油田采出水石油烃降解菌分离、鉴定及高通量选育复合诱变菌株

Isolation, identification and breeding of petroleum-degrading bacteria in oilfield produced water by UV-plasma complex mutagenesis

【目的】为解决石油烃类物质(Total petroleum hydrocarbons,TPHs)引起的环境污染问题,提高野生型菌株对TPHs的降解率。【方法】基于微生物修复技术,通过富集培养、初筛和复筛从油田采出水中分离TPHs优势降解菌株,经形态学、生理生化以及16S r RNA基因序列分析确定其种属。采用紫外线与等离子体复合诱变技术,以96孔板发酵法替代摇瓶培养发酵,采用以多功能酶标仪双波长紫外光谱法测定为基础的高通量筛选方法选育TPHs高效降解菌株,并通过方差分析检验突变株的遗传稳定性。【结果】经鉴定,分离自采出水的野生型菌株PW04为多食鞘氨醇杆菌(Sphingobacterium multivorum)。通过复合诱变获得了3株能够高效降解TPHs的突变菌株:S.multivorum PW04-H10、S.multivorum PW04-G9、S.multivorum PW04-A6,降解率分别为85.1%、82.7%、82.9%。遗传稳定性分析结果表明这3株菌遗传性能均稳定。【结论】经分离、诱变选育出的TPHs高效降解菌株其降解率最高达85.1%,与野生型菌株相比提高了48%,可显著降低环境中TPHs含量,有效修复原油污染环境。

[Objective] In order to solve the environmental pollution problem caused by total petroleum hydrocarbons（TPHs）, the wild type strains＇ degradation rate of TPHs was enhanced in this study. [Methods] Based on bioremediation technique, the strains, which can degrade TPHs effectively, were isolated from the oilfield produced water and selected out by enrichment culture, prescreening and rescreening. Then, the strains were identified through morphological, physiological, biochemical, and phylogenetic（16S rRNA gene） characterization. In order to obtain highly efficient THPs-degrading strains, after being mutated by UV-plasma and 96-pore plate fermentation, the isolated strains were screened by high-throughput screening method, with the use of multi-functional microplate reader based on the dual wavelength ultraviolet spectrum. At last, the mutants＇ hereditary stability was tested by analysis of variance. [Results] The wild strain PW04, which was isolated from produced water, was identified as Sphingobacterium multivorum. After the compound mutation, three highly efficient THPs-degrading strains（S. multivorum PW04-H10, S. multivorum PW04-G9 and S. multivorum PW04-A6） were obtained, which the degradation rate of THPs can reach to 85.1%, 82.7% and 82.9% respectively. As was shown in the result of hereditary stability analysis, the three mutants all had stable genetic stability. [Conclusion] Through the isolation and mutation breeding, the TPHs degradation rate of the highly efficient THPs-degrading strain reached to 85.1%, increased by 48% compared with wild type strains. The highly efficient THPs-degrading strain can reduce the TPHs contents in the environment significantly and restore the oil-contaminated environment effectively.