利用PCR-DGGE技术指导高温油藏中功能微生物的分离  

Isolation of Functional Bacteria Guided by PCR-DGGE Technology from High Temperature Petroleum Reservoirs

作　　者：王君[1] 马挺[1] 刘静[1] 刘清坤[1] 赵玲侠[1] 梁凤来[1] 刘如林[1] 

机构地区：[1]南开大学生命科学学院天津市功能基因组学重点实验室,天津300071

出　　处：《环境科学》2008年第2期462-468,共7页Environmental Science

基　　金：国家自然科学基金项目(50674058);天津市应用基础研究计划重点项目(07JCZDJC03200)

摘　　要：采用PCR-DGGE技术对高温油藏的微生物群落进行了多样性分析,并将得到的信息用于指导油藏微生物的分离.本研究通过PCR-DGGE技术对油藏水样中DNA的16S rDNA V3、V8、V9 3个高可变区的扩增产物进行了比较,确定采用可得到更多微生物多样性信息的V9区引物进行PCR扩增,优势条带序列分析表明,在高温油藏中存在的微生物与GenBank数据库中α,β,γ-变形杆菌和芽孢杆菌的序列相似性最高.利用多元细菌培养技术,以序列信息为指导,采用富集培养、直接培养和特殊培养的方法,从水样中分离出5株高温菌(而传统分离方法只能获得3株),其中3株高温解烃菌分别属于Bacillus属、Geobacillus属和Petrobacter属,它们能够在55℃以上兼性厌氧条件良好生长,对原油的降解率分别为56.5%7、0.01%和31.87%,对原油的降粘率分别为40%、54.55%和29.09%,使原油的凝固点分别降低3.7、5.2和3.1℃.因此,序列指导和改变培养条件是分离更多有效采油微生物的改进方法,这3株高温菌的对原油的作用效果证明其具备提高石油采收率的潜力.It is a brand-new method to isolate functional bacteria from high temperature petroleum reservoirs according to the sequence information obtained from PCR-DGGE patterns. Three-set primers of 16S rDNA high variable region, V3, V8, V9, were compared. The results showed that more microbial diversity information could be obtained from the PCR product of V9 region. Sequence analysis indicated that the dominant bacteria in the petroleum reservoir had high sequence similarity with bacteria from α, β, γ-Proteobacterias and Bacilli from the GenBank database. According to the sequences information, multi-cultivation technology including enrichment cultivation, special cultivation and direct cultivation methods were employed, and finally, five strains （three strains by traditional methods） were isolated from oil-water samples. Among them, three thermophilic hydrocarbon-degrading bacteria, which belonged to Bacillus sp., Geobacillus sp. and Petrobacter sp., respectively, could grow well under 55 ~C in obligate anaerobic condition. The crude oil could be utilized by these strains with the degradation rate of 56.5 %, 70.01% and 31.78 % respectively along with the viscosity reduction rate of 40 %, 54.55 % and 29.09 %, meanwhile the solidify points of crude oil were reduced by 3.7, 5.2 and 3.1 ℃ . Therefore, the combination of sequence information from PCR-DGGE and altering cultivation conditions is an available novel method to isolate more functional microorganisms which could be utilized for microbial enhanced oil recovery.

关 键 词：油藏 梯度凝胶变性电泳(DGGE) 16SrDNA 高温解烃菌 培养技术 

分 类 号：X172[环境科学与工程—环境科学] Q938.1[生物学—微生物学]