Reactive Fe(III) minerals can influence methane (CH ) emissions by inhibiting microbial methanogenesis or by stimulating anaerobic CH oxidation. The balance between Fe(III) reduction, methanogenesis, and CH oxidation in ferruginous Archean and Paleoproterozoic oceans would have controlled CH fluxes to the atmosphere, thereby regulating the capacity for CH to warm the early Earth under the Faint Young Sun. We studied CH and Fe cycling in anoxic incubations of ferruginous sediment from the ancient ocean analogue Lake Matano, Indonesia, over three successive transfers (500 days in total). Iron reduction, methanogenesis, CH oxidation, and microbial taxonomy were monitored in treatments amended with ferrihydrite or goethite. After three dilutions, Fe(III) reduction persisted only in bottles with ferrihydrite. Enhanced CH production was observed in the presence of goethite, highlighting the potential for reactive Fe(III) oxides to inhibit methanogenesis. Supplementing the media with hydrogen, nickel and selenium did not stimulate methanogenesis. There was limited evidence for Fe(III)-dependent CH oxidation, although some incubations displayed CH -stimulated Fe(III) reduction. 16S rRNA profiles continuously changed over the course of enrichment, with ultimate dominance of unclassified members of the order Desulfuromonadales in all treatments. Microbial diversity decreased markedly over the course of incubation, with subtle differences between ferrihydrite and goethite amendments. These results suggest that Fe(III) oxide mineralogy and availability of electron donors could have led to spatial separation of Fe(III)-reducing and methanogenic microbial communities in ferruginous marine sediments, potentially explaining the persistence of CH as a greenhouse gas throughout the first half of Earth history.

19Reactive Fe(III) minerals can influence methane (CH 4 ) emissions by inhibiting microbial 20 methanogenesis or by stimulating anaerobic CH 4 oxidation. The balance between Fe(III) 21 reduction, methanogenesis, and methane oxidation in ferruginous Archean and Paleoproterozoic 22 oceans would have controlled CH 4 fluxes to the atmosphere, thereby regulating the capacity for 23 CH 4 to warm the early Earth under the Faint Young Sun. We studied CH 4 and Fe cycling in 24 anoxic incubations of ferruginous sediment from the ancient ocean analogue Lake Matano, 25 Indonesia over three successive transfers (500 days total). Iron reduction, methanogenesis, 26 methane oxidation, and microbial taxonomy were monitored in treatments amended with 27 ferrihydrite or goethite. After three dilutions, Fe(III) reduction persisted only in bottles with 28 ferrihydrite. Enhanced CH 4 production was observed in the presence of goethite, highlighting the 29 potential for reactive Fe(III)-oxides to inhibit methanogenesis. Supplementing the media with 30 hydrogen, nickel and selenium did not stimulate methanogenesis. There was limited evidence for 31 Fe(III)-dependent CH 4 oxidation, although some incubations displayed CH 4 -stimulated Fe(III)-32 reduction. 16S rRNA profiles continuously changed over the course of enrichment, with ultimate 33 dominance of unclassified members of the order Desulfuromonadales in all treatments. Microbial 34 diversity decreased markedly over the course of incubation, with subtle differences between 35 ferrihydrite and goethite amendments. These results suggest that Fe(III)-oxide mineralogy and 36