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Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface.

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A methanogenic organism from the domain Archaea , designated strain T10T, was isolated from groundwater sampled from a deep diatomaceous shale formation located in Horonobe, Hokkaido, Japan. The strain utilized H2/CO2 and formate as substrates for methanogenesis. Cells were strictly anaerobic, Gram-negative-staining, flagellated, irregular coccoids, 0.7–1.6 µm in diameter, and occurred singly. The strain grew at 25–45 °C (optimum 37–42 °C), at pH 5.8–8.2 (optimum pH 6.7–6.8) and in the presence of 0–1.3 M NaCl (optimum 0.1–0.2 M NaCl). The G+C content of the genomic DNA was 62.9 mol%. 16S rRNA gene sequencing revealed that, although the strain is a member of the genus Methanoculleus , it clearly differed from all described species of this genus (95.5–98.3 % sequence similarity). Values for DNA–DNA hybridization with type strains of closely related Methanoculleus species were less than 50 %. Phenotypic and phylogenetic features of strain T10T clearly indicate that it represents a novel species of the genus Methanoculleus , for which the name Methanoculleus horonobensis sp. nov. is proposed. The type strain is T10T ( = DSM 21626T = JCM 15517T).