Characterization of archaeal and bacterial communities thriving in methane-seeping on-land mud volcanoes, Niigata, Japan.

Submarine mud volcanoes (MVs) have attracted significant interest in the scientific community for obtaining clues on the subsurface biosphere. On-land MVs, which are much less focused in this context, are equally important, and they may even provide insights also for astrobiology of extraterrestrial mud volcanism. Hereby, we characterized microbial communities of two active methane-seeping on-land MVs, Murono and Kamou, in central Japan. 16S rRNA gene profiling of those sites recovered the dominant archaeal sequences affiliated with methanogens. Anaerobic methanotrophs (ANME), with the subgroups ANME-1b and ANME-3, were recovered only from the Murono site albeit a greatly reduced relative abundance in the community compared to those of typical submarine MVs. The bacterial sequences affiliated to Caldatribacteriota JS1 were recovered from both sites; on the other hand, sulfate-reducing bacteria (SRB) of Desulfobulbaceae was recovered only from the Murono site. The major difference of on-land MVs from submarine MVs is that the high concentrations of sulfate are not always introduced to the subsurface from above. In addition, the XRD analysis of Murono shows the absence of sulfate-, sulfur-related mineral. Therefore, we hypothesize one scenario of ANME-1b and ANME-3 thriving at the depth of the Murono site independently from SRB, which is similar to the situations reported in some other methane-seeping sites with a sulfate-depleted condition. We note that previous investigations speculate that the erupted materials from Murono and Kamou originate from the Miocene marine strata. The fact that SRB (Desulfobulbaceae) capable of associating with ANME-3 was recovered from the Murono site presents an alternative scenario: the old sea-related juvenile water somehow worked as the source of additional sulfur-related components for the SRB-ANME syntrophic consortium forming at a deeper zone of the site. However, the reason for the differences between Murono and Kamou is still unknown, and this requires further investigation.