Isolation And Diversity Analysis Of Petroleum Degrading Bacteria In Coastal Zone

With the development of oil production and transportation, marine oil spill pollution incidents occur frequently, a lot of oil in a short period of time into the ocean, will have an impact on marine microbial ecology. There are three prevailing treatment of oil pollution: physical, chemical and biological treatment. Since bioremediation technology can deal with oil in situ with no secondary pollution, show a good application prospects, it has become an important direction in the treatment of marine oil pollution.Micro-organisms play a crucial role in the bioremediation of marine oil spills and it is an important method to reduce and remove marine oil pollution by using microbial degradation. By the technology of microbial separation matures, many micro-organisms have been cultivated. Simulated natural environment, together cultivation and molecular biology methods, with the study of rich varirty of micro-organisms in the environment, is a more in depth study of environmental microbes under a need and inevitable. Therefore, it has important theoretical significance and potential application value for the research on the petroleum degrading bacteria isolation, identification and the petroleum degrading microbial diversity.This study use the method of dilution coating to isolate the strain from oil pipeline leakage accident near contaminated seawater, which could utilize crude as the sole carbon source, and identification of the strains. The growth curve and degradation rate of each strain were studied, according to the degradation characteristics of crude oil, the strain was combined and the degradation effect of single strain and combination strain were compared. The structure and diversity of microbial flora in oil pollution seawater and natural seawater were studied by the method of clone library construction, PCR-RFLP technology and combined with sequence alignment and phylogenetic analysis. We could draw the following conclusion by researching:(1)Three kinds of strains were isolated from petroleum degrading bacteria. All those strains could utilize crude oil as the sole carbon source. After identification, three strains were Gallaecimonas pentaromativorans、Arthrobacter nicotianae、Acinetobacter venetianus,including Gallaecimonas pentaromativorans and Acinetobacter venetianus belong to γ- Proteobacteria and Arthrobacter nicotianae belongs to Actinobacteria. The degradation rate of Gallaecimonas pentaromativorans was the highest, which was 30.81%, and the degradation rate of Acinetobacter venetianus was the lowest, which was 9.11%.(2)Single bacteria composite degradation experiments showed that the degradation rate of A+B、A+C and A+B+C were 34.24%、31.04%、37.68%, which were higher than that of strain A(30.52%)、B(20.88%)、C(9.09%), the degradation rate of combination bacteria is not equal to the sum of each single strain degradation rate. Between various species due to various factors such as competition、antagonism and promotion, all kinds of factors work together to form the composite bacteria degradation.(3)16S r DNA clone library analysis results show that Proteobacteria and Flavobacteria are main bacteria in the oil pollution seawater. And in these bacteria, α-Proteobacteria and γ- Proteobacteria are main advantage bacteria. Alcanivorax app.、Marinomonas app.、Flavobacterium app. were the most predominant bacteria in oil pollution seawater; α- Proteobacteria and γ- Proteobacteria are main bacteria in natural seawater, Alcanivorax app. 、 Thalassospira app. 、 Novosphingobium app. and Pseudomonas app. were the most predominant bacteria in natural seawater.(4)By compared the clone library diversity index, it showed that microbial community in oil polluted seawater was lower than that of natural seawater, the richness index was higher than that of natural seawater and the distribution of bacteria in oil polluted seawater and natural seawater were relatively uniform, the dominant species and number of species were not much.Through the analysis of the oil degrading bacteria diversity in oil pollution seawater and natural seawater, it is indicated that Alcanivorax app. is the dominant species. This genus of bacteria is an important function of marine oil pollution in the environment.(5)The result of NCBI sequencing showed that 16 S r DNA clone library of oil pollution seawater and natural seawater were 63.74%、57.01%, the clones can be found in the NCBI and the strains similarity are more than 97%. Therefore, it was show that many oil-degrading micro-organisms had not yet been cultured, classified, and identified in oil pollution seawater and natural seawater.