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 Candidatus ( Ca.) 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 ( K 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 Ca. 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.