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Biotechnology Lecture Note
Biotechnology Lecture Note
6th Semester
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
from internet were consulted for preparing this document. The ownership of the
information lies with the respective authors or institutions. Further, this
document is not intended to be used for commercial purpose and the committee
faculty members are not accountable for any issues, legal or otherwise, arising
out of use of this document. The committee faculty members make no
representations or warranties with respect to the accuracy or completeness of
the contents of this document and specifically disclaim any implied warranties
of merchantability or fitness for a particular purpose.
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
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
27 Water purification
28 Water purification
29 Average disposal
30 Sewage purification
31 Problem discussion
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.
Applications of Biotechnology
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.
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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.
Various branches
medical microbiology
environmental microbiology
agricultural microbiology
food & diary microbiology
Classification
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.
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
He placed all the kingdom prokaryotes which again divided into four divisions
Application of microbiology
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
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:
e. Medical Microbiology:
f. Computer application:
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:
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 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
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:
Flagella
BIOTECHNOLOGY 15
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
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.
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
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
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.
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
3. Cultural characteristics
4. Metabolic characteristics
5. Antigenic characteristics
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
8. Ecological characteristics
Some organisms are widely distributed in nature, but others may be restricted to
a particular environment
CLASSIFICATION:
GOAL OF CLASSIFICATION:
1. Intuitive method:
2. Numerical taxonomy:
% similarity =
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:
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.
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.
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.
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.
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.
Carbon sources:
Energy sources:
Electron sources:
Four major nutritional classes based on their primary sources of carbon, energy
and electrons is known.
Growth factors:
Vitamins – small organic molecules that usually make up all or part of enzyme
cofactors, and only very small amounts sustain growth.
Coenzyme A – Brevibacterium
β- Carotene – Dunaliella
Vitamin D - Saccharomyces
BIOTECHNOLOGY 31
Module-2
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.
Beef extract – aqueous extracts from lean beef and contain amino acids,
peptides, nucleotides, organic acids and minerals and vitamins.
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.
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)
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.
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.
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.
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
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.
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.
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
(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
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
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.
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.
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.
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.
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:
Cultural Characteristics
1. Colony Appearance
2. Colony Form
3. Colony Elevation
4. Colony margin
5. Optical Density
6. Colour
7. Colony odour
8. Colony Consistency
BACTERIAL GROWTH
Growth Curve:
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
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
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.
G = t/n
Solve for n:
n=logb-logB/log2
n= logb-logB/ .301
n = 3.3 logb/B
G = t/n
Solve for G
G= t/3.3logb/B
G= t/3.3logb/B
G= 240minutes/3.3log107 /104
G= 240minutes/3.3x3
G = 24 minutes
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.
can be counted and the concentration of the bacterial cells in the original
sample is calculated as follows:
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.
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Population growth leads to increase in the total cell mass, as well as in cell
numbers. The following methods can be used.
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
Budding:
Fragmentation:
Binary fission:
Asexual reproduction
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.
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.
i. Aerobes
ii. Anaerobes
2. Based on carbon sources as energy:
i. Autotrops
ii. Heterotrops
3. Based on Temperature:
4. Based on habitat
Extremophiles thrive under conditions that would kill other creature. This
comes under the groups high and low pH loving microorganisms.
There are different ways by which the microorganisms derive nutrition for
their growth and development.
a. Saprophytism
b. Parasistism
c. Symbiosis
the parasite that have faculty to live as saprophyte in the absence of a suitable
host.
MICROBIAL INTERACTIONS
On the basics of the relative advantage to each partner i.e host and
microorganism, the relationships are basically of three types:
Types of interactions
I. Clay-Humus-Microbe interaction
II. Plant- Microbe Interaction
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a. Destructive Associations
b. Beneficial associations ( symbiosis)
a. Destructive Associations
b. Neutral associations (neutralism)
c. Symbiotic associations
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.
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a. Destructiveassociations (Diseases)
Algal disease- red rust of citrus, mango, guava, cocoa, sapota, tea.
Canker of citru.
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.
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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.
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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
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.
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:
b.Neutral Association:
c.Symbiotic Association:
Symbiotic association of bacteria, fungi and protozoans with insect, bird and
herbivorous mammals are below:
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.
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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.
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. Amensalism
2. Competition
3. Parasitism and predaction
2. Competition
Module-4
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
Aquatic microorganisms
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
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1. The bay serves as the receiving basin for nine major rivers, draining much of
southern New York State, penysylavania Maryland, virgina.
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.
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
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.
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
Chemical Characteristics:
1.pH
2.Acidity
3.Alkalinity
4.Hardness
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5.Chlorides
6.Sulphates
7.Iron
8.Solids
8.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.
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.
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
Bacteriological Characteristics:
The original test for the presence of coliform in water is done by standard test
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.
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.
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
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.
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.
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.
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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
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
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.
BIOTECHNOLOGY 86
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.
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(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.
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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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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.
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.
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.
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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,
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(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
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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.
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
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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.
Advantages
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
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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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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.
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
Questions
Module-1
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
3. What are growth factors and how are they important for microbial nutrition
a. Photolithoautotrophs. b. Photoorganoheterotrophs.
c. Chemolithoautotrophs. d. Chemoorganoheterotrophs.
e. Differential media
d. Chemoorganoheterotrophs e. Photolithoautotrophs
10. Define Growth. Explain the different phases in growth curve with proper
diagram.
a. Turbidostat b. Chemostat
c. Generation Time
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Module-3
Module-4
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
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Model Questions
b) Distinguish between 6
b) Mutualism of micro-organisms
c) Microbial mat
a) Bergy’ manual
b) rDNA sequencing
c) pure culture techniques