Dr.

Bibhas Rajkumar Paper name- Microbiology (BOT: DSC-101)

Assistant Professor Semester- 1st (FYUG)
Department of Botany, Unit-V: Water, Environmental and Medical
PDUAM, Eraligool. Microbiology

Water micro-flora
A large number of microorganisms both saprophytes and pathogens are found in water which fall
under the group’s bacteria, fungi, algae, protozoa and nematodes. Several animal viruses are also transmitted
through water. The majority of bacteria found in water belong to groups: fluorescent bacteria (e.g.
Pseudomonas, Alginomonas), chromogenic rods (Xanthomonas), coliform group (E. coil, Aerobacter),
Proteus group, non-gas forming, non-chromogenic and non-spore forming rods, spore formers of the genus
Bacillus, and pigmented and non-pigmented cocci (Micrococcus).

Microorganisms are also present in oceans and seas on the earth. Marine microbiology deals with
microorganisms living in sea. The bacteria growing in marine environment can be categorised in three
groups: (a) barotolerant (bacteria growing between 0 and 400 atm, but best at normal atmospheric
pressure), (b) moderate barophiles (bacteria growing optimally at 400 atm, but still grow at 1 atm) and (c)
extreme barophiles (bacteria growing only at higher pressure i.e. 6000 to 11,000 meter depth). Some
bacteria living in gut of deep-sea invertebrates (e.g. amphipods and holothurians) are truly basophilic. These
basophilic bacteria play a significant role in nutrient cycling. Recently, a very interesting group of bacteria
(e.g. archaeobacteria) has been discovered from the marine system. Archaeobacteria live in hostile
environment i.e. hot springs. The isolation and functional role of these bacteria are still to be studied in
detail.
 Lakes and rivers provide the major fresh water bodies which are used for potable water. The
nutrient-poor lakes are known as oligotrophic lakes, whereas the nutrient-rich lakes are called eutrophic
lakes. The eutrophic lakes support luxuriant growth of bacteria and algae. Eutrophication is a process by
which a water body becomes enriched with nutrients, typically nitrogen and phosphorus. This enrichment
often leads to an excessive growth of algae and other aquatic plants. The microorganisms growing in lakes
are the genera such as- Anabaena, Microcystis, Nostoc, Oscillatoria, Oedogonium, Spirulina, diatoms,
protozoa etc.

Role of microbes in sewage and domestic waste water treatment systems

Wastewater can be defined as water sample which is contaminated and no longer beneficial, and
must be treated before it can be used or released back into the environment. Four major types of wastewater
are as follows- domestic/municipal wastewater, industrial, urban runoff and agricultural runoff. Sewage is
defined as waste matter carried in sewers or drains. It is a “mixture of water and solids.” Following are the
main approaches of waste water treatment system-
(1) Fixed Film systems- This system usually grow microorganisms on substrates such as rocks, sand or
plastic. Then wastewater is spread over the substrate. Example- trickling filters, rotating biological
contactors.
(2) Suspended film systems- This system stir and suspend microorganisms in wastewater and settled out as
a sludge which then pumped back into the incoming wastewater. Example- activated sludge, extended
aeration. In activated sludge type, in the presence of oxygen aerobic bacteria break down organic matter into
simpler compounds through a process called oxidation. The activated sludge process is a common method
that utilizes aerobic bacteria to treat wastewater. In the absence of oxygen, anaerobic bacteria play a vital
role in breaking down complex organic compounds.

Determination of BOD and COD of water samples

Biological oxygen demand (BOD)- It is a measure of oxygen consumption required by the microbial
oxidation of organic matter in sewage. In the initial stage of decomposition of organic materials in sewage,
aerobic microorganisms are involved. These completely oxidise the organic materials under aerobic
condition rendering into complete stabilization of sewage. During this process, all the dissolved oxygen is
consumed for oxidation of organic materials. Consequently there develops anaerobic conditions in water
which results in death of water animals and emitting foul smell due to incomplete oxidation of organic
materials in sewage. The sewage of high organic material has high BOD value and vice versa, Thus, BOD is
the important indication of levels of biological pollution in water, and provides an index of the amount of
microbially oxidizable organic matter.
One can measure BOD in the laboratory. Test sample of water is taken in BOD bottles without air
space. Initially, the level of dissolved oxygen in the water sample is determined. Thereafter, bottles are
incubated at 20°C. After incubation for five days the amount of dissolved oxygen is again measured. The
difference in oxygen values is the BOD which denotes the amount of Oxygen utilized within five days. BOD
is expressed as parts per million.
Calculation-
BOD (mg/L) = Initial DO - Final DO

Chemical oxygen demand (COD)- In aquatic ecosystems, the chemical Oxygen demand (COD) is often
used as an index of the total organic carbon in the sample. The COD is determined by measuring the amount
of oxidizing reagent consumed during oxidation of organic matter with dichromate or permanganate.
Calculation-
COD (mg/L) = (Initial volume of oxidizing agent - Final volume of oxidizing agent) x Conversion
factor

Microorganisms as indicator of water quality

A microbiological water quality indicator is generally one specific species or group of
microorganisms, which can enter into water via faecal matter that pose a risk to human health. The
characteristic groups of bacteria are the coliforms (intestinal bacteria). Coliforms are defined as facultatively
anaerobic, Gram negative, non-sporing, rod-shaped bacteria that ferment lactose with gas formation within
48 hours at 35°C. Total coliform, faecal coliform, E. coli and faecal Streptococcus are presently being used
to assess contamination in water. Coliforms are the members of the family Enterobacteriaceae which
includes E. coli, Enterobacter aerogenes, Salmonella and Klebsiella pneumoniae. These bacteria make up
about 10% of intestinal microorganisms of humans and other animals, therefore have found widespread use
as indicator organisms for possible water contamination. If such bacteria are not detectable in water in 100
ml, the water can be said as potable water. The ideal indicator organism for assessing water quality should
have following characteristics-
(1) It must be easily detected by simple methods.
(2) It must not grow in natural water.
(3) The organism should be found in warm-blooded animals intestines.
(4) The organism should have a longer survival time.

Tests for coliforms

The original test for the presence of coliform in water is done by standard multiple tube fermentation
technique. This method involves the three routine standard tests- (a) The presumptive test, (b) The
confirmed test, and (c) The complete test.
(a) Presumptive Test- A series of fermentation tubes each containing lactose broth of known concentration
are inoculated with known amount of water. These tubes are incubated for 24 to 48 hours at 35°C.
Generally, five fermentation tubes containing single or double strength lactose broth are inoculated with 10
ml water, 5 tubes with 1 ml water and 5 with 0.1 ml water. At the end of 24 hours of incubation, the tubes
indicate that the coliforms are absent. These tubes are incubated for an additional 24 hour to be sure for the
absence of coliforms, which is gas production.
(b) Confirmed Test- If a positive test of gas production is obtained, it does not mean that coliforms are
present. The other organisms too also give false positive presumptive test because they are also capable of
fermenting lactose with formation of acid and gas. If yeasts, species of Clostridium and some other
microorganisms are present, gas is also produced. Therefore, a confirmed test is performed for the presence
of coliforms. All fermentation tubes showing gas within 48 hours at 35°C are used for confirmed test. It
is of two types as described below.
The positive presumptive fermentation tube is gently shaken. A drop of its culture is transferred to
brilliant green lactose bile broth fermentation tube (BGLB). The tubes are incubated for 48 hours at 35°C.
The appearance of gas within this period indicates for positive confirmed test. The dye (brilliant green)
inhibits the Gram-positive bacteria. The second confirmed test is done by Eosine methylene blue (EMB)
agar method. In eosine methylene blue agar method, a definite amount of two stains (eosin and methylene
blue) is added to a melted lactose agar. The medium is poured into Petri dishes. Over the surface of EMB
agar medium, a loopful culture from each positive fermentation tube is streaked. Plates are incubated at
35°C for 24 hours. The development of typical colonies (nucleated, with or without metallic sheen) shows
that the confirmed test is positive.
(c) Completed Test- The completed test is performed to ascertain about the presence of coliforms in water.
The purpose of the completed test is to determine whether- (a) the colonies growing on EMB agar are again
capable of fermenting lactose and forming acid and gas (b) the organisms transferred to agar slants show the
morphological appearance of coliform group. Each colony that gives positive confirmed test is transferred to
lactose fermentation tube and to nutrient agar slants. The tubes are incubated at 35°C for 48 hours.
Production of gas in fermentation tubes and demonstration of Gram-negative, non-spore forming rods on the
agar slants constitute a positive completed test for coliforms. The absence of gas and the rod production
confirms for negative test of coliforms.
The Most Probable Number of (MPN) of Coliforms- For the first time Hoskins (1934) computed the
MPN to evaluate number of coliforms present in water sample by fermentation tube method. By referring to
a MPN table, a statistical range of the coliform numbers is determined by observing the number of broth
tubes producing gas. Confirmed and positive tests are used to calculate the MPN.

Bioremediation of contaminated soil

Bioremediation is a waste management technique that involves the use of organisms to remove or
neutralize pollutants from a contaminated site. In other words, bioremediation is a treatment that uses
naturally occurring organisms to breakdown hazardous substances into less toxic or non-toxic substances. In
the process of bioremediation, microbes are provided with optimum conditions, fertilizers, stimulators etc.
so that they can breakdown the organic matter into its simplest form.
Role of microbes for In-situ remediation- It uses microorganisms to breakdown contaminants present
right at its point of origin. Instead of removing the contaminated soil from its place, it is treated right where
it is present. The process could be anaerobic whose end products are methane, hydrogen gas, sulphide and
elemental Sulphur. Examples- Escherichia coli, Staphylococcus, Clostridium. When it occurs in the
presence of oxygen (aerobic process), which results in the formation of CO2, H2O and salts. For example,
Pseudomonas putida is a hydrocarbon degrading bacterium which degrades oils spills in water, thus cleans
sea water.
Role of microbes for Ex-situ remediation- In ex-situ bioremediation, waste is removed from the place of
its origin and carried to treatment site. Ex-situ bioremediation used the microbes and added nutrients to
speed up the breakdown process of the environmental pollutants in bio-reactors. Different species of
Alternaria, Aspergillus, Penicillium, Trichoderma have been used in ex-situ bioremediation.

Enumeration of microorganisms in air

Air is not a natural environment for microorganisms as it doesn't contain enough moisture and
nutrients to support their growth and reproduction. One of the most common source of air microflora is the
soil. Soil microorganisms when disturbed by the blowing wind get liberated into the air and remain
suspended there for a long period of time. Man-made actions like digging or ploughing the soil may also
release soil borne microbes into the air. There are several methods, which require special devices, designed
for the enumeration of microorganisms in air. The most important ones are solid and liquid impingement
devices, filtration, sedimentation, centrifugation, electrostatic precipitation etc. Some of the methods are
described below-
(1) Impingement in liquids- In this method, the air is drawn through a very small opening or a capillary
tube and bubbled through the liquid. The organisms get trapped in the liquid medium. Aliquots of the liquid
are then plated to determine its microbial content.
(2) Impingement on solids- In this method, the microorganisms are collected, or impinged directly on the
solid surface of agar medium. Colonies develop on the medium where the organism impinges. Several
devices are used, of which the settling-plate technique is the simplest. In this method the cover of the
petridish containing agar medium is removed, and the agar surface is exposed to the air for several minutes.
A certain number of colonies develop on incubation of the petridish. Choice of the medium depends upon
the kind of microorganisms to be enumerated. For an overall count of pathogenic, commensal and
saprophytic bacteria, blood agar can be used. During sampling it is better to keep the plates about 1 metre
above the ground. Immediately after exposure for the given period of time, the plates are closed with the
lids. Then the plates are incubated for 24h at 37°C for aerobic bacteria and for 3 days at 22°C for
saprophytic bacteria. For molds, the incubation temperature varies from 10-25°C for 1-2 weeks. Since the
technique does not record the volume of air actually sampled, it gives only a rough estimate. However, it
does give information about the kind of microorganisms in a particular area.
(3) Filtration- The membrane filter devices are adaptable to direct collection of microorganisms by
filtration of air.

Control of air born microorganisms

(1) Physical Techniques
(a) Dust Control- As stated earlier, the dust particles contain large number of microbial agents that
cause diseases and allergies. Therefore, controlling dust in home, schools, factories, laboratories and
hospitals is extremely important to lower down microbial content.
(b) Ultraviolet Radiation- This technique has great potential value for reducing indoor airborne
microbial population. Various types of germicidal lamps are used for this purpose. These lamps emit
radiations in 250-260 nm region, the most effective bactericidal region.
(c) Laminar-air flow System- This is a new technique recommended for controlling indoor
microbial population. This technique represents unidirectional airflow system in which the air
passes high-efficiency particulate air (HEPA) filters.
(2) Chemical Techniques
Bactericidal Vapours (Chemical Agents)- Indoor air-borne microbial population can be
effectively reduced by vaporizing or spraying certain chemical substances into the air such as-
propylene glycol, triethylene glycol, resorcinol etc. Nearly all microorganisms present in a litre of
heavily contaminated air can be killed by vapour from 0.5 mg of propylene glycol.
 Triethylene glycol is nearly ten times effective in comparison to propylene glycol. However,
great care is required for safe and efficient application of chemical agents. Therefore, they may
be used only when rapid control of air-borne microbial content is essential.
(3) Vedic technology for air purification- The Rishis were aware of making their houses and surroundings
pure by using vedic technology, one of which is the Agnihotra. Gases are produced after burning of various
substances like- ghee, cereals, medicinal herbs, forest herbs etc., which diffused in surroundings. These
gases kill/inhibit various kinds of microorganisms present in air. Gupta and Singh (1996) at Gurukul Kangri
University performed Agnihotra and noticed that the total microbial population of bacteria, fungi and
actinomycetes decreased in the surrounding after Yajna fire. Nautiyal et al. (2007) observed the effect of a
mixture of medicinal herbs (havan samagri) on population of bacteria present in air. They recorded 94%
reduction in bacterial counts after exposure of 60 min.

Biogas production
Definition- Biogas is the mixture of gases produced by microorganisms, primarily consisting of methane
(CH4) and carbon dioxide (CO2) along with hydrogen sulphide (H2S). It is a renewable source of energy.
The production of biogas involves the breakdown of organic matter by microorganisms in the absence of
oxygen (anaerobic condition). The bacteria which produce the gaseous mixture are collectively known as
methanogens. Methanobacterium is one such methanogen.
India is one of the pioneer countries in biogas technology. Uttar Pradesh Government in the years
1957 and 1960 established a permanent station named 'Gobar gas Research Station' in Etawah district. One
of the major plants installed at Kanpur is the Rajapurwa Biogas Plant where about 1400 kg human waste
from 50 seat toilet complex is pooled through underground pipelines at one place. National sugar Institute,
Kanpur has developed methods for production of biogas from bagasse and other agricultural residues.
Feedstock Materials- There are two sources of biomass i.e. plant and animal. The biomass obtained from
plants is aquatic or terrestrial in origin, while biomass generated from animals includes cattle dung manure
from poultry, goat, sheep and slaughter houses, fisheries waste, etc. Cattle dung is most potent for biogas
production.
Biogas Production (Anaerobic Digestion)- The anaerobic digestion is carried out in an air tight cylindrical
tanks which is called digester. A digester is made up of concrete bricks and cement or steel. It has a side
opening (charge pit) into which organic materials for digestion are incorporated. There lies a cylindrical
container above the digester to collect the gas. after 50 days, sufficient gas is produced in gas tank, which is
used for house hold purposes. Anaerobic digestion is accomplished in three stages, solubilisation,
acidogenesis and methanogenesis.
(a) Solubilisation- It is the initial stage when feed stock is solubilized by water and enzyme to make
slurry. The complex polymers are hydrolysed into organic acids alcohols by hydrolytic fermentative
methanogenic bacteria.
(b) Acidogenesis- In this stage, facultative anaerobic bacteria convert the simple organic material via
oxidation/reduction reactions into acetate, H2 and CO2.
(c) Methanogenesis- This is the last stage of anaerobic digestion where acetate, H2 and CO2 are
converted into methane, carbon dioxide, water and other products by methane producing bacteria.

Advantages of Biogas
(1) Biogas is a safe, cheap, renewable source of energy.
(2) Biogas can be burnt in stoves to provide heat.
(3) It is used for domestic and street lighting, and cooking.
(4) It is eco-friendly and does not cause any pollution.
(5) It is also used for driving engines.
(6) It is easy to generate, transport and store.
(7) It improves the sanitation of the surroundings.
(8) The residue left after the production of biogas can be used as manure.
 Microbes in biodegradation of hydrocarbons
Microbes play a crucial role in the biodegradation of hydrocarbons, which are organic compounds
made up of hydrogen and carbon atoms. Hydrocarbons are found in various petroleum products, such as
crude oil and refined fuels. Several types of microorganisms such as- Pseudomonas produce enzyme
hydroxylases, which initiate the breakdown of hydrocarbons. Fungi and bacteria are the main agents which
decompose oil and oil products. Besides cyanobacteria, yeast and algae have shown to oxidise
hydrocarbons. The simplest hydrocarbon pollutant is methane. It is degraded by a specialized group of
bacteria called methanotrophic bacteria. Oil is insoluble in water and is less dense; it floats on the surface
and forms oil films. Hydrocarbon-oxidising microorganisms develop rapidly in such films.
In recent years, experimentations on biotechnology have been done for the production of potential
microbial strains capable of degrading pollutants. Dr. Anand Mohan Chakraborty (an India-borne American
Scientist) succeded in producing a genetically engineered strain of Pseudomonas putida that utilized
complex chemical compounds. It was called as super bug.

Microbial diseases (Aspergillosis, Tuberculosis, Tetanus, Gonorrhea, Syphilis, Leprosy)

(1) Aspergillosis-
Causes- Aspergillosis is caused by the species of Aspergillus especially A. fumigatus. For the first time in
1856, Virchow reported aspergillosis in man. Aspergillosis occurs in sinuses, bronchi, lungs, and other parts
of body as well. A. fumigatus generally causes pulmonary aspergillosis. The other species of Aspergillus
viz., A. niger, A. clavatus, A. terreus and A. phialiseptus are also known to be associated with pulmonary
aspergillosis.
Preventive measures- (1) Reduction of mold exposure.
(2) Antifungal prophylaxis can be given to patients. Posaconazole is given as
prophylaxis in case of severe immunocompromised patients.
(3) Avoid medication that may weaken immune system.
(4) Avoid forests, grain stores, piles of dead leaves.
(5) Dust control.

(2) Tuberculosis-
Causes- Robert Koch for the first time identified Mycobacterium tuberculosis as the causal agent of TB
(tuberculosis). During that period TB caused 1/7 of death in Europe. About 20% of the world's population is
suffering from TB and about 8 million people are victimised each year. Commonly tuberculosis occurs
among the homeless, malnourished persons or alcoholic drinkers. This bacterium spreads through droplet
nuclei and the respiratory route.
Preventive measurs- (1) Avoid close contact with people who have active TB disease.
(2) Eat a nutritious diet and exercise regularly to keep your immune system strong.
 (3) People serving in healthcare sector, follow protocols for wearing masks.
(4) People having latent TB infection, follow the entire treatment protocol.
(5) If you’re traveling to a high risk area and you have a compromised immune
system, talk with a doctor about preventive treatments.

(3) Tetanus-
Causes- Tetanus is caused by Clostridium tetani, is a Gram-positive, anaerobic and endospore forming
bacterium, found in sol, dust and faeces of many farm animals and humans. Transmission of the bacterium
takes place through skin wound. In the wound the neurotoxin, tetanospasmin is released after the death and
break down of bacteria.
Preventive measures- Tetanus is prevented by using tetanus toxoid. tetanus toxiod is given with DPT
program. After a few months of birth, initial dose is given. The second dose should be given after 4-6
months of the first dose. Finally, the reinforcing dose should be given 6 to 12 months after the second dose.
Between the age of 4 to 6 years, a final booster dose is given. A single booster dose can provide protection
for 10 to 20 years.

(4) Gonorrhea-
Causes- Gonorrhea is a sexually transmitted disease (STD) caused by the bacterium Neisseria gonorrhoeae
which enters into body through sexual fluids like semen or vaginal fluid. It is small Gram-negative, oxidase-
positive, diplococcus bacterium. The bacteria also referred to as gonococci. It primarily infects the genital
organ and reproductive tract, but it can also affect the mouth, throat, eyes, and rectum. The bacteria is named
after Albert L.S. Neisser who cultured the bacterium in 1879.
Preventive measures- (1) Always use a condom during sex.
(2) Don’t have sex with someone who has an active infection.
(3) Don’t have sex with someone who has gonorrhea symptoms.
(4) Limit number of sex partners.
(5) Consider regular gonorrhea screening.
(6) The recommended antibiotics for treatment of gonorrhea are penicillin G,
streptomycin, tetracycline etc.

(5) Syphilis-
Causes- Syphilis is a sexually transmitted disease (STD) caused by the bacterium Treponema pallidum. It
can have serious consequences if left untreated, affecting various organs and systems in the body. During the
end of 15th century, syphilis was recognized in Europe. According to one hypothesis syphilis is of New
World Origin. Christopher Columbus (1451-1506) and his crew members acquired this disease in West
Indies and introduced into Spain when returned from their historic journey.
Preventive measures- (1) Always use a condom during sex.
 (2) Don’t have sex with someone who has an active infection.
(3) Don’t have sex with someone who has Syphilis symptoms.
(4) Limit number of sex partners.
(5) Consider regular Syphilis screening.
(6) The recommended antibiotics for treatment of Syphilis are penicillin.
(6) Leprosy-
Causes- Leprosy is caused by the bacterium Mycobacterium leprae. It primarily affects the skin, peripheral
nerves, mucosa of the upper respiratory tract, and the eyes. Leprosy is also termed Hansen’s disease after the
name of the scientist Dr Gerhard Hansen who discovered this infectious disease in the year 1873.
Preventive Measures- (1) Early detection and treatment of leprosy is the best way to keep it from
spreading.
(2) Identifying and screening individuals who had close contact with a person
diagnosed with leprosy.
(3) Encouraging good personal hygiene practices including regular hand washing can
contribute to reducing the risk of infection.
(4) Engaging communities in leprosy control programs fosters awareness and early
detection of the disease.

Probiotics
Probiotics are live microorganisms primarily bacteria and yeast, that confer health benefits when
consumed in adequate amounts. They are often referred to as "good" or "friendly" bacteria because of their
positive impact on the digestive system and overall well-being.
Types of Probiotics- While both yeast and bacteria constitute probiotics, it is the latter that has the most
types and is most widely used. Here is a list of the most commonly used probiotics-
(a) Lactobacillus- It is a species of bacteria that produces lactase, an enzyme that breaks down
lactose or milk sugar. Lactobacillus produces lactic acid that helps control the population of bad
bacteria in our gut. Example- Lactobacillus acidophilus, Lactobacillus bulgarius.
(b) Bifidobacteria- Bifidobacteria is the species of bacteria that is most commonly used in food and
its supplements. This bacteria supports our immune system.
Benefits of Probiotics- (1) Stronger immune system.
(2) Improved digestion.
(3) Increased energy from production of vitamin B12.
(4) Maintain gut health.
(5) Healthier skin.
(6) Reduced cold and flu.
(7) Healing from leaky gut and inflammatory bowel disease.
(8) Weight loss.