Archaeal methanogenesis in a deep terrestrial aquifer driven by Methanothermobacter tomskensis sp. nov

Methanogenic archaea, capable of producing organic matter from hydrogen and CO₂ emanating from the mantle, constitute one of the key functional groups of microorganisms in the deep subsurface. Although the diversity of methanogenic inhabitants of the deep biosphere has been studied in detail, the activity of methanogens in these biotopes remains virtually uncharacterized. We have measured the rate of hyrogenotrophic methanogenesis in water from the model borehole 3P (2.7 km deep), located near the small town of Parabel in the Tomsk Region. The potential rate of hydrogenotrophic methanogenesis, measured using ¹⁴C-labeled sodium bicarbonate, was 5.45 ± 0.528 nmol CH₄/dm³/day (0.00545 ± 0.0005 nmol CH₄/cm³/day) - a value compatible to the methanogenic archaeal activity known for anaerobic sediments of surface water bodies. The 16S rRNA gene profiles of water from well 3P were dominated by s single phylotype assigned to the genus Methanothermobacter; it accounted for 25.54% of the total microbial community. A MAG (metagenome assembled genome) designated 3P-20-051 and belonging to the genus Methanothermobacter was assembled from the water metagenome of well 3P. According to GTDB taxonomy, this MAG represents a novel species within the genus. The closest validly described relative of MAG 3P-20-001 was Methanothermobacter marburgensis with an ANI of 92.66%. Following the characterization of this MAG, a strain of methanogenic archaea designated as K4^T was isolated in pure culture. Morphological, physiological, genotypic, and phylogenetic data indicate that this strain represents a new species of the genus Methanothermobacter, for which we propose the name Methanothermobacter tomskensis sp. nov. Comparison of the strain K4^T genome with MAG 3P-20-051 showed that they were nearly identical: the ANI value was 99.96%. Thus, M. tomskensis sp. nov. indeed, represents a dominant methanogenic lineage revealed by metagenomics. Strain K4^T is characterized by an obligately hydrogenotrophic metabolism and possibly represents the main producer of organic matter in the deep horizons of the thermal aquifer.