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Construction and evaluation of an engineered bacterial strain for producing lipopeptide under anoxic conditions.

LIANG Xiao-long1,2, ZHAO Feng3, SHI Rong-jiu1, BAN Yun-he4, ZHOU Ji-dong1,2, HAN Si-qin1, ZHANG Ying1   

  1. (1Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China;
    2University of Chinese Academy of Sciences, Beijing 100049, China; 3State Key Laboratory of Urban Water Resource and Environment, School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China; 4College of Chemistry and Life Sciences, Shenyang Normal University, Shenyang 110034, China)
  • Online:2015-08-18 Published:2015-08-18

Abstract: Biosurfactantfacilitated oil recovery is one of the most important aspects of microbial enhanced oil recovery (MEOR). However, the biosurfactant production by biosurfactantproducing microorganisms, most of which are aerobes, is severely suppressed due to the insitu anoxic conditions within oil reservoirs. In this research, we successfully engineered a strain JD-3, which could grow rapidly and produce lipopeptide under anoxic conditions, by protoplast confusion using a Bacillus amyloliquefaciens strain BQ-2 which produces biosurfactant aerobically, and a facultative anaerobic Pseudomonas stutzeri strain DQ-1 as parent strains. The alignment of 16S rDNA sequence (99% similarity) and comparisons of cell colony morphology showed that fusant JD-3 was closer to the parental strain B. amyloliquefaciens BQ-2. The surface tension of culture broth of fusant JD-3, after 36hour cultivation under anaerobic conditions, decreased from initially 63.0  to 32.5 mN·m-1. The results of thin layer chromatography and infrared spectrum analysis demonstrated that the biosurfactant produced by JD-3 was lipopeptide. The surfaceactive lipopeptide had a low critical micelle concentration (CMC) of 90 mg·L-1 and presented a good ability to emulsify various hydrocarbons such as crude oil, liquid paraffin, and kerosene. Strain JD-3 could utilize peptone as nitrogen source and sucrose, glucose, glycerin or other common organics as carbon sources for anaerobic lipopeptide synthesis. The subculture of fusant JD-3 showed a stable lipopeptideproducing ability even after ten serial passages. All these results indicated that fusant JD-3 holds a great potential to microbially enhance oil recovery under anoxic conditions.