Study On Metabolism Of Petroleum Biodegradation By Indigenous Microorganisms In Dagang Oil Reservoir

Characterization of petroleum biodegradation by indigenous microorganisms in"western58-8-3#" oil reservoirs lies in Gangxi district was inverstigated by using16S rDNA, real-time polymerase chain reaction (RT-PCR), microcalorimetry, gas chromatograph-mass spectrometer (GC-MS) and high performance liquid chromatography (HPLC) analysis. The content include:the degradation characteristics and metabolic pathways of components of crude oil by indigenous microorganisms, the energy metabolism of microorganism and the expression of functional gene in degrading. From the results, highly efficient oil-degrading bacterial strains were obtained and the degradation mechanism of crude oil was preliminary expounded.They provide more information for in situ oil decontamination and improve the oil recovery rate in a certain extent by using biological method.The chromatogram of oil sample showed that "western58-8-3#" oil reservoir was biodegraded. Twelve oil-degrading bacterial strains which belonged to the genus Staphylococcus、Pseudomonas、Rhizobium、Bacillus、Acinetobacter and Rhodococcus were isolated, then their degradation characteristics and metabolic pathways were studied. A bacterial strain named ustb-2has the highest degradation rate and was89.1%. The16S rDNA sequence of ustb-2showed100%similarity with that of Bacillus subtilis BSn5(accession number:KJ018725). Moreover, the strains having the highest degrading capacity for naphthalene, fluorene, phenanthrene, pyrene were selected by the screening test, they are YC-4(Pseudomonas balearica SP1402), YC-11(Rhodococcus erythropolis PR4), YC-10(Rhodococcus erythropolis MM30) and YC-12(Bacillus amyloliquefaciens BC7) respectively, the degradation rate to the corresponding PAH was99.1%,42.1%,18.7%and17.6%. The kinetics of PAHs degradation conformed to a first-order reaction kinetic model and the metabolic pathways belonged to phthalic acid pathway. The half inhibitory ratio (IC50) of hexadecane inhibited organisms, determined by microcalorimetry, was lower in ustb-2than in Bacillus subtilis BSn5, it indicated that the strain ustb-2degrades hexadecane to make it less toxic compared with the normal strain. RT-PCR was used to evaluate the expression of a new oil-degrading enzyme, specifically 4-hydroxyphenylacetate3-monooxygenase genes (HPMO). Three times increase in the expression of HPMO genes was observed for ustb-2, while the expression of HPMO genes in reference strain Bacillus subtilis BSn5remained relatively stable.The Dagnag oil reservoirs can be considered as a potentical and huge strains library, which includes amounts of indigenous microorganisms and genes, and can provide excellent conditions for controlling the oil contamination and microbial enhanced oil recovery.