不同油藏采油功能微生物菌群表征
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摘要
微生物提高原油采收率（Microbial Enhanced Oil Recovery，MEOR）技术具有工艺简单，投资少和无污染等优点，是目前最具发展前景的采油技术之一。采油功能微生物在矿场试验过程中表现出的微生物活性以及产生的代谢产物是提高原油采收率的基础，要探明油藏环境下MEOR机理，对油藏环境下采油功能微生物菌群的认识最为关键。为此本论文采用分子生态学研究方法如16S rRNA基因克隆文库和第二代DNA测序技术，对6个不同油藏MEOR矿场试验过程中的微生物菌群进行监测分析，主要研究成果如下：
新疆低温稠油油藏微生物驱油矿场试验，采用糖蜜、玉米浆和无机氮磷激活剂，激活的采油功能菌群主要为假单胞菌属（Pseudomonas）的铜绿假单胞菌（Pseudomonas aeruginosa）和施氏假单胞菌（Pseudomonas stutzeri）；大庆中温聚合物驱后油藏微生物驱油矿场试验，采用玉米浆干粉和无机氮磷激活剂，激活的采油功能菌群主要为P.aeruginosa和P.stutzeri、芽孢杆菌属（Bacillus）的耐盐芽孢杆菌（Bacillus halodurans）；大港中高温稠油油藏内源微生物驱油矿场试验，采用纤维素、酵母粉和糖蜜复合营养剂，激活的采油功能菌群主要为P.aeruginosa和P.stutzeri、肠杆菌属（Enterobacter）的霍氏肠杆菌（Enterobacter hormaechei）；辽河高温高凝油油藏微生物驱油矿场试验，采用的激活剂主要为玉米浆、糖蜜和无机氮磷，激活的采油功能菌群主要为P.aeruginosa和类产碱假单胞菌（Pseudomonas pseudoaligenes）；江汉超高温高盐油藏微生物清防蜡矿场试验，采用无机氮磷和改性淀粉类激活剂，激活的微生物菌群为P.aeruginosa和P.stutzeri、Enterobacter hormaechei和Bacillus的环状芽孢杆菌（Bacillus circulans）。总体而言，虽然研究的油藏在油藏特性如地质构造、温度、矿化度、原油性质、微生物激活体系和MEOR实施方案等方面都不同，各研究油藏MEOR矿场试验之后油藏中优势菌群却表现出高相似性，Pseudomonas几乎在所有MEOR矿场试验后的油藏中以优势菌群出现，其次是Enterobacter和Bacillus，这一结果的出现主要是因为这些MEOR矿场试验过程都采用的是复合有机无机激活剂体系。
P.stutzeri是一类具有功能和基因亚种（genomovar，gv.)多样性的微生物，油藏中P.stutzeri较为常见并具有采油功能，揭示油藏中P.stutzeri亚种类型将有助于分析油藏中P.stutzeri功能多样性。基于16S rRNA基因序列分析，油藏环境中P.stutzeri能划分到4个已知的亚种类型，gv.1、gv.2、gv.3和gv.20以及8个未知的基因类型，其中gv.1型的P.stutzeri在油藏环境中普遍存在。检测到8个未知的P.stutzeri基因类型表明，油藏中存在着大量的不同于其它环境的P.stutzeri亚种类型，这些基因类型的出现与某些油藏特殊环境有关。油藏环境中P.stutzeri基因亚种多样性从侧面反映了P.stutzeri功能多样性，体现了P.stutzeri作为MEOR矿场试验采油功能菌的潜力。
Bacillus是一类主要的采油功能菌，但不基于培养的分子生物学方法往往不能很好地揭示Bacillus在油藏中的多样性。本论文将传统的微生物学培养方法和分子生态学方法相结合，发明了一种快速检测油藏样品中Bacillus的方法，该方法能有效地揭示油藏环境中Bacillus多样性。实验结果表明在油藏环境中存在着大量还没有分离培养的Bacillus，以及一些功能不明确的Bacillus。本论文中提供的方法有望能加速油藏中Bacillus多样性研究，为采油功能Bacillus的发现奠定基础。
主要创新点：（1）探明了采用复合无机有机营养剂体系的MEOR矿场试验过程中的采油功能菌菌群为Pseudomonas，Enterobacter和Bacillus；（2）发现了油藏环境中可能存在多个P.stutzeri新亚种；（3）发明了快速检测油藏中Bacillus的新方法。
Microbial enhanced oil recovery (MEOR) technique is one of the most prospectivemethods for oil recovery due to its economic, environmentally-friendly and simpleadvantages. Principally, microbes that possess functional activities and productsresulting in the increase of oil production in MEOR field trials are the key for thediscovery of MEOR mechanisms. In the present thesis, microbial communities insamples collected from oil wells during MEOR field trials carried out in six different oilreservoirs were monitored using molecular biological techniques, such as16S rRNAgene clone library and the next generation DNA sequencing. The major contents andfindings are as follows:
Microbes stimulated by nutrients of molasses, corn steep liquor with N and P in thelow-temperature heavy oil reservoir of Xinjiang oil field were Pseudomonas aeruginosaand Pseudomonas stutzeri; Microbes stimulated by nutrients of corn steep powder withN and P in the mesophilic polymer flooded oil reservoir of Daqing oil field were P.aeruginosa, P.stutzeri and Bacillus halodurans; Microbes stimulated by nutirents ofcellulose bran, yeast powder and molasses with N and P in the high-temperature heavyoil reservoir of Dagang oil field were P.aeruginosa, P.stutzeri and Enterobacterhormaechei; Microbes stimulated by nutrients of molasses, corn steep liquor with N andP in the high-temperature high pour-point oil reservoir of Liaohe oil field were P.aeruginosa, P.pseudoaligenes; Microbes stimulated by nutrients of modified starch withN and P in the ultra-temperature hypersaline oil reservoir of Jianghan oil field wereP.aeruginosa, P.stutzeri, Bacillus circulans and Enterobacter hormaechei. In summary,although characteristics, field trials and nutrients of the oil reservoirs under study weresignificantly different, stimulated microbes after MEOR field trials showed significantlysimilar. Pseudomonas, Bacillus and Enterobacter were dominant in the samplescollected from the oil reservoirs after MEOR field trials, indicating that Pseudomonas,Bacillus and Enterobacter were contributing microbes for MEOR.
Additionally, diversity within P.stutzeri is not limited to physiological propertiesbut is also reflected at the genetic level. P.stutzeri is a species detected frequently insamples from oil reservoirs and possessing functions for MEOR. The thesis discovered the diversity and genomovar assignment of P.stutzeri in oil reservoirs for the first time.High diversity of P.stutzeri was observed, which was exemplified in the detection ofsequences assigned to four known genomovars1,2,3,20and eight unknown genomicgroups. The frequent detection and predominance of P.stutzeri belonging to genomovar1in most of the oil reservoirs under study indicated an association of genomovars ofP.stutzeri with the oil field environments.
Finally, Bacillus is a contributing group for MEOR, however diversity of Bacillusin oil reservoirs can not be well unveiled based on molecular biological methods. In thethesis, based on the advantages of both culture-dependent microbial methods andculture-independent molecular biological methods, a method for quick detection ofBacillus was invented. Many genera of Bacillus detected in the oil reservoirs understudy by the method were not reported previously, and most of sequences detected bythe method showed low similarities with cultured strains of Bacillus, which indicatedthat diversity of Bacillus in oil reservoirs was far from clear.
In summary, the main innovations are concluded as follows:(1) Pseudomonas,Bacillus and Enterobacter were the main microbial communities stimulated bycomposite organic and inorganic nutrients in oil reservoirs during MEOR field trials.(2)High diversity of P. stutzeri was observed in samples from oil reservoirs, newgenomovars of P. stutzeri habiting in oil reservoirs were proposed.(3) A new method forquick detection of Bacillus in oil reservoirs was invented.
新疆低温稠油油藏微生物驱油矿场试验，采用糖蜜、玉米浆和无机氮磷激活剂，激活的采油功能菌群主要为假单胞菌属（Pseudomonas）的铜绿假单胞菌（Pseudomonas aeruginosa）和施氏假单胞菌（Pseudomonas stutzeri）；大庆中温聚合物驱后油藏微生物驱油矿场试验，采用玉米浆干粉和无机氮磷激活剂，激活的采油功能菌群主要为P.aeruginosa和P.stutzeri、芽孢杆菌属（Bacillus）的耐盐芽孢杆菌（Bacillus halodurans）；大港中高温稠油油藏内源微生物驱油矿场试验，采用纤维素、酵母粉和糖蜜复合营养剂，激活的采油功能菌群主要为P.aeruginosa和P.stutzeri、肠杆菌属（Enterobacter）的霍氏肠杆菌（Enterobacter hormaechei）；辽河高温高凝油油藏微生物驱油矿场试验，采用的激活剂主要为玉米浆、糖蜜和无机氮磷，激活的采油功能菌群主要为P.aeruginosa和类产碱假单胞菌（Pseudomonas pseudoaligenes）；江汉超高温高盐油藏微生物清防蜡矿场试验，采用无机氮磷和改性淀粉类激活剂，激活的微生物菌群为P.aeruginosa和P.stutzeri、Enterobacter hormaechei和Bacillus的环状芽孢杆菌（Bacillus circulans）。总体而言，虽然研究的油藏在油藏特性如地质构造、温度、矿化度、原油性质、微生物激活体系和MEOR实施方案等方面都不同，各研究油藏MEOR矿场试验之后油藏中优势菌群却表现出高相似性，Pseudomonas几乎在所有MEOR矿场试验后的油藏中以优势菌群出现，其次是Enterobacter和Bacillus，这一结果的出现主要是因为这些MEOR矿场试验过程都采用的是复合有机无机激活剂体系。
P.stutzeri是一类具有功能和基因亚种（genomovar，gv.)多样性的微生物，油藏中P.stutzeri较为常见并具有采油功能，揭示油藏中P.stutzeri亚种类型将有助于分析油藏中P.stutzeri功能多样性。基于16S rRNA基因序列分析，油藏环境中P.stutzeri能划分到4个已知的亚种类型，gv.1、gv.2、gv.3和gv.20以及8个未知的基因类型，其中gv.1型的P.stutzeri在油藏环境中普遍存在。检测到8个未知的P.stutzeri基因类型表明，油藏中存在着大量的不同于其它环境的P.stutzeri亚种类型，这些基因类型的出现与某些油藏特殊环境有关。油藏环境中P.stutzeri基因亚种多样性从侧面反映了P.stutzeri功能多样性，体现了P.stutzeri作为MEOR矿场试验采油功能菌的潜力。
Bacillus是一类主要的采油功能菌，但不基于培养的分子生物学方法往往不能很好地揭示Bacillus在油藏中的多样性。本论文将传统的微生物学培养方法和分子生态学方法相结合，发明了一种快速检测油藏样品中Bacillus的方法，该方法能有效地揭示油藏环境中Bacillus多样性。实验结果表明在油藏环境中存在着大量还没有分离培养的Bacillus，以及一些功能不明确的Bacillus。本论文中提供的方法有望能加速油藏中Bacillus多样性研究，为采油功能Bacillus的发现奠定基础。
主要创新点：（1）探明了采用复合无机有机营养剂体系的MEOR矿场试验过程中的采油功能菌菌群为Pseudomonas，Enterobacter和Bacillus；（2）发现了油藏环境中可能存在多个P.stutzeri新亚种；（3）发明了快速检测油藏中Bacillus的新方法。
Microbial enhanced oil recovery (MEOR) technique is one of the most prospectivemethods for oil recovery due to its economic, environmentally-friendly and simpleadvantages. Principally, microbes that possess functional activities and productsresulting in the increase of oil production in MEOR field trials are the key for thediscovery of MEOR mechanisms. In the present thesis, microbial communities insamples collected from oil wells during MEOR field trials carried out in six different oilreservoirs were monitored using molecular biological techniques, such as16S rRNAgene clone library and the next generation DNA sequencing. The major contents andfindings are as follows:
Microbes stimulated by nutrients of molasses, corn steep liquor with N and P in thelow-temperature heavy oil reservoir of Xinjiang oil field were Pseudomonas aeruginosaand Pseudomonas stutzeri; Microbes stimulated by nutrients of corn steep powder withN and P in the mesophilic polymer flooded oil reservoir of Daqing oil field were P.aeruginosa, P.stutzeri and Bacillus halodurans; Microbes stimulated by nutirents ofcellulose bran, yeast powder and molasses with N and P in the high-temperature heavyoil reservoir of Dagang oil field were P.aeruginosa, P.stutzeri and Enterobacterhormaechei; Microbes stimulated by nutrients of molasses, corn steep liquor with N andP in the high-temperature high pour-point oil reservoir of Liaohe oil field were P.aeruginosa, P.pseudoaligenes; Microbes stimulated by nutrients of modified starch withN and P in the ultra-temperature hypersaline oil reservoir of Jianghan oil field wereP.aeruginosa, P.stutzeri, Bacillus circulans and Enterobacter hormaechei. In summary,although characteristics, field trials and nutrients of the oil reservoirs under study weresignificantly different, stimulated microbes after MEOR field trials showed significantlysimilar. Pseudomonas, Bacillus and Enterobacter were dominant in the samplescollected from the oil reservoirs after MEOR field trials, indicating that Pseudomonas,Bacillus and Enterobacter were contributing microbes for MEOR.
Additionally, diversity within P.stutzeri is not limited to physiological propertiesbut is also reflected at the genetic level. P.stutzeri is a species detected frequently insamples from oil reservoirs and possessing functions for MEOR. The thesis discovered the diversity and genomovar assignment of P.stutzeri in oil reservoirs for the first time.High diversity of P.stutzeri was observed, which was exemplified in the detection ofsequences assigned to four known genomovars1,2,3,20and eight unknown genomicgroups. The frequent detection and predominance of P.stutzeri belonging to genomovar1in most of the oil reservoirs under study indicated an association of genomovars ofP.stutzeri with the oil field environments.
Finally, Bacillus is a contributing group for MEOR, however diversity of Bacillusin oil reservoirs can not be well unveiled based on molecular biological methods. In thethesis, based on the advantages of both culture-dependent microbial methods andculture-independent molecular biological methods, a method for quick detection ofBacillus was invented. Many genera of Bacillus detected in the oil reservoirs understudy by the method were not reported previously, and most of sequences detected bythe method showed low similarities with cultured strains of Bacillus, which indicatedthat diversity of Bacillus in oil reservoirs was far from clear.
In summary, the main innovations are concluded as follows:(1) Pseudomonas,Bacillus and Enterobacter were the main microbial communities stimulated bycomposite organic and inorganic nutrients in oil reservoirs during MEOR field trials.(2)High diversity of P. stutzeri was observed in samples from oil reservoirs, newgenomovars of P. stutzeri habiting in oil reservoirs were proposed.(3) A new method forquick detection of Bacillus in oil reservoirs was invented.
引文
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