水稻土和湿地土壤短链脂肪酸互营氧化产甲烷机理的研究

The syntrophic oxidation of short-chain fatty acids plays a key role in the methanogenic decomposition of organic materials in anoxic environments. The aim of this proposal is to investigate the processes and mechanisms of direct interspecies electron transfer (DIET) in the syntrophic oxidation of short-chain fatty acids in rice paddy soil and Zoige wetland from Tibetan Plateau. It has been well known that syntrophic oxidation of fatty acids depends on the interspecies electron transfer. The classical theory considers interspecies H2 and formate transfers as the major mechanisms for interspecies electron transfer. This theory, however, has been challenged recently owing to the discovery of DIET in 2010. Many studies have been conducted on DIET syntrophy in Geobacter-involving ethanol oxidation. The syntrophic oxidation of short-chain fatty acids like butyrate and propionate, however, is thermodynamically much stricter than ethanol oxidation. Very little has been known on the DIET syntrophy in the syntrophic oxidation of short-chain fatty acids. Three research plans are designed in the present proposal to investigate the DIET syntrophy in the syntrophic oxidation of butyrate, propionate and acetate in rice paddy soil and Zoige wetland. DIET syntrophy in environments may have two forms, one facilitated by the biological-origin mediators like conductive pili in Geobacter and the other facilitated by the environmental-origin mediators like the naturally-occurring conductive minerals and carbon. We will focus on the later form of DIET in this project. In Plan A, we will investigate the occurrence of DIET syntrophy facilitated by the naturally occurring conductive nanomaterials including magnetite, pyrite and black carbon (named environmental nanowires hereafter). The composition and distribution of environmental nanowires in the selected soils will be determined. The effect of different type of nanowires will be compared and the modification of nanowires by humic acids will be investigated. The key organisms involved in the syntrophic interaction of fatty acid oxidation and methane production will be identified. In Plan B, we will investigate the formation of microbial aggregates during the syntrophic oxidation of fatty acids. Aggregate formation is common in the syntrophic interaction. It is however unknown how environmental nanowires affect the aggregate formation and its electric conductivity. We will employ the microfluidic chip technology to direct observe the syntrophic aggregation and to determine the effect of environmental nanowires. The electric conductivity of syntrophic aggregates will be determined using electrochemical technology. In Plan C, we will tempt to identify the pathways of electron transfer chains inside and outside the syntrophic organisms using a combination of stable isotope probing and environmental genomics and transcriptomics technologies. The diverse methodologies will be applied in this project including molecular ecological techniques, anaerobic enrichment and coculture cultivation, stable isotope labeling, microfluidic technology, microscopic techniques and micro-electrochemical technology. We expect the outcomes of this project will lay a new foundation for better understanding the methanogenesis and carbon cycling in anoxic environments.

短链脂肪酸互营氧化是有机质厌氧分解产甲烷过程的关键步骤。本申请项目将从三个方面研究水稻土和若尔盖湿地土壤脂肪酸互营氧化的直接种间电子转移机制。第一，研究土壤中导电性纳米颗粒包括铁氧化物、黄铁矿和黑炭等（统称环境纳米电线）的组成和分布特点，明确环境纳米电线诱导的直接种间电子转移在水稻土和湿地土壤中的广泛性。阐明环境纳米电线与土壤腐殖质复合对电子传递作用的影响。揭示参与直接种间电子转移的关键互营菌和产甲烷菌种类。第二，在微观尺度观察互营微生物与环境纳米电线的相互作用、团聚体的形成机制及其导电特征。第三，揭示脂肪酸互营氧化的电子传递途径，明确胞外电子传递的分子机理。研究将采用分子生态学技术、稳定同位素标记技术、厌氧微生物富集培养和纯培养技术、微流控芯片技术、显微技术和微电极电化学方法等一系列先进技术相结合的研究手段。预计研究成果可为理解缺氧环境的产甲烷过程和碳循环提供新的理论基础。

以全球变暖为主要特征的全球变化是目前人类可持续发展所面临的严峻挑战之一，甲烷是仅次于二氧化碳的重要温室气体，而陆地生态系统大量常年性或季节性缺氧环境是大气甲烷的主要排放源。本项目以水稻土和自然湿地为研究对象，选择丁酸、丙酸和乙酸互营产甲烷过程为研究目标，聚焦环境纳米电线介导的直接种间电子转移（DIET）过程和机理。水稻土和湿地富含各种铁氧化物矿物包括导电性磁铁矿等，生物炭则是近年来广泛应用于农田土壤的土壤质量调理剂，这些材料的共同特性是具有导电性。本项目开展了以下三方面研究：第一，水稻土和自然湿地土壤中不同脂肪酸互营氧化的DIET作用及其关键微生物；第二，环境导电材料对DIET的激发作用；第三，互营微生物团聚体的形成机制及功能效率。取得如下主要研究进展： 1）揭示了水稻土和湿地土壤有机质厌氧降解过程中短链脂肪酸产生动态、关键互营微生物种类及环境影响因子，发现磁铁矿和生物炭均可显著促进水稻有机残体的厌氧分解及产甲烷过程，并揭示可能与中间产物互营代谢的直接种间电子传递作用有关；2）系统研究了磁铁矿和碳纳米管等环境纳米电线对丁酸和丙酸互营氧化的调控机制，发现这些厌氧互营微生物能借助这些导电材料发生直接种间电子传递，促进脂肪酸分解及产甲烷过程；3）发现磁铁矿Fe(II)/Fe(III)氧化还原循环对乙酸型产甲烷过程的促进作用，并揭示氢型产甲烷螺菌也能进行胞外电子传递，显著拓展了对水稻土和湿地厌氧微生物胞外电子转移功能多样性的认识；4）本项目在执行期间自制构建了厌氧微生物单细胞微流控研究技术平台，为理解厌氧微生物团聚体形成机制和实时表征提供了技术支撑。这些研究成果可为理解缺氧环境中有机质厌氧分解和产甲烷过程的演化规律，揭示人类活动对厌氧微生物过程特别是互营产甲烷过程的影响，准确预测缺氧环境中甲烷排放和碳循环特征提供科学基础。项目已发表SCI论文13篇，英文专著章节1篇，培养博士后2名、博士和硕士研究生共8名。