Endolithic microbial habitats hosted in carbonate nodules currently forming within sediment at a high methane flux site in the sea of Japan

Concretionary carbonates in deep-sea methane seep fields are formed as a result of microbial methane degradation, called anaerobic oxidation of methane (AOM). Recently, active microorganisms, including anaerobic methanotrophic archaea, were discovered from methane seep-associated carbonate outcroppings on the seafloor. However sedimentary buried carbonate nodules are a hitherto unknown microbial habitat. In this study, we investigated the microbial community structures in two carbonate nodules collected from a high methane flux site in a gas hydrate field off the Oki islands in the Sea of Japan. The nodules were formed around sulfate-methane interfaces (SMI) corresponding to 0.7 and 2.2 m below the seafloor. Based on a geochemical analysis, light carbon isotopic values ranging from −54.91‰ to −37.32‰ were found from the nodules collected at the shallow SMI depth, which were attributed to the high contributions of AOM-induced carbonate precipitation. Signatures of methanotrophic archaeal populations within the sedimentary buried nodule were detected based on microbial community composition analyses and quantitative real-time PCR targeted 16S rRNA, and functional genes for AOM. These results suggest that the buried carbonate nodule currently develops AOM-related microbial communities, and grows depending on the continued AOM under high methane flux conditions

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO2 in the seabed. The emission of CO2 may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO2 and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO2-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO2 concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO2 concentration, indicating that microbial activity and community structure are sensitive to CO2 venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO2. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO2-seep sedimentary environment; however, CO2 and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community

Biogeochemical Cycle of Methanol in Anoxic Deep-Sea Sediments

The biological flux and lifetime of methanol in anoxic marine sediments are largely unknown. We herein reported, for the first time, quantitative methanol removal rates in subsurface sediments. Anaerobic incubation experiments with radiotracers showed high rates of microbial methanol consumption. Notably, methanol oxidation to CO2 surpassed methanol assimilation and methanogenesis from CO2/H2 and methanol. Nevertheless, a significant decrease in methanol was not observed after the incubation, and this was attributed to the microbial production of methanol in parallel with its consumption. These results suggest that microbial reactions play an important role in the sources and sinks of methanol in subseafloor sediments

Molecular and Isotopic Composition of Volatiles in Gas Hydrates and in Sediment from the Joetsu Basin, Eastern Margin of the Japan Sea

Hydrate-bearing sediment cores were retrieved from the Joetsu Basin (off Joetsu city, Niigata Prefecture) at the eastern margin of the Japan Sea during the MD179 gas hydrates cruise onboard R/V Marion Dufresne in June 2010. We measured molecular and stable isotope compositions of volatiles bound in the gas hydrates and headspace gases obtained from sediments to clarify how the minor components of hydrocarbons affects to gas hydrate crystals. The hydrate-bound hydrocarbons at Umitaka Spur (southwestern Joetsu Basin) primarily consisted of thermogenic methane, whereas those at Joetsu Knoll (northwestern Joetsu Basin, about 15 km from Umitaka Spur) contained both thermogenic methane and a mixture of thermogenic and microbial methane. The depth concentration profiles of methane, ethane, propane, CO2, and H2S in the sediments from the Joetsu Basin area showed shallow sulfate-methane interface (SMI) and high microbial methane production beneath the SMI depth. Relatively high concentrations of propane and neopentane (2,2-dimethylpropane) were detected in the headspace gases of the hydrate-bearing sediment cores obtained at Umitaka Spur and Joetsu Knoll. Propane and neopentane cannot be encaged in the structure I hydrate; therefore, they were probably excluded from the hydrate crystals during the structure I formation process and thus remained in the sediment and/or released from the small amounts of structure II hydrate that can host such large gas molecules. The lower concentrations of ethane and propane in the sediment, high delta C-13 of propane and isobutane, and below-detection normal butane and normal pentane at Umitaka Spur and Joetsu Knoll suggest biodegradation in the sediment layers

Ubiquity of Euglena mutabilis Population in Three Ecologically Distinct Acidic Habitats in Southwestern Japan

Three strains of Euglena mutabilis were isolated from sediments in acidic inland water systems (pH = 3.4–4.7), in Southwestern Japan—acid mine drainage in Sensui (Fukuoka), cold sulfidic spring in Bougatsuru (Oita), and a temporal pool in the Ebinokogen volcanic area (Miyazaki). All strains grew well in acidic media at pH 3.07. Phylogenetic analysis among these three strains showed high similarities to plastid SSU and nuclear SSU rRNA gene sequences (99.86% and 99.76%, respectively). They were closely related to the cultured isolates from other highly acidic habitats (pH = 2.0–5.9). Concentration of sulfate, aluminum, calcium, and iron had 7–70 fold of differences among the three studied habitats. Our results imply that the rRNA genes of E. mutabilis have compensated for their low genetic diversity by adapting to a wide pH range, as well as various water chemistry of habitats