Enhanced Extracellular\nElectron Transfer in Magnetite-Mediated\nAnaerobic Oxidation of Methane Coupled to Humic Substances Reduction:\nThe Pivotal Role of Membrane-Bound Electron Transfer Proteins

Humic substances are organic substances prevalent in\nvarious natural\nenvironments, such as wetlands, which are globally important sources\nof methane (CH<sub>4</sub>) emissions. Extracellular electron transfer\n(EET)-mediated anaerobic oxidation of methane (AOM)-coupled with humic\nsubstances reduction plays an important role in the reduction of methane\nemissions from wetlands, where magnetite is prevalent. However, little\nis known about the magnetite-mediated EET mechanisms in AOM-coupled\nhumic substances reduction. This study shows that magnetite promotes\nthe reduction of the AOM-coupled humic substances model compound,\nanthraquinone-2,6-disulfonate (AQDS). <sup>13</sup>CH<sub>4</sub> labeling\nexperiments further indicated that AOM-coupled AQDS reduction occurred,\nand acetate was an intermediate product of AOM. Moreover, <sup>13</sup>CH<sub>3</sub><sup>13</sup>COONa labeling experiments showed that\nAOM-generated acetate can be continuously reduced to methane in a\nstate of dynamic equilibrium. In the presence of magnetite, the EET\ncapacity of the microbial community increased, and <i>Methanosarcina</i> played a key role in the AOM-coupled AQDS reduction. Pure culture\nexperiments showed that <i>Methanosarcina barkeri</i> can\nindependently perform AOM-coupled AQDS reduction and that magnetite\nincreased its surface protein redox activity. The metatranscriptomic\nresults indicated that magnetite increased the expression of membrane-bound\nproteins involved in energy metabolism and electron transfer in <i>M. barkeri</i>, thereby increasing the EET capacity. This phenomenon\npotentially elucidates the rationale as to why magnetite promoted\nAOM-coupled AQDS reduction.