Pushing the upper temperature limit of methanotrophy in continental hydrothermal ecosystems, active biological methane oxidation in hot springs of Yellowstone National Park

Methane oxidation in terrestrial geothermal systems is an understudied process contributing to carbon cycling in extreme environments. We combined geochemical analyses, 16S rRNA gene amplicon sequencing, shotgun metagenome sequencing, and 14 CH 4 microcosm assays across 61 Yellowstone hot springs spanning pH 1.9–9.0 and temperatures of 28.6–92.2 °C to survey hydrothermal systems for methanotrophy. Bacterial aerobic methanotroph phylotypes were detected at multiple sites, including Verrucomicrobia (order S-BQ2-57) and Alphaproteobacteria , with the family Methylocystaceae having the highest relative abundance among bacterial methanotroph phylotypes. No known archaeal anaerobic methanotrophs were observed. Biological methane oxidation was widespread, occurring at 14 of 17 experimental sites under both ambient and air-amended conditions. Rates were highest at CH 4 -rich, NH 3 -poor sites dominated by bacterial methanotrophs, consistent with energy supply predictions integrating CH 4 /O 2 and CH 4 /NH 3 concentration ratios. Conversely, NH 3 -rich, energy-rich sites exhibited lower methane oxidation rates (MOR) and were dominated by archaeal ammonia oxidizers, primarily Candidatus Nitrosocaldus , suggesting chemical competitive inhibition of NH 3 on methanotrophy. Remarkably, significant methane oxidation occurred at eight sites where no known methanotrophs were detected, including a site at 89.9 °C—well above the previously reported upper growth temperature limit for methanotrophs from continental geothermal and hydrothermal systems—pointing to uncharacterized thermophilic lineages. These results suggest that biological methane oxidation in Yellowstone hot springs is influenced by the interplay of substrate availability and energy supply. By linking energy supply calculations with microbial distributions, we identify both known methanotrophs ( Verrucomicrobia , Alphaproteobacteria ) and archaeal ammonia oxidizers as potential active contributors, while highlighting the potential for novel thermophilic lineages, thereby expanding the ecological and thermal boundaries of methane oxidation in extreme terrestrial ecosystems.