互营烷烃降解菌Syntrophaceae的定向分离及其响应高压胁迫的机制研究

It’s one of promising technologies for converting hard-to-use crude oil in petroleum reservoirs into methane using anaerobic microcosms. Syntrophic metabolism is an essential step in methanogenic degradation of petroleum hydrocarbon. Uncultured bacteria of family Syntrophaceae played key roles in syntrophic alkane degradation and distributed widely in oil reservoirs, through culture-independent methods and stable isotope probing. However, the pure culture of syntrophic hydrocarbon-degrading bacteria has not been isolated and characterized, which not only hampers the research of the physiological and biochemical properties of the syntrophic bacteria, but also hinders studies on the molecular mechanism of syntrophic hydrocarbon degradation. .This proposal will employ a novel idea of high-throughput separation before co-culture incubation, and develop co-culture methods to incubate syntrophic microorganisms in microfluidic chips. The proposal will try to obtain the two-component culture of syntrophic alkane-degrading bacteria and methanogen using this technique, and characterize the physiological, biochemical and phylogenetic traits of the syntrophic alkane-degrading bacteria. The proposed plan will also analyze the hydrocarbon degradation potential of two-component culture under different high-pressure conditions, and make researches on the stress response mechanisms to high pressure. The understanding will provide basis for the molecular mechanism research of syntrophic hydrocarbon degradation, and provide clues to isolate uncultured microorganisms with specific ecological functions.

将油藏中残留的原油转化为甲烷，进行气态开采(油藏变气藏)，是未来低品位油藏开发的新方向。互营代谢是微生物降解石油烃产甲烷过程中的限速步骤，未培养结合同位素示踪方法，已证明油藏中广泛存在的未培养Syntrophaceae科细菌，是互营烷烃降解的关键微生物。迄今为止，国内外均未获得互营烃降解菌的纯培养物，这不仅限制了人们对其生理生化功能的认识，也是深入研究互营烃降解产甲烷机理的瓶颈。.本项目在前期研究基础上，拟采用先“高通量隔离”后“共培养”的思路，利用微流控芯片技术，建立互营微生物在芯片上的共培养方法，获得互营烃降解菌-产甲烷古菌共培养物，揭示互营烃降解菌的生理生化特征；利用互营烃降解与产甲烷古菌的二元培养物，分析其在高压胁迫下的烃降解能力及响应机制，阐明互营烃降解菌的环境适应性特征。本研究不仅为深入开展互营烃降解的分子机理研究奠定基础，而且能为未培养功能微生物的分离研究提供新思路和手段。

互营微生物是有机质厌氧降解产甲烷过程中的关键微生物。它们对氧敏感、生长苛刻，目前只分离了40多个物种。迄今，还没有专性互营烃降解菌的分离报道。.本项目总结了互营微生物分离培养的研究进展；研发了厌氧微生物操作平台、自动化挑取装备和MALDI-TOF厌氧微生物数据库，将厌氧微生物的分选效率提高了200-300株/小时.机器，解决了厌氧微生物难以高通量操作的技术瓶颈。从烃降解产甲烷富集物中分离鉴定了厌氧微生物新属3个（Petroclostridium gen. nov、Gudongella gen. nov.和Biomaibacter gen. nov），提出新科3个、新目1个(Thermosediminibacterales ord. nov.、Hungateiclostridiaceae fam. nov.、Tepidanaerobacteraceae fam. nov.、和Thermosediminibacteraceae fam. nov.)。从烃降解产甲烷富集物中拼接获得了未培养Smithella sp.的基因组，推测其不具有硫酸盐还原能力。在基金委备案调整为研究硫酸盐胁迫下烃降解菌系的响应特征。发现在25 mM硫酸盐胁迫下的甲烷产量和比产甲烷速率分别下降到44%和56%，并且起始硫酸盐浓度决定了石油烃的碳流向。添加0.5 - 4 mM硫酸盐组的δ13CH4为-61.5 ± 1.7‰，而10–25 mM硫酸盐组的δ13CH4只有-50.7 ± 1.2‰，表明不同浓度硫酸盐胁迫下的产甲烷途径发生了显著变化。发现Smithella和Methanoculleus的丰度与硫酸盐浓度呈正相关；Desulfobacteraceae和Desulfomonile分别是低和高浓度硫酸盐中的优势硫酸盐还原菌。.本项目发表论文8篇（其中IJSEM 4篇、Biotechnology for Biofuels 1篇、Microbes and Environments 1篇、微生物学报和应用环境学报各1篇），申请专利和著作权各2项，在国内外学术会议上作口头报告6次。虽然互营烃降解菌的分离之路坎坷，我仍心往之。