Anaerobic methane-oxidizing activity in a deep underground borehole dominantly colonized by Ca. Methanoperedenaceae.

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 C-labelled 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.