【作者】 任红燕；

【导师】 赵立平；

【作者基本信息】 上海交通大学 ， 微生物学， 2011， 博士

【摘要】 目前全球的多数油田都已处于二次开采阶段,水驱采油是二次开采中应用最广泛的技术之一,它大幅提高了采油效率,延长了油田的开采年限;但在实施注水的过程中,外界的微生物以及氧气的进入,导致其内部的生态环境受到影响。油藏内部的微生物群落结构在长期的循环注水开采过程中是否会与注入水中的微生物出现趋同性?它们的微生物群落又会呈现怎样的多态性?这些问题的研究对于了解油藏生态和制订合理的开发措施具有重要的意义;此外,油田在经历过各种采油措施之后,仍有一半以上的原油无法采出,而研究也表明,微生物可以将油藏中的原油原位转化为甲烷,如果可以实现将无法开采的残余油以天然气的形式进行开采,这将对于缓解石油供需矛盾具有重要意义,然而,要实现微生物的高效转化,还需对其机制进行深入地研究。在本研究中,我们首先结合传统的变性梯度凝胶电泳（DGGE）、克隆文库技术与新兴的焦磷酸测序技术比较了长期注水的孤岛油田注水井和采油井中的微生物组成。三种方法的结果均显示,注入水与产出液中的微生物组成差异显著（P<0.05）。利用克隆文库对微生物组成的分析结果发现,注入水中96.3%的细菌分类操作单元（OTU）和70.6%的古菌OTU都无法从采油井中检出;而焦磷酸测序的结果也表明注水井中超过80%的操作分类单元（OTUs）,其中包括两个最优势的OTU,均无法从采油井中检出。而利用UniFrac显著性检验发现拥有相同地层结构的采油井中的微生物结构无显著差异（P>0.05）,地层结构不同的采油井中细菌组成差异显著（P<0.05）,这一结果表明各采油井中的微生物组成可能与其地层结构相关,而并非主要受注入水中的微生物的影响。其次,我们利用焦磷酸测序的方法对胜利油田埕东、沾三和孤岛三个区块采油井中超过3万条的细菌及古菌序列进行了分析。从胜利油田检测到21个细菌门以及2个古菌门;还有14.2%的细菌以及26.8%的古菌序列分类地位仍不明确,说明我们所研究的高温注水油藏中蕴藏着丰富的未知微生物资源。系统发育学分析表明胜利油田除了具有油藏环境中常见的Proteobacteia、Firmicutes、Bacteroidetes等门的细菌以及产甲烷古菌之外,还检测到油藏环境中鲜有报道的Aquificae和Fusobacteria等微生物类型。这些结果也意味着在高温、高压、厌氧的地下深部生物圈中同样存在着复杂的微生物群落结构以及大量新的微生物资源。UniFrac分析结果表明,三个区块的细菌组成并无显著差异（P>0.05）,而古菌组成差异显著（P<0.05）。利用典范对应分析（CCA）对各环境因子与微生物组成的影响分析结果证实,温度与各区块的微生物组成关系密切;SO42-对细菌组成的影响大于其它离子,而HCO3-对古菌组成的影响大于其它离子。本论文还对高温条件下不同厌氧代谢方式对石油烃的降解以及菌群结构的影响进行了分析。在实验室人工构建了硫酸盐还原和产甲烷两种培养体系,利用驯化的活性污泥以及油藏产出液中的外源菌群对胜利油田的原油进行了为期540天的厌氧降解,并对培养不同时间原油的降解程度以及菌群的变化进行了检测。结果发现驯化的菌群在两种培养条件下均能够实现对原油的降解,且在60℃高温时产甲烷条件对芳香烃的降解效率高于硫酸盐还原条件。DGGE指纹图谱和基于焦磷酸测序的UniFrac分析结果均显示,虽然接种物来源相同,产甲烷培养和硫酸盐还原培养条件下的菌群组成存在显著的差异,说明硫酸盐对于石油烃降解过程中菌群的组成具有很大的影响;而对于同一种培养条件,古菌的结构随培养时间的变化大于细菌。系统发育学分析结果显示,在两种培养体系中均检测到丰富的互营细菌、发酵细菌以及硫酸盐还原菌。对古菌的分析结果发现在硫酸盐还原和产甲烷两种培养条件下CO₂还原产甲烷途径和乙酸发酵产甲烷菌途径同时存在,但以利用H₂还原CO₂产甲烷途径为主。综上所述,本研究利用传统的分子生物学手段以及新兴的高通量测序技术证明,虽然胜利油田经历了长时间的循环注水,但是注入水中的微生物并不是影响采油井内部微生物生态的主要因素;另外,同一油田的不同工作区块的微生物组成存在较大差异,而且这一差异与各区块的温度关系密切;在60℃高温条件下,人工驯化的外源菌群在硫酸盐还原和产甲烷两种培养系统中均可实现石油烃的降解,且产甲烷培养体系对芳香烃的降解效率高于硫酸盐还原体系;在两种培养系统中CO₂还原和乙酸发酵产甲烷途径同时存在,并以CO₂还原产甲烷途径为主。更多还原

【Abstract】 Most of reservoirs in the world have entered the secondary recovery after decades of exploitation, in which water flooding plays a significant role. Water-flooding dramatically improves the efficiency of oil recovery. However, exogenous microorganisms and dissolved oxygen which may affect the microbial structure will enter the reservoir environment flowed with flooded water. For long-term water-flooding reservoirs, whether microbial structure of reservoir will assimilate by that in injection water is still not clear. Otherwise, more than half of crude oil still left in the reservoirs even after the exploit by using various oil recovery methods. Previous studies have proved the potential of conversion of the residual oil into methane by microorganisms, the study of microbial community in reservoir has an important significance on resolving the conflicts of crude oil supply and extending demand.In this study, Gudao petroleum reservoir, which has been water-flooded for over 30 years, was selected. Using denaturing gradient gel electrophoresis （DGGE）, 16S rRNA gene clone library and pyrosequencing, we compared the microbial communities of injection well with that of production well. The results showed that microbial composition between injection well and production well were significantly different （P<0.05）. It indicated that microbial composition of production well and injection well were not converged during long-term water flooding. Clone library result showed that 96.3% of bacterial operational taxonomic units （OTUs） and 70.6% of archaeal OTUs in injected water cannot be detected from production well. Similar results were obtained by pyrosequencing, more than 80% of OTUs in the injection water, including two of the most predominant OTUs, were difficult to detect in the production wells. UniFrac significance analysis showed that bacterial structures in production wells with the same oil-bearing strata has no significant difference （P>0.05）, but bacterial structures were significantly different in the production wells with different oil-bearing strata （P<0.05）. This suggests that microbial composition of production well may be closely related with the stratigraphic structure of itself.In the second part, we compared the microbial community structures of three working blocks of Shengli petroleum reservoir （Chengdong, zhansan and Gudao） using bar-coded pyrosequencing. 21 bacterial phyla and 2 archaeal phyla, together with 14.2% bacterial and 26.8% archaeal unclassified sequences were detected from more than 30,000 sequences. Phylogenetic analysis showed that predominant phylotypes in Shengli petroleum reservoir were Proteobacteria, Firmicutes, Bacteroidetes, and methnogens, which were considered as common type of microorganisms in petroleum reservoir. Except that, we also found Aquificae and Fusobacteria and other rarely reported bacteria in oil reservoir environment. UniFrac significance test indicated that bacterial community structure of three working block has no significant difference （P>0.05）, but opposite result was found in archaea （P<0.05）. Canonical Correspondence Analysis （CCA） showed stronger effect of temperature than mineralization on microbial community, SO42- has greater effect on bacterial community than other ions, but HCO3- has greater effect on archaeal community than others.Finally, we constructed high temperature sulfate reducing and methanogenic anaerobic crude oil degrading systems, investigated the degradation of crude oil and microbial composition of two systems for period of 540 days incubation. The result showed that aromatic hydrocarbon of crude oil was degraded under both conditions and the degradation rate under methanogenic condition was higher than sulfate reducing condition at 60℃. Both DGGE and bar-coded pyrosequencing results showed that the microbial community structures in two systems were different although using the same inoculums, indicating that sulfate has great impact on microbial composition during petroleum degradation process. But in the same culture condition, the structure of archaea changed greater than bacteria over time. Phylogenetic analysis showed that fermentative bacteria, sulfate-reducing bacteria and syntrophic bacteria were detected from both systems. Moreover, there exist two pathways for methane formation in these systems, acetate fermentation and H₂/CO₂ reduction, and the dominant pathway was H₂/CO₂ reduction.In summary, in this study, using both traditional molecular biological methods and pyrosequencing, we proved that microorganisms in the injection well are not the main factor that affects the endogenous ecology although it suffered long time water flooding. In the same oil field, the microbial communities of different working block were different, and the influence of temperature is greater than salinity. Special exogenous microbial community can degrade petroleum hydrocarbons in both sulfate reducing and methanogenic conditions at 60℃, and the degradation rate of aromatic hydrocarbon in methanogenic condition is fast than sulfate reducing condition.更多还原