ITRODUCTION

Petroleum ( petroleum, from Greek: (rock) + Latin: oleum (oil)) naturally

occurring, yellow-to-black liquid found in geologic formations beneath
the Earth's surface, which is commonly refined into various types of
fuels. This liquid contains aliphatic hydrocarbons, or hydrocarbons
composed of nothing but hydrogen and carbon. The carbon atoms link
together in chains of different lengths. It turns out that hydrocarbon
molecules of different lengths have different properties and behaviors.
For example, a chain with just one carbon atom in it (CH
4
) is the lightest
chain, known as methane. Methane is a gas so light that it floats
like helium. As the chains get longer, they get heavier. The first four
chains CH
4
(methane), C
2
H
6
(ethane), C
3
H
8
(propane) and C
4
H
10
(butane)
are all gases, and they boil at -161, -88, -46 and -1 degrees F,
respectively (-107, -67, -43 and -18 degrees C). The chains up through
C
18
H
32
or so are all liquids at room temperature, and the chains above
C
19
are all solids at room temperature. The name petroleum covers
both naturally occurring unprocessed crude oil and petroleum products
that are made up of refined crude oil. A fossil-fuel, petroleum is formed
when large quantities of dead organisms,
usually zooplankton and algae, are buried underneath sedimentary
rock and subjected to intense heat and pressure.

Petroleum is refined to produce petrol and diesel. Fractional
Distillation process is used on petroleum and at various
temperatures, different by products are formed from it. Petrol and
diesel both are derived at varying temperatures during refining
process. Petrol is produced at temperature between 35 degrees to
200 degrees while diesel is produced at a boiling point of 250-350
degrees. After distillation, in order to use these byproducts as
commercially acceptable petrol and diesel, some blending with
other elements has to be done. Petrol is produced first in this
process as it is produced at a lower temperature than diesel.
The component of petroleum and petroleum products
number in the tens of thousands. They range in molecular weight
from methane (16) to very large uncharacterized components with
molecular weight in the thousands. The toxicity of the components
varies immensely. Overall, the components of these mixtures have
only two common properties: They are derived from petroleum and
they contain hydrocarbon functional groups (C-H). The more
common functional categories of compounds found in petroleum
products are n-alkanes, branched alkanes, cycloalkanes, and
aromatic compounds. In certain mixtures there are other functional
categories present (alkenes, mercaptans, porphorins, etc.). They
provide energy to heat our homes and places of work, fuel our
transportation systems and power manufacturing processes and
tools, as well as providing a source for the numerous synthetic
materials we take for granted in our lives. When they are used as
intended, PHCs provide great benefits to society. However, when
released to the soil environment as raw feed stocks or refined fuels
or lubricants, problems can result. These include fire and explosion
hazard, human and environmental toxicity, movement through soil
to air or water, odour and impairment of soil processes such as
water retention and nutrient cycling.

Diesel Fuel :

Diesel is composed of about 75% saturated hydrocarbons (primarily
paraffins including n, iso, and cycloparaffins), and 25% aromatic
hydrocarbons (including naphthalenes and alkylbenzenes). The
average chemical formula for common diesel fuel is C12H23,
ranging from approx. C10H20 to C15H28. Petrol consists of
hydrocarbons with between 5 and 12 carbon atoms per molecule
but then it is blended for various uses. Overall a typical petrol
sample is predominantly a mixture of paraffins (alkanes),
naphthenes (cycloalkanes), aromatics and olefins (alkenes). The
ratios vary based on a variety of factors.

Impacts on Animals and Human health :

Health effects from exposure to petroleum products vary
depending on the concentration of the substance and the length of
time that one is exposed. Breathing petroleum vapors can cause
nervous system effects (such as headache, nausea and dizziness)
and respiratory irritation. Very high exposure can cause coma and
death. Liquid petroleum products which come in contact with the
skin can cause irritation and some can be absorbed through the skin.
Chronic exposure to petroleum products may affect the nervous
system, blood and kidneys. Gasoline contains small amounts of
benzene, a known human carcinogen. Animals exposed to high
levels of some petroleum products have developed liver and kidney
tumors. Whether specific petroleum products can cause cancer in
humans is not known; however, there is evidence that
occupationally exposed people in the petroleum refining industry
have an increased risk of skin cancer and leukemia.
A spill that affects a home or business may affect the health of
people who live and work there. Thus, spill or leak should be
stopped and cleaned up. Health complaints should be taken
seriously.
Individuals who experience health problems that may be related to
a petroleum spill should see their family physician or health care
provider. Some individuals are more sensitive to petroleum odors
and to the effects of exposure than others. The county health
department or state health department may be able to help
provide additional health information to affected individuals and
their doctors.
In situations where the indoor air environment has become
contaminated to the extent that strong odors are present or air
monitoring indicates serious contamination, home owners,
businesses, and tents may be advised to relocate until the cleanup
is completed. Generally, a DEC inspector or a county health
department assessor determine whether relocation is
recommended. People, including residential tents who may not be
legally responsible for the spill, can request relocation financial
assistance from the Fund to cover reasonable expenses for lodging
and meals. The county health department should be contacted for
relocation assistance. Concerns about possible exposure via soil or
water contamination should also be directed to the county health
department (See attached list of county health department
contacts).

Environmental problems :

Worldwide industrial and agricultural developments have released a large
number of natural and synthetic hazardous compounds into the environment
due to careless waste disposal, illegal waste dumping and accidental spills.
There are numerous sites in the world that require cleanup of soils and
sludge. In the United States, it has been estimated that contaminated site
treatment costs may approach 1.7 trillion dollars over the next 30 years.

Increasing industrialization and associated oil exploration
has resulted in vast quantities of petroleum contaminants being
released into the environment. Accidental spills from the petroleum
industry are one of the leading causes of soil and groundwater
pollution. Soils contaminated by petrochemicals can rapidly lose
their function and stability, with severe contamination events
leading to considerable environmental deterioration and/or loss of
natural and agricultural productivity. This is a reverse-chronological
list of oil spills that have occurred throughout the world and spill(s)
that are currently ongoing. Quantities are measured in tonnes of crude
oil with one tonne roughly equal to 308 US gallons, or 7.33 barrels, or
1165 liters. This calculation uses a median value of 0.858 for the specific
gravity of light crude oil; actual values can range from 0.816 to 0.893,
so the amounts shown below are inexact. They are also estimates,
because the actual volume of an oil spill is difficult to measure exactly.

Traditional remediation techniques involving physical (washing,
excavation) and/or chemical treatments are expensive and can be
environmentally destructive. For example, chemical treatment
technologies such as flushing soil with strong oxidisers or
surfactants can exceed costs as high as US$300 yard3 soil (US
EPA,2006). There is, therefore, a growing need to develop green
technologies that reduce both financial and environmental costs.
Bio-remediation, whereby enhanced microbial degradation of
contaminants occurs within the soil (rhizosphere), is one such
technology with great potential for mitigating the effects of organic
pollutants in surface soils. The remediation method, bio-
augmentation, involving the introduction of microorganisms into
contaminated water or soil, is widely used for cleaning up
environments polluted with organic compounds, co-contaminated
with hydrocarbons and heavy metals, or polluted with heavy metals.
In bio-augmentation of petroleum-contaminated soil, hydrocarbon-
degrading bacteria are applied to polluted environments in order to
accelerate the degradation of toxic compounds. One of the main
problems that limit the effectiveness of this process is very low
solubility and high hydrophobicity of oil pollutants. These
compounds strongly bind to soil particles, and thereby they are
poorly available for bacterial cells. Solution to this problem is the
use of bio-surfactants or bio-surfactant producing microorganisms.
Bio-surfactants are a structurally diverse group of surface-active
substances produced by microorganisms that exhibit the ability to
reduce surface and interfacial tension. They may enhance
hydrocarbon bioremediation by two mechanisms. The first includes
the increase the substrate availability for microorganisms, while the
second involves interaction with the cell surface, which increases
the hydrophobicity of the surface, allowing.
MATARIALS AND METHODS:
Glass wares:
All glass wares include conical flasks; petriplates, test
tubes and measuring cylinder were rinsed with tap water
and then deionized water.
Cleaning:
All the glass wares used in this experiment was
cleaned with teepol, a detergent. Finally rinsed with
deionized water.
Deionized water: obtained from deionizer plant (bhanu
aqua DM 600, BHANU scientific company).it is used
for cleaning and media preparation.
Chemicals:
All chemicals are obtained from either sigma-aldrich
or S.D fine-chemical ltd or Himedia or qualigens or
Emerck.
Sterilization:
Sterilization was done by autoclaving at 121
:
Orbital shaker:

Centrifuge:
All isolates were centrifuged at 10,000rpm/10min for
emulsification test.
Pcr amplifier:

Agarose electrophoresis:
16:

Gas chromatography:

Reagents:
Include dichloromethane, sodium anhydrous sulfate.
Sample collection:
Three samples were collected from diesel oil
contaminated sites in Hyderabad.
Enrichment of culture:
Mineral salt media with 5% diesel oil as sole carbon
and energy source of 50ml in each 100ml conical flask,
and 1g of soil sample was inoculated then incubated at
37c for three days in orbital shaker.
Isolation:
Isolation was done with loop by the transfer of culture
from enriched flasks to petriplates.
Purification:
It is done by repeated streaking.
Characterization and identification:


SNo mor