Little is known about the microbial distribution patterns in subseafloor sediments. This study examines microbial diversity and activities in sediments of the Nankai Trough, where biogenic gas hydrates are deposited. Illumina sequencing of 16S rRNA genes revealed that the prokaryotic community structure is correlated with hydrate occurrence and depth but not with the sedimentary facies. The bacterial phyla 'Atribacteria' lineage JS1 and Chloroflexi dominated in all samples, whereas lower taxonomic units of Chloroflexi accounted for community variation related to hydrate saturation. In archaeal communities, 'Bathyarchaeota' was significantly abundant in the hydrate-containing samples, whereas Marine Benthic Group-B dominated in the upper sediments without hydrates. mcrA gene sequences assigned to deeply branching groups and ANME-1 were detected only in hydrate-containing samples. A predominance of hydrogenotrophic methanogens, Methanomicrobiales and Methanobacteriales, over acetoclastic methanogens was found throughout the depth. Incubation tests on hydrate-containing samples with a stable isotope tracer showed anaerobic methane oxidation activities under both low- and seawater-like salinity conditions. These results indicate that the distribution patterns of microorganisms involved in carbon cycling changed with gas hydrate occurrence, possibly because of the previous hydrate dissociation followed by pore water salinity decrease in situ, as previously proposed by a geochemical study at the study site.

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The motile cells of the autotrophic dinoflagellates Peridinium umbonatum var. inaequale, Akashiwo sanguinea, Scrippsiella tinctoria, and Prorocentrum micans commonly contain five major sterols: cholesterol, 4-methylcholestan-3-ol, 4,24-dimethylcholestan-3-ol, dinosterol, and dinostanol. The motile cell of the heterotrophic dinoflagellate Protoperidinium crassipes contains cholesterol, 4,24-dimethylcholestan-3-ol, dinosterol, dinostanol, and 4-tetramethylcholestan-3-ol as major free sterols. The dinosterol concentrations of heterotrophic dinoflagellates are about 4-12 times higher than those of autotrophic species, suggesting that the heterotrophic dinoflagellate is an important source of dinosterol in some sediments. 4-Tetramethylcholestan-3-ol has not been reported in cultured samples of other heterotrophic dinoflagellates. The difference in 4-methyl sterol composition in heterotrophic dinoflagellates may be related to the feeds used during the culture experiment. The resting cyst of P. umbonatum var. inaequale contains 4-methylcholestan-3-ol, 4,24-dimethylcholestan-3-ol, dinosterol, dinostanol, and C 31 ∆ 5,22 -sterol as major free sterols. The C 31 ∆ 5,22 -sterol has not been reported in cultured samples of motile cells from dinoflagellates. This compound might be produced during the resting stage, and thus may serve as a potential biomarker for the resting cysts of dinoflagellates.

Gas hydrates deposited in subseafloor sediments are considered to primarily consist of biogenic methane. However, little evidence for the occurrence of living methanogens in subseafloor sediments has been provided. This study investigated viable methanogen diversity, population, physiology and potential activity in hydrate-bearing sediments (1–307 m below the seafloor) from the eastern Nankai Trough. Radiotracer experiments, the quantification of coenzyme F430 and molecular sequencing analysis indicated the occurrence of potential methanogenic activity and living methanogens in the sediments and the predominance of hydrogenotrophic methanogens followed by methylotrophic methanogens. Ten isolates and nine representative culture clones of hydrogenotrophic, methylotrophic and acetoclastic methanogens were obtained from the batch incubation of sediments and accounted for 0.5–76% of the total methanogenic sequences directly recovered from each sediment. The hydrogenotrophic methanogen isolates of Methanocalculus and Methanoculleus that dominated the sediment methanogen communities produced methane at temperatures from 4 to 55 °C, with an abrupt decline in the methane production rate at temperatures above 40 °C, which is consistent with the depth profiles of potential methanogenic activity in the Nankai Trough sediments in this and previous studies. Our results reveal the previously overlooked phylogenetic and metabolic diversity of living methanogens, including methylotrophic methanogenesis.