Crude oil Less dense, light crude needs less refining to remove higher carbon content (crude oil

is a hydrocarbon). Heavy crude, with higher carbon content, needs more refining. Sour crude contains higher concentrations of sulfur than sweet crude. Again, more processing is needed to remove excess sulfur from the crude if it is sour. Sweet crude, containing less sulfur, would need less refining and would be less toxic in the event of a spill.

Light sweet crude was the type of oil released in the Gulf of Mexico by the BP Oil Disaster. Light sweet crude is a form of oil that contains little or no wax and is processed into high value kerosene, gas, heating oil, jet fuel and diesel fuels. Light sweet crude is desirable in the oil and gas industry because its low viscosity eases much of the distribution process; light crude is easier than heavy crude oil to move from the ground to the pump because it is less sticky. It is also purer than other forms of crude. This means it is easier to process while also yielding a higher quality product. This type of oil is often a refiners choice over heavier oils. Thus, it carries greater prices than other forms of crude.

Polycyclic Aromatic Hydrocarbons, (PAHs), are an important chemical found within this particular type of crude oil, although, in a lesser amount than other heavy crude oils. The polycyclic aromatic hydrocarbons are less volatile and water soluble than some of the other components of the crude oil. However, they are more fat-soluble and may be stored within the oils and fats of an organism, entering the food chain for long periods of time. Many of these substances have been proven to be carcinogenic, (cancer-causing), mutagenic, (causing mutations), and teragenic (causing birth defects).

Natural gas is also a contaminant coming from the wellbore and occurs in the formation as a gas cap, sitting atop the crude oil in the formation. Most people are not aware that oil wells normally produce natural gas also and that natural gas is also flowing from the well.

The availability of water for a cell depends upon its presence in the atmosphere (relative humidity) or its presence in solution or a substance (water activity). Pseudomonas is a nonhalophile while staphylococcus is a halo tolerant Pseudomonas .91 Salmonella/E. coli .91 Lactobacillus .90 Bacillus .90 Staphylococcus .85 The aims of this study were: To evaluate the process of biostimulation achieved by the addition of bacteria spp. to petroleum-polluted soils. To study the relation of the total heterotrophic bacterial counts to the optical density of crude oil

MATERIALS AND METHODS Collection of contaminated soil A bulk soil from an agriculture field (Shahid Chamran university, ahvaz, , Iran ) was taken, air dried and passed through 2mm sieve. Then, the bulk soil was contaminated with crude-oil artificially. The crude-oil from well No: 69 of Maroon oil field (which is paraffin oil with a rate of 1% weight) was sprayed in a way that the whole soil would be polluted homogenically. The soil samples were kept for two weeks and then divided in to 5Kg parts and stored in special containers. The containers were left undisturbed (i.e. in the open air) for 2 weeks. Then the treatments, including additional of different amounts of agricultural fertilizers (urea, ammonium

phosphate and potassium sulphate) were applied, but equal rates of tilling were used. The various treatment containers were tilled twice a week with cutlass and shovels to provide the necessary aeration and mixing of nutrients and microbes with the contaminated soil (Ayotamuno et al., 2006).

bacterial enumeration Hydrocarbon-degrading and heterotrophic bacteria were enumerated using a MPN method adapted from Wrenn and Venosa (1996). Bushnell Haas medium (composition: magnesium soleplate 0.2 g g/L; calcium chloride 0.02 g/L ; monopotassium phosphate 1 g/L; ammonium phosphate dibasic 1 g/L; potassium nitrate 1 g/L and ferric chloride 0.05 g/L) supplemented with 2% (w/w) NaCl was used as the growth medium in 96 well microstate plates. Hydrocarbon sources were added to stimulate the growth of hydrocarbon degraders. A sample of 3 g of Soil samples was placed in a vial containing 10 mL of Bushnell Haas medium supplemented with 2% (w/w) Nail and mixed to form slurry. One mL of this slurry was placed into a vial containing 9 ml of Bushnell Haas medium supplemented with 2% (w/w) NaCl. A dilution series was prepared from this sample, from 101 to 1012, and used to inoculate plates. and each rarity has three repetition, in a form that for each soil sample 36 experiment pipe should be prepared, after diluting Risasorin identifier in a rate of 90 l added to pipes and then sterilized crude oil in a rate of 0.2 mL should be added to each pipe and Plates for enumeration of hydrocarbon -degrading bacteria were incubated at room temperature (26-27C) for 2 weeks. After incubation, all plates were read using a MPN chart to determine positive or negative growth of bacteria, it is necessary to mention that all the needed instruments were sterilized.

Experimental design and crude oil treatment The crude-oil was distributed into 14 treatment sample-containers (250ml Erlenmeyer flasks). Two containers did not receive any treatment (control)

Twelve containers had 10ml of nutrient broth which had been inoculated with the treating bacteria and 90ml of crude-oil and incubated overnight at 37 degree Celsius to create appropriate environment for the activity of crude-oil decomposing microorganisms. In order to remove the effect of the lack of oxygen and maintain proper aerobic conditions, the setup was corked with dry cotton wool and were mixed thoroughly by using a shaker at the speed of 140rpm for five to eight minutes before every reading. The symbols of the treatments in the experiment were: S0A and S0 (control), S1Ai and S1Aii (inoculated with pure ); S2Ai and S2Aii ().

Determination of oil dispersion. To estimate the extent of oil dispersion, all culture flasks were shaken vigorously using a shaker for 5 8 minutes, and 1-ml samples were transferred immediately into a 1cm x 1cm cuvertte. After shaking the mixture again by turning upside down, the Optical density of the mixtures were determined using a spectrophotometer at 500nm, 540nm and 600nm which was blanked with a sterile distilled water. For bacteria count, 1ml of the sample was immediately transferred from the culture flasks, serially diluted to 10-2 and inoculated onto nutrient agar in petri dishes and incubated overnight at 37 degree Celsius. Bacterial colonies were counted using a
Staurt Scientific colony counter (UK).

DISCUSSION The effect of time on petroleum degradation was significant. The petroleum degradation rate decreased with increasing time (Fig. 2) and this observation corresponded with the bacteria growing results. The highest bacteria growing was determined after ; because of the presence of nutrition materials, bacteria activity and as a result petroleum degradation was in a maximum rate. But, with increasing of time, due to the oil-resistant components with high chain

and within less remaining nutrients, the bacteria growth and oil degradation decreased (Schaefer and Juliane, 2007).

In Fig. 1 the average of oil degradation in soil in the treatment samples is observed, which shows significant difference with the control from the statistical point of view. Oil degradation in the lack of treatment samples was less and the lack of organic feeding matters, will limit the oil degradation and C/N ratio will increase (Odokuma and Dickson. 2003).

Abstract the addition of oil-degrading microorganisms to supplement the indigenous populations, has been proposed as an alternate strategy for the bioremediation of oil contaminated environments. The rationale for this approach is that indigenous microbial populations may not be capable of degrading the wide range of potential substrates present in complex mixtures such as petroleum (Leahy and Colwell, 1990) or that they may be in a stressed state as a result of the recent exposure to the spill. Other conditions under which bioaugmentation may be considered are when the indigenous hydrocarbon-degrading population is low, the speed of decontamination is the primary factor, and when seeding may reduce the lag period to start the bioremediation process (Forsyth et al., 1995)

ACKNOWLEDGEMENTS The authors appreciate the Soil Biology and Geochemistry laboratories staffs and also deeply thank Dr Alizadeh for the support of analytical instruments. REFERENCES Ayotamuno, M. J., Kogbara, R.B., Ogaj, S.O.T., and Probert, S.D., (2006). Bioremidiation of a crude-oil polluted agricultural-soil at Port Harcourt, Nigeria. Applied energy, 83: 1249-1257.

Abraham Reisfeld, Eugene Rosenberg, And David Gutnick 1972 Microbial Degradation of Crude Oil: FactorsAffecting the Dispersion in Sea Water by Mixed and Pure Cultures

Filter Paper Spot Method 1. Use a loop and pick a well-isolated colony from a fresh (18- to 24-hour culture) bacterial plate and rub onto a small piece of filter paper (please see Comments and Tips section for notes on recommended media and loops). 2. Place 1 or 2 drops of 1% Kovcs oxidase reagent on the organism smear. 3. Observe for color changes. 4. Microorganisms are oxidase positive when the color changes to dark purple within 5 to 10 seconds. Microorganisms are delayed oxidase positive when the color changes to purple within 60 to 90 seconds. Microorganisms are oxidase negative if the color does not change or it takes longer than 2 minutes. Identification of cellulose bacteria Inoculate onto cellulose agar Incubate at 37 degrees Celcius Only positive ones would grow well Confirmatory test Grow bacteria in CMC (Carboxymethylcellulose) Incubate for 8 hours Flood plates with congo red for 15 minutes and pour off Flood plates with 1ml of NaCl for 15 minutes Zones of hydrolysis would be seen as clear areas Slide test Coagulase Test Slide coagulase test is run with a negative control to rule out auto agglutination. Two drops of saline are put on to the slide microscopic slide labeled with sample number, Test (T) and control

(C). The two saline drops are emulsified with the test organism by using wire loop, straight wire, or wooden stick. Place a drop of plasma (rabbit plasma anti-coagulated with EDTA is recommended[2] ) on the inoculated saline drop corresponding to test and mix well with wooden applicator stick. Rock the slide gently for about 10 seconds.

If positive: Macroscopic clumping would be observed in the plasma within 10 seconds and no clumping in the saline drop. If negative: No clumping will be observed.

NOTE: If the slide coagulase test is negative, a tube test should follow as a confirmation. Clumping in both drops is an indication of auto-agglutination and therefore a tube test have to be carried out.