Anaerobic oxidation of methane (AOM) is a key microbiological process mitigating the release of methane from natural environments into the atmosphere1. AOM is mediated by anaerobic methanotrophic archaea (ANME), which couple methane oxidation to the reduction of an exogenous electron-acceptor2. In marine environments, many ANME lineages are known to perform AOM coupled to sulphate reduction via direct interspecies electron transfer to syntrophic sulphate-reducing bacteria2. Conversely, members of the Methanoperedenaceae (formerly known as ANME-2d) can couple AOM to the direct reduction of nitrate and metal oxides independent of a syntrophic partner3-5. A recent comparative genomic study revealed members of the family can utilise diverse terminal electron transfer strategies, suggesting a remarkable ability of these microorganisms to adapt to their environment6. However, our understanding of this metabolic diversity and ecology is limited by poor genome coverage of the family, with all but 3 of the available metagenome-assembled genomes (MAGs) assigned to the type genus “Candidatus Methanoperedens”. As such, it is very likely further metabolic diversity across the family is yet to be revealed.
In this study, we performed a large-scale survey of the Methanoperedenaceae in which newly developed computational tools were applied to screen 250,557 publicly available metagenomic datasets from the NCBI Sequence Read Archive. Members of the Methanoperedenaceae were identified in 2,609 datasets up to an abundance of 38%, confirming their numerical importance to global methane cycling. Unlike the marine ANME lineages, the Methanoperedenaceae were almost exclusively found in freshwater environments. These sites included novel ecological niches for the family, such as urban landfill and toxic-waste sites, where members of the family may provide a previously overlooked methane sink. From these datasets, the family’s coverage was expanded to 61 high-quality Methanoperedenaceae MAGs (> 70% completeness; < 10% contamination) including representation of a novel genus. Comparative genomics across these MAGs confirmed conservation of the methane oxidation pathway, and substantially expanded on previous investigations on the family’s diverse respiratory pathways. Findings include the widespread potential for terminal electron transfer to arsenate and selenate, as well as uncharacterised terminal reductases that may represent novel respiratory strategies, importantly linking methane cycling to additional biogeochemical cycles. This study’s findings will direct future efforts to characterise the Methanoperedenaceae and demonstrates a powerful and novel approach to large-scale interrogation of environmental datasets.