红色荧光蛋白基因标记产鼠李糖脂铜绿假单胞菌SG及其在油藏中的应用

Chromosomal integration of red fluorescent protein gene in rhamnolipids-producer Pseudomonas aeruginosa SG and its application in petroleum reservoir

【背景】微生物在油田注采系统中的迁移直接影响到油藏微生物群落组成及其在油田生产中的应用。然而,由于缺少特异性标记,很难将目标微生物同众多的土著微生物区分开。因此,需要构建携带特异性基因的微生物菌株。【目的】为了有效追踪定位微生物在油田注采系统中的迁移,本文构建一株红色荧光蛋白标记假单胞菌。【方法】运用染色体同源重组的方法,将带有组成型表达启动子的红色荧光蛋白编码基因(red fluorescent protein gene,rfp)插入到一株分离自油藏环境且产鼠李糖脂的铜绿假单胞菌SG染色体上编码β-内酰胺酶基因内部,获得标记菌株SG-rfp。【结果】构建的菌株SG-rfp能够在非诱导条件下表达红色荧光蛋白,而且对氨苄青霉素、卡那霉素、链霉素和庆大霉素不具有耐受性。与野生型菌株SG相比,构建的SG-rfp菌株也能够在有氧和缺氧条件下产生鼠李糖脂,在岩芯驱油实验中能够较好地提高原油采收率。此外,应用菌株SG-rfp,本文研究并证实了微生物在含油多孔介质中的迁移扩散及所受限制。【结论】本文所构建的菌株SG-rfp为深入研究微生物在油田注采系统中的迁移及微生物在油田生产中的应用提供了有力工具。

[Background]Microbial transfer in oilfiled production facilities is closely related to reservoir microbial community compositions and microbiologically improved oil production process.However,it is hard to distinguish the targeted microorganisms from the numerous indigenous microorganisms containing the same specific genes.Therefore,constructing microorganisms with specific gene marks is necessary.[Objective]In order to effectively track microbial transfer in oilfiled production facilities,an engineering strain Pseudomonas aeruginosa SG-rfp containing specific mark gene was constructed.[Methods]The red fluorescent protein gene(rfp)with a constitutive promoter was inserted into amp gene encodingβ-lactamase on chromosome of rhamnolipids-producer P.aeruginosa SG isolated from petroleum reservoir by homologous recombination using integration vector p EX18 Gm-URD and amp gene.[Results]P.aeruginosa SG-rfp constitutively expressing rfp gene was constructed.Strain SG-rfp is intolerant of ampicillin,kanamycin,streptomycin,and gentamicin.As well as the wild-type strain SG,SG-rfp can produce biosurfactants in aerobic and anaerobic conditions and significantly enhance oil recovery efficiency from oil-bearing core.Using strain SG-rfp,diffusion-limited microbial transfer in oil-bearing porous media was investigated and demonstrated via core-flooding test.[Conclusion]This study provides a powerful tool for in-depth investigation of microbial transfer in oilfiled production facilities and microbiologically improved oil production process.