The family Ca. Methanoperedenaceae archaea mediates the anaerobic oxidation of methane (AOM) in different terrestrial environments. Using a newly developed high-pressure laboratory incubation system, we investigated 214-and 249-m deep groundwater samples at Horonobe Underground Research Laboratory, Japan, where the high and low abundances of Ca. Methanoperedenaceae archaea have been shown by genome-resolved metagenomics, respectively. The groundwater samples amended with 13 Clabelled methane and amorphous Fe(III) were incubated at a pressure of 1.6 MPa. After 3-7 days of incubation, the AOM rate was 45.8 ± 19.8 nM/day in 214-m groundwater. However, almost no activity was detected from 249-m groundwater. Based on the results from 16S rRNA gene analysis, the abundance of Ca. Methanoperedenaceae archaea was high in the 214-m deep groundwater sample, whereas Ca. Methanoperedenaceae archaea was undetected in the 249-m deep groundwater sample. These results support the in situ AOM activity of Ca. Methanoperedenaceae archaea in the 214-m deep subsurface borehole interval. Although the presence of Fe-bearing phyllosilicates was demonstrated in the 214-m deep groundwater, it needs to be determined whether Ca. Methanoperedenaceae archaea use the Fe-bearing phyllosilicates as in situ electron acceptors by high-pressure incubation amended with the Fe-bearing phyllosilicates.

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 13C-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.