Isolation, Identification And Community Construction Of High Effective Petroleum-Degrading Bacteria

Petroleum production, used wildly around the world, really causes increasing harm to environment and human health. Petroleum, a hydrocarbon, could be used by some of bacteria as carbon resource and energy. Therefore, it is important to isolate petroleum-degrading bacteria from environment and combine petroleum-degrading community.In this paper, we aimed to screen efficient oil-degrading bacteria and combine bacteria community. Through a lot of study , we get the main findings as follows:(1) Petroleum-Degrading bacterial strains were accumulated by three culture conditions from different samples of soil, according to colony characteristics, 40 pure strains were gained by conventional isolation and purification of microorganisms.(2) By Denaturing Gradient Gel Electrophoresis (DGGE), 40 bacterial strains were classifyed into 17 categories, and then the oil degradation rate of 17 petroleum-degrading strains from 17 categories were determinated respectively, the results show that the oil degradation rates of 17 strains were between 17.6% and 77.9%, including 10 strains higher than 40% and 3 strains higher than 70%.(3) Through PCR, sequencing and homology analysis, 7 efficient oil degradation strains have 100% similarity with Lysinibacillus sphaericus, Brevundimonas, Pseudomonas aeruginosa , Alcanivorax , Pseudomonas, Pseudomonas pseudooalcaligenes, Achromobacter respectively.(4) The dehydrogenase and catechol 2,3-dioxygenase (C23Oase) activity of 7 strains were determined, the results showed that: the dehydrogenase activities were in the range of 7.0~119.0 mg?L-1?h-1 and the C23Oase activities were in the range of 0.22~0.87U. Adding diesel increased C23Oase activities with range of 3.34~8.98U, the dehydrogenase activity of S-51 and C23Oase activity of S-17 is the highest among the seven tested strains. Under the same culture condition, there is significant difference between the enzyme activity cell lysis solution and supernatant fluid, which suggested that C23Oase was one kind of intracellular enzyme.(5) 12 groups of bacteria communities were obtained under different enrichment culture conditions, the range of oil degradation rate of which was between 44% to 86.8%. The ability of petroleum degradation of the different groups is obviously different with each other. The results of PCR-DGGE and homology analysis showed that: different soil samples cultivated in the same condition had little difference on the diversity of bacteria, on the contrary, different culture conditions had impact on the diversity of bacteria significantly, even though the soil samples came from different sources.(6)According to the comparison of DGGE characteristic of mixed bacteria and pure strains, the author selected pure bacteria to combine bacteria community, including S-10-15, S-10-51, S-13-15-36, of which the diesel degradation rates were respectively as high as 86.8%, 87.5%, 81.3%.