BIOTECHNOLOGY

Subject Code- FECH 6302

DEPARTMENT OF CHEMICAL ENGINEERING

6th Semester

Mrs Ipsita D. Behera

Asst. professor
I.G.I.T. Sarang
Mail id:[email protected]
 Disclaimer

This document does not claim any originality and cannot be used as a substitute
for prescribed textbooks. The information presented here is merely a collection
by the committee faculty members for their respective teaching assignments as
an additional tool for the teaching-learning process. Various sources as
mentioned at the reference of the document as well as freely available material
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 SYLLABUS
Module - I
Introduction and application of microbiology, Structure and functioning of
bacterial cell, Classification and Identification criteria for bacteria.Nutritional
requirements and nutritional types of bacteria.

Module – II
Isolation of microorganisms, pure culture techniques and cultural
characteristics. Bacterial growth, measurements and reproduction.

Module - III
Fundamentals of microbiology ecology and ecosystems, microbial associations
and interactions.

Module - IV
Types of bacteria in water, sanitary examination of water, water purification,
average disposal and sewage purification.

Recommended Books:
1. Reid, P., Microbiology, TMH.
2. Atlas, R. M. &Bartha, R., Microbiology Ecology - Fundamentals and
Applications.
 LESSON PLAN
Class No.
Brief description of the Topic/Chapter to be taught
1 Introduction of Microbiology

2 Application of Microbiology

3 Application of Microbiology

4 Structure of Bacterial cell

5 Functioning of Bacterial cell

6 Nutritional requirements of bacteria

7 Nutritional types of bacteria

8 Isolation of micro organisms

9 Isolation of pure culture techniques

10 Cultural characteristics

11 Cultural characteristics

12 Bacterial growth

13 Bacterial Reproduction

14
Bacterial Reproduction
15 Bacterial Measurements
16 Bacterial Measurements
17 Fundamentals of ecology

18 Fundamentals of ecology

19 Microbial interaction
20 Microbial interaction

21 Microbial association

22 Microbial association

23 Types of bacteria in water

24 Types of bacteria in water

25 Sanitary examination of water

26 Sanitary examination of water

27 Water purification

28 Water purification

29 Average disposal

30 Sewage purification

31 Problem discussion

32 Semester question discussion

 BIOTECHNOLOGY 1

Module-1

INTRODUCTION

Plant, animal and microbes have been used by humans for nutrition and
development of products for consumption such as beer or bread. Understanding
of Physical phenomenon has allowed the invention of different types of
electronic gadgets, machines, devices and altogether these have been used to
increase the efficiency of human activities. Technological advancement has also
allowed him to exploit plant, animal and microbial wealth to provide products
of commercial or pharmaceutical importance. All these activities (research and
development) fall under the big umbrella of biotechnology. In simpler word,
Biotechnology is the summation of activities involving technological tools and
living organism in such a way that it will enhance the efficiency of the
production. The ultimate goal of this field is to improve the product yield from
living organism either by employing principles of bio-engineering/bio-process
technology or by genetically modifying the organisms. For example, production
of bread or other bakery items from wheat flour after adding yeast as fermenting
organism (Figure 1.1). In India, from ancient times wheat flour has been used to
prepare “Roti” but yeast has been added to the wheat flour to make it porous by
CO2 generation during fermentation. Since then this process has been very
popular in bakery industry and is responsible for preparation of bread, cakes,
pizza, etc.

Needs of Biotechnology-The population of India is more than 1 billion

and as per projection it may cross 1.5 billion by 2030. This will bring huge
burden on biological resources (animal/plant) to provide food for all. Naturally
occurring animal, plant or microbial strains have few limitations for them to be
utilized for desired products due to following reasons-
1. Purity of the living stock
 BIOTECHNOLOGY 2

2. Production of undesired products

3. Secretion of toxic metabolic by-products
4. Inability to withstand harsh biochemical processes/treatments.
5. Higher production cost
6. Susceptible to disease and other environmental conditions

The existing technology today enables us to engineer plants and animals

making them suitable for maximum production. Living organism has a complex
cellular structure, metabolic pathways, genetic make-up, and behaviour in the
synthetic growth media and understanding these processes can help us to
modulate specific process/environmental condition or metabolic pathways to
achieve the goal of biotechnology. Advancement in different fields of science
has paved ways to solve several issues responsible for lower yield of products.
Few of the selected science research areas contributing into the development of
biotechnology are given in the Figure 1.2. The foundation of biotechnology
relies on the research & development activities in different areas of science and
interaction of interdisciplinary areas. The research in the field of plant
biotechnology allowed us to produce plants through micro-propagation but with
the evident advancement of genetic engineering, it is now possible to produce
plant with predefined characteristics imprinted at genetic level through genetic
engineering. The similar relationship may also exist for many other overlapping
areas and as a result biotechnological operation output is amplified several
folds.
 BIOTECHNOLOGY 3

Applications of Biotechnology

Biotechnology has influenced human life in many ways by inventions to

make his life more comfortable. Many scientific fields contribute to
biotechnology and in return it gives product for their advancement. Few of the
biotechnology applications are given in Figure 1.3.

Microbiology

(Micro-small, Bios-life)

Things aren’t always the way they seem. On the face of it, ‘microbiology’
should be an easy word to define: the science (logos) of small (micro) life
(bios), or to put it another way, the study of living things so small that they
cannot be seen with the naked eye. Bacteria neatly fit this definition, but what
about fungi and algae? These two groups each contain members that are far
from microscopic. On the other hand, certain animals, such as nematode worms,
can be microscopic, yet are not considered to be the domain of the
microbiologist. Viruses represent another special case; they are most certainly
microscopic (indeed, most are sub microscopic), but by most accepted
definitions they are not living. Nevertheless, these too fall within the remit of
the microbiologist.
 BIOTECHNOLOGY 4

Microbiology is the study of microscopic organisms which are defined as

any living organisms which are either a single cell (unicellular) or no cell at all
(a cellular) and this includes eukaryotes such as fungi, protista and study of
prokaryotes. Microbiology is a board terms virology, mycology,
parapsychology, bacteriology other branches. It is the study of microorganisms
which are not only microscopic an exists as single cell but also ultramicroscopic
organisms which are not only cellular and cannot exists independently like
virus. Microbiology deals with the study and functioning of cells, their
diversity, and evolution their interaction with environments and with other
organisms and also with the human beings.

Why is microbiology important?

To the lay person, microbiology means the study of sinister, invisible ‘bugs’
that cause disease. As a subject, it generally only impinges on the popular
consciousness in news coverage of the latest ‘health scare’. It may come as
something of a surprise therefore to learn that the vast majority of
microorganisms coexist alongside us without causing any harm. Indeed, many
perform vital tasks such as the recycling of essential elements, without which
life on our planet could not continue Other microorganisms have been exploited
by humans for our own benefit, for instance in the manufacture of antibiotics
and foodstuffs. To get some idea of the importance of microbiology in the world
today, just consider the following list of some of the general areas in which the
expertise of a microbiologist might be used:
 medicine
 environmental science
 food and drink production
 fundamental research
 agriculture
 pharmaceutical industry
 Genetic engineering.

So microbiology has involved as an important branch of science which is the

study with respect to two measure aspects

-Basic biological science

-Applied biological science

 BIOTECHNOLOGY 5

Basic biological science it provides systems to understand of life nature of life

process, principles behind it and generation is involved in the inheritance of
traits to next generation.

Various branches

 medical microbiology
 environmental microbiology
 agricultural microbiology
 food & diary microbiology

An applied biological science, it deals with study of useful microorganisms as

well as that of pathogenic organisms.

Classification

 Classification means orderly arrangements of units under study into

groups of larger units.
 Until 18th century ,the classification of living organisms divided into two
kingdoms
Animal kingdom
Plant kingdom

After this some organisms are there which predominantly plant likes and
animal likes. But some organisms share the characteristics of both plant and
animal. Since there are organisms that do not fall naturally into either plant or
animal kingdom. It was purposed that a new kingdom to be established to
include those organism which typically neither plant or animal.

Haeckel’s three kingdom of classification

In 1866 German Zoologist E H Haeckel suggested that a third kingdom

protista to be formed to include those unicellular organisms that are typically
neither plant nor animal. The organism protista included bacteria, algae &
protista. So again it can be divided into lower &higher protista.
 BIOTECHNOLOGY 6

R H Whittaker’s five kingdom classification

In 1969 R H Whittaker proposed that there are three levels of cellular

organisms based on three principles mode of nutrition

1. Photosynthesis
2. Absorption
3. Ingestion

 The prokaryotes are included in kingdom monera and they are lack of
injective mode of nutrition. Then unicellular eukaryotes are placed in
kingdom protista. The mode of nutrition of protozoa is injective and the
mode of nutrition of some protista are absorptive & also some micro
algae shows photosynthetic type of nutrition, so all are come under
protista.
 With some overlap to photosynthetic and injective mode the multicellular
and multinucleated eukaryotes are formed in kingdom plantae (green
plant & higher algae).
 The kingdom animalia includes multicellular animals.
 The fungi include multicellular higher fungi.
 BIOTECHNOLOGY 7

 So, in this type of classification microorganisms are found in three and

five kingdom classification in monera, protista, fungi.

Bergey’s manual of systematic bacteriology

He placed all the kingdom prokaryotes which again divided into four divisions

1. Gracillicutes-These bacteria having cell wall structure characteristics of gram

positive.

2. Firmicutes-These bacteria having cell wall structure characteristics of gram

negative.

3. Tenericutes-These bacteria having no cell wall.

4. Mendosicutes-These bacteria having the both characteristics of

gracillicutes&firmicutes.
 BIOTECHNOLOGY 8

Application of microbiology

 Microbes in food and dairy industries

 Microbes in production of industrial products
 Microbes in genetic engineering and biotechnology
 Microbes in environmental microbiology
 Microbes in medical microbiology
 Microbes in agriculture
 Microbes in bioterrorism

a. Food and dairy industry:

 Provides food due to high protein content.

 Food nutrient for their growth (deterioration of food), enzymatic changes,
contributing flavor.
 Certain molds used for manufacturing of food and ingredients of food.
 Some molds used in production of oriental food, soya sauce etc.
 Used for enzyme making like amylase.
 Yeast: These are used in manufacturing of foods such as bread, beer,
wines, vinegar, surface ripened, and cheese. Some yeasts are grown for
enzymes and food.
 Bacteria: Some pigmented bacteria cause changes in colour on the
surfaces of liquid food. Acetobacteria oxidises ethyl alcohol to acetic
acid. Some bacteria cause ropiness in milk and slimy growth cottage
cheese.

b. Microbes in production of industrial products:

 Enzymes amino acid, vitamins, antibiotics, organic acid and alcohol are
commercially produced by microorganism.
 Primary microbial product: These products are used by microorganism
for their growth. E.g. amino acids, enzymes, vitamins
 Secondary microbial products: Not used by cell for their growth. E.g.
alcohol, antibiotics, organics acids.
 Microbes produce some important amino acids such as glomatic acid,
lysine, and methionine.
 BIOTECHNOLOGY 9

c. Microbes in Genetic Engineering and Biotechnology:

 Microbes used for mammalian proteins such as insulin and human growth
factor.
 Making vaccine for microbial and viral genes and induce a new strain of
microbes.
 Vaccine and diagnostic kits also depend on the improved strains of
microorganism.
 Lactic acid as food preservative.
 Acetic acid plays a major role in tanning and textile industry.
 Interferons are produced in animal cell if included by viral infection.
These are used in testing interleukins (which stimulate T-lymphocytes).
 Production of viral, bacterial or protozoan, antigen for protecting against
dysentery, typhoid, bacteria etc.
 N2 fixing bacteria reduce nitrogen gas to ammonia required plant growth.

d. Environmental Microbiology:

 Plays an important role in recycling of biological elements such as

oxygen, carbon, N2, Sulphur, and phosphorous.
 Microbes in biochemical cycle: Photosynthesis and chemosynthesis
microorganism convert CO2 into organic carbon. Methane is generated
anaerobically from CO2 and H2 by metheogenicarchaea. The organic
forms of N2 are interconverted by metabolic activities of microorganism
which maintain the natural N2 balance.
 Microbes in pollution microbiology: Biological sewage treatment and
self-purification. Both results in mineralization of organic, pollutants and
in utilization of dissolved oxygen.

e. Medical Microbiology:

 Vaccination: Small pox, diphtheria, whooping cough.

f. Computer application:

 Optimization via computer: Computers are used on scale of, to store,

evaluate effects of individual parameters on metabolic behaviour of
culture.
 Control via computer: Control fermentation process.
 BIOTECHNOLOGY 10

g. Microbes in agriculture:

 During compost formation by the crop residue like wheat straw, rice
straw, sugar cane bagasses are very difficult to degrade due to presence of
highly resistant lignified tissues. So, breakdown of these complex organic
materials can be done by microbes by a short span.
 Biogas production through anaerobic fermentation is must reverent to
fulfil their energy demand in rural population.
 The productivity of leguminous crop largely depend on efficient and
suitable management of the ecosystem by specific rhizobial association.
 Some bacterial helps in killing a wide range of insects like beetles,
mosquitoes, flies, aunts, termites which is very useful for agricultural
industries

h. Microbes in Bioterrorism:
It has been defined as deliberate release of disease causing germs,
microorganism with the intent of killing large number of people.
Accordingly microorganisms are used as weapons of mass destruction of
people and causes small pox, plague, cholera and also anthrax.

Mode of transmission:

 Air droplets and dust

 Food fruits and vegetables
 Drinking water

STRUCTURE AND FUNCTIONS OF BACTERIA CELL

Based on the cellular organization, the entire cells can be divided into two
major categories. One is called the prokaryotic cell; another is called the
eukaryotic cell. Prokaryotic cell, Pro means primitive; karyon means nucleus;
that means, obviously, this group of microorganisms are very primitive in
nature and obviously, bacteria is one of such example.

Eukaryotes are the developed cell, where this nucleus has got its own
nucleus membrane, which is not there in case of the prokaryotic organism.
 BIOTECHNOLOGY 11

Now, the overview of this prokaryotic cell, then, this prokaryotic cell is the
earliest organism in this universe, and evolved alone for 1.5 billion years.
Prokaryotes can be found in, everywhere, in every niche available on this
planet. Based on its structure, based on its different cellular organization, or
structure, the prokaryotes can be divided into, can be divided into two major
groups. One is called, one is called the archebacteria; another is the eubacteria.
Archebacteria is considered to be the most primitive type of bacteria, which is
otherwise known as the extremophile. Eubacteria is the true bacteria.
The various structure of bacterial cell differ from one another not only in
their physical features but also in their chemical characteristics and in their
functions.

THE SIZE AND SHAPE OFBACTERIAL CELLS

Bacteria display a wide diversity of shapes and sizes, called morphologies.

Bacterial cells are about one-tenth the size of eukaryotic cells and are typically
0.5- 1 micro meter in diameter.
 Most bacterial species are either spherical, called cocci (sing. coccus),
 Elongation is associated with swimming and rod-shaped,
called bacilli (sing.bacillus),
 Some bacteria, called vibrio, are shaped like slightly curved rods or
comma-shaped;
 others can be spiral-shaped, called spirilla,
 Tightly coiled, called spirochaetes.
 A small number of species even have tetrahedral or cuboidal shapes.
 More recently, bacteria were discovered deep under Earth's crust that
grow as branching filamentous types with a star-shaped cross-section.

The large surface area to volume ratio of this morphology may give these
bacteria an advantage in nutrient-poor environments. This wide variety of
shapes is determined by the bacterial cell wall and cytoskeleton, and is
important because it can influence the ability of bacteria to acquire nutrients,
attach to surfaces, swim through liquids and escape predators.
 BIOTECHNOLOGY 12

STRUCTURE

Bacteria, despite their simplicity, contain a well-developed cell structure

which is responsible for many of their unique biological structures. Many
structural features are unique to bacteria and are not found
among archaea or eukaryotes. Because of the simplicity of bacteria relative to
larger organisms and the ease with which they can be manipulated
experimentally, the cell structure of bacteria has been well studied, revealing
many biochemical principles that have been subsequently applied to other
organism.

Examination of bacterial cell reveals various component structures. Some

of these are external to the cell wall and others are internal to the cell wall.
BIOTECHNOLOGY 13
 BIOTECHNOLOGY 14

STRUCTURES EXTERNAL TO THE CELL WALL

 Flagella
 Pilli(Fimbriae)
 Capsules
 Sheaths
 Prosthecae and Stalks

Flagella
Perhaps the most recognizable extracellular bacterial cell structures
are flagella. Flagella are whip-like structures protruding from the bacterial cell
wall and are responsible for bacterial motility (i.e. movement). The arrangement
of flagella about the bacterial cell is unique to the species observed. Common
forms include:

 Monotrichous - Single flagellum

 Lophotrichous - A tuft of flagella found at one of the cell pole
 Amphitrichous - Single flagellum found at each of two opposite poles
 Peritrichous - Multiple flagella found at several locations about the cell

The bacterial flagellum consists of three basic components:

 a whip-like filament,
 a motor complex, and
 a hook that connects them

Flagella
 BIOTECHNOLOGY 15

The filament is approximately 20 nm in diameter and consists of several

protofilaments, each made up of thousands of flagellin subunits. The bundle is
held together by a cap and may or may not be encapsulated. The motor complex
consists of a series of rings anchoring the flagellum in the inner and outer
membranes, followed by a proton-driven motor that drives rotational movement
in the filament.

A-Monotrichous; B-Lophotrichous; C-Amphitrichous; D-Peritrichous;

Pilli (Fimbriae)

Fimbriae are protein tubes that extend out from the outer membrane in
many members of the Proteobacteria. They are generally short in length and
present in high numbers about the entire bacterial cell surface. Fimbriae usually
function to facilitate the attachment of a bacterium to a surface or to other cells
(e.g. animal cells during pathogenesis). A few organisms (e.g. Myxococcus) use
fimbriae for motility to facilitate the assembly of multicellular structures such as
fruiting. Pili are similar in structure to fimbriae but are much longer and present
on the bacterial cell in low numbers. Pili are involved in the process
of conjugation where they are called conjugation pili or "sex pili".

Capsule
Some bacteria cells are surrounded by a viscous substance forming a
covering layer or envelope around the cell wall. If the layer is visualized by
light microscopy using special staining methods it is termed a capsule. If the
layer is too thin to be seen in light microscopy it is termed as microcapsule.
The cell capsule is a very large structure of some prokaryotic cells, such as
bacterial cells. It is a polysaccharide layer that lies outside the cell
 BIOTECHNOLOGY 16

envelope of bacteria, and is thus deemed part of the outer envelope of a

bacterial cell.
Composition
It usually consists of polysaccharides,]but can be composed of other
materials (e.g., polypeptide(D-glutamic acid) in B. anthracis). Because most
capsules are so tightly packed, they are difficult to stain using standard stains
because most stains cannot adhere to the capsule. For examination under the
microscope, the bacteria and their background are stained darker than the
capsule, which doesn't stain. When viewed, bacterial cells as well as the surface
they are on, are stained dark, while the capsule remains pale or colorless and
appears as a ring, or halo, around the cell.
Function
 The capsule is considered a virulence factor because it enhances the
ability of bacteria to cause disease (e.g. prevents phagocytosis).
 The capsule can protect cells from engulfment by eukaryotic cells,
such as macrophages.
 A capsule-specific antibody may be required for phagocytosis to
occur.
 Capsules also contain water which protects the bacteria
against desiccation.
 They also exclude bacterial viruses and most hydrophobic toxic
materials such as detergents.
 There are 14 different capsule types, which each impart their own
specific antigenicity.
 Immunity to one capsule type does not result in immunity to the
other types. Capsules also help cells adhere to surface.
Sheaths
Some species of bacteria, particularly those from freshwater and marine
environment, form chains or trichomes that are enclosed by a hollow tube called
seath. This structure is more readily migrated from it.

Prosthecae and stalks

Prosthecae are semirigid extensions of the cell wall and cytoplasmic

membrane and have a diameter that is always less than that of the cell. These
cellular appendages are neither pili nor flagella, as they are extensions of
the cellular membrane and contain cytosol. Prosthecae increase the surface area
 BIOTECHNOLOGY 17

of the cell for nutrient absorption, which is advantageous in dilute environment

medium. Some prosthecae bacteria may form a new cell (bud) at the end of the
prosthecae.

Stalk is anon-living ribbon like or tubular appendages that are excreted by

the cell. This stalk aid in attachment of the cell to surface.

Cell Wall
The cell envelope is composed of the plasma membrane and cell wall. As
in other organisms, the bacterial cell wall provides structural integrity to the
cell. In prokaryotes, the primary function of the cell wall is to protect the cell
from internal turgor pressure caused by the much higher concentrations of
proteins and other molecules inside the cell compared to its external
environment. The bacterial cell wall differs from that of all other organisms by
the presence of peptidoglycan which is located immediately outside of
the cytoplasmic membrane. Peptidoglycan is made up of a polysaccharide
backbone consisting of alternating N-Acetylmuramic acid (NAM) and N-
acetylglucosamine (NAG) residues in equal amounts. Peptidoglycan is
responsible for the rigidity of the bacterial cell wall and for the determination of
cell shape. It is relatively porous and is not considered to be a permeability
barrier for small substrates. While all bacterial cell walls (with a few exceptions
e.g. extracellular parasites such as Mycoplasma) contain peptidoglycan, not all
cell walls have the same overall structures. Since the cell wall is required for
bacterial survival, but is absent in eukaryotes, several antibiotics (notably
the penicillins and cephalosporins) stop bacterial infections by interfering with
cell wall synthesis, while having no effects on human cells which have no cell
wall only a cell membrane. There are two main types of bacterial cell walls,
those of gram-positive bacteria and those of gram-negative bacteria, which are
differentiated by their Gram staining characteristics. For both these types of
bacteria, particles of approximately 2 nm can pass through the peptidoglycan. If
the bacterial cell wall is entirely removed, it is called a protoplast while if it's
partially removed, it is called a spheroplast. β-Lactam antibiotics such as
penicillin inhibit the formation of peptidoglycan cross-links in the bacterial cell
wall. The enzymelysozyme, found in human tears, also digests the cell wall of
bacteria and is the body's main defence against eye infection.

The Gram-positive cell wall

Gram-positive cell walls are thick and the peptidoglycan ( also known
as murein) layer constitutes almost 95% of the cell wall in some gram-positive
bacteria and as little as 5-10% of the cell wall in gram-negative bacteria. The
cell wall of some gram-positive bacteria can be completely dissolved
 BIOTECHNOLOGY 18

by lysozyme, as this enzyme attacks the bonds between GA and MA. In other
gram-positive bacteria, such as Staphylococcus aureus, the walls are resistant to
the action of lysozyme. They have O-acetyl groups on carbon-6 of some MA
residues. The matrix substances in the walls of gram-positive bacteria may be
polysaccharides or teichoic acids. The latter are very widespread, but have been
found only in gram-positive bacteria. There are two main types of teichoic acid:
ribitolteichoic acids and glycerol teichoic acids. The latter one is more
widespread. These acids are polymers ofribitol phosphate and glycerol
phosphate, respectively, and only located on the surface of many gram-positive
bacteria. However, the exact function of teichoic acid is debated and not fully
understood. A major component of the gram-positive cell wall is lipoteichoic
acid. One of its purposes is providing an antigenic function. The lipid element is
to be found in the membrane where its adhesive properties assist in its
anchoring to the membrane.
The gram-negative cell wall
Gram-negative cell walls are thin and unlike the gram-positive cell walls,
they contain a thin peptidoglycan layer adjacent to the cytoplasmic membrane.
The chemical structure of the outer membrane's lipopolysaccharides is often
unique to specific bacterial sub-species and is responsible for many of
the antigenic properties of these strains. Lipopolysaccharides, also
called endotoxins, are composed of polysaccharides and lipid A which are
responsible for much of the toxicity of gram-negative bacteria. It consists of
characteristic lipopolysaccarides embedded in the membrane.
 BIOTECHNOLOGY 19

STRUCTURE INTERNAL TO THE CELL WALL

 The cytoplasmic membrane
 Protoplast
 Speroplast
 The cytoplasma
 Gas vacuoles
 Nuclear material spore and cysts

The cytoplasmic membrane

The plasma membrane or bacterial cytoplasmic membrane is composed of

a phospholipid bilayer and thus has all of the general functions of a cell
membrane such as acting as a permeability barrier for most molecules and
serving as the location for the transport of molecules into the cell. In addition to
these functions, prokaryotic membranes also function in energy conservation as
the location about which a proton motive force is generated. Unlike eukaryotes,
bacterial membranes (with some exceptions
e.g. Mycoplasmaand methanotrophs) generally do not contain sterols. However,
many microbes do contain structurally related compounds
called hopanoids which likely fulfill the same function.
Unlike eukaryotes, bacteria can have a wide variety of fatty acids within their
membranes. Along with typical saturated and unsaturated fatty acids, bacteria
can contain fatty acids with additional methyl, hydroxy or even cyclic groups.
As a phospholipid bilayer, the lipid portion of the outer membrane is
impermeable to charged molecules. However, channels called porins are present
in the outer membrane that allow for passive transport of
many ions, sugars and amino acids across the outer membrane. These molecules
are therefore present in the periplasm, the region between the cytoplasmic and
outer membranes. The periplasm contains the peptidoglycan layer and many
proteins responsible for substrate binding or hydrolysis and reception of
extracellular signals. The periplasm is thought to exist in a gel-like state rather
than a liquid due to the high concentration of proteins and peptidoglycan found
within it. Because of its location between the cytoplasmic and outer membranes,
signals received and substrates bound are available to be transported across
the cytoplasmic membrane using transport and signalling proteins imbedded
there.

Protoplast
It consists of cytoplasmic membrane and cell material bounded by it.
It prevents the cell from brusting in isotonic medium.
These are soft, fragile and spherical; regardless the original shape of the
bacteria.
 BIOTECHNOLOGY 20

Speroplast

These are round, somatically fragile forms of Gram-negative bacteria.In case of

gram-negative bacteria it possess an outer membrane.

The Cytoplasma

The cell material bounded by the cytoplasmic membrane & it divided into
 Ribosomes
 Chromatic area
 Fluid part

Gas vacuoles
Gas vacuoles are membrane-bound, spindle-shaped vesicles, found in
some planktonic bacteria and Cyanobacteria, that provides buoyancy to these
cells by decreasing their overall cell density. Positive buoyancy is needed to
keep the cells in the upper reaches of the water column, so that they can
continue to perform photosynthesis. They are made up of a shell of protein that
has a highly hydrophobic inner surface, making it impermeable to water (and
stopping water vapour from condensing inside) but permeable to most gases.
Because the gas vesicle is a hollow cylinder, it is liable to collapse when the
surrounding pressure increases.

Classification and Identification criteria for bacteria

Classification is a mean of bringing order to the bewilding variety of

organisms in nature. Once we learn the characteristics of an organism we can
compare with other organisms to discover similarities of and differences. The
human kind tends to arrange similar things together in groups and to distinguish
these groups from another.
In order to identify and classify microorganism, we must first learn their
characteristics. It is not feasible to study the characteristics of a single
microorganism, because of its small size; therefore we study the characteristics
of a culture i.e a population of microorganism.
A culture consists of a single kind of microorganism (one living species)
.regardless of the number of individuals, in an environment free of other living
organisms is called a pure culture.
 BIOTECHNOLOGY 21

Major characteristics of microorganisms

The major characteristics of microorganisms fall into the following categories.

1. Morphological characteristics-
2. Chemical composition
3. Cultural characteristics
4. Metabolic characteristics
5. Antigenic characteristics
6. Genetics characteristics
7. Pathogenicity characteristics
8. Ecological characteristics

1. Morphological characteristics
 This focus on cell shape, size, structure, cell arrangement, occurrence of
special structures, staining reactions, motility and flagellar movement.
 Unlike other kind of microbial characteristics differentiation of
morphological feature requires study of cells of a pure culture. This can
be done for use of microscope which provides magnification of 1000
times of its original structure.

2. Chemical composition

 These undergo various constituent of cell.

 Microbial cell consists of wide variety of organic compound & when
these cells are broken & their component subjected to chemical analysis,
it shows chemical composition both quantitavely and qualitatively.

3. Cultural characteristics

 Many organisms can be grown in or on a cultural medium (a mixture of

nutrients used in laboratory to support growth and multiplication
microorganism.
 Others generally grow in inorganic compound or organic compounds
(amino acids, sugars, purines, and pyrimidine).
 BIOTECHNOLOGY 22

 In addition to nutrients, each kind of organisms also requires physical

condition for growth. For example:-some bacteria grow best at high
temperature cannot grow below 20℃.
 The gaseous atmosphere required for growth is also important, for
instance, some bacteria require oxygen for growth, oxygen is lethal to
others & they can grow only in its absence.

4. Metabolic characteristics

 The life process of microbial cells is complex integrated series chemical

reactions collectively referred to as metabolism.
 The various chemical reactions of organisms are catalyzed by proteins
called enzymes possessed by one kind of organisms.

5. Antigenic characteristics

 Certain chemical compounds of microbial cells are called as antigen.

 If microbe cells enter the animal body the animal response to their
antigen by forming specific blood serum proteins called antibodies.
 Antibodies are highly specific for antigen that induced their formation.

6. Genetic characteristics

 The DNA of each kind of microorganism has certain features that are
constant & characteristics for organism & useful for its classification.
 DNA base composition-DNA molecule is made up of base pairs
(guanine-cytosine & adenine-thymine).
 Sequence of nucleotide bases in DNA-Sequence is unique for each kind
of organisms and is most fundamental of all characteristics of organisms.

7. Pathogenic characteristics

 Few species of microorganisms cause diseases, certain microorganism are

pathogenic for animals or plants & some organisms cause diseases in
other microorganisms.
 For example:-Bacteria known as bdellovibrioscan infect and destroy
bacterial cells.
 BIOTECHNOLOGY 23

8. Ecological characteristics

 The habitat of organisms is important in characteristics that organisms.

 For example:-Microorganisms normally found in marine environments
generally differs from those in fresh water environments.

Some organisms are widely distributed in nature, but others may be restricted to
a particular environment

CLASSIFICATION:

In microbiology taxa are initially construct from strains. A strain is made of

microorganism coming under same group in a pure culture. The basic
taxonomic group (taxon) i.e. a collection of strains having similar
characteristics. Bacterial species consist of special strain i.e. called type strain.

Family: a group of similar genera

Order: a group of similar family

Class: a group of similar order

Division: a group of similar class

Kingdom: a group of similar divisions

GOAL OF CLASSIFICATION:

It has 2 special qualities.

1. Stability: during classification if there is frequent radical changes

occurred then it causes confusions. So the classification must be on
focusing the stable characteristics. So that it will give minor changes to the
classification if any new information will available.
2. Predictability: by knowing the characteristics of one member of a
taxonomic group, it should be possible to assume that other members of
same group have similar characteristics and also in future they have same
genetic characteristics.
 BIOTECHNOLOGY 24

GENERAL METHOUDS OF CLASSIFICATION:

1. Intuitive method:

For classification of microorganisms a microbiologist has to study the

characters thoroughly. The trouble of this method is that of one character is
important to one person then that particular character may not be important to
another person. So a different group is arises. But some cases such type
classifications are also useful.

2. Numerical taxonomy:

During classification of bacteria a microbiologist determine many characters

usually 100 to 200 for each strain study, giving each characters equal weight.
Then using a computer he calculates similarity for any 2 strain.

% similarity =

NS=no. of similar characters (+ve or -ve)

ND= no. of dissimilar characters

Those strain having high % of “S” to each other are placed into a group. Those
group having high %S to each other, then they are places in a large group.

3. Genetic relatedness:

 This method is the moist objective of all is based on most fundamental

accept of organism i.e. they are hereditary materials.
 It is seen that two organism of same or similar species that are very
closely related, will have very % of GTC value.
 And it is also seen that two organisms quite different % of GTC value are
not very closely related.
But in future it is also prove that organisms that are completely unrelated may
have similar % of GTC value. So to avoid these difficulties modern technology
is discovered i.e. DNA homology method.

DNA homology experiment:

In this experiment double stranded DNA molecule from two organisms are
heated to convert into single stranded. The single strand of one organism is
mixed with those from other organisms and allows to cool. If the 2 organisms
 BIOTECHNOLOGY 25

are closely related then they will form hetroduplexes i.e. a strand from 1
organism will pair with strand of other organisms.

2. rRNAhomology and rRNA oligonucleotides cataloguing:

These are 2 modern method used to determine the degree of similarities

between the r RNA cystrons of different organisms. These technologies are
complex and being used different laboratories for determining the
characteristics.
 BIOTECHNOLOGY 26

Nutritional requirements and nutritional types of bacteria

To obtain energy and construct new cellular components, organisms,

must have a supply of raw materials or nutrients. Nutrients – are substances
used in biosynthesis and energy production.

Nutrient Requirements:

Microbial cell composition shows that 95% of cell dry weight is made up
of a few major elements: Carbon, oxygen, hydrogen, nitrogen, sulphur,
phosphorous, potassium, calcium, magnesium and iron.

Macronutrients or macro elements:

These are required by microorganisms in relatively large amounts. Carbon,

oxygen, hydrogen nitrogen, sulphurs and phosphorous are components of
carbohydrates, lipids, proteins and nucleic acids. The remaining four macro
elements (K, Ca, Mg and Fe) exist in the cell as cations.

K+ - is required for the activity by a number of enzymes, including those

involved in protein synthesis.

Ca2+ - contributes to the heat resistance of bacterial endospores. 15% of spore

contains dipicolinic acid and calcium.

Mg2+ - serves as a cofactor for many enzymes, complexes with ATP and
stabilizes ribosomes and cell membranes.

Fe2+ and Fe2+ - part of cytochromes and a cofactor for enzymes and electron-
carrying proteins.

Micronutrients or Trace elements:

These are manganese, zinc, cobalt, molybdenum, nickel and copper. These
are normally part of enzymes and cofactors, and they aid in the catalysis of
reactions and maintenance of protein structure.

Zn2+ - is present at the active site of some enzymes but is also involved in the
association of regulatory and catalytic subunits in E.coli aspartate
carbomoyltransferase.

Mn2+ - aids many enzymes catalysing the transfer of phosphate groups.

Mo2+ - required for nitrogen fixation.

 BIOTECHNOLOGY 27

Co2+ - is a component of Vitamin B12.

Besides macro and micro nutrients, some microorganisms may have

particular requirements that reflect the special nature of their morphology or
environment. Diatoms need silicic acid to construct their beautiful cell walls of
silica. Bacteria growing in saline lakes and oceans depend on the presence of
high concentrations of sodium ion. Microorganisms require a balanced mixture
of all the above nutrients for proper growth.

Requirements for carbon, hydrogen and oxygen:

Carbon is needed for the skeleton or backbone of all organic molecules and
molecules serving as carbon sources normally also contribute both oxygen and
hydrogen atoms. One important carbon source that does not supply hydrogen or
energy is CO2.

Autotrophs – can use CO2 as their sole or principal source of carbon.

Many microorganisms are autotrophic, and most of these carry out
photosynthesis and use light as their energy source. Some autotrophs oxidize
inorganic molecules and derive energy from electron transfer.

Heterotrophs – are organisms that use reduced pre-formed organic

molecules as carbon sources. Ex. Glycolytic pathway produces carbon skeleton
for use in biosynthesis and also releases energy as ATP and NADH.
Actinomycetes will degrade amyl alcohol, paraffin and even
rubber. Burkholderiacepacia can use over 100 different carbon compounds.
Some microorganisms can metabolize even relatively indigestible human-made
substances such as pesticides. Indigestible molecules can be oxidized and
degraded in the presence of a growth promoting nutrient that is metabolized at
the same time, a process called Co-metabolism. The products of this breakdown
can then be used as nutrients by other microorganisms.

Nutritional types of microorganisms:

In addition to Carbon, hydrogen and oxygen all organisms require sources of

energy and electrons for growth.

Carbon sources:

Autotrophs - CO2 sole or principal biosynthetic carbon source

Heterotrophs – reduced, preformed organic molecules from other organisms.

 BIOTECHNOLOGY 28

Energy sources:

Phototrophs – use light as their energy source.

Chemotrophs – obtain energy from the oxidation of chemical compounds (either

organic or in organic)

Electron sources:

Lithotrophs – use reduced inorganic substances as their electron source.

Organotrophs – extract electrons from organic compounds.

Four major nutritional classes based on their primary sources of carbon, energy
and electrons is known.

1. Phtotolithotrophic autotrophs or photoautotrophs or

photolithoautotrophs:

Source of energy – light energy

Source of electrons – Inorganic hydrogen/ electron

Carbon source - CO2

Example: Algae, purple and green sulphur bacteria and cyanobacteria.

2. Photoorganotrophic heterotrophy or photoorganoheterotrophy:

Source of energy – light energy

Source of electrons – organic hydrogen/ electron

Carbon source – organic carbon sources (CO2 may also be used)

Example: Purple and green nonsulphur bacteria (common inhabitants of lakes

and streams)

3. Chemolithotrophic autotrophs or chemolithoautotrophy:

Source of energy – Chemical energy source (inorganic)

Source of electrons – Inorganic hydrogen/ electron donor

Carbon source - CO2

 BIOTECHNOLOGY 29

Example: Sulphur-oxidizing bacteria, hydrogen bacteria, nitrifying bacteria,

iron-oxidizing bacteria.

4. Chemoorganotrophic heterotrophs or chemoorganoheterotrophy:

Source of energy – Chemical energy source (organic)

Source of electrons – Inorganic hydrogen/ electron donor

Carbon source – organic carbon source

Example: Protozoan, fungi, most non-photosynthetic bacteria (including most

pathogens)

The most common nutritional types are photolithoautotrophs and

chemoorganoheterotrophs. Bacteria Beggiatoa rely on inorganic energy sources
and organic (or sometimes CO2) carbon sources. These microbes are sometimes
called Mixotrophic because they combine chemolithoautotrophic and
heterotrophic metabolic processes.

Requirements for nitrogen, phosphorous and sulphur:

Nitrogen is needed for the synthesis of amino acids, purines, pyramidines,

some carbohydrates and lipids, enzyme cofactors and other substances. Most
phototrophs and many nonphotosynthetic microorganisms reduce nitrate to
ammonia and incorporate the ammonia in assimilatory nitrate reduction. A
variety of bacteria like many Cyanobacteria and Rhizobiium can reduce and
assimilate atmospheric nitrogen using the nitrogenase systems. Phosphorous is
present in nucleic acids, phospholipids, ATP, several cofactors, some proteins
and other cell components. All microorganisms use inorganic phosphate as their
phosphorous source and incorporate it directly. E.coli can use both organic and
inorganic phosphate. Organophosphates such as hexose 6- phosphate can be
taken up directly by transport proteins. Other organophosphates are often
hydrolyzed in the periplasm by the enzyme alkaline phosphatase to produce
inorganic phosphate which is then transported across the plasma membrane.
When inorganic phosphate is outside the bacterium, it crosses the outer
membrane by the use of a porin protein channel. Sulphur is needed for the
synthesis of substances like the amino acids cysteine and methionine, some
carbohydrates biotin and thiamine. Most of them use sulphate as a source of
sulphur and reduce it by assimilatory sulphate reduction; a few require a
reduced form of sulphur such as cysteine.
 BIOTECHNOLOGY 30

Growth factors:

Many microorganisms have the enzymes and pathways necessary to

synthesize all cell components. Many lack one or more enzymes and hence
require organic compounds because they are essential cell components or
precursors of such components and cannot be synthesized by the organisms are
called – growth factors. There are three major classes of growth factors:

Amino acids – needed for protein synthesis.

Purines and Pyramidines – for nucleic acid synthesis

Vitamins – small organic molecules that usually make up all or part of enzyme
cofactors, and only very small amounts sustain growth.

Knowledge of the specific growth factor requirements of many microorganisms

makes possible quantitative growth response assays for a variety of substances.
The observation that many microorganisms can synthesize large quantities of
vitamins has led to their use in industry. Several water-soluble and fat-soluble
vitamins are produced using industrial fermentations.

Ribofalvin – Clostridium, Candida, Ashbya, Eremothecium

Coenzyme A – Brevibacterium

Vitamin B12 – Streptomyces, Propionibacterium, Pseudomonas

Vitamin C – Gluconobacter, Erwinia, Corynebacterium

β- Carotene – Dunaliella

Vitamin D - Saccharomyces
 BIOTECHNOLOGY 31

Module-2

Isolation of micro organisms

In the laboratory, bacteria are normally grown or cultured in either liquid

medium, in flasks, bottles or large culture vessels called fermenters or solid
medium in Petri dishes which are round, normally plastic or glass dishes.
Introduction of microbes into or onto these media is called inoculation. The
nature of the medium depends on the microorganism's natural environment
because its nutrient requirements reflect its natural surroundings. The culture
medium must contain all the nutrients that the microorganism requires for
growth like water, a source of energy, carbon, nitrogen, essential inorganic ions
and a number of trace elements. Bacteria that can synthesize all they require
from the basic ingredients are called Prototrophs – and most microorganisms
that survive in the outside environment can do this. Microbes that have become
adapted to life in a situation rich with nutrients such as human body may require
other growth factors like vitamins, amino acids or nitrogenous bases to be
provided. These are called auxotrophs.

The media that are used in microbiology laboratories to culture bacteria are
referred to as artificial media or synthetic media, because they do not occur
naturally; rather they are prepared in the laboratory. There are number of ways
of categorizing the media, one way is to classify based on whether the exact
contents of the media are known or not.

Chemically defined media: Is one in which all the ingredients are known; and
was prepared in the laboratory by adding a certain number of grams of each of
the components (Carbohydrates, amino acids, salts etc). Particularly
photolithoautotrophs such as Cyanobacteria and eukaryotic algae can be grown
on relatively simple media containing CO2 as a carbon source (often added as
sodium carbonate or bicarbonate), nitrate or ammonia as a nitrogen source,
sulfate, phosphate and a variety of minerals. Chemoorganoheterotrophs can be
grown in a defined media with glucose as a carbon source and an ammonium
salt as a nitrogen source.

Complex media: contains undefined ingredients and the exact contents are not
known. Complex medium may be sufficiently rich and complete to meet the
nutritional requirements of many different microorganisms. They contain
undefined components like peptones, meat extract and yeast extract.

Peptones – are proteins hydrolysates prepared by partial proteolytic digestion

of meat, casein, soyameal, gelatin and other protein sources (Carbon, energy
and nitrogen).
 BIOTECHNOLOGY 32

Beef extract – aqueous extracts from lean beef and contain amino acids,
peptides, nucleotides, organic acids and minerals and vitamins.

Yeast extract – from brewer's yeast and contain an excellent source of B

vitamins, nitrogen and carbon compounds.

Three commonly used complex media are

1. Nutrient broth – Peptone (5.0g/L), Beef Extract (3.0g/L)

2. Tryptic soya broth – Tryptone (enzyme digest of casein), Peptone (enzyme

digest of soybean meal), glucose, NaCl and K2HPO4

3. MacConkey Agar – Pancreatic digest of gelatin, casein, peptic digest of

animal tissue, bile salts mixture, NaCl, neutral red, crystal red and agar.

These media are routinely used for cultivation of bacteria in the laboratory and
particularly useful for cultivation of bacteria whose growth requirements have
not been defined.

Culture media can also be categorized as liquid or solid. Liquid media (also
called broths) are contained in tubes, flasks and fermenters. Solid media are
prepared by adding agar to liquid medium and then pouring the media into tubes
or Petridishes where the media will solidify. Agar is a complex polysaccharide
that is obtained from red marine algae. Other solidifying agents are gelatin,
silica. A 1% or 2% agar can be used to solidify, but 1.5% is the most commonly
used.

Enriched Medium: Is a broth or solid medium containing a rich supply of

special nutrients that promotes the growth of fastidious organisms (that have
complex nutrition and environmental requirements). It is usually prepared by
adding extra nutrients to a medium called nutrient agar.

Blood agar (nutrient agar + 5% sheep red blood cells): It is bright red in color
and distinguishes between the hemolytic and non-hemolytic bacteria
( Streptococci and other pathogens)

Chocolate agar (nutrient agar + powdered hemoglobin): It is brown in color and

is considered more enriched than blood agar as hemoglobin is more readily
accessible in chocolate agar.Pathogens
likeNeisseriagonorrhoeae and Haemophillus influenza, which will not grow on
blood agar, can be cultured.
 BIOTECHNOLOGY 33

Types of Media:

Selective Media: Are designed to suppress the growth of unwanted bacteria and
encourage the growth of the desired microbes.

Examples: MacConkey agar inhibits growth of gram positive bacteria and thus
is selective for gram-negative bacteria. Phenylethyl alcohol (PEA) agar and
Colistinalidixic acid (CAN) agar are selective for gram positive bacteria.

Manitol salt agar contains a concentration of 7.5% NaCl that is quite inhibitory
to most human pathogens. Bile salts, a component of fecus, inhibit most gram
positive bacteria while permitting many gram negative rods to grow. This media
is used for selecting intestinal pathogens which contain bile salts. Dyes such as
methylene blue and crystal violet also inhibit certain gram positive bacteria
(Staphylococcus can produce acid from mannitol and turn the phenol red dye to
bright yellow.

A medium containing acetate as a carbon source would be selective for

organisms that grow on acetate. Sabaraud's dextrose agar is used to isolate fungi
and cellulose for cellulose digesting bacteria.

Differential media: Makes it easier to distinguish colonies of the desired

organism from other colonies growing on the same plate.

MacConkey agar is also a differential and selective medium to distinguish

between lactose fermenting organisms (e.g., E.coli) and other non-fermenters
(e.g., Shigella sp.). Lactose in the medium is fermented by E.coli producing acid
which causes an indicator dye to change colour to red and colonies that do not
ferment lactose are white. Dyes can be used as differential agents because many
of them are pH indicators that change colour in response to the production of an
acid or base. MacConkey agar contains neutral red, a dye that is yellow when
neutral and pink or red when acidic.

Mannitol salt agar is used to screen for Staphylococcus aureus, and it turns the
originally pink medium to yellow due to its ability to ferment mannitol. In a
sense, blood agar is also differential because it is used to determine the
hemolytic and non-hemolytic bacteria.

Blood agar is both differential and an enriched one. It distinguishes between

hemolytic and non-hemolytic bacteria. Hemolytic bacteria (eg.,
many Streptococci and Staphylococci) produce clear zones around their
colonies because of red blood cell destruction.
 BIOTECHNOLOGY 34

Isolation of pure cultures:

Microorganisms are generally found in nature (air, soil and water) as mixed
populations. Even the diseased parts of plants and animals contain a great
number of microorganisms, which differ markedly from the microorganisms of
other environments. To study the specific role played by a specific
microorganism in its environment, one must isolate the same in pure culture.
Pure culture involves not only isolation of individual microorganisms from a
mixed population, but also the maintenance of such individuals and their
progenies in artificial media, where no other microorganisms find way to grow.

However, it is not easy to isolate the individual microorganisms from natural

habitats and grow them under imposed laboratory conditions. For this, great
deal of laboratory manipulation is required. If inoculums from any natural
habitat is taken and allowed to grow in a culture medium, a large number of
diverse colonies may develop that, due to crowdedness, may run together and,
thereby, may lose individuality. Therefore, it is necessary to make the colonies
well-isolated from each other so that each appears distinct, large and shows
characteristic growth forms. Such colonies may be picked up easily and grown
separately for detailed study. Several methods for obtaining pure cultures are in
use. Some common methods are in everyday-use by a majority of
microbiologists, while the others are methods used for special purposes.

Common Methods of isolation of pure culture

Pure culture of microorganisms that form discrete colonies on solid media, e.g.,
yeasts, most bacteria, many other micro fungi, and unicellular microalgae, may
be most commonly obtained by plating methods such as streak plate method,
pour plate method and spread plate method.

But, the microbes that have not yet been successfully cultivated on solid media
and are cultivable only in liquid media are generally isolated by serial dilution
method. Various techniques are developed for the isolation of pure culture from
mixed culture. Some of which are given below:

1. Use of Micromanipulator

2. Isolation by expose to air

3. Isolation by streaking or streak plate technique

4. Isolation by inoculating in animals

5. Isolation by selective or enrichment media

 BIOTECHNOLOGY 35

6. Other methods.

1.Use of micromanipulator:
A single viable cell may be transferred on the culture medium to develop
axenic pure culture by using micromanipulator which is use with a microscope
for picking up a single colony from a mixed population.

2. Isolation by exposed to air:

The nutrient agar slide or culture media containing plate is exposed to the
atmosphere for few minutes.

After incubation (over night or more) small colonies appears on the surface of
medium which may be transferred on a fresh medium aseptically to obtain pure
culture, such technique is called sub-culturing.

When transfer is from solid medium (agar) to liquid medium (broth), the term is
known as picking off.

In such cases, the colour of colony, their size, shape, appearance, consistency
and optical properties are recorded.

3. Isolation by streaking or Streak Plate Method:

This method is used most commonly to isolate pure cultures of bacteria. A small
amount of mixed culture is placed on the tip of an inoculation loop/needle and is
streaked across the surface of the agar medium. The successive streaks "thin
out" the inoculums sufficiently and the microorganisms are separated from each
other. It is usually advisable to streak out a second plate by the same
loop/needle without reinoculation. These plates are incubated to allow the
growth of colonies. The key principle of this method is that, by streaking, a
dilution gradient is established across the face of the Petri plate as bacterial cells
are deposited on the agar surface. Because of this dilution gradient, confluent
growth does not take place on that part of the medium where few bacterial cells
are deposited.
 BIOTECHNOLOGY 36

Various methods of streaking

Presumably, each colony is the progeny of a single microbial cell thus

representing a clone of pure culture. Such isolated colonies are picked up
separately using sterile inoculating loop/ needle and restreaked onto fresh media
to ensure purity.

Pour Plate Method:

This method involves plating of diluted samples mixed with melted agar
medium. The main principle is to dilute the inoculum in successive tubes
containing liquefied agar medium so as to permit a thorough distribution of
bacterial cells within the medium. Here, the mixed culture of bacteria is diluted
directly in tubes containing melted agar medium maintained in the liquid state at
 BIOTECHNOLOGY 37

a temperature of 42-45°C (agar solidifies below 42°C).

The bacteria and the melted medium are mixed well. The contents of each tube
are poured into separate Petri plates, allowed to solidify, and then incubated.
When bacterial colonies develop, one finds that isolated colonies develop both
within the agar medium (subsurface colonies) and on the medium (surface
colonies). These isolated colonies are then picked up by inoculation loop and
streaked onto another Petri plate to insure purity.

Pour plate method has certain disadvantages as follows:

(i) the picking up of subsurface colonies needs digging them out of the agar
medium thus interfering with other colonies, and
(ii) the microbes being isolated must be able to withstand temporary
exposure to the 42-45° temperature of the liquid agar medium; therefore
this technique proves unsuitable for the isolation of psychrophilic
microorganisms.

However, the pour plate method, in addition to its use in isolating pure cultures,
is also used for determining the number of viable bacterial cells present in a
culture
 BIOTECHNOLOGY 38

Pour Plate Method

C. Colony
development after
incubation. Control
A. Media/dilution B. Pouring of the plate; and
consists of the
sterilized plating
medium alone

The isolated colonies are picked up and transferred onto fresh medium to ensure
purity. In contrast to pour plate method, only surface colonies develop in this
method and the microorganisms are not required to withstand the temperature of
the melted agar medium.
 BIOTECHNOLOGY 39

Spread Plate Method:

In this method the mixed culture of microorganisms is not diluted in the melted
agar medium (unlike the pour plate method); it is rather diluted in a series of
tubes containing sterile liquid, usually, water or physiological saline. A drop of
so diluted liquid from each tube is placed on the centre of an agar plate and
spread evenly over the surface by means of a sterilized bent-glass-rod.
The medium is now incubated. When the colonies develop on the agar medium
plates, it is found that there are some plates in which well-isolated colonies
grow. This happens as a result of separation of individual microorganisms by
spreading over the drop of diluted liquid on the medium of the plate.

Serial Dilution Method:

As stated earlier, this method is commonly used to obtain pure cultures of those
microorganisms that have not yet been successfully cultivated on solid media
and grow only in liquid media. A microorganism that predominates in a mixed
culture can be isolated in pure form by a series of dilutions.

Spread plate method:

 BIOTECHNOLOGY 40

The inoculum is subjected to serial dilution in a sterile liquid medium, and a

large number of tubes of sterile liquid medium are inoculated with aliquots of
each successive dilution. The aim of this dilution is to inoculate a series of tubes
with a microbial suspension so dilute that there are some tubes showing growth
of only one individual microbe. For convenience, suppose we have a culture
containing 10 ml of liquid medium, containing 1,000 microorganisms i.e., 100
microorganisms/ml of the liquid medium.

If we take out 1 ml of this medium and mix it with 9 ml of fresh sterile liquid
medium, we would then have 100 microorganisms in 10 ml or 10
microorganisms/ ml. If we add 1 ml of this suspension to another 9 ml. of fresh
sterile liquid medium, each ml would now contain a single microorganism. If
this tube shows any microbial growth, there is a very high probability that this
growth has resulted from the introduction of a single microorganism in the
medium and represents the pure culture of that microorganism.

4. Isolation by inoculating in animals:

The disease causing microbes are unable to grow in an artificial media. If the
impure culture is injected into the susceptile animals as guinea pig or rabbits,
 BIOTECHNOLOGY 41

turn those animals allow to grow the disease causing organism while rests of the
microorganism fail to grow.

5. Isolation by using selective and enrichment media:

Certain media are selective because these have chemicals or dyes which
enrich the media and also they have growth inhibiting effect on some other
microorganism that why such media separate the dominant species.
Chemical dyes- malachite green and crystal violet are used to inhibit the growth
of bacteria and yeast.
Sodium azide is a metal binding agent that inhibits the growth of anaerobic
bacteria but does not affect the anaerobic lactic acid bacteria.

6. Other methods:

The other methods to isolate the microorganism are:

(i) By controlling physical environment (like temperature, pH, and
moisture).

(ii) By culturing highly diluted microbial suspension by pore plate method

in which isolated population of microorganism growing from single
isolate can be easily separated (serial dilution technique).

Cultural Characteristics

1. Colony Appearance

Many fungi produce colonies with fluffy appearance similar to cotton

wool. The molds produce colonies which on aging develop a dry chalky
appearance. The colony characteristics are in the following picture.

2. Colony Form

The colony shape may be circular, filamentous, punctiform, irregular or

spindle shape.

3. Colony Elevation

This form is used to describe the depth of the colony developed by

microorganisms. A colony may be flat, railed, convex, umbonate or with
papilate surface.
 BIOTECHNOLOGY 42

4. Colony margin

The margins may be entire, undulate ( Wavy) , crenate, denate, lobate,

rhizoidal or filamentous.

5. Optical Density

The colony may be transparent or translucent (foggy in appearance) or

opaque ( not permitting light to pass through) or iridescent ( rainbow
colour).

6. Colour

Many microbes develop colonies which are pigmented. Such coloured

substances are either water soluble or in soluble. The soluble pigments
diffuse out in the medium.
 BIOTECHNOLOGY 43

7. Colony odour

Some microbes produce a characteristics smell which sometimes helps in

identifying the microbe. The actinomycetes produce an earthy odour
which is qua first a shower or rain.

8. Colony Consistency

The degree of thickness, solidity or firmness of the medium hould be

examined as given below.

a) If the entire broth appears milky cloudy, it is called turbid.

b) If a deposit of cells is present at the bottom of tube, the term
sediment is used.
c) When microbial or bacterial growth forms a continuous or
interrupted sheet over the broth, it is called pellicle.
d) If the growth of microbe is similar in appearance to that of butter, it
is called butyrour.
BIOTECHNOLOGY 44
 BIOTECHNOLOGY 45

BACTERIAL GROWTH

Growth is defined as an increase in cellular constituents which leads to a rise in

cell number. As we are aware, microorganisms reproduce by binary fission or
by budding. In order to study growth, normally one follows the changes in the
total population number. The cells copy their DNA almost continuously and
divide again and again by the process called binary fission.

Growth Curve:

The increase in cell number or growth in population is studied by analysing the

growth curve of a microbial culture. Bacteria can be grown or cultivated in a
liquid medium in a closed system or also called as batch culture. In this method,
no fresh medium is added and hence with time, nutrient concentration decreases
and an increase in wastes is seen. As bacteria reproduce by binary fission, the
growth can be plotted as the logarithm of the number of viable cells verses the
time of incubation. The curve plotted shows four basic phases of growth; the
lag, log, stationary, and death phase.

Lag Phase: As the cells are introduced into the new medium, no immediate
increase in cell number occurs. During this phase, the cells are undergoing a
period of intense metabolic activity involving synthesis of enzymes and various
other molecules required to divide in the coming phase. This phase can vary
considerably in length depending on the nature of the medium and the
microorganism. The medium may be different from the one the microorganism
was growing in previously. The cells may be old and depleted of ATP, essential
cofactors and ribosomes; these must be synthesized before growth can begin.
So, the microorganism requires time to recover and young, vigorously growing
cultures and fresh medium are to be used for the lag phase to be short.
 BIOTECHNOLOGY 46

Growth curve of a typical bacterial cell

Log Phase: In this phase the cell starts dividing in a logarithmic way and this is
also called as exponential phase and the growth is balanced. Cellular
reproduction is high during this period and the plot during this phase is a
straight line. The cells are most active metabolically during this phase and the
population is most uniform during this phase; therefore exponential phase
cultures are usually used in biochemical and physiological studies. But during
this phase, the microorganisms may be particularly sensitive to adverse
conditions. On the whole, in this phase the cells are growing and dividing and
increasing in cell number. The rate of exponential growth of a bacterial culture
is expressed as generation time , also the doubling time of the bacterial
population. Generation time (G) is defined as the time (t) per generation/ n (n =
number of generations). Hence, G=t/n is the equation from which calculations
of generation time can be derived.

Exponential phase or log phase is balanced growth. That is, all cellular
constituents are manufactured at constant rates relative to each other. If nutrient
levels or other environmental conditions change, unbalanced growth results.
The individual cells may take slightly longer than others to go from lag phase to
the log phase, and they do not all divided precisely together. If they divided
together and the generation time is same, the number of cells in a culture would
increase in a stair – step pattern, exactly doubling every 20 min or a particular
time – a hypothetical situation called Synchronous growth. In an actual culture,
each cell divides sometime during the 20 min generation time, with about 1/20
cells dividing each minute – a natural situation called nonsynchronous growth
or asynchronous growth which appears as a smooth line, not as steps.
 BIOTECHNOLOGY 47

Organisms in a tube of culture medium can maintain logarithmic growth for

only a limited time. As the number of organisms' increases, nutrients are used
up, metabolic wastes accumulate, living space may become limiting factor and
aerobes suffer from oxygen depletion

Stationary Phase: Exponential growth cannot be continued forever in a batch

culture (e.g. a closed system such as a test tube or flask). Population growth is
limited by one of three factors:

1. exhaustion of available nutrients;

2. accumulation of inhibitory metabolites or end products;

3. exhaustion of space, in this case called a lack of "biological space".

During the stationary phase, if viable cells are being counted, it cannot be
determined whether some cells are dying and an equal number of cells are
dividing, or the population of cells has simply stopped growing and dividing.
The stationary phase, like the lag phase, is not necessarily a period of
quiescence. Bacteria that produce secondary metabolites, such as antibiotics,
do so during the stationary phase of the growth cycle (Secondary metabolites
are defined as metabolites produced after the active stage of growth). It is
during the stationary phase that spore-forming bacteria have to induce or
unmask the activity of dozens of genes that may be involved in sporulation
process. Starving bacteria frequently produce a variety of starvation proteins,
which make the cell much more resistant to damage. They increase
peptidoglycan cross-linking and cell wall strength. The Dps (DNA- binding
proteins from starved cells) protein protects the DNA. Bacterial pathogens
like Salmonella typhymurium become more virulent when starved.

Death Phase: Due to the conditions during the stationary phase, the death phase
is seen as there is a decline in the number of viable cells. This phase also is like
the log phase where the cell number is declining in a logarithmic way. The cell
is said to be dead if it does not revive itself and reproduce when incubated again
in a fresh medium. In this phase, the number of live cells decreases at a
logarithmic rate, as indicated by the straight downward sloping diagonal line.
The duration of this phase is as highly variable as the duration of log phase.
Both depend primarily on the genetic characteristics of the organism.
 BIOTECHNOLOGY 48

Mathematics of Growth:

Microbial growth during the exponential phase is very important and of interest
to microbiologists and the analysis applies to microorganisms dividing by
binary fission. The time required by a cell to divide is called the generation time
or doubling time. In the laboratory, under favourable conditions, a growing
bacterial population doubles at regular intervals. Growth is by geometric
progression: 1, 2, 4, 8, etc. or 20, 21, 22, 23.........2n (where n = the number of
generations). This is called exponential growth. In reality, exponential growth is
only part of the bacterial life cycle, and not representative of the normal pattern
of growth of bacteria in Nature. This might vary from organism to organism
depending upon the environmental conditions etc. For example in E.coli the
generation time is 20 min and hence after 20 generations a single initial cell
would increase to over 1 million cells. This would require a little less than 7
hours. The population is doubling every generation; hence the increase in
population is always 2nwhere n is the number of generations. The resulting
population increase is exponential or logarithmic.

When growing exponentially by binary fission, the increase in a bacterial

population is by geometric progression. If we start with one cell, when it
divides, there are 2 cells in the first generation, 4 cells in the second generation,
and 8 cells in the third generation, and so on. The generation time is the time
interval required for the cells (or population) to divide.

G (generation time) = (time, in minutes or hours)/n(number of generations)

G = t/n

t = time interval in hours or minutes

B = number of bacteria at the beginning of a time interval

b = number of bacteria at the end of the time interval

n = number of generations (number of times the cell population doubles during

the time interval)

b = B x 2n (This equation is an expression of growth by binary fission)

 BIOTECHNOLOGY 49

Solve for n:

logb = logB + nlog2

n=logb-logB/log2

n= logb-logB/ .301

n = 3.3 logb/B

G = t/n

Solve for G

G= t/3.3logb/B

Example: What is the generation time of a bacterial population that

increases from 10,000 cells to 10,000,000 cells in four hours of growth?

G= t/3.3logb/B

G= 240minutes/3.3log107 /104

G= 240minutes/3.3x3

G = 24 minutes

BACTERIAL GROWTH MEASUREMENT

Measurement of Microbial Growth:

A number of techniques are available in order to measure growth of microbial

populations. Either population number of mass may be calculated ad growth
leads to increase in both.

Direct measurement of cell numbers:

Bacteria or microorganisms can be counted directly on the plate and also called
as plate counting. Advantage of this method is that it measures the number of
viable cells. Disadvantage is that, it is time consuming and expensive as one
needs media and other conditions need to be maintained. Bacteria counted on
 BIOTECHNOLOGY 50

plate counts are referred to as colony forming units as a single cell or a clump of
bacterial cells can lead to a colony which contains many cells. The colonies
when they are counted in plate count method are to be present sparsely for
accurate counting as overcrowding can lead to incorrect counting. To solve this,
one has to adapt the serial dilution method in order to get an accurate count.

Serial dilution and pour

and spread
plate: Supposing one has to
accurately count the number
of cells given in a solution,
then serial dilution needs to
be performed. A 1ml of the
sample is taken and
transferred to a tube
containing 9ml of sterile
water and this process can be
repeated until we reach a
considerable dilution (say
106 to 107). Once the original
inoculum is diluted one
needs to perform a pour plate
or a spread plate technique in
order to count the number of
bacteria present in the
diluted sample and then the
original sample. In pour
plate method Fig. 3. Serial dilution methodology the
diluted sample is poured into the petriplate and then the medium which is at
nearly 500C is poured over the inoculum and mixed by gentle agitation. With
this method, colonies grow within the nutrient agar as well as on the surface of
the agar plate. As certain disadvantages are encountered in this method like heat
sensitive microorganisms might not grow and also bacteria when they grow
within the nutrient medium might not be useful for diagnostic purposes. In order
to avoid these problems, spread plate method is mostly used (Fig. 3). A 0.1ml of
the diluted sample is added to the surface of the nutrient medium and spread
uniformly with the help of a glass spreader and after incubation, the colonies
 BIOTECHNOLOGY 51

can be counted and the concentration of the bacterial cells in the original
sample is calculated as follows:

Number of bacteria/ml = Number of colonies on plate x reciprocal of dilution of

sample

Membrane Filtration: This method can be used in order to study if the

quantity of the bacteria is very small as in aquatic samples like lakes, streams
etc. Membranes with different pore sizes are used to trap different
microorganisms. The sample is drawn through these special membrane filters
and placed on an agar medium or on a pad soaked with liquid media. After
incubation, the number of colonies can be counted and the number determined
in the original sample. Selective media or differential media can be used for
specific microorganisms. This is mostly used for analysing aquatic samples.

Microscopic count: The Petroff-Hausser counting chamber or slide is easy,

inexpensive and relatively quick method and also gives information about the
size and morphology of the microorganisms. These specially designed slides
have chamber of known depth with an etched grid on the chamber bottom
(Fig.4).

Bacteria/mm3 = (bacteria/square) (25 squares)/ (50)

Bacteria can be counted by taking into account the chamber's volume and any
sample dilution. The disadvantage encountered in this method is that fairly large
volume is required and also it is difficult to distinguish between living and dead
cells. Microorganisms of larger sizes can be counted by using electronic
counters such as coulter counter; where in the number of cells in a measured
volume of liquid is counted. This method gives accurate results with larger cells
and is extensively used in hospital laboratories to count red and white blood
cells.
 BIOTECHNOLOGY 52

Fig. 4. Direct microscopic count of bacterial cells

Indirect methods of measurement of cell mass:

Population growth leads to increase in the total cell mass, as well as in cell
numbers. The following methods can be used.

Turbidity: As bacteria grow/multiply in a liquid medium, the medium becomes

turbid (Fig. 5). Spectrophotometer is used in order to measure the turbidity. A

Fig. 5. Broth culture showing turbidit

beam of light is transmitted through a bacterial suspension to a light-sensitive

 BIOTECHNOLOGY 53

detector. The fact that microbial cells scatter light striking them, the amount of
scattering is directly proportional to the biomass of cells present and indirectly
related to cell number. The extent of light scattering can be measured and is
almost linearly related to bacterial concentration at low absorbance levels.

Dry weight: This method is mostly used for filamentous bacteria and moulds.
The microorganism is grown in liquid medium, filtered or centrifuged to
remove extraneous material, and dried in an oven and then weighted. It is time
consuming and hence not very sensitive.

BACTERIAL REPRODUCTION

Bacteria reproduce by
 Vegetative
 Asexual and
 Sexual methods

Vegetative reproduction includes Budding, Fragmentation and Binary fission.

Budding:

In this case, a small

protuberance, called bud,
develops at one end of the cell.
Genome replication follows,
and one copy of the genome
gets into the bud. Then the bud
enlarges, eventually become a
daughter cell and finally gets
separated from the parent cell.

Fragmentation:

Mostly during unfavourable

conditions, bacterial
protoplasm undergoes
compartmentalization and subsequent fragmentation, forming minute bodies
called gonidia. Under favourable conditions, each gonidium grows to a new
bacterium. It becomes apparent that prior to fragmentation the bacterial genome
has to undergo repeated replication so that each fragment gets a copy of it.
 BIOTECHNOLOGY 54

Binary fission:

It is the commonest type of reproduction under favourable conditions in which

cell divides into two similar daughter cells. During the process, the bacterial
chromosomes get attached to the cell membrane and replicates to the bacterial
chromosomes. As the cell enlarges the daughter chromosomes gets separated. A
cross wall is formed between the separating daughter chromosomes. It divides
the cell into two daughter cells. The daughter cells soon grow to maturity within
20-30 minutes. Under favourable conditions many bacteria divide once in 20-30
minutes.

Asexual reproduction

It takes place by endospore formation, conidia and zoo spores.

Endospore formation: Endospore are resting spores formed in some gram

positive bacteria (Bacillus and Clostridium) during unfavourable conditions.
They are formed within the cells. During this process a part of the protoplast
becomes concentrated around the chromosome. A hard resistant wall is secreted
around it. The rest of the bacterial cell degenerates; Endospore are very resistant
 BIOTECHNOLOGY 55

to extreme physical conditions and chemicals. During favourable conditions the

spore wall gets ruptured and the protoplasmic mass gives rise to a new
bacterium.

Sexual reproduction

It occurs in the form of genetic recombination. There are three main methods of
Genetic Recombination: Transformation, Transduction and Conjugation.

Transformation: Here genetic material of one bacterial cell goes into another
bacterial cell by some unknown mechanism and it converts one type of
bacterium into another type (non capsulated to capsulated form). This was first
studied by Griffith (1928) in Diplococcus pneumonia.

Transduction: In this method, genetic material of one bacterial cell goes to

other bacterial cell by agency of bacteriophages or phases (viruses, infecting
bacteria). It was first of all reported in Salmonella typhimeurium by Zinder and
Lederberg (1952).

Conjugation: It was first reported by Lederberg and Tatum (1946) in E.coli

bacteria. Cell to cell union occurs between two bacterial cells and genetic
material (DNA) of one bacterial cell goes to another cell lengthwise through
conjugation tube which is formed by sex pili.
BIOTECHNOLOGY 56
 BIOTECHNOLOGY 57

Module-3

Ecology
The study of reciprocal relations between organisms and their environment.

It is the study of organism in their natural habitat and ecology deals with
organism and environment. It includes the living component of the ecosystem
i.e. plant and animal. It can further sub divided into macroscopic and
microscopic.

Ecological group of microorganism

Microorganisms are an important component of an ecosystem. Several

ecological categories are made on certain grounds while grouping the
microorganisms.

1. Based on oxygen requirement:

i. Aerobes
ii. Anaerobes
2. Based on carbon sources as energy:

i. Autotrops
ii. Heterotrops
3. Based on Temperature:

a. Psychrophiles- Which are grow at a very low temp. Below 200C

b. Mesophiles- The microorganism growing optimally at temp between 250C to

350C.They have capacity can grow at 15 0C to 450C.

c.Thermophiles(Heat loving) -Microorganism growing readily at temp

450C to 650C
d. Hyper thermophiles and super hyperthermophiles-Grow above 800C to 1000C
(boiling point of water at sea level. These are
found at hot spring and water.

Super hyperthermophiles are grown at 2000C to 3000C.

 BIOTECHNOLOGY 58

4. Based on habitat

On the basics of habitat microorganism are divided into following categories.

a. Soil microorganism: - Microorganism dwelling in soil subsystem is

known as soil microorganism or soil microflora.
b. Aquatic microorganism: - Microorganism residing in water
subsystem irrespective of its quality or physical or chemical nature
are called aquatic microorganism.
c. Aeromicroflora: - Microorganism living in air generally known as
aeromicroflora or microorganism of air.

5. Microorganism living in Extreme environment i.e extremophiles

Extremophiles thrive under conditions that would kill other creature. This
comes under the groups high and low pH loving microorganisms.

6. On the basics of mode of nutrition or habit

There are different ways by which the microorganisms derive nutrition for
their growth and development.

a. Saprophytism
b. Parasistism
c. Symbiosis

a.Saprophytism:- It is aphenomenon which refers to getting nutrition from

dead organic materials, and such microorganism are known as saprophytes for
example Aspergillus, pencillium,Rhizopus, Mucor, etc. Some time in presence
of a living host a few saprophytes change their tendency and cause disease.
Such saprophytes are called facultative parasites. Facultative parasites are
basically saprophytes but have tendency to behave as parasite as well.

b. Parasitism:-Parasitism refers to deriving nutrition from a living plant or

animal host, and microorganisms associated with parasitism are known as
parasites. There are certain nutrients which are not found in dead organic
materials .For such nutrients the parasites have to infect the plant or animals. On
the other hand, it can be said that the parasitism is a tendency of parasites to
infect living hosts. When a parasite is very virulent and cannot live without a
living host, it is called obligate parasite such as puccina, powdery mildews, etc.
On the other hand a parasite, in the absence of a suitable living host can pass its
life as saprophyte. It is a second mode of leading the life and survival
mechanism. Such type of parasites are known as facultative saprophytes i.e. ,
 BIOTECHNOLOGY 59

the parasite that have faculty to live as saprophyte in the absence of a suitable
host.

c. Symbiosis: -In parasitism advantages are only to the microorganisms. The

host are the losers. Consequently, there develop disease. However in other case
both the microbes and the host s are benefited as far as nutrition is concerned.
Such association of mutual benefit is known as symbiosis. Symbiosis can be
seen in lichens, mycorrhiza, root nodules of legumes and non-leguminous
plants.

MICROBIAL INTERACTIONS

Microorganisms are ubiquitous in their occurrence. However, in natural

environment they interact among themselves with plants, with animals, and,
moreover, with their niche. Finally, different types of interrelations are
established. Reasons for microbial interactions are the competition for nutrients
and space in an ecological niche. Microbes may not affect the other, or may
affect by one or more of the following ways:

 By stimulation of growth and development of associate

 By inhibition of growth and development of associate.
 By stimulating the formation of resting bodies by the associate.
 By inhibiting the formation of resting bodies by associate.
 By enforcing the dormancy of the associate.
 By causing lysis of the associate.
 By harming the population of plants.
 By directly benefiting the plants and
 By getting influenced by its own micro environment.

On the basics of the relative advantage to each partner i.e host and
microorganism, the relationships are basically of three types:

a) Neutralism ( where host remain unaffected by the microbe)

b) Mutualism (where both partners get benefits from the association)
c) Parasitism ( where one partner get benefits and the second suffers from
damage)

Types of interactions

In the microbial world there are major four types of interactions.

I. Clay-Humus-Microbe interaction
II. Plant- Microbe Interaction
 BIOTECHNOLOGY 60

i. Interaction s above the ground parts

a. Destructive association ( Diseases)

b. Beneficial association ( symbiosis)

ii. Interaction below the ground parts

a. Destructive Associations
b. Beneficial associations ( symbiosis)

III. Animal –Microbe interaction

a. Destructive Associations
b. Neutral associations (neutralism)
c. Symbiotic associations

IV. Microbe-Microbe Interactions

a. Symbiosis between alga and fungus (Lichen)

b. Antagonistic Interactions (antagonism)

I. Clay- Humus-Microbe Interaction

Clay minerals and humic substances affect the activity, ecology and population
of microorganism in soil. Clays modify the physic-chemical environment of the
microbes which either enhance or attenuate the growth of individual microbial
population. After release from clays, the organic materials are either degraded
by microorganisms or again bind to clays. Microorganisms have a negative
change at the pH of most microbial habitats.

Clay minerals get adsorbed and bind with proteins, aminoacids, small peptides
and humic substrates. Microorganisms utilize the nutrients for their growth and
activity directly from clay-protein, clay-amino acids or peptides, and clay-
humic substrate complexes.
 BIOTECHNOLOGY 61

II. Plant-microbe Interaction

i. Interaction above the Ground Parts

a. Destructiveassociations (Diseases)

 Plants provide a substantial ecological niche for microorganisms.

However the abundance of this potential niche with respect to any
individual microbe apparent then real, since a few are able to grow on a
wide range of plant species. The microbe leads to destructive association
are called pathogen.
 Disease development is governed by the three important factors- host
susceptibility, congenial environment and virulent pathogen.
 Nematodes directly a slight mechanical injury on plant root. Their saliva
is toxic for host tissues which results in cellular hypertrophy, suppression
mitosis, growth stimulation etc.

Some examples of pathogen causing disease are:

Algal disease- red rust of citrus, mango, guava, cocoa, sapota, tea.

Bacterial Disease- fire blight of apple

Stem blight of pea

Canker of citru.

b.Beneficial association (symbiosis):

Symbiosis is the phenomenon of living together where both partners are

benefited. The excellence example of plant-microbe interaction resulting
beneficial association visualized on above the ground part is the
development of stem nodule. There are three known genera of legumes
which known to bear stem nodules are Aeschynomene, Sebacnia,
Neptunia.E

ii.Interaction on below the ground parts.

Similar to above ground parts, plant root- microbe interactions occurs in soil as
well which leads different types associations, i.e. destructive, associative or
symbiotic.
 BIOTECHNOLOGY 62

a.Destructive Association
 Like destructive association above the ground parts, roots also result in a
destructive association. The symptoms developed by the pathogens on
roots are damping off,wilt,rot,knot,scab,etc.
 The pathogens infect roots.Entry of pathogens takes place through
wounds caused by fungi or nematodes, cracks or root hairs.
 In most of the cases penetration is preceded by the formation of a specific
cushion like structure(appressorium) which exerts mechanical pressure on
root surface. Some pathogens directly penetrate the root tissues.
 A member of actontinomytes (streptomyces scabies) causes scab disease
of potato.
 Agrobacterium tumifaciens, a soil borne bacterium, causes crown gall of
fruit trees including roots.
 Pseudomonas solanacearum causing brown root and bacterial wilt of
tomato, potato and other solanaceous plant is well known pathogen.
 After cutting open the affected tubers and creamy viscous exudation from
open surface is observed and the dark brown discoloration of the vascular
region becomes distinct. Consequently the tuber formation and size of
tuber is greatly reduced.
b.Beneficial Association (Symbiosis)
 Symbiosis is the phenomena of living together where both partners are
benefited.
 The microsymbionts derive freshly prepared food from the host plant
which lack in soil.The macrosymbionts get certain nutrients from the soil
which are not easily available such as trace elements nitrogen,
phosphorus etc. Symbiotic associations with different groups of
microorganisms are discussed below-

Cyanobacterial Symbiosis:
 The term cynobacteria is of recent origin which includes the members of
cynaophyceae.
 They are both hetrocystous and non-hetrocytous form.
 Heterocyst is the site of nitrogen fixation.
 The non-heterocystous forms also fix nitrogen. Anabaenacycade is
associated with coralloid roots of cycas. It is present in well-defined
region which is known as algal zone.
 BIOTECHNOLOGY 63

Bacterial Symbiosis:

 Among bacteria there are two categories of symbiosis, one that does not
form apparent symbiotic structure(root noudle) and the second group
which form root nodules. However, there is a third group which enhances
without entering in symbiosis.

Legume-Rhizobium

 Rhizobium, a soil bacterium, enters in symbiosis with leguminous plant.

It develop root nodule which are the site of 2 fixation.

Actinomycete-Non-Legume Symbiosis:

 From this classthe species o Frankia are known to develop nodules which
are known as actinorhiza.
 These plants grow in such condition where the concentration of nitrogen
is low. One of the most extensively is the alder tree.
Ex-:alnus,hippophae.

Fungal Symbiosis (Mycorrhiza):

 Mycorrhiza has been defined as an apparent structure developed as a

result of roots. These types of symbiosis are discovered in both structure
and physiological functions.

III. Animal- Microbe Interactions

There are many kinds of microorganism that interacts with different groups
of animal and develop a variety of relationship like

a. Destructive association
b. Neutral association
c. Symbiotic association

a. Destructive association:

 Pathogenic microbes interact with animals including human and causes

many kinds of disease.
 Example: - Different types of diseases, we will get in medical
microbiology. Also it is found in between two microbes interaction such
as bacteria and fungai, fungi and nematodes etc.
 BIOTECHNOLOGY 64

b.Neutral Association:

 Normal microbes of human body: There is a large number of

microorganism that normally act as the resident of different body organs
of human such as skin, nose and nasopharynx, oropharynx, respiratory
tract, mouth, eyes, external ears, stomach, small intestine, and genito-
uniary tract. Reason for having information about the normal human
microbiotare:

a) To have an understanding of microorganism at specific site so

that greater insight into the possible infection can be provided.
b) To help the physician investigate so that he can understand the
causes and consequences of overgrowth of microorganism
normally absent at a specific body site
c) To increase awareness of the role of indigenous microbioant
that stimulate host immune response.

c.Symbiotic Association:

Symbiotic association of bacteria, fungi and protozoans with insect, bird and
herbivorous mammals are below:

i. Ectosymbiosis of protozoa, bacteria and fungi with insects and birds:

Most of the animals such as insects (terminates and cockroaches) cannot

utilize the cellulose and lignin components of woody tissues of tree due to
lack of cellulose and lignin degrading enzymes. Therefore several insects
develop ectosymbiotic association with cellulose and lignin decomposing
microorganism that can degrade these substrates. All termites and
cockroaches that eat upon wood, harbour flagellated protozoa in their guts.
These protozoa digest cellulose. In turn the protozoa develop symbiotic
association with certain N2-fixing bacteria and spirochetes which perhaps
also help in cellulose degradation.

ii.Endosymbisis of bacteria and Fungi with Birds and insects:

Moreover there is a group of birds belonging to the genus Indicator which are
commonly known as honey guides. These birds are found in Africa also in
India. These birds eat upon remnants of exposed honey comb but cannot
digest bees wax. Therefore they harbour in their intestine the two microbes,
Micrococcus cerolyticus and Candida albicans for carrying out the digestion
of bees wax.
 BIOTECHNOLOGY 65

Iii.Ruminant Symbiosis:

The herbivorous mammals (e.g.: cattle, sheep, goats, camels, etc.) are known
as ruminants because they have a special region of gut which is called rumen.
These animals use plant cellulose as the source of carbohydrate which is not
digested in normal gut. The cellulose material is digested in rumen which is
act as a incubation chamber teeming with protozoa and bacteria.

IV. Microbe- Microbe Interactions

Microorganisms interacts themselves and lead beneficial and harmful

relationships.

a.Symbiosis between alga and fungus (Lichens)

Lichen is a thallus of dual organism i.e. a fungus and alga that form a
self-supporting combination .The fungal component is called mycobiant
and algal partner as phycobiant. The two groups of organisms live in
close proximity and appears us a single plant.

Generally fungi derived nutrition from dead body and from living host by
saprophytically and parasitically respectively.

But lichen fungal mycelium derives nutrition from alga. The algal cells
form food themselves and/or fix N2 from the atmosphere which then are
diffused into fungal hyphae. This mode of nutrition is called
biotropicnutrition .

Classification:-

1. Ascolichen: - In which fungal component is an Ascomycete

2. Bascidiolichen: -In which the fungal component is a Basicidiomycete

On the basis of habitant lichens are divided into three groups

 Saxicolous: - Growing on rock or stones

 Corticolous:-Growing on leaves and bark of trees
 Terricolous:-Growing on soil
 BIOTECHNOLOGY 66

b. Antagonish interaction (Antagonisim)

The composition of micro flora of any habitant is governed by the

biological balance created through interaction and association of an
individuals present in a community.

Any inhibitory effect of an organism created by means to the other

organisms is known Antagonistic interaction and the phenomenon of this
activity is known as antagonism.

There are three types of Antagonistic actions:-

1. Amensalism
2. Competition
3. Parasitism and predaction

1. Amensalism(Antibiosis and lyses)

 It is a phenomenon where one microbial species is adversely

affected by the other species, where the other species is unaffected
by the first one.
 Generally amensalism is accomplished by the secretion inhibitory
substance such as antibiotic.
 Antibiosis is a situation where the metabolites secret by organism
A inhabits the organism B,but the organism A is unaffected. These
metabolites penetrate the cell wall and inhabit its activity by
chemical toxicity.

2. Competition

 Among the microorganism, competition exists for nutrients,

including oxygen and space but not for water potential,
temperature, or pH.
 Competition exists for limiting resources.
 The inadequate quantity of readily availability of carbon compound
is a more likely basis for competition.
 At low level of carbon, the first growers will often holds slow
growers in check when both are added to sterilized soil.

3. Parasitism and predation

 Parasitism is a phenomenon where one organism consumes another

organism in subtle and non-debilitating relationships.
 BIOTECHNOLOGY 67

 Predation is an apparent mode of antagonism where a living

organism is mechanically attached by the other with the
consequences of death of the former.
 It is violent and destructive relationship.
 These phenomena are dealt with the example of fungi, amoebae
and nematodes.

These interactions are of various types.

a) Mycoparasitism( Fungus-fungus interaction)

b) Mycophagy( feeding upon fungi by amoebae)
c) Nematophagy(eating upon nematodes by fungi)
 BIOTECHNOLOGY 68

Module-4

Raw Water Source

The various sources of water can be classified into two categories:

1. Surface sources, such as

a. Ponds and lakes;
b. Streams and rivers;
c. Storage reservoirs; and
d. Oceans, generally not used for water supplies, at present.
2. Sub-surface sources or underground sources, such as
a. Springs;
b. Infiltration wells ; and
c. Wells and Tube-wells.

Microorganism in the aquatic environment may occur at all depths ranging

from the surfaces region to the very bottom of ocean trenches. The top “layers
“especially the surface film and the bottom sediments harbour the higher
concentration of microorganisms, particularly in deep waters.

A large no of microorganism both saprophytes and pathogen are found in

water which falls under the groups bacteria, fungi, algae, protozoa and
nematodes.

1.The majority of bacteria found in water belong to groups

 Fluorescent bacteria
 Chromogenic rod
 Proteus group
 Non-gas forming
 Non-spore forming rod
 Spore formers
 Pigmented
 Non-pigmented cocci
 BIOTECHNOLOGY 69

Distribution of microorganisms in the aquatic environment

1. Planktons (phytoplankton, zooplankton)

The aggregation of floating and drifting microbial life in the surface region of
the aquatics ecosystem is called plankton. Plankton may be composed of
primarily of algae (phytoplankton), or it may be predominantly protozoa and
other protozoa minute animal life (zooplanktons).Phototrophic microorganisms
are regarded as the most important plankton since they are the primary
producers of organic matters via photosynthesis. Most phytoplanktonic
organism are motile possess some structural features, or contains oil droplet
which give them buoyancy: all these features aid the organisms in maintain their
location in photosynthetic zone.

2. Benthic microorganisms

Microbial inhabitants of the bottom region of a body of water are referred to as

the benthic organisms. The richest region of an aquatic system in terms of
numbers and kinds of organisms is the benthic region. Many aquatic
microorganisms inhabit the gut of marine animals, an even richer habitat.

3. Mixing of waters (upwelling)

The moment of water by wind, tide or currents accomplishes some

redistribution of the microbial flora. a phenomenon called upwelling occurs in
an ocean when water rises from a deeper to a shallower depth, usually as a
result of divergence of offshore currents or winds .In this process the bottom
water carries with it a rich supply of nutrients that are delivered to the surface
region .Upwelling occurs off the coasts of California and Peru and is
responsible for the high productivity of these regions. Geothermal vents also
contribute to the total nutrients budget of the oceans. It has been calculated that
vents such as the one near the Galapagos island account for most of the
nutrients results in the ocean of the world .prior to discoverer of this vents,
oceanographers were unable to account for nutrients on the basis of
precipitations input from the rivers and springs, and other obvious source.
Another interesting feature of the oceans is the gyre, large spiralling surface
currents in the oceans that tend to aggregate and retain nutrients, wastesand
microorganisms.
 BIOTECHNOLOGY 70

Aquatic microorganisms

As previously stated, aquatic microbiology is the study of microbial life in

fresh, estuarine and marine waters .it includes the microbiology of lakes, ponds,
streams estuaries and the sea .it is an all-inclusive term for the study of
microorganisms in naturals waters. The microbiology of fresh waters constitutes
a part of the science of limnology, which the study of the flora and the
conditions for life in lakes, ponds and streams

1. Lakes and ponds

Lakes and ponds have a characteristics zonation and stratification. There is

usually a fairly large littoral zone along the shore, which has considerable
rooted vegetation and include regions where light penetrates to the bottom .in
open areas, the limnetic zone is determined by the light –compensation levels
(depth of effective light penetration). Photosynthetic activity decreases
progressively in the deeper regions of the open water (profoundly zone).The
benthic zone is composed of soft mud or ooze at the bottom together with the
profoundlyzone, the benthic region is largely populated by heterotrophic
organisms. The greatest variety of physiological types is found in the limnetic
and littoralzones, and in addition they constitute the most productivity regions.
In the summer top layer tends to be warmer than the lower regions but in winter
its reverse is happening, due to lesser density the ice are coming to the top so
the algae are growing massively due to proper mixing.

2. Stream

Streams are obtains a majority of nutrients from the flow of inorganic and
organic materials from the surrounding terrestrial system or lakes or ponds .To a
major extent, the microbial flora reflects the immediate terrestrial conditions ,
including the effects of agricultural and industrial practice. The drastically
conditions rises in pond and streams due to rapidly expanding of urbanizations
and changes in farming practice.

3. Estuaries

An estuary is semi enclosed coastal body of water which has a free connection
with the open sea .stated differently, it is the coastal adjunct of the marine
ecosystem .estuaries are commonly receive the many thing in input from
 BIOTECHNOLOGY 71

different sources (temperature, salinity, turbidity, nutrient load, and other

conditions )

The major estuarine systems are

1. The bay serves as the receiving basin for nine major rivers, draining much of
southern New York State, penysylavania Maryland, virgina.

2. It has a shoreline of 4600 miles, which includes highly industrialized areas,

farmlands and uninhabited marshlands.

3. The salinity varies from less than 1% in the tributes to 3-5 %( normal sea
water) at the mouth of the bay .It is estimated that the estuary system is filled
with river water and the sea water.

4. The bay, directly or indirectly, is subjects to the activities of a million

population with in the regions. Practice associated with agricultural, commerce,
industry and recreations influence the conditions of the bay.

From these observations it is apparent that the microbial flora of the estuary is
subjects to considerable fluctuations .some species are indigenous to specific
ecological niches of the estuary; other are transient, having been aided from
domestics, agriculture, industrial or atmospheric sources.

4. Sea

Microorganisms are found at all depths and at all latitude in sea waters .they
occurs in planktons and in the sediments of the oceans floor. They great volume
of the open sea provides an environment with less variation in conditions than
the other aquatic waters discussed. Due to some technical problem the same
value will be changed.

5. Marine planktons

The phytoplankton populations comprises numerous species of diatoms,

cyanobacteria, dinoflagellates, coccolithophores, silicoflagellates,
chrysomonads, and chlamydomands. This group of microorganisms is chiefly
responsible for the conversion of radiant energy to chemical energy – energy
stored i chemical substances that accumulate in the sea. The magnitude of this
accomplishments is revealed by a calculation that suggests a requirements of 50
 BIOTECHNOLOGY 72

billion metric tons of phytoplankton to support the growth of the potential world
fish catch, estimated at 50 million metric tons.

Plankton and algae can grow into enormous population with resultant
discoloration of the water condition like bloom. Due to bloom they are
appearing like red tides, brown, amber, or greenish-yellow in the sea.

The beneficial effect of the plankton may be attributed to both the organic
substances they elaborate and the solid surface provided for bacterial
aggregation .Bacterial populations differ widely with prevailing conditions. The
temperature of the marine environment and the degree of salinity would be
suitable for the growth of psychrophilic and halophilic physiological types.

Gram negative bacteria are having the lipopolysaccharide of the outer

membrane affords gram negative bacteria protection from certain toxic
molecules e.g. fatty acids and antibiotics and it may serve to sequester important
nutrients from the water. The microbial population is spare near the surface of
the sea because the intensity of illumination is inhibitory. Beneath the region of
photic activity there exists another regions inhabited during the day by
vertically migrating zooplanktons. The bacterial populations is distributed more
or less uniformly throughout and below these layers , feeding on descending
organic material and other nutrients.`

Water Quality

The raw or treated water is analysed by testing their physical, chemical and
bacteriological characteristics:

Physical Characteristics:

1. Turbidity
2.Colour
3.Taste and Odour
4.Temperature

Turbidity: - If a large amount of suspended solids are present in water, it will

appear turbid in appearance. The turbidity depends upon fineness and
concentration of particles present in water. Originally turbidity was determined
by measuring the depth of column of liquid required to cause the image of a
candle flame at the bottom to diffuse into a uniform glow. This was measured
 BIOTECHNOLOGY 73

by Jackson candle turbidity meter. The calibration was done based on

suspensions of silica from Fuller's earth. The depth of sample in the tube was
read against the part per million (ppm) silica scale with one ppm of suspended
silica called one Jackson Turbidity unit (JTU). Because standards were prepared
from materials found in nature such as Fuller's earth, consistency in standard
formulation was difficult to achieve. These days turbidity is measured by
applying Nephelometry, a technique to measure level of light scattered by the
particles at right angles to the incident light beam. The scattered light level is
proportional to the particle concentration in the sample. The unit of expression
is Nephelometric Turbidity Unit (NTU). The IS values for drinking water is 10
to 25 NTU.

Colour: - Dissolved organic matter from decaying vegetation or some inorganic

materials may impart colour to the water. It can be measured by comparing the
colour of water sample with other standard glass tubes containing solutions of
different standard colour intensities. The standard unit of colour is that which is
produced by one milligram of platinum cobalt dissolved in one litre of distilled
water. The IS value for treated water is 5 to 25 cobalt units.

Taste and Odour: - Odour depends on the contact of a stimulating substance

with the appropriate human receptor cell. Most organic and some inorganic
chemicals, originating from municipal or industrial wastes, contribute taste and
odour to the water. Taste and odour can be expressed in terms of odour intensity
or threshold values. A new method to estimate taste of water sample has been
developed based on flavour known as 'Flavour Profile Analysis' (FPA). The
character and intensity of taste and odour discloses the nature of pollution or the
presence of microorganisms.

Temperature: - The increase in temperature decreases palatability, because at

elevated temperatures carbon dioxide and some other volatile gases are
expelled. The ideal temperature of water for drinking purposes is5 to 12 °C -
above 25 °C, water is not recommended for drinking.

Chemical Characteristics:

1.pH
2.Acidity
3.Alkalinity
4.Hardness
 BIOTECHNOLOGY 74

5.Chlorides
6.Sulphates
7.Iron
8.Solids
8.Nitrates

pH:-pH value denotes the acidic or alkaline condition of water. It is expressed

on a scale ranging from 0 to 14, which is the common logarithm of the
reciprocal of the hydrogen ion concentration. The recommended pH range for
treated drinking waters is 6.5 to 8.5.

Acidity:-The acidity of water is a measure of its capacity to neutralise bases.

Acidity of water may be caused by the presence of uncombined carbon dioxide,
mineral acids and salts of strong acids and weak bases. It is expressed as mg/L
in terms of calcium carbonate. Acidity is nothing but representation of carbon
dioxide or carbonic acids. Carbon dioxide causes corrosion in public water
supply systems.

Alkalinity:-The alkalinity of water is a measure of its capacity to neutralise

acids. It is expressed as mg/L in terms of calcium carbonate. The various forms
of alkalinity are (a) hydroxide alkalinity, (b) carbonate alkalinity, (c) hydroxide
plus carbonate alkalinity, (d) carbonate plus bicarbonate alkalinity, and (e)
bicarbonate alkalinity, which is useful mainly in water softening and boiler feed
water processes. Alkalinity is an important parameter in evaluating the optimum
coagulant dosage.

Hardness:-If water consumes excessive soap to produce lather, it is said to be

hard. Hardness is caused by divalent metallic cations. The principal hardness
causing cations are calcium, magnesium, strontium, ferrous and manganese
ions. The major anions associated with these cations are sulphates, carbonates,
bicarbonates, chlorides and nitrates.

The total hardness of water is defined as the sum of calcium and magnesium
concentrations, both expressed as calcium carbonate, in mg/L. Hardness are of
two types, temporary or carbonate hardness and permanent or non-carbonate
hardness. Temporary hardness is one in which bicarbonate and carbonate ion
can be precipitated by prolonged boiling. Non-carbonate ions cannot be
precipitated or removed by boiling, hence the term permanent hardness. IS
value for drinking water is 300 mg/L as CaCO3.

Chlorides:-Chloride ion may be present in combination with one or more of the

cations of calcium, magnesium, iron and sodium. Chlorides of these minerals
are present in water because of their high solubility in water. Each human being
 BIOTECHNOLOGY 75

consumes about six to eight grams of sodium chloride per day, a part of which
is discharged through urine and night soil. Thus, excessive presence of chloride
in water indicates sewage pollution. IS value for drinking water is 250 to 1000
mg/L.

Sulphates:-Sulphates occur in water due to leaching from sulphate mineral and

oxidation of sulphides. Sulphates are associated generally with calcium,
magnesium and sodium ions. Sulphate in drinking water causes a laxative effect
and leads to scale formation in boilers. It also causes odour and corrosion
problems under aerobic conditions. Sulphate should be less than 50 mg/L, for
some industries. Desirable limit for drinking water is 150 mg/L. May be
extended up to 400 mg/L.

Iron:-Iron is found on earth mainly as insoluble ferric oxide. When it comes in

contact with water, it dissolves to form ferrous bicarbonate under favourable
conditions. This ferrous bicarbonate is oxidised into ferric hydroxide, which is a
precipitate. Under anaerobic conditions, ferric ion is reduced to soluble ferrous
ion. Iron can impart bad taste to the water, causes discolouration in clothes and
incrustations in water mains. IS value for drinking water is 0.3 to 1.0 mg/L.

Solids:-The sum total of foreign matter present in water is termed as 'total

solids'. Total solids are the matter that remains as residue after evaporation of
the sample and its subsequent drying at a defined temperature (103 to 105 °C).
Total solids consist of volatile (organic) and non-volatile (inorganic or fixed)
solids. Further, solids are divided into suspended and dissolved solids. Solids
that can settle by gravity are settle able solids. The others are non-settle able
solids. IS acceptable limit for total solids is 500 mg/L and tolerable limit
is 3000 mg/L of dissolved limits.

Nitrates:-Nitrates in surface waters occur by the leaching of fertilizers from soil

during surface run-off and also nitrification of organic matter. Presence of high
concentration of nitrates is an indication of pollution. Concentration of nitrates
above 45 mg/L causes a disease methemoglobinemia. IS value is 45 mg/L.

Microbial analysis of water purity

The natural water body such as lakes, streams, rivers contain sufficient amount
of nutrients that support the ground of microorganisms. However, there are
different ways by which microorganisms enter in water supply, for example

 Broken sewer lines,

 Congested centers,
 Inappropriate treatment,
 BIOTECHNOLOGY 76

 Unhygienic environment at public places of water collection

 People suffering from communicable diseases such as dyentenry, typhoid
etc.

Bacteriological Characteristics:

Bacterial examination of water is very important, since it indicates the degree of

pollution. Water polluted by sewage contains one or more species of disease
producing pathogenic bacteria. Pathogenic organisms cause water borne
diseases, and many non-pathogenic bacteria such as E.Coli, a member of
coliform group, also live in the intestinal tract of human beings. Coliform itself
is not a harmful group but it has more resistance to adverse condition than any
other group. So, if it is ensured to minimize the number of coliforms, the
harmful species will be very less. So, coliform group serves as indicator of
contamination of water with sewage and presence of pathogens.

The faecal Coliforms:

On the basic of microbiological examination of water, its potability may be

ascertained. Intestinal bacteria present in water generally do not survive in
aquatic environment due to physiological stress but if entered human system in
the mean while they cause serious problem. The characteristic groups of
intestinal bacteria are the coliforms.

Coliforms are defined as facultative anaerobic, gram –negative, non-sporing,

rod shaped bacteria. The coliform groups are present in water due to faecal
contamination i.e discharge of faeces by human and other animals in water.

1. Sanitary tests of coliforms

The original test for the presence of coliform in water is done by standard test

a. The presumptive test

b. The confirmed test
c. The complete test

a.The Presumptive Test

A series of fermentation tube each containing lactose broth or lauryl trypoe

broth of known concentration, are incubated with known amount of water.
These tubes are inoculated with known amount of water. Then these tubes are
incubated for 24 to 48 hours at 350C. Generally five fermentation tubes
 BIOTECHNOLOGY 77

containing single or double strength broth are inoculated with 10ml water, 5tube
with 1 ml water and another 5 tube with 0.1 ml water. At the end of the 24
hours of incubation, the tubes indicate that the coliforms are absent. These tubes
are incubated for an additional 24 hours to be sure for absence of coliform,
i.e.gas production.

b.Confirmed Test

If a positive test of gas production is obtained, it does not mean that coliform
are present. The other organisms too also give false positive presumptive test
because they are also capable of fermenting lactose with formation of acid and
gas. All the tubes showing gas within 48 hours at 350C are used for the presence
of coliforms. It is of two types.

a. The positive presumptive fermentation tube is gently shaken. A drop of

its culture is transferred to brilliant green lactose bile broth fermentation.
The tubes are incubated f 48hours at 350C. The appearance of gas within
this period indicates for positive confirmed test.
b. The second confirmed test is done by eosine methylene blue (EMB) agar
or endo agar method. In this method a definite amount of two strains
(eosine and methylene blue) is added to a melted lactose agar. The
medium is poured into Petri dices. Over the surface of EMB agar medium
a loopful culture from each positive fermentation tube is streaked. Plates
are incubated at 350C for 24 hours keeping them in inverted position.
There develops three types of colony, a. typical colony, b. atypical
colony, c. negative colony. The development of typical colony shows a
positive confirmed test.

c.Complete Test

In the last the complete test is performed to ascertain about the presence of
coliforms in water. The purpose of complete test is to determine whether (a) the
colonies growing on EMB or endo agar are again capable of fermenting lactose
, formic acid and gas and (b) the organisms transferred to agar slant shows the
morphological appearance of coliform group.

2.TheMost Probable Number

Most probable number is a number which represents the bacterial density which
is most likely to be present. E.Coli is used as indicator of pollution. E.Coli
ferment lactose with gas formation with 48 hours incubation at 35°C. Based on
this E.Coli density in a sample is estimated by multiple tube fermentation
 BIOTECHNOLOGY 78

procedure, which consists of identification of E.Coli in different dilution

combination. MPN value is calculated as follows:

Five 10 ml (five dilution combination) tubes of a sample is tested for E.Coli. If

out of five only one gives positive test for E.Coli and all others negative. From
the tables, MPN value for one positive and four negative results is read which
2.2 is in present case. The MPN value is expressed as 2.2 per 100 ml. These
numbers are given by Maccardy based on the laws of statistics.

Membrane Filter Technique

In this test a known volume of water sample is filtered through a membrane

with opening less than 0.5 microns. The bacteria present in the sample will be
retained upon the filter paper. The filter paper is put in contact of a suitable
nutrient medium and kept in an incubator for 24 hours at 35°C.The bacteria will
grow upon the nutrient medium and visible colonies are counted. Each colony
represents one bacterium of the original sample. The bacterial count is
expressed as number of colonies per 100 ml of sample.

3. IMViC Tests

The IMViC tests are a group of individual tests used in microbiology lab testing
to identify an organism in the coliform group.Except for the lowercase "i",
which is added for ease of pronunciation, each of the letters in "IMViC" stands
for one of these tests. "I" is for indole test; "M" is formethyl red test; "V" is
for Voges-Proskauer test, and "C" is for citrate test. The lower case "i" is merely
for "in", as the Citrate test requires coliform samples to be placed "in Citrate".

Indole Test

In this test, the organism under consideration is grown in peptone water broth. It
contains tryptophan, which under the action of enzyme tryptophanase is
converted to an Indole molecule, pyruvate and carbon dioxide. The indole is
then extracted from the broth by means of xylene. To test the broth for indole
production, Kovac's reagent is added after incubation. A positive result is
indicated by a pink/red layer forming on top of the liquid.

Methyl red and Voges–Proskauer test

These tests both use the same broth for bacterial growth. The broth is called
MRVP broth. After growth, the broth is separated into two different tubes, one
for the methyl red (MR) test and one for the Voges-Proskauer (VP) test.
The methyl red test detects production of acids formed during metabolism
using mixed acid fermentation pathway using pyruvate as a substrate. The pH
 BIOTECHNOLOGY 79

indicator Methyl Red is added to one tube and a red colour appears at pH's
lower than 4.2, indicating a positive test (mixed acid fermentation is used). The
solution remaining yellow (pH = 6.2 or above) indicates a negative test,
meaning the butanediol fermentation is used.
The VP test uses alpha-naphthol and potassium hydroxide to test for the
presence of acetylmethylcarbinol (acetoin), an intermediate of the 2,3-
butanediol fermentation pathway. After adding both reagents, the tube is shaken
vigorously then allowed to sit for 5-10 minutes. A pinkish-red colour indicates a
positive test, meaning the 2; 3-butanediol fermentation pathway is used.

Citrate test
This test uses Simmon's citrate agar to determine the ability of a microorganism
to use citrate as its sole carbon source. The agar contains citrate and ammonium
ions (nitrogen source) and bromothymol blue as an indicator. The citrate agar is
green before inoculation, and turns blue as a positive test indicator, meaning
citrate is utilized.

Water Purification, Average Disposal and Sewage Purification.

Introduction
In urban areas for domestic and industrial uses the source of water is generally
reservoir, river, lake, and wells. Out of this total water supplied, generally 60 to
80% contributes as a wastewater. In most of the cities, wastewater is let out
partially treated or untreated and it either percolatesinto the ground and in turn
contaminates the ground water or it is discharged into the natural drainage
system causing pollution in downstream water bodies.
Water scarcity is a function not only of volumetric supply, but also of quality
sufficient to meet the demand. The drinking water demand is perhaps the largest
demand for high quality water apart from many industrial uses which also
require high quality water. Agriculture, by far the largest consumer of water,
also suffers when water supplies become saline. In India, water pollution comes
from the main sources such as domestic sewage, industrial effluents, leachets
from landfills, and run-off from solid waste dumps and agriculture land.
 BIOTECHNOLOGY 80

Domestic sewage (black water) and sullage (grey water) is the main source of
water pollution in India, especially in and around large urban centres.
In the past disposal of waste from water closets was carried out manually and
wastewatergenerated from kitchen and bathrooms was allowed to flow along the
open drains. Thisprimitive method was modified and replace by a water carriage
system, in which these wastesare mixed with sufficient quantity of water. This
waste is carried through closed conduitsunder the conditions of gravity flow.
This mixture of water and waste products is known as sewage.

1. Waste water
Industrial wastewater: It is the wastewater generated from the industrial and
commercial areas. This wastewater contains objectionable organic and
inorganic compounds that may not be amenable to conventional treatment
processes.
Sanitary sewage: Sewage originated from the residential buildings comes
under this category. This is very foul in nature. It is the wastewater generated
from the lavatory basins, urinals andwater closets of residential buildings, office
building, theatre and other institutions. It is alsoreferred as domestic
wastewater.
Sewage: It indicates the liquid waste originating from the domestic uses of
water. It includes sullage, discharge from toilets, urinals, wastewater generated
from commercial establishments, institutions, industrial establishments and also
the groundwater and storm water that may enter into the sewers. Its
decomposition produces large quantities of malodorous gases, and it contains
numerous pathogenic or disease producing bacteria, along with high
concentration of organic matter and suspended solids.
Sewage Treatment Plant: It is a facility designed to receive the waste from
domestic, commercial and industrial sources and to remove materials that
damage water quality and compromise public health and safety when
 BIOTECHNOLOGY 81

discharged into water receiving systems or land. It is combination of unit

operations and unit processes developed to treat the sewage to desirable
standards to suit effluent norms defined by regulating authority.
Sewer: It is an underground conduit or drain through which sewage is carried to
a point of discharge or disposal. There are three types of sewer systems that are
commonly used for sewage collection. Separate sewers are those which carry
the house hold and industrial wastes only. Storm water drains are those which
carry rain water from the roofs and street surfaces.
Combine sewers are those which carry both sewage and storm water together in
the same conduit. House sewer (or drain) is used to discharge the sewage from a
building to a streetsewer. Lateral sewer is a sewer which collects sewage
directly from the household buildings.
Branch sewer or submain sewer is a sewer which receives sewage from a
relatively small area. Main sewer or trunk sewer is a sewer that receives sewage
from many tributary branches and sewers, serving as an outlet for a large
territory.
Sewerage: The term sewerage refers the infrastructure which includes device,
equipment and appurtenances for the collection, transportation and pumping of
sewage, but excluding works for the treatment of sewage. Basically it is a water
carriage system designed and constructed for collecting and carrying of sewage
through sewers.
Storm water: It indicates the rain water of the locality.
Subsoil water: Groundwater that enters into the sewers through leakages is
called subsoil water.
Sullage: This refers to the wastewater generated from bathrooms, kitchens,
washing place and wash basins, etc. Composition of this waste does not involve
higher concentration of organic matter and it is less polluted water as compared
to sewage.
 BIOTECHNOLOGY 82

Wastewater: The term wastewater includes both organic and inorganic

constituents, in soluble or suspended form, and mineral content of liquid waste
carried through liquid media. Generally the organic portion of the wastewater
undergoes biological decompositions and the mineral matter may combine with
water to form dissolved solids.
2. Sources of Sewage
The wastewater generated from the household activities contributes to the major
part of the sewage. The wastewater generated from recreational activities,
public utilities, commercial complexes, and institutions is also discharged in to
sewers. The wastewater discharged from small and medium scale industries
situated within the municipal limits and discharging partially treated or
untreated wastewater in to the sewers also contributes for municipal wastewater.
3. Effect of Untreated Wastewater Disposal
The daily activities of human beings produce both liquid and solid wastes. The
liquid portion of the wastewater is necessarily the water supplied by the
authority or through private watersources, after it has fouled by variety of uses.
The sources of wastewater generation can bedefined as a combination of the
liquid or water-carried wastes removed from residences, institutions, and
commercial and industrial establishments, together with groundwater, surface
water, and storm water as may be present.
If the untreated wastewater is allowed to accumulate, it will lead to highly
unhygienic conditions. The organic matter present in the wastewater will
undergo decomposition with production of large quantities of malodorous gases.
If the wastewater is discharged without treatment in the water body, this will
result in the depletion of Dissolved Oxygen (DO) from the water bodies. Due to
depletion of DO, the survival of aquatic life will become difficult, finally
leading to anaerobic conditions in the receiving waters. The nutrients present in
the wastewater can stimulate the growth of aquatic plants, leading to problems
 BIOTECHNOLOGY 83

like eutrophication. In addition, the untreated domestic wastewater usually

contains numerous pathogenic ordisease causing microorganisms, that dwell in
the human intestinal tract or it may be present incertain industrial wastewaters.
Apart from this, the wastewater contains inorganic grittymaterials. The
continuous deposition of this inorganic material may reduce the capacity of
water body considerably over a period.

4. Objectives of Sewage Collection and Disposal

The objective of sewage collection and disposal is to ensure that sewage
discharged from communities is properly collected, transported, treated to the
required degree so as not to cause danger to human health or unacceptable
damage to the natural environment and finally disposed of without causing any
health or environmental problems. Thus, efficient sewerage scheme can achieve
the following:
• To provide a good sanitary environmental condition of city protecting public
health.
• To dispose the human excreta to a safe place by a safe and protective means.
• To dispose of all liquid waste generated from community to a proper place to
prevent a favourable condition for mosquito breeding, fly developing or bacteria
growing.
.To treat the sewage, as per needs, so as not to endanger the body of water or
groundwater or land to get polluted where it is finally disposed of. Thus, it
protects the receiving environment from degradation or contamination.

5. Sewage Characteristics
Characterization of wastes is essential for an effective and economical waste
management programme. It helps in the choice of treatment methods deciding
the extent of treatment, assessing the beneficial uses of wastes and utilizing the
 BIOTECHNOLOGY 84

waste purification capacity of natural bodies of water in a planned and

controlled manner.
Domestic sewage comprises spent water from kitchen, bathroom,
lavatory, etc. The factors which contribute to variations in characteristics of the
domestic sewage are daily per capita use of water, quality of water supply and
the type, condition and extent of sewerage system, and habits of the people.
Municipal sewage, which contains both domestic and industrial wastewater,
may differ from place to place depending upon the type of industries and
industrial establishment. The important characteristics of sewage are:
a. Temperature
The observations of temperature of sewage are useful in indicating solubility of
oxygen, which affects transfer capacity of aeration equipment in aerobic
systems, and rate of biological activity. Extremely low temperature affects
adversely on the efficiency of biological treatment systems and on efficiency of
sedimentation. In general, under Indian conditions the temperature of the raw
sewage is observed to be between 15 and 350C at various places in different
seasons.
b. The pH
The hydrogen ion concentration expressed as pH, is a valuable parameter in the
operation of biological units. The pH of the fresh sewage is slightly more than
the water supplied to the community. However, decomposition of organic
matter may lower the pH, while the presence of industrial wastewater may
produce extreme fluctuations. Generally the pH of raw sewage is in the range
5.5 to 8.0.
c. Colour and Odour
Fresh domestic sewage has a slightly soapy and cloudy appearance depending
upon its concentration. As time passes the sewage becomes stale, darkening in
colour with a pronounced smell due to microbial activity.
 BIOTECHNOLOGY 85

d. Solids
Though sewage generally contains less than 0.5 percent solids, the rest being
water, still the nuisance caused by the solids cannot be overlooked, as these
solids are highly degradable and therefore need proper disposal. The sewage
solids may be classified into dissolved solids, suspended solids and volatile
suspended solids. The estimation of suspended solids, both organic and
inorganic, gives a general picture of the loadon sedimentation and grit removal
system during sewage treatment. Dissolved inorganic fraction is to be
considered when sewage is used for land irrigation or any other reuse is
planned.
e. Nitrogen and Phosphorus
The principal nitrogen compounds in domestic sewage are proteins, amines,
amino acids, and urea. Ammonia nitrogen in sewage results from the bacterial
decomposition of these organic constituents. Nitrogen being an essential
component of biological protoplasm, its concentration is important for proper
functioning of biological treatment systems and disposal on land. Generally, the
domestic sewage contains sufficient nitrogen, to take care of the needs of the
biological treatment. For industrial wastewater if sufficient nitrogen is not
present it is required to be added externally.
Phosphorus is contributing to domestic sewage from food residues containing
phosphorus and their breakdown products. The use of increased quantities of
synthetic detergents adds substantially to the phosphorus content of sewage.
Phosphorus is also an essential nutrient for the biological processes. The
concentration of phosphorus in domestic sewage is generally adequate to
support aerobic biological wastewater treatment.
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f. Chlorides
Concentration of chlorides in sewage is greater than the normal chloride content
of water supply. The chloride concentration in excess than the water supplied
can be used as an index of the strength of the sewage.
g. Organic Material
Organic compounds present in sewage are of particular interest for
environmental engineering. A large variety of microorganisms (that may be
present in the sewage or in the receiving water body) interact with the organic
material by using it as an energy or material source. The utilization of the
organic material by microorganisms is called metabolism. The conversion of
organic material by microorganism to obtain energy is called catabolism and the
incorporation of organic material in the cellular material is called anabolism.
To describe the metabolism of microorganisms and oxidation of organic
material, it is necessary to characterize quantitatively concentration of organic
matter in different forms.
There are two standard tests based on the oxidation of organic material:
1) The Biochemical Oxygen Demand (BOD) and
2) The Chemical Oxygen Demand (COD) tests.
In both tests, the organic material concentration is measured during the test. The
essential differences between the COD and the BOD tests are in the oxidant
utilized and the operational conditions imposed during the test such as
biochemical oxidation and chemical oxidation. The other method for measuring
organic material is the development of the Total Organic Carbon (TOC) test as
an alternative to quantify the concentration of the organic material.
Biochemical Oxygen Demand (BOD): The BOD of the sewage is the amount
of oxygen required for the biochemical decomposition of biodegradable organic
matter under aerobic conditions. The oxygen consumed in the process is related
to the amount of decomposable organic matter.
 BIOTECHNOLOGY 87

Chemical Oxygen Demand (COD): The COD gives the measure of the
oxygen required for chemical oxidation. It does not differentiate between
biological oxidisable and nonoxidisable material. However, the ratio of the
COD to BOD does not change significantly for particular waste and hence this
test could be used conveniently for interpreting performance efficiencies of the
treatment units.
In COD test, the oxidation of organic matter is essentially complete within two
hours, whereas, biochemical oxidation of organic matter takes several weeks.
h.Toxic Metals and Compounds
Some heavy metals and compounds such as chromium, copper, cyanide, which
are toxic may find their way into municipal sewage through industrial
discharges. The concentration of these compounds is important if the sewage is
to treat by biological treatment methods or disposed of in stream or on land. In
general these compounds are within toxic limits insanitary sewage; however,
with receipt of industrial discharges they may cross the limits in municipal
wastewaters.
6 .Classification and Application of Wastewater Treatment Methods
The degree of treatment required can be determined by comparing the influent
wastewater characteristics to the required effluent characteristics, adhering to
the regulations. Number of different treatment alternatives can be developed to
achieve the treated wastewater quality.
Classification of Treatment Methods
The individual treatment methods are usually classified as:
 Physical unit operations
 Chemical unit processes
 Biological unit processes.
Physical Unit Operations: Treatment methods in which the application of
physical forces predominates are known as physical unit operations. Most of
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these methods are based on physical forces, e.g. screening, mixing, flocculation,
sedimentation, flotation, and filtration.
Chemical Unit Processes: Treatment methods in which removal or conversion
of contaminant is brought by addition of chemicals or by other chemical
reaction are known as chemical unit processes, for example, precipitation, gas
transfer, adsorption, and disinfection.
Biological Unit Processes: Treatment methods in which the removal of
contaminants is brought about by biological activity are known as biological
unit processes.
 This is primarily used to remove biodegradable organic substances from
the wastewater, either in colloidal or dissolved form.
 In the biological unit process, organic matter is converted into gases that
can escape to the atmosphere and into bacterial cells, which can be
removed by settling.
 Biological treatment is also used for nitrogen removal and for
phosphorous and sulphate removal from the wastewater.
The different treatment methods used in wastewater treatment plant are
classified in three different categories as:
 Primary Treatment: Refers to physical unit operations.
 Secondary Treatment: Refers to chemical and biological unit processes.
 Tertiary Treatment: Refers to any one or combination of two or all three
i.e., physical unit operations and chemical or biological unit processes,
used after secondary treatment.
Primary Treatment Units:
The primary treatment incorporates unit operations for removal of floating and
suspended solids from the wastewater. They are also referred as the physical
unit operations. The unit operations used are screening for removing floating
papers, rages, cloths, plastics, cansstoppers, labels, etc.; grit chambers or
 BIOTECHNOLOGY 89

detritus tanks for removing grit and sand; skimming tanks for removing oils and
grease; and primary settling tank for removal of residual settleable suspended
matter.
Screens
Screen is the first unit operation in wastewater treatment plant. This is used to
remove larger particles of floating and suspended matter by coarse screening.
This is accomplished by a set of inclined parallel bars, fixed at certain distance
apart in a channel. The screen can be of circular or rectangular opening. The
screen composed of parallel bars or rods is called a rack.
The screens are used to protect pumps, valves, pipelines, and other
appurtenances from damage or clogging by rags and large objects.
Industrial wastewater treatment plant may or may not need the screens.
However, when packing of the product and cleaning of packing bottles/
containers is carried out, it is necessary to provide screens even for industrial
wastewater treatment plant to separate labels, stopper, cardboard, and other
packing materials.
Types of Screens
Screens can be broadly classified depending upon the opening size provided as
coarse screen (bar screens) and fine screens. Based on the cleaning operation
they are classified as manually cleaned screens or mechanically cleaned screens.
i) Coarse Screen
It is used primarily as protective device and hence used as first treatment unit.
Common type of these screens are bar racks (or bar screen), coarse woven-wire
screens, and comminutors.
Bar screens are used ahead of the pumps and grit removal facility. This screen
can be manually cleaned or mechanically cleaned. Manually cleaned screens are
used in small treatment plants. Clear spacing between the bars in these screens
may be in the range of 15 mm to 40 mm.
 BIOTECHNOLOGY 90

(ii)Grinder or Comminutor
It is used in conjunction with coarse screens to grind or cut the screenings. They
utilize cutting teeth (or shredding device) on a rotating or oscillating drum that
passes through stationary combs (or disks). Object of large size are shredded
when it will pass through the thin opening of size 0.6 to 1.0 cm.
(iii) Fine Screen
Fine screens are mechanically cleaned screens using perforated plates, woven
wire cloths, or very closely spaced bars with clear openings of less than 20 mm,
less than 6 mm typical.
Commonly these are available in the opening size ranging from 0.035 to 6 mm.
Fine screens are used for pre-treatment of industrial wastewaters and are not
suitable for sewage due to clogging problems, but can be used after coarse
screening. Fine screens are also used to remove solids from primary effluent to
reduce clogging problem of trickling filters. Various types of micro screens
have been developed that are used to upgrade effluent quality from secondary
treatment plant.
Grit Chamber
Grit chamber is the second unit operation used in primary treatment of
wastewater and it is intended to remove suspended inorganic particles such as
sandy and gritty matter from the wastewater. This is usually limited to
municipal wastewater and generally not required for industrial effluent
treatment plant, except some industrial wastewaters which may have grit.
The grit chamber is used to remove grit, consisting of sand, gravel, cinder, or
other heavy solids materials that have specific gravity much higher than those of
the organic solids in wastewater. Grit chambers are provided to protect moving
mechanical equipment from abrasion and abnormal wear; avoid deposition in
pipelines, channels, and conduits; and to reduce frequency of digester cleaning.
 BIOTECHNOLOGY 91

Skimming Tank
The floating solid materials such as soap, vegetables, debris, fruit skins, and
pieces of corks, etc. and oil and grease are removed from the wastewater in
skimming tanks. A skimming tank is achamber designed so that floating matter
rises and remains on surface of the wastewater until removed, while the liquid
flows continuously through outlet or partition below the waterlines. The
detention time in skimming tank is 3 minutes. To prevent heavy solids from
settling at the bed, compressed air is blown through the diffusers placed in the
floor of the tank. Due to compress air supply, the oily matters rise upward and
are collected in the side trough, from where they are removed.
Primary Sedimentation Tank
Effluent of the grit chamber, containing mainly lightweight organic matter, is
settled in the primary sedimentation tanks. The objective of treatment by
sedimentation is to remove readily settleable solids and floating material and
thus to reduce the suspended solids content when they are used as preliminary
step to biological treatment, their function is to reduce the load on the biological
treatment units.
Secondary Treatment
The effluent from primary treatment is treated further for removal of dissolved
and colloidal organic matter in secondary treatment. This is generally
accomplished through biochemical decomposition of organic matter, which can
be carried out either under aerobic or anaerobic conditions. In these biological
units, bacteria’s decompose the fine organic matter, to produce clearer effluent.
The end products of aerobic decomposition are mainly carbon dioxide and
bacterial cells, and that for anaerobic process are CH4, CO2 and bacterial cells.
The biological reactor in which the organic matter is decomposed (oxidized) by
aerobicbacteria may consist of:
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1) Filters (tricking filters),

2) Activated Sludge Process (ASP),
3) Oxidation ponds, etc.
Trickling Filter
A trickling filter is a fixed film attached growth aerobic process used for
removal of organic matter from the wastewater. The surface of the bed is
covered with the biofilm and as the wastewater trickles over this media surface,
organic matter from the wastewater comes in contact with the aerobic bacteria
and oxidation of organic matter occurs. In the past rock was used as a bed
material with size ranging from 25 mm to 100 mm. Now plastic media which
offers higher surface area per unit volume is used. The media is randomly
packed in the reactor and the wastewater is applied on the top through rotary
arm which trickles down over the filter media surface (Figure 4.1). Hence, this
reactor is known as trickling filter. Since, the wastewater is applied through the
rotary arm from the top of the reactor the biofilm grown on the media surface
receives wastewater intermittently. As the wastewater trickles down leaving the
wet biofilm, the biofilm is exposed to the air voids present in the media, and
thus oxygen from the air, after getting dissolved in the water adhering on
biofilm, is made available to aerobic bacteria grown in the biofilm by diffusion
through the biofilm. The end product CO2 diffuses out of the biofilm into the
flowing liquid. Treated wastewater is collected from the bottom of the bed
through an under-drainage system and is settled in the final settling tank.
The biological film or slime forms on the surface of the filter media after
application of wastewater. Organic matter is adsorbed on the slime layer and it
is degraded by the aerobic microorganisms present in the slime. As the
thickness of the slime layer increases the condition near the surface of the media
becomes anaerobic because of limitations of availability of oxygen. At this
stage the microbes loose their ability to cling to the surface of the media and the
 BIOTECHNOLOGY 93

slime layer gets detached and washed out along with flowing liquid. This
phenomenon is called as ‘sloughing’. Soon after the sloughing the new slime
layer formation starts. Hence secondary sedimentation tank (SST) is provided to
settle this washed out biomass.

Fig 4.1: trickling filter

Activated Sludge Process
It is aerobic biological treatment system. The settled wastewater is aerated in an
aeration tank for a period of few hours. During the aeration, the microorganisms
in the aeration tank stabilize the organic matter. In this process part of the
organic matter is synthesized into new cells and part is oxidized to derive
energy. The synthesis reaction followed by subsequent separation of the
resulting biological mass and the oxidation reaction is the main mechanism of
BOD removal in the activated sludge process.
The biomass generated in the aeration tank is generally flocculent and it is
separated from the aerated wastewater in a secondary settling tank and is
recycled partially to the aeration tank.
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The mixture of recycled sludge and wastewater in the aeration tank is referred
as mixed liquor. The recycling of sludge helps in the initial built up of a high
concentration of active microorganism in the mixed liquor, which accelerates
BOD removal. Once the required concentration of microorganism in the mixed
liquor has been reached its further increase is prevented by the regulating
quantity of sludge recycled and wasting the excess sludge from the system.
Aeration units are main units of activated sludge process, the main aim of which
is to supplyoxygen to the wastewater to keep the reactor content aerobic and to
mix up the return sludge with wastewater thoroughly. The usual practice is to
keep the detention period between 6 to8 hours for treatment of sewage or
similar industrial wastewater. The volume of aeration tank is also decided by
considering the return sludge, which is about 25 to 50% of the wastewater
volume. The mode of air supply in aeration tank can be either diffused air
aeration, by supplementing compressed air from tank bottom, or by mechanical
aerators provided at surface or by both diffused aeration and mechanical
aerators.

Figure 4.2 Conventional Activated Sludge Process

Oxidation Ponds
Oxidation ponds are the stabilization ponds, which received partially treated
sewage. It is an earthen pond dug into the ground with shallow depth. The pond
should be at least 1.0 m deep to discourage growth of aquatic weeds and should
not exceed 1.8 m. The detention time in the pond is usually 1 to 4 weeks
depending upon sunlight and temperature. Better efficiency of treatment is
 BIOTECHNOLOGY 95

obtained if several ponds are placed in series so that the sewage flows
progressively from one to another unit until it is finally discharged.
Tertiary Treatment
Secondary treatment removes 85 to 95 percent of BOD and TSS and minor
portions of nitrogen, phosphorus, and heavy metals. Tertiary treatment is the
next wastewater treatment process after secondary treatment. This treatment is
sometimes called as the final or advanced treatment and consists of removing
the organic load left after secondary treatment for removal of nutrients from
sewage and particularly to kill the pathogenic bacteria. The effluents from
secondary sewage treatment plants contain both nitrogen (N) and phosphorus
(P). N and P are ingredients in all fertilizers. When excess amounts of N and P
are discharged, plant growth in the receiving waters may be accelerated which
results in eutrophication in the water body receiving such waste. Algae growth
may be stimulated causing blooms which are toxic to fish life as well as
aesthetically unpleasing. Secondary treated effluent also contains suspended,
dissolved, and colloidal constituents which may be required to be removed for
stipulated reuse or disposal of the treated effluent.
The purpose of tertiary treatment is to provide a final treatment stage to raise the
effluent quality before it is discharged to the receiving environment such as sea,
river, lake, ground, etc., or to raise the treated water quality to such a level to
make it suitable for intended reuse. This step removes different types of
pollutants such as organic matter, SS, nutrients, pathogens, and heavy metals
that secondary treatment is not able to remove. Wastewater effluent becomes
even cleaner in this treatment process through the use of stronger and more
advanced treatment systems. It includes sedimentation, coagulations, membrane
processes, filtration, ion exchange, activated carbon adsorption, electrodialysis,
nitrification and denitrification, etc. Tertiary treatment is costly as compared to
primary and secondary treatment methods.
 BIOTECHNOLOGY 96

Need of Tertiary treatment

Tertiary treatment may be provided to the secondary effluent for one or more of
the following contaminant further.
 To remove total suspended solids and organic matter those are present in
effluents after secondary treatment.
 To remove specific organic and inorganic constituents from industrial
effluent to make it suitable for reuse.
 To make treated wastewater suitable for land application purpose or
directly discharge it into the water bodies like rivers, lakes, etc.
 To remove residual nutrients beyond what can be accomplished by earlier
treatment methods.
 To remove pathogens from the secondary treated effluents.
 To reduce total dissolved solids (TDS) from the secondary treated
effluent to meet reuse quality standards.
Sludge Management
In the context of wastewater treatment residual is used to refer “sludge”. The
term sludge refers to the solids that are settled and separated during wastewater
treatment. It is necessary to treat properly or dispose the sludge generated
during the various stages of wastewater treatment like primary sedimentation,
secondary sedimentation and sludge generated from advanced (tertiary)
treatment. The quantity of sludge generated depends upon the degree of
treatment or quality of treated effluent required i.e., higher the degree of
wastewater treatment, the larger the quantity of sludge to be treated and
handled. Because of strict rules and regulations involving the handling and
disposal of sludge, it has become necessary to reduce the volume of sludge in
order to reduce the operating costs (approximately 50% of the plant cost) of
 BIOTECHNOLOGY 97

treatment plants. Hence a properly designed and efficiently operated sludge

processing and disposal system is essential to the overall success of the
wastewater treatment plant. The sludge generated during the wastewater
treatment can be classified into three categories:
Primary Sludge: Sludge settled in primary settling tanks comes under this
category which contains 3% to 7% solids out of which approximately 60% to
80% are organic. Primary sludge solids are usually gray in colour, slimy, fairly
coarse, and with highly obnoxious odours. This sludge is difficult to dewater
without treatment, hence digestion is necessary. This type of sludge can be
digested readily by aerobic or anaerobic bacteria under favourable operating
conditions.
Secondary Sludge: This type of sludge from secondary settling tanks has
commonly a brownish, flocculent appearance and an earthy odour. It consists
mainly of microorganism containing 75% to 90% organic fraction and
remaining inert materials.
Tertiary Sludge: The nature of sludge from the tertiary (advanced) treatment
process depends on the unit process followed like membrane processes or
chemical methods, etc. Chemical sludge from phosphorus removal is difficult to
handle and treat. Tertiary sludge from biological nitrification and denitrification
is similar to waste activated sludge.
The sludge is generated in the wastewater treatment plant in the form of already
present settleable solids, when settled in the PST, and in the form of biological
cell mass, generated in the biological secondary treatment that settled in SST.
The water content of the sludge is very high, and solids constitute very small
part of it. Therefore before final disposal further treatment is required for this
sludge to reduce water content and oxygen demand.
 BIOTECHNOLOGY 98

Sludge is stabilized to
(i) reduce pathogens,
(ii) eliminate odours,
(iii) inhibit, reduce, or eliminate the potential for decomposition, and
(iv) improve dewatering characteristics of the sludge to reduce volume for
disposal.

There are four means to eliminate this nuisance condition through stabilization.
They are
(1) biological reduction of volatile solids,
(2) chemical oxidation of volatile solids,
(3) addition of chemicals to make conditions not suitable for bacterial growth,
(4) application of heat to disinfect or sterile the sludge.
Disposal of the sludge presents problems due to
(i) the solids present are mostly organic and undergo decomposition, and
(ii) volume of the sludge is many times than the solids constituents. Hence,
further treatment is required for reducing volume of the sludge, stabilizing
organic matter present in the sludge, and improving its filtration ability for easy
dewatering. The reduction in volume of the sludge can be achieved by
thickening, dewatering and drying; and stabilization of organic matter can be
obtained by employing digestion (aerobic or anaerobic), incineration,
composting, heat treatment, and chlorine oxidation or lime stabilization.
Sludge Thickening
Sludge thickening or dewatering is adopted for reducing the volume of sludge
and increasing the solid contents. This will help in following:
(i) Increasing the loading on the digester, requiring lesser digester volume,
(ii) Increase feed solids concentration to vacuum filters,
 BIOTECHNOLOGY 99

(iii) Economize transport and handling cost of sludge within the plant and final
disposal,
(iv)Minimize land required and handling cost for final disposal of the digested
sludge on land, and
(v) Save fuel if incineration is practiced.
In sludge thickeners, greater amount of water is removed from the sludge than
what could obtain from sedimentation tank. This reduces overall volume of the
sludge considerably. The thickening of the sludge can be achieved either by
gravity thickening, application of air floatation or by centrifugation.
Gravity thickening
Gravity thickening is accomplished in a tank similar in design to a
sedimentation tank. This is most commonly used for concentrating the sludge
for achieving saving in the digester volume and sludge handing cost. This is
used for primary sludge and for combine primary and secondary sludge, and it
is not suitable for ASP sludge alone. When the ASP sludge is more than 40%
(weight ratio) of the total combined sludge, gravity thickening is not effective
and other methods of thickening have to be considered.
Gravity thickeners can be operated either as continuous flow or fill and draw
type, with or without chemical addition. The thickened sludge is withdrawn
from the bottom of the tank and pumped to the digester. The supernatant is
returned to the PST. Use of slow stirring improves efficiency. Continuous feed
tanks are circular in shape with central feeding and overflow at the periphery.
The side water depth is kept about 3.0 m. Due to relatively high concentration
of the solids, as compared to PST or SST, the settling in thickeners will follow
hindered settling in the beginning and compaction at later stage. Concentration
of the underflow solids is governed by the depth of sludge blanket up to 1 m
beyond which there is very little influence of the blanket. Thickeners are
designed for hydraulic loading of 20 to 25 m3/m2.d. Loading rates lesser than
 BIOTECHNOLOGY 100

12 m3/m2.d are likely to give very high solids concentration, which may require
dilution with plant effluent for transporting. The underflow solid concentration
will increase with increase in detention time, and detention time of about 24 h
will produce maximum compaction. During peak condition, lesser detention
time is allowed to keep the sludge blanket sufficiently below the overflow weirs
to prevent excessive solids carryover.

Figure 4.3: Schematic diagram of a gravity thickening unit

Air floatation
By applying air under pressure or vacuum the thickening of the sludge can be
achieved. This is normally preferred for ASP sludge. This requires additional
equipment, power for operation, skilled supervision for operation and
maintenance, hence it is costly. However, better removal of oil and grease,
solids, and odour control are the advantages offered by this method. Addition of
alum, polyelectrolytes can increase the efficiency of the flotation unit. Alum
will increase the sludge but polyelectrolyte will not increase the solids
concentration but improves solids capture from 90 to 98%.

Centrifugation
Thickening by centrifugation is used only when the land available is limited and
sludge characteristics will not permit adoption of other methods. This will
require high maintenance and operational cost. A centrifuge acts both ways to
 BIOTECHNOLOGY 101

thicken and to dewater sludge. The centrifuge process separates liquid and solid
by the influence of centrifugal force which is typically 50 to 300 times that of
gravity.

Anaerobic Sludge Digestion

In anaerobic digestion process the organic material, in mixture of primary
settled sludge and biological sludge from secondary clarifier, is converted to
CH4 and CO2 under anaerobic conditions. This is carried out in an air tight
reactor in absence of oxygen. Sludge is introduced continuously or
intermittently and retained in the reactor for varying periods of time. Two basic
processes involved in anaerobic digestion are liquefaction and gasification. The
stabilized sludge which is withdrawn continuously or intermittently from the
process, is non putrescible, and its pathogen content is also greatly reduced.
Anaerobic digestion is defined as being biological oxidation of degradable
organic sludge by microbes under anaerobic condition. It occurs in absence of
oxygen and organic matter acts as food source for microorganisms. Most
microbes used in this digestion are obligate anaerobes or facultative type. This
process is employed for treatment of the organic sludge
During oxidation of organic matter anaerobically following reaction occurs
Organic matter CO2 + CH4+ new cell + energy for cells + other products
(Anaerobic bacteria) (H2S, H2, N2 etc.)
Microbial action by anaerobic bacteria consists of three stages as
1) liquefaction of solids,
2)digestion of soluble solids,
3) gas production.
Organic acid forming heterotrophs use complexorganic substrate such as
carbohydrates, proteins, fats, oils and their degradation products andproduce
organic acids. The breakdown of three major organic matters is shown below
 BIOTECHNOLOGY 102

Carbohydrates Simple sugars Alcohols aldehydes

Organic acids
Proteins Amino acids Organic acids + NH3
Fats and oils organic acids
Most of these organic acids forming bacteria are facultative anaerobes easily
found in soil and works in relatively wide pH range. Methane producing
heterotrophs, obligately anaerobes, use organic acids in narrow pH range of 6.7
to 7.4 to produced CO2 and CH4.

Organic acids CO2 + CH4 + H2S, H2, N2 etc. in traces

(55-75%) (35-45%)
The first group of microorganisms hydrolyses the complex organic substances
to soluble end products and is called as hydrolytic bacteria. The second group of
microorganisms called acidogenic bacteria converts the product of first group of
bacteria into simple end product primarily VFA and alcohols. The third group
called methanogenic bacteria converts the produced acid by the second group
into methane and carbon dioxide. The reactor content should be free from
oxygen. Alkalinity present in the reactor should be sufficient for proper
functioning of the digester to maintain the pH between 6.5 to 8.0. Temperature
has got tremendous effect in the functioning of a digester. It has been
established that two types of bacteria, mesophilic (200 to 400C) and
thermophilic (450 to 650C) are responsible for biodegradation. Therefore, the
digester can be operated either at mesophilic or thermophilic temperature range.
Places where the temperature is less than 200C, the digesters are required to be
heated externally to bring the temperature to the mesophilic range.
Advantages and disadvantages of anaerobic digestion

Advantages

 Methane recovered can be used as alternate fuel source.

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 Reduce production of landfill greenhouse gases when otherwise these

untreated sludge is disposed on landfill, which then broken down
anaerobically to release methane into atmosphere.
 Reduction in volume of sludge and improving dewatering characteristics
of the sludge makes it easy to dry.
 Reduces odour/ flies problem.
 Low operating cost, since energy is not require to supply oxygen being
anaerobic process.
Disadvantages
 Accumulation of heavy metal and recalcitrant contaminants in the sludge.
 Narrow operating temperature control range.
 When heating is to be provided safely handling is required with electrical
grid based heat management.

Aerobic digestion
Aerobic digestion is affected by biosolids temperature, rate of biosolids
oxidation, biosolids loading rate, system oxygen requirements, biosolids age,
and biosolids characteristics. Theprocess converts organic sludge solids to
carbon dioxide, ammonia, and water by aerobic bacteria with reduction of
volatile solids, pathogens, and offensive order. It can be used to treat only
(i) waste activated sludge,
(ii) mixture of ASP sludge (or trickling filter sludge) and PST sludge, and
(iii) waste sludge of ASP designed without PST.
It is similar to ASP with HRT of 10 to 12 days. The oxygen requirement in
aerobic digestion for the complete oxidation of the BOD is about 2 kg/kg of
cells. To ensure proper operation, the contents of the aerobic digestion should
be well mixed. During this extended aeration, the microorganisms enter a phase
(the endogenous stage) where materials previously stored by the cell oxidize,
reducing the biologically degradable organic matter. During this endogenous
stage, food supplies to microbial life are depleted to the point where the
microorganisms begin to consume their own protoplasm, oxidizing it to carbon
dioxide, water, and ammonia. As the digestion process continues, the ammonia
 BIOTECHNOLOGY 104

is further converted to nitrates. Eventually, the oxygen uptake rate levels off and
thebiosolids matter is reduced to inorganic matter and relatively stable volatile
solids.
In a conventional aerobic digester, concentration of influent VSS must not be
more than 3% for retention times of 15 to 20 days. In the batch basis, the
digester is filled with raw sludge and aerated for 2 to 3 weeks, then stopped.
The supernatant is decanted and the settled solids are removed. For the semi
batch basis, raw sludge is added every couple of days; the supernatant is
decanted periodically, and the settled solids are held in the digester for a long
time before being removed.

Sludge Conditioning
Sludge is conditioned to improve its dewatering characteristics. Two methods
are commonly used for sludge conditioning
(i) addition of chemicals and
(ii) heat treatment.
Chemical conditioning results in coagulation of the solids and release of the
absorbed water. Conditioning is used in advance of vacuum filtration and
centrifugation. Chemicals used include ferric chloride, lime, alum and organic
polymers. The chemical dosage required is determined in the laboratory test.
The sludge which is difficult to dewater requires higher dose.
Heat Treatment of sludge is both stabilization and a conditioning process. This
involves heating of sludge for short period (30 min) under pressure (1.0 to 1.4
MN/m2). The temperature is kept in the range of 140 to 200 oC. The treatment
coagulates solids, breaks down the gel structure and reduces the water affinity
of sludge solids. As a result the sludge is sterilized, deodorized, and is
dewatered readily on vacuum filter or filter presses, without addition of
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chemicals. The heat treatment process is most applicable to biological sludges

that may be difficult to stabilize or condition by other means.
Sludge Dewatering
The digestion of the primary or mixed sludge will bring down the water content
to about 90%; however, treatment is necessary to reduce the water content
further. When digested sludge is applied on the sludge drying beds, the water
content of the sludge can be reduced to around 70%. Presence of excess oil and
grease will interfere with this process. Sludge drying beds require large land
area (nearly 40% of the total area required for sewage treatment plant), hence at
the places where land is not available other alternatives such as, mechanical
dewatering on vacuum filters, filter press or centrifuge followed by heat drying
or incineration could be used after sludge conditioning. In India, most of the
parts of the country there is favourable climate for open sludge drying, hence
sludge drying beds are preferred as an economical way and easy to manage.
Sludge Drying Beds
This is used where land available is adequate and the dried sludge is used for
soil conditioning. The sludge is applied on the bed of sand, which is supported
on gravel. Major portion of the liquid drains off in the first few hours after
which drying occur due to evaporation. Sludge cake shrinks, producing cracks
which further accelerate evaporation from the sludge surface. In dry region
generally the sludge will get dried within two weeks. The drying period will
depend on sunshine, rainfall, wind velocity, and relative humidity, apart from
sludge characteristics. Under adverse weather condition, it may take up to four
weeks.
Final Disposal of Sludge
Final disposal of the sludge from the treatment plant generally involves some
form of land disposal. The most common methods of land disposal include
spreading on land, lagooning, dumping, and landfilling.
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Spreading on land: Dewatered and composted sludge can be disposed of by

spreading overfarm lands, and plowing under after it has dried. Wet dewatered
sludge can be incorporated into soil directly by injection. The humus in the
sludge conditions the soil and improves its moisture retentiveness.
Lagooning: It is an economical mode of disposal in remote area. A lagoon is an
earthen basin into which untreated or digested sludge is deposited. Stabilization
of untreated sludge can be carried out in a lagoon which gives objectionable
odours. The stabilized sludge settles to the bottom of the lagoon and
accumulates. Excess liquid from the lagoon, if there is any, is returned to the
wastewater treatment plant at PST. Sludge may be stored indefinitely in a
lagoon, or it may be removed periodically after draining and drying.
Dumping: Dumping in an abandoned mine quarry is a suitable disposal method
only for the sludges and solids that have been stabilized, so that no
decomposition or nuisance condition will results. Digested sludge, clean grit
and incinerator residue can be disposed of safely by this method.
Landfilling: A sanitary landfill can be used for disposal of sludge, grease, grit
and other solids, whether it is stabilized or not. The sanitary landfill method is
most suitable if it is also used for disposal of the other solid wastes of the
community. In a sanitary landfill, the wastes are deposited in a designated area,
compacted in place with a tractor or roller and covered with a 30 cm layer of
clean soil. With daily coverage of the newly deposited wastes, nuisance
condition such as odour and flies are minimized.
Onsite Sanitation
Rural areas and the outskirts of the urban areas may have insufficient population
and infrastructure to support the sewer system and central treatment plant.
Hence, onsite sanitation becomes necessary to maintain hygienic living
conditions. For environmentally safe onsite sanitation, satisfactory wastewater
management techniques should ensure that:
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 water body used for water supplies are not contaminated;

 flies and vermin have no access to excreta;
 surface water bodies are not polluted by runoff; and
 Nuisance conditions such as odour are minimized.

Septic Tanks
This is basically a sedimentation tank with some degree of solid destruction due
to sedimentation and subsequent anaerobic digestion. Septic tanks are ordinarily
designed for 24 h liquid retention time at average daily flow. Considering the
volume required for sludge and scum accumulation, the septic tank may be
designed for wastewater retention time of 1 to 2 days. Septic tanks can be made
from concrete, masonry or fiberglass. Prior two are of rectangular shape and
later is generally of circular shape. The inlet and outlet are baffled so that the
floating matter and grease will be retained in the tank. Heavy solids settle at the
bottom of the tank, where the organic fraction will decompose following
anaerobic pathway. The production of biogas may interfere with the
sedimentation of the solids. Every septic tank should be provided with the
ventilation pipe with the top of the pipe covered with suitable mosquito proof
wire mesh.

The sludge accumulated in the tank is cleaned at the frequency of once in 2 to 3

years. Minimum of 300 mm of free board should be provided in the tank. The
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effluent of the septic tank is offensive and potentially dangerous. Hence, further
treatment for septic tank effluent is necessary to protect the receiving
environment. Due to inadequate treatment offered to the sewage, septic tanks
are recommended for individual houses and for cluster of houses or institutes
where contributing population is not exceeding 300 persons.

References

1. Microbiology By M.J Pelczar,jrm

2. Reid, P., Microbiology, TMH.

3. Atlas, R. M. &Bartha, R., Microbiology Ecology - Fundamentals and
Applications.

4. R.C Dubey &D.K maheshwari, A text book of microbiology. S. Chand

 BIOTECHNOLOGY 109

Questions

Module-1

1. What is the importance of microbiology and why study it?

2. Briefly describe the scope of microbiology?
3. Differentiate between Gram-positive and Gram-negative bacteria?
4. Draw and explain the bacterial growth curve
5. Explain distinguishing characteristics of prokaryotic cells.
6. What are the different shapes of the bacteria? Explain with structures.
7. Write a short note on bacterial cell wall.
8. Differentiate between gram positive and gram negative cell wall.
9. Write down the names of different structures present external to the cell
wall of bacterial cell.
10.Write a short note on the following:
o a. Flagella b. Fimbriae c. Pili

11.How will you differentiate bacterial species on the basis of their patterns
of flagella distribution?
12. Explain the structure of Flagella.
13.Define the term Chemotaxis.
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Module-2

1. Name the different nutritional types of microorganisms?

2. Write short notes on the nutritional requirements of microorganisms

a. Macronutrients b. Micronutrients c. Requirement of carbon,

hydrogen and oxygen d. Requirement of nitrogen, sulphur and phosphorus

3. What are growth factors and how are they important for microbial nutrition

4. Write a short note on the following:

a. Photolithoautotrophs. b. Photoorganoheterotrophs.

c. Chemolithoautotrophs. d. Chemoorganoheterotrophs.

5. Write short notes on the following:

a. Chemically defined media b. Complex media

c. Enriched media d. Selective media

e. Differential media

6. What are the different methods of isolation of pure cultures? Explain

7. Define the terms:

a. Prototrophs b. Auxotrophs c. Inoculation

d. Chemoorganoheterotrophs e. Photolithoautotrophs

8. Differentiate between Chemically defined media and Complex media.

9. Write a short note on Colony morphology and growth.

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10. Define Growth. Explain the different phases in growth curve with proper
diagram.

11. Explain Generation time or Doubling time of the bacterial population

mathematically.

13. What are the different ways to measurement of cell numbers?

14. Differentiate between Batch culture and Continuous culture.

15. Define the following terms:

a. Turbidostat b. Chemostat

c. Generation Time
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Module-3

1. Classify the microorganisms on the basics of carbon sources and

temperature.
2. Write a brief note on extremophiles.
3. Classify the microorganism on the basics of nutrition.
4. What do you know about plant-microbe interactions? Discuss in brief
destructive associations.
5. Write an extended note on plant-microbe interactions with emphasis on
symbiosis.
6. What do you know about symbiosis? Write in brief different type of
symbiotic interaction in plants.
7. What do you know about microbe-microbe interactions? Write in details
about antagonism.
8. What do you known about predation and parasitism?
9. Discuss the nutritional requirement of bacteria.
10.Write short notes on the following.
i. Psychrophiles
ii. Hyperthermophiles
iii. Super-hyper-thermophiles
iv. Extremophiles
v. Saprophytes
vi. Symbiosis
vii. Antagonism
viii. Rumen symbiosis
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Module-4

1. Write a brief note on different types of water found in the environment.

2. Write an essay on microorganisms of water.
3. What is MPN? Write in brief the different tests used to measure the MNP.
4. Write in details the different methods of water purification.
5. Discuss in details the sanitary tests of coliforms.
6. Write short notes on the following.

i. Sedimentation
ii. Fresh water microbiology
iii. Marine water microbiology
iv. Faecal bacteria
v. Presumptive test
vi. Membrane filter technique
vii. IMViC test
viii. Filtration of water
ix. Disinifection
 BIOTECHNOLOGY 114

Model Questions

Semeter Question in2011

1. Answer all the following 2x10

a) Draw the figure for reproduction of bacteria by budding.

b) What is the basic of five kingdom classification scheme according
to Whittaker?
c) What are the general methods used for classifying the bacteria?
d) Why staining is carried out and why it is done?
e) What is freeze-Etching?
f) What do you mean by bacterial chemotaxis?
g) What are the physical conditions required for bacterial growth?
h) Name some species of pathogenic organisms which may be present
in polluted water.
i) What do you mean by biochemical oxygen demand?
j) What are the differences between prokaryotic and eukaryotic cells?

2. Discuss the major characteristics of microorganisms. 10

3. Compare the structure and chemistry of the cell walls of Gram-positive
eubacteria versus those of Gram –negative eubacteria. List some major
different between the cell walls of archeobacteria those of eubacteria. 10
4. Explain the modern advanced waste water treatment with neat flow sheet.
10
5. Compare the advantages and disadvantages of the various techniques for
isolation of microorganisms in pure culture. 10
6. a) If a bacteria count increases from 103 to 109 in hours, determine the
generation (doubling) time, G. 6

b) Briefly explain denitrification. 4

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7. a) Briefly explain the methods used to evaluate the microbial ecosystem.4

b) Distinguish between 6

i. phototrops and chemotrops

ii. lithotrop and organotrops
iii. autotrops and heterotrops

8. Answer any two. 2x5

a) Activated sludge process

b) Mutualism of micro-organisms

c) Microbial mat

Semeter Question in 2012

1. Answer all the questions 2x10

a) What is germ theory of diseases?

b) Describe briefly about plasmid.
c) What is endospore? How it become the adverse environmental
conditions.
d) What do you mean by specialized transuduction?
e) What is generation time of bacteria? How it is related to growth of
bacteria?
f) Describe briefly about the composition of bacterial cell wall?
g) What is synchronous culture?
h) What is nucleoid? How it differ from nucleus?
i) What are coliform bacteria? Where they found?
j) Describe briefly about the rhizosporic microorganism in plant
growth.
 BIOTECHNOLOGY 116

2. Give an account of ultra-structure of bacterial cell. Distinguish between

gram positive and gram negative bacterial cell wall. 10
3. Discus the nutritional classification of bacteria with suitable example. 10
4. What is bacterial growth? Describe briefly about the kinetic of bacterial
growth. 3+ 7
5. What are microorganisms found in contaminated water? Describe briefly
about the purification process of sewage water? 5+5
6. What is microbial association? Discuss the microbial interactions. 10
7. Distinguish between the following (any two) 5+5

a) Gram positive and gram negative bacteria

b) Streak plate and pour plate technique
c) Rhizospore and phylospore

8. Write short notes on any two 5x2

a) Bergy’ manual
b) rDNA sequencing
c) pure culture techniques