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The biological cleavage of methylphosphonate (MPn), which releases both phosphate and methane, is one pathway that may contribute to this paradox. Here, we explore the genomic and functional potential for oxic methane production (OMP) via MPn in Flathead Lake, a large oligotrophic freshwater lake in northwest Montana. Time series and depth profile measurements show that epilimnetic methane was persistently supersaturated despite high oxygen levels, suggesting a possible\n            <jats:italic>in situ<\/jats:italic>\n            oxic source. Metagenomic sequencing indicated that 10% of microorganisms in the lake, many of which are related to the Burkholderiales (Betaproteobacteria) and Actinomycetota, have the genomic capacity to cleave MPn. We experimentally demonstrated that these organisms produce methane stoichiometrically with MPn consumption across multiple years. However, methane was only produced at appreciable rates in the presence of MPn when a labile organic carbon source was added, suggesting that this process may be limited by both MPn and labile carbon supply. Members of the genera\n            <jats:italic>Acidovorax<\/jats:italic>\n            ,\n            <jats:italic>Rhodoferax<\/jats:italic>\n            , and\n            <jats:italic>Allorhizobium<\/jats:italic>\n            , organisms which make up less than 1% of Flathead Lake communities, consistently responded to MPn addition. We demonstrate that the genomic and physiological potential for MPn use exists among diverse, resident members of Flathead Lake and could contribute to OMP in freshwater lakes when substrates are available.\n          <\/jats:p>\n          <jats:sec>\n            <jats:title>IMPORTANCE<\/jats:title>\n            <jats:p>\n              Methane is an important greenhouse gas that is typically produced under anoxic conditions. We show that methane is supersaturated in a large oligotrophic lake despite the presence of oxygen. Metagenomic sequencing indicates that diverse, widespread microorganisms may contribute to the oxic production of methane through the cleavage of methylphosphonate. We experimentally demonstrate that these organisms, especially members of the genus\n              <jats:italic>Acidovorax<\/jats:italic>\n              , can produce methane through this process. However, appreciable rates of methane production only occurred when both methylphosphonate and labile sources of carbon were added, indicating that this process may be limited to specific niches and may not be completely responsible for methane concentrations in Flathead Lake. This work adds to our understanding of methane dynamics by describing the organisms and the rates at which they can produce methane through an oxic pathway in a representative oligotrophic lake.\n            <\/jats:p>\n          <\/jats:sec>","DOI":"10.1128\/aem.01097-23","type":"journal-article","created":{"date-parts":[[2023,11,30]],"date-time":"2023-11-30T14:02:11Z","timestamp":1701352931000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":19,"title":["Oxic methane production from methylphosphonate in a large oligotrophic lake: limitation by substrate and organic carbon supply"],"prefix":"10.1128","volume":"89","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0163-2769","authenticated-orcid":false,"given":"Logan M.","family":"Peoples","sequence":"first","affiliation":[{"name":"Flathead Lake Biological Station, University of Montana, Polson, Montana, 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