High-efficiency methane consumption by atmospheric methanotrophs in subsurface karst caves: The irrefutable methane sink

Subsurface karst systems represent substantial but underexplored methane sinks, yet the identities and activities of cave-dwelling methanotrophs remain poorly characterized. We detected increased methane oxidation rates from 2.9 ± 0.1 to 90.7 ± 4.5 ng·g^-1·hour^-1 while supplied with 2 to 500 parts per million (ppm) CH₄ to cave sediments. Atmospheric methanotroph Upland Soil Clusters γ (USCγ), responsible for this oxidation, was further assigned to three genera within the family <i>Candidatus</i> (<i>Ca</i>.) Methyloligotrophaceae, including two previously unrecognized genera. Nano-scale secondary ion mass spectrometry (NanoSIMS) imaging and the produced ¹³C-PLFAs (phospholipid fatty acids) and ¹³CO₂ in ¹³CH₄-fed microcosm confirmed methane as both carbon and energy sources. These methanotrophs exhibited low half-saturation constant (<i>K</i>_m; 138.8 ± 15.8 ppm), high carbon assimilation efficiency (>50%), and metabolic versatility, as revealed by metagenomics and metatranscriptomics analyses. By extrapolating global distribution of <i>Ca.</i> Methyloligotrophaceae and comparing methane oxidation rates between caves and soil ecosystems, we conservatively estimate that subsurface karst in southwest China sequester ~0.56 Tg CH₄ annually. These findings highlight the ecological importance of karst ecosystems as a previously overlooked methane sink.