Summary The family Methanoperedenaceae archaea mediate anaerobic oxidation of methane (AOM) in various terrestrial environments. In this study, we newly developed a high-pressure laboratory incubation system by controlling hydraulic pressure ranging from ambient to 5 MPa. Using the system, we investigated groundwater from 214- and 249-m deep boreholes at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Methanoperedenaceae archaea have been revealed by genome-resolved metagenomics, respectively. We incubated the groundwater samples amended with or without amorphous Fe(III) as an electron acceptor and 13 C-labelled methane at an in-situ pressure of 1.6 MPa. After three to seven-day incubation, AOM activities were not detected from the 249-m deep groundwater but from the 214-m deep groundwater. The AOM rates were 93.7 ± 40.6 and 27.7 ± 37.5 nM/day with and without Fe(III) amendment. To clarify the differences in AOM activity between the 214- and 249-m deep groundwater samples, we characterized Fe(III) contents in suspended particulates collected by filtration. The particulates were not visible in the 249-m deep groundwater on the filter, while they were abundant and contained Fe(III)-bearing phyllosilicates in the 214-m deep groundwater. These results support the in-situ activity of Fe(III)-dependent AOM in the deep subsurface borehole.

Fe(III)-dependent anaerobic methane-oxidizing activity in a deep underground borehole demonstrated by in-situ pressure groundwater incubation