, many of which are used in
food. Simple sugars, also called monosaccharides, include glucose, fructose, and galactose.
Compound sugars, also called disaccharides or double sugars, are molecules made of two
bonded monosaccharides; common examples are sucrose (glucose + fructose), lactose (glucose
+ galactose), and maltose (two molecules of glucose). White sugar is a refined form of sucrose.
In the body, compound sugars are hydrolysed into simple sugars.
Longer chains of monosaccharides (>2) are not regarded as sugars, and are called
oligosaccharides or polysaccharides. Starch is a glucose polymer found in plants, and is the
most abundant source of energy in human food. Some other chemical substances, such as
glycerol and sugar alcohols, may have a sweet taste, but are not classified as sugar.
Scientifically, sugar loosely refers to a number of carbohydrates, such as monosaccharides,
disaccharides, or oligosaccharides. Monosaccharides are also called "simple sugars", the most
important being glucose. Most monosaccharides have a formula that conforms to CnH2nO
n with n between 3 and 7 (deoxyribose being an exception). Glucose has the molecular formula
C6H12O6. The names of typical sugars end with -ose, as in "glucose" and "fructose". Sometimes
such words may also refer to any types of carbohydrates soluble in water. The acyclic mono-
and disaccharides contain either aldehyde groups or ketone groups. These carbon-oxygen
double bonds (C=O) are the reactive centres. All saccharides with more than one ring in their
structure result from two or more monosaccharides joined by glycosidic bonds with the
resultant loss of a molecule of water (H2O) per bond.
Monosaccharides in a closed-chain form can form glycosidic bonds with other
monosaccharides, creating disaccharides (such as sucrose) and polysaccharides (such as starch
or cellulose). Enzymes must hydrolyse or otherwise break these glycosidic bonds before such
compounds become metabolized. After digestion and absorption, the principal
monosaccharides present in the blood and internal tissues include glucose, fructose, and
galactose. Many pentoses and hexoses can form ring structures. In these closed-chain forms,
the aldehyde or ketone group remains non-free, so many of the reactions typical of these groups
cannot occur. Glucose in solution exists mostly in the ring form at equilibrium, with less than
0.1% of the molecules in the open-chain form.
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Proteins: Proteins are polypeptides. They are linear chains of amino acids linked by peptide
bonds.
Each protein is a polymer of amino acids. As there are 20 types of amino acids (e.g., alanine,
cysteine, proline, tryptophan, lysine, etc.), a protein is a heteropolymer and not a homopolymer.
A homopolymer has only one type of monomer repeating ‘n’ number of times. This information
about the amino acid content is important as later in your nutrition lessons, you will learn that
certain amino acids are essential for our health and they have to be supplied through our diet.
Hence, dietary proteins are the source of essential amino acids. Therefore, amino acids can be
essential or non-essential. The latter are those which our body can make, while we get essential
amino acids through our diet/food. Proteins carry out many functions in living organisms, some
transport nutrients across cell membrane, some fight infectious organisms, some are hormones,
some are enzymes. Collagen is the most abundant protein in animal world and Ribulose
bisphosphate Carboxylase-Oxygenase (RuBisCO) is the most abundant protein in the whole of
the biosphere. Some Proteins and their Functions are follows:
SN Protein Functions
1 Collagen Intercellular ground substance
2 Trypsin Enzyme
3 Insulin Hormone
4 Antibody Fights infectious agents
5 Receptor Sensory reception (smell, taste, hormone, etc.)
6 GLUT-4 Enables glucose transport into cells

Proteins are complex organic compounds. The basic structure of protein is a chain of amino
acids. They provide energy for the body.
Protein is an important component of every cell in the body. Hair and nails are mostly made of
protein. Your body uses protein to build and repair tissues. You also use protein to make
enzymes, hormones, and other body chemicals. Protein is an important building block of bones,
muscles, cartilage, skin, and blood.
Along with fat and carbohydrates, protein is a “macronutrient,” meaning that the body needs
relatively large amounts of it. Vitamins and minerals, which are needed in only small quantities,
are called “micronutrients.” But unlike fat and carbohydrates, the body does not store protein,
and therefore has no reservoir to draw on when it needs a new supply.

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Types of protein
Proteins are made up of amino acids. Some of these amino acids can’t be made by our bodies,
so these are known as essential amino acids. It’s essential that our diet provide these. In the
diet, protein sources are labelled according to how many of the essential amino acids they
provide:
➢ A complete protein source is one that provides all of the essential amino acids. You
may also hear these sources called high quality proteins. Animal-based foods; for
example, meat, poultry, fish, milk, eggs, and cheese are considered complete protein
sources.
➢ An incomplete protein source is one that is low in one or more of the essential amino
acids. Complementary proteins are two or more incomplete protein sources that
together provide adequate amounts of all the essential amino acids.
➢ Every function in the living cell depends on proteins.
➢ Motion and locomotion of cells and organisms depends on contractile proteins.
[Examples: Muscles]
➢ The catalysis of all biochemical reactions is done by enzymes, which contain protein.
➢ The structure of cells, and the extracellular matrix in which they are embedded, is
largely made of protein. [Examples: Collagens] (Plants and many microbes depend
more on carbohydrates, e.g., cellulose, for support, but these are synthesized by
enzymes.)
➢ The transport of materials in body fluids depends of proteins. [Blood]
➢ The receptors for hormones and other signalling molecules are proteins.
List of High-Protein Foods and Amount of Protein in Each
Beef
➢ Hamburger patty, 4 oz – 28 grams protein
➢ Steak, 6 oz – 42 grams
➢ Most cuts of beef – 7 grams of protein per ounce
Chicken
➢ Chicken breast, 3.5 oz - 30 grams protein
➢ Chicken thigh – 10 grams (for average size)
➢ Drumstick – 11 grams
➢ Wing – 6 grams
➢ Chicken meat, cooked, 4 oz – 35 grams

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Fish
➢ Most fish fillets or steaks are about 22 grams of protein for 3 ½ oz (100 grams) of
cooked fish, or 6 grams per ounce
➢ Tuna, 6 oz can - 40 grams of protein
Pork
➢ Pork chop, average - 22 grams protein
➢ Pork loin or tenderloin, 4 oz – 29 grams
➢ Ham, 3 oz serving – 19 grams
➢ Ground pork, 1 oz raw – 5 grams; 3 oz cooked – 22 grams
➢ Bacon, 1 slice – 3 grams
➢ Canadian-style bacon (back bacon), slice – 5 – 6 grams
Eggs and Dairy
➢ Egg, large - 6 grams protein
➢ Milk, 1 cup - 8 grams
➢ Cottage cheese, ½ cup - 15 grams
➢ Yogurt, 1 cup – usually 8-12 grams, check label
➢ Soft cheeses (Mozzarella, Brie, Camembert) – 6 grams per oz
➢ Medium cheeses (Cheddar, Swiss) – 7 or 8 grams per oz
➢ Hard cheeses (Parmesan) – 10 grams per oz
Beans (including soy)
➢ Tofu, ½ cup 20 grams protein
➢ Tofu, 1 oz, 2.3 grams
➢ Soy milk, 1 cup - 6 -10 grams
➢ Most beans (black, pinto, lentils, etc) about 7-10 grams protein per half cup of cooked
beans
➢ Soy beans, ½ cup cooked – 14 grams protein
➢ Split peas, ½ cup cooked – 8 grams
Nuts and Seeds
➢ Peanut butter, 2 Tablespoons - 8 grams protein
➢ Almonds, ¼ cup – 8 grams
➢ Peanuts, ¼ cup – 9 grams
➢ Cashews, ¼ cup – 5 grams
➢ Pecans, ¼ cup – 2.5 grams
➢ Sunflower seeds, ¼ cup – 6 grams
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 ➢ Pumpkin seeds, ¼ cup – 8 grams
➢ Flax seeds – ¼ cup – 8 grams
Recommendations
It’s recommended that 10–35% of your daily calories come from protein. Below is the
Recommended Dietary Allowances (RDA) for different age groups. Recommended Dietary
Allowance for Protein.
SN Age groups Grams of protein needed each day
1 Children age 1 – 3 13
2 Children age 4 – 8 19
3 Children age 9 – 13 34
4 Girl’s age 14 – 18 46
5 Boys age 14 – 18 52
6 Women ages 19 – 70+ 46
7 Men age 19 – 70+ 56

Sports Nutrition - Protein

Athletes need protein primarily to repair and rebuild muscle that is broken down during
exercise and to help optimizes carbohydrate storage in the form of glycogen. Protein isn’t an
ideal source of fuel for exercise, but can be used when the diet lacks adequate carbohydrate.
This is detrimental, though, because if used for fuel, there isn’t enough available to repair and
rebuild body tissues, including muscle.
Recommended Daly Protein Intake
➢ The average adult needs 0.8 grams per kilogram (2.2lbs) of body weight per day.
➢ Strength training athletes need about 1.4 to 1.8 grams per kilogram (2.2lbs) of body
weight per day
➢ Endurance athletes need about 1.2 to 1.4 grams per kilogram (2.2lbs) of body weight
per day.
Structure of Proteins
Proteins, as mentioned earlier, are heteropolymers containing strings of amino acids. Structure
of molecules means different things in different contexts. In inorganic chemistry, the structure
invariably refers to the molecular formulae (e.g., NaCl, MgCl2, etc.). Organic chemists always
write a two-dimensional view of the molecules while representing the structure of the
molecules (e.g., benzene, naphthalene, etc.). Physicists conjure up the three-dimensional views

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of molecular structures while biologists describe the protein structure at four levels. The
sequence of amino acids i.e., the positional information in a protein – which is the first amino
acid, which is second, and so on – is called the primary structure of a protein. A protein is
imagined as a line, the left end represented by the first amino acid and the right end represented
by the last amino acid. The first amino acid is also called as N-terminal amino acid. The last
amino acid is called the C-terminal amino acid. A protein thread does not exist throughout as
an extended rigid rod. The thread is folded in the form of a helix (similar to a revolving
staircase). Of course, only some portions of the protein thread are arranged in the form of a
helix. In proteins, only right-handed helices are observed. Other regions of the protein thread
are folded into other forms in what is called the secondary structure. In addition, the long
protein chain is also folded upon itself like a hollow woollen ball, giving rise to the tertiary
structure. This gives us a 3-dimensional view of a protein. Tertiary structure is absolutely
necessary for the many biological activities of proteins. Some proteins are an assembly of more
than one polypeptide or subunits. The manner in which these individual folded polypeptides
or subunits are arranged with respect to each other (e.g. linear string of spheres, spheres
arranged one upon each other in the form of a cube or plate etc.) is the architecture of a protein
otherwise called the quaternary structure of a protein. Adult human haemoglobin consists of 4
subunits. Two of these are identical to each other. Hence, two subunits of a type and two
subunits of b type together constitute the human haemoglobin (Hb).

Various levels of Protein Structure

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Amino acid:
An amino acid contains both a carboxylic group and an amino group. Amino acids that have
an amino group bonded directly to the alpha-carbon are referred to as alpha amino acids. Every
alpha amino acid has a carbon atom, called an alpha carbon, Cα; bonded to a carboxylic acid,
–COOH group; an amino, –NH2 group; a hydrogen atom; and an R group that is unique for
every amino acid.

Classification of amino acids

There are 20 amino acids. Based on the nature of their ‘R’ group, they are classified based on
their polarity as:

Classification based on essentiality: Essential amino acids are the amino acids which you
need through your diet because your body cannot make them. Whereas non-essential amino
acids are the amino acids which are not an essential part of your diet because they can be
synthesized by your body.

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Peptide bonds
Amino acids are linked together by ‘amide groups’ called peptide bonds. During protein
synthesis, the carboxyl group of amino acid at the end of the growing polypeptide chain
reacts with the amino group of an incoming amino acid, releasing a molecule of water. The
resulting bond between the amino acids is a peptide bond.

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