煤层产甲烷菌高产CH4限速步骤代谢调控机理研究

Coalbed methane (CBM) is a new type of clean energy. Considering the continuous rise in price of energy sources and shortage in supply of petroleum and gas, exploration and development of CBM would bring favourable social, economic and environmental benefits. Biogenic coalbed methane is renewable and thus its generation mechanismes and processes have been a focus of research in the field of exploration and development. In this study, Methanogenium of coal seam is selected as the specimen. Through the clone and functional verification of methyl coenzyme M reductase (MCR), which controlled the rate-limiting step of the CH4 synthesis pathway in Methanogenium of Coal seam, bioinformatic analysis and molecular docking, construction of a constitutive promoter and a recombinant expression vector, methanogens genetic transformation, detection of gene expression by means of Q-PCR, and GC-MS analysis of metabolites, we aim to reveal the relationship between methanogens MCR expression levels/activity and CH4 synthesis, and to clarify the factors and elements that affect MCR activity. Ultimately, we hope to reveal the regulation mechanism of CH4 anabolism at molecular level and to establish fermentation regulation strategy for CH4 high yield. The results will not only provide more theoretical supports for the CBM formation mechanism, but also establish an operational approach for development and utilization CBM resource.

煤层气是一种新型洁净能源。在当前全球能源价格持续上涨、油气供应日趋紧张的形势下，勘探开发煤层气资源具有很好的社会效益、经济效益和环境效益。生物成因煤层气具有可再生的特点，研究其生成机理与过程是勘探评估和开发煤层气资源领域的一个重点和热点问题。本课题以煤层产甲烷菌为研究对象，拟通过CH4合成途径限速步骤的甲基辅酶M还原酶（MCR）基因克隆与功能验证、生物信息学分析与分子对接、组成型启动子及重组表达载体构建、产甲烷菌的遗传转化、基因表达Q-PCR监测、代谢产物GC-MS分析等技术集成，解析产甲烷菌MCR表达量或活性与CH4合成之间的关系，明晰CH4合成过程中影响MCR的各种因素和因子，从分子水平上揭示CH4合成代谢调控机理，建立CH4高产发酵调控策略，为生物成因气的形成机理提供更多的理论支持，也为开发和利用煤层气资源奠定一定的技术基础。

生物成因煤层气具有可再生的特点，研究其生成机理与过程是勘探评估和开发煤层气资源领域的一个重点和热点问题。本课题以无烟煤和煤层水为研究对象，根据煤样工业分析、煤岩性质分析和水质分析结果，判定煤层是适合微生物生存的环境。采用宏基因组学的方法，研究了不同煤矿区煤层微生物菌群的多样性，分析了培养基对煤层微生物菌群组成与丰度的影响。从煤层水中分离获得羧状芽孢杆菌和甲烷八叠球菌菌株，对羧状芽孢杆菌的生理特性和代谢特征做了较深入研究，克隆了甲烷八叠球菌甲基辅酶M还原酶基因的3个亚基，并初步分析了α亚基的空间结构，同时，利用荧光定量PCR的方法，确定了甲烷菌基因表达与培养基氧化还原电位呈负相关。利用正交设计方法，证明酵母粉是甲烷菌培养基中关键组分，羧状芽孢杆菌和甲烷八叠球菌的组合实验也初步证明培养基组分对甲烷菌生长的重要调控作用。这些结论为生物成因气的形成机理提供了更多的理论支持。