Gaoshiqia hydrogeniformans sp. nov., a novel hydrogen-producing bacterium isolated from a deep diatomaceous shale formation.

In the deep subsurface Miocene groundwater of Horonobe, Hokkaido, Japan, we isolated strain Z1-71ᵀ, an obligately anaerobic, Gram-stain-negative, non-motile bacterium with rod-shaped morphology (2.7-4.8 µm × 0.4 µm). This strain could grow at 10-42 °C (optimum, 30-35 °C), over a pH range of 6.0-9.0 (optimum, pH 7.0-7.2) and in the presence of 0-30 g l^-1 NaCl (optimum, 5-15 g l^-1). Physiologically, strain Z1-71ᵀ displayed positive catalase activity but negative oxidase reaction, with notable hydrogen production during d-glucose metabolism. Chemotaxonomic analysis revealed MK-7 as the sole respiratory quinone, while cellular lipid profiling identified four unidentified polar lipids, one unidentified phospholipid, one unidentified aminolipid and one unidentified glycolipid. The predominant fatty acids comprised C_17:0 (23.4%), C_17:1  ω6c (13.8%), anteiso-C_15 :0 (6.9%) and iso-C_17:0 3-OH (6.6%). Genomic characterization determined a genome size of 5.7 Mb with a G+C content of 45.9 mol%. Comprehensive phylogenetic analysis of 16S rRNA gene sequences positioned strain Z1-71ᵀ within the family Prolixibacteraceae, showing the highest sequence similarity to Gaoshiqia sediminis A06^T (95.0%), followed by Mangrovibacterium diazotrophicum SCSIO N0430^T (94.6%), Mangrovibacterium lignilyticum BM_7^T (94.3%) and Mangrovibacterium marinum FA423^T (93.6%). Digital DNA-DNA hybridization and orthologous average nucleotide identity tool using USEARCH (OrthoANIu) analyses between strain Z1-71ᵀ and G. sediminis A06^T yielded values of 20.7% and 75.8%, respectively, confirming genomic distinction. Based on these phylogenetic and phenotypic characteristics, we propose strain Z1-71ᵀ (=DSM 117644ᵀ=JCM 36072ᵀ) as the type strain of a novel species, Gaoshiqia hydrogeniformans sp. nov. This discovery not only expands our understanding of microbial diversity in deep terrestrial subsurface environments but also highlights the ecological significance of hydrogen-producing anaerobes in these previously underexplored habitats.