分子生态学方法对油藏细菌群落结构分析的比较
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摘要
目前对油藏细菌群落结构分析方法繁多,为得到准确全面的群落分析结果,必须选取合适的方法。群落分析方法的优劣进行比较分析,选取从新疆陆梁油田注水井筛出的11株单菌,测定其16srDNA序列,制成包含7个菌属、9个菌种的混合菌液。应用基于PCR扩增的三种分子生态技术DGGE、T-RFLP、建立16SrDNA文库比较和分析了混合菌液细菌多样性。使用PCR-DGGE方法时发现,DGGE方法可灵敏地检测到序列1 bp的变化,能将不同菌株分开,但该方法经过切胶测序后的的目标序列较短,信息量不大且有时有一定误差,较适合用于定性对比;而用于菌群结构的分析时应结合其他方法。T-RFLP法可区分大部分菌属,但数据库不够完整,不能确定细菌群落组成;因此也不适合单独用在细菌群落检测中,可用作多个样品种群多样性的对比或结合其他方法对样品进行系统透彻解析。建立16srDNA克隆文库法成功鉴定到7个菌属8个菌种16SrDNA序列,更适用于油藏细菌群落结构的分析。
There are many methods for analysis of bacterial community structure in reservoir now. It must select a appropriate method for accurate and comprehensive analysis of bacterial community. The study was comparison of advantages and disadvantages of methods of community analysis. 11 strains from the bacteria obtained from water injection well in Xinjiang Luliang oil well are selected and sequenced. A mixture of bacterium suspension compounded by the 11 strains contains 7 different bacteria species and 9 different strains. Using three PCR-based molecular ecological techniques,i. e. DGGE,T-RFLP and construction of 16 S rDNA clone library,the bacterial community diversity of the mixture was investigated and compared. In the DGGE profiles,16 S rDNA high variable region from the same specie or strain cannot reach the same stripe,while the high variable region from a variety bacteria of different species of which similarity had multiple differences can reach the same stripe. So the best use of DGGE in the examination is as an initial means of gene mutation detection but not the analysis of bacterial community. TRFLP can separating a great mount of bacteria species. But the database for it is not complete,so that T-RFLP cannot determine the bacteria species that sample contains. So T-RFLP is not suitable for detection of the bacterial community alone,it can use for contrast of community structure diversity in multiple samples or analysis detailedly in combination with other methods. Through the construction of 16 S rDNA clone library,16 S rDNA sequences of 7different bacteria species and 8 different stains are identified successful. In summary,the method of 16 S rDNA clone library construction is more appropriate for analysis of bacterial community in reservoir.
There are many methods for analysis of bacterial community structure in reservoir now. It must select a appropriate method for accurate and comprehensive analysis of bacterial community. The study was comparison of advantages and disadvantages of methods of community analysis. 11 strains from the bacteria obtained from water injection well in Xinjiang Luliang oil well are selected and sequenced. A mixture of bacterium suspension compounded by the 11 strains contains 7 different bacteria species and 9 different strains. Using three PCR-based molecular ecological techniques,i. e. DGGE,T-RFLP and construction of 16 S rDNA clone library,the bacterial community diversity of the mixture was investigated and compared. In the DGGE profiles,16 S rDNA high variable region from the same specie or strain cannot reach the same stripe,while the high variable region from a variety bacteria of different species of which similarity had multiple differences can reach the same stripe. So the best use of DGGE in the examination is as an initial means of gene mutation detection but not the analysis of bacterial community. TRFLP can separating a great mount of bacteria species. But the database for it is not complete,so that T-RFLP cannot determine the bacteria species that sample contains. So T-RFLP is not suitable for detection of the bacterial community alone,it can use for contrast of community structure diversity in multiple samples or analysis detailedly in combination with other methods. Through the construction of 16 S rDNA clone library,16 S rDNA sequences of 7different bacteria species and 8 different stains are identified successful. In summary,the method of 16 S rDNA clone library construction is more appropriate for analysis of bacterial community in reservoir.
引文
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15 吴翔,曲毅,程国军,等.一株芽孢杆菌的分子鉴定及初步应用条件探究.化工学报,2013;64(4):1459—1465Wu X,Qu Y,Cheng G J,et al.Molecular identification and conditions for preliminary application of a Bacillus sp..CIESC Journal,2013;64(4):1459—1465
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19 Muyzer G,Dewaal E C,Uitterlinden A G.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes encoding for 16S rRNA.Applied and Environmental Microbiology,1993;59:695 —700
20 李伟光,田文德,康晓荣,等.强化生物除磷工艺微生物种群结构分析.化工学报,2011;62(12):3532—3538Li W G,Tian W D,Kang X R,et al.Analysis of microbial community structure in enhanced biological dephosphorization.CIESC Journal,2011;62(12):3532—3538
2 包木太,孔祥平,宋永亭,等.胜利油田S12块内源微生物群落选择性激活条件研究.石油大学学报:自然科学版,2004;28(6):44 —48Bao M T,Kong X P,Song Y T,et al.Selective activation condition of stratal microflora of S12 block in Shengli Oilfield.Journal of the University of Petroleum,China,2004;28(6):44—48
3 Lazer I,Petrisor I G,Yen T F.Microbial enhanced oil recovery(MEOR).Petroleum Science and Technology,2007;25(11):1353—1366
4 高配科,马挺,赵玲侠,等.胜利油田沾3区块內源微生物激活剂的筛选、优化及效果评价.化工学报,2011;62(7):2005—2012Gao P K,Ma T,Zhao L X,et al.Screening,optimization and evaluation of indigenous microorganism activation system in Zhan 3 block,Shengli Oilfield.CIESC Journal,2011;62(7):2005—2012
5 Newman D K,Banfield J F.Geomicrobiology:how molecular-scale interactions underpin biogeochemical systems.Science,2002;296:1071—1077
6 Kaeberlein T,Lewis K,Epstein S S.Isolating“uncultivable”microorganisms in pure culture in a simulated natural environment.Science,2002;96:1127—1129
7 杨永华,姚健.分子生物学方法在微生物多样性研究中的应用.生物多样性,2000;8(3):337—342Yang Y H,Yao J.Molecular techniques and their application to the study of microbial diversity.Chinese Biodiversity,2000;8(3):337—342
8李辉,牟伯中.油藏微生物多样性的分子生态学研究进展.微生物学通报,2008;35(5):803—808Li H,Mu B Z.Recent advances in molecular microbial ecology of petroleum reservoirs.Microbiology,2008;35(5):803—808
9 Fu B,Liao X Y,Ding L L.Characterization of microbial community in an aerobic moving bed biofilm reactor applied for simultaneous nitrification denitrification.World Microbiol Biotechnol,2010;26(11):1981—1990
10 Wei Mannan,Zhang Rubing,Wang Yuguang,et al.Microbial community structure and diversity in deep-sea hydrothermal vent sediments along the Eastern Lau Spreading Centre.Acta Oceanol,Sin.,2013;32(2):42—51
11 罗剑飞,林炜铁,任杰,等.T-RFLP技术及其在硝化细菌群落分析中的应用.微生物学通报,2008;35(3):456—461Luo J F,Lin W T,Ren J,et al.T-RFLP technique and its application on community analysis of nitrifying bacteria.Microbiology,2008;35(3):456—461
12 余素林,吴晓磊,钱易.环境微生物群落分析的T-RFLP技术及其优化措施.应用与环境生物学报,2006;12(6):861—868Yu S L,Wu X L,Qian Y.Application and Optimization of T-RFLP Method for Microbial Community Analysis.Chinese Journal of Applied&Environmental Biology,2006;12(6):861—868
13 程海鹰,肖生科,汪卫东,等.变形梯度凝胶电泳方法在內源微生物驱油研究中的应用.石油学报,2005;26(6):82—89Cheng H Y,Xiao S K,Wang W D,et al.Application of denaturing gradient gel electrophoresis method to study of oil recovery using indigenous microorganism.Acta Petrolei Sinica,2011;32(1):8—17
14 王中华,梁静儿,杨建强,等.原油微生物群落构成及降解菌降解特性的研究.生物技术通报,2013;1:178—185Wang Z H,Liang J,Yang J Q,et al.Microbial diversity of the crude oil and characteristics of a hydrocarbon-degrading bacterium.Biotechnology Bulletin,2013;1:178—185
15 吴翔,曲毅,程国军,等.一株芽孢杆菌的分子鉴定及初步应用条件探究.化工学报,2013;64(4):1459—1465Wu X,Qu Y,Cheng G J,et al.Molecular identification and conditions for preliminary application of a Bacillus sp..CIESC Journal,2013;64(4):1459—1465
16 Kim K Y,Chun J,Lee J H,et al.The analysis of oral microbial communities of wild-type and toll-like receptor 2-deficient mice ucing a 454 GS FLX titanium pyrosequencer.BMC Microbiol,2010;10:101
17 Tamura K,Dudley J,Nei M,et al.MEGA4:molecular evolutionary genetics analysis(MEGA)software version 4.0.Mol Biol Evol,2007;24:596—1599
18 宋智勇,郭辽原,袁书文,等.高温油藏內源微生物的堵调及种群分布.石油学报,2010;31(6):975—979Song Z Y,Guo L Y,Yuan S W,et al.Microbial plugging and community distribution of indigenous thermophilic microbes in high-temperature oil reservoirs.Acta Petrolei Sinica,2010;31(6):975—979
19 Muyzer G,Dewaal E C,Uitterlinden A G.Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction amplified genes encoding for 16S rRNA.Applied and Environmental Microbiology,1993;59:695 —700
20 李伟光,田文德,康晓荣,等.强化生物除磷工艺微生物种群结构分析.化工学报,2011;62(12):3532—3538Li W G,Tian W D,Kang X R,et al.Analysis of microbial community structure in enhanced biological dephosphorization.CIESC Journal,2011;62(12):3532—3538