典型湿地好氧甲烷氧化过程的微生物固氮潜力及其环境驱动机制

Methanotrophs consume more than half of methane produced in wetland that would otherwise have doubled the global methane budget; however, it remains unclear how methanotrophs overcome nitrogen constraints for this vast amount of carbon sequestration. It has long been postulated that nitrogen fixation by methanotrophs play important roles in counteracting methane emission from wetland, but the diazotrophic methanotrophy remains poorly understood in complex environments. This project aims to decipher the mechanisms underlying microbial nitrogen fixation process driven by methane oxidation in wetland by elucidation of physiological and metabolic functions of nitrogen fixation by methane-oxidizing bacteria in both pure culture system and complex wetland environment. The 13CH4 and 15N tracer would be employed to probe diazotrophy of methanotrophic type strains, in combination with nano secondary ion mass spectrometry (NanoSIMS) imaging technology and the metatranscriptomics. The output of this project is: (1) to reveal the genetic mechanisms of diazotrophic methanotrophs during nitrogen fixation and to establish the nitrogen fixation potential budget of all known representative aerobic methane oxidizers from wetland, (2) to elucidate the adaptive mechanism of diazotrophic methanotrophs during ecological degradation of typical natural wetland, and during the conversion of typical natural wetland to flooded rice fields; (3) to decipher the biogeographic patterns of diazotrophic methanotrophs in natural wetlands and artificial wetland of rice field, and to identify the driving force responsible for niche differentiation of diazotrophic methanotrophs across hierarchical scales; (4) to obtain a comprehensive understanding of methanotrophic diazotrophy in rice paddy soil across China; (5) to explore the interactive mechanisms of diazotrophic methanotrophs in complex soils by microcosm incubation of type strain mixtures and of artificial soils mixture. The ultimate goal of this project will be to provide a clear picture of diazotrophic methane oxidation both in pure culture and wetland environments by using a combination suite of molecular techniques at unprecedented resolutions.

全球湿地每年甲烷排放量数以亿万吨计，导致微生物甲烷氧化过程中面临着强烈的氮素环境胁迫并演化出了固氮功能，但迄今为止，好氧甲烷氧化固氮的微生物过程机制仍不清楚。 本项目拟针对湿地甲烷氧化驱动的微生物固氮过程，以碳氮生物耦合为核心，围绕纯培养体系和复杂湿地环境中甲烷氧化菌的固氮生理功能，利用13CH4和15N2同位素示踪技术，结合基因组、转录组、蛋白组等多组学技术和NanoSIMS成像技术，研究湿地好氧甲烷氧化菌模式菌株的生理固氮机制，解析已知所有湿地代表性好氧甲烷氧化菌株的固氮潜力；阐明好氧甲烷氧化固氮过程对典型自然湿地退化的响应模式；进一步针对开垦于自然湿地及旱地的>20种稻田人工湿地，解析好氧甲烷氧化固氮的活性微生物地理分异规律，评估我国主要稻作区甲烷氧化固氮潜力；同时选择代表性菌株及湿地样品两两混合培养，探讨湿地好氧甲烷氧化近源菌株的共存机制及其固氮功能分异规律。

甲烷氧化过程驱动的固氮作用是湿地氮素循环微生物调控机制的一个重要研究内容。项目组针对性的研究了我国典型湿地生态系统的甲烷氧化过程和固氮过程的微生物耦合机制，取得了系列创新成果。主要包括：（1）揭示了我国典型水稻土的氮矿化速率差异，明确了氮矿化速率低的水稻土的好氧甲烷氧化固氮潜力更强，并且甲烷氧化驱动固氮过程需要一定的甲烷氧化量阈值。（2）揭示了缺氧条件下我国典型水稻土、青藏高原沼泽湿地以及南海底泥中甲烷氧化驱动固氮过程的动力学过程、主导甲烷氧化菌类型以及关键影响因子。证明Methylobacter是各类湿地环境中缺氧条件下驱动固氮过程的主导甲烷氧化菌类型。土壤碳氮含量是缺氧条件下固氮潜力的关键影响因子，而尿素的施用对甲烷氧化驱动的固氮过程的影响在不同水稻土中并不一致，可以表现为促进、抑制以及无显著影响。（3）揭示了甲烷氧化微生物对维持稻田氮素水平、提高作物生产力的作用。发现不同生育周期的水稻根系均可在缺氮条件下通过甲烷氧化过程驱动生物固氮，同时还可以激发21.7%的根系有机氮矿化为无机氮，缓减了水稻根生物降解的氮素限制。证明甲烷氧化菌不仅是温室气体减排的生物“过滤器”，而且是稻田生物有效氮源的“发动机”。（4）揭示了氧气浓度是各类湿地源甲烷氧化菌固氮活性的关键影响因子，并且固氮的氧气阈值因种属的不同而不同。在无氮培养基中，Methylobacter在5%氧气下无法生长，Methylocystis和Methylocaldum在5%氧气下生长良好，而Methylosinus可以在10%氧气下固氮生长。目前项目组成员在环境微生物生态学主流刊物发表论文30篇，其中SCI论文22篇。项目负责人入选 2019 年科技部中青年科技创新推进人才计划，2020年入选中组部万人计划，2020 年应邀参加了美国国家科学院战略咨询研讨会。项目执行期间培养了博士后1名，研究生12名（博士4名，硕士8名）。

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