Diversity and Function of Methanotrophic Bacteria in Caves

Despite representing the second largest sink for the atmospheric greenhouse gas methane (CH4), the methanotrophic organisms responsible for atmospheric CH4 consumption have eluded cultivation. High-throughput studies of methanotrophic communities present an opportunity to learn novel details about the organisms responsible, yet such studies have rarely been conducted. Recent observations of subatmospheric CH4 concentrations in cave-air have led to the hypothesis that methanotrophs are active over large spatial scales in the subsurface. Karst terrains cover between 10 - 20 % of the terrestrial surface area and offer abundant cave-related methanotrophic habitat due to the exchange of air with the atmosphere. We collected 42 cave soil samples from 20 caves to test the hypothesis that subterranean methanotrophy removes CH4 from cave-air. Methanotrophs were found in 90 % of samples, notably in locations with subatmospheric CH4­ concentrations. Methylocystaceae were present in caves and accounted for 92 % of the methanotrophic community on average (median), however almost all of the observations were of unidentified Methylocystaceae. Abundances of uncultured and unidentified members of the Methylococcales were correlated with cave-air CH4 concentrations suggesting that some Methylococcales may contribute to atmospheric CH4 oxidation. Individual caves had a strong influence on the observed methanotrophic community composition accounting for 77 % of the variance in the assemblage. Nevertheless, cave-air CH4 concentrations were predictive of the methanotrophic community composition accounting for 5 % of the variation. Our findings also are suggestive of CH4-fueled microbial food webs. For example, abundances of known methylotrophic organisms were correlated with cave-air CH4 concentrations. This may suggest that some methylotrophs contribute to atmospheric CH4 oxidation or that molecules produced in the CH4 oxidation pathway, like methanol, are leaked from methanotrophic cells allowing for the growth of methylotrophs. Our results suggest that uncultivated and unidentified methanotrophs are responsible for subatmospheric CH4 concentrations in caves and have secondary influences on the cave-microbial community structure.