![INTRODUCTION:
Our biosphere is under constant threat from continuing environmental pollution. Impact on its
atmosphere, hydrosphere and lithosphere by anthropogenic activities cannot be ignored. Man
made activities on water by domestic, industrial, agriculture, shipping, radio-active,
aquaculture wastes; on air by industrial pollutants, mobile combustion, burning of fuels,
agricultural activities, ionization radiation, cosmic radiation, suspended particulate matter;
and on land by domestic wastes, industrial waste, agricultural chemicals and fertilizers, acid
rain, animal waste have negative influence over biotic and abiotic components on different
natural ecosystems. Some of the recent environmental issues include green house effect, loss
in bio-diversity, rising of sea level, abnormal climatic change and ozone layer depletion etc.
In recent years, different approaches have been discussed to tackle man made environmental
hazards. Clean technology, eco-mark and green chemistry are some of the most highlighted
practices in preventing and or reducing the adverse effect on our surroundings.
Among many engineering disciplines – Civil Engineering, Mechanical Engineering,
Electrical Engineering etc., Textile Engineering has a direct connection with environmental
aspects to be explicitly and abundantly considered. The main reason is that the textile
industry plays an important role in the economy of the country like India and it accounts for
around one third of total export. Out of various activities in textile industry, chemical
processing contributes about 70% of pollution. It is well known that cotton mills consume
large volume of water for various processes such as sizing, desizing, scouring, bleaching,
mercerization, dyeing, printing, finishing and ultimately washing. Due to the nature of
various chemical processing of textiles, large volumes of waste water with numerous
pollutants are discharged. Since these streams of water affect the aquatic eco-system in
number of ways such as depleting the dissolved oxygen content or settlement of suspended
substances in anaerobic condition, a special attention needs to be paid. Thus a study on
different measures which can be adopted to treat the waste water discharged from textile
chemical processing industries to protect and safeguard our surroundings from possible
pollution problem has been the focus point of many recent investigations.[5]
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![Agricultural waste:
Pesticides are widely used by farmers to control plant pests and enhance production, but
chemical pesticides can also cause water quality problems. Farms with
large livestock and poultry operations, such as factory farms, can be a major source of point
source wastewater.[25]
Hazards:
Water pollution has number of effects. The effects could be classified as:
1] Effects on ecosystem
2] Effects on animal health
3] Effects on human health
Virtually all types of water pollution are harmful to the health of humans and animals. Water
pollution may not damage our health immediately but can be harmful after long term
exposure. Different forms of pollutants affect the health of animals in different ways: Heavy
metals from industrial processes can accumulate in nearby lakes and rivers. These are toxic to
marine life such as fish and shellfish, and subsequently to the humans who eat them. Heavy
metals can slow development; result in birth defects and some are carcinogenic. Industrial
waste often contains many toxic compounds that damage the health of aquatic animals and
those who eat them. Some of the toxins in industrial waste may only have a mild effect
whereas other can be fatal. They can cause immune suppression, reproductive failure or acute
poisoning. Microbial pollutants from sewage often result in infectious diseases that infect
aquatic life and terrestrial life through drinking water. Microbial water pollution is a major
problem in the developing world, with diseases such as cholera and typhoid fever being the
primary cause of infant mortality. Organic matter and nutrients causes an increase in aerobic
algae and depletes oxygen from the water column. This causes the suffocation of fish and
other aquatic organisms. Sulfate particles from acid rain can cause harm the health of marine
life in the rivers and lakes it contaminates, and can result in mortality. Suspended particles in
freshwater reduces the quality of drinking water for humans and the aquatic environment for
marine life. Suspended particles can often reduce the amount of sunlight penetrating the
water, disrupting the growth of photosynthetic plants and micro-organisms. Some
organochlorine pesticides (like DDT, BHC, Endrin) are known for bioaccumulative and
biomagnifiable characters. [21]
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![Bioremediation:
By definition, bioremediation is the use of living organisms, primarily microorganisms, to
degrade the environmental contaminants into less toxic forms. It uses naturally occurring
bacteria and fungi or plants to degrade or detoxify substances hazardous to human health
and/or the environment. The microorganisms may be indigenous to a contaminated area or
they may be isolated from elsewhere and brought to the contaminated site. Contaminant
compounds are transformed by living organisms through reactions that take place as a part of
their metabolic processes. Biodegradation of a compound is often a result of the actions of
multiple organisms.[10]
Types of Bioremediation:
Biostimulation
Bioaugmentation
Intrinsic Bioremediation
Advantages of bioremediation
Bioremediation is a natural process and is therefore perceived by the public as an
acceptable waste treatment process for contaminated material such as soil. Microbes able
to degrade the contaminant increase in numbers when the contaminant is present; when
the contaminant is degraded, the biodegradative population declines.
Theoretically, bioremediation is useful for the complete destruction of a wide variety of
contaminants. Many compounds that are legally considered to be hazardous can be
transformed to harmless products. This eliminates the chance of future liability associated
with treatment and disposal of contaminated material.
Instead of transferring contaminants from one environmental medium to another, for
example, from land to water or air, the complete destruction of target pollutants is
possible.
Bioremediation can often be carried out on site, often without causing a major disruption
of normal activities. This also eliminates the need to transport quantities of waste off site
and the potential threats to human health and the environment that can arise during
transportation. Bioremediation can prove less expensive than other technologies that are
used for clean-up of hazardous waste.[10]
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![LITERATURE REVIEW:
Textile Effluent:
Sources and Causes of Generation of Textile Effluent:
Textile industry involves wide range of raw materials, machineries and processes to engineer
the required shape and properties of the final product. Waste stream generated in this industry
is essentially based on water-based effluent generated in the various activities of wet
processing of textiles. The main cause of generation of this effluent is the use of huge volume
of water either in the actual chemical processing or during re-processing in preparatory,
dyeing, printing and finishing. In fact, in a practical estimate, it has been found that 45%
material in preparatory processing, 33% in dyeing and 22% are re-processed in finishing. The
fact is that the effluent generated in different steps is well beyond the standard and thus it is
highly polluted and dangerous.[5]
Textile wastewater includes a large variety of dyes and chemicals additions that make the
environmental challenge for textile industry not only as liquid waste but also in its chemical
composition (Venceslau et al., 1994). Main pollution in textile wastewater came from dyeing
and finishing processes. These processes require the input of a wide range of chemicals and
dyestuffs, which generally are organic compounds of complex structure. Because all of them
are not contained in the final product, became waste and caused disposal problems. Major
pollutants in textile wastewaters are high suspended solids, chemical oxygen demand, heat,
colour, acidity, and other soluble substances. The removal of colour from textile industry and
dyestuff manufacturing industry wastewaters represents a major environmental concern. In
addition, only
47% of 87 of dyestuff are biodegradable (Pagga and Brown, 1986). It has been documented
that
residual colour is usually due to insoluble dyes which have low biodegradability as reactive
blue
21, direct blue 80 and vat violet with COD/BOD ratio of 59.0, 17.7, and 10.8, respectively.[32]
The textile sector has a high water demand. Its biggest impact on the environment is related
to primary water consumption (80–100 m3/ton of finished textile) and waste water discharge
(115– 175 kg of COD/ton of finished textile, a large range of organic chemicals, low
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![biodegradability, colour, salinity). Therefore, reuse of the effluents represents an economical
and ecological challenge for the overall sector (Li Rosi et al., 2007).[6]
Table 1. Properties of Waste Water from Textile Chemical Processing:
[5]
Table 2. Composite textile industry wastewater characteristics:
[2]
Categorization of Waste Generated in Textile Industry:
Textile waste is broadly classified into four categories, each of having characteristics that
demand different pollution prevention and treatment approaches. Such categories are
discussed in the following sections:
1. Hard to Treat Wastes
This category of waste includes those that are persistent, resist treatment, or interfere with the
operation of waste treatment facilities. Non-biodegradable organic or inorganic materials are
the chief sources of wastes, which contain colour, metals, phenols, certain surfactants, toxic
organic compounds, pesticides and phosphates.
The chief sources are:
Colour & metal-dyeing operation
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![ Phosphates-
Non-biodegradable organic materials-
Since these types of textile wastes are difficult to treat, the identification and elimination of
their sources are the best possible ways to tackle the problem. Some of the methods of
prevention are chemical or process substitution, process control and optimization,
recycle/reuse and better work practices
2. Hazardous or Toxic Wastes
These wastes are a subgroup of hard to treat wastes. But, owing to their substantial impact on
the
environment, they are treated as a separate class. In textiles, hazardous or toxic wastes
include metals, chlorinated solvents, non-biodegradable or volatile organic materials. Some
of these materials often are used for non-process applications such as machine cleaning.
3. High Volume Wastes
Large volume of wastes is sometimes a problem for the textile processing units. Most
common large volume wastes include:
• High volume of waste water
• Wash water from preparation and continuous dyeing processes and alkaline wastes from
preparatory processes.
• Batch dye waste containing large amounts of salt, acid or alkali
These wastes sometimes can be reduced by recycle or reuse as well as by process and
equipment modification.
4. Dispersible Wastes:
The following operations in textile industry generate highly dispersible waste:
• Waste stream from continuous operation (e.g. preparatory, dyeing, printing and finishing)
• Print paste (printing screen, squeeze and drum cleaning)
• Lint (preparatory, dyeing and washing operations)
• Foam from coating operations
• Solvents from machine cleaning
• Still bottoms from solvent recovery (dry cleaning operation)
• Batch dumps of unused processing (finishing mixes) [5]
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![Diagram 1: Cotton Fabric Production & Associated Water Pollutants:
[18]
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![Table 3.Effluent Characteristics From Textile Industry:
[18]
Heavy metals and its hazards:
Metal pollution is a global concern. Input of chemical substances constitutes a great
pollution burden on natural ecosystems. When considering their impact, it is necessary to
distinguish between poisonous substances, that have a toxic effect on organisms even at low
concentrations, and burdening substances that bring about undesirable changes in eco systems
at higher concentration or load. The level of metals in all environments including air, water
and soil are increasing in some cases to toxic levels with contribution from a wide variety of
industrial sources.
Heavy metals are roughly defined as elements having a density over 6g/cm3. Among
these elements, Co, Cu, Mn, Ni, Se and Zn are important in small amounts. some metals such
as Cu,Fe and Zn are essential at low concentrations and are toxic at high levels. The main
cause of water pollution are household waste (As, Cr, Cu, Mn and Ni), coal fed power
stations (As, Hg and Se), iron and steel production (Cr, Mo, Zn and Sb), and metal smelters (
Cd, Ni, Sb and Se). Of these metals, 25% are thought to enter rivers and lakes, and their
surrounding soils are also heavily pollulated by these metals.
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![Table 4. Substances present in industrial effluents:
[23]
Metals in the environment can be divided into two classes, bioavailable (soluble, nonsorbed
and mobile) and non-bioavailable (precipitated, complexed, sorbed and non-mobile). It is the
bioavailable metal concentration that is taken up and is thus toxic to biological systems.
Some metals like Hg and Pb are highly toxic but many other metals are also of concern,
including As, Br, B, Cd, Cr, Cu, Ni, Mn, Se, Ti and Zn. Elevated metal concentration in the
environment has a wide ranging on animals, plants and microbial species. Toxicity to an
organism can be defined as the inherent potential or capacity of a material to cause adverse
on living organisms and depends on the bioavailability of the toxicant.
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![In textile industry wet processing, a large number of chemicals are used, such as caustic
soda, hydrogen peroxide, formic acid, sodium nitrate, azo dyes, direct dyes, reactive dyes,
and mordants. Correia et al. (1994), reported that problems associated with textile processing
effluents are heavy metal ions, which might rise from metal used in the dyeing process, or, in
considerably higher amounts from metal containing dyes. For cotton, azoic, direct azo dye
and mordant are used, which contains metals in complexed form. Azo and direct azo dye
contains sodium salts, fixing agent and metallic compounds of Cu and Cr, whereas, the
mordants are the metallic salts of Al, Cr and Fe, which are used on cotton silk fibre.
Commonly used metal containing pigments are chrome yellow (Cr), zinc yellow (Zn),
chrome oxide green (Cr) and iron blue pigment (Fe) in textile industries.[14]
It can be seen that of all of the heavy metals chromium is the most widely used and
discharged to the environment from different sources. However, it is not the metal that is
most dangerous to living organisms. Much more toxic are cadmium, lead and mercury. These
have a tremendous affinity for sulphur and disrupt enzyme function by forming bonds with
sulphur groups in enzymes. Protein carboxylic acid (-CO2H) and amino (-NH2) groups are
also chemically bound by heavy metals. Cadmium, copper, lead and mercury ions bind to cell
membranes, hindering transport processes through the cell wall. Heavy metals may also
precipitate phosphate bio-compounds or catalyse their decomposition.
Mercury:
Because of its toxicity, its mobilisation as methylated forms by anaerobic bacteria, and
other pollution factors, mercury generates a great deal of concern as a heavy-metal pollutant.
Mercury is found as a trace component of many minerals, with continental rocks containing
an average of around 80 ppb, or slightly less, of this element. Cinnabar, red mercuric
sulphide, is the chief commercial mercury ore. Metallic mercury is used as an electrode in the
electrolytic generation of chlorine gas, in laboratory vacuum apparatuses and in other
applications. Organic mercury compounds used to be widely applied as pesticides,
particularly fungicides. Mercury enters the environment from a large number of
miscellaneous sources related to human use of the element. These include discarded
laboratory chemicals, batteries, broken thermometers, lawn fungicides, amalgam tooth
fillings and pharmaceutical products. Sewage effluent sometimes contains up to 10 times the
level of mercury found in typical natural waters. The toxicity of mercury was tragically
illustrated in the Minamata Bay area of Japan during the period of 1953-1960. A total of 111
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![cases of mercury poisoning and 43 deaths were reported among people who had consumed
seafood from the contaminated bay. Among the toxicological effects of mercury were
neurological damage, including irritability, paralysis, blindness, insanity, chromosome
breakage and birth defects. The milder symptoms of mercury poisoning, such as depression
and irritability, have a psychopathological character and may escape detection. The
unexpectedly high concentrations of mercury found in water and in fish tissues result from
the formation of soluble monomethylmercury ion, CH3Hg+, and volatile dimethylmercury,
(CH3)2Hg, by anaerobic bacteria in sediments. Mercury from these compounds becomes
concentrated in fish lipid (fat) tissue and the concentration factor from water to fish may
exceed 103.[23]
Zinc:
Zinc is a trace element that is essential for human health. When people absorb too little zinc
they can experience a loss of appetite, decreased sense of taste and smell, slow wound
healing and skinsores. Zinc-shortages can even cause birth defects. Although humans can
handle proportionally large concentrations of zinc, too much zinc can still cause eminent
health problems, such as stomach cramps, skin irritations, vomiting, nausea and anaemia.
Very high levels of zinc can damage the pancreas and disturb the protein metabolism, and
cause arteriosclerosis. Extensive exposure to zinc chloride can cause respiratory disorders.
In the work place environment zinc contagion can lead to a flu-like condition known as metal
fever. This condition will pass after two days and is caused by over sensitivity.
Zinc can be a danger to unborn and newborn children. When their mothers have absorbed
large concentrations of zinc the children may be exposed to it through blood or milk of their
mothers.[33]
Manganese:
Manganese effects occur mainly in the respiratory tract and in the brains. Symptoms of
manganese poisoning are hallucinations, forgetfulness and nerve damage. Manganese can
also cause Parkinson, lung embolism and bronchitis. When men are exposed to manganese
for a longer period of time they may become impotent. A syndrome that is caused by
manganese has symptoms such as schizophrenia, dullness, weak muscles, headaches and
insomnia.
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![Manganese compounds exist naturally in the environment as solids in the soils and small
particles in the water. Manganese particles in air are present in dust particles. These usually
settle to earth within a few days. Humans enhance manganese concentrations in the air by
industrial activities and through burning fossil fuels. Manganese that derives from human
sources can also enter surface water, groundwater and sewage water. Through the application
of manganese pesticides, manganese will enter soils.
For animals manganese is an essential component of over thirty-six enzymes that are used for
the carbohydrate, protein and fat metabolism. With animals that eat too little manganese
interference of normal growth, bone formation and reproduction will occur. For some animals
the lethal dose is quite low, which means they have little chance to survive even smaller
doses of manganese when these exceed the essential dose. Manganese substances can cause
lung, liver and vascular disturbances, declines in blood pressure, failure in development of
animal foetuses and brain damage.When manganese uptake takes place through the skin it
can cause tremors and coordination failures. Finally, laboratory tests with test animals have
shown that severe manganese poisoning should even be able to cause tumor development
with animals.[34]
Chromium:
Chromium(VI) is known to cause various health effects. When it is a compound in leather
products, it can cause allergic reactions, such as skin rash. After breathing it in chromium(VI)
can cause nose irritations and nosebleeds.
Other health problems that are caused by chromium(VI) are:
- Skin rashes
- Upset stomachs and ulcers
- Respiratory problems
- Weakened immune systems
- Kidney and liver damage
- Alteration of genetic material
- Lung cancer
- Death
The health hazards associated with exposure to chromium are dependent on its oxidation
state. The metal form (chromium as it exists in this product) is of low toxicity. The
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![hexavalent form is toxic. Adverse effects of the hexavalent form on the skin may include
ulcerations, dermatitis, and allergic skin reactions. Inhalation of hexavalent chromium
compounds can result in ulceration and perforation of the mucous membranes of the nasal
septum, irritation of the pharynx and larynx, asthmatic bronchitis, bronchospasms and edema.
Respiratory symptoms may include coughing and wheezing, shortness of breath, and nasal
itch.[35]
Aluminium :
The water-soluble form of aluminum causes the harmful effects, these particles are called
ions. They are usually found in a solution of aluminum in combination with other ions, for
instance as aluminum chlorine. The uptake of aluminum can take place through food, through
breathing and by skin contact. Long lasting uptakes of significant concentrations of
aluminum can lead to serious health effects, such as:
- Damage to the central nervous system
- Dementia
- Loss of memory
- Listlessness
- Severe trembling
The concentrations of aluminum appear to be highest in acidified lakes. In these lakes the
number of fish and amphibians is declining due to reactions of aluminum ions with proteins
in the gills of fish and the embryo's of frogs. High aluminum concentrations do not only
cause effects upon fish, but also upon birds and other animals that consume contaminated fish
and insects and upon animals that breathe in aluminum through air. The consequences for
birds that consume contaminated fish are eggshell thinning and chicks with low birth-
weights. The consequences for animals that breathe in aluminum through air may be lung
problems, weight loss and a decline in activity. Another negative environmental effect of
aluminum is that its ions can react with phosphates, which causes phosphates to be less
available to water organisms. High concentrations of aluminum may not only be found in
acidified lakes and air, but also in the groundwater of acidified soils. There are strong
indications that aluminum can damage the roots of trees when its located in groundwater.[36]
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![Lead:
Inorganic lead arising from a number of industrial and mining sources occurs in water
in the +2 oxidation state. Lead from leaded gasoline used to be a major source of atmospheric
and terrestrial lead, much of which eventually enters natural water systems. Despite greatly
increased total use of lead by industry, evidence from hair samples and other sources
indicates that body burdens of this toxic metal have decreased during recent decades. This
may be the result of less lead used in plumbing and other products that commonly come in
contact with food or drink. Acute lead poisoning in humans causes severe dysfunction in the
kidneys, reproductive system, liver, and the brain and nervous system. [23]
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![Table 5. Heavy Metals Found in Major Industries
[23]
Table 6. Comparative strengths of wastewater from industry:
[23]
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![microbial biomass, including fungal mycelium, can also adsorb metal ions, often in larger
quantities than living biomass.
Biodegradation is the microbially mediated decomposition of not only organic but also
xenobiotic pollutants. Biodegradation can be a minor change in the molecule, fragmentation
or complete mineraization. A range of current approaches to biodegradation are available,
starting from identification of microbial strains that have the ability to break down or alter
environmental pollutants such as petroleum products, polychlorinated biphenyls, halogenated
aromatic compounds,toluene, and nitrogen containing heterocyclic aromatic analogues of
polycyclic aromatic hydrocarbons to screening for these isolates for degradation abilities by
providing target substances as carbon source or electron acceptor. [14]
FACTORS OF BIOREMEDIATION:
The control and optimization of bioremediation processes is a complex system of many
factors. These factors include: the existence of a microbial population capable of degrading
the pollutants; the availability of contaminants to the microbial population; the environment
factors (type of soil, temperature, pH, the presence of oxygen or other electron acceptors, and
nutrients).
MICROBIAL POPULATIONS FOR BIOREMEDIATION PROCESSES
Microorganisms can be isolated from almost any environmental conditions. Microbes will
adapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in water,
with an excess of oxygen, and in anaerobic conditions, with the presence of hazardous
compounds or on any waste stream. The main requirements are an energy source and a
carbon source. Because of the adaptability of microbes and other biological systems, these
can be used to degrade or remediate environmental hazards. We can subdivide these
microorganisms into the following groups:
Aerobic.: In the presence of oxygen. Examples of aerobic bacteria recognized for their
degradative abilities are Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and
Mycobacterium. These microbes have often been reported to degrade pesticides and
hydrocarbons, both alkanes and polyaromatic compounds. Many of these bacteria use the
contaminant as the sole source of carbon and energy.
Anaerobic.: In the absence of oxygen. Anaerobic bacteria are not as frequently used as
aerobic
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![bacteria. There is an increasing interest in anaerobic bacteria used for bioremediation of
polychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solvent
trichloroethylene (TCE), and chloroform.
Ligninolytic fungi.: Fungi such as the white rot fungus Phanaerochaete chrysosporium have
the
ability to degrade an extremely diverse range of persistent or toxic environmental pollutants.
Common substrates used include straw, saw dust, or corn cobs.
Methylotrophs.: Aerobic bacteria that grow utilizing methane for carbon and energy. The
initial
enzyme in the pathway for aerobic degradation, methane monooxygenase, has a broad
substrate range and is active against a wide range of compounds, including the chlorinated
aliphatics trichloroethylene and 1,2-dichloroethane.
For degradation it is necessary that bacteria and the contaminants be in contact. This is not
easily
achieved, as neither the microbes nor contaminants are uniformly spread in the soil. Some
bacteria are mobile and exhibit a chemotactic response, sensing the contaminant and moving
toward it. Other microbes such as fungi grow in a filamentous form toward the contaminant.
It is possible to enhance the mobilization of the contaminant utilizing some surfactants such
as sodium dodecyl sulphate (SDS).
ENVIRONMENTAL FACTORS:
Although the microorganisms are present in contaminated soil, they cannot necessarily be
there in the numbers required for bioremediation of the site. Their growth and activity must
be stimulated. Biostimulation usually involves the addition of nutrients and oxygen to help
indigenous microorganisms. These nutrients are the basic building blocks of life and allow
microbes to create the necessary enzymes to break down the contaminants. All of them will
need nitrogen, phosphorous, and carbon. Carbon is the most basic element of living forms
and is needed in greater quantities than other elements. In addition to hydrogen, oxygen, and
nitrogen it constitutes about 95% of the weight of cells. Phosphorous and sulfur contribute
with 70% of the remainders. The nutritional requirement of carbon to nitrogen ratio is 10:1,
and carbon to phosphorous is 30:1.[10]
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![Table 7. Environmental conditions affecting degradation:
[10]
Types of Bioremediation:
The main types of bioremediation are as follows:
Biostimulation -- Nutrients and oxygen - in a liquid or gas form - are added to
contaminated water or soil to encourage the growth and activity of bacteria already
existing in the soil or water. The disappearance of contaminants is monitored to ensure
that remediation occurs.
Bioaugmentation -- Microorganisms that can clean up a particular contaminant are
added to the contaminated soil or water. Bioaugmentation is more commonly and
successfully used on contaminants removed from the original site, such as in municipal
wastewater treatment facilities. To date, this method has not been very successful when
done at the site of the contamination because it is difficult to control site conditions for
the optimal growth of the microorganisms added. Scientists have yet to completely
understand all the mechanisms involved in bioremediation, and organisms introduced into
a foreign environment may have a hard time surviving.
Intrinsic Bioremediation -- Also known as natural attenuation, this type of
bioremediation occurs naturally in contaminated soil or water. This natural
bioremediation is the work of microorganisms and is seen in petroleum contamination
sites, such as old gas stations with leaky underground oil tanks. Researchers are studying
whether intrinsic bioremediation happens in areas with other types of chemical
contamination. Application of this technique requires close monitoring of contaminant
degradation to ensure that environmental and human health are protected.
All three types of bioremediation can be used at the site of contamination (in situ) or on
contamination removed from the original site (ex situ). In the case of contaminated soil,
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![sediments, and sludges, it can involve land tilling in order to make the nutrients and oxygen
more available to the microorganisms. [26]
Table 8: Bioremediation strategies:
[10]
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![Zinc:
Alcaligenes eutrophus and Pseudomonas aeruginosa are Zn tolerant Gram negative
bacteria.Strains of Pseudomonas putida, Pseudomonas putida 06909, Pseudomonas putida
06909s21x Pseudomonas putida 0690s22x, , Pseudomonas putida 0690 s23 were evaluated for
MRLof Zn on mannitol glutamate agar. These strains of Pseudomonas putida exhibited
different values values, which were, Pseudomonas putida 06909(11.5mM), Pseudomonas
putida 06909s21x(11.5mM), Pseudomonas putida 0690s22x(7.0mM),Pseudomonas putida
0690s23(10.5mM).
At Ingurtosa (southwestern Sardinia, Italy), due the mining in the past, mine tailings
were deposited along the Rio Naracaini creek. Rio Naracainli stream water was highly
polluted by heavy metals like Zn, Pb, Cd, Fe, Mn, Cu,and Ni. Podda et al.reported natural
bioremediation of heavy metals by a consortium of a photosynthetic filamentous bacterium,
classified as scytonema spp, strain ING-1, associated with microalga chlorella spp, strain
SA1. This microbial community was responsible for the natural polishing of heavy metals in
the water of stream Rio Naracainli by coprecipitation with hydrozincite [Zn 5(CO3)2(OH)6].
Scytonema spp was responsible for formation of hydrozinciteduring photosynthesisof
chlorella spp, thus removing Zn2+ in addition to Pb, Cd, Ni and Cu.[14]
Metal Tolerance Mechanisms
In high concentrations, heavy metal ions react to form toxic compounds in cells (Nies, 1999).
To have a toxic effect, however, heavy metal ions must first enter the cell. Because some
heavy metals are necessary for enzymatic functions and bacterial growth, uptake mechanisms
exist that allow for the entrance of metal ions into the cell. There are two general uptake
systems — one is quick and unspecific, driven by a chemiosmotic gradient across the cell
membrane and thus requiring no ATP, and the other is slower and more substrate-specific,
driven by energy from ATP hydrolysis. While the first mechanism is more energy efficient, it
results in an influx of a wider variety of heavy metals, and when these metals are present in
high concentrations, they are more likely to have toxic effects once inside the cell (Nies and
Silver, 1995). To survive under metal-stressed conditions, bacteria have evolved several types
of mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the
efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside
the cell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999).[1]
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![MATERIALS AND METHODS:
A] Collection of wastewater and soil sample:
Location:
Wastewater and soil sample was collected from koperkhairane nullah near MIDC
industrial area besides Furnace fabrica Ltd & Alok industry- Navi Mumbai.
Procedure:
Water sample was collected in a plastic bottle and soil sample was collected in sterile
Petri plates.
pH and temperature of the water sample was checked.
Water sample and soil sample was transported to college laboratory.
Water sample and soil sample were kept in refrigerator (40C) until further analysis was
carried out.
B] Waste water analysis:
1] Determinaton of total hardness of water:
AIM: To determine the hardness of water by EDTA titrimetric method.
Principle:
EDTA forms chelated soluble complex when added to a solution of certain metal cations. If
small amount of dye such as Eriochrome black T or calmagite is added to an aqueous solution
containing calcium and magnesium ions at pH 10.0 the solution turns wine red .If EDTA is
added as a titrant, the calcium and magnesium will be complexed. When all of the calcium
and magnesium has been complexed, the solution turns wine red to blue, marking the end
point of titration.
Requirements:
Equipments: Conical flask, burette, beakers etc.
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![Reagents:
Buffer solution: Dissolve 16.9 gm of ammonium chloride in 143 ml conc Ammonium
hydroxide and dilute to 250ml with distilled water.
Eriochrome black T indicator
Ethylene diamino tetra acetic acid [EDTA][0.01M]:weigh 3.723gm of disodium
EDTA and dissolved in 50ml of distilled water and make the volume to 1000ml and
store in a brown bottle.
Standard calcium :weigh 1gm of calcium carbonate powder into a 500ml conical
flask. Place a funnel in the neck of the flask and slowly add conc. HCL drop wise and
shake the flask repeatedly until the entire CaCO3 dissolved . Add 200ml D/W and boil
for few minutes to expel CO2.
Procedure:
Take 10ml of sample in a conical flask.
Add 1ml of buffer solution and shake well.
Add 1 to 2 drops of Eriochrome black T indicator, shake well to get wine red colour.
Titrate it against EDTA by adding drop wise from the burette until the colour changes
wine red to blue.
Calculation:
1ml of 1m of EDTA =40.08 of Ca+2
Since atomic weight of Ca+2 is 40.08, 1ml of 0.01M=0.4008mg of Ca+2
When standardization is done with Std .Calcium,
Hardness(EDTA) as mg Ca+2/L= BR x Normality of EDTA x 40.08 x1000
10[volume of sample taken]
= BR x 0.01 x 40.08 x1000
10
= BR x 40.08mg Ca+2/L
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![2] Determination of total alkalinity of water:
Principle:
Generally pH of water remains neutral. Alkalinity of water represents the presence of
hydroxyl ions[-OH] in water; hence, it is capacity of water to neutralize a strong acid. In
natural or waste water alkalinity is due to the presence of free hydroxyl ions which cause
through hydrolysis of salts by weak acids and strong bases (e.g. carbonate and bicarbonates).
Requirements:
Equipments: conical flasks, titration assembly etc.
HCL solution(0.1N)
Methylene orange indicator
Procedure:
Take 50ml of water sample in flask and add 2-3 drops of methyl orange into it. Clour
turns to orange .
Transfer 0.1N HCL solution ito burette in titration assembly and titrate with the water
sample[methylene orange added] untl yellow colour changes to pink. Note the end
point.
Calculation:
Methylene orange alkalinity (mg/litre) =volume of 0.1 N HCL solution used as titrant x 1000
Volume of water sample
3] Determination of chlorine in water:
Principle:
The potable water is chlorinated to make the water free from microorganisms. However,
some times the concentration of chloride ions in water is increased than what is normally
required. Apart from this water also receives chloride ions from multifarious sources. The
chloride ions (Cl-) can be estimated by titrating with silver nitrate solution.
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![4] Determination of chemical oxygen demand(COD) of water:
Principle:
Due to gradually increasing population the number of industries is also increasing. This
results in increased pollution. The chemically oxidisable organic substances discharged in
water depletes the amount of oxygen. Estimation of BOD alone could not give the exact idea
of pollutants present in water, therefore , COD is estimated. COD refers to the oxygen
consumed by the oxidisable organic substances. The values of COD cannot be compared
directly with that of BOD.
The chemical oxidants such as potassium dichromate (K2Cr2O7) or potassium
permanganate (KMnO4) are used to measure the oxidisability of the organic matter of water
where the oxidants oxidize the constituents (or the hydrogen but not nitrogen). Then
potassium iodide (KI) is added. The excess amount of oxygen reacts with KI and liberates
equal amount of iodine. By using starch indicator, iodine is titrated with sodium thiosulfate
and amount is estimated.
Requirements:
Potassium dichromate solution (0.1N): 3.676gm in 1 litre of distilled water.
Sodium thiosulphate (0.1N): 15.811gm of sodium thiosulfate in 2 litre of distilled water.
Sulphuric acid (2M): 10.8 ml of concentrated H2SO4 in 100ml of distilled water.
Potassium iodide solution (10%)
Starch solution (1%),water bath,conical flask,titration assembly etc.
Procedure:
Take three 100m conical flask and pour 50ml of water sample in each.
Simultaneously run distilled water blanks standards (also in triplicates)
Add 5ml of K2Cr2O7 solution in each of the six flask.
Keep the flask in waterbath at 1000C (boiling temperature) for one hour.
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![ Allow the sample to cool for 10 minutes.
Add 5ml of potassium iodide in each flask.
Add 10ml of H2SO4 in each flask.
Titrate the contents of each flask with 0.1M sodium thiosulphate until the appearance of
pale yellow colour.
Add 1 ml of starch solution to each flask (solution turns blue).
Titrate it again with 0.1 M sodium thiosulfate until the blue colour disappears.
Result:
COD of sample mg/litre= 8 x C x(A-B)
S
C = concentration of titrant (mmol/litre)
A = volume of titrant used for blank (ml)
B = volume of the totrant used for sample (ml)
S = volume of water sample taken (ml)
5] Determination of dissolved oxygen (DO) of water:
Some amount of oxygen is dissolved in water which is used by the aquatic plants and
animals. The sources of dissolved oxygen in water are the autotrophic aquatic plants which as
a result of photosynthesis evolve oxygen,and air where from oxygen is dissolved in water
depending o salinity, temperature and water movement. Moreover, in an oligotrophic lake the
amount of dissolved nutrients salts remains low, therefore ,it supports sparse plant and animal
lives. Tis results in high dissolved oxygen gradually increasing with depth. In addition, in
eutrophic water reservoirs e.g. lakes, ponds, pools, etc. the organic nutrients accumulate
abundantly which in turn are subjected to microbial decomposition. More growth of
microorganisms, plants and animals depletes oxygen.
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![ Again titrate against thiosulfate solution until the blue colour disappears. Note the volume
of sodium thiosulfate solution used in titration.
Calculation:
Calculate the amount of dissolve oxygen (DO) (mg/litre) by sing the formula:
DO (mg/l) = 8 x 1000 x N x v
V
Where, V = volume of water sample for titration
v = volume of sodium thiosulfate (titrant)
N = normality of the titrant
8 = it is a constant since 1ml of 0.025N sodium thiosulfate solution is equivalent to
0.2
mg oxygen.
6] Determination of biological oxygen demand (BOD) of water :
Principle:
Biochemical Oxygen Demand (BOD) is the amount of oxygen, expressed in mg/L or parts
per million (ppm), that bacteria take from water when they oxidize organic matter. The
carbohydrates (cellulose, starch, sugars), proteins, petroleum hydrocarbons and other
materials that comprise organic matter get into water from natural sources and from pollution.
They may be dissolved, like sugar, or suspended as particulate matter, like solids in sewage.
Organic matter can be oxidized (combined with oxygen) by burning, by being digested in the
bodies of animals and human beings, or by biochemical action of bacteria. Because organic
matter always contains carbon and hydrogen, oxidation produces carbon dioxide (the oxygen
combining with the carbon) and water (the oxygen combining with the hydrogen). Bacteria in
water live and multiply when organic matter is available for food and oxygen is available for
oxidation. About one-third of the food bacteria consumed becomes the solid organic cell
material of the organisms. The other two-thirds is oxidized to carbon dioxide and water by
the biochemical action of the bacteria on the oxygen dissolved in the water. To determine
BOD, the amount of oxygen the bacteria use is calculated by comparing the amount left at the
end of five days with the amount known to be present at the beginning, whereas the same can
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![be incubated at 270C for 3 days in tropical and subtropical regions where metabolic activities
are higher. At room temperature, the amount of oxygen dissolved in water is 8 mg/L. At
freezing, it increases to 14.6 mg/L; it also increases at high barometric pressures (low
altitudes). At the boiling point, the solubility of oxygen is zero. [13][12]
Requirements:
BOD-free water
BOD bottles ,Erlenmeyer flask, pipette, BOD incubator, pH meter
Phosphate buffer solution (pH 7.4)
Allythiourea (0.5%)
Alkaline KI solution: Dissolve 100g of KOH and 50g of KI in 200ml of distilled water.
Sodium thiosulphate (0.025N)
Manganese sulfate(MnSO44H2O) solution: Dissolve 100g of manganese sulfate in 200ml
of distilled water, filter and keep in stoppered bottle.
Starch indicator: Dissolve 5g of starch in 100ml hot distilled water(boiled) and a few
drops of formaldehyde(HCHO).
Concentrated H2SO4
Procedure:
Add 1nacid/1N alkali in the water sample to adjust the pH to 7.0
Gently transfer this water inti BOD bottles so as bubbles should not come out.
Add 1 ml of allylthiourea to each bottle to avoid nitrification.
Measure dissolved oxygen following the steps as described for dissolved oxygen
Incubate the other BOD bottle at 270C for 3 days in a BOD incubator.
Measure the amount of oxygen as done earlier.
Calculation:
Calculate the BOD of water by using the following formula:
BOD (mg/l) = D1-D2
Where, D1 = initial dissolved oxygen (mg/l) in the first sample
D2 = dissolved oxygen (mg/l) in the second sample after 3 days of incubation. [15]
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![C] ISOLATION OF MICROORGANISMS:
Suspensions of the soil sample were prepared using physiological saline. Spread plate
technique on Nutrient Agar plates was used for the isolation of microorganisms.
Requirements:
Nutrient Agar medium.
Composition/litre:
SR NO. COMPOSITION g/l
1. AGAR 15.0
2. PEPTONE 5.0
3. NaCl 5.0
4. YEAST EXTRACT 2.0
5. BEEF EXTRACT 1.0
Table 9: Nutrient Agar Composition.
The pH is adjusted to 7.4(+/-) 0.2 at 250C.
Preparation: Add components to Distilled water and bring the volume to 1.0L. Mix
thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15
mins at 15 psi pressure and -1210C. Pour into sterile Petri plates.
Physiological saline.
Add 0.85g of NaCl in 100ml Distilled water. Autoclave for 15 mins at 15 psi
pressure
and -1210C
Procedure:
0.1ml of soil suspensions were spread onto the Nutrient Agar plates under aseptic conditions
using a sterile glass spreader. These Petri plates were incubated overnight at room
temperature.
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![D] CHARACTERIZATION OF THE MICROORGANISMS:
GRAM STAINING:
Gram staining (or Gram's method) is an empirical method of differentiating
bacterial species into two large groups: Gram-positive and Gram-negative, based on the
chemical and physical properties of their cell walls. The Gram stain named after the
Danish scientist Hans Christian Gram, is almost always the first step in the identification of a
bacterial organism.
Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-
90% of cell wall), which stains purple while Gram-negative bacteria have a thinner layer
(10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer
membrane which contains lipids, and is separated from the cell wall by the periplasmic space.
There are four basic steps of the Gram stain, which include applying a primary stain (crystal
violet) to a heat-fixed smear of a bacterial culture, followed by the addition of a trapping
agent (Gram'siodine), rapid decolorization with alcohol or acetone,
and counterstaining with safranin. Basic fuchsin is sometimes substituted for safranin since it
will more intensely stain anaerobic bacteria but it is much less commonly employed as a
counterstain.
Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – )
ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and
Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial
cells and stains the cells purple.
Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and
iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as
a mordant, but is a trapping agent that prevents the removal of the CV-I complex and
therefore color the cell. After decolorization, the gram-positive cell remains purple and the
gram-negative cell loses its purple color. Counterstain, which is usually positively charged
safranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or
red color.
Requirements:
Nutrient Agar slants of the isolates.
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![ Reagents: Crystal violet, Gram’s iodine, 95% ethyla alcohol and safranin.
Eqiupments: Inoculating loop, glass slides, Bunsen burner, staining tray,bibulous
paper, lens paper, immersion oil and microscope.
Method:
A bacterial suspension is prepared and loopful of suspension is used to make a smear
on a clean glass slide under sterile conditions.
The smear is allowed to air dry, then it is heat fixed and the slide is transferred to a
staining tray.
On the tray, smear is gently flood with crystal violet for 1 min.Wash briefly in water to
remove excess crystal violet.
Flood with Gram’s iodine 1 min. Wash briefly in water, to remove excess of stain.
Decolourise with 95% ethanol until the moving dye front has passed the lower edge of
the section. Wash immediately in tap water.
Counterstain with safranine for 45 sec. and wash with tap water.
The slide is blotted dry using bibulous paper and examined under oil immersion lens
of microscope.[37]
E] BIOCHEMICAL CHARACTERIZATION:
1]Catalase test:
Catalase is a common enzyme found in nearly all living organisms that are exposed to
oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to
water and oxygen.
H2O2 ------catalase--------> H2O + O2
Catalase test is used to detect the presence of catalse enzyme. Hydrogen peroxide is
formed by some bacteria as an oxidative end product of the aerobic breakdown of sugars. If
allowed to accumulate it is highly toxic to bacteria and can result in cell death. Catalase either
decomposes hydrogen peroxide or oxidizes secondary substrates, but it has no effect on other
peroxides.
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![Catalase production is indicated bubbles of free oxygen gas upon addition of substrate to a
culture. The absence of bubble formation is a negative test.
Requirements:
Nutrient Aagr slants of the soil isolates.
3% Hydrogen peroxide solution.
Equipments: Inoculating loop, Cavity slide.
Method:
A drop of 3% Hydrogen peroxide solution is placed onto a watch glass.
A loopful of culture is taken out under sterile conditions and placed on the drop and
results are observed. [38]
2] Citrate Utilization test:
Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source.
Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium
dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator
bromthymol blue.
Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or
citrate-permease to transport the citrate into the cell. These organisms also convert the
ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an
alkaline environment in the medium. At pH 7.5 or above, bromthymol blue turns royal blue.
At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media.
If the medium turns blue, the organism is citrate positive. If there is no color change, the
organism is citrate negative. Some citrate negative organisms may grow weakly on the
surface of the slant, but they will not produce a color change.
Requirements:
Simmon’s Citrate Agar slants.
Composition/l:
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![SR NO. COMPOSITION g/l
1. AMMONIUM DIHYDROGEN 1.0
PHOSPHATE
2. DIPOTASSIUM PHOSPHATE 1.0
3. NaCl 5.0
4. SODIUM CITRATE 2.0
5. MAGNESIUM SULFATE 0.2
6. AGAR 15.0
7. BROMOTHYMOL BLUE 0.08
Table 10: Simmon’s Citrate Agar Composition.
Nutrient Agar slants of the soil isolates.
Equipments: Inoculating loop, Bunsen burner.
Method:
A loopful of culture is taken and streaked on Simmon’s citrate agar slant.
After 24 hr. incubation period, results are observed. [39]
3] Nitrate Utilization Test:
Nitrate may be reduce to multiple compounds by two processes. Anaerobic respiration
and denitrification. In anaerobic respiration the bacterium uses nitrate as it's terminal electron
acceptor, reducing nitrate to a variety of compounds, while denitrification reduces nitrate
solely to molecular nitrogen. Sulfanilic acid and dimethyl 1-naphthylamine are added to
detect nitrite, which will complex with these molecules forming a red color. If no red color is
observed there are two possibilities; the nitrate has not been reduced, or it has been reduced
further than nitrite. To differentiate between these two possibilities, zinc powder is added,
which will complex with nitrate forming a red color. Thus if the tube turns red after zinc,
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![nitrate has not been reduced and the result is negative. If no red color is observed then the
nitrate has been reduced further than nitrite and the result is positive.
Requirements:
Nutrient Agar slants of the soil isolates.
Equipments: Inoculating loop, Bunsen burner.
Compounds: Nitrate broth, sulfanilic acid, dimethyl 1-naphthaline and zinc powder.
Method:
Inoculate a trypticase-nitrate tube with the organism to be tested.
Incubate for 24 hours, test for results.
Add 1ml of sulfanilic acid to each tube, then add 1ml of dimethyl 1-naphthylamine
solution, Mix
Development of a red color indicates a positive test.
If no red color develops add a small amount of zinc powder.
If a red color develops, nitrate is present and the test is negative. If no red color
develops, nitrate has been reduced and the test is positive.
4] MRVP Test:
This test is used to determine two things. The MR portion (methyl red) is used to
determine if glucose can be converted to acidic products like lactate, acetate, and formate.
The VP portion is used to determine if glucose can be converted to acetoin.
Methyl red-positive organisms produce high levels of acid during fermentation of
dextrose, overcome the phosphate buffer system and produce a red color upon the addition of
the methyl red pH indicator.
In the Voges-Proskauer test, the red color produced by the addition of potassium
hydroxide to cultures of certain microbial species is due to the ability of the organisms to
produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of
dextrose.3 The acetoin is oxidized in the presence of oxygen and alkali to produce a red
color.3 This is a positive Voges-Proskauer reaction.
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![These tests are performed by inoculating a single tube of MRVP media with a transfer
loop and then allowing the culture to grow for 1 day. After the culture is grown, about half of
the culture is transferred to a clean tube. One tube of culture will be used to conduct the MR
test, the second tube serves as the VP test.
Requirements:
Nutrient Agar slants of the soil isolates.
Equipments: Inoculating loop, Bunsen burner.
Chemicals: MRVP medium, Methyl Red(MR), 5% alpha-naphthol, 40% potassium
hydroxide.
Method:
Inoculate the tubes with MRVP media with Organism to be tested.
Incubate the tubes at room temperature for 24hrs.
Methyl Red Test: Add 5 drops of methyl red indicator to the broth under sterile
conditions. Interpret the colour result immediately.
Voges-Proskauer’s Test: First 5% alpha-naphthol and then 40% potassium hydroxide
are added to the VP tube. The culture is allowed to sit for 15 mins for colour
development to occur. Interpret the result. [40]
5] Indole Test:
The Indole test determines the ability of an organism to produce indole from the
degradation of amino acid trytophan. Tryptophan is hydrolysed by trytophanase to produce
three possible end products- one of which is indole.
Organisms possessing the enzyme trytophanase cleave tryptophan, producing 3 end
products. Amyl alcohol in Kovac’s reagent acts as a solvent for indole which then reacts with
p-dimethylaminobenzaldehyde to produce a red rosindole dye. Organisms which don’t
produce the enzyme, produce no colour change in the medium upon addition of Kovac’s
reagent.
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![Requirements:
Nutrient Agar slants of the soil isolates.
Equipment: Bunsen burner, Inoculating loop.
Chemicals: Tryptic Soy Broth, Kovac’s reagent.
Method:
Inoculate the Tryptic soy broth with organism to be tested and incubate at room
temperature for 24hrs.
Add 5 drops of Kovac’s reagent and gently agitate.
Examine the upper layer of liquid.
Positive result: red/pink colour.
Negative result: No colour change.[41][42]
6] Gelatin Test:
Gelatin causes liquids to solidify at temperatures below 28 degrees Celsius. At
temperatures above 28 degrees C. gelatin is a liquid. Some bacteria produce gelatinase, a
proteolytic enzyme that hydrolyzes gelatin. The hydrolyzed gelatin no longer has the ability
to gel and thus remains a liquid, even if placed at temperatures below 28 degrees. The
presence or absence of gelatinase can be used to aid in identification of certain bacteria.
Requirements:
Nutrient agar slants of the soil isolates.
Equipments: Bunsen burner, Inoculating loop.
Medium for gelatin: Nutrient gelatin deep tubes containing 12% gelatin
Method:
An inoculum of test organism is stabbed into the nutrient gelatin deep tubes.
The media is incubated at room temperature for 24 hrs. and hen refrigerated for
approximately 30 mins.
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![ If the gelatin is still intact ( organism did not produce gelatinase), the media will
solidify in the refrigerator and a negative result is recorded.
If organism has produced sufficient gelatinase then the tube will remain liquid
(atleast partially) and a positive result is recorded.
7] Utilization of carbon sources:
Carbohydrates are the chief energy providers of the organism. They are made up of
carbon, hydrogen and oxygen. The bonds between carbon and hydrogen are broken down
with the help of oxygen to give energy (oxidation). Bacteria produce acidic products when
they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect
the change in pH which would occur if fermentation of the given carbohydrate occurred.
Acids lower the pH of the medium which will cause the pH indicator (Andrade’s solution) to
turn dark pink. If the bacteria do not ferment the carbohydrate then the media remains light
pink.[43]
Requirements:
Nutrient agar slants of the soil isolates.
Equipments: Bunsen burner, Inoculating loop.
Chemicals: Different sugars- Glucose, Lactose, Xylose, Mannitol, Fructose, Sucrose,
Inositol, Galactose (All 5g): Peptone (10g), NaCl (5g).
Preparation of media: 1% peptone, 0.5% NaCl, 1% sugar. After autoclaving the media, add 2-
3 drops of Andrade’s indicator.
Method:
The media is prepared as mentioned above.
The media is then inoculated with the test organism and incubated at room
temperature for 24 hrs.
Results are observed. [44]
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![8] Starch hydrolysis test:
Principle:
Starch agar is a differential medium that tests the ability of an organism to produce certain
exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starch
molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to
degrade starch into subunits that can then be utilized by the organism.
Starch agar is a simple nutritive medium with starch added. Since no color change occurs in
the medium when organisms hydrolyze starch, we add iodine to the plate after incubation.
Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence
of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed
starch.
In this test, starch agar is inoculated with the species in question. After incubation at an
appropriate temperature, iodine is added to the surface of the agar. Iodine turns blue-black in
the presence of starch. Absence of the blue-black color indicates that starch is no longer
present in the medium. Bacteria which show a clear zone around the growth produce the
exoenzyme amylase which cleaves the starch into di- and monosaccharides. These simpler
sugars can then be transported into the cell to be catabolized. Bacillus species are known to
produce the exoenzyme, amylase.[27][28]
Requirements:
Sterile starch agar plates.
Nutrient Agar slants of the soil isolates.
Equipments: Inoculating loop, Bunsen burner.
Iodine solution.
Starch agar medium
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![Composition per litre
Sr No. Chemicals Qty(gms)
1. Beef Extract 3.0
2. Soluble Starch 10.0
3. Agar 12.0
[29]
Table 11: Starch agar medium
Method:
Starch media are prepared according to the above mentioned composition.
It is then autoclaved at 15lbs at 1210C for 20 minutes.
Sterile starch agar plates are prepared
The plates are inoculated with the culture on surface by spot inoculation.
They are incubated for 24 hrs at room temperature.
After incubation iodine solution is added to surface of the plates and colour is observed
9] Casein Hydrolysis Test:
Principle:
Casein hydrolysis test is used to determine if an organism can produce the exoenzyme
casesase.
Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein
is a large protein that is responsible for the white color of milk. This test is conducted on milk
agar which is a complex media containing casien, peptone and beef extract. If an organism
can produce casein, then there will be a zone of clearing around the bacterial growth.
Requirements:
Nutrient Agar slants of the soil isolates.
Equipments: Inoculating loop, Bunsen burner
Sterile petri plates
Microbial cultures.
Milk agar medium :
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![Composition per litre:
Sr No Chemicals Qty (gms)
1. Agar 15.0
2. Peptone 5.0
3. Yeast extract 3.0
4. Milk solids 1.0
The pH is adjusted to 7.2 +/- 0.2 at 250c
Table 12: Milk agar medium
Method:
Milk agar media are prepared according to the above mentioned composition.
It is then autoclaved at 15lbs at 1210C for 20 minutes.
Sterile milk agar plates are prepared
The plates are inoculated with the culture on surface by spot inoculation.
They are incubated for 24 hrs at room temperature, observe results.
Clearing of the agar around the bacterial growth indicates casein hydrolysis.[30]
10] Macconkey agar test:
MacConkey Agar (MAC) is a selective and differential medium designed to isolate and
differentiate enterics based on their ability to ferment lactose. Bile salts and crystal violet
inhibit the growth of Gram positive organisms. Lactose provides a source of fermentable
carbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red at a pH
below 6.8 and is colorless at any pH greater than 6.8.
Organisms that ferment lactose and thereby produce an acidic environment will appear pink
because of the neutral red turning red. Bile salts may also precipitate out of the media
surrounding the growth of fermenters because of the change in pH. Non-fermenters will
produce normally-colored or colorless colonies.[31]
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![Requirements :
Macconkey agar medium
Composition per litre:
Sr No. Chemicals Qty(gms)
1. Peptic digest of animal tissue 17.0
2. Proteose peptone 3.0
3. Lactose 10.0
4. Bile salts 1.5
5. Sodium chloride 5.0
6 Neutral red 0.03
7 Agar 15.0
[19]
The pH is adjusted to 7.1
Table 13: Macconkey agar medium
Method:
Macconkey media are prepared according to the above mentioned composition.
It is then autoclaved at 15lbs at 1210C for 20 minutes.
Sterile macconkey agar plates are prepared
The plates are inoculated with the culture on surface by spot inoculation or streaking
They are incubated for 24 hrs at room temperature, observe results.
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![11] Protease test:
This test is performed to determine the presence of enzyme protease in the bacterial
cell.
Requirements:
Gelatin Agar medium.
Sr No. Chemicals Qty(grams)
1. Gelatin 30
2. Agar Agar 15
3. Pancreatic digest of casein 10
4. Sodium Chloride 10
Table 14: Gelatin Agar medium
Method:
Gelatin Agar media was prepared according to the above mentioned composition.
It was then autoclaved at 15lbs at 1210C for 20 minutes.
Sterile Gelatin Agar plates were prepared.
The plates were inoculated with the culture on surface by spot inoculation or streaking.
They were incubated for 24 hrs at room temperature.
10% HgCl2 was then added to the plates and the clear zone formation around growing
colony was considered as positive.
12] Lipase test:
Many microbes hydrolyse fats, resulting in rancidity.This is accomplished by an
enzyme, Lipase. Fats are usually broken down to glycerol and fatty acids. Lipase is one of
the most important class of industrial enzymes and has been explored in oleochemical,
paper, organic chemical processing, agrochemical industry,etc.
This test qualitatively determines the presence or absence of lipase in the bacterial
cell.
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![Requirements:
Peptone Agar medium.
Sr No. Composition Qnty(grams)
1. Peptone 5
2. Agar Agar 15
Table 15: Peptone Agar medium.
Method:
Peptone Agar media was prepared according to the above mentioned composition.
It was then autoclaved at 15lbs at 1210C for 20 minutes.
Sterile Peptone Agar plates were prepared.
The plates were inoculated with the culture on surface by spot inoculation or streaking.
They were incubated for 24 hrs at room temperature.
Formation of opaque whitish zone around colony was taken as a positive result.
F] STUDY OF EFFECTS OF VARIOUS PARAMETERS ON THE SOIL ISOLATES.
1] Effect of different temperatures:
Overview:
An effort was taken to study the effects of varying temperatures on the growth of the
soil isolates. The temperatures selected for this purpose were 4 0C, 100C, 280C(R.T), 370C
and 550C.
Method:
Nutrient broth was prepared with its pH adjusted to 7.2 and was dispensed into
test-tubes.
The test-tubes containing the media were then autoclaved at 121 0C at 15lbs for
15mins.
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![ Keeping one control tube for every temperature, rest of the tubes were inoculated
with the 14 soil isolates under sterile conditions and incubated for 24hrs. at
respective temperatures.
2] Effect of different pH:
Overview:
An effort was made to study the effects of varying pH on growth of the soil isolates. The
dfifferent pH selected for this purpose were 3 and 5
Method:
Nutrient broth was prepared in 2 conical flasks and pH of the media in each conical
flask was adjusted to 3 and 5 respectively.
The media from all the flasks was then dispensed into test-tubes.
The test-tubes containing the media were then autoclaved at 1210C at 15lbs for
15mins.
Keeping one control tube for every pH, rest of the tubes were inoculated with the soil
isolates under sterile conditions and incubated for 24hrs.
3] Effect of varying concentrations of different heavy metals:
An effort was made to study the effects of varying concentrations of different heavy
metals on the growth of the soil isolates. The heavy metals used for the purpose were
aluminium(AlCl3), chromium(KCrO4), manganese(MnSO4), zinc(ZnSO4) and
mercury(HgCl2).
Method:
15ml of Nutrient agar media was prepared per butt tube.
The butt tubes were then autoclaved at 1210C at 15lbs for 20 mins.
The media was then slightly cooled and following concentrations of heavy metals
were added per tube as follows:
A stock of 10,000ppm of heavy metal was prepared. (10mg/ml)
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![ It was diluted to 1000ppm by adding 9ml of distilled water. (making it to
10mg/10ml). Thn using this as the stock solution, required quantities of heavy metal
concentrations wee prepared using the formula RT/G.
For eg: To make 10ppm conc. of a heavy metal,
R= Required concentration, T= Total quantity and G= given conc. of heavy metal.
Therefore, 10*15/1000 = 0.15.
After adding the heavy metal, the media was poured into petri plates and allowed
to solidify.
The soil isolates were then streaked onto the plates under sterile conditions and
incubated at room temperature for 24 hrs.
The results were observed.
4] To study the growth curve of the organisms at different concentrations of phenol:
The growth curve is studied of the organisms at various concentrations of 50 ppm and
100ppm of phenol
Method :
Three flask of 100ml capacity were taken and nutrient broth is prepared (100ml) in each
flask.
The media was then autoclaved at 1210C at 15lbs for 20 mins.
Phenol solution of 50 ppm and 100 ppm concentration is prepared by formula RT/G and
added to respective flask, and 7ml isolate suspension of 0.05 O.D at 540nm is inoculated
in each flask.
The flask were kept in shaker at room temperature for 24 hours.
The growth curve is studied for period of 24 hours at regular time intervals and
absorbance is measured at 540nm.
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![RESULTS:
A] Waste water analysis:
Sr No. Test Parameters Results Units
1 Total Dissolve Solids 1423 mg/l
2 COD 1963 mg/l
3 BOD 3 days, 270C 1076 mg/l
4 Cyanide Absent mg/l
5 Zinc 0.59 mg/l
6 Copper BDL mg/l
7 Aluminium 58 mg/l
8 Manganese BDL mg/l
9 Mercury 0.017 mg/l
10 Iron 0.19 mg/l
11 Chromium 0.23 mg/l
12 Oil & Grease 12 mg/l
13 Methylene orange alkalinity 66 mg/l
14 Chloride 23252.5 mg/l
15 Hardness (EDTA) 148.2 mg Ca+2/L
Table 16: Waste water analysis
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![Some of the parameters of wastewater analysis like heavy metal ion detection were given in
laboratory and reports were obtained. pH of water was 9 and temperature around 300C. The
colour of wastewater was found to be black and reddish brown.
B] Isolation of microorganisms:
1] Soil microorganisms:
Sr No. Dilutions cfu/ml Average cfu/ml
1 10-3 3570 x 104
2 10-4 2840 x 104 ≈ 10 x 107
3 10-5 2740 x 104
Table 17: Soil microorganisms
2] Waste water microorganisms:
Sr No. Dilutions cfu/ml Average cfu/ml
1 10-3 880 x 104
2 10-4 790 x 104 ≈ 20 x 106
3 10-5 500 x 104
Table 18: Wastewater microorganisms
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![C] Gram staining:
Organism No. Gram Nature Morphology
KN1. Negative Rods
KN2. Positive Rods
KN3. Positive Rods
KN4. Positive Cocci
KN5. Positive Rods
KN6. Positive Cocci
KN7. Positive Rods
KN8. Positive Rods
KN9. Negative Rods
Table 19: Gram staining
D] Biochemical Tests:
Sr Test KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
1. Citrate Utilization + - + + - + + + -
2. Nitrate Utilization + + + - - - + + -
3. Indole test - - - - - - - - -
4. Gelatin - - - - - - + + +
5. Methyl red test + - - - - - - - -
6. Voges-Proskauer - - - - - - - - -
test
Table 20:Biochemical Tests
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![E] Acid production:
Sr Sugars KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
1. Glucose - + + - - - + + +
2. Lactose - - - - + - - - -
3 Xylose + - - - - - - - -
4 Manitol + + + - - + - - -
5 Inositol - - - + - - - - -
6 Fructose + + + + - + + + +
7 Sucrose + + + + - + - - -
8 Galactose + + + + - - - - -
Table 21: Acid production
F] Enzyme assay:
Sr Test KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
1. Starch + + - + - - - - -
hydrolysis
2. Casein + + - - + + + - -
Hydrolysis
3. Catalase + + - + + + + + -
test
4. Protease - - - - - - - - -
5. Lipase - - - - - - - - -
Table 22: Enzyme assay
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![G] Probable organisms:
The biochemical test, acid production test and enzyme assay were then compared to different
species in the bergy’s manual and following organisms characteristics were matched with
studied isolates.
Pseudomonas spp
Bacillus spp
Micrococcus spp
Arthrobacter spp
H] Heavy Metal Tolerance:
1] ZnSO4Tolerance:
The soil isolates were tested against varying concentrations of different heavy metals to
check their tolerance against these heavy metals. The results were confirmed by comparing
turbidity of the inoculated tubes with the control tubes.
Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
(ppm)
1 10 + + + + + + + + +
2 50 + + + + + + + + +
3 100 + + + + + + + + +
4 300 + + + + + + + - -
5 500 + + + + - - - - -
6 700 - + - + - - - - -
7 1000 - - - - - - - - -
Table 23: ZnSO4 Tolerance:
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![2] KCrO4Tolerance:
Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
(ppm)
1 10 + + + + + + + + +
2 50 + + + + + + + + +
3 100 + + + + + + + + +
4 300 + + + + + + + + +
5 500 + + + + + + + + +
6 700 - + + + + + + + +
7 1000 - + + + + + + + +
Table 24: KCrO4 Tolerance
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![3] MnSO4Tolerance:
Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
(ppm)
1 10 + + + + + + + + +
2 50 + + + + + + + + +
3 100 + + + + + + + + +
4 300 + + + + + + + + +
5 500 + + + + - + + - +
6 700 - + + + - + + - +
7 1000 - + + + - + - - +
Table 25: MnSO4Tolerance
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![4] HgCl2Tolerance:
Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
(ppm)
1 10 + + + + + + - + +
2 50 - + + - + - - - +
3 100 - + - - + - - - +
4 300 - + - - + - - - -
5 500 - + - - - - - - -
6 700 - - - - - - - - -
7 1000 - - - - - - - - -
Table 26: HgCl2 Tolerance
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![5] AlCl3 Tolerance:
Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
(ppm)
1 10 + + + + + + + + +
2 50 + + + + + + + + +
3 100 + + + + + + + + +
4 300 - - + + + - + - -
5 500 - - - - - - - - -
6 700 - - - - - - - - -
7 1000 - - - - - - - - -
Table 27: AlCl3 Tolerance
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![I] Growth at different pH:
Sr pH KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No.
1 5 + + + + + + + + +
2 3 - - - - - - + + +
Table 28: Growth at different pH
J] Growth at different Temperatures:
Sr Temperature KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9
No. [0C]
1 4 - - - - - - - - -
2 10 - - - - - - - - -
3 28 + + + + + + + + +
4 37 + + + + + + + + +
5 55 - - - - - - - - -
Table 29: Growth at different Temperatures
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![K] Effect of various concentration of phenol on growth curve of the organism:
Time (Hrs) Optical Optical Optical
density(540nm) density(540nm) density(540nm)
Without Phenol Phenol(50ppm) Phenol(100ppm)
0 0.0 0.0 0.0
2 0.0 0.0 0.0
4 0.20 0.14 0.10
6 0.35 0.23 0.13
8 0.56 0.41 0.29
10 0.77 0.65 0.56
12 0.85 0.73 0.65
14 0.95 0.79 0.73
16 1.00 0.86 0.83
18 1.05 0.94 0.89
20 1.06 0.95 0.89
22 1.09 1.03 0.98
24 1.09 1.06 1.01
Table 30: Effect of various concentration of phenol on growth curve of the organism
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![DISCUSSION:
Due to rapid industrialization, different types of pollution have emerged and recognized,
which include air, soil and ground water pollution, caused by gaseous, liquid and solid forms
of chemicals. Soil degradation from various inorganic and organic contaminants, is not only
an ecological risk, but simultaneously it is also a social, health and economic issue. Such soils
become poor in physiochemical properties, susceptible to erosion; result in loss of production
and sustainability, diminished food chain quality, and tainting of water resources. Restoration
of such degraded land depends on one hand upon action of nature itself, which is time
consuming, and on the other hand, it also depends upon human intervention. Among
available remediation technologies, bioremediation is a useful approach to clean up
contaminated soil and wastewaters, which is based upon utilization of natural or engineered
biota( Rani Faryal 2003)[14]
In the present study, an attempt has been made to analyze physiochemical properties of
wastewater collected from koperkhairane nullah near MIDC industrial area besides Furnace
fabrica Ltd and Alok industry, Maharashtra. Analysis of effluent showed that effluent
samples were highly coloured like dark black and reddish brown, foul smelling, alkaline and
having temperature approximately 300C. Colour is the first contaminant to be recognized in
the textile industry waste water, due to presence of unused dye in water, which affects
aesthetic as well as biological activity of the ecosystem (sarnaik and kanekar, 1995; bant et al
1996). Total dissolved solids (TDS) of the effluent were high (1423 mg/l) as compared to the
Indian standard of specification requirement but in the permissible limit. BOD of the effluent
was exceeding Indian standard specifications(appendix 1). These results were in line with the
work of Gowrisankar et al.(1997) on textile mill effluent in India, with BOD ranging between
400-1000mg/L. In general high BOD reflects high concentrations of substances that can be
biologically degraded, thereby consuming oxygen and potentially resulting in dissolved
oxygen. (Rani Faryal 2003)[14]
COD of the effluents was also high (1963mg/L) as compared to Indian standard
specifications. In textile effluent, pollution is aggravated by the presence of free chorine and
toxic heavy metals, which causes rapid depletion of dissolved oxygen leading to an “oxygen
sag” in receiving waters. In other words, a high degree of pollution is associated with high
COD.[14] Alkalinity and Hardness of water were under normal range, but chlorides were
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![To survive under metal-stressed conditions, bacteria have evolved several types of
mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the efflux
of metal ions outside the cell, accumulation and complexation of the metal ions inside the
cell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999). [1]
All the isolated strains (KN 1-9) were tolerant towards five different heavy metals. Most of
the isolates are tolerant to ZnSO4 up to 300ppm-500ppm, only two KN2 & KN3 tolerated up
to 700ppm(Table 15). For K2CrO4 all isolates tolerates up to 1000ppm except KN1(Table 16).
5 isolates KN 2, 3, 4, 6& 9 are tolerant towards MnSO4 up to 1000ppm(Table 17). The
isolates are least tolerant to HgCl2, only KN 5 tolerates up to 500ppm(Table 18). Most of the
isolates are tolerant towards AlCl3 up to 100ppm. Only KN 3, 4, 5 AND 7 tolerates up to
300ppm(Table 19).
All the isolates showed full growth at 280C and 370C suggesting these temperatures to be
their optimum temperatures. None could tolerate freezing temperatures of 4 0C and 100C and
thermophilic range of 550C (Table21). The pH study was carried for pH 5 & 3. All the
isolates showed growth at pH 5, only KN 7, 8 and 9 showed growth at pH 3(Table 21).
Effects of various concentration of phenol on growth curve of the microorganism were
observed. The growth curve was optimum for organism without phenol concentration. The
growth curve slightly decreased with increasing concentrations of phenol (Graph 1).
Thus the said project paves the ways to study the wastewater of this region further.
Analysis of various dyes and attempt to study the degradation of dyes with soil
microorganisms would be helpful to decrease the pollution of water.
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![Conclusions:
1] Textile effluents are highly polluted. Such polluted effluents must be treated properly
before their discharge into the drainage channel to minimize the effect of various
pollutants on the environment.
2] Although some heavy metals are important and essential trace elements, at high
concentrations, such as those found in many environments today, most can be toxic to
microbes. Microbes have adapted to tolerate the presence of metals or can even use them to
grow. Thus, a number of interactions between microbes and metals have important
environmental and health implications. Some implications are useful, such as the use of
bacteria to clean up metal-contaminated sites.
3] Wastewater collected from Koperkhairane Nullah is a source of heavy metal
Contamination, containing Aluminium, Iron, Mercury.
4] High BOD and COD of effluent samples is an indicator of high load of organic pollutants.
5] The isolated microbes showed remarkable tolerance against various heavy metals.
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![BIBLIOGRAPHY:
1] Anne Spain, Communicated by: Dr. Elizabeth Alm (2003). Implications of Microbial
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organic pollutants present in textile industry.
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18] R.O. YUSUFF, J.A. SONIBARE (2004). CHARACTERIZATION OF TEXTILE
INDUSTRIES’ EFFLUENTS IN KADUNA, NIGERIA AND POLLUTION
IMPLICATIONS. Global Nest: the Int. J. Vol 6, No 3, pp 212-221, 2004.
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treatment Process.
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Chemical Characterization of Effluents of Local Textile Industries of Faisalabad–
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Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah.
The document is a project dissertation submitted by Priyesh Waghmare to Padmashree Dr. D.Y. Patil University for a Bachelor's degree in Biotechnology. The project involved wastewater analysis and study of soil microorganisms from Koparkhairane Nullah near industrial areas in Navi Mumbai. Physiochemical analysis of wastewater from the nullah showed high levels of color, foul smell, alkalinity, temperature, BOD and COD indicating pollution from nearby industries. Soil samples from the nullah were then used to isolate microorganisms and characterize them. Studies were also conducted to evaluate the tolerance of isolated soil microorganisms to heavy metals for their potential use in
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Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah.
- 1. Wastewater Analysis andStudy of Soil Microorganisms of Koparkhairane Nullah. The Project Dissertation submitted to Padmashree Dr D.Y.Patil University Department of Biotechnology and Bioinformatics For the degree of Bachelors of Technology In Biotechnology By PRIYESH V. WAGHMARE (BBT-2-06055) Research Guide: Mr. Manish Bhat Padmashree Dr.D.Y.Patil University Department of Biotechnology and Bioinformatics C.B.D. Belapur, Navi Mumbai 400 614 2010 1
- 2. DECLARATION I, Mr Priyesh Vijay Waghmare, student of Dr. D. Y. Patil Institute for Biotechnology and Bioinformatics, Padmashree Dr. D. Y. Patil University, Navi Mumbai hereby state that to the best of my knowledge and belief, the project report entitled “Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah.” was carried out under the guidance of Mr. Manish R.Bhat, Assistant Professor and being submitted for partial fulfillment of Bachelor’s of Technology In Biotechnology (2007- 2010). This research project is an original work and has not been submitted in part/ full for any other degree/ diploma to any other University to the best of my knowledge. Place: Navi Mumbai Date: 30th June, 2010 Mr Priyesh Vijay Waghmare BBT-2-06055 Research Student Padmashree Dr.D.Y.Patil University Department of Biotechnology and Bioinformatics C.B.D Belapur Navi Mumbai 2
- 3. CERTIFICATE This is tocertify that the project entitled “Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah.” has been carried out by Mr Priyesh Vijay Waghmare in partial fulfillment of his degree of Bachelor of Technology in Biotechnology (B.Tech Biotechnology) at the Dr D. Y. Patil Institute for Biotechnology and Bioinformatics, CBD Belapur, Navi Mumbai-400614. This work has been carried out under my supervision and has not been submitted in part/ full for any other degree/ diploma to any other University. Date: 30th June, 2010 Place: Navi Mumbai (Mr. Manish R.Bhat) Asst Professor Research Guide 3
- 4. ACKNOWLEDGEMENTS In the firstplace, I thank God Almighty for His grace and all the blessings that have been showered upon me. I am deeply indebted to a number of people who enabled the completion of my project, all of whom especially need to be acknowledged for all the support and assistance redeemed to me through each stage of this project. I owe my thanks to Dr. D.A. Bhiwgade, Dean of the Department of Biotechnology and Bioinformatics, Padmashree Dr. D.Y. Patil University, for providing us the opportunity to take our first steps in exploring the field of biotechnology through this project. I avail this opportunity to express sincere reverence and deep sense of gratitude to my guide and mentor, Prof. Manish Bhat for his valuable and indispensable guidance throughout my project tenure. I am much obliged to him for charting out the course of my work and for his constant words of encouragement and inordinate patience. His supervision over the laboratory practicals and invaluable contribution to the writing of this thesis is greatly acknowledged. I wish to thank Leena, Hrishikesh, Akshay, Jay, Abhishek, Manish, Vikas, Amit, Sid and to all my peers for their kind co-operation. My thanks are due to all teaching and non-teaching staff and especially to the laboratory staff for their timely technical support. I am very much obliged to my family, my pillars of strength, for their unwavering faith and whole hearted support for giving me a choice to pursue a wonderful career. Priyesh Vijay Waghmare. 4
- 5. ABSTRACT: Physiochemical analysis ofwaste water was carried out from the effluent sample collected from koparkhairane nullah near MIDC industrial area besides Furnace fabrica Ltd and Alok industry-Navi Mumbai. Analysis of effluent showed that effluent samples were highly coloured like dark black and reddish brown, foul smelling, alkaline and having temperature approximately 300C with high BOD and COD values. Traces of heavy metal contamination was found in textile wastewater Then the isolation and characterization of soil microorganisms from koparkhairane nullah and heavy metal tolerance studies of soil isolates was carried out. The organisms isolated from the soil could belong to Pseudomonas spp, Bacillus spp, Micrococcus spp or Arthobacter spp. Optimization of various parameters like pH, Temperature, growth curve studies and evaluation of heavy metal tolerance of soil isolates was carried out for bioremediation. Key Words:Textile wastewater; BOD; Bioremediation; 5
- 6. INDEX: Sr. No. Topic Page No. 1 Introduction 11 2 Aims and Objectives 16 3 Literature Review 18 4 Materials and Methods 39 5 Results 66 6 Discussion 85 7 Conclusion 89 8 Future prospects 91 9 Bibliography 93 10 Appendix 98 6
- 7. LIST OF TABLESAND GRAPHS: 1) Table 1. Properties of Waste Water from Textile Chemical Processing. (Page no 20) 2) Table 2. Composite textile industry wastewater characteristics. (Page no 20) 3) Table 3.Effluent Characteristics From Textile Industry. (Page no 23) 4) Table 4. Substances present in industrial effluents. (Page no 24) 5) Table 5. Heavy Metals Found in Major Industries. (Page no 30) 6) Table 6. Comparative strengths of wastewater from industry. (Page no 30) 7) Table 7. Environmental conditions affecting degradation. (Page no 34) 8) Table 8: Bioremediation strategies. (Page no 35) 9) Table 9: Nutrient Agar Composition. (Page no 49) 10) Table 10: Simmon’s Citrate Agar Composition. (Page no 53) 11) Table 11: Starch agar medium. (Page no 59) 12) Table 12: Milk agar medium. (Page no 60) 13) Table 13: Macconkey agar medium. (Page no 61) 14) Table 14: Gelatin Agar medium. (Page no 62) 15) Table 15: Peptone Agar medium. (Page no 63) 16) Table 16: Waste water analysis. (Page no 67) 17) Table 17: Soil microorganisms. (Page no 68) 18) Table 18: Wastewater microorganisms. (Page no 68) 19) Table 19: Gram staining. (Page no 70) 20) Table 20: Biochemical Tests. (Page no 70) 21) Table 21: Acid production. (Page no 74) 22) Table 22: Enzyme assay. (Page no 74) 23) Table 23: ZnSO4 Tolerance. (Page no 76) 24) Table 24: KCrO4 Tolerance. (Page no 77) 7
- 8. 25) Table 25:MnSO4Tolerance. (Page no 78) 26) Table 26: HgCl2 Tolerance. (Page no 80) 27) Table 27: AlCl3 Tolerance. (Page no 81) 28) Table 28: Growth at different pH. (Page no 82) 29) Table 29: Growth at different Temperatures. (Page no 82) 30) Table 30: Effect of various concentration of phenol on growth curve of the organism. (Page no 83) 31) Graph 1: Effect of phenol on growth curve ( Page no 84) 8
- 9. LIST OF FIGURES,DIAGRAMSAND PHOTOS: 1) Diagram 1: Cotton Fabric Production & Associated Water Pollutants(Page no 22) 2) Figure 1: Citrate Utilization test.(Page no. 69) 3) Figure 2: Glucose Utlization Test( Page no. 69) 4) Figure 3: Sucrose Utlization Test ( Page no. 71) 5) Figure 4: Nitrate Utlization Test ( Page no. 71) 6) Figure 5: Casein Hydrolysis Test (Page no. 72) 7) Figure 6: Casein Hydrolysis Test (Page no. 72) 8) Figure 7: Starch Hydrolysis Test (Page no. 73) 9) Figure 8: ZnSO4Tolerance. (Page no. 75) 10) Figure 9: KCrO4Tolerance. (Page no. 75) 11) Figure 10: HgCl2Tolerance. (Page no. 79) 12) Figure 11: HgCl2Tolerance. (Page no. 79) 9
- 10. LIST OF ABBREVATIONS: 1)pH- negative log of H- ion concentration 2) ˚C- Degree celcius 3) MRL- Maximum resistivity levels 4) MIC- Minimum inhibitory concentration 5) Mg/L- Milligram per liter 6) COD- Chemical oxygen demand 7) BOD- Biochemical oxygen demand 8) As- Arsenic 9) Mn- Manganese 10) Ni- Nickel 11) Cd- Cadmium 12) Cu- Copper 13) CO- Cobalt 14) Cr-Chromium 15) Pb- Lead 16) Fe- Iron 17) Zn- Zinc 18) Hg- Mercury 19) Al- Aluminium 20) spp.- Species 21) µ- Micro 22) M- Molar 10
- 11. INTRODUCTION 11
- 12. INTRODUCTION: Our biosphere isunder constant threat from continuing environmental pollution. Impact on its atmosphere, hydrosphere and lithosphere by anthropogenic activities cannot be ignored. Man made activities on water by domestic, industrial, agriculture, shipping, radio-active, aquaculture wastes; on air by industrial pollutants, mobile combustion, burning of fuels, agricultural activities, ionization radiation, cosmic radiation, suspended particulate matter; and on land by domestic wastes, industrial waste, agricultural chemicals and fertilizers, acid rain, animal waste have negative influence over biotic and abiotic components on different natural ecosystems. Some of the recent environmental issues include green house effect, loss in bio-diversity, rising of sea level, abnormal climatic change and ozone layer depletion etc. In recent years, different approaches have been discussed to tackle man made environmental hazards. Clean technology, eco-mark and green chemistry are some of the most highlighted practices in preventing and or reducing the adverse effect on our surroundings. Among many engineering disciplines – Civil Engineering, Mechanical Engineering, Electrical Engineering etc., Textile Engineering has a direct connection with environmental aspects to be explicitly and abundantly considered. The main reason is that the textile industry plays an important role in the economy of the country like India and it accounts for around one third of total export. Out of various activities in textile industry, chemical processing contributes about 70% of pollution. It is well known that cotton mills consume large volume of water for various processes such as sizing, desizing, scouring, bleaching, mercerization, dyeing, printing, finishing and ultimately washing. Due to the nature of various chemical processing of textiles, large volumes of waste water with numerous pollutants are discharged. Since these streams of water affect the aquatic eco-system in number of ways such as depleting the dissolved oxygen content or settlement of suspended substances in anaerobic condition, a special attention needs to be paid. Thus a study on different measures which can be adopted to treat the waste water discharged from textile chemical processing industries to protect and safeguard our surroundings from possible pollution problem has been the focus point of many recent investigations.[5] 12
- 13. Sources of industrialwastewater: Iron and steel industry: The production of iron from its ores involves powerful reduction reactions in blast furnaces. Contamination of waste streams includes gasification products such as benzene, naphthalene, anthracene, cyanide, ammonia, phenols, cresols together with a range of more complex organic compounds known collectively as polycyclic aromatic hydrocarbons (PAH). Mines and quarries: Some specialized separation operations, such as coal washing to separate coal from native rock using density gradients, can produce wastewater contaminated by fine particulate haematite and surfactants. Oils and hydraulic oils are also common contaminants. Food industry: Wastewater generated from agricultural and food operations has distinctive characteristics, it is biodegradable and nontoxic, but that has high concentrations of biochemical oxygen demand (BOD) and suspended solids (SS). Complex organic chemicals industry: A range of industries manufacture or use complex organic chemicals. These include pesticides, pharmaceuticals, paints and dyes, petro- chemicals, detergents, plastics, paper pollution, etc. Waste waters can be contaminated by feed-stock materials, by-products, product material in soluble or particulate form, washing and cleaning agents, solvents and added value products such as plasticisers. Water treatment: Water treatment produces organic and mineral sludges from filtration and sedimentation. Ion exchange using natural or synthetic resins removes calcium, magnesium and carbonate ions from water, replacing them with hydrogen and hydroxyl ions. Regeneration of ion exchange columns with strong acids and alkalis produces a wastewater rich in hardness ions which are readily precipitated out, especially when in admixture with other wastewaters. 13
- 14. Agricultural waste: Pesticides arewidely used by farmers to control plant pests and enhance production, but chemical pesticides can also cause water quality problems. Farms with large livestock and poultry operations, such as factory farms, can be a major source of point source wastewater.[25] Hazards: Water pollution has number of effects. The effects could be classified as: 1] Effects on ecosystem 2] Effects on animal health 3] Effects on human health Virtually all types of water pollution are harmful to the health of humans and animals. Water pollution may not damage our health immediately but can be harmful after long term exposure. Different forms of pollutants affect the health of animals in different ways: Heavy metals from industrial processes can accumulate in nearby lakes and rivers. These are toxic to marine life such as fish and shellfish, and subsequently to the humans who eat them. Heavy metals can slow development; result in birth defects and some are carcinogenic. Industrial waste often contains many toxic compounds that damage the health of aquatic animals and those who eat them. Some of the toxins in industrial waste may only have a mild effect whereas other can be fatal. They can cause immune suppression, reproductive failure or acute poisoning. Microbial pollutants from sewage often result in infectious diseases that infect aquatic life and terrestrial life through drinking water. Microbial water pollution is a major problem in the developing world, with diseases such as cholera and typhoid fever being the primary cause of infant mortality. Organic matter and nutrients causes an increase in aerobic algae and depletes oxygen from the water column. This causes the suffocation of fish and other aquatic organisms. Sulfate particles from acid rain can cause harm the health of marine life in the rivers and lakes it contaminates, and can result in mortality. Suspended particles in freshwater reduces the quality of drinking water for humans and the aquatic environment for marine life. Suspended particles can often reduce the amount of sunlight penetrating the water, disrupting the growth of photosynthetic plants and micro-organisms. Some organochlorine pesticides (like DDT, BHC, Endrin) are known for bioaccumulative and biomagnifiable characters. [21] 14
- 15. Bioremediation: By definition, bioremediationis the use of living organisms, primarily microorganisms, to degrade the environmental contaminants into less toxic forms. It uses naturally occurring bacteria and fungi or plants to degrade or detoxify substances hazardous to human health and/or the environment. The microorganisms may be indigenous to a contaminated area or they may be isolated from elsewhere and brought to the contaminated site. Contaminant compounds are transformed by living organisms through reactions that take place as a part of their metabolic processes. Biodegradation of a compound is often a result of the actions of multiple organisms.[10] Types of Bioremediation: Biostimulation Bioaugmentation Intrinsic Bioremediation Advantages of bioremediation Bioremediation is a natural process and is therefore perceived by the public as an acceptable waste treatment process for contaminated material such as soil. Microbes able to degrade the contaminant increase in numbers when the contaminant is present; when the contaminant is degraded, the biodegradative population declines. Theoretically, bioremediation is useful for the complete destruction of a wide variety of contaminants. Many compounds that are legally considered to be hazardous can be transformed to harmless products. This eliminates the chance of future liability associated with treatment and disposal of contaminated material. Instead of transferring contaminants from one environmental medium to another, for example, from land to water or air, the complete destruction of target pollutants is possible. Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. This also eliminates the need to transport quantities of waste off site and the potential threats to human health and the environment that can arise during transportation. Bioremediation can prove less expensive than other technologies that are used for clean-up of hazardous waste.[10] 15
- 16.
- 17. AIMS & OBJECTIVES: 1)Collection of wastewater & soil sample from KoparKhairane Nullah. 2) Physiochemical Analysis of wastewater. 3) Isolation and characterization of soil microorganisms. 4) Study of heavy metal tolerance of soil isolates. 5) Optimization of various parameters for bioremediation. 17
- 18.
- 19. LITERATURE REVIEW: Textile Effluent: Sourcesand Causes of Generation of Textile Effluent: Textile industry involves wide range of raw materials, machineries and processes to engineer the required shape and properties of the final product. Waste stream generated in this industry is essentially based on water-based effluent generated in the various activities of wet processing of textiles. The main cause of generation of this effluent is the use of huge volume of water either in the actual chemical processing or during re-processing in preparatory, dyeing, printing and finishing. In fact, in a practical estimate, it has been found that 45% material in preparatory processing, 33% in dyeing and 22% are re-processed in finishing. The fact is that the effluent generated in different steps is well beyond the standard and thus it is highly polluted and dangerous.[5] Textile wastewater includes a large variety of dyes and chemicals additions that make the environmental challenge for textile industry not only as liquid waste but also in its chemical composition (Venceslau et al., 1994). Main pollution in textile wastewater came from dyeing and finishing processes. These processes require the input of a wide range of chemicals and dyestuffs, which generally are organic compounds of complex structure. Because all of them are not contained in the final product, became waste and caused disposal problems. Major pollutants in textile wastewaters are high suspended solids, chemical oxygen demand, heat, colour, acidity, and other soluble substances. The removal of colour from textile industry and dyestuff manufacturing industry wastewaters represents a major environmental concern. In addition, only 47% of 87 of dyestuff are biodegradable (Pagga and Brown, 1986). It has been documented that residual colour is usually due to insoluble dyes which have low biodegradability as reactive blue 21, direct blue 80 and vat violet with COD/BOD ratio of 59.0, 17.7, and 10.8, respectively.[32] The textile sector has a high water demand. Its biggest impact on the environment is related to primary water consumption (80–100 m3/ton of finished textile) and waste water discharge (115– 175 kg of COD/ton of finished textile, a large range of organic chemicals, low 19
- 20. biodegradability, colour, salinity).Therefore, reuse of the effluents represents an economical and ecological challenge for the overall sector (Li Rosi et al., 2007).[6] Table 1. Properties of Waste Water from Textile Chemical Processing: [5] Table 2. Composite textile industry wastewater characteristics: [2] Categorization of Waste Generated in Textile Industry: Textile waste is broadly classified into four categories, each of having characteristics that demand different pollution prevention and treatment approaches. Such categories are discussed in the following sections: 1. Hard to Treat Wastes This category of waste includes those that are persistent, resist treatment, or interfere with the operation of waste treatment facilities. Non-biodegradable organic or inorganic materials are the chief sources of wastes, which contain colour, metals, phenols, certain surfactants, toxic organic compounds, pesticides and phosphates. The chief sources are: Colour & metal-dyeing operation 20
- 21. Phosphates- Non-biodegradable organic materials- Since these types of textile wastes are difficult to treat, the identification and elimination of their sources are the best possible ways to tackle the problem. Some of the methods of prevention are chemical or process substitution, process control and optimization, recycle/reuse and better work practices 2. Hazardous or Toxic Wastes These wastes are a subgroup of hard to treat wastes. But, owing to their substantial impact on the environment, they are treated as a separate class. In textiles, hazardous or toxic wastes include metals, chlorinated solvents, non-biodegradable or volatile organic materials. Some of these materials often are used for non-process applications such as machine cleaning. 3. High Volume Wastes Large volume of wastes is sometimes a problem for the textile processing units. Most common large volume wastes include: • High volume of waste water • Wash water from preparation and continuous dyeing processes and alkaline wastes from preparatory processes. • Batch dye waste containing large amounts of salt, acid or alkali These wastes sometimes can be reduced by recycle or reuse as well as by process and equipment modification. 4. Dispersible Wastes: The following operations in textile industry generate highly dispersible waste: • Waste stream from continuous operation (e.g. preparatory, dyeing, printing and finishing) • Print paste (printing screen, squeeze and drum cleaning) • Lint (preparatory, dyeing and washing operations) • Foam from coating operations • Solvents from machine cleaning • Still bottoms from solvent recovery (dry cleaning operation) • Batch dumps of unused processing (finishing mixes) [5] 21
- 22. Diagram 1: CottonFabric Production & Associated Water Pollutants: [18] 22
- 23. Table 3.Effluent CharacteristicsFrom Textile Industry: [18] Heavy metals and its hazards: Metal pollution is a global concern. Input of chemical substances constitutes a great pollution burden on natural ecosystems. When considering their impact, it is necessary to distinguish between poisonous substances, that have a toxic effect on organisms even at low concentrations, and burdening substances that bring about undesirable changes in eco systems at higher concentration or load. The level of metals in all environments including air, water and soil are increasing in some cases to toxic levels with contribution from a wide variety of industrial sources. Heavy metals are roughly defined as elements having a density over 6g/cm3. Among these elements, Co, Cu, Mn, Ni, Se and Zn are important in small amounts. some metals such as Cu,Fe and Zn are essential at low concentrations and are toxic at high levels. The main cause of water pollution are household waste (As, Cr, Cu, Mn and Ni), coal fed power stations (As, Hg and Se), iron and steel production (Cr, Mo, Zn and Sb), and metal smelters ( Cd, Ni, Sb and Se). Of these metals, 25% are thought to enter rivers and lakes, and their surrounding soils are also heavily pollulated by these metals. 23
- 24. Table 4. Substancespresent in industrial effluents: [23] Metals in the environment can be divided into two classes, bioavailable (soluble, nonsorbed and mobile) and non-bioavailable (precipitated, complexed, sorbed and non-mobile). It is the bioavailable metal concentration that is taken up and is thus toxic to biological systems. Some metals like Hg and Pb are highly toxic but many other metals are also of concern, including As, Br, B, Cd, Cr, Cu, Ni, Mn, Se, Ti and Zn. Elevated metal concentration in the environment has a wide ranging on animals, plants and microbial species. Toxicity to an organism can be defined as the inherent potential or capacity of a material to cause adverse on living organisms and depends on the bioavailability of the toxicant. 24
- 25. In textile industrywet processing, a large number of chemicals are used, such as caustic soda, hydrogen peroxide, formic acid, sodium nitrate, azo dyes, direct dyes, reactive dyes, and mordants. Correia et al. (1994), reported that problems associated with textile processing effluents are heavy metal ions, which might rise from metal used in the dyeing process, or, in considerably higher amounts from metal containing dyes. For cotton, azoic, direct azo dye and mordant are used, which contains metals in complexed form. Azo and direct azo dye contains sodium salts, fixing agent and metallic compounds of Cu and Cr, whereas, the mordants are the metallic salts of Al, Cr and Fe, which are used on cotton silk fibre. Commonly used metal containing pigments are chrome yellow (Cr), zinc yellow (Zn), chrome oxide green (Cr) and iron blue pigment (Fe) in textile industries.[14] It can be seen that of all of the heavy metals chromium is the most widely used and discharged to the environment from different sources. However, it is not the metal that is most dangerous to living organisms. Much more toxic are cadmium, lead and mercury. These have a tremendous affinity for sulphur and disrupt enzyme function by forming bonds with sulphur groups in enzymes. Protein carboxylic acid (-CO2H) and amino (-NH2) groups are also chemically bound by heavy metals. Cadmium, copper, lead and mercury ions bind to cell membranes, hindering transport processes through the cell wall. Heavy metals may also precipitate phosphate bio-compounds or catalyse their decomposition. Mercury: Because of its toxicity, its mobilisation as methylated forms by anaerobic bacteria, and other pollution factors, mercury generates a great deal of concern as a heavy-metal pollutant. Mercury is found as a trace component of many minerals, with continental rocks containing an average of around 80 ppb, or slightly less, of this element. Cinnabar, red mercuric sulphide, is the chief commercial mercury ore. Metallic mercury is used as an electrode in the electrolytic generation of chlorine gas, in laboratory vacuum apparatuses and in other applications. Organic mercury compounds used to be widely applied as pesticides, particularly fungicides. Mercury enters the environment from a large number of miscellaneous sources related to human use of the element. These include discarded laboratory chemicals, batteries, broken thermometers, lawn fungicides, amalgam tooth fillings and pharmaceutical products. Sewage effluent sometimes contains up to 10 times the level of mercury found in typical natural waters. The toxicity of mercury was tragically illustrated in the Minamata Bay area of Japan during the period of 1953-1960. A total of 111 25
- 26. cases of mercurypoisoning and 43 deaths were reported among people who had consumed seafood from the contaminated bay. Among the toxicological effects of mercury were neurological damage, including irritability, paralysis, blindness, insanity, chromosome breakage and birth defects. The milder symptoms of mercury poisoning, such as depression and irritability, have a psychopathological character and may escape detection. The unexpectedly high concentrations of mercury found in water and in fish tissues result from the formation of soluble monomethylmercury ion, CH3Hg+, and volatile dimethylmercury, (CH3)2Hg, by anaerobic bacteria in sediments. Mercury from these compounds becomes concentrated in fish lipid (fat) tissue and the concentration factor from water to fish may exceed 103.[23] Zinc: Zinc is a trace element that is essential for human health. When people absorb too little zinc they can experience a loss of appetite, decreased sense of taste and smell, slow wound healing and skinsores. Zinc-shortages can even cause birth defects. Although humans can handle proportionally large concentrations of zinc, too much zinc can still cause eminent health problems, such as stomach cramps, skin irritations, vomiting, nausea and anaemia. Very high levels of zinc can damage the pancreas and disturb the protein metabolism, and cause arteriosclerosis. Extensive exposure to zinc chloride can cause respiratory disorders. In the work place environment zinc contagion can lead to a flu-like condition known as metal fever. This condition will pass after two days and is caused by over sensitivity. Zinc can be a danger to unborn and newborn children. When their mothers have absorbed large concentrations of zinc the children may be exposed to it through blood or milk of their mothers.[33] Manganese: Manganese effects occur mainly in the respiratory tract and in the brains. Symptoms of manganese poisoning are hallucinations, forgetfulness and nerve damage. Manganese can also cause Parkinson, lung embolism and bronchitis. When men are exposed to manganese for a longer period of time they may become impotent. A syndrome that is caused by manganese has symptoms such as schizophrenia, dullness, weak muscles, headaches and insomnia. 26
- 27. Manganese compounds existnaturally in the environment as solids in the soils and small particles in the water. Manganese particles in air are present in dust particles. These usually settle to earth within a few days. Humans enhance manganese concentrations in the air by industrial activities and through burning fossil fuels. Manganese that derives from human sources can also enter surface water, groundwater and sewage water. Through the application of manganese pesticides, manganese will enter soils. For animals manganese is an essential component of over thirty-six enzymes that are used for the carbohydrate, protein and fat metabolism. With animals that eat too little manganese interference of normal growth, bone formation and reproduction will occur. For some animals the lethal dose is quite low, which means they have little chance to survive even smaller doses of manganese when these exceed the essential dose. Manganese substances can cause lung, liver and vascular disturbances, declines in blood pressure, failure in development of animal foetuses and brain damage.When manganese uptake takes place through the skin it can cause tremors and coordination failures. Finally, laboratory tests with test animals have shown that severe manganese poisoning should even be able to cause tumor development with animals.[34] Chromium: Chromium(VI) is known to cause various health effects. When it is a compound in leather products, it can cause allergic reactions, such as skin rash. After breathing it in chromium(VI) can cause nose irritations and nosebleeds. Other health problems that are caused by chromium(VI) are: - Skin rashes - Upset stomachs and ulcers - Respiratory problems - Weakened immune systems - Kidney and liver damage - Alteration of genetic material - Lung cancer - Death The health hazards associated with exposure to chromium are dependent on its oxidation state. The metal form (chromium as it exists in this product) is of low toxicity. The 27
- 28. hexavalent form istoxic. Adverse effects of the hexavalent form on the skin may include ulcerations, dermatitis, and allergic skin reactions. Inhalation of hexavalent chromium compounds can result in ulceration and perforation of the mucous membranes of the nasal septum, irritation of the pharynx and larynx, asthmatic bronchitis, bronchospasms and edema. Respiratory symptoms may include coughing and wheezing, shortness of breath, and nasal itch.[35] Aluminium : The water-soluble form of aluminum causes the harmful effects, these particles are called ions. They are usually found in a solution of aluminum in combination with other ions, for instance as aluminum chlorine. The uptake of aluminum can take place through food, through breathing and by skin contact. Long lasting uptakes of significant concentrations of aluminum can lead to serious health effects, such as: - Damage to the central nervous system - Dementia - Loss of memory - Listlessness - Severe trembling The concentrations of aluminum appear to be highest in acidified lakes. In these lakes the number of fish and amphibians is declining due to reactions of aluminum ions with proteins in the gills of fish and the embryo's of frogs. High aluminum concentrations do not only cause effects upon fish, but also upon birds and other animals that consume contaminated fish and insects and upon animals that breathe in aluminum through air. The consequences for birds that consume contaminated fish are eggshell thinning and chicks with low birth- weights. The consequences for animals that breathe in aluminum through air may be lung problems, weight loss and a decline in activity. Another negative environmental effect of aluminum is that its ions can react with phosphates, which causes phosphates to be less available to water organisms. High concentrations of aluminum may not only be found in acidified lakes and air, but also in the groundwater of acidified soils. There are strong indications that aluminum can damage the roots of trees when its located in groundwater.[36] 28
- 29. Lead: Inorganic lead arising from a number of industrial and mining sources occurs in water in the +2 oxidation state. Lead from leaded gasoline used to be a major source of atmospheric and terrestrial lead, much of which eventually enters natural water systems. Despite greatly increased total use of lead by industry, evidence from hair samples and other sources indicates that body burdens of this toxic metal have decreased during recent decades. This may be the result of less lead used in plumbing and other products that commonly come in contact with food or drink. Acute lead poisoning in humans causes severe dysfunction in the kidneys, reproductive system, liver, and the brain and nervous system. [23] 29
- 30. Table 5. HeavyMetals Found in Major Industries [23] Table 6. Comparative strengths of wastewater from industry: [23] 30
- 31. Bioremediation: Heavy metals contaminatethe aquatic environment and sources of potable water because of their known accumulation in food chain and their persistence in nature, when they are discharged in small quantities by numerous industrial activities. The need for economical, effective and safe methods for removing heavy metals from waste water has resulted in the search for unconventional materials that may be useful in reducing the levels or accumulation of heavy metals in the environment. The newly developed metal sequestering properties of certain types of microbial biomass of fungi, yeast, bacteria, algae and plants, offer considerable promise in terms of providing a cost effective process, for removing toxic heavy metals from the industrial effluents. Microbial activities in the natural environment are the main process that remove, mobilize or detoxify heavy metals and radionuclides. The activities can be harnessed to clean up toxic metal wastes, before they enter the wider environment and such biotechnological processes are used to control pollution from diverse sources. The study of microorganisms that are capable of resisting and surviving in polluted environments provides the basic knowledge of bioremediation. Natural and genetically engineered species are used for protection and remediation of organic contaminants. Due to their non-biodegradability heavy metals cannot be treated biologically in situ and must instead be extracted from contaminating streams. The utilization of microbial biomass for removal of metals from industrial waste water and polluted waters is a well recognized method of remediation. Metals adversely influence microorganisms, effecting their growth, morphology and biochemical activities, resulting in decreased biomass and diversity. Despite these toxic stresses, numerous microorganisms develop metal resistance and detoxification mechanisms, which includes volatilization, extracellular precipitation and exclusion, intracellular sequestration, membrane associated metal pumps, entrapment of cellular component, cation exchange or complextion, adsorption and degradation of organometals. The term biosorption is used to encompass uptakes by the whole biomass living or dead, via physiochemical mechanisms, adsorption, coordination and covalent bonding and ion exchange. Where living biomass is used, metabolic uptake mechanisms may also contribute to the process. The main chemical groups in biomass, which are able to partake in biosorption are, electro negative groups such as hydroxyl or sulphydrl groups, anionic groups such as carboxyl or phosphate groups and nitrogen containing groups such as amino groups. Dead 31
- 32. microbial biomass, includingfungal mycelium, can also adsorb metal ions, often in larger quantities than living biomass. Biodegradation is the microbially mediated decomposition of not only organic but also xenobiotic pollutants. Biodegradation can be a minor change in the molecule, fragmentation or complete mineraization. A range of current approaches to biodegradation are available, starting from identification of microbial strains that have the ability to break down or alter environmental pollutants such as petroleum products, polychlorinated biphenyls, halogenated aromatic compounds,toluene, and nitrogen containing heterocyclic aromatic analogues of polycyclic aromatic hydrocarbons to screening for these isolates for degradation abilities by providing target substances as carbon source or electron acceptor. [14] FACTORS OF BIOREMEDIATION: The control and optimization of bioremediation processes is a complex system of many factors. These factors include: the existence of a microbial population capable of degrading the pollutants; the availability of contaminants to the microbial population; the environment factors (type of soil, temperature, pH, the presence of oxygen or other electron acceptors, and nutrients). MICROBIAL POPULATIONS FOR BIOREMEDIATION PROCESSES Microorganisms can be isolated from almost any environmental conditions. Microbes will adapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in water, with an excess of oxygen, and in anaerobic conditions, with the presence of hazardous compounds or on any waste stream. The main requirements are an energy source and a carbon source. Because of the adaptability of microbes and other biological systems, these can be used to degrade or remediate environmental hazards. We can subdivide these microorganisms into the following groups: Aerobic.: In the presence of oxygen. Examples of aerobic bacteria recognized for their degradative abilities are Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium. These microbes have often been reported to degrade pesticides and hydrocarbons, both alkanes and polyaromatic compounds. Many of these bacteria use the contaminant as the sole source of carbon and energy. Anaerobic.: In the absence of oxygen. Anaerobic bacteria are not as frequently used as aerobic 32
- 33. bacteria. There isan increasing interest in anaerobic bacteria used for bioremediation of polychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solvent trichloroethylene (TCE), and chloroform. Ligninolytic fungi.: Fungi such as the white rot fungus Phanaerochaete chrysosporium have the ability to degrade an extremely diverse range of persistent or toxic environmental pollutants. Common substrates used include straw, saw dust, or corn cobs. Methylotrophs.: Aerobic bacteria that grow utilizing methane for carbon and energy. The initial enzyme in the pathway for aerobic degradation, methane monooxygenase, has a broad substrate range and is active against a wide range of compounds, including the chlorinated aliphatics trichloroethylene and 1,2-dichloroethane. For degradation it is necessary that bacteria and the contaminants be in contact. This is not easily achieved, as neither the microbes nor contaminants are uniformly spread in the soil. Some bacteria are mobile and exhibit a chemotactic response, sensing the contaminant and moving toward it. Other microbes such as fungi grow in a filamentous form toward the contaminant. It is possible to enhance the mobilization of the contaminant utilizing some surfactants such as sodium dodecyl sulphate (SDS). ENVIRONMENTAL FACTORS: Although the microorganisms are present in contaminated soil, they cannot necessarily be there in the numbers required for bioremediation of the site. Their growth and activity must be stimulated. Biostimulation usually involves the addition of nutrients and oxygen to help indigenous microorganisms. These nutrients are the basic building blocks of life and allow microbes to create the necessary enzymes to break down the contaminants. All of them will need nitrogen, phosphorous, and carbon. Carbon is the most basic element of living forms and is needed in greater quantities than other elements. In addition to hydrogen, oxygen, and nitrogen it constitutes about 95% of the weight of cells. Phosphorous and sulfur contribute with 70% of the remainders. The nutritional requirement of carbon to nitrogen ratio is 10:1, and carbon to phosphorous is 30:1.[10] 33
- 34. Table 7. Environmentalconditions affecting degradation: [10] Types of Bioremediation: The main types of bioremediation are as follows: Biostimulation -- Nutrients and oxygen - in a liquid or gas form - are added to contaminated water or soil to encourage the growth and activity of bacteria already existing in the soil or water. The disappearance of contaminants is monitored to ensure that remediation occurs. Bioaugmentation -- Microorganisms that can clean up a particular contaminant are added to the contaminated soil or water. Bioaugmentation is more commonly and successfully used on contaminants removed from the original site, such as in municipal wastewater treatment facilities. To date, this method has not been very successful when done at the site of the contamination because it is difficult to control site conditions for the optimal growth of the microorganisms added. Scientists have yet to completely understand all the mechanisms involved in bioremediation, and organisms introduced into a foreign environment may have a hard time surviving. Intrinsic Bioremediation -- Also known as natural attenuation, this type of bioremediation occurs naturally in contaminated soil or water. This natural bioremediation is the work of microorganisms and is seen in petroleum contamination sites, such as old gas stations with leaky underground oil tanks. Researchers are studying whether intrinsic bioremediation happens in areas with other types of chemical contamination. Application of this technique requires close monitoring of contaminant degradation to ensure that environmental and human health are protected. All three types of bioremediation can be used at the site of contamination (in situ) or on contamination removed from the original site (ex situ). In the case of contaminated soil, 34
- 35. sediments, and sludges,it can involve land tilling in order to make the nutrients and oxygen more available to the microorganisms. [26] Table 8: Bioremediation strategies: [10] 35
- 36. Microorganisms and itsrole: Level of microbial resistance is important criteria for evaluation of an isolate from metal contaminated site, for use in bioremediation. Lead: Konopka et al (1999) analyzed Pb contaminated soil for microbial community diversity potential microbial activity and metal resistance in three soils, having Pb in the range of 0.00039-48.00 mM/kg of soil. In all samples, 10-15% of the total culturable bacteria where Pb resistant and had MRL of Pb of 100-150 µM. The Pb resistant isolates were all gram positive that were members of the genus bacillus Coryneform or Actinomycetes. Pb resistant bacteria were isolated from P contaminated sites of silver valley, Iadho (United States), containing 17.20-108.00 µM Pb/g dry soil. Pb resistant genera isolated included Pseudomonas, Bacillus, Corynebacterium and Enterobacter species. Among these isolates, Pseudomonas marginalis had a higher resistance at 2.5 mM total Pb, as compared to 0.625 mM for Bacillus megaferium. However, resistance to soluble Pb was much low, 0.3 and 0.1 mM, respectively. The degree of Pb resistance, and the mechanism of resistance for these two isolates correspond with their environmental exposure. Pb biosorption by Pseudomonas aeruginosa PU21 was investigated along with effects of environmental factorsand growth conditions. The results showed that, at pH 5.5, resting cells were able to uptake Pb, 110mg/g dry cell. The resting cells held optimal Pb absorption capacity at the early stationary phase. By adjusting pH to 2.0, Pseudomonas aeruginosa PU21 biomass removed 98% Pb. Niu te al. (1993), studied removal of lead ions from aqueous solution by adsorption on non living Penicillum Chrysogenum. Biosorption of the Pb2- ion was strongly affected by pH. Within a pH range of 4.0-5.0 the saturated sorption was 116mg/g dry biomass. This strain was applied to treat industrial lead contaminated wastewater, in which lead concentration was reduced to less than 1mg/l, after treatment. Chromium: Cr is the widespread industrial and nuclear waste, and several waste dumps serve as a reservoir for Cr tolerant and accumulator microorganisms. A consortium of bacteria with tolerance towards high concentrations of Cr 6- (up to 2,500mg/L), and other toxic metal was obtained from metal refinishing waste water in Chengdu, People ’s Republic of china. The 36
- 37. consortium comprised ofa range of Gram positive and Gram negative rods, and these rods had capacity to reduce Cr6- to Cr3- . Cr removal was 80-90% from concentrations ranging from 50-2000mg/L. Removal was not totally passive because killed and gamma irradiated cells could immobilize much of the metal concentration. This consortium was named sulphate reducing bacteria SRB III. Several chromate reducing bacteria have been reported, from the genera Pseudomonas, Achromobacter, Aeromonas, Basillus, Enterobacter, Escherichia and Micrococcus. Experiments with free cell biomass (cells plus exoploysaccharides) of rhizobium BJVr12 (mungbean isolate) showed that amount of Cr 3- ion sorbed was influenced by amount of biomass to Cr3- concentration ratio and time of contact. A reduction rate of 49.7% for Cr3- was observed for free cells, 95.6% for cells immobilized in ceramic bead and 94.6% for cells in aquacell ( a porous cellulose career with a charged surface), was achieved after 48 hours under shaking conditions. Manganese: A large number of bacteria are reported to be involved in the oxidation of Mn2 and include the genera Leptothrix, Pedomicrobium, Hypomicrobium, Caulobacter, Arthrobacter, Micrococcus, Bacillus, Chromobacterium, Pseudomonas, Vibrio, Oceanospiralium. Biosorption of toxic metals( Mn, Cu, Ni, Pb) by species of Arthrobacter showed highest value of specific uptake (mg/g) in case of Mn(406) followed by Cu(148), Pb(30), and the least value observed was that of Ni, which was 13 mg/g. Pyrobaculum islandicum is a hyperthermophilic microorganism, which has the capacity to reduce Mn(IV) oxide to white precipitate of manganese carbonate, when washed cell suspension was used at 100 0C. Four strains of Pseudomonas putida, Pseudomonas putida 06909, Pseudomonas putida 0690 s22x, Pseudomonas putida 06909s21x, Pseudomonas putida 0690 s23 were determined for their MRL for Mn. All four strains showed MRL of more than 2.5mM. Saccharomyces cerevisiae, exhibits greater metal accumulation capacity, as compared to bacteria nd accumulate Mn 2 cation. Immobilized Rhizopus arrhizus has also been shown to accumulate Mn2. 37
- 38. Zinc: Alcaligenes eutrophus andPseudomonas aeruginosa are Zn tolerant Gram negative bacteria.Strains of Pseudomonas putida, Pseudomonas putida 06909, Pseudomonas putida 06909s21x Pseudomonas putida 0690s22x, , Pseudomonas putida 0690 s23 were evaluated for MRLof Zn on mannitol glutamate agar. These strains of Pseudomonas putida exhibited different values values, which were, Pseudomonas putida 06909(11.5mM), Pseudomonas putida 06909s21x(11.5mM), Pseudomonas putida 0690s22x(7.0mM),Pseudomonas putida 0690s23(10.5mM). At Ingurtosa (southwestern Sardinia, Italy), due the mining in the past, mine tailings were deposited along the Rio Naracaini creek. Rio Naracainli stream water was highly polluted by heavy metals like Zn, Pb, Cd, Fe, Mn, Cu,and Ni. Podda et al.reported natural bioremediation of heavy metals by a consortium of a photosynthetic filamentous bacterium, classified as scytonema spp, strain ING-1, associated with microalga chlorella spp, strain SA1. This microbial community was responsible for the natural polishing of heavy metals in the water of stream Rio Naracainli by coprecipitation with hydrozincite [Zn 5(CO3)2(OH)6]. Scytonema spp was responsible for formation of hydrozinciteduring photosynthesisof chlorella spp, thus removing Zn2+ in addition to Pb, Cd, Ni and Cu.[14] Metal Tolerance Mechanisms In high concentrations, heavy metal ions react to form toxic compounds in cells (Nies, 1999). To have a toxic effect, however, heavy metal ions must first enter the cell. Because some heavy metals are necessary for enzymatic functions and bacterial growth, uptake mechanisms exist that allow for the entrance of metal ions into the cell. There are two general uptake systems — one is quick and unspecific, driven by a chemiosmotic gradient across the cell membrane and thus requiring no ATP, and the other is slower and more substrate-specific, driven by energy from ATP hydrolysis. While the first mechanism is more energy efficient, it results in an influx of a wider variety of heavy metals, and when these metals are present in high concentrations, they are more likely to have toxic effects once inside the cell (Nies and Silver, 1995). To survive under metal-stressed conditions, bacteria have evolved several types of mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999).[1] 38
- 39.
- 40. MATERIALS AND METHODS: A]Collection of wastewater and soil sample: Location: Wastewater and soil sample was collected from koperkhairane nullah near MIDC industrial area besides Furnace fabrica Ltd & Alok industry- Navi Mumbai. Procedure: Water sample was collected in a plastic bottle and soil sample was collected in sterile Petri plates. pH and temperature of the water sample was checked. Water sample and soil sample was transported to college laboratory. Water sample and soil sample were kept in refrigerator (40C) until further analysis was carried out. B] Waste water analysis: 1] Determinaton of total hardness of water: AIM: To determine the hardness of water by EDTA titrimetric method. Principle: EDTA forms chelated soluble complex when added to a solution of certain metal cations. If small amount of dye such as Eriochrome black T or calmagite is added to an aqueous solution containing calcium and magnesium ions at pH 10.0 the solution turns wine red .If EDTA is added as a titrant, the calcium and magnesium will be complexed. When all of the calcium and magnesium has been complexed, the solution turns wine red to blue, marking the end point of titration. Requirements: Equipments: Conical flask, burette, beakers etc. 40
- 41. Reagents: Buffer solution: Dissolve 16.9 gm of ammonium chloride in 143 ml conc Ammonium hydroxide and dilute to 250ml with distilled water. Eriochrome black T indicator Ethylene diamino tetra acetic acid [EDTA][0.01M]:weigh 3.723gm of disodium EDTA and dissolved in 50ml of distilled water and make the volume to 1000ml and store in a brown bottle. Standard calcium :weigh 1gm of calcium carbonate powder into a 500ml conical flask. Place a funnel in the neck of the flask and slowly add conc. HCL drop wise and shake the flask repeatedly until the entire CaCO3 dissolved . Add 200ml D/W and boil for few minutes to expel CO2. Procedure: Take 10ml of sample in a conical flask. Add 1ml of buffer solution and shake well. Add 1 to 2 drops of Eriochrome black T indicator, shake well to get wine red colour. Titrate it against EDTA by adding drop wise from the burette until the colour changes wine red to blue. Calculation: 1ml of 1m of EDTA =40.08 of Ca+2 Since atomic weight of Ca+2 is 40.08, 1ml of 0.01M=0.4008mg of Ca+2 When standardization is done with Std .Calcium, Hardness(EDTA) as mg Ca+2/L= BR x Normality of EDTA x 40.08 x1000 10[volume of sample taken] = BR x 0.01 x 40.08 x1000 10 = BR x 40.08mg Ca+2/L 41
- 42. 2] Determination oftotal alkalinity of water: Principle: Generally pH of water remains neutral. Alkalinity of water represents the presence of hydroxyl ions[-OH] in water; hence, it is capacity of water to neutralize a strong acid. In natural or waste water alkalinity is due to the presence of free hydroxyl ions which cause through hydrolysis of salts by weak acids and strong bases (e.g. carbonate and bicarbonates). Requirements: Equipments: conical flasks, titration assembly etc. HCL solution(0.1N) Methylene orange indicator Procedure: Take 50ml of water sample in flask and add 2-3 drops of methyl orange into it. Clour turns to orange . Transfer 0.1N HCL solution ito burette in titration assembly and titrate with the water sample[methylene orange added] untl yellow colour changes to pink. Note the end point. Calculation: Methylene orange alkalinity (mg/litre) =volume of 0.1 N HCL solution used as titrant x 1000 Volume of water sample 3] Determination of chlorine in water: Principle: The potable water is chlorinated to make the water free from microorganisms. However, some times the concentration of chloride ions in water is increased than what is normally required. Apart from this water also receives chloride ions from multifarious sources. The chloride ions (Cl-) can be estimated by titrating with silver nitrate solution. 42
- 43. Requirements: Equipments : conical flask,pipette, titration assembly etc Reagents: Silver nitrate (AgNO3) solution (0.025N) –dissolve 3.4g dried AgNO3 in distilled water and dilute to make 1 litre.store in coloured bottle Potassium dichromate solution (5%)- dissolve 5g of K2Cr2O7 in 100ml of distilled water. Procedure: Take 50ml of sample in a conical flask and 2ml of K2Cr2O7 solution. Pour 0.025N AgNO3 solution into burette set with titration assembly. Titrate the water sample with AgNO3 solutio until reddish tinge appears. Note the end point (AgNO3 reacts with Cl- ions and forms very slightly solble white precipitate of AgCl2(silver chloride). Free silver ions (Ag++) react with chromate ions (Cr2O7) to form silver chromate of reddish rown colour). Calculation: Chloride (mg/litre) =volume of AgNO3 solution x 1000 x35.5 Volume of the water sample used 43
- 44. 4] Determination ofchemical oxygen demand(COD) of water: Principle: Due to gradually increasing population the number of industries is also increasing. This results in increased pollution. The chemically oxidisable organic substances discharged in water depletes the amount of oxygen. Estimation of BOD alone could not give the exact idea of pollutants present in water, therefore , COD is estimated. COD refers to the oxygen consumed by the oxidisable organic substances. The values of COD cannot be compared directly with that of BOD. The chemical oxidants such as potassium dichromate (K2Cr2O7) or potassium permanganate (KMnO4) are used to measure the oxidisability of the organic matter of water where the oxidants oxidize the constituents (or the hydrogen but not nitrogen). Then potassium iodide (KI) is added. The excess amount of oxygen reacts with KI and liberates equal amount of iodine. By using starch indicator, iodine is titrated with sodium thiosulfate and amount is estimated. Requirements: Potassium dichromate solution (0.1N): 3.676gm in 1 litre of distilled water. Sodium thiosulphate (0.1N): 15.811gm of sodium thiosulfate in 2 litre of distilled water. Sulphuric acid (2M): 10.8 ml of concentrated H2SO4 in 100ml of distilled water. Potassium iodide solution (10%) Starch solution (1%),water bath,conical flask,titration assembly etc. Procedure: Take three 100m conical flask and pour 50ml of water sample in each. Simultaneously run distilled water blanks standards (also in triplicates) Add 5ml of K2Cr2O7 solution in each of the six flask. Keep the flask in waterbath at 1000C (boiling temperature) for one hour. 44
- 45. Allow the sample to cool for 10 minutes. Add 5ml of potassium iodide in each flask. Add 10ml of H2SO4 in each flask. Titrate the contents of each flask with 0.1M sodium thiosulphate until the appearance of pale yellow colour. Add 1 ml of starch solution to each flask (solution turns blue). Titrate it again with 0.1 M sodium thiosulfate until the blue colour disappears. Result: COD of sample mg/litre= 8 x C x(A-B) S C = concentration of titrant (mmol/litre) A = volume of titrant used for blank (ml) B = volume of the totrant used for sample (ml) S = volume of water sample taken (ml) 5] Determination of dissolved oxygen (DO) of water: Some amount of oxygen is dissolved in water which is used by the aquatic plants and animals. The sources of dissolved oxygen in water are the autotrophic aquatic plants which as a result of photosynthesis evolve oxygen,and air where from oxygen is dissolved in water depending o salinity, temperature and water movement. Moreover, in an oligotrophic lake the amount of dissolved nutrients salts remains low, therefore ,it supports sparse plant and animal lives. Tis results in high dissolved oxygen gradually increasing with depth. In addition, in eutrophic water reservoirs e.g. lakes, ponds, pools, etc. the organic nutrients accumulate abundantly which in turn are subjected to microbial decomposition. More growth of microorganisms, plants and animals depletes oxygen. 45
- 46. Dissolved oxygen ismeasured by titrimetric method. The theory behind this method is that the dissolved oxygen combines with magnous hydroxide which in turn liberates iodine (equivalent to oxygen fixed) after acidification with H 2SO4. The iodine can be titrated with sodium thiosulfate solution by using starch indicator. Requirements: BOD bottles (250 ml capacity),pippetes,titration set etc. Alkaline KI solution: Dissolve 100g of KOH and 50g of KI in 200ml of distilled water. Sodium thiosulphate (0.025N) Manganese sulfate(MnSO44H2O) solution: Dissolve 100g of manganese sulfate in 200ml of distilled water, filter and keep in stoppered bottle. Starch indicator: Dissolve 5g of starch in 100ml hot distilled water(boiled) and a few drops of formaldehyde(HCHO). Concentrated H2SO4 Procedure: Collect water sample in a BOD glass bottle (250ml) in such a way that water bubble should not come out. Pipette separately 2ml of manganese sulfate and 2ml of alkaline iodine-azide solutions. Add these solutions in succession at the bottom of bottle and place the stopper of bottle. Shake the bottle upside down for about 6-8 times .There develops brown precipitate. Leave the bottle for few minutes , the precipitate settles down. Add 2ml of concentrated H2SO4 in the bottle. Shake properly so that brown precipitate may dissolve. Take a clean flask and pour 50 ml of this water sample. Titrate it against 0.025 N sodium thiosulphate solution taking in a burette until pale starw colour develops. Add 2 drops of starch solution to the flask. Colour of contents chages from ple to blue. 46
- 47. Again titrate against thiosulfate solution until the blue colour disappears. Note the volume of sodium thiosulfate solution used in titration. Calculation: Calculate the amount of dissolve oxygen (DO) (mg/litre) by sing the formula: DO (mg/l) = 8 x 1000 x N x v V Where, V = volume of water sample for titration v = volume of sodium thiosulfate (titrant) N = normality of the titrant 8 = it is a constant since 1ml of 0.025N sodium thiosulfate solution is equivalent to 0.2 mg oxygen. 6] Determination of biological oxygen demand (BOD) of water : Principle: Biochemical Oxygen Demand (BOD) is the amount of oxygen, expressed in mg/L or parts per million (ppm), that bacteria take from water when they oxidize organic matter. The carbohydrates (cellulose, starch, sugars), proteins, petroleum hydrocarbons and other materials that comprise organic matter get into water from natural sources and from pollution. They may be dissolved, like sugar, or suspended as particulate matter, like solids in sewage. Organic matter can be oxidized (combined with oxygen) by burning, by being digested in the bodies of animals and human beings, or by biochemical action of bacteria. Because organic matter always contains carbon and hydrogen, oxidation produces carbon dioxide (the oxygen combining with the carbon) and water (the oxygen combining with the hydrogen). Bacteria in water live and multiply when organic matter is available for food and oxygen is available for oxidation. About one-third of the food bacteria consumed becomes the solid organic cell material of the organisms. The other two-thirds is oxidized to carbon dioxide and water by the biochemical action of the bacteria on the oxygen dissolved in the water. To determine BOD, the amount of oxygen the bacteria use is calculated by comparing the amount left at the end of five days with the amount known to be present at the beginning, whereas the same can 47
- 48. be incubated at270C for 3 days in tropical and subtropical regions where metabolic activities are higher. At room temperature, the amount of oxygen dissolved in water is 8 mg/L. At freezing, it increases to 14.6 mg/L; it also increases at high barometric pressures (low altitudes). At the boiling point, the solubility of oxygen is zero. [13][12] Requirements: BOD-free water BOD bottles ,Erlenmeyer flask, pipette, BOD incubator, pH meter Phosphate buffer solution (pH 7.4) Allythiourea (0.5%) Alkaline KI solution: Dissolve 100g of KOH and 50g of KI in 200ml of distilled water. Sodium thiosulphate (0.025N) Manganese sulfate(MnSO44H2O) solution: Dissolve 100g of manganese sulfate in 200ml of distilled water, filter and keep in stoppered bottle. Starch indicator: Dissolve 5g of starch in 100ml hot distilled water(boiled) and a few drops of formaldehyde(HCHO). Concentrated H2SO4 Procedure: Add 1nacid/1N alkali in the water sample to adjust the pH to 7.0 Gently transfer this water inti BOD bottles so as bubbles should not come out. Add 1 ml of allylthiourea to each bottle to avoid nitrification. Measure dissolved oxygen following the steps as described for dissolved oxygen Incubate the other BOD bottle at 270C for 3 days in a BOD incubator. Measure the amount of oxygen as done earlier. Calculation: Calculate the BOD of water by using the following formula: BOD (mg/l) = D1-D2 Where, D1 = initial dissolved oxygen (mg/l) in the first sample D2 = dissolved oxygen (mg/l) in the second sample after 3 days of incubation. [15] 48
- 49. C] ISOLATION OFMICROORGANISMS: Suspensions of the soil sample were prepared using physiological saline. Spread plate technique on Nutrient Agar plates was used for the isolation of microorganisms. Requirements: Nutrient Agar medium. Composition/litre: SR NO. COMPOSITION g/l 1. AGAR 15.0 2. PEPTONE 5.0 3. NaCl 5.0 4. YEAST EXTRACT 2.0 5. BEEF EXTRACT 1.0 Table 9: Nutrient Agar Composition. The pH is adjusted to 7.4(+/-) 0.2 at 250C. Preparation: Add components to Distilled water and bring the volume to 1.0L. Mix thoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15 mins at 15 psi pressure and -1210C. Pour into sterile Petri plates. Physiological saline. Add 0.85g of NaCl in 100ml Distilled water. Autoclave for 15 mins at 15 psi pressure and -1210C Procedure: 0.1ml of soil suspensions were spread onto the Nutrient Agar plates under aseptic conditions using a sterile glass spreader. These Petri plates were incubated overnight at room temperature. 49
- 50. D] CHARACTERIZATION OFTHE MICROORGANISMS: GRAM STAINING: Gram staining (or Gram's method) is an empirical method of differentiating bacterial species into two large groups: Gram-positive and Gram-negative, based on the chemical and physical properties of their cell walls. The Gram stain named after the Danish scientist Hans Christian Gram, is almost always the first step in the identification of a bacterial organism. Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50- 90% of cell wall), which stains purple while Gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the periplasmic space. There are four basic steps of the Gram stain, which include applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture, followed by the addition of a trapping agent (Gram'siodine), rapid decolorization with alcohol or acetone, and counterstaining with safranin. Basic fuchsin is sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly employed as a counterstain. Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – ) ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple. Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to as a mordant, but is a trapping agent that prevents the removal of the CV-I complex and therefore color the cell. After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color. Counterstain, which is usually positively charged safranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink or red color. Requirements: Nutrient Agar slants of the isolates. 50
- 51. Reagents: Crystal violet, Gram’s iodine, 95% ethyla alcohol and safranin. Eqiupments: Inoculating loop, glass slides, Bunsen burner, staining tray,bibulous paper, lens paper, immersion oil and microscope. Method: A bacterial suspension is prepared and loopful of suspension is used to make a smear on a clean glass slide under sterile conditions. The smear is allowed to air dry, then it is heat fixed and the slide is transferred to a staining tray. On the tray, smear is gently flood with crystal violet for 1 min.Wash briefly in water to remove excess crystal violet. Flood with Gram’s iodine 1 min. Wash briefly in water, to remove excess of stain. Decolourise with 95% ethanol until the moving dye front has passed the lower edge of the section. Wash immediately in tap water. Counterstain with safranine for 45 sec. and wash with tap water. The slide is blotted dry using bibulous paper and examined under oil immersion lens of microscope.[37] E] BIOCHEMICAL CHARACTERIZATION: 1]Catalase test: Catalase is a common enzyme found in nearly all living organisms that are exposed to oxygen, where it functions to catalyze the decomposition of hydrogen peroxide to water and oxygen. H2O2 ------catalase--------> H2O + O2 Catalase test is used to detect the presence of catalse enzyme. Hydrogen peroxide is formed by some bacteria as an oxidative end product of the aerobic breakdown of sugars. If allowed to accumulate it is highly toxic to bacteria and can result in cell death. Catalase either decomposes hydrogen peroxide or oxidizes secondary substrates, but it has no effect on other peroxides. 51
- 52. Catalase production isindicated bubbles of free oxygen gas upon addition of substrate to a culture. The absence of bubble formation is a negative test. Requirements: Nutrient Aagr slants of the soil isolates. 3% Hydrogen peroxide solution. Equipments: Inoculating loop, Cavity slide. Method: A drop of 3% Hydrogen peroxide solution is placed onto a watch glass. A loopful of culture is taken out under sterile conditions and placed on the drop and results are observed. [38] 2] Citrate Utilization test: Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source. Simmons citrate agar contains sodium citrate as the sole source of carbon, ammonium dihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicator bromthymol blue. Organisms which can utilize citrate as their sole carbon source use the enzyme citrase or citrate-permease to transport the citrate into the cell. These organisms also convert the ammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates an alkaline environment in the medium. At pH 7.5 or above, bromthymol blue turns royal blue. At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium turns blue, the organism is citrate positive. If there is no color change, the organism is citrate negative. Some citrate negative organisms may grow weakly on the surface of the slant, but they will not produce a color change. Requirements: Simmon’s Citrate Agar slants. Composition/l: 52
- 53. SR NO. COMPOSITION g/l 1. AMMONIUM DIHYDROGEN 1.0 PHOSPHATE 2. DIPOTASSIUM PHOSPHATE 1.0 3. NaCl 5.0 4. SODIUM CITRATE 2.0 5. MAGNESIUM SULFATE 0.2 6. AGAR 15.0 7. BROMOTHYMOL BLUE 0.08 Table 10: Simmon’s Citrate Agar Composition. Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner. Method: A loopful of culture is taken and streaked on Simmon’s citrate agar slant. After 24 hr. incubation period, results are observed. [39] 3] Nitrate Utilization Test: Nitrate may be reduce to multiple compounds by two processes. Anaerobic respiration and denitrification. In anaerobic respiration the bacterium uses nitrate as it's terminal electron acceptor, reducing nitrate to a variety of compounds, while denitrification reduces nitrate solely to molecular nitrogen. Sulfanilic acid and dimethyl 1-naphthylamine are added to detect nitrite, which will complex with these molecules forming a red color. If no red color is observed there are two possibilities; the nitrate has not been reduced, or it has been reduced further than nitrite. To differentiate between these two possibilities, zinc powder is added, which will complex with nitrate forming a red color. Thus if the tube turns red after zinc, 53
- 54. nitrate has notbeen reduced and the result is negative. If no red color is observed then the nitrate has been reduced further than nitrite and the result is positive. Requirements: Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner. Compounds: Nitrate broth, sulfanilic acid, dimethyl 1-naphthaline and zinc powder. Method: Inoculate a trypticase-nitrate tube with the organism to be tested. Incubate for 24 hours, test for results. Add 1ml of sulfanilic acid to each tube, then add 1ml of dimethyl 1-naphthylamine solution, Mix Development of a red color indicates a positive test. If no red color develops add a small amount of zinc powder. If a red color develops, nitrate is present and the test is negative. If no red color develops, nitrate has been reduced and the test is positive. 4] MRVP Test: This test is used to determine two things. The MR portion (methyl red) is used to determine if glucose can be converted to acidic products like lactate, acetate, and formate. The VP portion is used to determine if glucose can be converted to acetoin. Methyl red-positive organisms produce high levels of acid during fermentation of dextrose, overcome the phosphate buffer system and produce a red color upon the addition of the methyl red pH indicator. In the Voges-Proskauer test, the red color produced by the addition of potassium hydroxide to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation of dextrose.3 The acetoin is oxidized in the presence of oxygen and alkali to produce a red color.3 This is a positive Voges-Proskauer reaction. 54
- 55. These tests areperformed by inoculating a single tube of MRVP media with a transfer loop and then allowing the culture to grow for 1 day. After the culture is grown, about half of the culture is transferred to a clean tube. One tube of culture will be used to conduct the MR test, the second tube serves as the VP test. Requirements: Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner. Chemicals: MRVP medium, Methyl Red(MR), 5% alpha-naphthol, 40% potassium hydroxide. Method: Inoculate the tubes with MRVP media with Organism to be tested. Incubate the tubes at room temperature for 24hrs. Methyl Red Test: Add 5 drops of methyl red indicator to the broth under sterile conditions. Interpret the colour result immediately. Voges-Proskauer’s Test: First 5% alpha-naphthol and then 40% potassium hydroxide are added to the VP tube. The culture is allowed to sit for 15 mins for colour development to occur. Interpret the result. [40] 5] Indole Test: The Indole test determines the ability of an organism to produce indole from the degradation of amino acid trytophan. Tryptophan is hydrolysed by trytophanase to produce three possible end products- one of which is indole. Organisms possessing the enzyme trytophanase cleave tryptophan, producing 3 end products. Amyl alcohol in Kovac’s reagent acts as a solvent for indole which then reacts with p-dimethylaminobenzaldehyde to produce a red rosindole dye. Organisms which don’t produce the enzyme, produce no colour change in the medium upon addition of Kovac’s reagent. 55
- 56. Requirements: Nutrient Agar slants of the soil isolates. Equipment: Bunsen burner, Inoculating loop. Chemicals: Tryptic Soy Broth, Kovac’s reagent. Method: Inoculate the Tryptic soy broth with organism to be tested and incubate at room temperature for 24hrs. Add 5 drops of Kovac’s reagent and gently agitate. Examine the upper layer of liquid. Positive result: red/pink colour. Negative result: No colour change.[41][42] 6] Gelatin Test: Gelatin causes liquids to solidify at temperatures below 28 degrees Celsius. At temperatures above 28 degrees C. gelatin is a liquid. Some bacteria produce gelatinase, a proteolytic enzyme that hydrolyzes gelatin. The hydrolyzed gelatin no longer has the ability to gel and thus remains a liquid, even if placed at temperatures below 28 degrees. The presence or absence of gelatinase can be used to aid in identification of certain bacteria. Requirements: Nutrient agar slants of the soil isolates. Equipments: Bunsen burner, Inoculating loop. Medium for gelatin: Nutrient gelatin deep tubes containing 12% gelatin Method: An inoculum of test organism is stabbed into the nutrient gelatin deep tubes. The media is incubated at room temperature for 24 hrs. and hen refrigerated for approximately 30 mins. 56
- 57. If the gelatin is still intact ( organism did not produce gelatinase), the media will solidify in the refrigerator and a negative result is recorded. If organism has produced sufficient gelatinase then the tube will remain liquid (atleast partially) and a positive result is recorded. 7] Utilization of carbon sources: Carbohydrates are the chief energy providers of the organism. They are made up of carbon, hydrogen and oxygen. The bonds between carbon and hydrogen are broken down with the help of oxygen to give energy (oxidation). Bacteria produce acidic products when they ferment certain carbohydrates. The carbohydrate utilization tests are designed to detect the change in pH which would occur if fermentation of the given carbohydrate occurred. Acids lower the pH of the medium which will cause the pH indicator (Andrade’s solution) to turn dark pink. If the bacteria do not ferment the carbohydrate then the media remains light pink.[43] Requirements: Nutrient agar slants of the soil isolates. Equipments: Bunsen burner, Inoculating loop. Chemicals: Different sugars- Glucose, Lactose, Xylose, Mannitol, Fructose, Sucrose, Inositol, Galactose (All 5g): Peptone (10g), NaCl (5g). Preparation of media: 1% peptone, 0.5% NaCl, 1% sugar. After autoclaving the media, add 2- 3 drops of Andrade’s indicator. Method: The media is prepared as mentioned above. The media is then inoculated with the test organism and incubated at room temperature for 24 hrs. Results are observed. [44] 57
- 58. 8] Starch hydrolysistest: Principle: Starch agar is a differential medium that tests the ability of an organism to produce certain exoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starch molecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes to degrade starch into subunits that can then be utilized by the organism. Starch agar is a simple nutritive medium with starch added. Since no color change occurs in the medium when organisms hydrolyze starch, we add iodine to the plate after incubation. Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presence of starch. A clearing around the bacterial growth indicates that the organism has hydrolyzed starch. In this test, starch agar is inoculated with the species in question. After incubation at an appropriate temperature, iodine is added to the surface of the agar. Iodine turns blue-black in the presence of starch. Absence of the blue-black color indicates that starch is no longer present in the medium. Bacteria which show a clear zone around the growth produce the exoenzyme amylase which cleaves the starch into di- and monosaccharides. These simpler sugars can then be transported into the cell to be catabolized. Bacillus species are known to produce the exoenzyme, amylase.[27][28] Requirements: Sterile starch agar plates. Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner. Iodine solution. Starch agar medium 58
- 59. Composition per litre Sr No. Chemicals Qty(gms) 1. Beef Extract 3.0 2. Soluble Starch 10.0 3. Agar 12.0 [29] Table 11: Starch agar medium Method: Starch media are prepared according to the above mentioned composition. It is then autoclaved at 15lbs at 1210C for 20 minutes. Sterile starch agar plates are prepared The plates are inoculated with the culture on surface by spot inoculation. They are incubated for 24 hrs at room temperature. After incubation iodine solution is added to surface of the plates and colour is observed 9] Casein Hydrolysis Test: Principle: Casein hydrolysis test is used to determine if an organism can produce the exoenzyme casesase. Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Casein is a large protein that is responsible for the white color of milk. This test is conducted on milk agar which is a complex media containing casien, peptone and beef extract. If an organism can produce casein, then there will be a zone of clearing around the bacterial growth. Requirements: Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner Sterile petri plates Microbial cultures. Milk agar medium : 59
- 60. Composition per litre: Sr No Chemicals Qty (gms) 1. Agar 15.0 2. Peptone 5.0 3. Yeast extract 3.0 4. Milk solids 1.0 The pH is adjusted to 7.2 +/- 0.2 at 250c Table 12: Milk agar medium Method: Milk agar media are prepared according to the above mentioned composition. It is then autoclaved at 15lbs at 1210C for 20 minutes. Sterile milk agar plates are prepared The plates are inoculated with the culture on surface by spot inoculation. They are incubated for 24 hrs at room temperature, observe results. Clearing of the agar around the bacterial growth indicates casein hydrolysis.[30] 10] Macconkey agar test: MacConkey Agar (MAC) is a selective and differential medium designed to isolate and differentiate enterics based on their ability to ferment lactose. Bile salts and crystal violet inhibit the growth of Gram positive organisms. Lactose provides a source of fermentable carbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red at a pH below 6.8 and is colorless at any pH greater than 6.8. Organisms that ferment lactose and thereby produce an acidic environment will appear pink because of the neutral red turning red. Bile salts may also precipitate out of the media surrounding the growth of fermenters because of the change in pH. Non-fermenters will produce normally-colored or colorless colonies.[31] 60
- 61. Requirements : Macconkey agar medium Composition per litre: Sr No. Chemicals Qty(gms) 1. Peptic digest of animal tissue 17.0 2. Proteose peptone 3.0 3. Lactose 10.0 4. Bile salts 1.5 5. Sodium chloride 5.0 6 Neutral red 0.03 7 Agar 15.0 [19] The pH is adjusted to 7.1 Table 13: Macconkey agar medium Method: Macconkey media are prepared according to the above mentioned composition. It is then autoclaved at 15lbs at 1210C for 20 minutes. Sterile macconkey agar plates are prepared The plates are inoculated with the culture on surface by spot inoculation or streaking They are incubated for 24 hrs at room temperature, observe results. 61
- 62. 11] Protease test: This test is performed to determine the presence of enzyme protease in the bacterial cell. Requirements: Gelatin Agar medium. Sr No. Chemicals Qty(grams) 1. Gelatin 30 2. Agar Agar 15 3. Pancreatic digest of casein 10 4. Sodium Chloride 10 Table 14: Gelatin Agar medium Method: Gelatin Agar media was prepared according to the above mentioned composition. It was then autoclaved at 15lbs at 1210C for 20 minutes. Sterile Gelatin Agar plates were prepared. The plates were inoculated with the culture on surface by spot inoculation or streaking. They were incubated for 24 hrs at room temperature. 10% HgCl2 was then added to the plates and the clear zone formation around growing colony was considered as positive. 12] Lipase test: Many microbes hydrolyse fats, resulting in rancidity.This is accomplished by an enzyme, Lipase. Fats are usually broken down to glycerol and fatty acids. Lipase is one of the most important class of industrial enzymes and has been explored in oleochemical, paper, organic chemical processing, agrochemical industry,etc. This test qualitatively determines the presence or absence of lipase in the bacterial cell. 62
- 63. Requirements: Peptone Agar medium. Sr No. Composition Qnty(grams) 1. Peptone 5 2. Agar Agar 15 Table 15: Peptone Agar medium. Method: Peptone Agar media was prepared according to the above mentioned composition. It was then autoclaved at 15lbs at 1210C for 20 minutes. Sterile Peptone Agar plates were prepared. The plates were inoculated with the culture on surface by spot inoculation or streaking. They were incubated for 24 hrs at room temperature. Formation of opaque whitish zone around colony was taken as a positive result. F] STUDY OF EFFECTS OF VARIOUS PARAMETERS ON THE SOIL ISOLATES. 1] Effect of different temperatures: Overview: An effort was taken to study the effects of varying temperatures on the growth of the soil isolates. The temperatures selected for this purpose were 4 0C, 100C, 280C(R.T), 370C and 550C. Method: Nutrient broth was prepared with its pH adjusted to 7.2 and was dispensed into test-tubes. The test-tubes containing the media were then autoclaved at 121 0C at 15lbs for 15mins. 63
- 64. Keeping one control tube for every temperature, rest of the tubes were inoculated with the 14 soil isolates under sterile conditions and incubated for 24hrs. at respective temperatures. 2] Effect of different pH: Overview: An effort was made to study the effects of varying pH on growth of the soil isolates. The dfifferent pH selected for this purpose were 3 and 5 Method: Nutrient broth was prepared in 2 conical flasks and pH of the media in each conical flask was adjusted to 3 and 5 respectively. The media from all the flasks was then dispensed into test-tubes. The test-tubes containing the media were then autoclaved at 1210C at 15lbs for 15mins. Keeping one control tube for every pH, rest of the tubes were inoculated with the soil isolates under sterile conditions and incubated for 24hrs. 3] Effect of varying concentrations of different heavy metals: An effort was made to study the effects of varying concentrations of different heavy metals on the growth of the soil isolates. The heavy metals used for the purpose were aluminium(AlCl3), chromium(KCrO4), manganese(MnSO4), zinc(ZnSO4) and mercury(HgCl2). Method: 15ml of Nutrient agar media was prepared per butt tube. The butt tubes were then autoclaved at 1210C at 15lbs for 20 mins. The media was then slightly cooled and following concentrations of heavy metals were added per tube as follows: A stock of 10,000ppm of heavy metal was prepared. (10mg/ml) 64
- 65. It was diluted to 1000ppm by adding 9ml of distilled water. (making it to 10mg/10ml). Thn using this as the stock solution, required quantities of heavy metal concentrations wee prepared using the formula RT/G. For eg: To make 10ppm conc. of a heavy metal, R= Required concentration, T= Total quantity and G= given conc. of heavy metal. Therefore, 10*15/1000 = 0.15. After adding the heavy metal, the media was poured into petri plates and allowed to solidify. The soil isolates were then streaked onto the plates under sterile conditions and incubated at room temperature for 24 hrs. The results were observed. 4] To study the growth curve of the organisms at different concentrations of phenol: The growth curve is studied of the organisms at various concentrations of 50 ppm and 100ppm of phenol Method : Three flask of 100ml capacity were taken and nutrient broth is prepared (100ml) in each flask. The media was then autoclaved at 1210C at 15lbs for 20 mins. Phenol solution of 50 ppm and 100 ppm concentration is prepared by formula RT/G and added to respective flask, and 7ml isolate suspension of 0.05 O.D at 540nm is inoculated in each flask. The flask were kept in shaker at room temperature for 24 hours. The growth curve is studied for period of 24 hours at regular time intervals and absorbance is measured at 540nm. 65
- 66. RESULTS 66
- 67. RESULTS: A] Waste wateranalysis: Sr No. Test Parameters Results Units 1 Total Dissolve Solids 1423 mg/l 2 COD 1963 mg/l 3 BOD 3 days, 270C 1076 mg/l 4 Cyanide Absent mg/l 5 Zinc 0.59 mg/l 6 Copper BDL mg/l 7 Aluminium 58 mg/l 8 Manganese BDL mg/l 9 Mercury 0.017 mg/l 10 Iron 0.19 mg/l 11 Chromium 0.23 mg/l 12 Oil & Grease 12 mg/l 13 Methylene orange alkalinity 66 mg/l 14 Chloride 23252.5 mg/l 15 Hardness (EDTA) 148.2 mg Ca+2/L Table 16: Waste water analysis 67
- 68. Some of theparameters of wastewater analysis like heavy metal ion detection were given in laboratory and reports were obtained. pH of water was 9 and temperature around 300C. The colour of wastewater was found to be black and reddish brown. B] Isolation of microorganisms: 1] Soil microorganisms: Sr No. Dilutions cfu/ml Average cfu/ml 1 10-3 3570 x 104 2 10-4 2840 x 104 ≈ 10 x 107 3 10-5 2740 x 104 Table 17: Soil microorganisms 2] Waste water microorganisms: Sr No. Dilutions cfu/ml Average cfu/ml 1 10-3 880 x 104 2 10-4 790 x 104 ≈ 20 x 106 3 10-5 500 x 104 Table 18: Wastewater microorganisms 68
- 69. Figure 1: CitrateUtilization test Figure 2: Glucose Utlization Test 69
- 70. C] Gram staining: OrganismNo. Gram Nature Morphology KN1. Negative Rods KN2. Positive Rods KN3. Positive Rods KN4. Positive Cocci KN5. Positive Rods KN6. Positive Cocci KN7. Positive Rods KN8. Positive Rods KN9. Negative Rods Table 19: Gram staining D] Biochemical Tests: Sr Test KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. 1. Citrate Utilization + - + + - + + + - 2. Nitrate Utilization + + + - - - + + - 3. Indole test - - - - - - - - - 4. Gelatin - - - - - - + + + 5. Methyl red test + - - - - - - - - 6. Voges-Proskauer - - - - - - - - - test Table 20:Biochemical Tests 70
- 71. Figure 3: SucroseUtlization Test Figure 4: Nitrate Utlization Test 71
- 72. Figure 5: CaseinHydrolysis Test: Figure 6: Casein Hydrolysis Test: 72
- 73. Figure 7: StarchHydrolysis Test: 73
- 74. E] Acid production: Sr Sugars KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. 1. Glucose - + + - - - + + + 2. Lactose - - - - + - - - - 3 Xylose + - - - - - - - - 4 Manitol + + + - - + - - - 5 Inositol - - - + - - - - - 6 Fructose + + + + - + + + + 7 Sucrose + + + + - + - - - 8 Galactose + + + + - - - - - Table 21: Acid production F] Enzyme assay: Sr Test KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. 1. Starch + + - + - - - - - hydrolysis 2. Casein + + - - + + + - - Hydrolysis 3. Catalase + + - + + + + + - test 4. Protease - - - - - - - - - 5. Lipase - - - - - - - - - Table 22: Enzyme assay 74
- 75. Figure 8: ZnSO4Tolerance: Figure9: KCrO4Tolerance: 75
- 76. G] Probable organisms: Thebiochemical test, acid production test and enzyme assay were then compared to different species in the bergy’s manual and following organisms characteristics were matched with studied isolates. Pseudomonas spp Bacillus spp Micrococcus spp Arthrobacter spp H] Heavy Metal Tolerance: 1] ZnSO4Tolerance: The soil isolates were tested against varying concentrations of different heavy metals to check their tolerance against these heavy metals. The results were confirmed by comparing turbidity of the inoculated tubes with the control tubes. Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. (ppm) 1 10 + + + + + + + + + 2 50 + + + + + + + + + 3 100 + + + + + + + + + 4 300 + + + + + + + - - 5 500 + + + + - - - - - 6 700 - + - + - - - - - 7 1000 - - - - - - - - - Table 23: ZnSO4 Tolerance: 76
- 77. 2] KCrO4Tolerance: Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. (ppm) 1 10 + + + + + + + + + 2 50 + + + + + + + + + 3 100 + + + + + + + + + 4 300 + + + + + + + + + 5 500 + + + + + + + + + 6 700 - + + + + + + + + 7 1000 - + + + + + + + + Table 24: KCrO4 Tolerance 77
- 78. 3] MnSO4Tolerance: Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. (ppm) 1 10 + + + + + + + + + 2 50 + + + + + + + + + 3 100 + + + + + + + + + 4 300 + + + + + + + + + 5 500 + + + + - + + - + 6 700 - + + + - + + - + 7 1000 - + + + - + - - + Table 25: MnSO4Tolerance 78
- 79. Figure 10: HgCl2Tolerance: Figure11: HgCl2Tolerance: 79
- 80. 4] HgCl2Tolerance: Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. (ppm) 1 10 + + + + + + - + + 2 50 - + + - + - - - + 3 100 - + - - + - - - + 4 300 - + - - + - - - - 5 500 - + - - - - - - - 6 700 - - - - - - - - - 7 1000 - - - - - - - - - Table 26: HgCl2 Tolerance 80
- 81. 5] AlCl3 Tolerance: Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. (ppm) 1 10 + + + + + + + + + 2 50 + + + + + + + + + 3 100 + + + + + + + + + 4 300 - - + + + - + - - 5 500 - - - - - - - - - 6 700 - - - - - - - - - 7 1000 - - - - - - - - - Table 27: AlCl3 Tolerance 81
- 82. I] Growth atdifferent pH: Sr pH KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. 1 5 + + + + + + + + + 2 3 - - - - - - + + + Table 28: Growth at different pH J] Growth at different Temperatures: Sr Temperature KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9 No. [0C] 1 4 - - - - - - - - - 2 10 - - - - - - - - - 3 28 + + + + + + + + + 4 37 + + + + + + + + + 5 55 - - - - - - - - - Table 29: Growth at different Temperatures 82
- 83. K] Effect ofvarious concentration of phenol on growth curve of the organism: Time (Hrs) Optical Optical Optical density(540nm) density(540nm) density(540nm) Without Phenol Phenol(50ppm) Phenol(100ppm) 0 0.0 0.0 0.0 2 0.0 0.0 0.0 4 0.20 0.14 0.10 6 0.35 0.23 0.13 8 0.56 0.41 0.29 10 0.77 0.65 0.56 12 0.85 0.73 0.65 14 0.95 0.79 0.73 16 1.00 0.86 0.83 18 1.05 0.94 0.89 20 1.06 0.95 0.89 22 1.09 1.03 0.98 24 1.09 1.06 1.01 Table 30: Effect of various concentration of phenol on growth curve of the organism 83
- 84. Graph 1: Effectof phenol on growth curve 84
- 85. DISCUSSION 85
- 86. DISCUSSION: Due to rapidindustrialization, different types of pollution have emerged and recognized, which include air, soil and ground water pollution, caused by gaseous, liquid and solid forms of chemicals. Soil degradation from various inorganic and organic contaminants, is not only an ecological risk, but simultaneously it is also a social, health and economic issue. Such soils become poor in physiochemical properties, susceptible to erosion; result in loss of production and sustainability, diminished food chain quality, and tainting of water resources. Restoration of such degraded land depends on one hand upon action of nature itself, which is time consuming, and on the other hand, it also depends upon human intervention. Among available remediation technologies, bioremediation is a useful approach to clean up contaminated soil and wastewaters, which is based upon utilization of natural or engineered biota( Rani Faryal 2003)[14] In the present study, an attempt has been made to analyze physiochemical properties of wastewater collected from koperkhairane nullah near MIDC industrial area besides Furnace fabrica Ltd and Alok industry, Maharashtra. Analysis of effluent showed that effluent samples were highly coloured like dark black and reddish brown, foul smelling, alkaline and having temperature approximately 300C. Colour is the first contaminant to be recognized in the textile industry waste water, due to presence of unused dye in water, which affects aesthetic as well as biological activity of the ecosystem (sarnaik and kanekar, 1995; bant et al 1996). Total dissolved solids (TDS) of the effluent were high (1423 mg/l) as compared to the Indian standard of specification requirement but in the permissible limit. BOD of the effluent was exceeding Indian standard specifications(appendix 1). These results were in line with the work of Gowrisankar et al.(1997) on textile mill effluent in India, with BOD ranging between 400-1000mg/L. In general high BOD reflects high concentrations of substances that can be biologically degraded, thereby consuming oxygen and potentially resulting in dissolved oxygen. (Rani Faryal 2003)[14] COD of the effluents was also high (1963mg/L) as compared to Indian standard specifications. In textile effluent, pollution is aggravated by the presence of free chorine and toxic heavy metals, which causes rapid depletion of dissolved oxygen leading to an “oxygen sag” in receiving waters. In other words, a high degree of pollution is associated with high COD.[14] Alkalinity and Hardness of water were under normal range, but chlorides were 86
- 87. present beyond thepermissible limit. Except for Aluminum, all other heavy metals like Zinc, Copper, Manganese, Mercury, Iron, and Chromium are under desirable limits of Indian standard specifications. An attempt has been made to isolate and enrich microbes from soil of koperkhairane nullah near MIDC industrial area besides Furnace fabrica Ltd and Alok industry, Maharashtra. Identification and characterization of the isolates was based on gram staining, biochemical tests like citrate utilization, nitrate utilization, indole test, gelatin test, MR-VP test and acid production. All the isolates appeared to be Gram positive with most of them being purple rods, a few purple cocci and only two isolates KN 1 & 2 were Gram negative rods (Table 19). Most of the isolates were found to be citrate positive indicating that they could use sodium citrate as a carbon source.All the isolates showed negative results for Indole and MRVP test. This could infer that these organisms did not possess the enzyme tryptophanase and do not produce high levels of acid during fermentation of glucose. However, few organisms did show the presence of gelatinase and hence tested positive for gelatin test. Out of 9 isolates 5 isolates showed positive nitrate reduction test and rest were negative. Out of the various sugars used as carbon sources like, glucose, lactose, xylose, manitol, inositol, fructose, sucrose, galactose, the isolated colonies successfully fermented most of the sugars except lactose, inositol and xylose. Only few isolates were able to ferment lactose, inositol and xylose.( Table 20, 21). Most of the result for Qualitative enzyme assay i.e. strach hydrolysis, casein hydrolysis and catalase test were positive which indicates that isolates can produce enzyme required for degradation of respective substrates. But results were negative for protease and lipase test.(Table22). All the biochemical characteristics were compared with Bergey’s Manual chart, from which few organisms were matching in their characteristics with the studied isolates in the said work Hence, it could be said that the organisms isolated from soil nearby waste water could belong to any one of Pseudomonas spp, Bacillus spp, Micrococcus spp, or Arthobacter spp. Isolates of the said project can be further confirmed upto species level by sequencing and morphological study. In the next half of the study, an attempt was made to study heavy metal tolerance of the soil isolates and optimize various parameters like pH, temperature and growth curve studies of the soil microorganism. 87
- 88. To survive undermetal-stressed conditions, bacteria have evolved several types of mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the efflux of metal ions outside the cell, accumulation and complexation of the metal ions inside the cell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999). [1] All the isolated strains (KN 1-9) were tolerant towards five different heavy metals. Most of the isolates are tolerant to ZnSO4 up to 300ppm-500ppm, only two KN2 & KN3 tolerated up to 700ppm(Table 15). For K2CrO4 all isolates tolerates up to 1000ppm except KN1(Table 16). 5 isolates KN 2, 3, 4, 6& 9 are tolerant towards MnSO4 up to 1000ppm(Table 17). The isolates are least tolerant to HgCl2, only KN 5 tolerates up to 500ppm(Table 18). Most of the isolates are tolerant towards AlCl3 up to 100ppm. Only KN 3, 4, 5 AND 7 tolerates up to 300ppm(Table 19). All the isolates showed full growth at 280C and 370C suggesting these temperatures to be their optimum temperatures. None could tolerate freezing temperatures of 4 0C and 100C and thermophilic range of 550C (Table21). The pH study was carried for pH 5 & 3. All the isolates showed growth at pH 5, only KN 7, 8 and 9 showed growth at pH 3(Table 21). Effects of various concentration of phenol on growth curve of the microorganism were observed. The growth curve was optimum for organism without phenol concentration. The growth curve slightly decreased with increasing concentrations of phenol (Graph 1). Thus the said project paves the ways to study the wastewater of this region further. Analysis of various dyes and attempt to study the degradation of dyes with soil microorganisms would be helpful to decrease the pollution of water. 88
- 89. CONCLUSION 89
- 90. Conclusions: 1] Textile effluentsare highly polluted. Such polluted effluents must be treated properly before their discharge into the drainage channel to minimize the effect of various pollutants on the environment. 2] Although some heavy metals are important and essential trace elements, at high concentrations, such as those found in many environments today, most can be toxic to microbes. Microbes have adapted to tolerate the presence of metals or can even use them to grow. Thus, a number of interactions between microbes and metals have important environmental and health implications. Some implications are useful, such as the use of bacteria to clean up metal-contaminated sites. 3] Wastewater collected from Koperkhairane Nullah is a source of heavy metal Contamination, containing Aluminium, Iron, Mercury. 4] High BOD and COD of effluent samples is an indicator of high load of organic pollutants. 5] The isolated microbes showed remarkable tolerance against various heavy metals. 90
- 91.
- 92. Future Prospects: Further research can be carried out to enhance detoxification and biodegradation capabilities of these strains. Genetic engineered strains can be developed from these isolates, to enhance bioremediative abilities. Analysis of various dyes and attempt to study the degradation of dyes with soil microorganisms would be helpful to decrease the pollution of water. 92
- 93. BIBLIOGRAPHY 93
- 94. BIBLIOGRAPHY: 1] Anne Spain,Communicated by: Dr. Elizabeth Alm (2003). Implications of Microbial Heavy Metal Tolerance in the Environment. Reviews in Undergraduate Research, Vol. 2, 1-6,2003. 2] ADEL AL-KDASI, AZNI IDRIS, KATAYON SAED, CHUAH TEONG GUAN(2004). TREATMENT OF TEXTILE WASTEWATER BY ADVANCED OXIDATION PROCESSES – A REVIEW. Global Nest: the Int. J. Vol 6, No 3, pp 222- 230, 2004 3] Asamudo, N. U., A.S. Daba and O.U. Ezeronye (2005). Bioremediation of textile effluent using Phanerochaete chrysosporium. African Journal of Biotechnology Vol. 4 (13), pp. 1548-1553, December 2005. 4] Clifford C. Hach, Robert L. Klein, Jr., Charles R. Gibbs. Introduction to BIOCHEMICAL OXYGEN DEMAND Technical Information Series—Booklet No. 7. 5] Dr. Subrata Das. Textile Effluent Treatment – A Solution to the Environmental Pollution. 6] Irina-Isabella Savin, Romen Butnaru(2008). WASTEWATER CHARACTERISTICS IN TEXTILE FINISHING MILL. November/December 2008, Vol.7, No.6, 859-864. 7] John G. Holt, Noel R. Krieg, Peter H.A. Sneath, James T. Staley, Stanley T. Williams. Bergey’s Manual of Determinative Bacteriology. 93-94,530-583. 8] Lars Järup (2003). Hazards of heavy metal contamination. British Medical Bulletin 2003; 68: 167–182. 94
- 95. 9] Muhammad MasudAslam, M A Baig2, Ishtiaq Hassan, Ishtiaq A Qazi, Murtaza Malik, Haroon Saeed (2004). TEXTILE WASTEWATER CHARACTERIZATION AND REDUCTION OF ITS COD & BOD BY OXIDATION. Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377. 804-811. 10] M. Vidali (2001). Bioremediation. An overview. Pure Appl. Chem., Vol. 73, No. 7, pp. 1163-1172. 11] Naeem Ali (2006). Biotechnological Approaches for the Treatment of Textile Dyes by Indigenous Microorganisms. 12] Nupur Mathur, Anuradha Singh. Industrial Microbiology A Laboratory Manual. 217- 220 13] Olukanni O. D., Osuntoki, A. A. and Gbenle, G. O.( 2006). Textile effluent biodegradation potentials of textile effluent-adapted and non-adapted bacteria. 14] Rani Faryal (2003). Role of microorganisms in bioremediation of heavy metal ions and organic pollutants present in textile industry. 15] R.C.Dubey, D.K.Maheshwari. Practical Microbiology.162-182,286-301. 16] RONALD M. ATLAS, Edited by Lawrence C.Parks. Handbook of Microbiological Media. 17] R. Baskar, K.M. Meera Sheriffa Begum, S.Sundaram (2006). CHARACTERIZATION AND REUSE OF TEXTILE EFFLUENT TREATMENT PLANT WASTE SLUDGE IN CLAY BRICKS. Journal of the University of Chemical Technology and Metallurgy, 41, 4, 2006, 473-478. 18] R.O. YUSUFF, J.A. SONIBARE (2004). CHARACTERIZATION OF TEXTILE INDUSTRIES’ EFFLUENTS IN KADUNA, NIGERIA AND POLLUTION IMPLICATIONS. Global Nest: the Int. J. Vol 6, No 3, pp 212-221, 2004. 95
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- 98. APPENDIX 98
- 99. Appendix: 99
- 100.
- 101.
- 102.
- 103.
- 104.
- 105.