Research Starter Grant: Enrichment Culture Studies of Anaerobic Methane Oxidation

甲烷厌氧氧化是一种微生物介导的过程，通过该过程硫酸盐的还原与甲烷的氧化相耦合。这一过程在全球范围内普遍存在，估计将消耗现代大气甲烷净通量的12%至50%(每年70至300Tg)。负责这一过程的微生物很重要，因为甲烷是大气中一种强有力的温室气体，它们的新陈代谢阻止了地球上大量的甲烷进入大气。尽管这一过程很重要，但负责任的微生物从未在实验室中培养过。我们实验室最近进行的研究表明，如果条件合适，特别是甲烷压力升高(50至150个大气压)，这些微生物在实验室中持续生长是可能的。因此，我们建议继续培养这些富集菌，表征富集物的微生物群落组成和生理学，并分离甲烷氧化菌。具体地说，我们建议：1)表征连续培养物生长过程中微生物群落结构以及甲烷和硫酸盐代谢的变化，2)表征甲烷氧化古生菌在浓缩培养物中的生长，包括对生长速率、碳转化效率、生长形态和甲烷分压的影响的特定物种的分析，3)量化与甲烷氧化和脂肪生物合成相关的同位素分馏系数(13C和2H)，以及4)在(100个大气)甲烷水平升高的情况下使用改进的卷管分离负责的微生物。更广泛的影响除了上述建议的研究外，这项工作还将导致开发在高压低温下严格培养厌氧细菌和古生菌的新方法。这些方法有望加强微生物研究的基础设施。建议的研究也有望增进我们对厌氧甲烷氧化的理解，并为未来的研究奠定基础。在实验室中培养负责任的生物体的能力将允许对这一环境重要过程进行各种生化、遗传学、生理学和分子研究。未来研究中可能解决的关键问题包括：1)在没有氧气的情况下，稳定的甲烷分子如何被激活？2)这种化学能应用于工业环境中的甲烷裂解吗？3)厌氧甲烷氧化的进化史是什么，它与地球气候系统的演变有何关系？4)分离株的生理学与厌氧甲烷氧化的环境观察是否一致？了解这些生物体的生理学并在实验室中分离它们是回答这些和其他重要问题的第一步。

Intellectual Merit The anaerobic oxidation of methane is a microbially-mediated process by which the reduction of sulfate is coupled to the oxidation of methane. This process is prevalent on a global scale and is estimated to consume between 12 and 50% the net modern atmospheric methane flux (70 to 300 Tg per year). The microbes responsible for this process are important as methane is a potent greenhouse gas in the atmosphere, and their metabolism prevents large quantities of methane in the Earth from reaching the atmosphere. Despite the importance of this process, the responsible organisms have never been cultured in the laboratory. Recent studies conducted in our lab indicate that sustained growth of these organisms is possible in the laboratory given the appropriate conditions, especially elevated pressures of methane (50 to 150 atmospheres). We therefore propose to continue growth of these enrichment cultures, to characterize the microbial community composition and physiology of the enrichments, and to isolate the methane oxidizers. Specifically, we propose to: 1) Characterize changes in microbial community structure as well as in methane and sulfate metabolism during growth of successive enrichment cultures, 2) Characterize growth of the CH4-oxidizing archaea in the enrichment cultures including species-specific analyses of growth rates, carbon conversion efficiencies, growth morphologies, and the impact of methane partial pressure, 3) Quantify isotope fractionation factors (for 13C and 2H) associated with methane oxidation and lipid biosynthesis, and 4) Isolate the responsible organisms using modified roll tubes at elevated (100 atmosphere) methane levels. Broader Impacts In addition to the studies proposed above, this work will also lead to development of new methods for culturing strictly anaerobic bacteria and archaea at low temperatures with high gas pressures. These methods promise to enhance infrastructure for microbiological research. The proposed studies also promise to enhance our understanding of anaerobic methane oxidation and to lay a foundation for future studies. The ability to grow the responsible organisms in the laboratory will allow for a variety of biochemical, genetic, physiological and molecular studies of this environmentally important process. Key questions that may be addressed in future studies include: 1) how is the stable methane molecule activated in the absence of oxygen?, 2) can this chemistry be applied to methane cracking in an industrial setting?, 3) what is the evolutionary history of anaerobic methane oxidation and how does it relate to the evolution of Earth.s climate system?, and 4) is the physiology of the isolate consistent with environmental observations of anaerobic methane oxidation? Understanding the physiology of these organisms and isolating them in the laboratory are the first steps in answering these and other important questions.