FUNDAMENTALS OF MICROBIOLOGY

Course Teacher: Prof. T. J. Abraham

DEPARTMENT OF AQUATIC ANIMAL HEALTH

FACULTY OF FISHERY SCIENCES,
WEST BENGAL UNIVERSITY OF ANIMAL AND FISHERY SCIENCES,
CHAKGARIA, KOLKATA - 700 094, WEST BENGAL

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 History of microbiology, microbial world and their classification
1.Milestones in Microbiology
Microbiology is a science that deals with the study of living organisms and agents that are
too small to be seen clearly by the naked eye. (i.e the study of microorganisms). The term
microbiology was derived from Greek – ‘Mikros’ means small, ‘Bios‘ means life and ‘Logos’
means science.
Microorganisms include bacteria, fungi, viruses, many algae and protozoa. These can be
seen with the aid of microscopes. Many scientists have contributed to the development of the
science of microbiology.
In 1590, Zacharias Janssen, a Dutch astronomer developed a prototype of the present-day
telescope and the compound microscope. Galileo also constructed a microscope at around the same
time (1610), who later improved upon the earlier type by devising mechanisms by which focusing
could be done.The first person to observe and describe microorganisms was the amateur
microscopist, Anton Van Leeuwenhoek (1632–1723) of Holland. He constructed a simple
microscope composed of double convex glass lenses held between two silver plates. His
microscopes could magnify up to 300 times. He described moving microscopic organisms as
animalcules. His descriptions of animalcules were made in 1673.

Figure of earliest microscope

Robert Hooke (1665) constructed a compound microscope, considered the forerunner of the
present-day compound microscope. He published the drawings of the fruiting bodies of fungi and
confirmed several reports of Leeuwenhoek.
Theory of Spontaneous generation:
From the earliest times, people believed that living organisms could develop from non-living
matter. Experiments by Italian physician Francesco Redi disproved this theory in 1aboutd to
macroscopic organisms. His experiments on decaying meat and the development of maggots

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discredited the theory of spontaneous generation for larger organisms. Leeuwenhoek’s discovery of
microorganisms once again developed the controversy of spontaneous generation that
microorganisms develop spontaneously. The belief in the spontaneous generation of
microorganismswas disproved by:
- Italian Priest and naturalist Lazzaro Spallanzani (1729 – 1799)
- Louis Pasteur (1822 – 1895), a French Chemist and
- John Tyndall (1820 – 1893), English Physicist.

Spallanzani put the broth into four flasks

Flask 1 was left open - Microbes found

Flask 2 was sealed - Microbes found Flask 4 was boiled and then sealed -
Flask 3 was boiled and then left open - Microbes were not found
Microbes found
Swan neck flask Experiment of Louis Pasteur

Of these, the swan-neck-flask experiment of Louis Pasteur in 1861 conclusively disproved the theory
of spontaneous generation. Besides this, Louis Pasteur contributed to microbiology in several other
ways and is considered to be the “Father of Microbiology”
- He discovered that alcohol production from grape juice was due to yeast
- He found that contamination of grape juice with rod-shaped bacteria resulted in the bitter taste
of wine
- He found that fermentation took place in the absence of air
- He coined the terms aerobicto describe those organisms requiring air and anaerobic to describe
organisms that do not require air for their growth
- He also developed the anthrax vaccine rabies vaccine

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The germ theory of Disease:
Agostino Bassi, in 1835 was the first one to demonstrate that microorganism cause disease
(silk-work disease – by fungi). In 1867, Joseph Lister, the Father of antibiotic surgery introduced
antibiotic principles in surgical practice. He used carbolic acid as an antiseptic. The first direct
demonstration of the role of bacteria in causing anthrax disease was by the German Physician Robert
Koch (1843 – 1910). (Anthrax caused by Bacillus anthracis). His criteria for providing a causal
relationship between a microorganism and a specific disease are known as Koch’s postulates:
1: The microorganism must be present in every case of the disease but absent from the healthy host
2. The suspected microorganism must be isolated and grown in a pure culture
3. The same disease must result when the isolated microorganism is inoculated into a healthy host
4. The same microorganism must be isolated again from the experimentally diseased host
Robert Koch also developed the techniques required to grow bacteria on solid media and to
isolate a pure culture of pathogenic microorganisms. He first tried to grow bacteria on sterile surfaces
of cut, boiled potatoes. He then used gelatin to solidify the liquid medium. (However, the media
liquefied when the temperature exceeded 28oC). Fannie Eilshemius Hesse suggested the use of agar as
a solidifying agent in culture media. Agar has several advantages:
- Agar is not digested by most bacteria
- Agar remains solid until temperature reaches above 98oC and medium solidifies at around 44oC
One of Koch’s assistants, Richard Petri developed the Petri dishfor the cultivation of bacteria
Vaccination was successfully used against smallpox by Edward Jenner in 1798. He administered
the cowpox virus to protect individuals against the smallpox virus.
Elie Metchnikoff discovered some blood leucocytes that could engulf disease-causing bacteria.
He called these cells as phagocytes and the process phagocytosis.
Winogradsky (1856 – 1953) and Beijerinck (1851 – 1931) investigated the ecological role of
microorganisms and revealed the role of microorganisms in carbon, nitrogen and sulfur cycles. They
developed the enrichment–culture technique and the use of selective media, which have been of great
importance in microbiology.
The development of microbiology in the twentieth century in close relationship with other
biological disciplines like genetics and biochemistry contributed to the rise of molecular biology – the
branch of biology dealing with the physical and chemical aspects of living matter and its function.
Other significant developments in microbiology
1796 – Edward Jenner – Vaccination – Smallpox
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1873 –Lister - obtained pure culture of bacteria by serial dilution
1881 – Paul Ehrlich - Staining of bacteria with methylene blue
1881 – 1883 – Robert Koch – Simple methods for isolation and maintenance of microorganisms on
chemically defined solid medium
1884 – Hans Christian Gram – differential staining
1884 – Elie Metchnikoff – Phagocytosis – WBC engulfing foreign particles
1884 – Charle Chamberland – Invention of bacterial filters for sterilization of liquids. Invention of
autoclave
1887 – Richard Petri – Petridishes
1892 – Iwanowski – observed filterable particles responsible for tobacco mosaic disease (viruses)
1929 – Alexander Flemming – Antibiotics (Penicillium notatum)
Fifty years from 1860 to 1910 marked by the works of Pasteur on alcohol fermentation to the
death of Robert Koch is termed as the Golden Age of Microbiology.

2. Microscopy
Light Microscopy
Refraction: The deflection of a light ray from a straight path as it passes from one medium to another
(The ray is bent)
The refractive index is a measure of how greatly a substance slows the velocity of light. When light
passes from air into glass, it is slowed and bent toward the normal. As light leaves glass intoair, it
accelerates and is bent away from the normal. Lenses act like a collection of prisms operating as a unit.
When parallel rays of light strike the lens, a convex lens will focus these rays at a specific point, called
the focal point (F). The distance between the centre of the lens and the focal point is called the focal
length. A lens with a short focal length will magnify an object more than a lens with a longer focal
length.
Bright-field microscope
An ordinary compound microscope is called bright–field microscope, because it forms a dark image
against a brighter background.
Image formation is shown in the figure (the path of light is shown)

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The objective lens forms an enlarged real image within the microscope and the eyepiece (ocular) lens
further magnifies this primary image into the final virtual image (which appears to lie about 25 cm
away beyond the stage.
The total magnification is calculated by multiplying the objective and eyepiece magnification together.
(For example, if a 45x objective lens is used with a 10x ocular, the magnification of the specimen will
be 450x.)
Resolution of a microscope
Resolution is the ability of a lens to separate or distinguish between small objects that are close
together. The minimum distance (d) between two objects that reveals them as separate entities is given
by Abbe’s equation:
0.5 λ Where λ is the wavelength of light used for illumination
d = ------------ n sin θ Is the numerical aperture (NA)
n sin θ
As d becomes smaller, the resolution increases and final details can be seen in a specimen.
The numerical aperture may be termed as the light-gathering ability of a lens.
Numerical aperture = n sin θ
Theta (θ) is defined as ½ the angle of the cone of light entering an objective. It is important to
illuminate the specimens properly to have higher resolution. A wide cone of light through the slide
(specimen) and into the objective lens increases the numerical aperture thereby improving the
resolution of the microscope. The angle of the cone of light that can enter a lens depends on the
refractive index (n) of the medium in which the lens works. The refractive index for air is 1.0. Since
sin θ cannot be greater than 1.0 (the max is 90o and sin 90o is 1.0), no lens working in the air can have
a numerical aperture greater than 1.0. The only way to raise NA above 1.0 to have a higher resolution
is to increase the refractive index with immersion oil (same refractive index as glass).

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The maximum theoretical resolving power of a microscope with an oil immersion objective (NA =
1.25) and blue-green light is approximately 0.2 µm
(0.5) 530 nm
d = ----------------------- = 212 nm or 0.2 µm
1.25
The working distance of an objective lens is the distance between the front surface of the lens and the
surface of the cover glass or the specimens when it is in sharp focus. Objectives with large NA and
great resolving power have short working distances.
Normally a microscope is equipped with three or four objectives ranging in magnifying power from 4x
to 100x
Property Objectives Oil immersion
Scanning Low power High power
Magnification 4x 10x 40 – 45x 90 – 100x
NA 0.10 0.25 0.55 – 0.65 1.25 – 1.4
Approx. focal length 40 mm 16 mm 4mm 0.1 mm
Approx. resolving power with 2.3 µm 0.9 µm 0.35 µm 0.18 µm
the light of 450 nm (blue light) (0.2 µm)
Ideally, a microscope should be parfocal – that is, the image should remain in focus when objectives
are changed.
Darkfield Microscopy
Darkfield microscope is used for examining live microorganisms which are invisible in the ordinary
light microscope or that cannot be stained (some organisms are distorted by staining). A darkfield
microscope uses a dark field condenser that contains an opaque disc. The disc blocks light that would
enter the objective directly. Only the light that is reflected off the specimen reaches the objective lens.
The specimen appears bright against a black background.
Phase–contrast microscopy
A phase contrast microscope can be used to study internal structures in living microorganisms. There is
no need to fix or stain the specimen.
The principle of phase-contrast microscopy is based on slight variations in refractive index. As rays
pass from the light source through the specimen, their velocity may be altered by differences in the
thickness and physical properties of various structures of the specimen. Light rays passing through the

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specimen are diffracted differently and travel in different pathways to reach the eye of the viewer.
These phase differences are seen through the microscope as different degrees of brightness. The
internal details of a cell appear as degrees of brightness against a dark background.

Darkfield Phase contrast

A phase contrast microscope uses a special condenser that contains a ring-shaped annular diaphragm.
The diaphragm allows a ring of light to pass through the condenser, focusing light on the specimen and
a ring-shaped phase plate in the objective lens. The diffracted and undiffracted rays are then brought
into phase with each other to produce the image that meets the eye.
Fluorescence Microscope
A fluorescence microscope exposes a specimen to ultraviolet. Violet or blue light forms an image of
the object with the resulting fluorescent light. Usually, a mercury vapour arc lamp produces an intense
beam, which passes through a special infrared filter which reduces heat transfer. The light passes
through an exciter filter that transmits only the desired wavelength. A dark field condenser provides a
black background against which the fluorescent objects glow.
The microscope is used to visualize objects that emit light when light of a different wavelength strikes
the object. Usually, the specimens are stained with fluorescent dyes, called fluorochromes that
fluoresce brightly upon exposure to light of a specific wavelength. Some microorganisms can auto-
fluoresce also.

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The fluorescence microscope has applications in several fields and can be used for direct detection of
microorganisms using fluorescent antibody techniques (FAT) and for direct microscope counts (DMC)
after staining them with fluorochromes in ecological studies.

Electron Microscope
In this microscope, electrons are used to illuminate the specimen for magnification (instead of light).
The electrons have a wavelength of over 1000 times shorter than visible light; therefore, the resolving
power increases by about a thousand-fold. The resolving power of an electron microscope is about 0.3
nm – 0.5 nm.
There are two different types of electron microscopes:
1. Transmission electron microscope (TEM)
2. Scanning electron microscope (SEM)
Transmission Electron Microscope
A heated tungsten filament in the electron gun generates a beam of electrons that is focused on the
specimen by the condenser. Electrons cannot pass through a glass lens and therefore, magnetic lenses
(electromagnets) are used to focus the beam. Electrons deflect by collisions with air molecules; hence,
the column and specimen must be kept under a high vacuum to obtain a clear image. The specimen
scatters electrons passing through it and the beam is focused by magnetic lenses to form an enlarged
visible image of the specimen on a fluorescent screen. A denser region in the specimen scatters more
electrons and appears darker in the image since fewer electrons strike that area of the screen. In

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contrast, electron-transparent regions are brighter. The image can also be captured on photographic
film.
The Scanning Electron Microscope
This microscope is used to study the external surface features of microorganisms. The specimen need
not be sectioned. SEM provides three-dimensional views of specimens. In SEM also, an electron gun
provides a finely focused beam of electrons called the primary electron beam. These electrons pass
through electromagnetic lenses and are directed over the surface of the specimen. The primary electron
beam knocks electrons out of the surface of the specimen. These secondary electrons are transmitted to
an electron collector, amplified and used to produce an image on a viewing screen or photographic
plate. SEM has a resolution of about 20 nmand a useful magnification of about 10000x.

TEM SEM
There are also other advanced microscopes with higher resolution and magnification.
1. Acoustic microscopes
There are microscopes that employ sound waves with a frequency of 3000 megahertz which have a
resolution equal to that of the optical microscope. Acoustic waves easily propagate through materials
impervious to optical waves.
2. Scanning tunnelling microscopes (STM)
STM is used for studying surface characteristics. Even the arrangement of individual atoms on the
surfaces of metals can be accurately revealed.

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3. Microbial taxonomy: Classification of Living organisms
Grouping and Description of living organisms
All organisms were earlier classified into two groups: Plants and animals based on mode of
nutrition and the presence or absence of cell wall. As more and more information about unicellular
organisms became known, it became impossible to assign organisms like bacteria, slime molds, etc. to
either of these groups. Thus, a third kingdom called Protista was formed by Haeckel (1866) consisting
of unicellular organisms. As science progressed, differences in cell structure and cell wall came to be
known and Copeland (1956) created a fourth kingdom Monera, containing unicellular prokaryotic
bacteria. Gradually all unicellular prokaryotes having a peptidoglycan cell wall, irrespective of their
mode of nutrition were kept in Monera and a fifth kingdom Mycetae was created by Whittaker (1969)
to accommodate fungi.
Two fundamentally different types of cells exist.
1. Prokaryotic cells:
Cells that lack a true membrane–delimited nucleus eg. All bacteria
2. Eukaryotic cells:
Cells that have a membrane-enclosed nucleus. Algae, fungi, protozoa, higher plants and animals are
eukaryotic
Description of Microorganisms:
Bacteria: Bacteria are prokaryotes, unicellular organisms generally lacking chlorophyll, capable of
reproduction by binary fission. The size of bacteria varies from 0.1 µ m to 18 µ m (most pathogenic
bacteria measure 0.2 - 10 µ m)
Viruses: Viruses are infectious agents having a simple organization with a protein coat and a single
type of nucleic acid (either RNA / DNA). They lack independent metabolism and reproduce only
within living hosts. Size ranges from 20 to 350 nm.
Fungi: (Fungus in Latin means mushroom)
Fungi are eukaryotic, spore-bearing organisms with absorptive nutrition. They do not have chlorophyll
and reproduce sexually or asexually.
Algae: (Algae means simple aquatic plants) Algae are eukaryotic organisms that lack roots and leaves
but have chlorophyll and other pigments for carrying out oxygen-producing photosynthesis.
Protozoa: (In Greek Protos means first and zoon means animal)
Protozoan can be defined as a usually eucaryotic protist. Protozoa are not multicellular.

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Although there are several classifications of organisms, the five-kingdom classification by Robert H.
Whittaker in 1969, placed organisms into five kingdoms based on three major criteria.
i. Cell type (Procaryotic / Eucaryotic)
ii. Level of the organization (Solitary / colonial; Uni or multicellular)
iii. Nutritional type
Accordingly
1. Kingdom: Monera or Procaryote
All procaryotes
2. Kingdom: Protista
- Unicellular or colonical eucaryotic organisms that lack true tissues eg. Protozoa, lower fungi and
smaller algae
3. Kingdom: Fungi
- Eucaryotic organisms with absorptive nutrition and multinucleate
4. Kingdom: Animalia
- Multicellular animals with ingestive nutrition
5. Kingdom: Plantae
- Multicellular plants with walled eucaryotic cells and photosynthesis

The 5-kingdom system is not accepted by all microbiologists. A major problem is its lack of distinction
between archaebacteria and eubacteria. Various alternatives have been suggested. Another two empires
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and eight kingdom classification (or) three domain classification have also been proposed for better
clarity.
Cavalier – Smith’s classification:
Empire : Bacteria
Kingdom – Eubacteria
- Arahaebacteria
Empire :Eucaryota
Kingdom - Archaozoa
- Protozoa
- Chromista
- Plantae
- Fungi
- Animalia
Carl Woeseemployed the comparison of RNA sequences and developed a three domain or empire
classification. Accordingly, organisms are classified into Eubacteria, Archaebacteria (Archaea) and
Eucaryotes, which are placed above kingdom level. The traditional Kingdoms are distributed among
these domains.
Later a super kingdom Prokaryotae was created having a single kingdom Monera consisting of all
unicellular prokaryotic organisms. Microbiology usually deals with organisms belonging to kingdom
Monera now comprised of Eubacteria, Archaea, Cyanobacteria, Mycoplasma, Phytoplasma, Rickettsia,
L-forms, Spiroplasma, Viruses, Viroids, Prions, etc. are also studied under this branch.
The artificial system of bacterial classification groups prokaryotes based on similarities. This system is
designed for use as a Determinative key, the best example of which is Bergey’s Manual of Systematic
Bacteriology (1984). It contains names, descriptions, literature citations and determinative keys for the
classification of a new isolate. In Bergey’s Manual prokaryotes are grouped on the basis of
characteristics such as Gram stain, morphology (rods, cocci, etc.), motility, structural features (e.g.
spores, filaments, sheaths, appendages, etc.), and on distinguishing physiological features (e.g.
anoxygenic photosynthesis, methanogenesis, lithotrophy, etc.).
Nowadays, organisms including prokaryotes are grouped on a genetic basis, i.e. by comparison of the
nucleotide sequences of the small subunit ribosomal RNA (rRNA) that is contained in all cellular
organisms. The molecular analyses of organisms show that life forms can be grouped into three
domains: Bacteria, Archaea and Eukarya. C. Woese developed the phylogenetic tree of prokaryotes by
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analysis and comparison of highly conserved markers such as 16S rRNA sequence. 16S rRNA of 30S
ribosomes yield highly conserved sequences, which indicate the similarities with almost all bacteria
and dissimilarities with eukaryotes and some large groups of bacteria.
Bergey’s Manual of Systematic Bacteriology contains the phenotypic characteristics used to classify
bacteria by conventional taxonomy and keys that can be used to identify unknown strains from their
phenotypic characters. According to Bergey’s Manual of Systematic Bacteriology, all bacteria can be
classified into four divisions or phyla according to the characteristics of cell walls. Each division is
further subdivided into sections based on:
· Grams stain reaction
· Cell shape
·Cell arrangements
· Oxygen requirement
· Motility
· Nutritional requirements
· Metabolic properties

4. Prokaryotes: Structure,Ultrastructure and Function of Prokaryotic Cell

Prokaryotic Cell Structure
Bacteria vary in size and shape. The most common shapes are coccus, bacillus and spirillum.
Coccus: Roughly spherical, Cocci cells exist as individual cells or in characteristic arrangements.
1. In pairs which is called diplococcus. eg.Neisseria.sp.
2. as long chains – eg.Streptococcus sp.
3. in irregular grape-like clumps eg.Staphylococcus sp.
4. in tetrads eg.Micrococcus sp.
Bacillus: rod shape eg.Bacillus spp.
Bacilli differ in their length – to width ratio. Coccobacilli – resemble cocci as they are short and wide.
The shape of the rod’s end often varies – may be flat, rounded and cigar-shaped or bifurcated. Rods
may occur in pairs or chains. Rods may also are curved to form commas or incomplete spirals.
Some bacteria form long multinucleate filaments or hyphae – eg.Actinomycetes.
Spirillum: Some are shaped like long rods twisted into spirals or helix. They are called spirilla (rigid)
and spirochetes (if flexible). There are also other shapes like oval or pear-shaped bacteria, or even flat,

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square to rectangular box-shaped. There are some bacteria which are variable in shape and lack a
characteristic form. They are called pleomorphic.

Size: Some members of Mycoplasma are only 100 - 200 nm in diameter.

E. coli is 1.1 to 1.5 µ m wide by 2.0 to 6.0 µ m. long. There are a few large bacteria whose size reaches
500 µm. in length – eg. Spirochetes.
Epulopisciumfishelsoniis an exceptionally long bacteriummeasuring 80 µm x 600 µm.
Structure and function of prokaryotes: Membrane systems
Both prokaryotic and eukaryotic membranes are similar in the overall structure. Membranes of
eukaryotic microorganisms serve to compartmentalize cell contents into organelles such as
mitochondria, thus allow the concentration of specific metabolites at certain locations. Prokaryotic
organisms contain only a single membranous structure, the cytoplasmic membrane or plasma
membrane. The cytoplasmic membrane separates the cell contents from the environment and measures
4 – 5 nm thick. The cytoplasmic membrane also constitutes the permeability barrier of the cell and
allows it to concentrate desired nutrients and excrete waste products. Membranes are also involved in
complex biochemical processes such as respiration. All membranes are formed of a lipid bilayer,
made of phospholipids. Phospholipid molecules consist of two parts, a fatty acid portion which is
hydrophobic (water-repelling) and a glycerol phosphate part which is hydrophilic water-lovingg).
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In such a bilayer, the hydrophobic fatty acid portions point towards each other and produce a
hydrophobic environment. The hydrophilic parts are exposed to the aqueous external environment. The
proteins of the membrane may be associated with only one side of the membraneor may be completely
embedded in the phospholipid matrix. Because of this, the inner and outer sides of the cytoplasmic
membrane have different properties. This property of ‘sidedness’ is of great importance in membrane
function.
The overall structure of a membrane is maintained by hydrogen bonds and hydrophobic interactions.
Positively charged ions (Mg2+, Ca2+) help to stabilize the structure by forming ionic bonds with
negatively charged phospholipids.
Eucaryotic membranes can be differentiated from those of prokaryotes with the presence of sterols.
(Sterols are flat, rigid molecules, whereas lipids are flexible). Sterols help the membrane to withstand
greater physical stress. The only group of prokaryotes that contain sterols in their membranes are
mycoplasmas, as they lack cell wall and the cytoplasmic membrane has to withstand all stress.
Photosynthetic membranes of bacteria include a.Chlorobium vesicles in Green sulphur bacteria b.
Lamellae in Purplesulphur bacteria and c. Chromatophores chlorosomes in Non-sulphur bacteria
Cell walls
Protozoans lack a cell wall, whereas bacteria, algae and fungi have one. The bacterial cell wall
is unique and two broad categories can be recognized depending upon the appearance of cells upon
staining, Gram-positive and Gram-negative. Gram-positive bacteria have a thick, single-layered wall
whereas the gram-negative have a complex multilayered wall which is relatively thin.
The peptidoglycan layer is present in the cell walls of both Gram-positive and Gram-negative
eubacteria. In Gram-positive bacteria, the bulk of the wall is peptidoglycan whereas in Gram-negative
it accounts for only the innermost layer and is relatively thin. Peptidoglycan consists of alternate layers
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of amino sugars N-acetylmuramic acid (NAM) and N-acetylgucosamine (NAG) which are linked by
bonds described as β1-4 linkages (since carbon atoms 1 and 4 of adjacent residue are involved). Short
peptide chains of four amino acids are linked to the N- acetylmuramic acid (NAM). Peptidoglycan
forms a three-dimensional network, a single bag–shaped molecule (a murein sacculus). The
peptidoglycan structure is completed by interpeptide cross–bridges, which join together pairs of
adjacent peptide chain (normally from 3rd AA in one chain to the 4th in another chain).

There is a group of bacteria (Archaea) which lack peptidoglycan, but have six types of cell wall
namely pseudopeptidoglycan, polysaccharide, sulphated polysaccharides, glycoprotein, proteins and a
unique cytoplasmic membrane.
Gram-positive cell walls also contain large amounts of another polymer called teichoic acid,
made up of glycerol or ribitol joined by phosphate groups. Some genera including Mycobacterium,
Corynebacterium contain waxy esters of mycolic acids, which are complex fatty acids.
Among eukaryotes, algal cell wall is made fibres of cellulose which form a strong wall surrounding the
whole cell. Cellulose is a straight-chain polymer of glucose. In addition to cellulose, hemicellulose
(polysaccharide of glucose and other sugars) and Pectin may be present. Some algae contain a number
of additional polysaccharides like xylans, mannans and alginic acids. Fungal hyphae have a thick,
multilayered cell wall composed of chitin (fibrillar carbohydrate-based polymer).
Fimbriae and Pili:
Some bacteria possess additional hair-like structures on their cell surface, called fimbriae.
These are shorter than flagella but are numerous. They enable the organism to stick to a surface. Pili
are similar to fimbriae but are longer and fewer in number. The function of sex pilus is to bring
together two cells during the conjugation process before the transfer of genetic material where it acts as

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a conjugation tube. Pili of a different type are involved with the attachment of pathogenic bacteria to
human tissues.
Glycocalyx (Slime/capsule)
The glycoclyx consists of several polysaccharides, in association with glycoprotein. It is
secreted onto the outer surface of the bacterial cell wall. It may aid the invasion of a host organism by
binding a bacterium to a specific tissue. It also hinders the engulfing (phagocytosis) of the bacterium
by a host organism’s phagocytic immune defence cells. Glycocalyx also prevents desiccation.
Cytoplasm
The cytoplasm is the entire content of the cell. It has a gel-like consistency where small
molecules move rapidly. The liquid component of cytoplasm is called cytosol and 80% of it is water.
The cytoplasm consists of the following components: Protein and enzymes, ribosomes, storage
granules, bacterial chromosomes and plasmids. Bacterial cytoplasm also contains helical actin-like
proteins that contribute to cell shape.
Ribosomes
The ribosome is a cytoplasmic nucleoprotein particle with RNA accounting for 2/3 of the mass.
It consists of two subunits denoted 30S and 50S. Together the ribosome has a sedimentation coefficient
of the 70S. Ribosomes serve as the site of mRNA translation and protein synthesis. The 30S subunit
has 16S rRNA and 21 proteins. 50S subunit contains 5S and 23S rRNA and 31 proteins. A typical
bacterium may have as many as 15,000 ribosomes.

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Nucleoid
Prokaryotic DNA is in circular form. Prokaryotes lack a nuclear envelope. Bacterial DNA is
not associated with proteins, therefore it is described as ‘naked DNA’. The DNA is highly coiled
(supercoiled). It may be concentrated in the centre of the cell as a nucleoid. In addition to the major
DNA (bacterial chromosome), some accessory DNA materials called plasmids may be present in
bacteria. They are extra-chromosomal circular DNA which confers certain specific characteristics to
the cell such as resistance to antibiotics and other agents.
Eukaryotic organisms possess a nuclear envelope which contains the genetic material. In
eukaryotes, the DNA exists as a single linear molecule to which histones and other proteins are
attached. The number of chromosomes can vary from just a few to many hundreds.
Flagella
Bacteria move by means of hair-like structures called flagella. Bacterial flagella are thin (14 –
20 nm diameter) and rigid and rotate like a ship’s propeller. Flagella are made up of a protein called
Flagellin, which is organized into flagellar subunits. At the base of the flagellum, there is a basal body
which rotates the flagellum to cause movement of the cell. Through the action of the basal body, each
flagellum is caused to rotate in an anticlockwise direction at about 12000 rpm giving a speed of 20 µm
to 80 µm per second. This is about 10 lengths per second. Can move up to 60 cell length per second.
Each flagellum is a semi-rigid structure that moves by rotation like a propeller. The rotary motion is
given from the basal body (which functions as the motor). Mot complex drives rotation of the
flagellum deriving energy through proton movement across the membrane. The arrangement of
flagellain a bacterial cell is important in classification. Accordingly, bacteria are designated as follows:

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 A. Atrichous: Bacteria without flagella are called
atrichous.
B. Monotrichous: Bacteria with the single polar
flagellum. Eg.Vibrio cholerae
C. Amphitrichous: Single flagellum at each end of the
cell. eg.Spirillum volutans
D. Lophotrichous: Two or more (a tuft) flagella at each
end of the cell. eg.Alcalegenes faecalis
E. Peritrichous: Flagella all over the entire cell. eg.E.coli
Chemotaxis and Motility
Chemotaxis is the movement of an organism towards or away from a chemical. Positive chemotaxis is
the movement towards a chemical (attractant); negative chemotaxis is the movement away from a
chemical (repellent). Bacterial movement is controlled by the presence of these compounds such that
where there is no gradient of attractant or repellent in the environment, the organism moves in a
random way. However, in the presence of a concentration gradient, the net movement of the bacterium
is in one direction. Bacteria detect the presence of a gradient through the action of membrane-bound
chemoreceptors. Bacterial movement is characterized by runs and tumbles. But when an attractant
(gradient of a chemical attractant) is present it is marked by larger runs and less frequent tumbles.
Bacterial endospores
Many living organisms like filamentous fungi produce structures called spores. Their function
is a reproductive one. The bacterial endospore is not a reproductive structure. Bacterial cell produces
only one endospore, which is resistant to harsh environmental conditions including high temperatures
and toxic chemicals. Bacteria of the genera Bacillus and Clostridium produce endospores.

20
 The structure of the endospore is more complex than the vegetative cell which produces it.
Dipicolinic acid (DPA) with high concentrations of calcium ions is found in all endospores. A complex
of calcium and DPA is responsible for their heat resistance. Sporulation occurs in the environment (in
soil and on nutrient media) but not in human/animal tissues.
The sporulation process occurs in four success stages:
1. Preparatory stage
2. Forespore stage
3. Stage of cell wall formation
4. Maturation stage
Under unfavourable conditions (eg. Nutrient depletion) structural changes take place inside the
cell. It is characterized by the thickening of the cytoplasm in a certain region and the formation of a
forespore, which becomes surrounded by a thick poorly permeable multilayered wall. The rest of the
cell gradually disappears, and a spore is produced.
Other structures:
Inclusion bodies may be present in prokaryotes which are meant for the storage of materials. The most
common storage materials are: 1. Poly-β hydroxybutyric acid (PHB), a lipid-like molecule surrounded
by proteins. 2. Granules of polyphosphate, sometimes called volutin granules or metachromatic
granules, which are used by the cells for thegeneration of ATP and other cell constitutions.

5. Viruses, structure, classification, characters and their economic importance

General characteristics of viruses
Viruses may be regarded as exceptionally complex aggregations of nonliving chemicals or
exceptionally simple living microbes. Because viruses are inert outside living host cells but once
viruses enter a host cell, they become active and viral multiplication occurs. Therefore, viruses can be
termed obligatory intracellular parasites. The virus is the Latin word for poison.
Viruses contain a single type of nucleic acid (either DNA or RNA) and a protein coat,
sometimes enclosed by an envelope composed of lipids, proteins and carbohydrates. Viruses are
obligatory intracellular parasites. They multiply by the host cells synthesizing mechanisms. A
complete, fully developed viral particle composed of nucleic acid surrounded by a coat is called a
Virion.
Host range:

21
 Host range refers to the spectrum of host cells in which a virus can multiply. Viruses are host
specific. There are viruses that infect invertebrates, vertebrates, plants, protists, fungi and bacteria.
Most viruses infect specific types of cells of only one host species. Host range is determined by the
specific attachment site on the host cells’ surface and the availability of host cellular factors.
Size:
Viral size is measured by electron microscopy. Viruses range from 20 nm to 300 nm in length.
Viral Structure

Nucleic acid
A virus contains a single kind of nucleic acid, either DNA or RNA which is the genetic material. The
nucleic acid can be single-stranded or double-stranded. The nucleic acid can be linear or circular.
Capsid and Envelope
The nucleic acid of a virus is surrounded by a protein coat called the capsid. Each capsid is composed
of protein subunits called capsomeres, which can be a single type of protein or several types. The
capsid of some viruses is enclosed by an envelope consisting of lipids, proteins and carbohydrates.
Some envelopes are covered with carbohydrate–protein complexes called spikes.
Morphology
Viruses may be classified into several morphological types based on their capsid architecture.
1. Helical viruses - Eg. Tobacco mosaic virus: These resemble long rods, and their capsids are
hollow cylinders surrounding the nucleic acid.
2. Polyhedral viruses – eg. Adenoviruses: These have many sides and usually the capsid is an
icosahedron.

22
 3. Enveloped viruses are covered by an envelope and are roughly spherical but highly
pleomorphic. There are also enveloped helical viruses (influenza virus) and enveloped
polyhedral viruses (herpes virus).
4. Complex viruses: These have complex structures for example many bacteriophages have a
polyhedral capsid with a helical tail.
Virus Symmetry
The capsids of virions tend to have one of two symmetries – helical or cuboid. Helical
symmetry can be described as having a spriral ‘staircase’ structure. The structure has an obvious axis
down the center of the helix. The subunits are placed between the turns of the nucleic acid. Example:
tobacco mosaic virus. Animal viruses with a similar capsid structure include measles, rabies and
influenza. Most animal viruses have spherical or cuboid symmetry. The ‘closed shell’ capsid is usually
based on the structure referred to as icosahedrons. A regular icosahedron, formed from assembly of
identical subunits, consists of 20 equilateral triangular faces, 30 edges and 12 vertices and exhibits 2-,
3- and 5- fold symmetry. The minimum number of capsomers required to construct an icosahedron is
12, each composed of five identical subunits.

Taxonomy of Viruses
Viruses are classified based onthe type of nucleic acid, morphological class and presence or,
absence of an envelope. Virus family names end in --------- viridae and genus names end in -------
virus. A viral species is a group of viruses sharing the same genetic information and ecological niche.
Isolation, cultivation and identification of viruses
Viruses must be grown in living cells.

23
Growth of viruses in the laboratory
Bacteriophages can be cultivated by the number estimated by plaque assay. The plaque assay
mixes bacteriophages with host bacteria and nutrient agar. After several viral multiplication cycles, the
bacteria in the area surrounding the original virus are destroyed. The area of lysis is called a plaque.
Each plaque originates with a single viral particle. The concentration of viruses is given as plaque-
forming units (pfu)

Plaque assay 1A and 1B - Cell line without and with CPE

Cultivation of some animal viruses requires whole animals. Some animal viruses can be
cultivated in embryonated eggs. Viruses can also be grown on cell cultures (cell cultures are cells
growing in culture media in the laboratory); Primary cell lines grow for a short time while continuous
cell lines can be maintained indefinitely. Viral growth causes Cytopathic effect (CPE) in the cell
culture.
Viral identification
Serological tests are used to identify viruses. Viruses may be identified by techniques like
polymerase chain reaction (PCR) methods, restriction enzyme fragments, the appearance of host cells
following infection and electron microscopy.
Multiplication of viruses
Viruses invade a host cell and direct the host’s metabolic machinery to produce viral enzymes
and components. The multiplication cycle of viruses can be divided into five distinct stages, namely:
Attachment, Penetration, Biosynthesis, Maturation and Release.
Phages can multiply by two mechanisms: the lytic cycle or the lysogenic cycle. The lytic cycle
results in the lysis and death of the host cell, whereas the host cell remains alive in the lysogenic cycle.

24
Lytic cycle of T – even bacteriophage
1. Attachment:
During this process, an attachment site on
the virus attaches to a complementary
receptor site on the bacterial cell.
Attachment is established by the formation
of weak bonds between the tail fibres of
T–even phages and complementary
receptor sites on the bacterial cell wall.

2. Penetration
After attachment, the phage injects its DNA into the bacterium. An enzyme, phage lysozyme is
released from the phage’s tail, which breaks down a portion of the bacterial cell wall; the tail sheath
contracts to force the tail core through the cell wall and phage DNA enters the bacterial cell. The
capsid remains outside.
3. Biosynthesis
Once the phage DNA has entered the cytoplasm of the host cell, host protein synthesis is
arrested. Phage uses the host cell’s nucleotides and its enzymes to synthesize phage DNA. Phage DNA
is transcribed into mRNA and synthesis of enzymes and capsid protein occurs. During this stage, only
separate components (DNA, protein) are formed. This period during viral multiplication where
complete virions are not yet present is called the eclipse period.
4. Maturation
In this process, DNA and capsids are assembled into complete virions
5. Release
During this final stage, lysozyme is synthesized within the cell which causes the bacterial cell
wall to breakdown. This results in the release of bacteriophages. The time from phage attachment to

25
release is known as burst time and is usually from 20 to 40 min. The number of newly synthesized
phage particles released from a single cell is called burst size and it ranges from 50 to 200.

Lysogenic cycle of Bacteriophage

Some phages begin a lysogenic cycle by
incorporating their DNA into the host cell’s DNA.
During this state, called lysogeny, the phage remains
latent. Upon penetration into a bacterial cell, the
linear phage DNA becomes a circle. The circular
DNA of the phage may recombine with and become
part of the circular bacterial DNA. The inserted
phage DNA is called a prophage. Every time the
host cell replicates the bacterial chromosome, the
prophage DNA also gets replicated. The prophage
remains latent within the progeny cells. Under some
circumstances, or due to the action of UV light or
certain chemicals excision of phage DNA occurs
which initiates the lytic cycle.

Multiplication of Animal viruses

The process is essentially the same as that of bacteriophages. Animal viruses attach to the
plasma membrane of the host cell and penetration occurs by endocytosis. Animal viruses are uncoated
by either viral or host cell enzymes. The DNA is released into the nucleus of the cell, where several
copies of DNA are synthesized. Capsid protein is synthesized in the cytoplasm. (DNA viruses include
members of the families Adenoviridae, Poxviridae, Herpesviridae etc.) Multiplication of RNA viruses
(Picornaviridae, Rhabdoviridae, Togaviridae) occurs in the cytoplasm of the host cell.
After maturation, viruses are released. One method of release is budding. Naked viruses are released
through ruptures in the host cell membrane.

26
6. Cyanobacteria, actinomycetes, archaea, mycoplasma rickettsia etc
Actinomycetes (The Filamentous Bacteria)
The actinomycetes (sing. actinomycete) are a large group of aerobic, high G-C percentage gram-
positive bacteria that form branching filaments or hyphae and asexual spores. These bacteria closely
resemble fungi in overall morphology. When grown on an agar surface, the actinomycetes branch
forming a network of hyphae growing both on the surface and under-surface of the agar. The on-the-
surface hyphae are called aerial hyphae and the under-surface hyphae are called substrate hyphae.
Septa normally divide the hyphae into long cells (20 mm and longer) possessing many bacterial
chromosomes (nucleoids). These are the aerial hyphae that extend above the substratum and reproduce
asexually. Most actinomycetes are non-motile. Actinomycetes are particularly useful because they
break down hard organic materials like newspaper, tree bark and woody stems.
Actinomycete colony on agar surface
1. Chain of conidiospores (Conidia)
2. Aerial hyphae
3. Agar surface
4. Substrate hyphae
The composition of the cell wall in actinomycetes varies greatly among different groups and is of
considerable taxonomic significance. Four major cell wall types are distinguished in these filamentous
bacteria based on the features of peptidoglycan composition and structure.

27
Spirochaetes (also Spirochetes) belong to a phylum of distinctive Gram-negative bacteria, which have
long, helically coiled (spiral-shaped) cells. Spirochetes are chemoheterotrophic in nature, with lengths
between 5 and 250 µm and diameters around 0.1-0.6 µm. Spirochaetes are distinguished from other
bacterial phyla by the location of their flagella, sometimes called axial filaments which run lengthwise.
These cause a twisting motion which allows the spirochaete to move about. When reproducing, a
spirochaete will undergo asexual transverse binary fission. Most spirochaetes are free-living and
anaerobic, but there are numerous exceptions.

Spirochaete Cyanobacteria
The spirochetes are divided into three families: (Brachyspiraceae, Leptospiraceae, and
Spirochaetaceae), all placed within a single order (Spirochaetales). Some of the disease-causing
members include the following: 1. Leptospira species, which causes leptospirosis; 2. Borrelia
recurrentis, which causes relapsing fever; 3. Treponema pallidum - causes syphilis; 4. Treponema
pertenue, which causes yaws.
Cyanobacteria
Cyanobacteria, also known asblue-green algae, blue-green bacteriaorcyanophyta, is a phylum
of bacteria that obtain their energy through photosynthesis. The name "cyanobacteria" comes from the
colour of the bacteria. They are a significant component of the marine nitrogen cycle and an important
primary producer in many areas of the ocean, but are also found in habitats other than the marine
environment; in particular, cyanobacteria are known to occur in both freshwater, hypersaline inland
lakes and in arid areas where they are a major component of biological soil crusts. Chloroplasts in
plants and eukaryotic algae have evolved from cyanobacteria via endosymbiosis.

Mycoplasma is a genus of bacteria which lack a cell wall. Without a cell wall, they are unaffected by
many common antibiotics such as penicillin. They can be parasitic or saprotrophic. Several species are
pathogenic in humans, including M. pneumoniae, which is an important cause of atypical pneumonia
and other respiratory disorders. The genus Mycoplasma is restricted to vertebrate hosts. Despite the
lack of a cell wall, the cells often present a certain shape. These cell shapes presumably contribute to

28
the ability of mycoplasmas to thrive in their respective environments. For example, the members of the
genus Spiroplasmaassume an elongated helical shape. M. pneumoniae cells possess an extension, the
so-called 'tip-structure', protruding from the coccoid cell body. M. pneumoniae cells are of small size
and pleomorphic, but with a rough shape in longitudinal cross-section resembling that of a round-
bottomed flask. Mycoplasmas are unusual among bacteria in that most require sterols for the stability
of their cytoplasmic membrane.

Rickettsiae
Rickettsia is a genus of motile, Gram-negative, non-sporeforming, highly pleomorphic bacteria that
can present as cocci (0.1 μm in diameter), rods (1-4 μm long) or thread-like (10 μm long). Obligate
intracellular parasites, the Rickettsiasurvival depends on entry, growth, and replication within the
cytoplasm of eukaryotic host cells (typically endothelial cells). Because of this, Rickettsia cannot live
in artificial nutrient environments and are grown either in tissue or embryo cultures. MostRickettsia
bacteria are susceptible to antibiotics of the tetracycline group. Rickettsia species are carried as
parasites by many ticks, fleas, and lice, and cause diseases such as typhus, rickettsialpox, Boutonneuse
fever, African Tick Bite Fever etc., in human beings. They have also been associated with a range of
plant diseases.
Archaebacteria (Archaea)
Archaea are a group of prokaryotic organisms, which although look like bacteria cytologically, they
are not closely related to them. The archaea can be divided into two evolutionary lineages based on
rRNA sequences, namely the Crenarchaeotae and the Euryarcheotae.
The crenarchaeotaeare organisms that grow at high temperatures (thermopiles) and metabolize
elemental sulfur. The euryarchaetoes have several different phenotypes. Many are methanogens
(methane-producing anaerobes) and some grow aerobically at very high concentrations of salt called
halophiles.
Archaea possess unusual membrane lipids composed of branched-chain hydrocarbons (based on 5 –
carbon isoprene unit) bound to one or two glycerol molecules by ether bonds. In the case of most
bacteria and all eukaryotes, the membranes are composed of straight-chain hydrocarbons bound to a
single glycerol molecule by ester-bonds.

29
7. Eukaryotes
Basic structure and organization of Eukaryotic cell in Comparison to Prokaryotic cell

Cell Membrane
 Eukaryotes resemble prokaryotes basically in terms of membrane structure and functions.

 It is a fluid phospholipid bilayer embedded with proteins and glycoproteins.

glycoproteins
 In addition, it contains glycolipids as well as complex lipids called sterols,, such as the cholesterol
molecules found in animal cell memb
membranes,
ranes, that are not found in prokaryotic membranes (except
for some mycoplasmas). The sterols make the membrane less permeable to most biological
molecules, help to stabilize the
Cell wall
• Cell wall is quite simple.
• In algae and plant cells, the cell w
wall is usually composed of cellulose.
• In molds it is composed of chitin and/or cellulose.
• Animal cells and protozoans lack cell walls.
30
Flagella and Motility in eukaryotes
Many eukaryotic microorganisms also move by means of flagella and cilia. Cilia are shorter
than flagella but structurally similar. Eukaryotic flagella are long, flexible structures which move in a
whiplash fashion. They have a complex structure consisting of a number of microtubules. Both consist
of 9 fused pairs of protein microtubules (tubulin) with side arms of the motor molecule dynein that
originate from a centriole. These form a ring around an inner central pair of microtubules that arise
from a plate near the cell surface. The arrangement of microtubules is known as a 2X9+2 arrangement.
This complex of microtubules is surrounded by a sheath continuous with the cytoplasmic
membrane.Each microtubule is composed of protein, tubulin, with subunits arranges in a helical
fashion along the tubule axis. Eukaryotic microbes move at a rate of 30 to 250 µ m per second. In
contrast to flagella, cilia are more in numbers, more or less rigid and beat in a coordinated fashion
called metachronal rhythm.
Amoeba and slime moulds do not possess
flagella or cilia but show a movement produced
by their cytoplasm, called cytoplasmic
streaming. Such cytoplasmic flow produces
projections, called pseudopodia which cause the
cell to move.

Mitochondria: Mitochondria are approximately the same size as bacteria (2 – 3 µ m long and 1 µ m in
dia). The number of mitochondria varies in cells from just 2 (in yeast cell) to up to 1000 (in a liver
cell). The process of respiration and oxidative phosphorylation (the mechanism of ATP synthesis
during respiration) are localized in membrane bound organelles (mitochondria) in eukaryotic
organisms. The mitochondrial membranes lack sterols and are more permeable so that ATP
synthesized within the mitochondrion can move into the cytoplasm. The inner mitochondrial
membrane is highly folded to produce cristae. Cristae project into the mitochondrial lumen. The
enzymes involved in ATP production and specific transport proteins which regulate the passage of
metabolites into and out of the matrix are located within the inner membrane.

31
 Enzymes of TCA cycle and β – oxidation are located in the matrix. Complexes of the enzyme
ATP synthetase are present as small round particles attached to the cristae. These small sphere shaped
particles are called F1 particles and they synthesize ATP during cellular respiration. Mitochondria use
its DNA and ribosomes to produce some of its own proteins. Mitochondria reproduce by binary fission
Chloroplasts
All photosynthetic eukaryotic organisms, including microscopic algae possess chlorophyll
containing organelles called chloroplasts. The structure of chloroplast reveals thylakoids. The
thylakoid membrane is impermeable to ions and other metabolites and well suited for its role in protein
translocation and the generation of proton motive force for the synthesis of ATP. The thylakoids are
stacked to produce structural units called grana. The stroma of chloroplasts contains enzymes for
reduction of CO2 to organic material. The outer chloroplast membrane is highly permeable and allows
ATP and glucose synthesised during photosynthesis to diffuse into the cytoplasm. Although some
bacteria are photosynthetic, they do not possess chloroplasts. Instead, components of the
photosynthetic mechanism are located on membranous structures formed by modification of the
cytoplasmic membrane.
Nuclei
Eukaryotic organisms possess a nuclear envelope which contains the genetic material. The
envelope consists of two membranes which fuse at several places to produce pores. Contact between
the nuclear contents and the cytoplasm is maintained through the pores. The outer membrane of the
nuclear envelope carries ribosomes and forms a continuous structure with the endoplasmic reticulum
and cytoplasmic membrane. The nuclear envelope encloses several chromosomes in association with
chromosomal proteins. These are called histones. Histones are positively charges proteins which
neutralize the negatively charged phosphate groups of the DNA. Eukaryotic cells contain much more

32
DNA than do bacteria. The nucleus divides by mitosis, a process that ensures each daughter cell
receives the same number of chromosomes as the original parent cell. Haploid sex cells are produced
from diploid cells by meiosis
Endoplasmic reticulum (ER) and Golgi bodies
The endoplasmic reticulum or ER is a maze of parallel membranous tubules and flattened sacs
surrounding the nucleus that connects with the nuclear membrane and runs throughout the cytoplasm.
The functions are i) provide a surface area for protein and lipid synthesis; ii) form a pathway for
transporting molecules within the cell; and iii) provide a storage area for molecules the cell has
synthesized.
Golgi bodies
The Golgi is principally responsible for directing molecular traffic in the cell - nearly all
molecules pass through the Golgi complex at some point in their existence. The sorting is mediated by
the vesicles. When proteins bind with their appropriate receptor on the vesicle, they are encoated in the
vesicle and transported away.
The cytoskeleton
The cytoplasm of eukaryotic cells contains an extensive network of microfilaments and
microtubules. These filaments contain a number of proteins including actin and myosin. The main
proteins of the microtubules are called tubulins. The cytoskeleton has a number of functions, including
the maintenance of cell shape positioning cell organelles and cell motility (Spirochactes is an exception
among Prokaryotes in having cytoskeleton).

Lysosomes:
Lysosomes are cellular organelles that contain acid hydrolase enzymes to break down waste
materials and cellular debris. They are found in animal cells, while in yeast and plants the same roles
are performed by lytic vacuoles. Lysosomes digest excess or worn-out organelles, food particles, and
engulfed viruses or bacteria.
33
Vacuoles:
Vacuoles are large membranous sacs; vesicles are smaller. Vacuoles are often used to store
materials used for energy production such as starch, fat, or glycogen. Plant cells often contain large
vacuoles filled with water. Vacuoles and vesicles also transport materials within the cell and form
around particles that enter by endocytosis.
Differences between Prokaryotes and Eukaryotes
Characters Prokaryotes Eukaryotes
Groups where found Bacteria, Cyanobacteria, Archaea Algae, Fungi, Protoza, Plants and Animals
Size 1-2 by 1-4 µm or less Greater than 5 µm
Cell wall Made up of peptidoglycan Made of cellulose in algae and plants, of
fungal cellulose or chitin in fungi, absent
in protozoan and animals
Osmotic control Wall possesses mechanical strength Maintained by contractile vacuole
necessary to counterbalance turgor
pressure of cytoplasm
Plasma membrane Does not contain sterols except in Sterol present
Mycoplasma
Fatty acids, exclusively saturated or All lipids are polyunsaturated
monounsaturated
No inter membrane structure is Complex network of inter membrane
connected to plasma membrane connected with plasma membrane
Plasma membrane is the only Several cell organelles also covered by
membrane membrane
Plasma membrane folded to form Mesosomes are absent.
mesosomes.
Contains the respiratory machinery E.T.S. is present on mitochondrial
i.e. E.T.S., enzymes etc. and carries membrane.
out respiration.
Photosynthetic machinery present Photosynthetic pigments and ETS present
on bacterial membrane. In in the chloroplast membrane.
cyanobacteria membrane forms
34
 vesicles, which carry pigments.
Cytoplasm Granular due to presence of Fibrillar made up of fibrous proteins
ribosomes, fibrous cytoskeleton which form fibrous cytoskeleton
absent
Pinocytosis, Exo- Absent, intracellular digestion. Present.
cytosis, Endocytosis,
Phagocytosis
Gas vacuoles Present Absent
Ribosomes 70S, freely distributed in cytoplasm 80S free as well bounded to ER. 70S in
mitochondria and chloroplast.
Mitochondria Absent Present
Chloroplast Absent Present in plant cells
Golgi apparatus Absent Present
Lysosomes Absent Present
Endoplasmic Absent Present
Reticulum
Peroxisomes Absent Present
Centrioles Absent Present
Nucleolus Present Absent
True vacuoles Absent Present
Gas vesicles and Present Absent
carboxysomes
Flagella Made up of flagellin sub-units Made up of tubulin microtubules arranged
in 9+2 formation
Pili Present Absent
Cilia Absent Present in protozoans
Replication of DNA Throughout cell cycle Only during ‘S’ phase
Synthesis of histone Absent Only during ‘S’ phase
proteins
Cell division Immediately after replication or During mitosis (M) phase

35
 simultaneously
Meiosis or reduction Absent Present
division
Chromosome number Usually 1 Variable, 2 to many
DNA Circular, single chromosome, single Single, linear, double stranded, helically
or double stranded, helically coiled, coiled, chromosome diploid.
usually haploid chromosome.
Extranuclear DNA Present in bacteria (plasmid 0.1-5% Absent except in yeasts
of bacterial chromosome)
Histone proteins Absent Present
DNA position, Usually central, not surrounded by Surrounded by nuclear membrane
nuclear membrane nuclear membrane
Chromosomal Unidirectional from donor to Exchange of chromosome between two
movement recipient; at times only exchange of cells (gametes)
plasmids.
Structure of mRNA Polycistronic Monocistronic
N2 fixation Present in some bacteria and Absent
cyanobacteria
Respiration Performed by plasma membrane Performed by mitochondria
Anaerobic respiration Present in many bacteria Shown by some e.g. yeasts, few
protozoans, muscle cells
Locomotion Present in some bacteria, by Absent in plants, algae, fungi, except in
flagella. zoospores and few unicellular algae and
fungi, present in animals and protozoans.
Reproduction Simple fragmentation or binary Complex, meiosis present, also by
fission, no meiosis, asexual spores fragmentation, binary fission and asexual
produced in some. spores in some plants. Algae and fungi
and few protozoans.
Genetic Present in some but chromosomal By chromosomal cross over during
recombination cross over absent. meiosis.

36
Fungi
Fungi are eukaryotic, spore-bearing organisms with absorptive nutrition. They lack chlorophyll
and usually are filamentous and multicellular with definite cell. Differentiation exists between
reproductive and somatic structures. Fungi are primarily terrestrial organisms; grow on acidic medium
(pH 6.0). It consists of yeasts and molds.
Structure:
The vegetative structure of a fungus is called Thallus. Thallus varies in size and complexity
from unicellular yeast to multicellular mold and macroscopic mushrooms. The fungal cell is encased in
a cell wall made of chitin (polysaccharide of N – acetyl glucosamine residues).

Yeast is a unicellular fungus with a single nucleus, reproduces by budding and transverse division or
sexually through spore formation.
Mold consists of long, branched filaments called the hyphae, which aggregate to form mycelium.
These hyphae are either coenocytic (lack cross walls) or septate (have cross walls). If septate, they
have either a single pore or multiple pores that allow protoplasmic streaming.
Nutrition
Fungi grow best in dark, moist habitats. Based on the mode of nutrition they are classified as parasites
and saprophytes. 1. Obligate saprophytes – that live on dead organic matter not infecting living
organisms. 2. Facultative parasites or saprophytes – that are capable of living on dead organic matter or
live organisms. 3. Obligate parasites – that require living protoplasm. They are chemo-
organoheterotrophs and use organic material as a source of carbon, electrons and energy. Fungi are
usually aerobic. Some yeasts are facultatively anaerobic and can obtain energy by fermentation.

37
Reproduction in Fungi
Fungi reproduce either sexually or asexually. Asexual reproduction may be accomplished in
several ways:
1. A parent cell can divide into two daughter cells by central constriction and formation of a new cell
wall
2. Somatic vegetative cells may bud to produce new organisms ( eg. Yeast)
3. By the production of asexual spores
a. Arthrospores: A hypha can fragment (through splitting of the cross wall or septum) to forms
cells that behave like spores.
b. Chlamydospores: The cells are surrounded by a thick wall before separation.
c. Sporangiospores: Spores that develop with a sac called sporangium at a hyphal tip.
d. Conidiospores: Spores that are not enclosed in a sac, but produced at the tips or sides of the
hypha.
e. Blastospores: Spores produced from a vegetative mother cell by budding.

38
Sexual Reproduction
Sexual reproduction involves the union of compatible nuclei. Sexually compatible gametes are
produced on the same mycelium in some species. Some other species require crossing between
different but sexually compatible mycelia. Therefore, sexual fusion may occur between haploid
gametes or hyphae. Sexual reproduction may also yield spores (like zygospore, ascospore or
basidiospore). Details of sexual reproduction are discussed under different divisions of fungi.
Taxonomic classification of fungi
Division Common Name Approx. No. of species
Zygomycota Zygomycetes 600
Ascomycota Sac fungi 35,000
Basidiomycota Club fungi 30,000
Deuteromycota Fungi imperfecti 30,000
The above classification is based primarily on variations in sexual reproduction.

39
Division Zygomycota
The zygomycetes are coenocytic. Most are saprophytic. A few are parasites of plants, insects,
animals and humans. Eg.Rhizopus stolonifer, the bread mold. It grows on moist, carbohydrate rich
foods such as breads, fruits and vegetables. Special hyphae called rhizoids extend into the substrate
and absorb nutrients. Some hyphae produce asexual sporaniga at the tip. Each spore when released can
start a new mycelium.
Rhizopus usually reproduces asexually, but if food becomes scarce, it begins sexual
reproduction. Sexual reproduction requires compatible strains of opposite mating types (usually
designated + and – strains). When two mating strains come close, hormones are produced and the
hyphae form projections called progametangia which mature to gametangia. After fusion of the
gametangia, the nuclei of the two gametes fuse to form Zygote. The zygote develops a thick, rough,
black coat and becomes zygospore. Zygospore produces an asexual sporangium and the cycle begins.

Division Ascomycota
The fungi of this division are called ascomycetes or sac fungi. Most of the red, brown and blue
green molds that cause food spoilage belong to this division. This division contains several yeasts.
Many ascomycetes are parasites of higher plants. Ergotism is a disease (toxic) condition in humans
and animals who eat grains infected with the fungus, Claviceps purpurea. The ascomycetes are named
for their characteristic reproductive structure, the club or sac-shaped ascus. The mycelium of
ascomycetes is composed of septate hyphae. Asexual reproduction is common in the ascomycetes and
takes place be way of conidiospores.

40
 Sexual reproduction involves the formation of an ascus containing ascospores. Sexual
reproduction starts with the development of special ascogenous hyphae. One nucleus from a male
mycelium (antheridium) and the other from a female mycelium (ascogonium) migrate to form the
ascogenous hyphae. The paired nuclei divide in such a way that there is one pair of nuclei in each cell.
After the ascogenous hyphae have matured, nuclear fusion occurs at the hyphal tips in the ascus
mother cells. The diploid zygote nucleus then undergoes meiosis, resulting in four haploid nuclei.
These divide mototically to produce a row of eight nuclei in each developing ascus. These nuclei
become ascospores. Once they mature, they are released from the asci with great force. Upon reaching
suitable environments, each ascospore germinates. Some of the yeasts also reproduce sexually in this
manner and are classified in ascomycota.

Division: Basidiomycota:
Basidiomycetes are commonly called club fungi. It includes mushrooms etc. Basidiomycetes
are named for their characteristic structure, the basidium, involved in sexual reproduction. The
members of basidiomycetes affect humans in many ways. Most are saprophytic and decompose plant
debris, especially cellulose and lignin. Many mushrooms are used as food. Many mushrooms produce
specific alkaloids that act either as poisons or hallucinogens. There are several other plant pathogens
and human pathogens under badiomycetes.
The life cycle of a typical basidiomycete starts with a basidiospore germinating to produce a
monokaryotic mycelium (single nucleus in each septate cell). The mycelium spreads through the soil
41
and when this primary mycelium meets another monokaryotic, mycelium of a different making type,
the two mycelia fuse to initiate a new dikaryotic secondary mycelium. The secondary mycelium
consists of cells with two nuclei, one of each mating type. This mycelium is stimulated to produce a
solid mass of hyphae, as a button that pushes through the soil, elongates and develops a cap. The entire
structure is called basidiocarp. The cap contains plate like gills, each coated with basidia. The two
nuclei in the tip of basidium fuse to form a diploid zygote nucleus. This zygote undergoes meiosis to
form four haploid nuclei. These nuclei move into the developing basidiospores. These are released
upon maturity.

Division Deuteromycota
When a fungus lacks the sexual phase or if this phase has not been observed, it is placed in the
division Deuteromycota, commonly called Fungi Imperfeci. Most Fungi Imperfecti are terrestrial.
They are either saprophytes or parasites of plants. A few are parasitic on other fungi. Several human
pathogenic forms causing diseases like athlete’s foot, ringworm etc., belong to these group.
Slime molds and water molds
These molds resemble fungi in appearance and life-style. In their cellular organization,
reproduction and life cycles, they are related most closely to the protists.
Division: Myxomycota
Plasmodial slime molds exist as thin, streaming masses of colourfulpeoroplasm that creep
along in an amoeboid fashion over moist, rotting logs, leaves and other organic matter. Feeding is by
phagocytosis. They lack cell wall and so, called plasmodium. Plasmodium contains many diploid
nuclei. When it matures (or when food and moisture are scarce, it develops delicate fruiting bodies;
where spores are formed with cellulose walls that are resistant to environmental conditions. The spores
germinate to release either non-flogellaled amoeboid myxamoeba or flagellated swarm cells. They fuse

42
eventually to form diploid zygote. The zygote feeds, grows and multiplies its nuclei by mitosis to form
the multinucleate plasmodium.
Division Acarasiomycota
Cellular slime molds consist of irregular amoeboid cells called myxamoeba which feed by
phagocytosis on bacteria and yeasts. When food is plenty, they divide repeated by mitosis and
cytokinesis, producing daughter myxamoebae. When food is scarce, myxamoebae secrete cyclic
adenosine monophosphate (cAMP) which attracts other myxamoebae and aggregate themselves to
form pseudoplasmodium becomes sedentary and differentiates into prestalk cells and prespore cells. A
structure called sorocarp forms and matures into sporangiun to produce spores. Spores release and
germininate to haploid amoebae under favourable conditions.

43
Division Oomycota
Oomycetes or water molds resemble fungi, consists of finely branched filaments called haphae.
Its cell wall is made of cellulose. Several parasitic and saprophytic forms are present in oomycetes.
Some have a mode of sexual reproduction where a large egg cell is fertilized by a sperm cell or smaller
antheridium. Oomycetes also produce asexual zoospores that have two flagella.
Algae
Algae are classified under the group thallophyta in the division cryptogams. This group
includes plants which are not differentiated into various parts like root, stem, leaves and flowers. In
other words they lack tissues. Algae are autotrophic and carryout oxygenic photosynthesis, found both
in freshwater as well as seawater and moist soil. They may be single celled, colonial or filamentous.
They form the green scum found floating on surface of stagnant water or ponds. Algae are usually
green because of the presence of chlorophyll. But the colour of some algae may be blue – green or red
or brown due to the presence of other pigments. Their cell wall is made up of cellulose and they store
the reserve food as starch. Examples:Chlamydomonas (single – celled), Volvox (colonial), Spirogyra
(filamentous).

Lichens are a symbiotic combination of an algae and a fungus where both are mutually
benefited. The fungal partner provides water, mineral and physical protection while the alga
synthesizes food lichens are found growing on rocks and barks of trees. E.g.Rhizocarpon, Collema etc.
Properties of major groups of algae
Groups Morphology Pigments/carbon Habitats Examples
reserve/cell wall features
Chlorophyta (Green Unicellular to Chlorophylls aandb; starch; Freshwater, soil Chlamydomonas
algae) leafy cell walls of cellulose and marine
Chrysophyta Unicellular Chlorophylls a, c and e; Freshwater, soil Navicula

44
Golden-brown Lipids; silica and marine
algae, diatoms
Phaeophyta (Brown Filamentous to Chlorophylls aandc Marine Laminaria
algae) leafy Xanthophylls; Laminarin,
mannitol; cellulose
Pyrrophyta Unicellular Chlorophylls aandc; starch; Freshwater, Gonyaulux
(Dinoflagellates) flagellated cellulose marine
Rhodophyta (Red Unicellular, Chlorophylls aandd, Marine Polysiphonia
algae) filamentous to phycocyanin,
leafy phycoerythrin; starch;
fluoridoside, cellulose

Protozoa
Protozoa are characterized by the features summarized below:
They are unicellular or colonicaleurkaryotes. They possess protoplasmic level of organization.
The body may be covered by simple plasma membrane or cuticle or pellicle. The cell wall is absent.
They have various membrane bound cell organelles. Locomotory organelles may be pseudopodia (as in
Amoeba) cilia (Paramecium) or flagella (Euglena). Parasitic forms do not possess any of these
locomotory structures. Nutrition may be holophytic or holozoic. They may be autotrophic,
heterotrophic or myxotrophic. Respiration and excretion take place through body surface. In freshwater
forms contractile vacuoles serve as osmoregulatory organelles. Reproduction is generally asexual by
binary or multiple fission. Sexual reproduction by conjugation is seen in some cases. Cyst formation is
common in freshwater forms to overcome dry, unfavourable conditions.
Major groups of protozoa and their diseases
Group Habitats Common diseases Examples
Mastigophora Freshwater; parasites of animals Sleeping sickness Trypanosoma
(Flagellates) Leishmaniasis Leishmania
Sarcodina Freshwater and marine; animal Amoebic dysentery Entamoeba
(Amoebas) parasites
Ciliophora Freshwater and marine; animal Dysentery Paramecium
(Ciliates) parasites

45
 Sporozoa Primarily animal parasites Malaria Plasmodium
(Sporozoans)
Euglenoids Freshwater, some marine (Also --- Euglena
(Phototrophic considered with algae)
flagellates)

8. Microbial techniques: Culture Media

Specialized media are essential in isolation and identification of microbes, although all
microorganisms require sources of energy, carbon, nitrogen, phosphorus, sulfur and minerals.
Synthetic / Defined media
Medium in which all components are known chemically is called a defined medium or
synthetic medium.
Complex media
Media that contain some ingredients of unknown chemical composition. These are useful as the
exact nutritional requirements of many microorganisms are not known and use of rich nutrients will
facilitate their growth. For ex. Yeast extract is a good source of B vitamins and Carbon and Nitrogen
compounds. Some examples of complex media are tryptic soya agar (TSA), Nutrient agar (NA) and
Plate count agar (PCA). These media are also referred to as general purpose media or non-selective
media. Agar is a solidifying agent not degraded by most bacteria and it is incorporated at the level of
1.5% for solid media. The solidifying point of agar is 42 oC – 44oC and melting point is 90oC.
Selective media
Media that favour growth of particular group of microorganisms. Specific salts / dyes are used
to suppress organisms other than the target microorganisms. For eg. Bile salts or dyes like basic
fuchsin and crystal violet favour the growth of G-ve bacteria by inhibiting Gram positive bacteria.
46
Some examples are Bismuth sulphate agar (BSA) – for Salmonella spp, Baird Parkar agar (BPA) – for
Staphylococcus spp.
Differential media
Media that distinguish between different groups of bacteria and even permit tentative
identification of microorganisms based on their biological characteristics.
Quantification of microorganisms / Measurement of cell numbers
Direct microscopic count (DMC)
The microbial load in a sample can be quantified by direct counting. Using a counting chamber
is easy, quick and inexpensive. It also gives information about the size and morphology of
microorganisms. Petroff–Hausser counting chamber can be used for counting bacteria.
Haemocytometer can be used for larger eukaryotic microorganisms. These are specially designed
slides, that have chambers of known depth with an etched grid on the chamber bottom. The number of
microorganisms in a sample can be calculated by considering the chamber’s volume and the dilution of
the sample.
The bacteria in several of the central squares are counted (usually at 400x to 500x
magnification). The average no. of bacteria in these squares is used to calculate the concentration of
cells in the original sample. There are 25 squares covering an area of 1 mm 2. The total number of
bacteria in an area of 1 mm2 is calculated / counted. The chamber is 0.02 mm deep and therefore,
Bacteria / mm3 = number / square x 25 squares x 50
The number of bacteria per cm2 is 1000 times this value.
For eg. You have counted 20 bacteria in 1 square
20 x 25 x 50 = 25000 x 1000 = 2.5 x 106 / ml
Disadvantages of this method
1. Very small volume is sampled and therefore the results are not accurate, unless the microbial
population is large.
2. It is not possible to distinguish live and dead cells.
Plating techniques
In c, mixture of cells is diluted and spread out on an agar surface or mixed with agar medium in
a petridish, so that every cell grows into a completely separate colony (macroscopically visible growth
or cluster of microorganisms on a solid medium). There are two common plating techniques followed
for enumeration of bacteria and fungi.
1. Spread plate
47
 2. Pour plate
In spread plating, 0.1 or 0.5 ml of diluted samples are spread out on an agar surface so that
every cell grows into a colony and the colonies are counted and the load in original sample estimated.
In pour plating, the original sample is diluted several times and small volumes (1 ml) of diluted
samples are mixed with liquid agar that has been cooled to about 45 oC and the mixtures are poured into
sterile culture dishes. After the agar has solidified, each cell is fixed in place and forms an individual
colony. The total number of colonies equals the number of viable microorganisms in the diluted
sample. The results of plating technique are always expressed as colony forming units rather than the
no. of microorganisms. Low counts will result if clumps of cells are not broken up and dispersed, and
it is not certain that each colony arose from an individual cell.
Membrane Filtration Technique
In this technique, a sample is drawn through a special membrane filter. The filter is then placed
on an agar medium or on a pad soaked with liquid media and incubated until each cell forms a separate
colony. A colony count gives the no. of microorganisms in the filtered sample.
Indirect methods
Measurement of cell mass
During population growth, there is increase in the total cell mass, as well as cell numbers. By
determination of microbial dry weight, the mass and numbers can be determined. Cells growing in
liquid medium are collected by centrifugation, washed, dried in an oven and weighed. This is useful
for measuring the growth of fungi, as bacteria weigh very little, and large volumes have to be
centrifuged.
Turbidimetry
This technique depends on the fact that microbial cells scatter light striking them. Because
microbial cells in a population are of roughly constant size, the amount of scattering is proportional to
the concentration of cells present. When the concentration of bacteria reaches about 10 7/ml the
medium appears slightly cloudy or turbid. Further increase in concentration / density results in greater
turbidity and less light is transmitted through the medium. The extent of light scattering can be
measured by a spectrophotometer and is almost linearly related to the bacteria concentration at low
absorbance levels. Thus, bacterial growth can be easily measured by turbidimetry.

9. Sterilization and Disinfection

48
Sterilization and Disinfection
Sterilization means destruction of all microorganisms including spores. Disinfection means the
destruction of vegetative organisms which might cause disease or putrefaction.
Sterilization Methods
1. Red heat (Flaming)
2. Dry heat (Hot air)
3. Moist heat
a. Steam under pressure (autoclaving)
b. Steam not under pressure (Tyndallization)
4. Filtration.
The above methods are commonly practiced in microbiological laboratories. Besides, there are
methods like (a) irradiation (b) Aquoustics (Ultrasonic sound waves) which can also bring about
sterilization.
Red Heat
Instruments such as inoculating wires and loops are sterilized by holding them in a flame until
they are red hot. Incineration is routinely followed for destroying the microbial load in hospital and
laboratory wastes. The contaminated materials (cloths, bandages etc) and lab animals are burnt off in
incinerator. Flaming of instruments eg. Glass spreaders, forceps, scissors are subjected to flaming after
dipping in alcohol and then allowing the alcohol to flame and burn off. (This can destroy only
vegetative microorganisms).
Dry Heat
The sterilizing effect of dry heat is due to oxidation of intracellular components and extreme
dehydration. This requires very high temperature to achieve the required effect. Dry heat is applied to
sterilize materials which include glass petridishes, pipettes, flasks and metal objects in hot air oven,
which is electrically heated and thermostatically controlled (oils, waxes and powder are also sterilized
in hot air oven). The operation or use of hot air oven includes (i) the heating up period which is the
time taken for the entire load to reach the sterilization temperature (ii) the holding period – the time
taken for complete sterilization which is normally about 1 h at 160 oC or 2 hours at 140oC and (iii) the
cooling down period, the time required for gradual fall of temperature (usually 2 h or more). The
temperature should be brought down to less than 80 oC before opening the oven to prevent glasswares
from cracking as a result of too rapid fall in temperature.

49
Moist Heat
The sterilizing effect of moist heat is due to coagulation of proteins.
Steam under pressure
This is done by autoclaving. Bacteria are more readily killed by moist heat than dry heat. The
safe condition for sterilization is to use steam at a pressure of 15 psi which corresponds to a
temperature of 121oC for 15 min. This is suitable for culture media, aqueous solutions, treatment of
discarded cultures and specimens etc. Air has an important influence on the efficiency of steam
sterilization because its presence changes the pressure temperature relationship. The boiling point of
water increases with increasing pressure of steam.
Steam pressure Temperature
0 100oC
5 109oC
10 115oC
15 121oC
Therefore, the first step in autoclaving is evacuation of air so that the pressure temperature relation will
hold good.
Laboratory autoclaves
There are two different types
1. Pressure cooker type 2. Gravity displacement models

50
Pressure cooker type laboratory autoclave
This device for boiling water under pressure consists of a vertical metal chamber with a strong
metal lid which can be fastened down and sealed with a rubber gasket. An air and steam discharge tap
(bleeder), pressure gauge and safety valve are filled in the lid. Water in the bottom of the autoclave is
heated by external gas burners or electric immersion heater. The process of sterilization involves three
cycles or steps, namely, the come up cycle where the temperature of the autoclave reaches the
operating temperature; the holding cycle where the operating temperature or pressure is maintained for
the specified time; and the cooling cycle where the pressure is brought down to that of the atmosphere
by gently letting off the steam.

Steam not under pressure (at 100oC)

(i) Moist heat at 100oC
This is achieved by boiling water or by directly letting steam into sterilizers. eg. Sterilizers in
clinics, Arnold’s steam sterilizer. In laboratories, tyndallization is performed. Tyndallization is also
called fractional sterilization. The materials to be sterilized are subjected to heating at 100 oC for 20 –
30 min for three successive days. On the first occasion vegetative bacteria are killed. Any spores that
survive will germinate in the nutrient medium. The surviving spores that have grown overnight into
vegetative forms are killed by the second or third steaming.

(ii) Moist heat below 100oC (Pasteurization)

It is not a sterilization method in strict sense.
Pasteurization is adopted in preserving milk intended to destroy the vegetative pathogenic organisms.
This can be performed in two ways.
1. Holder method – The process is at 62oC for 30 min.
51
 2. Flash method – The process is at 72oC for 30 sec.
The total bacterial count is generally reduced by 97 to 99%. Pasteurization is effective because
the common milk-borne pathogens (tubercle bacillus, Salmonella, Streptococcus and Brucella) do not
form spores.
Filtration
Bacteria can be removed from liquids by passing them through filters with very small pores
that trap bacteria. This method is useful for sterilizing heat labile fluids like vitamins, antibiotics, sera,
urea, protein solution etc.
There are four types of filters
1. Earthernware candle filters – made of diatomaceous earth or porcelain, eg.Berkefeld filter,
Chamberland filter
2. Asbestos pad filters eg. Seitz filters
3. Sintered glass filter – made of finely ground glass fused together (fused jena glass)
4. Membrane filters
There are made from cellulose acetate or cellulose nitrate etc. These membranes can be
sterilized by autoclaving and a range of pore size is available. Usually, membranes of 0.45 µ m or 0.22
µ m are used.
Radiation
Many forms of electromagnetic radiation are harmful to microorganisms. As the wavelength of
the electromagnetic radiation decreases, the energy of the radiation increases. Gamma rays and x-rays
are more energetic than visible light or infra-red waves. Radiation is the emission and propagation of
energy through a space or material medium.

52
Radiation can be of two types:
1. Ionising radiation 2. Non ionising radiation
Ionising radiations can cause atoms to lose electrons or ionize, so called ionising radiation. Two
major forms are 1) X-rays, which are artificially produced and 2) γ (gamma) rays – which are emitted
during radioisotope decay. Low levels of ionising radiation produce mutations and indirectly result in
death, whereas higher levels are directly lethal. Destruction of DNA is the most important cause of
death of organisms. There are some bacteria for eg.Deinococcusradiodurans can survive large doses of
ionising radiation.
Ultra–violet (UV) radiation
It is called non-ionising radiation UV rays can kill all kinds of microorganisms due to its short
wavelength and high energy UV has maximum bactericidal effect or is lethal at a wavelength of 260
nm. The primary mechanism of UV damage is the formation of thymine dimers in DNA. Two adjacent
thymines in a DNA strand are covalently joined to inhibit DNA replication and function. The
sterilizing effect of sun light is attributed to its UV light (300 - 400 nm). Killing is appreciable at 330
nm and below. Use: Low pressure mercury vapour lamps emitting radiation at 254 nm are used to
reduce airborne infection.
Sterilization using radiation is called cold sterilization as it does not generate heat but brings about
sterilization.
Photodynamic sensitization
In the presence of certain fluorescent dyes, strong visible light denatures proteins and sterilizes
bacteria and viruses. These dyes retain an absorbed quantum for a long time, during which the energy
is transferred to another molecule instead of being emitted as fluorescence. The transfer leads to
oxidation of certain residues in proteins and in nucleic acids. Even in the absence of dyes, intense
visible light can kill bacteria, via physiologically occurring photosensitizing substances such as
riboflavin and porphyrins.
Ultrasonic and Sonic waves
In ultrasonic range (frequency of 15,000 and more) sound waves denature proteins, disperse a variety
of materials and sterilize and fragment bacteria. This method is not used as means of sterilization, but
it is useful for disrupting cells for experiments (Sonication).

53
Freezing
When frozen, the crystallization of the water results in the formation of tiny pockets of
concentrated solution of salts, which gets crystallized only below the eutectic point (about –20 oC for
NaCl). The localised high salt concentration and ice crystals damage the bacteria. Only some of the
cells are killed. Repeated freezing and thawing reduce the viability of most cells.
Chemical sterilization
Ethylene oxide (EtO) - It is effective microbicidal and sporicidal agent. It combines with cell
protein and causes denaturation. It is capable of penetrating packaging materials. Used for sterilization
of plasticwares, tubings, etc. The materials to be treated are exposed to EtO at the concentration of 700
mg / lit for 5 to 8 hours at 38oC or 3 to 4 hours at 54oC. Excessive aeration is required after the
treatment to remove the EtO residue in treated materials. Other chemical sterilizing agents are
Betapropiolactone (BPL) - sterilizing gas and vapour phase of H2O2.
Disinfection
There are different chemicals used in laboratories and processing plants for disinfection. Most
disinfectants are effective against vegetative bacteria but not spores. Viruses are also less susceptible to
disinfectants. While selecting disinfectants, factors such as their toxicity and harmful effects on the
skin, eyes and respiratory tract should be considered.
Desirable properties of a disinfectant
1. Effective and capable of rapidly killing microorganisms
2. Reasonably stable
3. Non-corrosive and non-staining
4. Odourless or have an inoffensive odour
5. Non-toxic and non-irritating to skin and eyes
6. Readily soluble in water and readily rinsable
7. Cost – effective

The commonly used disinfectants are clear phenolics and hypochlorites. Others are aldehydes,
alcohols, iodophores and quaternary ammonium compounds.
Clear phenolics
Phenolics are effective against bacteria and fungi but inactive against spores. In laboratory, it
can be used for disinfection of surfaces and discard jars. Dilutions should be prepared daily and used.
Usually 2 – 5% solutions are recommended.

54
Hypochlorites
The activity is due to chlorine and is effective against bacteria, spores and fungi. It is used at
following concentrations. Reasonably clean surfaces – 1000 ppm; Pipette and discard jars - 2500 ppm.

Aldehydes
Formaldehyde and glutaraldehyde are good disinfectants. They are active against bacteria,
spores and fungi. Formalin is diluted to 1:10 and used. Aldehydes are, however, toxic and cause eye
and skin irritation.
Alcohol and Alcoholic mixtures
Ethanol and propanol at 70 – 80% concentrations in water are effective against vegetative
bacteria. Effectiveness is enhanced by the addition of formaldehyde or hypochlorite. A 10% fomalin in
70% alcohol or 2000 ppm of available chlorine in alcohol is effective.
QAC (Quaternary Ammonium Compounds)
These are cationic detergents effective against vegetative bacteria and some fungi and are used
at 1 – 2% dilutions for cleaning surfaces in food hygiene laboratories.
Iodophores
Iodines are effective against vegetative bacteria, spores, fungi and virus. They are used at 75 –
150 ppm iodine for disinfecting surfaces and used in discard jar.
Chemical Disinfectants
Agent Mode of Action Uses
Halogens Oxidation of proteins and enzymes A general disinfectant and sanitizer
Chlorine and its compounds Protein inactivation by iodination Antiseptic
Iodine and iodophores
Heavy metals Enzyme inactivation by coupling to A disinfectant for surface
Mercuric chloride and sulphydryl groups of proteins sterilization of bench tops;
organomercurials Denaturation of proteins and organomercurials as antiseptic for
Silver nitrate enzymes skin; In antiseptic eyedrops
Phenolic compounds Disruption of cell membrane, Germicidal agent not inactivated by
inactivation of proteins and organic matter
enzymes
Alcohols Solubilization of lipids, Mostly as a skin antiseptic

55
 denaturation of proteins and
inactivation of enzymes
Quarternary Ammonium Disruption of cell membranes; Disinfectants of utensils, skin
compounds denaturation of proteins and antiseptic
inactivation of enzymes
Formaldehyde Strong reducing agent, inactivates A penetrating disinfectant
enzymes
Elthylene oxide Inactivates enzymes For sterilization of heat labile
materials.

10. Stains and Staining Reactions

Stains and Staining Reactions
Bacteria are semi-transparent and consist of a clear protoplasmic matter that differs slightly in
refractive index from the medium in which they are growing. It is difficult to observe the bacteria in
unstained state, except when special methods of illumination are used, to see them in the unstained
state. Stains are useful for the following reasons.
 It makes the microscopic semi-transparent objects visible
 To study the shape and size
 To reveal the presence of various internal and external structures
 To produce specific chemical and physical reaction
The term stain and dye are not the same. Acolouring agent that is used for general purposes is
called a dye. The one that is used for biological purposes is called a stain. Based on their chemical
behavior, the dyes are classified as acidic, basic and neutral.
An acid (or anionic) dye has a negative change. Eg., Eosin, Rose Bengal and Acid fuchsin. The
negatively charged groups are carboxyls (-COOH) and Phenolic hydroxyls (-OH). Since they are
negatively charged, bind to positively charged cell structures. pH plays an important role in the
effectiveness of staining because the nature and the degree of the charge on cell components change
with pH. The anionic dyes stain better under acidic conditions, where the proteins and may other
molecules carry a positive charge. A basic dye (or cationic) carries a positive charge. eg., Methylene
Blue, basic fuchsin, crystal violet, malachite green, safranin. Basic dyes bind to negatively charged
molecules like nucleic acid and many proteins. Since the bacterial cells surfaces are negatively
56
charged, basic dyes are most often used in Bacteriology. Basic dyes are normally available as chloride
salts.
A neutral dye is a complex salt of a dye acid with a dye base. The dyes used in bacteriology
have two features in common.
1. They have chromophore groups, groups with double bonds that give the dye its colour
2. They can bind with cells by ionic, covalent or hydrophobic bonding.
Relationship between the type of the dye and its charge when associated is summarized.
Dye salt Dye type
Organic ion (dye base) Inorganic ion
Positively charged (Cation) Negatively charged (anion) Basic
Negatively charged (anion) Positively charged (cation) Acidic
In positive staining procedure, a stain that has a positively charged chromophore (coloured
portion of the stain molecule) is attracted to the negatively charged outer surface of the microbial cell.
A stain such as methylene blue has a positively charged blue portion of the molecule that stains the
microorganism. In negative staining procedures, a negatively charged chromophore is repelled by the
negatively charged microorganisms, resulting in negative or indirect staining of the microbial cell.
Nigrosin and Indian ink are frequently used for negative staining of microbial cells, and this type of
staining is particularly useful for viewing some structures such as capsules that surround some
bacterial cells.

The interaction of a cell with negative and positive stain reagents: The outer layer of a cell is
negatively charged and a positive stain is attracted to the cell, whereas a negative stain is repelled.

57
Staining reactions
When the pH of the surroundings of the microbial cells is either neutral or alkaline, all
microbial cells have a negative charge on their surface, called the surface charge. Many bacterial
cultures produce acids, thereby adding hydrogen ions to a culture medium and decreasing it pH. These
hydrogen inos (𝐻 ) interact with the surface of the negative charges on the surface. When this
happens, the cell surface no longer strongly attracts positively charged dye ions (basic dyes). Thus, the
microbes from acidic environments stain poorly with basic dyes. For this reason, the basic dyes are
made up as alkaline solutions. For example, potassium hydroxide (OH) is added to solutions of
methylene blue to form the stain called Loeffler’s Methylene blue.
Some bacteria excrete alkaline materials during growth and this decreases the number of
available hydrogen ions in the culture medium. Under such conditions, the cell surface has a greater
negative charge, which is more attractive to basic dyes and therefore, allows greater binding,
penetration and internal staining of the microbe. Basic dyes stain microorganisms better under neutral
or alkaline conditions.
If the dye base molecule has a negative charge, it is repelled by the cell’s negatively charged
surface. Thus, negatively charged dyes neither bind to the cell’s surface nor are they able to penetrate
into the cell. These are called acid dyes.
The negatively charged cells are not stained by the negatively charged dye, and they appear as
clear area surrounded by a coloured background. Negatively charged dyes used in this way are called
negative stains.
Negative stains are of limited usefulness for those using light microscopes, but they can be used
to avoid some of the disadvantages of staining with basic dyes.
Simple staining
A simple staining solution contains only one stain, which is dissolved in a solvent. It is applied
to the microorganism in one application. The microorganisms give the colour characteristic of the
staining solution. The purpose of simple staining is to reveal the size and shape of the microorganism.
The simple stains that are commonly used for routine purposes are dilute solution of carbol fuchsin,
crystal violet and methylene blue.
Methylene blue is more frequently used than any other stain in bacteriology. It is because of its
strong nature and it stains nuclei and nucleic acid granules very intensively. Methylene blue is used for
the diagnosis of Diphtheria.

58
Differential Staining
In this procedure, more than one dye is employed. Differential staining procedure helps to
divide the bacteria into separate groups based on staining characteristics. The two most important
differential stains used bacteriologists are Gram stain and Acid-fast stain.
Gram Staining
The simple staining procedure makes to visualize bacteria clearly, but it does not distinguish
between organisms of similar morphology. In 1884, a Danish Physician named, Christian Gram
discovered a new technique to differentiate the bacteria of similar morphology. He used two dyes in
sequence, each of a different colour. The organisms that retain the colour of the first dye are called
Gram positive and those that cannot retain the first dye when washed with a decolourizing solution, but
then take on the colour of the second dye are called Gram negative.
Principle: The Gram-positive bacteria will retain the crystal violet and appear deep violet in colour.
The Gram-negative bacteria lose the crystal violet on decolorization and are counter stained by the
safranine and appear red in colour. Iodine solution is used as a mordant that fixes the primary stain in
or on a substrate by combining with the dye to form an insoluble compound-mordant, for the first
stain.
The reactions are associated with the
structure and composition of the cell wall. The
cell walls of Gram-negative bacteria are thinner
than that of Gram-positive bacteria and contain
a higher percentage of lipid content. During the
staining of Gram-negative bacteria, the alcohol
treatment extracts the lipid. This results in
increased porosity or permeability of the cell
wall. The crystal violet-iodine complex thus can
be extracted and the Gram-negative bacteria is
decolorized. The cells subsequently take up the
colour of the counter stain safranin.

59
 The cell walls of Gram-positive bacteria with lower lipid content become dehydrated during
alcohol treatment. The pore size decreased, permeability is reduced and the CV-I complex cannot be
extracted. Therefore, the Gram-positive cells remain purple.
Endospore Staining
Endospore formation is a distinguishing feature of the family Bacillaceae, which includes
members of the aerobic genus, Bacillus and the anaerobic genus, Clostridium. Endospore resists
adverse environmental conditions such as dryness, heat and poor nutrient supply. The endospore is a
highly retractile body formed within the vegetative bacterial cell at a certain stage of growth. The size,
shape, and position of the spore are relatively constant characteristics of a given species and are
thereof, of some value in distinguishing the kind of bacillus from another. The position of the spore in
the cell may be central, sub terminal or terminal. It may be the same diameter as the cell, smaller, or
larger causing a swelling of the cell.
Endospores strongly resist application of simple dyes, but once stained are quiet resistant to
decolorization. This character suggests one way to make the structure visible. If simple stains are used,
the body of the bacillus is deeply colored, whereas the spore is unstained and appears as a clear area in
the organism. By vigorous staining procedures the dye can be introduced into the substance of the
spore. When thus stained, the spore tends to retain the dye after treatment with decolorizing agents. To
make the distinction clear between the spore and the vegetative portion of the cell, a contrasting
counter stain is usually applied in the ordinary fashion and the resulting picture shows the initial stain
taken up by the spore and the second stain appear in the cytoplasm. Thus, it makes for a very simple
method of distinguishing the endospore from the vegetative cell.

11. Bacterial metabolism: nutritional requirements, nutritional types and their

ecological significance
Nutritional Pattern among Organisms
Microbes are distinguished by their great metabolic diversity. All organisms, including
microbes, can be classified metabolically according to their nutritional pattern—that is, their source of
energy and their source of carbon. Considering the energy source, we can generally classify organisms
as photototrophs or chemotrophs. Phototrophs use light as their primary energy source, whereas
chemotrophs depend on oxidation-reduction reactions of inorganic or organic compounds for energy.
For their principal carbon source, autotrophs (“self-feeders”) use carbon dioxide, and heterotrophs

60
(feeders on others) require an organic carbon source. Autotrophs are also referred to as lithotrophs
(rock eating), and heterotrophs are referred to as organotrophs. By combining the energy and carbons
sources, organisms can be classified as: Photoautotrophs, photoheterotrophs, chemoautotrophs, and
chemoheterotrophs.
Photoautotrophs
Photoautotrophs use light as a source of energy and carbon dioxide as their chief source of
carbon. They include photosynthetic bacteria (green sulfur and purple sulfur bacteria and
cyanobacteria), algae, and green plants. In the photospynthetic reactions of cyanobacteria, algae, and
green plants, the hydrogen atoms of water are used to reduce carbon dioxide, and oxygen gas is given
off. Because this photosynthetic process produces O2, it is sometimes called oxygenic. Some bacteria
cannot use H2O to reduce CO2 and cannot carry on photoysyntheis when oxygen is present (they must
have an anaerobic environment). Consequently, their photosynthetic process does not produce O 2 is
called anoxygenic. Two of the families are photoatuotrophs: the green sulfur and purple sulfur bacteria.
The green sulfur bacteria, such as Chlorobium use sulfur (S), sulfur compounds (such as hydrogen
sulfide, H2S), or hydrogen gas (H2) to reduce carbon dioxide and form organic compounds. Applying
the energy from light and the appropriate enzymes, these bacteria oxidize sulfide (S 2-) or sulfur (S) to
sulfate (SO42-), or hydrogen gas to water (H2O). The purple sulfur bacteria, such as Chromatium, also
use sulfur, sulfur compounds, or hydrogen gas to reduce carbon dioxide. They are distinguished from
the green sulfur bacteria by their type of chlorophyll and the location of stored sulfur.
The chlorophyll used by these photosynthetic bacteria are called bacteriochlorophylls, and they
absorb light at longer wavelengths than that absorbed by chlorophyll a. Bacteriochlorophylls of green
sulfur bacteria are found in vesicles called chlorosomes or chlorobium vesicles underlying and
attached to the plasma membrane. In the purple sulfur bacteria, the bacteriochlorophylls are located in
invaginations of the plasma membrane (intracytoplasmic membrane)
Photoheterotrophs
Photoheterotrophs use light as a source of energy but cannot convert carbon dioxide to sugar;
rather, they use organic compounds, such as alcohols, fatty acids, other organic acids, and
carbohydrates, as sources of carbon. They are anoxygenic. Among the photoheterotrophs are the green
non-sulfur bacteria, such as Chloroflexus, and purple non-sulfur bacteria, such as Rhodopseudomonas.
Chemoautotrophs
Chemoautotrophs use the electrons from reduced inorganic compounds as a source of energy,
and they use CO2 as their principal source of carbon. Inorganic sources of energy for these organisms
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include hydrogen sulfide (H2S) for Beggiatoa; elemental sulfur (S) for Thiobacillus thiooxidans;
ammonia (NH3) for Nitrosomonas; nitrite ions (NO-2) for Nitrobacter; hydrogen gas (H2) for
Hydrogenomonas; and ferrous iron (Fe2+) for Thiobacillus ferrooxidans. The energy derived from the
oxidation of these inorganic compounds is eventually stored in ATP, which is produced by oxidative
phosphorylation.
Chemoheterotrophs
In chemoheterotrophs, the distinction of energy and is not so clear because the energy source
and carbon source are usually the same organic compound, glucose, for example. Chemoheterotrophs
specifically use the electrons from hydrogen atoms in organic compounds as their energy source.
Heterotrophs are further classified according to their source of organic molecules: saprophytes
live on dead organic matter, and parasites derive nutrients from a living host. Most bacteria, and all
fungi, protozoa, and animals are chemoheterotrophs.
Chemical requirements
Carbon
One of the most important requirements for microbial growth is the source of carbon.
According to their source of energy and source of carbon microorganisms can be classified.
Combining the energy and carbon source the organisms can be classified as follows:
Nutritional type Energy source Carbon source Example
Photoautotrophs Light CO2 Photosynthetic bacteria
cyanobacteria, algae, plants
Photoheterotrophs Light Organic compounds Green non-sulphur bacteria
Chemoautotrophs Electrons from inorganic CO2 Nitrifying bacteria, sulphur
compounds bacteria, iron bacteria
Chemoheterotrophs Electrons from organic Organic compounds Most bacteria all fungi, protozoa
compounds and animals.
Nitrogen, Sulfur and Phosphorus
Nitrogen, sulfur and phosphorus are needed by microbes for synthesis of cellular materials.
Protein synthesis requires nitrogen and sulfur. DNA or RNA synthesis requires nitrogen and
phosphorus. ATP synthesis requires nitrogen and phosphorus. Nitrogen makes up about 12 – 15% of
the dry weight of bacterial cells; sulfur and phosphorous together constitute 3%. Many bacteria derive
nitrogen by decomposing protein containing material. Some bacteria derive nitrogen from ammonium

62
ions in organic material. Some bacteria derive nitrogen from nitrates. Some bacteria derive nitrogen
directly from the atmosphere (N-fixation).Important natural sources of sulfur include the sulphate ion,
H2S and sulfur containing amino acids. Important source of phosphorous is the phosphate ion (PO 4- -).
Other elements that are required by microorganisms are potassium, magnesium, calcium, etc.
Trace elements
Microbes require small amounts of other minerals such as iron, copper, molybdenum and zinc.
These are referred to as trace elements. Most of the trace elements are essential for activity of enzymes.
Oxygen
Based on oxygen requirements, organisms are classified into five different categories.
1. Obligate aerobes
2. Facultative anaerobes
3. Obligate anaerobes
4. Aerotolerant anaerobes
5. Microaerophiles
Organisms that require oxygen to live are called obligate aerobes. Facultative anaerobes are
those that can grow both in the presence and absence of oxygen. In fact, they do not require oxygen for
growth but grow better in its presence. Eg.E.coli. Obligate anaerobes are those that do not tolerate
oxygen at all and die in its presence. Only in the absence of oxygen they can grow. Eg.Clostridium
spp. Aerotolerant anaerobes do not use oxygen but can tolerate it. They grow equally well whether it is
present or not. Microaerophilic organisms are basically aerobes but grow only in oxygen
concentrations lower than those in air. The normal atmosphere oxygen level is about 20%. These
microaerophiles grow at a range of 2 - 10%.
Generally, organisms can be harmed by oxygen. Oxygen is toxic because of the production of
toxic forms like Hydrogen peroxide (H2O2), super oxide radical (O2-) and hydroxyl radical (OH).
Aerobes, facultative anaerobes, and microaerophiles must possess enzymes that destroy the toxic forms
of oxygen namely superoxide dismutase and either catalase or peroxidase
2O2- + 2H O2 + H2O2
Super oxide dismustase
2H2O2 2H2O + O2
Catalase
H2O2 + NADH+H+ 2H2O + NAD+
Peroxidase
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Organic growth factors
Organic growth factors are essential organic compounds that cannot be synthesized by
organisms. They must be directly obtained from the environment. There are three major classes of
growth facts:
1) amino acids
2) purines and pyrimidines
3) vitamins
Bacterial Photosynthesis
Essentially, photosynthesis is the conversion of light energy from the sun into chemical energy.
The chemical energy is then used to convert CO 2 from the atmosphere to more reduced carbon
compounds, primarily sugars.
Cyanobacteria, algae, and green plants all contribute to this vital recycling with photosynthesis.
Photosynthesis can be summarized as follows:
6CO2 + 12 H2O + Light energy  C6H12O6 +6O2 + 6H2O
Photosynthesis takes place in two stages. In the first stage, called the light reactions, light energy is
used in a process that converts ADP and P to ATP. In additions, in the predominant form of the light
reactions, the electron carrier NADP is reduced to NADPH. The coenzyme NADPH, like NADH, is an
energy-rich carrier of electrons. In the second stage, called the dark (light-independent) reactions, these
electrons are used along with energy from ATP to reduce CO 2 to sugar.

12. Microbial growth– measurement of cell growth

Growth of Bacterial Culture
Microbial growth refers to increase in number of cells, not the size of the cells. The
requirements for microbial growth can be divided into two main categories, chemical and physical.
Chemical requirements include water, sources of carbon and nitrogen, minerals, oxygen and organic
growth factors. Physical aspects include temperature, pH and osmotic pressure.
Binary Fission
The microbial population can become large in a very short time. Bacteria normally reproduce
by binary fission. The first step in the division is cell elongation and duplication of the chromosomal
DNA. The cell wall and cell membrane then begin to grow inward from all sides at a point between the

64
two regions of the chromosomal DNA. The in-growing cell walls meet forming a cross wall and two
individual cells are formed.
A few bacterial species reproduce by budding. They form a small outgrowth that enlarges in
size to that of the parent cell and then it separates. Some filamentous bacteria (Actinomycetes)
reproduce by producing spores or by fragmentation, and fragments grow as new cells.
Generation Time
Bacterial numbers increase exponentially. The population will double in number during a
specific length of time. The time required for a cell to divide is called the generation time or
doubling time. For example, a culture tube is inoculated with one bacterium that divides every 20
minutes. The population would be 2 after 20 min, 4 cells after 40 minutes and so on. The increase in
population is always 2n where n is the number of generations.
∴ After 6 generations the number of cells will be:
Population = 26 = 64
After 20 generations = 220 = 1,048,576
This can be expressed as Nt = N0 x 2n
Nt = the population at time ‘t’
N0 = the initial number
log Nt – log N0
n = number of generation in time‘t’ n = -----------------------
log 2
The rate of growth in a batch culture can be expressed in terms of mean growth rate constant
(k). k is the no. of generation per unit time, expressed as the generation per hour.
n log Nt – long N0
k = --------- = ----------------------
t log2 t
The mean generation time or mean doubling time (g) can be calculated. If a population doubles (t = g)
Nt = 2 N0

log (2 N0) - log N0 log 2 + log N0 – log N0

K = ------------------------ = ---------------------------
Log2 g log2 g

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 1
K = -------
g
The mean generation time is the reciprocal of the mean growth rate constant
1
g = -------
K
The mean generation time (g) can be calculated from the growth data. For example a bacterial
population increases from 103 cells to 109 in 10 h.
log 109 - log 103 9–3
K = ------------------------------- = --------------- = 2 generations/ h
(0.301) (10h) 3.01
1
g = -------------------- = 0.5h or 30 minutes / generation
2.0 generation / h
The growth Curve
When microorganisms are cultured in a liquid medium, they are grown in a batch culture or
closed system. In this closed system or batch culture, they are incubated in a closed culture vessel with
a single batch of medium. The growth of microorganisms reproducing by binary fission can be plotted
as the logarithm of cell number versus the incubation time. The graph or curve has four distinct phases.
(i) Lag Phase:
When microorganisms are inoculated into fresh culture medium, usually no immediate increase
in cell number occurs. Therefore, this period is called the lag phase. Lag phase is also called tooling up
phase, as the cells prepare themselves for growth in the new medium. The cells synthesize new
compounds prior to start of cell division. The lag phase varies considerably in length depending upon
factors like (1) the Nature of the medium and (2) the condition of microorganisms.
(ii) Exponential phase:
During the exponential or log phase, microorganisms grow and divide at a rapid rate depending
upon their genetic potential, the nature of the medium and growth conditions. The rate of growth is
constant during the exponential phase. The population is uniform in terms of chemical and
physiological properties during this phase and therefore exponential phase cultures are usually used in
biochemical and physiological studies.
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(iii) Stationary phase
Population growth ceases and growth curve becomes horizontal. This phase usually is attained
by bacteria at a population density of around 109 cells per ml. Protozoans and algal cultures have
maximum densities of about 106 per ml. This occurs as the number of cells dividing would equal cell
death and the number of viable microorganisms remains constant. Microbial populations enter the
stationary phase due to
i. Nutrient limitation
ii. Depletion of oxygen (in case of aerobes)
iii. Production of toxic metabolites
(iv) Declining phase (or) Death Phase:
Changes like nutrient depletion and buildup of toxic waste lead to decline in the number of
viable cells. The death of the population is usually logarithmic similar to its growth.
Continuous culture of microorganisms
A microbial population can be maintained in the exponential growth phase and at a constant
biomass concentration for extended periods in a continuous culture system. Two major types of
continuous culture systems are commonly used: (1) chemostats and (2) turbidostats.
In a chemostat, a sterile culture medium is fed into the culture vessel. This fresh medium
contains a limiting amount of an essential nutrient. Growth rate is determined by the rate of flow of
medium through the culture vessel. The medium is fed into the vessel at the same rate as the media
containing microorganisms is removed.
The Turbidostat
The turbidostat has a photocell that measures the absorbance or turbidity of the culture in the
growth vessel. The flow rate of media through the vessel is automatically regulated to maintain
predetermined turbidity or cell density. The dilution rate in a turbidostat varies and the culture medium
lacks a limiting nutrient.
Synchronous culture
Synchronous culture is composed of cells which are at the same stage of the cell cycle.
Synchronous culture of bacteria can be obtained by several techniques. Synchrony in bacteria is
accomplished either by repetitive shifts of temperature or by furnishing fresh nutrients to cultures that
have just entered the stationary phase. A synchronous population can be selected from a random
population by the physical separation of cells that are at the same stage of development. An excellent
method of obtaining synchronous culture is the Helmstetter – Cummings technique – which is based
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on certain bacteria that stick tightly to cellulose nitrate filter. This technique involves filtering an
unsynchronized culture of bacteria through filter, then inverting the filter and allowing fresh medium
to flow through it. After loosely associated bacteria have been washed from the filter, the only bacterial
cultures in the effluent stream of medium are those which arise through division. Hence all cells in the
effluent are newly formed and at the same stage of cell cycle. Synchronous cultures rapidly lose
synchrony because various cells of a population do not divide at the same size, time.

13. Factors affecting microbial growth

Physical requirements of growth: Temperature
Microorganisms grow best at warm ambient temperatures. Some bacteria are capable of growth
at extreme temperatures unlike eukaryotic organisms. Microorganism is divided into three primary
groups, based on their preferred range of growth temperature.
Type Growth range Optimum
1. Psychrophilic (cold loving): -50C to 200C 150C
2. Mesophilic (moderate-temp loving): 150C to 400C 35-370C
3. Thermophilic (heat loving) : 400C – 700 55 – 600C

Each bacterial species grows at minimum, optimum and maximum temperatures (Cardinal
temperature). Optimal growth temperature is usually near the top of the range, above that temperature,
growth drops off rapidly. Temperature influences growth in two ways: 1) as temperature increases
enzymes and chemical reactions proceed rapidly; but beyond a range, irreversible changes occur such

68
as protein denaturation and collapse of membranes 2) as temperature decreases the transport processes
slow down such that growth cannot occur due to membrane gelling.
There is another group of bacteria, other than psychrophiles that can grow at 0 0C and has
higher optimal temperature usually between 200C and 300C. These are called psychrotrophs and are
important in food spoilage. The optimal temperature for human pathogenic bacteria is about 37 0C.
Thermophilic bacteria are important in organic compost, where temperature can rise rapidly to 50 0 or
600C. Some microbes, members of archaebacteria have optimal temperature of 80 0C or higher. These
are called hyperthermophiles or extreme thermophiles. These live in hot springs.

pH
Most bacteria grow best in a narrow range of pH near neutrality (6.5 – 7.5) and are called
neutrophiles. Very few bacteria grow at an acidic pH below 4.0. Therefore, food preservation employs
lower pH. Some bacteria called acidophiles are tolerant of acidity. One type of chemoautotrophic
bacteria, found in drainage waters of coal mines oxidizes sulphur to sulphuric acid and can survive at a
pH of 1 (Thiobacillussp). Molds and yeast grow over a wide range of pH, and their optimal range is
about pH 5-6. Bacteria that grow over a pH range of 8.5 to 11.5 are called alkalinophiles, for eg. Some
of the Bacillusspp - live in soda lakes (high carbonate solutions). When bacteria are cultured in
laboratory certain chemicals called buffers are included in the medium. Peptones and amino acids can
act as buffers. Phosphate salts exhibit buffering effect and are non-toxic and provide phosphorous, an
essential nutrient.

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Osmotic pressure:
Microbes obtain their nutrients in solution from the surrounding water. Higher osmotic
pressures remove water from a cell. When a cell is kept in a hypertonic (higher solute concentration
than in the cell) solution, the cellular water passes out through the plasma membrane causing shrinkage
of the membrane and growth is inhibited. The phenomenon is called plasmolysis.
Therefore, in food preservation, addition of salts helps to prevent microbial growth. A low
molecular weight compound such as NaCl has a greater effect than higher molecular weight compound
such as sucrose. Some bacteria, extreme halophiles have good adaptation at high salt concentrations.
There are facultative halophiles (halotolerant) that can grow normally up to 2% salt. A few facultative
halophiles can tolerate 15% salt for eg.Staphylococcus aureus. Some microbes are designated as
Osmophiles (those that can grow in sugar solutions) and some as Xerophiles (those that grow in low
moisture conditions)
The term ‘water activity’ is generally used to explain this function the amount of water
available to microorganism can be reduced by interaction with solute molecules. Water activity of a
solution is 1/100 of the relative humidity of the solution. Water activity is the ratio of the vapour
pressure of air in equilibrium with a substance or a solution (P soln) to that of pure water (P water) at
the same temperature.
Water activity is inversely related to osmotic pressure. If a solution has high osmotic pressure
it’s aw is low. (aw of pure water is 1.0; Seawater – 0.98; Fruit jams – 0.80; Salted fish - 0.75). Most
microorganism grows well at aw of 0.98.Osmotolerant organisms like Staphylococcuscan grow up to aw
0.8. Some yeasts and molds grow up to aw of 0.6.
Oxygen requirement / tolerance of microbes

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Fermentation: Types and significance
Fermentation can be defined in several ways, it is a process that:
1. Releases energy from sugars or other organic molecules, such as amino acids, organic acids, purines,
and pyrimidines.
2. Does not require oxygen.
3. Does not require use of the Krebs cycle or an electron transport chain.
4. Uses an organic molecule as the final electron acceptor.
5. Produces only small amounts of ATP (only one or two ATP molecules for each molecule of starting
material) because much of the original glucose energy remains in the chemical bonds of the organic
end-products, such as lactic acid or ethanol.
Lactic acid Fermentation
During glycolysis, which is the first phase of lactic acid fermentation, a molecule of glucose is
oxidized to two molecules of pyruvic acid. This oxidation generates the energy that is used to form the
two molecules of ATP. In the next step, the two molecules of pyruvic acid are reduced by two
molecules of NADH to form molecules of lactic acid. Because lactic acid is the end-product of the
reaction, it undergoes no further oxidation, and most of the energy produced by the reaction remains
stored in the lactic acid. Thus, this fermentation yields only a small amount of energy. The overall
reaction is:
Glucose + 2ADP + 2P—> 2 Lactic acid + 2 ATP
Two important genera of lactic acid bacteria are Streptococcus and Lactobacillus. Because
these microbes produce only lactic acid, they are referred to as homolactic (or homofermetative).
Lactic acid fermentation can result in food spoilage. However, the process can also produce yogurt
from milk, sauerkraut from fresh cabbage, and pickles from cucumbers.
Alcohol Fermentations
Alcohol fermentation also begins with the glycolysis of a molecule of glucose to yield two
molecules of pyruvic acid and two molecules of ATP. In the next reaction, the two molecules of
pyruvic acid are converted to two molecules of acetaldehyde and two molecules of CO 2. The two
molecules of acetaldehyde are next reduced by two molecules of NADH to form two molecules of
ethanol. Alcohol fermentation is carried out by a number of bacteria and yeasts. Ethanol made by
yeasts is the alcohol in alcoholic beverages, and carbon dioxide made by yeasts causes bread dough to
rise. Organisms that produce lactic acid as well as other acids or alcohols are known as heterolactic (or

71
heterofermentative) and often use the pentose phosphate pathway. The overall reaction for typical
heterolactic fermentations is:
Glucose + ADP + P —>Lactic acid + Ethanol + CO2 + ATP

14. Microbial genetics; general principles, genetic recombination, transformation,

transduction and conjugation
Microbial Genetics
Glossary
1. Strain or clone: A clone is a population of cells that are genetically ideal. Sometimes a clone is
called a pure culture.
2. Genome : Genome refers to all the genes present in a cell or virus. Bacteria have one set of genes
(haploid- 1N) while eukaryotic microorganisms have two sets of gene (Diploid – 2N)
3. Phenotype: Phenotype is the collection of characteristics that are observable.
4. Genotype:Genotype of an organism is the specific set of genes it possess.
5. Gene: A gene is a nucleotide sequence that code for a polypeptide, tRNA or rRNA. Most bacterial
genes have at least four major parts each with different functions: promoters, leaders, coding regions
and trailers.
6. Genetic recombination is the process by which genetic elements contained in two separate
genomes are brought together in one unit. Gene transfer and recombination are important research
tools that allow analysis of genetic structure of an organism.
7. Mutation is an inherited change in the base sequence of the nucleic acid comprising the genome of
an organism.
Importance of microbial genetics
1. Gene function is the basis of cell functions
2. Microorganisms provide simple systems for studying genetic phenomena
3. Microorganisms are used for isolation and duplication of specific genes from other organisms
by molecular cloning
4. Microorganisms produce valuable substances such as antibiotics, vitamins, hormones etc., and
can be used for large scale industrial processes
5. Genes of higher organisms can be transferred to microorganisms by cloning and can be used
for study and industrial processes

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 6. Disease causing mechanisms can be studied and prevention measures can be arrived at.
Genetic recombination or Gene Transfer
In prokaryotes, genetic recombination occurs because fragments of homologous DNA from a
donor chromosome are transferred to a recipient cell by any of the three following processes.
1. Transformation – Transfer of bacterial genes involving free DNA
2. Transduction – Transfer of host genes from one cell to another medicated by a virus
3. Conjugation – Transfer or genes from one cell to another involving cell to cell contact and a
plasmid.
Transformation
A cell that is able to take up a molecule of DNA and be transferred is called competent cell.
Bacteria differ in the form in which DNA is taken up. In Gram negative bacteria (eg.Haemophilus)
only ds-DNA is taken up into the cell, however only ss–DNA segment is incorporated into the genome.
In Gram positive bacteria (Streptococcus sp. and Bacillus) only ss-DNA is taken up. Transforming
DNA is bound at the cell surface by DNA binding protein, after which either double stranded DNA is
taken up or nuclease degrades one strand and the other is taken up. After uptake, the DNA is attached
to Rec A protein and gets integrated into the genome.
Transduction
Not all phages can transducer and not all bacteria are transducible. In generalized transduction
host DNA derived from any portion of host genome becomes a part of the DNA of the mature virus
particle in place of the virus genome, which gets integrated into another cell upon entry. In specialized
transduction, when a lysogenized cell reverts to lytic cycle, a part of host DNA is exchanged for phage
DNA, which replicates and forms phage, which when transduced, the new gene gets into another cell.
Bacterial conjugation
Conjugation or mating – involves the transfer of DNA from a donor to a recipient by cell to cell
contact through the F (Fertility) pilus, followed by recombination within the recipient bacterial cell.
Pili are filamentous appendages of Gram-negative bacteria that project from the cell’s surface and are
involved in attachment processes. F pili specifically join mating bacteria. When an F pilus joins with
the mate, there is a change in plasma membrane permeability so that DNA can move from one cell to
another. Bacteria that produce F pili are donors and are designated F+ strains if the F plasmid (which
codes for F pilus production) is independent.
During mating, a single strand of donor DNA is replicated, and this copy is transferred to the
recipient where the complimentary strand is synthesized. Bacteria are designated Hfr (high frequency
73
recombinant) if the F plasmid DNA is incorporated into the bacterial chromosome. Bacteria lacking F
pili are recipient strains and are designated F – strains. When F + cell mates with F – cell, the F plasmid
DNA is copied and transferred from donor to the recipient. This results in F + strains.
The F plasmid confers the genetic information for acting as a donor strain.When a Hfr strain
–
mates with a F strain, the genes of the bacterial chromosome are transferred to the recipient cell
before the genes meant for F pilus production. The F plasmid is often not near the beginning of the
DNA that is transferred. Mostly there will not be sufficient mating time for complete transfer of the
complete bacterial chromosome, the recipient cell normally remains F -. Thus there is a relatively high
–
frequency of recombination of genes of the bacterial chromosome when Hfr strains are mated with F
strains.

15. Plasmids and Mutation

Some bacterial cells contain one or more small circular macromolecules of DNA that store
additional specialized information. These are called plasmids (extra chromosomal DNA). Plasmids
contain only 1 – 5% as much DNA as in the bacterial chromosome (roughly about 20 genes) which
supplement the essential genetic information contained in the bacterial chromosome. However, the
genetic information contained in plasmids can be important, in establishing characters such as
resistance to antibiotics and tolerance to heavy metals. Thus, the gene products of plasmids may permit
the survival of bacteria under conditions that are normally unfavourable for growth and survival.
Plasmids can be transferred from one bacterial cell to another, sometimes even from one bacterial
species to another.
Protoplasts and Spheroplasts
When the peptidoglycan layer of the cell wall is digested with lysozyme or when its synthesis is
blocked, the cell ordinarily lyses. However, in a hypertonic medium (eg. 20% of sucrose or 0.5M KCl),
the cell survives as an osmotically sensitive sphere. With gram-positive organisms, this product is free
of wall constituents and is called a protoplast. With gram-negative bacteria, these osmotically sensitive
spheres retain much of the outer membrane and are called spheroplasts.

Mutation

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 i. Mutation is an inherited change in the base sequence of the nucleic acid comprising the
genome of an organism. A strain carrying such a change is called a mutant. A mutant may
differ from its parent strain in genotype (sequence of nucleotides in the DNA of the genome)
and sometimes in phenotype (observable properties from its parent) also. A nutritional mutant
that has a requirement for a growth factor is called an auxotroph and the wild-type parent from
which the auxotroph was derived is called a prototroph.
ii. Mutation can be either spontaneous or induced. Spontaneous mutation occurs naturally
(natural radiation or due to error in pairing of bases during replication). Mutation involving one
or a very few base pairs are referred to as point mutations. Mutation involving change in base
pairs without causing change in the amino acid that code for is called silent mutation. (For eg.
Change in UAC to UAU would not account for change as both code for tyrosine). Mutation
involving change in base pair which codes for a different amino acid is called missense
mutation (UAC - Tyrosin; AAC– asparagine). Sometimes a mutation may result in premature
termination of translation (as the base pair alteration contribute to stop codon, TAG or UAG
resulting in incomplete protein – such is called non-sense mutation.
iii. Agents that induce mutations are called mutagens which may be chemical or physical agents.
Eg. chemical mutagens – Nitrous acid (HNO3), Hydroxylamine (NH2OH), alkylating agents.
Physical mutagens – UV and ionizing radiation (x-rays)

16. Microbial Ecology, Aquatic and Environmental Microbiology

Microbial ecology
Interactions of organisms with each other and with their physical environment contribute to the
functioning of ecosystems.
Symbiosis
Symbiosis is an association of two or more different species. Microorganisms can be physically
associated with other organisms in several ways:
1. Ectosymbiosis-microorganism remains outside the other organism;
2. Endosymbiosis-microorganism is found within the other organism;
3. Ecto/ endo symbiosis-microorganism lives both on the inside and the outside of the other organism.
Mutualism

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 An obligatory association that provides some reciprocal benefit to both partners. For example:
Lichens-an association between a fungus and an alga or cyanobacterium, Fungal partner (mycobiont)
obtains nutrients from alga by hyphal projections that penetrate the algal cell wall as well as oxygen
for respiration. Algal partner (phycobiont) is protected from excess light intensity and is provided with
water, minerals, and a firm substratum in which it can grow protected from environmental stress.
Endosymbiotic chemolithotrophic bacteria provide the main energy source in the community
through the oxidation of hydrogen sulfide. The endosymbiotic bacteria are maintained in specialized
cells (trophosome) of the tube worm. The tube worm binds hydrogen sulfide to hemoglobin and
transports it to the bacteria; the bacteria use the energy from hydrogen sulfide oxidation to synthesize
reduced organic material that is supplied to the tube worm.
Syntrophism
Syntrophism -a mutually beneficial relationship in which each organism provides one or more
growth factors, nutrients, or substrates for the other organism; also referred to as cross-feeding or the
satellite phenomenon; an important example is interspecies hydrogen transfer, which occurs in
anaerobic environments. Quorum sensing allows microorganisms to communicate as they form
association with plants and animals
Commensalism
The microorganism (commensal) benefits, while the host is neither harmed nor helped; often
the microorganism shares the same food source with the host. Occurs in situations in which waste
products of one microorganism serves as the substrate for another; also occurs in situations where one
microorganism modifies the environment making it better suited for another microorganism (some
examples are Nitrification-requires the activity of two different species; one oxidizes ammonia to
nitrite and the other oxidizes nitrite to nitrate. The common nonpathogenic strain of Escherichia coli
lives in the human colon; this facultative anaerobe uses oxygen creating an anaerobic environment in
which obligate anaerobes (e.g., Bacteroides) can grow; coli derives no obvious benefit or harm
Predation
Predator organism engulfs or attacks a prey organism; prey can be larger or smaller than
predator; normally results in death of prey. Predatory bacteria are known (e.g., Bdellovibrio,
Vampirococcus, and Daptobacter); may cause lysis of prey, release of cell contents while attached to
surface of prey, or penetrate cytoplasm of prey.

Parasitism
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 One organism (parasite) benefits from another (host); there is a degree of coexistence between
the host and parasite that can shift to a pathogenic relationship (a type of predation)
Amensalism
An organism releases a specific compound that harms another organism. Ex. Antibiotic
production by bacterium
Competition:
Different organisms within a population or community try to acquire the same resources (e.g.,
nutrients, location, etc). Competitive exclusion principle is that if two populations overlap too much in
terms of their resource use, then one of the populations is excluded.
Extremophiles and their significance
Prokaryotes are known to thrive in harsh environments where other eukaryotes do not exist.
Extreme halophiles
Prokaryotes that inhabit highly saline environments such as salt pans and natural salt lakes or
heavily salted foods are called extreme halophiles. These organisms require 1.5 to 4M (9-23%) NaCl
for optimal growth, and can grow up to the limit of saturation of NaCl (32%). Extreme halophiles
belong to Archaea and are collectively called halobacteria. Examples are Halobacterium.
Halobacterium sp. are well adopted to saline environments. The cell wall is stabilized by sodium ions.
Na+ binds to the outer surface of the well thus maintaining cellular integrity. Further cells of
Halobacterium pump large amounts of K+ from the environment into the cell to counter the Na+
outside cell, thereby remain in positive water balance. The cell wall is made up of glycoprotein which
has high content of acidic amino acids. The cytoplasmic proteins are also highly acidic and contain low
levels of hydrophobic amino acids. All extremely halophilic Archaea are chemo-organotrophs and
most of them are obligate aerobes.
Deep sea bacteria
Organisms that inhabit the deep sea have to overcome three major environmental extremes.
They are low temperatures, high pressure and low nutrient levels. There are two groups of deep sea
bacteria. Barotolerant groups that tolerate up to 400 atm. pressure and barophilic groups growing
optimally at pressures of above 400atm. Barophilic and barotolerant bacteria are also psychrophilic.
These bacteria are adopted to these harsh pressure conditions that their enzymes must be folded in such
way to retain the binding capacity. They have increased proportion of unsaturated fatty acids in their
cytoplasmic membranes and their cell wall also possesses specific outer membrane protein called
OmpH to overcome the high-pressure conditions.
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Hydrothermal bacteria
Hydrothermal vents are thermal springs on ocean floor where hot basalt and magma lie very
near the floor causing cracks through which seawater mixes with hot mineral and emitted, forming a
hot nutrient rich habitat for organisms to thrive. Sulphur–oxidizing chemolithotrophs such as
Thiobacillus, Thiothrixand Beggiatoa are present in and around such vents. Other populations also
include nitrifying bacteria, hydrogen, iron and manganese oxidizing bacteria. Most of these bacteria
live in association with other invertebrate communities of the thermal vents. At great depths,
hydrothermal fluid is emitted at temperatures of 270-380 oC and hyperthermophilic bacteria such as
Methanopyrusexist in walls of such vents also. Such vents are called black smokers.

17. Aquatic microbial groups

Aquatic microbial groups
The study of microorganisms and their activities in natural waters is called aquatic
microbiology. Natural waters include lakes, ponds, streams, rivers, estuaries and the sea. Generally, the
concentration of bacteria in water is proportional to the amount of organic material in the water.
Freshwater Microbiota
The number and location of freshwater microbiota depend on the availability of oxygen and
light. The littoral zone along the shore has rooted vegetation and light penetrates through it. The
limnetic zone is the open surface water area away from the shore. The profundal zone is the deeper
water below the limnetic zone. The benthic zone is the deepest zone with sediment at the bottom.
Photosynthetic algae are the primary producers of a lake. They are found in limnetic zone.
Pseudomonas, Cytophaga, Caulobacter andHyphomicrobiumare found in the zone where O2 is
abundant. Microbial growth in stagnant water uses available oxygen and cause eutrophication leading
to fish death. Purple and green sulfur bacteria are found in the profundal zone where there are light and
H2S but no oxygen. In Benthic zone Desulfovibrio reduces SO4 to H2S in benthic mud. Methane
producing bacteria are also found in this zone.
In small water bodies, nutrients such as phosphates cause algal blooms, this can lead to
eutrophication of aquatic ecosystems. Eutrophication is the result of addition of pollutants or natural
nutrients. The use of microorganisms to remove pollutants is called bioremediation.

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Bioremediation:
Bioremediation is defined as the use of microbes to remove pollutants from the environment.
Due to industrialization and other anthropogenic activities, soil and ground water leaching from
industrial and municipal toxic waste dumps contaminate ground water and other water bodies making
them polluted and dangerous. Bioremediation involves two steps: i) isolate microbes that can degrade
or utilize a particular pollutant and ii) to provide conditions that facilitate the microbial activities
effective thereby eliminating the pollutants.
Seawater Microbiota:
The characteristics of the open ocean are that it exerts relatively high osmotic pressure,
contains low level of nutrients and is very cold at great depths. The pH also is slightly higher than
optimal for microorganisms. Therefore, bacterial populations in oceans are much lesser than in
estuaries and in small freshwater bodies. Much of the microscopic life in ocean is composed of
photosynthetic diatoms and other algae; these plankton community forms the basis of the oceanic food
chain. Ocean bacteria benefit from the death and decomposition of phytoplankton and also attach to
their living bodies.
Microbial luminescence is seen in deep-sea life. Luminescent bacteria produce light flashes
when they are agitated by wave action. Many of these luminescent bacteria have established symbiotic
relationships with benthic fish. These fish sometimes use the light of their resident bacteria to attract
and capture prey in dark deep ocean depths. As in freshwater lakes and small water bodies, the
microbes in marine environments also play major roles in the mineralization of nutrients especially
more pronounced in shallow continental waters.

18. Aquatic environment as habitat: Distribution of microorganisms and their

biomass in rivers, lakes, sea and sediment
Ecology is the scientific study of how organisms interact with each other and with their
environment. This includes relationships between individuals of the same species, between different
species, and between organisms and their physical and chemical environments. Aquatic
ecology includes the study of these relationships in all aquatic environments, including oceans,
estuaries, lakes, ponds, wetlands, rivers, and streams.

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 An ecosystem is a community of living organisms and their physical and chemical
environment, linked by flows of energy and nutrients. Ecosystems function as a discrete ecological
unit, and can be defined at a variety of scales.
The physical characteristics of aquatic habitats affect the types of organisms found there.
Living organisms in a particular environment are directly affected by environmental characteristics
such as nutrient concentrations, temperature, water flow, and shelter. Only the organisms that are able
to survive in the conditions of a particular habitat and use the resources available there will thrive.
Interactions between living organisms also affect the type of organisms found in an aquatic ecosystem,
as competition for resources (e.g., food, habitat) and predation affects species abundance and diversity.
Understanding the basic components of aquatic ecosystems and the interaction among living organisms
and their environment can lead to better management of human impacts on these systems.
Organisms living in aquatic ecosystems are dependent on the resources of their environment.
Biological communities—including the types of animals present and their relative abundance—are
also shaped through interactions with other organisms.
Energy and Food
Every organism must acquire energy to live, grow and reproduce. In aquatic ecology, biologists
often classify organisms according to how they obtain energy. Because sunlight is the ultimate source
of energy used by organisms on the earth's surface, a basic distinction lies between those who use its
energy directly—autotrophs—and those who receive it indirectly by consuming other organisms—
heterotrophs.
Autotrophs
Autotrophs, or producers, are organisms that can manufacture their own organic material from
inorganic sources. Most autotrophs carry out this process using photosynthesis, the process by which
plants and algae use solar energy to combine carbon dioxide with water to produce starch, sugars and
oxygen. Photosynthesis is the most important biological process on the planet, and its products drive
the biological activity of nearly all ecosystems, including aquatic environments. The oxygen produced
is available to be used by other organisms, making photosynthesis an important controller of carbon
dioxide and oxygen in the environment.
Photosynthesis in aquatic systems is carried out by a wide variety of autotrophs, which range in
size from microscopic single-celled organisms to large aquatic plants called macrophytes. Autotrophs
are primary producers because they produce the first level of organic carbon from inorganic

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compounds. Ultimately, all other types of organisms (heterotrophs) are dependent on the organic
carbon produced by autotrophs
Because photosynthesis depends on sunlight, the distribution of autotrophs is reliant in part on
the amount of light available in an aquatic ecosystem. In shallow, stony rivers, periphyton (or biofilm)
– especially diatoms and cyanobacteria – are the main source of primary production, but shade from
riparian vegetation can limit photosynthesis; nutrients may also be in short supply in these habitats. In
wider rivers, reduced shading from riparian vegetation allows the river surface to receive more light.
However, in deep or turbid sections, light penetration may be insufficient to sustain growth of
autotrophs.
Heterotrophs
Heterotrophs, or consumers, are organisms that must obtain energy by consuming other
organisms (autotrophs or other heterotrophs) as food. From the perspective of energy flow in
ecological systems, heterotrophs can be classified according to what they eat:
 Herbivores are called primary consumers because they eat only plants.
 Carnivores are called secondary consumers because they feed on other animals.
 Omnivores feed both on autotrophs and on other heterotrophs; that is, they eat both plants and
animals. Many aquatic organisms, including fish, are omnivorous.
 Detritivores consume dead organic matter (detritus). Detritivores include many bacteria and
fungi, invertebrates such as worms and insects, and some scavenging vertebrates. Aquatic
insects, for instance, shred dead leaves, but also consume bacteria and fungi growing on the
leaves.
Heterotrophs can also be classified according to how they obtain food energy (i.e., functional
feeding groups), and by their specific roles in the aquatic ecosystem.
The grazer-scraper category includes herbivores that feed on periphyton and biofilm.
Shredders are detritivores feeding on coarse organic particles, especially leaf litter derived from the
riparian zone.
 Collectors eat fine organic particles and can be subdivided according to whether the food
particles they collect are suspended in the water (e.g., filtering-collectors or filter-feeders), or
have been deposited on the substratum (collector-gatherers).
 Deposit-feeders ingest fine bottom sediments and the organic material that they contain.
 Predators are species that eat other animals.

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Food Chains and Food Webs
The energy and matter produced by plants and other autotrophs are distributed to other
organisms in an ecosystem through pathways known as food chains and food webs.
A food chain is a simple linkage of producers to consumers through feeding relationships. For
example, when a small fish eats an aquatic insect, and a larger fish eats the small fish, the two fish and
the insect are linked in a food chain.
Food webs are more complex, and consist of a network of linked food chains. Organisms
commonly consume, and are consumed by, more than one other type of organism. Each organism has
characteristic feeding preferences and patterns, and can itself be prey to other consumers. Food webs
connect autotrophs, at the lowest feeding level, to the herbivores (primary consumers) and then to
various carnivores (secondary consumers).
The trophic level is an organism's position in the food chain as determined by the number of
energy-transfer steps required to reach that level. A fish that has consumed an insect, which itself has
just consumed algae, is at a higher trophic level than the insect.
In rivers, as in the majority of other aquatic and terrestrial systems, the energy at the base of a
food web comes from the solar energy fixed by plants (through photosynthesis) growing in the water
or on land.
Energy derived from terrestrial plants enters the water in the form of plant parts, such as leaves
or twigs, or in the form of dissolved organic matter. This material is used as a source of energy by
microorganisms such as fungi and bacteria, and by invertebrates. Plants in the river are also important
in food webs—microscopic algae are often eaten while alive, while larger aquatic plants mainly enter
food chains after they have died.
Biomass and Production
Organisms use energy to maintain biological functions and to enable growth and reproduction.
The organic matter produced by autotrophs and heterotrophs, in excess of what they need to sustain
life, adds to the ecosystem's total biomass. The biomass in an ecosystem includes the mass of all living
and dead organic matter. Production is the incremental increase in biomass produced by organisms
over a period of time. Estimates of biomass and production are one measure that can be used to assess
the health of aquatic ecosystems.
Primary production refers to the production of organic matter, such as body tissue, produced
mainly by photosynthetic plants. It is expressed as a rate of biomass production—for example, the
amount of wood produced each year.
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 Secondary production is the assimilation of organic material and building of tissue by
heterotrophs, and may involve animals eating plants, animals eating other animals, or microorganisms
decomposing dead organisms to obtain the resources (material, energy, nutrients) needed for producing
biomass. Secondary production is also expressed as a rate of biomass production, such as the amount
of meat produced by grazing cattle each year.
In a productive environment, living plant or animal tissue will accumulate over time. Biomass
is the amount of this accumulated material at a given moment, while production is the rate of
increase in the total biomass. In a river system, biomass may be lost by export (such as downstream
transport of biomass), or gained by import from other systems (such as leaves falling into a stream).
Aquatic microorganisms
Microorganisms include members of the plant kingdom, protozoa, bacteria, and fungi. These
organisms differ radically, and share only their small size; most are not visible without a microscope,
though colonies of some can be seen with the naked eye. Microorganisms are present in large
quantities everywhere and can survive extreme physical and chemical conditions. Many
microorganisms play foundational roles in aquatic ecosystems, capturing the sun’s energy through
photosynthesis and, through their role in decomposition, releasing nutrients stored in organic tissue.
Bacteria
Some of the smallest and most ancient organisms on earth, bacteria are present in virtually
every environment and are abundant in all aquatic systems. In rivers and streams, many of the bacteria
wash in from the surrounding land, and their abundance can increase dramatically after the rainfall.
The abundance of bacteria is typically in the millions per millilitre (mL), and in the hundreds of
millions per millilitre in especially productive or polluted waters.If conditions are right, bacteria
reproduce extremely rapidly by simple division to produce very large numbers in a short period of
time. Bacteria can be found suspended in the water, associated with decaying material (such as dead
wood or leaves), or coating the surface of rocks, stones and sand grains as part of the biofilm (the
slippery coating on hard surfaces in rivers). They can make up a large fraction of the living material in
aquatic systems.
Bacteria display the greatest range in metabolic ability of any group of organisms. There are
both autotrophic and heterotrophic bacteria. Heterotrophic bacteria are a crucial link in the
decomposition of organic matter and the cycling of nutrients in aquatic systems.Autotrophic bacteria
are primary producers in aquatic systems as are true algae. For this reason, autotrophic bacteria
(predominantly cyanobacteria) are often categorized as 'algae', though the organisms are by no means
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closely related. Cyanobacteria used to be mistakenly called 'blue-green algae'. Ecologically, much of
what applies to algae is relevant to autotrophic bacteria.
Fungi
Fungi occur as single cells, and in filaments called hyphae. Most aquatic fungi are microscopic;
those known as hyphomycetes are the most abundant and important. Fungi are heterotrophic, and, like
heterotrophic bacteria, obtain their nutrition by secreting exoenzymes into their immediate
environment, which break compounds down into simpler substances the fungi can absorb. Fungi are
critical to the decomposition of plant matter in aquatic systems, because they are among the few
organisms that can break down certain plant structural compounds such as cellulose and lignin.
Protozoa
Protozoa are microscopic, single-celled organisms that sometimes group together into colonies.
There are both autotrophic and heterotrophic types of protozoa. Unlike bacteria and fungi, which
absorb dissolved organic compounds from their environment, heterotrophic protozoa (such as the
amoebas and Paramecium) consume other organisms such as algae, bacteria, or other protists.
Together with other microorganisms, protozoa make up the biofilm coating sediments and hard
surfaces on riverbeds, though some protozoa are free-swimming. Certain protozoa are parasites and
cause diseases such as giardia (beaver fever).
Algae and Phytoplankton
Several groups of largely autotrophic protists are referred to as algae. Like the term
'microorganisms' it is an informal term, used for convenience to describe microorganisms that carry out
photosynthesis; the cyanobacteria are often included as algae. Algae vary in size from microscopic to
large colonies that can be considered macrophytes. Several types of algae—including phytoplankton—
play an important role in supplying the energy at the base of many aquatic food webs.
Phytoplankton are small, microscopic plants that live suspended in the open water.
Phytoplankton are generally more abundant in lakes than rivers, and are absent from fast-flowing
streams, or where the rate at which the plants are washed downstream is greater than the rate at which
they reproduce. Damming a river leads to still-water conditions more suitable for phytoplankton, and
nuisance algal blooms may develop in reservoirs. Inputs of nutrients, including nitrogen and
phosphorus, can also lead to algal blooms.Phytoplankton can exist as single cells or in chains or
colonies. Phytoplankton are direct food sources for many zooplankton and some fish, and are the base
of the food web in deep waters. Phytoplankton vary in their requirements for nutrients, light, and other
conditions. Water bodies support a complex mixture of phytoplankton that can change markedly with
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environmental conditions. In rivers containing significant amounts of phytoplankton, the concentration
of algal cells (number per unit volume) is generally highest when flows are lowest, while elevated
suspended sediment loads during high flows can lead to reduced light and photosynthesis. Some
phytoplankton can cause taste and odour problems in water, and anoxic conditions that can kill fish.
Some cyanobacteria produce toxins lethal to various fish, wildlife, and domestic species.
Periphyton and Biofilm
Algae, bacteria, fungi, protozoa, and the breakdown products of dying cells form layers on
submerged surfaces, including bottom sediment, rocks, submerged leaves and branches,
and macrophytes. The term periphyton refers to a layer consisting mainly of algae, but the entire
assemblage of layers is often known as biofilm. Periphyton is an important food source in shallow,
stony rivers with adequate light penetration. Heterotrophic organisms, including larger invertebrates
such as snails and insects, scrape the biofilm from surfaces, while some larger animals, such as fish,
also feed on biofilm. Biofilm can be important in absorbing or breaking down chemical contaminants
as well. Seasonal changes in the abundance of periphyton reflect fluctuations in river discharge, as
layers of algal cells build up in times of low or decreasing flow, and wash away during flood periods.

19.Factors Affecting Aquatic Ecosystems

Variability and change are natural processes in aquatic ecosystems, and ecosystem
communities and individual organisms have in many cases adapted to different environmental
conditions. Human effects on aquatic ecosystems can result from pollution, changes to the landscape or
hydrological systems, and larger-scale impacts such as global climate change. The complexity of
aquatic ecosystems and the linkages within them can make the effect of disturbances on them difficult
to predict. These linkages mean that damage to one component of the ecosystem can lead to impacts on
other ecosystem components.
Flooding can be part of the natural hydrological cycle, and is essential to the ecosystems it
affects. Hydrological connectivity between floodplains and rivers is maintained through flooding.
When a river floods, it deposits nutrient rich sediment on the banks, and in turn washes bits of
vegetation into the river that become food for aquatic organisms. Floods can also replenish lakes and
ponds found within the floodplain and can raise the water table.
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 When flooding is introduced through damming or extreme events it can be detrimental to an
aquatic system until a balance is reached. In the case of extreme flood events, the balance often occurs
once the flood has receded, and the aquatic organisms can rebuild their habitat in a more nutrient-rich
environment.
Human Influences on Aquatic Ecosystems
Human activities affecting aquatic ecosystems are more likely to disrupt natural patterns and
processes because species do not have the ability to adapt to the rapid changes to their environment
that can occur. Some contaminants that enter aquatic systems are preferentially stored in organisms,
usually in fat tissue, rather than being released or excreted. This results in an accumulation of the
contaminant over time in a process known as bioaccumulation.Biomagnification refers to the higher
concentrations of contaminants in organisms at higher trophic levels within food webs. While an
organism in a low trophic level of a food web may contain low levels of a contaminant, its consumer
will concentrate the contaminant as it consumes many of these individuals over its lifetime. At each
trophic level in the food web, contaminants become more concentrated. Contaminant accumulation is
higher in food webs with more steps to the top predator. Therefore, the top predator in systems with
longer food webs usually has higher contaminant concentrations than those in shorter food webs, all
else being equal.

20. Microbial biofilms

A biofilm is any group of microorganisms in which cells stick to each other and often also to a
surface. These adherent cells become embedded within a slimy extracellular matrix that is composed
of extracellular polymeric substances (EPS). The EPS components are produced by the cells within the
biofilm and are typically a polymeric conglomeration of extracellular DNA, proteins, and poly-
saccharides. Biofilms are frequently described metaphorically as "cities for microbes."
Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial
and hospital settings. The microbial cells growing in a biofilm are physiologically distinct from
planktonic cells of the same organism, which, by contrast, are single cells that may float or swim in a
liquid medium. Biofilms can be present on the teeth of most animals as dental plaque, where they may
cause tooth decay and gum disease.

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 The formation of a biofilm begins with the attachment of free
free-floating
floating microorganisms to a
surface. It is thought that the first colonist bacteria of a biofilm adhere to the surface initially through
weak, reversible adhesion via van der Waals forces and hydrophobic effects. If the colonists are not
immediately separated from the surface, they can anchor themselves more permanently
pe using cell
adhesion structures such as pili.
During surface colonization
colonization, bacterial cells are able to communicate using quorum
sensing (QS) products such as N
N-acyl homoserine lactone (AHL). Once colonization has begun, the
biofilm grows through a combination of cell division and recruitment. Polysaccharide matrices
typically enclose bacterial biofilms. In addition to the polysaccharides, these matrices may also contain
material from the surrounding environment, including but not limited to minerals,
mineral soil particles, and
blood components, such as erythrocytes and fibrin. The final stage of biofilm formation is known as
dispersion, and is the stage in which the biofilm is established and may only change in shape and size.
The development of a biofilm may allow for an aggregate cell colony (or colonies) to be
increasingly resistant to antibiotics. Cell
Cell-cell communication or quorum sensing has been shown to be
involved in the formation of biofilm in several bacterial species.

Biofilms are the product of a microbial developmental process. The process is summarized by
five major stages of biofilm development
1. Initial attachment
2. Irreversible attachment
3. Maturation I
4. Maturation II
5. Dispersion

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 Dispersal of cells from the biofilm colony is an essential stage of the biofilm life cycle.
Dispersal enables biofilms to spread and colonize new surfaces. Enzymes that degrade the biofilm
extracellular matrix, such as dispersin B and deoxyribonuclease, may play a role in biofilm
dispersal. Biofilm matrix degrading enzymes may be useful as anti-biofilm agents. Recent evidence
has shown that a fatty acid messenger, cis-2-decenoic acid, is capable of inducing dispersion and
inhibiting growth of biofilm colonies. Secreted by Pseudomonas aeruginosa, this compound induces
cyclo heteromorphic cells in several species of bacteria and the yeast Candida albicans. Nitric oxide
has also been shown to trigger the dispersal of biofilms of several bacteria species at sub-toxic
concentrations. Nitric oxide has the potential for the treatment of patients that suffer from chronic
infections caused by biofilms.
Biofilms are usually found on solid substrates submerged in or exposed to an aqueous solution,
although they can form as floating mats on liquid surfaces and also on the surface of leaves,
particularly in high humidity climates. Given sufficient resources for growth, a biofilm will quickly
grow to be macroscopic (visible to the naked eye). Biofilms can contain many different types of
microorganism, e.g. bacteria, archaea, protozoa, fungi and algae; each group performs specialized
metabolic functions. However, some organisms will form single-species films under certain conditions.
The social structure (cooperation/competition) within a biofilm depends highly on the different species
present.
Biofilms are ubiquitous in organic life. Nearly every species of microorganism have
mechanisms by which they can adhere to surfaces and to each other. Biofilms will form on virtually
every non-shedding surface in non-sterile aqueous or humid environments. Biofilms can grow in the
most extreme environments: from, for example, the extremely hot, briny waters of hot springs ranging
from very acidic to very alkaline, to frozen glaciers.
Biofilms can be found on rocks and pebbles at the bottom of most streams or rivers and often
form on the surface of stagnant pools of water. In fact, biofilms are important components of food
chains in rivers and streams and are grazed by the aquatic invertebrates upon which many fish feed.
In the human environment, biofilms can grow in showers very easily since they provide a moist
and warm environment for the biofilm to thrive. Biofilms can form inside water and sewage pipes and
cause clogging and corrosion. Biofilms on floors and counters can make sanitation difficult in food
preparation areas. Biofilm in soil can cause bioclogging. Biofilms in cooling- or heating-water systems
are known to reduce heat transfer. Biofilms in marine engineering systems, such as pipelines of the
offshore oil and gas industry, can lead to substantial corrosion problems. Corrosion is mainly due to
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abiotic factors; however, at least 20% of corrosion is caused by microorganisms that are attached to the
metal subsurface (i.e., microbially influenced corrosion).
Bacterial adhesion to boat hulls serves as the foundation for biofouling of seagoing vessels.
Once a film of bacteria forms, it is easier for other marine organisms such as barnacles to attach. Such
fouling can reduce maximum vessel speed by up to 20%, prolonging voyages and consuming fuel.
Time in dry dock for refitting and repainting reduces the productivity of shipping assets, and the useful
life of ships is also reduced due to corrosion and mechanical removal (scraping) of marine organisms
from ships' hulls.
Stromatolites or stromatoliths are layered bio-chemical accretionary structures formed in
shallow water by the trapping, binding and cementation of sedimentary grains by biofilms (microbial
mats) of microorganisms, especially cyanobacteria. Fossilized stromatolites provide ancient records
of life on Earth by these remains, some of which may date from 3.7 billion years ago.Accretion is a
process by which material is added to a tectonic plate or a landmass.
Dental plaque is an oral biofilm that adheres to the teeth and consists of many species of both
bacteria and fungi (such as Streptococcus mutans and Candida albicans), embedded in salivary
polymers and microbial extracellular products. The accumulation of microorganisms subjects the teeth
and gingival tissues to high concentrations of bacterial metabolites, which results in dental disease.
Biofilm on the surface of teeth is frequently subject to oxidative stress and acid stress. Dietary
carbohydrates can cause a dramatic decrease in pH in oral biofilms to values of 4 and below (acid
stress). The dental plaque biofilm can result in the disease dental caries if it is allowed to develop over
time. By preventing the dental plaque biofilm from maturing or by returning it back to a non-
cariogenic state, dental caries can be prevented and arrested. This can be achieved though the
behavioural step of reducing the supply of fermentable carbohydrates (i.e. sugar intake) and frequent
removal of the biofilm (i.e. tooth brushing).
Taxonomic diversity
Many different bacteria form biofilms, including Gram-positive (e.g. Bacillus spp., Listeria
monocytogenes, Staphylococcus spp, and lactic acid bacteria, including Lactobacillus plantarum
and Lactococcus lactis) and Gram-negative species (e.g. Escherichia coli, or Pseudomonas
aeruginosa). Cyanobacteria also form biofilms in aquatic environments. Along with bacteria, biofilms
are also generated by archaea and by a rangeof eukaryotic organisms,
including fungi e.g.Cryptococcuslaurentii and microalgae. Among microalgae, one of the main
progenitors of biofilms are diatoms, which colonise both fresh and marine environments worldwide.
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Infectious diseases
Biofilms have been found to be involved in a wide variety of microbial infections in the body,
by one estimate 80% of all infections. Infectious processes in which biofilms have been implicated
include common problems such as bacterial vaginosis, urinary tract infections,
catheterinfections,middle-ear infections, formation of dental plaque, gingivitis, coating contact
lenses, and less common but more lethal processes such as endocarditis, infections in cystic fibrosis,
and infections of permanent indwelling devices such as joint prostheses, heart valves, and
intervertebral disc.
In aquaculture
In shellfish and algae farms, biofouling species tend to block nets and cages and ultimately
outcompete the farmed species for space and food. Bacterial biofilms start the colonization process by
creating microenvironments that more favorable for biofouling species. In the marine environment,
biofilms could reduce the hydrodynamic efficiency of ships and propellers, lead to pipeline blockage
and sensor malfunction, and increase the weight of appliances deployed in seawater.Numerous studies
have shown that biofilm can be a reservoir for potentially pathogenic bacteria in freshwater
aquaculture. Biofilms can be difficult to eliminate even when antibiotics or chemicals are used in high
doses.
22. Role of microorganisms in the production and breakdown of organic matter,
sedimentation and mineralization process
The bottom of an aquatic ecosystem is covered by mud due to sedimentation of particles in the
water, which comprise inorganic silt derived from surrounding lands and organic particles of dead
plants and animals sinking down the water column. These act as food for the bottom dwelling animals
and source chemicals to the water. For example, in many lakes, bottom sediments are principal sites of
decomposition. Sediments with high organic content contain large numbers of bacteria, which break
them into smaller inorganic molecules. These bacteria require oxygen and when the sediment becomes
totally devoid of oxygen, they start to adsorb it from the overlying water. If the lake is stratified, then
there is no mixing of hypolimnion and hence no replenishment of oxygen to the lower layers. This
leads to anoxic conditions and death of animals. If the lake is large and regularly has a deoxygenated
hypolimnion, only those animals able to endure the absence of oxygen will survive.
When the sediments are devoid of oxygen, some processes of decomposition cannot occur, but
other chemical reactions that occur only in the absence of oxygen take over. Anaerobic bacteria can

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operate without oxygen and break down large quantities of organic matter in the mud to produce
methane gas (made up of carbon and hydrogen CH4). The released methane is used as energy by other
bacteria, which are aerobic and can live only in oxygenated conditions. Other anaerobic bacteria can
use sulphates and the by-product of their activity is hydrogen sulphide. Many lakes have a layer of
deoxygenated water at the bottom during summer, above which a layer of oxygenated water is present.
The balance between these two layers and between the intensity of aerobic and anaerobic activities is
essential to the health of the lake ecosystem.
Organic matter in freshwater arises from living material (directly from photosynthesis or
indirectly from terrestrial organic matter) and as a constituent of many waste materials and effluents.
The total organic matter can be a useful indication of pollution. In surface waters, concentration of
total organic carbon is less than 10 mg/L.

Chemical Oxygen Demand (COD) is a measure of the oxygen equivalent of the organic matter
in a water sample that is susceptible to oxidation by strong chemical oxidant such as dichromate. The
concentration of COD in surface water ranges from 20mg/L oxygen or less in unpolluted waters to
greater than 200 mg/L (in waters receiving industrial effluents). Biochemical Oxygen Demand (BOD)
is an approximate measure of the biochemically degradable organic matter present in the water sample.
It is defined as the amount of oxygen required for aerobic microorganisms in the sample to oxidise the
organic matter to a stable inorganic form. Unpolluted water typically has BOD value of 2mg/L, but
those receiving effluent may have more than 10mg/L.
Organic matter from flora and fauna makes a major contribution to the natural quality of
surface water, the composition of which is extremely diverse. Natural organic matter is not toxic but
exerts major influence on biochemical and hydrochemical processes in the water body. Humus is

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formed by biochemical and chemical decomposition of vegetative residues and microorganism
activity. It enters directly from the soil or is a result of biochemical transformations within the lake.
Humus is divided into humic and fulvic acids. These concentrations are highly dependent on the
physical-geographical conditions and range from 10 to 100 µg of carbon/litre.
Plants and microorganisms convert inorganic nitrogen to organic nitrogen. The inorganic
compounds include nitrite, nitrate, ammonium ions and molecular nitrogen. These undergo biological
and non-biological transformations in the environment. The major non-biological transformations
include sorption (absorption and adsorption), voltalisation and sedimentation. Biological
transformations include: a) assimilation of inorganic ions (ammonia and nitrate) by plants and
microorganisms to form organic nitrogen (amino acids); b) reduction of nitrogen gas to organic
nitrogen and ammonia by microorganisms; c) oxidation of ammonia to nitrite and nitrate
(nitrification); d) conversion of organic compounds to ammonia during the decomposition of organic
matter; e) bacterial reduction of nitrate to nitrous oxide and molecular nitrogen under anoxic conditions
(denitrification).
Bacteria in the sediments at the bottom of the lake break down organic content of dead plants
and animals and phosphate is released into the water in the spaces between the sediment particles. This
process is rapid in sediments devoid of oxygen. In a lake with oxygenated water, even a thin layer of
sediment having oxygen will act as a barrier and prevent the release of phosphate from the sediments
below (which is deoxygenated). If water becomes deoxygenated (as in summer), phosphate is slowly
released from the sediments. In a shallow lake, however, the phosphate may stay locked in the deep
layers, but due to the lack of depth the wind mixes the water and releases the phosphate from the
sediment. Aquatic plants and algae absorb the released phosphate in the water and their population’s
increase. This enhances the death and decomposition of more phosphate containing materials in the
water, which in turn reduces the oxygen levels and speeds up the release of more phosphate. This is a
cyclical process and explains why shallow lakes are productive.

23. Nutrient cycles: Carbon, nitrogen, sulfur, phosphorus iron and manganese
cycles
Role of microorganisms in the cycling of elements

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 Decomposition and photosynthesis are the two important process of an ecosystem.
Microorganisms depending on substrate specificity colonize the organic matter and decompose it.
However, the organic matter serves two functions for microflora. Firstly, it provides energy for growth
and secondly it provides carbon source for the formation of new cells. During this process, certain
waste products are also produced by microorganisms e.g. organic acids, carbon-dioxide, methane, etc.
The process of conversion of substrate to protoplasmic carbon is known as assimilation. About 20-40%
substrate is assimilated and rest is released as carbon-dioxide or accumulated as waste. When the
carbon assimilation occurs, the other inorganic chemicals such as nitrogen, phosphorus, potassium and
sulphur are also taken up for the formation of new cells. By this process, microorganisms accumulate
inorganic substances in their cells and reduce the concentration of nutrients for plants in soil. This
event of accumulation of inorganic substance by the microorganisms and making the plants, nutrient-
deficient is known as immobilization. Microorganisms of different groups colonize the substrate
depending upon its chemical composition. Thus microbial succession occurs on the decomposing
material till it fully disappears in elemental forms. The events of sequential appearance of
microorganisms on a substrate with respect to time is called succession.
Biogeochemical cycle
The major plant nutrients derived from soil are nitrogen, phosphorus and potassium, because
these are made biologically available to plants. Biogeochemical cycling associated with
microorganisms is very important for the maintenance of soil fertility.
Nitrogen cycle
Nitrogen is the highest concentration in the atmosphere. It is also an essential constituent of
proteins and chlorophyll found in organisms. The key processes of biogeochemical cycling of nitrogen
are nitrogen fixation, ammonification, nitrification and denitrification.
Nitrogen fixation
The conversion of molecular nitrogen into a nitrogenous compound is known as nitrogen
fixation. There are free living and symbiotic microorganisms, which fix nitrogen into proteins. The
nitrogen fixing microorganisms are called diazotrophs.
Ammonification
Ammonification is a process in which organic nitrogen is converted to ammonia. The amino
acids are utilized as nutrients by microorganisms. Under aerobic conditions, the amino groups are
removed from aminoacids with the liberation of ammonia.
Nitrification
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 It is process in which ammonia is oxidized nitrate. The process consists of two steps. In the
first step, ammonia is oxidized to nitrite. This is called nitrosofication.
2N𝐻 + 3𝑂 2𝐻𝑁𝑂 + 2𝐻 𝑂
The oxidation reaction provides energy, and this is carried out in the bacterial genera
Nitrosomonas and Nitrosococcus. In the second step, the nitrite is oxidized to nitrate
2𝐻𝑁𝑂 + 𝑂 2𝐻𝑁𝑂 + 2𝐻 𝑂
This oxidation also provides energy, and this is carried out in the bacterial genus Nitrobacter.

Denitrification
Denitrification is a process in which nitrates are reduced to nitrites and subsequently to gaseous
nitrogen.
𝑁𝑂 𝑁𝑂 𝑁𝑂 𝑁
In denitrification, organic compounds serve as hydrogen donors and nitrate serves as an
electron acceptor. Denitification occurs under anaerobic conditions eg. during seasonal flooding on the
land. It is carried out under the influence of bacterial genera like Thiiobacillusdenitrificans,
Micrococcus dentrificationand Clostridium sp.
Phosphorus cycle
Phosphorus is only second to nitrogen as a mineral nutrient required for plants, animals and
microorganisms. It is a major constituent of nucleic acids and in all living systems, it is essential for
the accumulation and release of energy. This element is generally added to the soil as a chemical
fertilizer or in the form of organic phosphates present in plant residues.
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 Microorganisms play a key role to bring about several transformations of this element. These
include (i) altering its solubility, ii) mineralization of organic phosphate into inorganic phosphate, (iii)
oxidation and reduction of phosphorus compounds. Of these mobilization and immobilization are the
most important.
The phosphate requirement of plant is met by the uptake of phosphate ions which are then
utilized for the synthesis of organic phosphates within the cell. By this, a fraction of the phosphate gets
immobilized. Upon the death of the plants, the organic phosphate is rapidly released by enzymatic
hydrolysis. In many instances, phosphate becomes limiting factor for plant growth, because much of it
in the soil is bound as insoluble calcium, iron or aluminium phosphates.
The availability of phosphates therefore depends on the degree of solubilization of insoluble
phosphates by various organic and inorganic acids produced by microorganisms. Several soil
microorganisms, particularly fungi are known to produce substantial amounts of these acids and
thereby solubilize insoluble phosphates and make it available to the plants. Important microorganisms
active in solubilization of inorganic phosphates include both bacteria and fungi such as species of
Bacillus, Pseudomonas, Micrococcus, Aspergillus, Penicillium and Fusarium. The enzyme
phosphatase plays key role in the solubilization of organic phosphates.
Carbon cycle
In nature, carbon exists in the form of inorganic and complex organic compounds. In
atmosphere the concentration of CO2 is only 0.32%, which is less than what is required by plants for
photosynthesis. The CO2 is the main source of carbon required to build the organic world. The CO2
returns back into the atmosphere through the process of respiration by all groups of organisms. The
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other method of returning carbon is through degradation (decomposition) of organic matter by
microorganisms. A simplified carbon cycle is given in Figure.

Sulfur cycle
The cyclic movements of sulfur between the living organisms and the environment are referred
as sulfur cycle. Sulfur is an essential element for all living organisms. It is present in free as well as
combined states. Plant, animal and microbial proteins, aminoacids – cystine and methionine contain
sulphur. In soil, it occurs both in organic (sulfur amino acids, vitamins, etc.) as well as in the inorganic
form (sulfur and sulfates) and is readily metabolized. Four distinct transformations are recognized;
these are: (i) decomposition of larger organic sulfur compounds to smaller units and their version into
inorganic compounds (mineralization), (ii) microbial associated immobilization, (iii) oxidation of
organic ions and compounds such as sulphides, thiosulphates and sulfur, (iv) reduction of sulphates to
sulphides.

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Decomposition of sulfur compounds
Plants obtain their sulfur requirement from sulfur compounds, while animals do so by feeding
on plant materials or other animals. In these systems sulfur is found mostly as a component of sulfur
containing amino acids such cystine and methionine. The dead organic matter contains large
molecules. The decomposers like bacteria, actinomycetes and fungi excrete digestive enzymes. These
enzymes convert large molecules into small ones. Sulfur containing amino acids are converted to
inorganic compounds like H2S and NH3. For example, the amino acid cysteine releases H2S and NH3
as follows:

Microbial associated assimilation or immobilization

Many compounds serve as sulphur sources for microbial assimilation. Sulphur is present in
inorganic and organic sources. Whatever its sources, sulfur in its elemental form cannot be utilized by
plants and animals. Sulfur in soluble form, mostly as SO 4, is absorbed through plant roots. In the plants
SO4 is incorporated into amino acids and then to proteins. Plants utilize sulfur in the form of sulphates
and then reduce it within the cells to H2S before it is utilized mainly in the synthesis of sulfur amino
acids and vitamins.

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Oxidation of sulfur compounds
Some microorganisms oxidize reduced sulfur compounds. They are known as sulfur oxidizers.
Members of the genus Thiobacillus are the main organisms involved in the oxidation of elemental
sulphur. The following reactions are catalyzed by some of the thiobacilli.

The ability oxidize sulphur is not restricted to only the genus Thiobacillus. Heterotrophic
bacteria, actinomycetes, and fungi are also able to oxidize sulphur compounds. Sulfur is first converted
enzymatically to sulphite, which is then oxidized to sulphate.
Reduction of sulfur compounds
Under anaerobic conditions, sulfate is reduced to H2S by sulfate reducing bacteria. The
inorganic compounds are reduced by bacteria, called sulfate reducing bacteria. Among the bacteria,
Desulfovibriodesulfuricansseems to be the most important. The mechanism by which sulfate is
reduced involves the conversion of sulphate to sulphite, a reaction that needs ATP. The sulphite is
reduced to H2S.
𝑆𝑂 𝑆𝑂 𝑆 𝑂 S

24. Sewage microbiology: Self-purification in natural water and sewage treatment

Sewage / wastewater Treatment
Wastewater that is water after being used is called sewage. Sewage includes household water,
toilet waste, industrial waste and rainwater. Sewage has to be treated before it is discharged into rivers
and sea. Sewage treatment is done in three steps, namely: Primary Treatment, Secondary Treatment
and Tertiary Treatment.
Primary Treatment
It is the process of removal of solid matter called sludge from sewage. About 40 ~ 60% of
suspended solids are removed by passing the sewage through sedimentation tanks. Sometimes
flocculating chemicals are added to remove more solids. Primary treatment removes approximately
25% to 35% of the biochemical oxygen demand (BOD) of the sewage. BOD is a measure of the

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biologically degradable organic matter in water. BOD is determined by the amount of oxygen required
by bacteria to metabolize the organic matter.
Secondary Treatment
Secondary treatment is the biological degradation or organic matter in sewage after primary
treatment. In this process, the sewage is aerated vigorously to encourage growth of aerobic bacteria
and other microorganisms that oxidize the dissolved organic matter to CO 2 and water. Two commonly
used methods of secondary treatment are activated sludge systems and trickling filters. Activated
sludge system consists of aeration tank where air or pure oxygen is mixed with the effluent from
primary treatment and some of the sludge from a previous batch is added to the incoming water.
Sludge inoculum contains large number of metabolizing bacteria, together with yeasts, molds and
protozoa. Species of Zoogloea bacteria are important as they form flocculent masses in the tank and
oxidize the organic matter. As these flocs settles out organic matter is removed along. Most of the
settled sludge is removed for treatment in an anaerobic sludge digester. Then the effluent is sent on for
final treatment. Activated sludge systems remove 75% to 95% of the BOD from sewage.
Trickling filters
In this method sewage is sprayed over a bed of rocks or molded plastic. The rocks provide
enough surface area for microbial activity and the space between rocks are large enough to provide air
to the bottom. A slimy gelatinous film of aerobic microorganisms grows on rock surface and
metabolizes the organic matter in water when it passes along into CO 2 and water. Trickling filters
remove 80 to 85% of BOD.
Final Treatment
Usually, the effluent from secondary treatment is disinfected and discharged onto land or into
water. Sometimes, it is subjected to tertiary treatment. The tertiary treatment uses physical filtration
and chemical precipitation to remove all the BOD, nitrogen and phosphorus from water. The tertiary
treatment provides drinkable water, when as secondary treatment provides water for irrigation. The
sludge is further digested in an anaerobic sludge digester. Bacteria degrade organic matter and produce
methane and CO2. In the first stage of anaerobic sludge digestion, there is the production of CO 2 and
organic acids by anaerobic fermentation. In the next stage, organic acids are metabolized to form
hydrogen and CO2 and some organic acids like acetic acid. In the third stage, there are converted into
methane by methane-producing bacteria
CO2 + 4H2 CH4 + 2H2O
CH3 COOH CH4 + CO2
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25. Drinking water microbiology
Drinking water, also known as potable water or improved drinking water, is water that is safe
to drink or to use for food preparation, without risk of health problems. Globally, 91% of people had
access to water suitable for drinking. Many people still use an unsafe drinking water source which may
be contaminated by faeces. This can result in infectious diarrhea such as cholera and typhoid among
others. Reduction of waterborne diseases and development of safe water resources is a major public
health goal in developing countries. Bottled water is sold for public consumption in most parts of the
world. Parameters for drinking water quality typically fall under three categories:
 physical
 chemical
 microbiological
Physical and chemical parameters include heavy metals, trace organic compounds, total
suspended solids (TSS), and turbidity.
Microbiological parameters include coliform bacteria, E. coli, and specific pathogenic species
of bacteria (such as cholera-causing Vibrio cholerae), viruses, and protozoan parasites.
Chemical parameters tend to pose more of a chronic health risk through build-up of heavy
metals although some components like nitrates/nitrites and arsenic can have a more immediate impact.
Physical parameters affect the aesthetics and taste of the drinking water and may complicate the
removal of microbial pathogens.
Originally, faecal contamination was determined with the presence of coliform bacteria, a
convenient marker for a class of harmful faecalpathogens. The presence of faecal coliforms (like E.
coli) serves as an indication of contamination by sewage. Additional contaminants include
protozoan oocysts such as Cryptosporidium sp., Giardia lamblia, Legionella, and enteric viruses.
Microbial pathogenic parameters are typically of greatest concern because of their immediate health
risk. Throughout most of the world, the most common contamination of raw water sources is from
human sewage and in particular human faecal pathogens and parasites. Waterborne pathogens may be
killed or inactivated by boiling but it is difficult to store boiled water in sterile conditions. Other
techniques, such as filtration, chemical disinfection, and exposure to ultraviolet radiation (including
solar UV) have been demonstrated to significantly reduce levels of water-borne disease. Another type
of water treatment is called desalination and is used mainly in dry areas with access to large bodies of
saltwater.

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Indicator organisms
The use of indicator organisms, in particular the coliform group, as a means of assessing the
potential presence of water-borne pathogens has been paramount to protecting public health. These are
based upon the principle of the detection of selected bacteria that are indicative of either contamination
or deterioration of water quality using simple bacteriological tests. Indicator organisms are used to
assess the microbiological quality of water. For many pathogens, such as viruses and protozoan
parasites, reliable indicators are not available. The use of indicator bacteria, in particular Escherichia
coli (E. coli) and the coliform bacteria, as a means of assessing the potential presence of water-borne
pathogens have been paramount to protecting public health.
Coliform bacteria
Coliform bacteria belong to the family Enterobacteriaceae and share similar cultural
characteristics. Typical genera encountered in water supplies are Citrobacter, Enterobacter,
Escherichia, Hafnia, Klebsiella, Serratia and Yersinia. Coliform bacteria are defined as Gram-
negative, non-spore-forming, rod-shaped bacteria which are capable of aerobic and facultative
anaerobic growth in the presence of bile-salts or other surface-active agents with similar growth-
inhibiting properties. They usually ferment lactose at 37 °C within 48 hours, possess the enzyme β-
galactosidase and are oxidase-negative. Faecal coliform bacteria possess the characteristics of coliform
bacteria but can carry out lactose fermentation at 44 ⁰C. The term “faecal coliform” is not precise and
has been used to describe coliform bacteria thought to be of faecal origin. The term “thermotolerant
coliform” has been used to describe presumptive faecal coliform bacteria.
When coliform bacteria are isolated from drinking water supplies it is often useful to determine
which species of coliform bacteria are present. The potential source of coliform bacteria in water
supplies results from sub-optimal operation of water treatment processes or ingress of contamination
from breaches in the integrity of the water distribution system. Coliform bacteria can be present in
domestic plumbing systems with kitchen taps and sinks being recognised sources of these organisms.
Escherichia coli
E. coli is a coliform bacterium and has historically been regarded as the primary indicator of
faecal contamination of both treated and untreated water. As a coliform bacterium it is a member of the
family Enterobacteriaceae and is capable of fermenting lactose or mannitol at 44 ⁰C, usually within 24
hours, and produces indole from tryptophan. Most of the E. coli strains possess the enzyme β-
glucuronidase, which can be detected using specific fluorogenic or chromogenic substrates. E. coli

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occurs in the faeces of all mammals, often in high numbers (up to 10 9/g of faeces). This widespread
faecal occurrence, coupled with methods that for the recovery and enumeration of E. coli are relatively
simple to conduct, has contributed to the detection of this bacterium as the cornerstone of
microbiological water quality assessment for over 100 years. The survival characteristics and
susceptibility to disinfection of E. coli are similar to those of many other bacterial pathogens,
particularly Salmonella and Shigella, and it does not multiply in temperate surface water or in treated
waters. Many tests for E. coli rely upon selective isolation at 44 °C. Some of the strains of E. coli,
however, do not grow well at this temperature but will be isolated at 37 °C. These isolates, when
identified as E. coli still have the same sanitary and operational significance with regard to their faecal
origin.
Microbiological standards for drinking water
Escherichia coli (E. coli): Not detectable (MPN <1/100 ml)
Total coliforms: Not detectable (MPN <1/100 ml)
During treatment, the drinking water is initially held in a holding tank / reservoir for setting the
suspended matter. Then, the colloidal materials such as clay are removed from water by using
flocculant chemicals such as aluminium potassium sulfate (Alum). After flocculation treatment, water
is treated by filtration that is passing it through beds of sand or diatomaceous earth. Some
microorganisms including protozoan cysts are removed during this process. Finally, water is
disinfected with chlorine to kill remaining pathogenic bacteria.

26. Sanitary quality of water for aquaculture

Water quality is a critical factor when culturing any aquatic organism. Optimal water quality
varies by species and must be monitored to ensure growth and survival. The quality of the water in the
production systems can significantly affect the organism's health and the costs associated with getting a
product to the market. Water quality parameters that are commonly monitored in the aquaculture
industry include temperature, dissolved oxygen, pH, alkalinity, hardness, ammonia, and nitrites.
Depending on the culture system, carbon dioxide, chlorides, and salinity may also be monitored. Some
parameters such as alkalinity and hardness are fairly stable, but others like dissolved oxygen and pH
fluctuate daily. It is important to establish a standardized water quality testing protocol for a particular
situation. Know the tolerance range for the culture species, establish critical levels, and be prepared to

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act if a problem occurs. The chart below indicates the water quality preferences for some commonly
cultured species.

Water Quality Tolerance by Species

Dissolved
Temperature Alkalinity Ammonia Nitrite
Species Oxygen pH
°F mg/L mg/L mg/L
mg/L

Baitfish 60-75 4-10 6-8 50-250 0-0.03 0-0.6

Catfish/Carp 65-80 3-10 6-8 50-250 0-0.03 0-0.6

Hybrid Striped Bass 70-85 4-10 6-8 50-250 0-0.03 0-0.6

Perch/Walleye 50-65 5-10 6-8 50-250 0-0.03 0-0.6

Salmon/Trout 45-68 5-12 6-8 50-250 0-0.03 0-0.6

Tilapia 75-94 3-10 6-8 50-250 0-0.03 0-0.6

Tropical Ornamentals 68-84 4-10 6-8 50-250 0-0.03 0-0.5

Water quality is determined by physical, chemical and microbiological properties of water.

These water quality characteristics throughout the world are characterized with wide variability.
Organic Indicators of Water Quality
For quantity assessment of concentrations of organic materials, the indicator Total Oxygen Demand is
used. The Total Oxygen Demand includes Chemical Oxygen Demand (COD); Biochemical Oxygen
Demand and Nitrogenous Biochemical Oxygen Demand and can be shown as:
CaHbOcNdSe + xO2 aCO2 + ½ bH2O + dNO3- + eSO4-
Biological Oxygen Demand
Biological oxygen demand (BOD), the most widely used parameter, is a measure of the amount
of oxygen used by the indigenous microbial population in water in response to the introduction of
degradable organic material. This parameter depends on water characteristics: dilution, essential
nutrients (N, P, K, Fe, etc), and bacteria seed. The 5-day BOD (BOD 5) is the most widely used. The
BOD5 of natural water is related to the dissolved oxygen concentration, which is measured at zero time
and after 5 days of incubation at 20°C. The difference is the dissolved oxygen used by the

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microorganisms in the biochemical oxidation of organic matter. The BOD 5 can be calculated as
BOD5 = D0 - D1, in which the BOD5 is in mg/L and D0 and D1 are the dissolved oxygen concentration
in mg/L at time 0 and 5 days, respectively. Typical concentration of BOD5 for streams and rivers
throughout the world are <2 to 15 mg/L and the observed range is < 2 to 65 mg/L.
Chemical Oxygen Demand
The chemical oxygen demand (COD) test of natural water yields the oxygen equivalent of the
organic matter that can be oxidized by strong chemical oxidizing agent in an acidic medium. Potassium
permanganate is an oxidizing chemical. Silver sulfate is added as a catalyst to minimize the
interference of chloride on the COD test. Mercuric sulfate is also added to inhibit the interferences of
metals on the oxidation of organic compounds. The reaction of the dichromate with organic matter is
presented here in a general way:
Organic matter (CaHbOc) + Cr2O72- + H- 2Cr3+ + CO2 + H2O
The COD observed in natural streams and rivers is < 2 mg/L to 100 mg/L.
Microbiological Characteristics
The principal groups of microorganisms in natural water include protists, plants and animals.
Some of the physical and biological characteristics of organisms important for water quality
considerations are presented below:
Simplified Classification of Microorganisms in Water
Environmentally resistant
Representative members Size, mm Shape
stage
Protozoa 100-102 Variable Cysts
Algae
Fungi (molds and yeasts) 100-102 Filamentous, coccoid Spores
0
Blue-green algae 10 Coccoid, filamentous Cysts
Rod, coccoid, spiral
Bacteria 10-1-101 Spores, cyst-like
comma
Many bacteria, viruses and protozoa are causative organisms for some of the more virulent
diseases transmitted to humans directly through water and indirectly through contaminated food. Assay
and confirmation of the presence of the causative agent of waterborne diseases are lengthy and time-
consuming. Instead of specific analyses, coliform organisms have been used to determine the
biological characteristics of natural waters. The coliform group of bacteria are aerobic and/or
facultative Gram-negative, non-spore-forming, rod-shaped bacteria that ferment lactose to

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gas. Escherichia coli is commonly used as an indicator organism. This organism is present in the
intestine of warm-blooded animals, including humans. Therefore, the presence of Escherichia coli in
water samples indicates the presence of fecal matter and then the possible presence of pathogenic
organisms of human origin. The concentration of indicator organisms is reported in MPN/100 mL
(MPN = most probable number) or in CFU/100 mL (CFU = colony forming units). Other enteric
organisms that are also considered indicator organisms are fecal streptococci (Streptococcus faecalis)
and clostridia (Clostridium perfringens).
Microbial quality of shellfish waters

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27. Bioremediation
Bioremediation is a natural process that involves the use of biological entities to neutralize the
contaminated site. According to the United States Environmental Protection Agency, (USEPA)
bioremediation is a "treatment that uses naturally occurring organisms to break down hazardous
substances into less toxic or non-toxic substances". Technologies can be generally classified as in
situ or ex situ. In situ bioremediation involves treating the contaminated material at the site, while ex
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situ involves the removal of the contaminated material to be treated elsewhere. Some examples of
bioremediation related technologies are phytoremediation, mycoremediation, bioventing,
bioleaching, landfarming, bioreactor, composting, bioaugmentation, rhizofiltration, and biostimulation.
Bioremediation may occur on its own (natural attenuation or intrinsic bioremediation) or may
only effectively occur through the addition of fertilizers, oxygen, etc., that help in enhancing the
growth of the pollution-eating microbes within the medium (biostimulation). For example, it has
been demonstrated that windrowing and aeration of petroleum-contaminated soils enhanced
bioremediation using the technique of land farming. Recent advancements have proven successful via
the addition of matched microbe strains to the medium to enhance the resident microbe population's
ability to break down contaminants. Microorganisms used to perform the function of bioremediation
are known as bioremediators.
Not all contaminants are easily treated by bioremediation using microorganisms. For
example, heavy metals such as cadmium and lead are not readily absorbed or captured by
microorganisms. Other contaminants, such as aromatic hydrocarbons as are common in petroleum, are
relatively simple targets for microbial degradation, and some soils may even have some capacity to
autoremediate, as it were, owing to the presence of autochthonous microbial communities capable of
degrading these compounds.
The use of genetic engineering to create organisms specifically designed for bioremediation has
great potential. The bacterium Deinococcusradiodurans (the most radio-resistant organism known) has
been modified to consume and digest toluene and ionic mercury from highly radioactive nuclear
waste. In aquaculture systems also a wide variety of bioremediators are used to remove toxic ammonia,
nitrite, nitrate or other wastes
Bioaugmentation. The injection of a small amount of oil-degrading microbes into an affected area
Biostimulation. The addition of nutrients to stimulate the growth of innate oil-degrading microbes to
increase the rate of remediation

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28. Economic significance of microbes including aquatic microbes
Economic importance of fungi
Fungi are both useful as well as harmful to humankind in several ways, some of which are summarized
below:
1. Useful activities of fungi
1.1. Food Industry
i. Mushrooms are a type of fungi. Many of its varieties are edible. They have high nutritive value as
they are rich in proteins and vitamins. They are cultivated on a commercial scale. Agaricus bisporous
(white button mushroom) is the most common of the edible mushrooms
ii. Lecanoraesculatais lichen and is regarded as bread of heavens by Jews.
iii. Reindeer moss – lichen in tundra constitutes the staple food of reindeer, musk, ox etc.
iv. Food yeast contains vitamins of the B-group, E-group and 15% proteins. Used as single cell protein
(SCP).
v. Industrial applications: several industries particularly breweries and confectionaries depend upon
various species of fungi such as:
a. In breweries: During anaerobic respiration (fermentation) of glucose, yeasts yield ethyl alcohol and
CO2. Alcohol is the main constituent of wine, whiskey, beer etc. CO 2 obtained as a by-product could
be solidified and sold as dry-ice.
b. In bakeries: Sponginess in bread and other items of bakery is brought out by the air spaces. Air
spaces formed when CO2 rises through the dough due to fermentation by yeasts. Saccharomyces
cervisiae is referred to as baker’s yeast.
c. Cheese processing: several species of Penicillium and Aspergillus are used to provide different
flavours and ripening of cheese.
d. Chemical industry: Many acids like citric acid, gallic acid, gluconic acid are manufactured using
many species of Aspergillus.
e. Others: Delicate perfumes are obtained from lichens such as Labularia pulmonaria and
Everiniaprunastri. Orecin – a biological stain is obtained from lichen Roulla tinctoria.
vi. Role in agriculture: Saprophytic fungi help in the decay of dead animals and plants. They convert
complex organic compounds into simple organic compounds such as nitrates, nitrites, sulphates,
phosphates etc., which increase the fertility of soil and help in increasing the yield of crops.

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 vii. Recycling and mineralization of materials: The saprophytic fungi and other decomposers
decompose the dead bodies of animals and plants and their waste. It is mineralized and returned to the
soil for reuse.
viii. Medical uses:
a. Anibiotics: The first antibiotic was obtained from Penicillium by Alexander Fleming in 1928 AD.
Other antibiotics derived from fungi are as follows
Patulia– Aspergillus claratus
Fumigati – Aspergillus fumigatus
Cephalosporin – Emericellopsis minimum
Viridin – Gliocladium virens
b. Synthesis of vitamins and enzymes: Vitamin B-complex, Vitamin E etc., are obtained from yeasts.
Vitamin A is obtained from Rhodotorulagracilis. Enzymes such as amylase, pectinase, glucose oxidase
and invertase are synthesized from some fungi such as Penicillium and Aspergillus.
c. Synthesis of proteins and fats: Proteins are synthesized from yeasts, where as fats from Penicillium
and Aspergillus sp.
2. Harmful activities of fungi
i. Human diseases: Many skin diseases are due to fungal infection. High fever and allergies also result
from fungal infections.
ii. Plant diseases: Plant diseases like the blight of potato, rusts in wheat, powdery mildews, white rust
of crucifers and smuts in wheat, maize and other cereal crops are caused by fungi.
iii. Food spoilage: Fruits, vegetables, meat, dairy products bread etc., are spoiled by fungi, especially
moulds such as Rhizopus, Mucor, Penicillium, Aspergillus sp.
iv. Tropical deterioration: Warm and moist conditions of tropical regions especially during the
monsoon are favourable for the rapid growth of fungi. They grow on and spoil a variety of articles
such as leather goods, clothes, electrical equipment etc. This phenomenon is called tropical
deterioration.
Economic Significance of Algae
Algae are useful to us in many ways and are of much economic significance.
 Phytoplankton (microscopic algae) is a vast food source for most aquatic organisms such as fish. They
also produced a huge supply of oxygen in the environment.

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 The brown algae also contain an alginic acid on their cell walls. This alginic acid is a good source of
alginites, polysaccharides that are used to make gels as food.
 A sticky polysaccharide called carrageenan can also be found on the algae’s cell walls. This
polysaccharide is often used to produce cosmetics and gelatin capsules.
 Calcium carbonate is found in the cell walls of corraline algae. Calcium carbonates are important
components of coral reefs. These remains are good sources of fertilizers, detergents, paint removers,
insultants and other commercial products.
Harmful Effect:
When there is a huge increase in the algae (dinoflagellates) population, there will be what we called
“red tide“. It is characterized by the discolouration of some parts of the ocean. The water will be
covered with red pigments that come from the algae. Gonyaulax is a common type of dinoflagellate
which produces toxins. The toxins produced byGonyaulax can bring respiratory paralysis in
vertebrates. Shelled organisms, mussels, clams and others that are exposed on dinoflagellates are
poisonous due to the toxins that affect the neuro system. This could be dangerous to humans upon
consumption. This is called shellfish poisoning.
Other economic Significance:
Protozoa are also useful in some ways:
 Food: Protozoa provide food for insect larvae, crustaceans and worms, which are taken by large
animals like fishes, lobsters, clams, and crabs.
 Symbiotic Protozoa: Certain protozoa like Trichonympha and Colonymphya etc. live in the gut of
termites which help in the digestion of cellulose.
 Insect Control: Several protozoa control harmful insects by persisting their bodies.
 Sanitation: A large number of protozoa living in polluted water feed upon waste organic matter and
thus purify it. Many protozoa feed upon bacteria and play important role in the sanitary betterment
 Industry: The skeletal deposits of marine protozoa (Foraminifera and Radiolaria) form oceanic ooze
at the sea bottom. These skeletal deposits are put to many uses such as filtering agents, chalk and for
abrasives. The skeletal deposits in due course of time change into the limestone rock that is used as a
building material.

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What are the Important Impacts of Microbes on Ecosystems?
(1) Generate Oxygen in the Atmosphere.
Almost all the production of oxygen by bacteria on earth today occurs in the oceans by the
cyanobacteria or "blue-green algae.
(2) Recycle nutrients stored in organic matter to an inorganic form.
Decomposition releases the mineral nutrients (e.g., N, P, K) bound up in dead organic matter in
an inorganic form that is available for primary producers to use. Without this recycling of inorganic
nutrients, primary productivity on the globe would stop. On land, most of the decomposition (also
called "mineralization") of dead organic matter occurs at the soil surface, and the rate of decomposition
is a function of moisture and temperature. Fungi are important in terrestrial systems, but not in aquatic.
They are present even before the leaves and twigs enter the soil and so decomposition starts in the
living or senescent plant material. Fungi are the most important decomposers of structural plant
compounds. The fungi invade the organic matter in soils first and are then followed by bacteria.
In water, the decomposition of organic matter is mostly oxic in streams and in the ocean and
anoxic in the bottoms of lakes or in swamps. Oxic decomposition proceeds faster than decomposition
in environments where there is no oxygen. In the open ocean, the water is so deep (average 3900 m)
and contains so much oxygen, that most of the algal-formed organic matter at the surface decomposes
aerobically before it reaches the bottom. For example, only 2% of the primary productivity in the upper
ocean sinks to a depth of 3500 m. Most of the world is ocean, and most of the ocean is deep, so most of
the aquatic decomposition must be aerobic. But in shallow waters, coastal oceans, lakes and estuaries,
25-60% of the organic matter produced may settle out of the upper waters rapidly and be decomposed
anaerobically. Another important impact of decomposition besides generating inorganic nutrients is to
produce CO2 and CH4 that are released into the atmosphere.
(3) Fix nitrogen from the Atmosphere into a Useable Form.
The only organisms capable of removing N2 gas from the atmosphere and "fixing" it into a
usable nitrogen form (NH3) are bacteria. The specific bacteria that can perform N fixation are scattered
throughout the groups including the cyanobacteria. All organisms that fix nitrogen use the same
mechanisms and the same enzymes – this ability probably evolved only once and early in the history of
life. SymbioticN2 fixation costs the plant photosynthate to support the fixation and the NH 3
assimilation; this cost could be from 15-30% of the total carbon assimilated by the plant. In fact, fixing
one molecule of N2 requires about 25 molecules of ATP, so it is expensive from the bacterial

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standpoint, and that means that the plant must support that energy requirement. In return, the plant
receives nitrogen, which may otherwise be a limiting nutrient.
Another difficulty for the bacteria is that one of the enzymes necessary for nitrogen fixation is
destroyed by oxygen. One solution to this problem is to form symbiotic relationships with other
organisms that can provide carbohydrates; these include diatoms, the fungi of certain lichens,
shipworms, termites, and certain plants, especially in nodules of the roots.
(4) Allow Herbivores to Consume Poor Quality Food
In the ocean, most of the primary productivity is consumed by herbivores. In contrast, in
terrestrial systems, most of the primary productivity is not consumed by herbivores. The reasons for
this difference are: (1) animals lack digestive enzymes capable of using cellulose and lignin and other
structural plant compounds; (2) plants often have anti-grazing toxins, aromatic resins, or thorns; (3)
most land plant tissue is poor in mineral nutrients (especially N and P) compared to the tissue in the
herbivore.

Plant Community % of primary production consumed by herbivores

Phytoplankton (open water) 60 – 90

Grasslands 12 – 45

Kelp beds 10

Salt marshes 7

Mangroves 5

Deciduous forests 1.5 – 5

In a ruminant animal (cattle, deer, giraffe) the ingested food, possibly regurgitated and re-
chewed, passes into the rumen together with saliva (60-100 liters produced per day). The rumen is
really a continuous fermenter where the complex carbohydrates of the plant are fermented into
methane, carbon dioxide, and fatty acids. The biota of the rumen are found in about equal biomasses of
bacteria (1011/mL), protozoans (105/mL to 106/mL), and fungi (poorly known biomass). About 60-65%
of the total energy removed from the plant food that is ingested by the animal comes from rumen
fermentation. Plant tissues passing from the rumen undergo secondary fermentation in the caecum and
large intestine where an additional 8-30% of the total energy is provided.
In addition, many termites contain protozoans and bacteria in their guts that perform similar
operations. The protozoans are capable of digesting cellulose, and bacteria in the gut generate
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CH4 from the organic compounds released from the cellulose degradation. Finally, some termites also
have bacteria in their guts that are capable of fixing nitrogen from the atmosphere, providing a usable
nitrogen source for the termite.
(5) Give Plant Roots Access to Nutrients in the Soil.
Plant roots create a zone of nutrient depletion around themselves. To have access to new
sources of nutrients, a plant can either grow more roots and small root hairs (some as small as 10 um)
or form an association with a fungus whose hyphae provide an even more efficient absorptive
structure. Most vascular plants can form such associations, which are called "mycorrhizae".
Mycorrhizal fungi include those living on the surface of plants (ectotrophic or sheathing) and those
which enter the host (endotrophic or vesicular-arbuscular or simply "V-A"). The added advantage to
the plant is that the hyphae can secrete enzymes that break down organic molecules and make
inorganic nutrients available. While the plants gain nutrients, the fungi gain carbohydrate food from
the plant. There is also a cost to the plant in this association; one study reported that mycorrhizal
biomass was only 1% of a fir forest ecosystem but used 15% of the net primary production.

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