Isolation of oil- degrading microorganisms from soil contaminated with petroleum hydrocarbons

َAbstract

The extensive use of petroleum products leads to the contamination of almost all compartments of the environment. This problem must be mentioned by countries which produce or use this type of energy. Petroleum pollution is a major concern for Tehran refinery in Iran. Oil spills as a result of pipeline ruptures, tank failures and various production storage and transportation accident caused the pollution of soil and groundwater in this region. Bioremediation is a new method which has been proposed for remediation of petroleum pollutants using of indigenous or allochthonous microorganisms to detoxify and degrade environmental contaminants. Thus this study conducted to identify and isolate of microorganisms which have the potential for degradation of petroleum pollutants in soil media. Soil samples were taken from soil surface (0-30cm) at 8 points in Tehran refinery,Iran and kept at 4 ^oC until analysis. Ten grams of each sieved soil (<2mm) added to 25 ml oil broth media for isolation of hydrocarbon adopted bacteria. This media involves KH₂PO₄ 1gl^-1, K₂HPO₄ 1gl^-1, (NH₄)₂SO₄ 1gl^-1, MgSO₄,7H₂O 0.04 gl^-1, FeCl₂,6H₂O 0.004 gl^-1 and 0.1 of solute containing 100 mg/l  H₃BO₄, MnSO₄, ZnSO₄, CuSO₄ and COCl₂. All samples incubated in a shaker incubator at 30^oC and 140 rpm for 2 days. In next stage, 25 ml of each flask was removed and added to new flask which involved oil broth media and incubated for 2 days. This stage repeated for 2 weeks. Finally 0.1 ml of last flasks spread on Petri dishes that contained nutrient agar (NA). Fungi were isolated with serial dilution of soils in 10 ml sterile distilled water. 0.1 ml of each dilution being spread on Petri dishes that contained potato dextrose agar (PDA). After growing the fungi and bacteria in growth media, they were purified. The morphological characterization of each isolate was first performed, including color, size and colony characteristics. Gram strain test was performed for each isolate. The following biochemical tests were used in the bacterial identification process: oxidase. catalase, glucose fermentation and  gas production. Bacterial strains were identified based on Bergey’s manual of systematic bacteriology. Fungi were identified according to general principles of fungal classification, using selective media and macro and microscopic examination of morphological characters. Results showed that bacterial strains belong to the genus Micrococcus,Rhodococcus,Nocardia,Corinebacterium, Acinetobacter, Pseudomonas and Alcaligenes.  Fungi which have been identified were Aspergillus, Fusarium and Zygopus. Growing these strains in oil broth media with 400 micro liter crude oil, proved their potential for degradation of petroleum pollutants.