厌氧甲烷氧化古菌的电化学特性及胞外电子传递机制

Anaerobic oxidation of methane (AOM) driven by archaea acts as a main methane sink in anaerobic environments. The interspecies electron transfer between archaea and the partner bacteria is a key scientific issue for understanding the process and mechanism of AOM. However, most previous studies are mainly focused on the syntrophic and metabolic interactions of microbial consortia, few investigations have been carried out to explore the mechanisms of AOM by archaea alone under decoupled conditions. Based on previous observations that direct electron transfer between archaea and the partner bacteria is a principal mechanism of AOM, we link the processes of AOM and extracellular electron transfer (EET) and reduction of solid electron acceptors, and propose a hypothesis that outer membrane cytochrome proteins of archaea play a dominant role in mediating EET of AOM. This project aims to investigate systematically the EET property and electrochemical activity of AOM archaea enrichment culture. Using Fe(III) oxides and solid electrodes as electron acceptors and methane as an electron donor, the efficiency and pathway of EET by AOM archaea will be elucidated via many techniques including electrochemical in situ spectroscopy, scanning electrochemical microscopy. The role of cytochrome proteins and the molecular mechanisms of EET in the AOM process will be illuminated. Results of the project will provide new insights into the AOM principle and its relationship with reductive transformation of soil minerals, and provide a theoretical basis for controlling the greenhouse gas emission.

厌氧甲烷氧化(AOM)是厌氧环境中甲烷消耗的主要途径。其中，古菌/细菌间的互营代谢（即古菌呼出电子/细菌吸入电子的过程）是AOM的核心科学问题。然而，目前对于AOM的研究主要集中在古菌/细菌聚集体，解耦联状态下单独研究古菌的甲烷氧化机制的报道还非常少见。在现有AOM种间胞外电子传递理论的基础上，本项目将“AOM古菌与Fe(III)/电极还原”联系起来，提出AOM古菌外膜细胞色素蛋白直接介导的胞外电子传递假说。以AOM富集培养物为研究对象，以铁氧化物和固体电极为电子受体，甲烷为唯一电子供体，采用电化学原位光谱、扫描电化学显微镜等技术解析AOM古菌胞外电子传递能力和分子机制，从胞外呼吸角度系统研究AOM古菌的电化学活性和胞外电子传递效率，揭示细胞色素蛋白介导的古菌胞外电子传递机制，为理解厌氧环境中甲烷氧化机理及其与土壤矿物还原的关系提供新的线索，为削减温室气体排放提供理论依据。

厌氧甲烷氧化(AOM)是厌氧环境中甲烷消耗的主要途径。其中，古菌/细菌间的互营代谢（即古菌呼出电子/细菌吸入电子的过程）是AOM的核心科学问题。然而，目前对于AOM古菌/细菌聚集体或解耦联的AOM古菌如何驱动AOM耦合胞外固体还原仍不清楚。本项目以AOM古菌富集培养物和AOM纯菌为研究对象，以铁氧化物和固体电极为电子受体，甲烷为唯一电子供体，研究了厌氧甲烷氧化古菌催化AOM耦合固体还原过程的电化学活性及胞外电子传递机制。结果发现，在微生物电化学池中富集得到的AOM功能菌主要是Methanobacterium古菌，它能够与电活性细菌（如Geobacter、Desulfovibrio）合作，高效地将甲烷氧化过程的化学能转化成电能，产生的最大电流密度和最大输出电压分别为1130.2 mA/m2、0.64 V。同时，该富集物可有效进行AOM依赖的水铁矿还原，其亚铁生成速率为5.5 μmol/L/d；电子穿梭体（如AQS）可将显著提高AOM-水铁矿还原速率（高达7.4倍）。胞外电子传递机制分析表明，Methanobacterium古菌可能通过两种方式进行AOM耦合固体还原：（1）单独进行AOM并将电子转移至胞外固体；（2）Methanobacterium古菌释放一些中间产物（如有机酸），电活性微生物氧化中间产物后将电子传递给胞外固体。在解耦合条件下，本项目证实甲烷八叠球菌（Methanosarcina barkeri）可以逆转产甲烷代谢， 单独完成AOM和胞外电子传递过程，并实现产电。产电抑制分析发现，M.. barkeri的AOM产电过程依赖Mcr酶、镍铁氢酶和细胞色素。这些结果表明，产甲烷古菌的驱动AOM耦合胞外受体还原是厌氧环境甲烷降解的重要方式。该结果为削减温室气体排放、推动甲烷生物发电技术发展和应用提供了理论依据。