EFFECTS OF HEAVY METALS ON BACTERIAL GROWTH

Dissertation submitted to MATS University towards partial fulfillment for

the award of Bachelor of Science in Biotechnology

By
Abhishek Banjare
B. Sc. (Biotechnology), Semester IV

2014

Under the guidance of

Miss M.Rukmini

School of Life Sciences, MATS University, Aarang-Kharora Highway,

Aarang, Raipur, Chhattisgarh-493 441.

1
2
 DECLARATION

I do here by declare that the dissertation entitled “Effects of heavy heavy

metals on Bacterial growth” submitted towards partial fulfillment for the
award of degree of Bachelor of Sciences in Biotechnology of MATS University
is based on the results of studies carried out by me under the guidance and
supervision of Miss M. Rukmini at niTza biological research lab Pvt Ltd
Secunderabad, Hyderabad . This dissertation or no part of this has been
submitted elsewhere for the award of any degree or diploma.

Place: Raipur Abhishek Banjare

Date:

3
Vishwaprakash Roy
Head of the Department
School of Life Sciences
E-mail: vproy@matsuniversity.ac.in

Date: …………….

APPROVAL CERTIFICATE

This is to approve that the work presented in this dissertation entitled “Effects
of heavy heavy metals on Bacterial growth” was carried out by Abhishek
Banjare and submitted for the partial fulfillment of the requirements of the
award of the degree of bachelor of Science in Biotechnology in MATS
University is the original work carried out under the guidance and supervision
of Miss M. Rukmini at niTza biological research , secunderabad and that no
part of this dissertation has been submitted elsewhere for the award of any
degree or diploma.

(Vishwaprakash Roy)

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 ACKNOWLEDGEMENT

I have taken efforts in this project. However, it would not have been possible without the
kind support and help of many individuals and organizations. I would like to extend my
sincere thanks to all of them

I am highly indebted to Head of Department of Life Science , MATS University Mr.

Vishwaprakash Roy for their guidance and information regarding the project.

My special thanks to our guide Miss M. Rukhmini , niTza biological lab for their constant
guidance and supervision during the course of project. I also thank the rest of the staff of
niTza biological lab for their Co-operation.

I would like to thank the whole faculty of life science department, MATS University for their
encouragement and support towards my project.
I would like to express my gratitude to my parents and friends for their constant support and
help in completion of the project.

Abhishek Banjare
B.sc Biotechnology
MATS University

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 CONTECT
01. INTRODUCTION 6-17

02. REVIEW OF LITERATURE 18-20

03. AIM AND OBJECTIVE 21-22

04. MATERIALS AND METHODOLOGY 24-32

05. OBSERVATION AND RESULT 33-40

06. PHOTOPLATES 42-44

07. DISCUSSION 46-47

08. CONCLUSION 49

09. BIBLIOGRAPHY 51-52

10. ABSTRACT 54

11. APENDIX 56

6
INTRODUCTION

7
1 . INTRODUCTION
Wastewater is any water that has been adversely affected in quality by
anthropogenic influence. It comprises liquid waste discharged by domestic residences,
commercial properties, industry, and/or agriculture and can encompass a wide range of
potential contaminants and concentrations. In the most common usage, it refers to the
municipal wastewater that contains a broad spectrum of contaminants resulting from the
mixing of wastewaters from different sources

1.1 What are the sources and effects of water pollutants?

There are many sources of the water pollution and have a multiple effects on the body. It has
main adverse effects in the villages. Nearly, half of our population suffers from the water
pollution along with its consequences and is responsible for the death of one third of our
population. The water pollution causes the de oxygenation of water, addition of poisonous
and suspended particles; non toxic salts are also added. Heating is also a vital factor
responsible for it. The domestic wastes and sewages are the main source of water pollution.
It occurs from the boats, ships and municipalities. The canning industries and slaughter
house also add the organic waste into the water. The people in their villages wash their
utensils, animals and hands in the same pond. They also use this water for bathing and
drinking. This water is an agent of cholera, typhoid and jaundice. It also causes the skin
disease in few cases. There are many industrial wastes and effluents present in the municipal
sewers. They cause the degradation of sewage with the help of micro organisms. They eat up
all the oxygen present in the water. There are pathogens present in the raw sewage. The
intestinal bacteria are good indicators of the pollution in the raw sewage. The more is the
number of intestinal bacteria more is the pollution in the sewage. The river Yamuna has
more number of coliforms which are followed by the enterococci. The sewage also increases
the activity of decomposers which are referred as a sewage fungus. The bacteria, algae along
with the blue green algae constitute the sewage fungus. The oxygen to the sewage flora and
fauna is given by the algae. There are other plants present in the sewage which provides the

8
oxygen. There are many protozoa’s in the sewage which eat the bacteria. The algal
component also supports the fungal and bacterial count. If there is decrease in the algal
component the fungal and bacterial count also reduces. It affects the overall population
except the blood and sludge worms. There is a foul odor from the sewage. It makes the
sewage to look oily and water brown. A scum and sludge is formed from the organic waste
and makes the water unfit for recreational and industrial purposes. The blooms are formed by
the algae which decreases the level of oxygen. It adds more organic matter and cause the
fouling of water. There are new detergents used these days which provide white and bright
color to the clothes. These degrade slowly and aggregate making the water unfit for human
consumption. There are phosphates present in them which allow the algae to grow and
increase the overall organic content. There are many secondary pollutants which are formed
by the process of anaerobiosis. It includes the hydrogen disulphide, ammonia and methane.
The hydrogen disulphide has the ability to combine with the other elements and form a black
color sulphide that can float.

There are certain pollutants which are present on the surface of land and they are passed into
the water reservoirs during rains. The fertilizers are added to it to increase the yield of crop
which is also passed to the water reservoirs. This fertilizer rich water goes into the streams
and lakes leading to the eutrophication. It leads to the increase growth of blooms and aquatic
plants. The plants which give rise to the bloom occur on the surface. The submerged plants
do not produce the oxygen and cut of the light. The oxygen produced by the bloom goes into
the atmosphere. The respiration of plants and animals during the night further decreases the
level of oxygen. When the plant dies it increases the organic matter and it further decreases
the level of oxygen. It causes the death of aquatic animals. It leads to the formation of unfit
water for recreational and industrial processes. So, the excess of fertilizers must not be used.
However, the small doses of fertilizers and manure must be used. They are helpful in the
minimizing of surface run off. The ammonium sulphate and urea are considered the best
fertilizers for the paddy. Some research institutes have recommended the use of urea along
with the mud to form mud balls. They are situated below the soil surface. The ammonium
sulphate also replaces the nitrates in tea plantations.

9
The unethical agriculture and forest practices lead to the soil erosion. It may also occur due
to rain. It helps to make water muddy. It does not support the growth of plant as it cuts off
the light. So, the animal population also gets affected. They also decrease the water ways as
the mud and silt are deposited. The industrial wastes are moved into the water. They contain
the mercury, lead, cadmium and other metals. The mercury is liberated as the result of
combustion of coal. It is also produced by the smelting of ores and in the paper and paint
industries. It is persistent and in the water it changes into the water soluble form which is
known as the dimethyl. It has the ability to enter the food chain. It also causes the biological
and ecological amplification. It kills the fishes and contaminates the rest of fauna. If the
human beings feed on these fauna it leads to the condition known as a minamata disease
which shows a crippling deformity which is accompanied by the numbness of lips along with
the limbs, it affects the vision, hearing, taste, speech and ultimately leads to the death of an
individual. The genetic changes are brought by the mercury also. It was supported by the
Ramel in 1974. The lead occurs from the smelters, battery, industry, paint and the
automobile exhausts. It causes mutations and may lead to the anemia, headache,
discoloration in the gums, loss of muscle power, and irritation. The cadmium occurs from the
industries, electroplating, phosphates and the pesticide industry. It also causes the biological
amplification. It has the ability to accumulate inside the kidneys, liver and pancreas. It may
cause the renal damage, hypertension, anemia, necrosis of the testes and an injury to the
placenta. It can enter through the food chain and is entered via wheat and rice. It was
supported by the Nath in 1986. The other metals involved are copper, zinc, nickel and
titanium. They alter the function of enzymes. The liquid effluents are moved into the water
which consists of the acids, bases and toxic substances. They kill the fish and other aquatic
animals. They also cause an adverse effect on the human beings. They also involve many
rivers like Gomati, Yamuna, Gang and Hooghly. They are present in the different parts of
India. The continental shelf is a good source of oil. It is transported from one part of the
country to other by the use of sea. The tankers which carry the fuel are washed after
unloading in the sea. They are filled with the water. So, oil spills are very common in the sea.
They occur near the ports and shore lines. During transportation accidents may happen which
can spread the oil over few hundred kilometers. Nearly, 10 million tons of oil is spilled into
the ocean per year. The other source of spillage of oil in the river is the refineries. They
discharge in the form of effluents. There was a Barauni refinery which has an adverse effect
10
on the flora and fauna of the river Ganges in the late 20th century. It leads to the spreading of
oil on the surface of water which leads to the prevention of oxygenation. It also depletes the
oxygen present in the water and leads to the degradation. It does not allow the growth of
plankton and inhibits the photosynthetic activity of the other organisms in water. The
decreased availability of food, oxygen leads to the destruction of animal life. The oil present
on the surface of water may catch fire and can kill the organic matter present in water. The
sea birds which are smeared with the oil get sick and die. The detergents which are used to
clean the spill oil are also harmful and it was established in the Torrey Canyon accident
along the British coast. There are hot bodies and effluents which may lead to the thermal
pollution. The water which is warm contains less amount of oxygen as compared to the cold
water. As the water is warmer the rate of decomposition is less. There are many animals
which fail to divide and the green alga is replaced by the blue green algae. The trot fails to
hatch the eggs at higher temperature and the salmon does not spawn. There are industries
which are present in the coastal area which dump their wastes directly into the oceans and
rivers. There are certain inland waters in some areas which are rich in the insecticides and
pesticides. The sewage in the coastal cities does reach the sea directly. The products from the
ship like garbage, sewage, detergents also pollute the water. Around one fourth of the people
living in south Asia survive on this contaminated water and their products. There are people
in the south pacific region which survive on sea food. They suffer from cholera and hepatitis
frequently. There are certain segments of the people in Nigeria which use beaches as the
place of toilet. The population becomes prone to water borne diseases along with the viruses
and parasites. In the coastal waters the oxygen level becomes so dim that it is difficult for the
flora and fauna to survive.

1.2 Effluent:

Effluent is an out flowing of water from a natural body of water, or from a man made
structure.

11
Definition: Materials generally discarded from industrial operations or derived from
manufacturing processes.
Contaminations of drinking water supplies from industrial waste is a result of
various types of industrial processes and disposal practices .Industries that use large amounts
of water for processing have the potential to pollute waterways through the discharge of their
waste into streams and rivers, or by run off and seepage of stored wastes into near by water
sources. Other disposal practices, which cause water contamination ,include deep well
injection and improper disposal of wastes in surface impoundments .
Industrial wastes consists of both organic and inorganic compounds .Organic wastes include
pesticide residues , solvents ,cleaning fluids ,dissolved fluids fruit and vegetables and lignin
from pulp and paper .Effluents can also contain some inorganic wastes such as brine salts
and metals .Industries which use large amounts of water in their processes include chemical
manufacturers ,steel plants ,metal processes ,textile manufacturers .

1.2.1 Paper industry:

Industries are spread in all over the world .The production ,use and recycling of paper has a
no. of adverse affects on the environment ,which are known collectively as paper
pollution .Discarded paper is a major component of many landfill sites ,accounting for about
35% by weight of municipal solid waste (before recycling) .Even paper recycling can be as
source of pollution due to the sludge produced during drinking .
Pulp and paper is the third largest industrial polluter to air ,water and land in both Canada
and USA ,and releases well over hundred million kg of pollutant each year .
The world pulp and paper industry continues to expand production and increasingly , plants
are being built in newly industrialized countries .The dominant process is the sulfate
process .Historically ,substantial pollution problems have been associated with pulp
manufacturing operations. Following the recognition of large scale environ mental
contamination by organochlorines ,the industries implemented a no. of process internal
changes and continued to develop process external treatment process .

1.2.1.1 Microorganisms present in paper industry effluents :

The paper industries microorganism belonging from genera bacillus , pseudomonas ,
coliform bacteria ,some klebsiella species .They resist from oil ,hydrocarbon and other
12
chemical .The fungi present in these industries are belonging from Aspergillus and
dermatophytes species .

1.2.2 Effluents of paint industry:

Heavy metals can cause health hazards to man and aquatic lifes if their concs exceed
allowable limits .Concs of heavy metals below this limits even have potential for long term
contamination , because heavy metals are known to be accumulative within biological
system .Lead contamination of the environment is primarily due to anthropogenic
activities ,making it the most ubiquitous toxic metals in the environment .
Lead heavily accumulated in the humous rich surface layer of the soils due to its complexity
with organic matter and it was reported to the least mobile heavy metals in soils under
reducing and non reducing condition .Lead has been implicated to be the most common
heavy metal contaminants in urban (Abdus, salam and Adekola 2005) .The release of
industrial waste waters to the environment causes several adverse effects .These waste
waters commonly include Cd , Pb, Cu, Ni, Co .These heavy metals are not biodegradable and
their presence in streams and lakes leads to bioaccumulation in living organisms ,causing
health problems in animals ,plants and human beings .Paint industries are among those
industries that discharge effluents containing heavy metals .Paint compounds are
pigments ,solvents ,blotters ands auxiliary additives .Heavy metals are mainly usd as
pigments and blotters in the paint .The presence of these heavy metals are directly and
indirectly hazardous for human and other beings .Several methods have been developed for
decontamination of municipal and industrial waters and wastewaters .Among different heavy
metal emoval methods ,chemical precipitation ,membrane filtration (reverse osmosis and
electro dialysis ),electrolytic processes .
Microorganisms present in paint effluents are Listeria , Pseudomonas ,cocci etc.

1.2.3 Textile industry:

Textile industries have very important position in this era , several textile industries flows
their effluents in rivers which causes water pollution because in effluents several
chemicals ,dyes, and oils are present .
Textile industry can be classified into 3 categories viz ,cotton ,woolen and
synthetic fibers depending upon the raw materials used .The cotton textile industry is one of

13
the oldest industries in the countries with over 1000 industries (or mills) mainly centered in
Mumbai, Surat, Ahmadabad, Coimbatore and Kanpur .The water consumption and waste
water generation from a textile industry depends upon the processing operations employed
during the conversion of fibre to textile fabric .On the basis of waste and wastewater
generation ,the textile mills can be classified (ISPCH,1995) into 2 main groups viz,dry
processing mill and woven fabric finishing mills .In the dry processing mill mainly solid
water is generated due to the rejects of cotton In the other groups desizing, scouring,
bleaching, mercerizing, dying, printing and packing are the main processing stages .
The organisms present in it arew similar to paint industry like pseudomonas, Listeria ,etc.

1.2.4 Pharma industry:

Medicine is very essential for us ,several pharmaceutical industries
manufactured large amount of medicines but it also creates pollution ,which is very
hazardous for us .In dumping area the effluents mix with water and soil and polluted it .The
effluents contain several organic and inorganic chemicals ,solid waste materials and several
microorganisms .
Pharmaceutical effluents are wastes generated by pharmaceutical industry during the process
of drugs manufacturing. Some pharmaceutical effluents contain high concentration organic
compounds such as Phenol, Toluene, Toluene, Benzene, Xylene, Nitro Benzene, Ethyl
acetate, Isopropyl Ether and other aliphatic compounds. It also contains heavy metals like
mercury, cadmium, isomers of hexachlorocylohexane,1,2-dichloroethane and non-aqueous
solvents. The increasing awareness for the protection of environment, treatment & disposal
of industrial wastes has acquired great significance. Phenol and Toluene are the common
pollutants in several pharmaceutical industrial effluents. (3,4) Phenol is a toxic compound
even at low concentrations and the phenolic compounds are introduced in the environment in
the waste streams of several industrial operations, through its use as biocides or as
byproducts of other industrial operations, such as pulp bleaching with chlorine, Water
disinfections or even waste incineration.(5,6,7) The contamination of water and soils by
phenol and its derivatives is a matter of serious environmental concern due to their
toxic effects. Phenol is highly carcinogenic compound recognized by Environmental
Protection Agency. Therefore the treatment of phenol effluent is very important, since the
toxic effects of phenol pronounced even at very low concentrations. (8,9) Although physical
14
and chemical methods are in vogue for removal phenol and its derivatives, biodegradation
methods are less expensive and can potentially degrade these contaminants to innocuous
products. The effluent containing phenol in the range of 5 - 500 mg/L are considered suitable
for treatment by biological processes. The literature on phenol biodegradation by mixed or
pure cultures reports that many bacteria, but likely not all of them, metabolize phenol.(10)
Toluene is a toxic aromatic compound found as a component of petroleum hydrocarbons.
Often Toluene enters the environment in the form of industrial discharges from petroleum
refining, plastics, resins and pharmaceutical industrial effluents or oil spills. (11-15) In the
present work Bacillus sp., Pseudomonas sp., Staphylococcus sp., were taken for the
degradation of aromatic organic compounds especially phenol and Toluene. This is
recognized that these bacteria harbor specialized genes, which are capable of detoxifying
phenol and Toluene. Some Pseudomonas species have been identified efficient for
degradation of these compounds. Phenol and toluene were obtained from Merck Chemicals
Ltd. All other chemicals used were of highest purity and available commercially. Samples
were collected from aeropac of effluent treatment plant at Orchide.
Indian p-harmaceutical industry today is in front rank of India’s science based
industries with wide ranging capabilities in the complex field of drug manufacture and
technology .It ranks very high in the 3rd world in terms of technology ,quality and range of
medicines manufactured .From simple headache pills to sophisticated antibiotics and
complex cardiac compounds ,almost every type of medicine is now made indigeneously .
The pharmaceutical industries microorganisms belonging from gram negative species ,some
Listeria species ,Pseudomonas aureginosa ,Staphylococcus aureus .The fungi present in
these industries are penicillium , aspergillus species and some species of dermatophytes .

1.3 Heavy metals :

There are 35 metals that concern us because of occupational or residential exposure, 23 of
these are heavy elements or ‘HEAVY METALS’ : arsenic, bismuth, antimony, cadmium,
cerium, nickel, platinum, zinc.
Interestingly, small amounts of these elements are common in our environment and diet and
are actually necessary for good health, but large amount of any of them may cause acute or
pronic toxicity. Heavy metal toxicity may result in damage or reduced mental and central
nervous system and vital organs. Long term exposure may result in slowly progressing
15
physical, muscular and neurological degenerative processes that mimic Alzheimer’s disease,
Parkinson’s disease, muscular dystrophy. Allergies are not uncommon and repeated long
term contact with some metals or their compounds may even cause cancer. For some heavy
metals toxic level scan be just above the background concentrations naturally found in
nature. Therefore it is important for us to inform ourselves about benefits and hazardous
effects of heavy metals.

Beneficial heavy metals:

In some quantities, certain heavy metals are nutritionally essential for a healthy life.Some of
these are reffered to as the trace elements i.e iron, copper, manganese and zinc. These
elements, or some form of them, are commonly found naturally in foodstuffs, in fruits and
vegetables and in commercially available multivitamin products. Diagnostic medical
applications include direct injection of gallium during radiological production, dosing with
chromium in parenteral nutrition mixtures, and the use of lead as radiation shield around x-
ray equipment. Heavymetals are also common in industrial applications such as in the
manufacture of pesticides, batteries, alloys, electroplated metal parts, textile dyes, steel, and
so forth. Many of these products are in our homes and actually add to our quality of life
when properly used.

Toxic heavy metals:

Heavy metals become toxic when they are not metabolized by the body and accumulate in
the soft tissues. Heavy metals may enter the human body through food, water, air or
absorption through the skin when they come in contact with humans in agriculture and in the
manufacturing, pharmaceutical, industrial or residential settings. Industrial exposure
accounts for a common route of exposure for adults. Ingestion is the most common route of
exposure in children. Children may develop toxic levels from the normal hand to mouth
activity of small children who come in contact with the contaminated soil or by actually
earing objects that are not food. Less common routes of exposure are during parenteral
nutrition, from a broken thermometer, or from a suicide or attempt.

There are many minerals which are considered nutrients and are vital for the propewr
functioning of the body .These are generally splits into the macro minerals such as calcium,
16
magnesium, sodium, potassium, and zink and the trace minerals including seleniuym, iodine,
boron and molybdenum.
Equally, there are a no. of minerals that are toxic to the human body and interfere with its
functioning and undermine health. The group of most concern is known as the heavy metals
and includes mercury, cadmium, lead, copper and arsenic. The definitoion of what
constituents a heavy metal is vague and various criteria have been proposed based on density
,atomic weight or atomic no. or various chemical properties and toxicity. The term toxic
metyal have been proposed as an alternative sib\nce under many definition nutrient metals
such as zink ,copper and molybdenum actually fall under the heading of heavy metals .of
concern here is the fact that metals commonly reffered to as heavy metals or toxic metals are
determined to health for a variety of reasons and unfortunately are prevelant in the
environment due to considerable part to the activities of modern society .Individuals may be
exposed occupationally or due to factors such as consuming contaminated food and water .

1.3.1 Copper:-
Copper toxicity is a condition that that is increasingly common in this day and due to the
widespread occurance of copper in our food ,our hot water pipes ,along with the nutritional
deficiencies in zinc,manganeseand other trace minerals that keep levels of copper from
getting too high.
The use of birth control pills increases a woman’s risk of having a copper toxicity conbdition
due to estrogen’s effect of increasing copper retention in the kidney .Estrogen overt
stimulates aldosterone receptors in the kidneys ,increasing sodium ,copper and water
retention .Both estrogen and copper tend to to raise theblood pressure by increasing water
retention ,raising the blood volume .
Copper builds up first in the liver and disrupts the liver’s ability to detoxify the blood in
general .This copper toxicity in the liver therefore disrupts the liver’s ability to detoxify
excess estrogen from the blood .Copper is a very stimulating mineral to the nerves and
nervous system.Copper increases the production of epinephrine ,norepinephrine,and
dopamine while also implicated in a decreasec the histamine .These effecys on
neurotransmitter levels can give rise to manypsychological imbalances such as mood swings,
depressions, mental agitation, feeling over stimulated, restlessness, anxiety, insommia and a
racing mind with too many thoughts are all hallmarks of elevated copper toxicity.
17
Elevated copper in the body acts like caffeine or even amphetamines. It constantly keeps the
conversion of dopamine into norepinephrine going so that you have a constant adrenaline
rush to help you be on the go, but you also are unable to settle down or turn off your mind .

1.3.2 Cadmium:-
Cadmium is an extremely toxic metal commonly found in ndustrial workplaces, particularly
where any one is being processed or smelted .Several deaths from acute exposure have
occurred among welders who have unsuspectingly welded on cadmium containing alloys or
working with silver solders.

1.3.3 Mercury:-
Mercury is considered by many to be even more toxic than lead. Although mercury is poorly
absorbed from the gastrointestinal tract, mercury vapour is easily takenin through the lungs
and readily passes into the brain. Once in the body, mercury also concentrares in the nerves,
liver and esp[ecially the kidneys . Mercury is a potent cellular toxin and is known to decrease
neurotransmitter production, disrupt important processes within the nerve cells and
decreaseimportant hormones such as thyroid and testosterone.

1.3.4 Lead:-
Dangerous elements that can be harmful even in small amounts. Lead enters the human body
in many ways. It can be inhaled in dust from Lead paints, or waste gases from leaded
gasoline. It is found in trace amounts in various foods, notably fish, which are heavily
subjected to industrial pollution. Some old homes may have Lead water pipes , which can
easily contaminate drinking water. Most of the lead we take in is removed from our bodies in
urine, however, there is still risk of buildup, particularly in children. If lead buildup occurs,
health problems including damage to the nervous system, mental retardation, and even death,
can ensue.
Lead poisoning may be hard to detect at first because children who appear healthy can have
high levels of lead in their bodies. The accumulation of lead in the body usually is gradual,
building up unnoticed until levels are dangerous.
The signs and symptoms of lead poisoning in children are nonspecific and may include
irritability, loss of appetite, weight loss, sluggishness, abdominal pain, vomiting,

18
constipation, and anemia. Although children are primarily at risk, lead poisoning is also
dangerous to adults. The signs and symptoms of lead poisoning in adults may include: pain,
numbness or tingling of the extremities, muscular weakness, headache, abdominal pain,
memory loss, reproductive impairment in men, constipation and anemia.

1.4 Antibiotics:
Antibiotics are a substance or any compound that kills or inhibits the growth of bacteria.
They belong to the border group of antimicrobial compounds, used to treat infection caused
by the microorganisms including fungi and protozoa. The commonly used antibiotics are
penicillin, ampicillin, tetracycline and streptomycin.

1.4.1 Penicillin:
Penicillin is a group of antibiotics derived from penicillium fungi. The term penicillium
refers to the mixture of substance that naturally and organically produced. All penicillin are
beta-lactam antibiotics and are used in the treatment of bacterial infection caused by
susceptible, usually gram positive bacteria.

1.4.2 Ampicillin:
Ampicillin is also a beta-lactam driven group of antibiotics. Ampicillin also demonstrated
activity against gram negative bacteria. Ampicillin is a part of aminopectin family.

1.4.3 Tetracyclin:
Tetracyclin is broad spectrum polyketide antibiotic. It is a protein synthesis inhibitor.

1.4.4 Streptomycin:
It is derived from the actinobacterium streptomyces griceus . This is a bactericidal antibiotic.
The antibiotic and metal resistance in microorganisms is due to the continuous exposure of
them to the compounds. The commonly isolated gene that is responsible for the showing the
resistivity in microorganisms is pocA gene is isolated from the microbes found in the
effluent of industries. This gene has been isolated from the genomic DNA of the
microorganisms.

19
 REVIEW
OF LITERATURE

20
2.REVIEW OF LITERATURE

Metals are present in very high amount in industrial effluents ,it also contain chemical
agents which causes pollution .
The waste water from textile industries is difficult to treat (Fewson , 1988) . This is
because dyes usually have a synthetic origin and complex aromatic structures which make
them more stable and more difficult to biodegrade (Seshadri et.al .,1994).
The growing use of a variety of dyes besides pollution by dye waste water is becoming
increasingly serious (Mustafa and Hatue ., 2003) .
Currently there are approximately 30,000type of dye on the world market , more than
60,000 tons of dyes are released into the environment in industrial effluents from two major
sources ,the textile and dye stuffs industries (Keharia and Madamuar.,2003) .It is estimated
that 10 to 15% of the effluent is lost during the dyeing processing (Mustafa and Hatiu.,2003).
Isolation of bacteria from paper effluents:
Chlorophenols are major environmental pollutants discharged from paper
mills ,Tenneries, Distilleries, Dye and Paint manufacturing and pharmaceuticals industry
(Crosby.,1981).
The U.S Environmental Protection Agency regulates penta chlorophenol as a
priority pollutant and also consider 1.0mg/l of penta chlorophenol is hazardous for land and
disposal (Feeman.,1989;Sittuig.,1981).

Biochemical characterization of isolated bacterial strains:

The biochemical characterization of potential bacterial isolates was performed
as per standard protocol for biochemical tests (Barrow and Feltham,1993;Williams
etal.,1993).

Methods for Heavy metal removal from industrial effluents:

Various methods to reduce metals from the waste water stream includes physical
and chemical methods such as ion exchange, Filtration, Precipitation ,Electrochemical
treatment, Adsorption, Membrane technologies and Evaporation recovery
21
(Patterson ,1985.,Nyer ,1992.,Camargo et.al.,2003and Rama Krishna et.al.,2005.,Ahluwalia
and Goyal,2007 Sikaily et.al.,2007).
Removal of Heavy metals from paint industries:
Heavy metals are not Biodegradable and tend to accumulation in organisms and
cause numerous diseases and disorders (Cozer and Pirincci 2006).

22
 AIM
AND OBJECTIVE

23
2.1 AIM AND OBJECTIVE

The main aim of this project is to isolate different microorganisms from

various industrial effluents like paint, textile, pharma and paper industry and to identify the
microorganisms by microscopic observation and various biochemical tests, finally to check
their sensitivity towards various metal ions and perform molecular characterization.

2.1.1 OBJECTIVE

1. To decrease the environmental pollution especially heavy metal pollution.

2. To isolate the microorganisms resistant to heavy metal toxicity.
3. To check at which metal ion concentration the microorganisms are effective in their
growth.
4. To check the antibiotic sensitivity of isolated microorganisms .
5. To clone the gene responsible for resistant to toxicity in E.coli.
6. To check at which metal ion concentration the microorganisms are effective in their
growth.

24
 MATERIALS
AND
METHODOLOGY

25
3.1 Materials

3.1.1 Glasswares:
1. Beaker
2. Conical flask
3. Petriplates
4. Test tubes
5. Pipette
6. Glass rod
7. Measuring cylinder
8. Slide

3.1.2 Chemicals:
1. Agar
2. Beef extract
3. yeast extract
4. Peptone
5. Tryptone
6. NaCl
7. Dipotassium ortho phosphate
26
 8. Mannitol
9. Starch
10. Glucose
11. Phenol red
12. Agarose
13. Bromophenol blue
14. Hydrogen peroxide
15. V.P and M.R reagent

3.1.3 Instruments:
1. Laminar air flow
2. Weighing balance
3. Incubator
4. Microscope
5. Autoclave
6. Hot air oven
7. Water bath
8. Heater
9. PCR
10. Electrophoretic system

3.1.4 Others:
1. Cotton
2. Tissue paper
3. Spirit lamp
4. Rubber band
5. Test tube stand
6. Detergent
7. Match box
8. Test tube holder
9. Cylinder
10. Spreader and inoculating loop

27
3.2: METHODOLOGY
3.2.1 ISOLATION OF BACTERIA FROM INDUSTRIAL EFFLUENTS:
Effluents were collected from different industries and bacteria are isolated by
different isolation techniques on specific media .

3.2.1.1 nutrient agar media was prepared:

Steps:
1. NAM ingradients were weighed in appropriate quantities .
2. These were dissolved in approamount of water and mixed thoroughly until all the
components were dissolved completely .
3. The media was sterilized in an autoclave at 121ºc and at 15lb pressure for 15 min .
4. The autoclaved media was then poured into the petriplates and allowed to solidify .

3.2.1.2 Spread plate method was used:-

The spread plate technique is used for the separation of the dilute , mixed population
of microorganisms so that individual colonies can be isolated . In this technique
microorganisms are spread over the solidified agar medium with a sterile L-shaped glass rod
while the petridish is spun on a turntable .The theory behind this technique is that as the
petridish spuns ,at some stage ,single cells will be deposited with the bent glass rod on to the
agar surface .Some of these cells will be separated from each other by a distance sufficient to
allow the colonies to be free from each other .

3.2.1.3 Procedure:
1. 0.1ml of the effluent water was placed on nutrient agar media plates .
2. The sample water was spreaded on the plates with a sterile bent glass rod .

3. The plates were incubated at 37ºc for 24 hours.

28
3.2.2 SUBCULTURING OF BACTERIA BY STREAK PLATE METHOD:-

3.2.2.1 Principle:
The streak plate method offers a most practical method of obtaining discrete
colonies and pure cultures .It was originally developed by two bacteriologists ,Loeffer and
Gaffkey in the laboratory of Robert koch .In this method . a sterilized loop or transfer needle
is dipped into a suitable diluted suspension of organism which is then streaked on surface of
a already solidified agar plate to make a series of parallel ,non-overlaoping streaks .The aim
of this exercise is to obtain colonies of microorganisms that are pure ,i.e. growth derived
from a single cell/spore .

3.2.2.2 Procedure:
In this method ,a sterilized loop wsa taken and a loopful of inoculum was taken .The
inoculum was spreaded on one corner of the petriplate containing appropriate medium .From
this non overlapping streaks were made based on the mode of streaking such as zigzag
streaking ,radial streaking ,quadrant streaking etc.

3.3 IDENTIFICATION OF BACTERIA BY STAINING:

The isolated organisms were identified by two types of stainig procedures
i.e.Gram’s staining and Endospore staining .

3.3.1 Gram’s staining:

3.3.1.1 Principle:
It is a kind of differential staining technique first developed by the scientist
Christian Gram a Danish physician in 1884 .It helps in differentiating bacteria into two types
- Gram positive and Gram negative based on the chemical and physical differences in the
cell walls .
The bacteria which retain the primary stain and not decolorized by treatment with
95% alcohol and appear as purple or violet or gram positive and those that loose the crystal
violet and counter stain by safrinin are referred as gram negative
The gram-negative bacterial cell wall is thin complex and multilayered
structure and in addition to protein and mucopeptides. The higher amount of lipid is readily
29
dissolved by alcohol resulting in the formation of large pores in the cell wall which do not
close appreciately on dehydration of cell wall protein thus facilitating the leakage of crystal
violet iodine complex and resulting in the decolourisation of the bacterium which later takes
the counter stain and appear red in contrast gram-positive cell walls are thick and chemically
simple compounds mainly protein and cross linked mucopeptides. When treated with
alcohol, it cause dehydration and closer of cell walls pore. Therefore does not loss
crystallviollet-iodine complex and remain purple .

3.3.1.2 Procedure:
1. A clean slide was taken and wiped with alcohol.
2. One drop of distilled water was taken on the slide with the help of inoculation loop.
3. One loop full of culture was taken and was mixed with water drop taken on the slide
and thin smear was prepared.
4. The smeared was allowed to air dry and was heat fixed

3.3.1.3 Staining :

1. The heat fixed bacterial smear was covered with crystal violet for 30 second.
2. Excess stain was rinsed off with tap water for a few second until no more stain is
seen to flow off .
3. One or two drops of Gram’s iodine was added and was left for 30 second.
4. Excess stain was washed with tap water.
5. Then the smear was decolourised with 70% ethanol.
6. Then the slide was washed gently using tap water
7. The slide was counter stained with safrinine and was left for 30 to 60 second
8. The slide was then washed with tap water and was allowed to air dry
9. Smear was examined under oil immersion
10. Gram’s nature of bacteria were determined according to the colored observed under
microscope

30
3.3.2 Endospore staining:

3.3.2.1 Principle:
Endospores are produced by very few types of bacteria. These protective structures
are made through a process known as sporulation in response to extreme environmental
conditions, such as high temperatures, desiccation, chemicals, changes in pH and lack of
food.
In the dormant, inert endospore state, bacteria do not metabolize or reproduce, but
exist in a type of suspended animation, much like the seeds of plants do. When
environmental conditions again become favorable, the endospore germinates, returning the
bacterium to its normal active and reproducing metabolic state.
Normal water-based techniques, such as the Gram stain, will not stain these tough,
resistant structures. In order to stain endspores, malachite green must be forced into the spore
with heat (steam).

3.3.2.2 Procedure:
1. A clean slide was taken and wiped with alcohol .
2. One drop of distilled water was taken on the slide with the help of inoculation loop.
3. One loop full of culture was taken and was mixed with water drop taken on the slide
and thin smear was prepared.
4. The smeared was allowed to air dry and was heat fixed

3.3.2.3 Staining :

1. Place the heat-fixed bacterial slide over screened water bath and then apply the
primary stain malachite green.
2. Allow the slide to sit over the steaming water bath for 5 minutes, reapplying stain if it
begins to dry out.
3. Remove the slide from the water bath and rinse the slide with water until water runs
clear.
4. Flood slide with the counter stain safranin for 20 seconds and then rinse.
5. View specimen under oil immersion (magnification of 1000xTM) with a light
microscope.
31
 6. After this staining procedure, the endospores will appear green, having retained the
primary stain, malachite green. The vegetative cells (bacteria are in the active,
metabolizing state) will appear pink, having retained the counter stain, safranin.

3.4 BIOCHEMICAL TESTS FOR IDENTIFICATION OF BACTERIA:-

The sum of all the chemical reaction that occur within all living
organisms is termed as metabolism, the major new concequences of these reaction in
microorganisms is the suynthesis of a new cell.Metabolic reactions that release energy(ATP)
from the breakdown or degradation of a substrate are catabolic reaction, while the one that
use energy to assemble smaller molecules and produce biosynthetic building blocks are
called anabolic reaction, and when there is production of ATP and precursors of biosynthetic
building blocks are called amphibolic reaction. All these biochemical reactions that occur
both outside and inside the cell are precisely controlled by some governing factors, the
enzymes. An enzyme is a biological catalyst, a substance that accelerate the rate of specific
chemical reaction. The enzyme is either exoenzyme(extracellular) or endoenzymes (intra
cellular).
Exoenzymes, which are a few in numbers, are released from the cell and act on the substrate.
The cell utilizes endoenzymes for further metabolic degradation of carbohydrates and are
mainly responsible for synthesis of new protoplasmic requirements and production of
cellular energy from assimilated material.To identify the spcies of bacteria, following
biochemical tests were done.

3.4.1 Catalase test:

3.4.1.1 Principle
During aerobic respiration in the presence of oxygen microbes produce hydrogen
peroxide which is lethal to the cell.The enzyme catalase present in some microbes
breakdown hydrogen peroxide to water and o2.
Reaction= 2H2O2→2H2O+O2
Release of free O2 gas bubbles is a positive catalase test
No gas bubbles means negative catalase test

32
3.4.1.2 Procedure:
1. A clean glass slide was taken and wiped with alcohol
2. One drop of H2O2 was taken and a loopfull of culture was mixed with the H 2O2 inside
the laminar flow chamber.
3. Then the slides was observed for gas bubbles

3.5 METAL TOXICITY:

3.5.1 Principle:
The effect of metals are both stimulatory and inhibitory on the microorganisms. For example
iron is essential for growth of microorganisms. Althoughj while some ions (mercury silver)
are toxic in small conc. Certain metals (copper) are inhibitor at moderate conc., as the conc,
of metal ions decreases growth starts and at a particular conc.normal growth occurs. The
ability of certain metals to exert a lethal action on the bacteria is termed as the oligodynamic
effect.
A conc. Gradient of the metal ions is produced if a metal solution is placed broth. Depending
on the conc. Of metal ion and the ability of microorganisms grow at that particular conc. The
growth of the culture can be obtained.

3.5.2 Procedure:
1. NAM broth and 10ml of each metal solution having 1M concentration were prepared.
2. From the 1M metal solution 4 different conc.(10mM, 20mM, 100mM and 200mM)
of solution were prepared for each metal solution.
3. 0.1ml of suspension culture of each sample were added to 4 metal solution.
4. The test tubes were incubated for 24hrs and 48 hrs.
5. O.D was taken at 24hr and 48hr for growth analysis.

33
3.6 ANTIBIOTIC SENSITIVITY TEST:

3.6.1 Principle:
Antibiotics are the substances or any compound that kills or inhibits the growth
of bacteria .They belong to the border group of any microbial compounds ,used to treat
infection caused by the microorganisms including fungi and protozoa .
Antibiotic sensitivity is a term used to describe the susceptibility of bacteria to
antibiotics ,Antibiotic susceptibility test (AST) is usually carried out to determine which
antibiotic will be most succesful in treating a bacterial infection in vivo .Testing for
antibiotic sensitivity is often done by the Kirby-Bauer method .Small wafers containing
antibiotics are placed on to a plate upon which bacteria are growing .If the bacteria are
sensitive to the antibiotic ,a clean ring ,or zone of inhibition is seen around the wafer
indicating poor growth .Other methods to test antimicrobial susceptibility include the strokes
method ,E-test (also based on antibiotic diffusion ) .A microorganism is judged sensitive or
resistant accordingly to the diameter of the zone of inhibition .Some antibiotics actually kill
the bacteria (bactericidal) ,where as others merely prevent the bacteria from multiplying
(bacteriostatic)so that the host’s immune system can overcome them .

3.6.2 Procedure:
1. NAM was prepared as per the composition and sterilized by autoclaving .
2. The media was poured on to the petriplates and allowed to solidify .
3. 100µl of the suspension was then spreaded on the solidified agar plates .
4. The paper strips were then dipped in the antibiotic suspension and placed on the
spreaded plates .
5. The plates were then incubated at 37ºC for 24-48 hours to view the zone of inhibition
.

34
 RESULT
AND
OBSERVATION

35
4.OBSERVATION AND RESULTS
4.1 Gram staining:
The purified bacteria were identified microscopically by gram staining and by endospore
staining.
Observation: The colonies obtained from pure culture were identified as Gram positive
bacteria having rod or cocci shape.

TABLE-1: IDENTIFICATION OF ORGANISM BY GRAM STAINING:

Colony no. Colour Shape/surface Edge Gram Endospore
staining staining
S1 Off white Watery Circular Positive Positive
S2 Off white Watery Circular Positive Negative
P1 Watery Watery Circular Positive Positive
white
T1 Off white Watery Endulate Positive Negative
T2 Off white Watery Lobule Positive Positive

4.2 EFFECT OF METAL IONS ON DIFFERENT MICROORGANISMS:

The effect of metals are both stimulatory and inhibitory on the microorganisms.

READINGS FOR METAL TOXIITY GROWTH ANALYSIS USING “PB”

TABLE-4.2.1: METAL TOXICITY ASSAY OF S1
Culture broath Negative 5 mm 10 20 40mm Positive

36
 Control mm mm control
Inhibition
time
0 hour 1.11 .91 1.20 1.18 1.64 .05
24 hours 1.79 1.62 1.55 1.31 1.56 .56
48 hours 1.82 1.69 1.56 1.18 1.35 .56
72 hours 1.83 1.70 1.61 1.02 1.16 .71
96 hours 1.82 1.60 1.53 1.04 1.18 .67
TABLE-4.2.2: METAL TOXICITY ASSAY OF S2
Culture broath Negative 5 mm 10 mm 20 mm 40 mm Positive
control control
Inhibition
Time
0 hour .81 1.00 1.24 1.29 1.62 .12
24 hours 1.38 1.54 1.76 1.11 1.62 .38
48 hours 1.46 1.60 1.82 1.09 1.39 .79
72 hours 1.47 1.62 1.82 1.15 1.22 .96
96 hours 1.47 1.61 1.76 1.15 1.21 .96

TABLE-4.2.3: METAL TOXICITY ASSAY OF P1

Culture broath Negative 5 mm 10 mm 20 mm 40 mm Positive
control control
Incubation
Time
0 hour 1.32 1.39 1.43 1.45 1.68 .24
24 hours 1.33 1.37 1.35 1.08 1.34 .39
48 hours 1.31 1.32 1.25 1.11 1.31 .94
72 hours 1.32 1.26 1.25 1.15 1.28 1.20
37
96 hours 1.38 1.32 1.28 1.14 1.20 1.45

TABLE-4.2.4: METAL TOXICITY ASSAY OF T1

Culture broath Negative 5 mm 10 mm 20 mm 40 mm Positive
control control
Incubation
Time
0 hour 1.32 1.41 1.30 1.49 1.75 .30
24 hours 1.54 1.51 1.65 1.69 1.35 .72
48 hours 1.57 1.58 1.74 1.66 1.24 1.04
72 hours 1.60 1.60 1.74 1.53 1.15 1.24
96 hours 1.60 1.64 1.70 1.35 1.16 1.35

TABLE-4.2.5: METAL TOXICITY ASSAY OF T2

Culture broath Negative 5 mm 10 mm 20 mm 40 mm Positive
control control
Incubation
Time
0 hour 1.37 1.32 1.36 1.39 1.70 .22
24 hours 1.53 1.41 1.47 1.21 1.50 .71
48 hours 1.60 1.46 1.47 1.12 1.29 1,04
72 hours 1.58 1.52 1.40 1.07 1.06 1.20
96 hours 1.62 1.55 1.37 1.08 1.10 1.33

38
 4.3 GRAPHS FOR METAL TOXICITY TEST

FIGURE- 4.3.1: METAL TOXICITY ASSAY OF S1

FIGURE-4.3.2: METAL TOXICITY ASSAY OF S2

39
FIGURE-4.3.3: METAL TOXICITY ASSAY OF P1

FIRUGE-4.3.4: METAL TOXICITY ASSAY OF T1

40
FIGURE-4.3.5: METAL TOXICITY ASSAY OF T2

41
4.4 ANTIBIOTIC SENSITIVITY TEST
BACTERIAL ZONE OF INHIBITION IN MM
STRAIN A E N S
T1 33MM NO ZONE NO ZONE 12 MM
T2 40 MM 11MM NO ZONE 27 MM
S1 15 MM 15 MM NO ZONE 10 MM
S2 32 MM NO ZONE NO ZONE 24 MM
P1 22 MM 23 MM 16 MM NO ZONE

5. RESULTS
5.1: METAL TOXICITY TEST
As we can see that bacteria show some changes in their growth when they are applied to
heavy metals (lead) .

5.2: ANTIBIOTIC SENSITIVITY TEST

42
 Here we can see that when antibiotics are applied to bacterial strain, some bacteria show
no zone and some show zone.

43
 PHOTOPLATES

6.0: ANTIBIOTIC TEST

PHOTO-6.1: Antibiotic assey of S1

44
PHOTO-6.2: Antibiotic assey of S2

PHOTO-6.3: Antibiotic assey of P1

45
PHOTO-6.4: Antibiotic assey of T1

PHOTO-6.5: Antibiotic assey of T2

46
DISCUSSION

47
 DISCUSSION
Industrialisation is vital to a Nation’s economy because it serves as a vehicle
for development. Moreover, there are associated problems resulting from the introduction of
industrial waste products into the environment. Many of these products are problematic
because of persistence low biodegradability and toxicity. Implementation of cleaner
production processes and pollution prevention measures can yield both economic and
environmental benefits.
In this era of industrial development the industries are developed day by day and the
pollution has also increased .If we take advantage from this pollution this is very beneficial
for us.
A major environmental concern due to dispersal of industrial and urban wastes generated by
human activities is the contamination of soil. A wide range of inorganic and organic
compounds cause contamination. Major components of inorganic contaminates are heavy
metals they present a different problem than organic contaminants.
Many microorganisms have evolved an extensive suite of adaptive responses to heavy metal
toxicity. These include the immobilization, exclusion, chelation and compartmentalization of
metal ions and often involve metal binding ligands. A number of such ligands have been
identified in plants and include organic acids, amino acids, peptides and polypeptides.

48
The industrial effluents contain hazardous chemicals and several contaminants.This project
is based on microbes present in industrial effluents and studied those genes which are
responsible for their resistivity against metals .
The significance of this project is :-
1. To manipulate the microorganisms present in industries for our benefit by isolating
them from different industrial effluents.
2. Several microbiologists found new species of microorganisms by industrial effluents
because they are slowly adapted to highly contaminated environment .Most microbes
of industrial effluents produce organic acids, gas, reduce nitrate and break organic
acid into ethanol and actosin .They are endospore forming both gram positive and
gram negative bacteria are present ,they are commonly pathogenic .
3. For molecular analysis of metals resistance and antibiotic resistant microorganisms .
4. We use the degradative quality microorganisms present in industrial effluents
because we have microbiological molecular information of these organisms .
5. If organisms have metal resistance capacity its metal resistance gene used for those
bacteria which is used in biological treatment of waste water.

The pollution control Institute used several techniques to identify and inhibit the
growth of microorganisms , manipulate them for our benefits present in industrial
effluents ,there are several pollution control institute which control and regularly monitor
the pollution .The environment institutes are affiliated by government NEERI ,AP
pollution control board ,CECB (IN Chattisghar) , they are funded by government and
private sector .
In order to check the compatibility of microorganisms to degrade the toxic metals,
antibiotics and to use them as their nutrient source 8 microorganisms have been isolated
from different industries. They have been identified on the basis of the various
biochemical test performed and by observing their cultural characteristics.
The organism isolated in this project showed resistance to . These organisms are
susceptible The organisms isolated in this project is checked with 4 metals such as
copper, mercury, zinc, cobalt. Among all organisms Staphyllococcus epidermidis showed
high resistance against copper at 100 mM concentration.

49
The genomic DNA was isolated from Staphyllococcus epidermidis, Micrococcus
luteus,Staphyllococcus saprophyticus and Bacillus cereus and PCR amplification was
done for these four genomic DNA.
Now the DNA was restricted with EcoR1and then ligated with PUC18 plasmid
This was cloned in E.coli and the culture is checked for blue white screening .
The colonies were isolated and grown in media and checked for copper toxicity.
The transformed colonies showed high resistance against copper metal solution. .

SUMMARY
AND
CONCLUSION 50
 CONCLUSION
According to our findings, both heavy metals separately and combining

with reduced bacterial growth significantly considering to control depended on dose and

time. When compared the effects of heavy metal alone and combinations with on bacterial

number considering.

Although soil bacteria was more inhibited, when heavy metals doses were applied separately,

heavy metals doses combined and bacterial growth was inhibited.

Comparisons of whole treatments revealed that total bacterial growth was more inhibited,

This incubation experiment suggested that enhance the metal toxicity to bacteria in soils.

Considering the low solubility of and its strong adsorption to organic material, it seems that

deleterious effects of heavy metals enhanced by the presence of in contaminated soils with

heavy metals such as pb. This is also supported by Gogolev and Wilke.

51
stated that hydrophobic pollutants such interact with lipophilic compounds of cytoplasmatic

membranes of microorganisms. This could cause changes in the membrane structure and

might alter the permeability of the membranes. Therefore, we assume that may enhance the

toxicity of the metals because they can penetrate into the perforated microbial cells more

easily in soil.

BIBLIOGRAPHY

52
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55
ABSTRACT

56
 ABSTRACT
BY
Abhishek Banjare
Now a days environmental pollution has become major threat to
environment. Especially heavy metal pollution causing major damage to the existing
environment. Heavy metal pollution can cause many health hazards to humans and plants.
One of the cause of this pollution is industrial effluents. Many industries like textile, paint,
pharma, paper etc. contain heavy meytals in their effluents in toxic form. Some
microorganisms are capable of releasing those heavy metals from binding form. To isolate
those microorganisms from industrial effluents and manipulate those organisms in beneficial
nature.
In this project, isolation and identification of different bacteria from various
industrial effluents were done to check their metal toxicity against several metal ions .
Isolated bacteria were identified following standard cultural and biochemical tests . To
examine the metal ion toxicity , selected isolates were subjected to metal toxicity test. A
total of four samples from different industrial effluents paint , paper , textile and pharma
industries were analysed to detect the presence of microorganisms and all the samples were
found to be positive for the presence of microorganisms which were further confirmed by
various biochemical tests .
From the industrial samples obtained from effluents from textile industry
Staphyllococcus aureus and Staphyllococcus epidermidis and from paint industry
Micrococcus luteus and Staphyllococcus epidermidis and from pharma industry
Staphyllococcus aureus and Staphyllococcus saprophyticus and from paper industry Bacillus
cereus and Corynbacterium xerosis were isolated and identified . These microorganisms
were tested for metal ion toxicity at different concentrations and these organisms were
subjected to molecular characterization .
57
APPENDIX

58
APPENDIX
MICROBIOLOGICAL MEDIA
NAM media composition (pH 6.9)
 Peptone 5.0g
 Beef extract 3.0g
 Agar 18.0g
 NaCl 5.0g
 Distilled water 1000ml
Starch hydrolysis
 Peptone 5.0g
 Beef extract 3.0g
 NaCl 5.0g
 Agar - 18g
 Starch - 1%

59