Sulfate-catalyzed denitrifying anaerobic methane oxidation process mediated by Methanobacterium sp. from digestion sludges.

The reversibility of methanogenesis suggests a broader ecological role for methanogens beyond methane production. However, the mechanisms of reverse methanogenesis in methanogens remain elusive, particularly in aquatic ecosystems with coexisting sulfate and nitrate. Here, a 423-day enrichment in a methane-fed bioreactor emerged a sulfate-catalyzed denitrifying anaerobic methane oxidation (S-DAMO) process under conditions of sulfate and nitrate coexistence. Integrated multi-omics analyses suggested Methanobacterium sp. (bin031) as the most likely archaeon responsible for anaerobic oxidation of methane (AOM), while Limnobacter sp. and Chiayiivirga sp. mediated sulfate reduction and sulfide-driven denitrification, respectively. This tripartite synergy coupled AOM to complete nitrate reduction at a 5:8 molar ratio via cryptic sulfur cycling. Metatranscriptomics analysis revealed that Methanobacterium sp. (bin031) facilitated reverse methanogenesis without depending on the conventional multi-heme c-type cytochromes, while Limnobacter sp. expressed an outer-membrane multi-heme cytochromes-like protein. Quantitative FISH imaging revealed close physical associations between Methanobacterium sp. (bin031) to Limnobacter sp., suggesting a potential for electron exchange; however, proximity alone does not confirm directional direct interspecies electron transfer. Therefore, this study provides potential evidence for a fundamentally distinct AOM pathway involving a tripartite consortium, redefining the catalytic role of sulfate in methane-driven nitrate removal.