采油功能菌绿色荧光蛋白标记及其应用基础研究

A large number of oil fields in China have already been into the high water cut, and they now confront with the difficulty and great pressure of difficult exploitation, high cost and low recovery, and so on. Microbial enhanced oil recovery （MEOR）has some advantages such as simple process, good benefits and free pollution compared with other traditional tertiary EOR technologies. In order to obtain more analyses and estimate correctly and accurately to improve MEOR, goal strains for MEOR will be marked and tracked to indicate that how and where they moved and reproduced in oil reservoir and show their amount and rate in oil-field microorganism. As the most simple and convenient gene marker, green fluorescent protein (GFP) whose fluorescence can be excited without substrate and do not harm to host cell will be used in this research. Two receptor strains are Pseudomonas aeruginosa and Acinetobacter sp. producing biosurfactant which were isolated from oil reservoir and have been proved to be excellent qualified oil-field profile modification bacteria. Research on marking receptor bacteria with GFP gene and tracking its Green fluorescence will benefit for the study of MEOR theory greatly. In this study, GFP gene will be inserted into the chromosomal DNA of the microorganisms by triparental mating. Therefore the receptor strains will be labeled and achieve genetic stability of the GFP expression without select pressure. Meanwhile, they will produce biosurfactant after accepting the foreign fragment. Furthermore, the genetic stability of marked engineering bacteria, the compatibility with indigenous microorganisms, the effect of reservoir environment and environmental safety will be studied. Finally, the migration rule of MEOR bacteria in reservoir and oil recovery mechanism will be revealed through physical simulation experiment.

传统的微生物提高原油采收率（MEOR），很难考察目的菌种在油藏中的繁殖与生长状况，为了准确分析和客观评价MEOR现场试验效果，充分认识目的优势菌种经油藏运移后在产出水中分布的规律与数量，需要对其进行标记及跟踪监控，揭示MEOR机理。绿色荧光蛋白(GFP)分子，是现有遗传标记中最简单方便的一种基因标记，其荧光的激发无需底物作用，对宿主菌无毒害。铜绿假单胞菌和不动杆菌为油田常用的产生物表面活性剂的菌株。本课题将通过三亲本杂交的方法将GFP基因插入上述两株菌染色体DNA上达到对该采油功能菌进行标记目的，使GFP基因可以在受体菌中无选择压力及诱导物诱导的条件下稳定遗传表达，且受体菌在接受外源片段后自身代谢活动不受影响。同时考察标记菌株的遗传稳定性、与内源菌的配伍性、地层环境对标记菌株的影响、标记菌株的环境安全性等。最后利用标记菌株的物理模拟实验揭示采油功能菌在油藏中的运移规律及采油机理。

目前我国很多油田已进入高含水阶段，油田开采面临着开采难度大、开发成本高以及采出效率低等难题。微生物提高原油采收率与其它三次采油技术相比具有操作简单、环保无二次污染等优势。但在微生物采油过程中很难考察目的菌种在油藏中的繁殖与生长状况，为了准确分析和客观评价MEOR现场试验效果，充分认识目的优势菌种经油藏运移后在产出水中分布的规律与数量，本研究对油田常用的产生物表面活性剂菌株铜绿假单胞菌进行绿色荧光标记。绿色荧光蛋白(GFP)分子，是现有遗传标记中最简单方便的一种基因标记，其荧光的激发无需底物作用，对宿主菌无毒害。本课题通过三亲本杂交的方法使GFP基因插入铜绿假单胞菌染色体DNA上达到对该采油功能菌进行标记目的。GFP基因可以在受体菌中无选择压力及诱导物诱导的条件下稳定遗传表达，且受体菌在接受外源片段后自身代谢活动不受影响，对原油有很好的乳化效果。GFP标记后的铜绿假单胞菌与油藏内源微生物有较好的竞争性和配伍性，且GFP基因不发生水平转移。通过小鼠毒性毒理实验证明该工程菌对人类及其它生物无危害性。将标记菌株接种油田采出水，在厌氧油藏条件下，标记菌株与野生铜绿假单胞具有同样的原油乳化效果。岩芯实验结果表明，标记后的铜绿假单胞菌与野生菌增加的原油采收率效率分别为6.67%和6.25%。收集标记菌株采出液，利用荧光显微镜可迅速直观检测出荧光标记菌株的数量随采出程度的变化。