水稻田微生物催化的铁还原型甲烷厌氧氧化过程及其机理研究

Recently, two great breakthroughs have been made in the field of anaerobic oxidation of methane: first, the mechanisms of extracellular electron transfer between anaerobic methanotrophic archaea and sulfate-reducing bacteria were revealed; second, the bioprocess of iron-dependent anaerobic oxidation of methane (Fe-AOM) was finally confirmed by laboratory evidences. At present, some sporadic studies suggest that Fe-AOM may occur in many natural habitats, such as paddy soil, and may be mediated by archaea or by archaea and bacteria together. However, due to the lack of the “true” Fe-AOM culture, microbial mechanisms of Fe-AOM, including microbial metabolism and extracellular electron transfer between microorganisms and iron minerals, remain unclear. In this project, using isotope tracer and high-throughput sequencing, the distribution of Fe-AOM microorganisms in paddy will be investigated; the paddy soil with the highest Fe-AOM activity will be collected as inoculum to enrich and cultivate Fe-AOM microorganisms. Using spectrum analysis and high-throughput sequencing, the physical, chemical and biological features of the Fe-AOM culture will be characterized; substrate affinity, substrate diversity and the response to environmental factors of Fe-AOM microorganisms will also be investigated. Using isotope tracer and metagenomic sequencing, the metabolic pathway and extracellular electron transfer of Fe-AOM microorganisms will be revealed. The results of this project will not only enrich our knowledge of Fe-AOM process, but also provide more ideas for reducing methane emission from paddy fields.

近年来，在微生物甲烷厌氧氧化领域获得了两项重大突破，其一是揭示了甲烷厌氧氧化古菌与硫酸盐还原细菌之间的电子传递方式，其二是实验室证实了铁还原型甲烷厌氧氧化（Fe-AOM）过程。目前零星的研究表明，Fe-AOM过程可能存在于包括水稻土在内的许多生境中，Fe-AOM过程可能由古菌单独或古菌与细菌协作完成，但由于至今未获得“真正”的富集物，Fe-AOM过程机理尚不明确。本项目拟采用同位素示踪、高通量测序等技术，调查Fe-AOM微生物在水稻田中的分布特征，并以高Fe-AOM活性的水稻土为接种物，富集培养Fe-AOM微生物；采用光谱分析、高通量测序等技术，对富集物进行物理、化学、生物特性表征；以Fe-AOM富集物为实验材料，采用同位素示踪、宏基因组测序等技术，揭示Fe-AOM过程的代谢途径和电子传递方式。该项研究不仅能丰富人们对Fe-AOM过程的认识，还能为水稻田甲烷减排提供更多思路。

铁依赖型甲烷厌氧氧化（iron-dependent anaerobic oxiation of methane, Fe-AOM）是微生物介导的三价铁氧化甲烷的过程，广泛存在于多种自然生境中，在温室气体甲烷控制方面具有重要意义。由于同时存在高浓度甲烷和铁氧化物，水稻土被认为是Fe-AOM重要的生境，但相关研究较少。本项目通过长期培养水稻土，获得了Fe-AOM富集物，并研究了其物化特性、生物活性、影响因素和微生物机理。取得主要研究成果如下：（1）以不溶性水铁矿和可溶性EDTA-Fe(III)为电子受体，培养420天后获得了2种Fe-AOM富集物。铁盐的溶解性显著影响Fe-AOM活性，EDTA-Fe(III)可短期刺激Fe-AOM活性，但长期效果不佳。根据抑制试验和群落结构分析结果，推测Fe-AOM过程主要由甲烷厌氧氧化古菌和铁还原细菌共同完成。（2）以水铁矿、针铁矿、赤铁矿和磁铁矿等常见铁矿物为电子受体，研究其对Fe-AOM过程的长期影响。培养380天后，Fe-AOM活性顺序为针铁矿>水铁矿>赤铁矿>磁铁矿≈对照，说明水稻土中丰富的针铁矿在甲烷循环方面具有重要作用。（3）研究金属螯合剂EDTA和电子穿梭体HA对水铁矿依赖Fe-AOM的长期影响，发现EDTA和HA都能短期刺激Fe-AOM活性，说明铁生物可利用性和电子传递效率是Fe-AOM的两个限速步骤。然而，HA会大量消耗基质，刺激HA降解微生物，因此不能用于长期培养。（4）研究硫酸盐对水铁矿依赖Fe-AOM的长期影响，发现水稻土Fe-AOM活性远高于硫酸盐依赖型甲烷厌氧氧化（S-AOM）。抑制试验和微生物群落分析表明，当硫酸盐和水铁矿共存时Fe-AOM活性最高，“神秘的硫循环”途径贡献率可达47％。上述研究成果丰富了Fe-AOM方面的知识，并有助于对水稻田甲烷汇的了解。