厌氧产脂肽工程菌的构建及其代谢活性评价

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

利用微生物产生生物表面活性剂驱油,是微生物采油的重要技术之一.但油藏的缺氧环境使得大多数生物表面活性剂产生菌的代谢活性受到限制.本研究以筛选自油田采出水中的好氧产脂肽类表面活性剂的解淀粉芽孢杆菌BQ-2和兼性厌氧的施氏假单胞菌DQ-1为亲本菌,通过原生质体融合的方法构建了一株能在厌氧条件下迅速生长并能产脂肽类表面活性剂的融合子JD-3.融合子JD-3的菌落形态与亲本菌株BQ-2相似,其16S r DNA序列与BQ-2的相似度达99%;在厌氧条件下培养36 h,融合子JD-3发酵液的表面张力由初始的63.0m N·m-1降至32.5 m N·m-1;薄层层析及红外光谱分析结果显示,JD-3在厌氧条件下的产物为脂肽类表面活性剂;该表面活性剂的临界胶束浓度(CMC)为90 mg·L-1,且对原油、液体石蜡、煤油等有较好的乳化活性.JD-3在厌氧条件下可以蔗糖、葡萄糖或甘油为碳源,以蛋白胨等为氮源合成脂肽类表面活性剂,且经多次厌氧传代培养仍保持稳定的产生物表面活性剂功能,显示该菌株在油藏厌氧条件下对提高原油采收率具有较好的应用潜力.

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 16 S r DNA 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. 5m N·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^- 1and 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.