Microbial methanogenesis in subseafloor sediments is a key process in the carbon cycle on the Earth. However, the cultivation-dependent evidences have been poorly demonstrated. Here we report the cultivation of a methanogenic microbial consortium from subseafloor sediments using a continuous-flow-type bioreactor with polyurethane sponges as microbial habitats, called down-flow hanging sponge (DHS) reactor. We anaerobically incubated methane-rich core sediments collected from off Shimokita Peninsula, Japan, for 826 days in the reactor at 10 1C. Synthetic seawater supplemented with glucose, yeast extract, acetate and propionate as potential energy sources was provided into the reactor. After 289 days of operation, microbiological methane production became evident. Fluorescence in situ hybridization analysis revealed the presence of metabolically active microbial cells with various morphologies in the reactor. DNA-and RNA-based phylogenetic analyses targeting 16S rRNA indicated the successful growth of phylogenetically diverse microbial components during cultivation in the reactor. Most of the phylotypes in the reactor, once it made methane, were more closely related to culture sequences than to the subsurface environmental sequence. Potentially methanogenic phylotypes related to the genera Methanobacterium, Methanococcoides and Methanosarcina were predominantly detected concomitantly with methane production, while uncultured archaeal phylotypes were also detected. Using the methanogenic community enrichment as subsequent inocula, traditional batch-type cultivations led to the successful isolation of several anaerobic microbes including those methanogens. Our results substantiate that the DHS bioreactor is a useful system for the enrichment of numerous fastidious microbes from subseafloor sediments and will enable the physiological and ecological characterization of pure cultures of previously uncultivated subseafloor microbial life.

Hydrogen and carbon isotope systematics of H 2 O-H 2 -CO 2 -CH 4 in hydrogenotrophic methanogenesis and their relation to H 2 availability were investigated. Two H 2 -syntrophic cocultures of fermentatively hydrogenogenic bacteria and hydrogenotrophic methanogens under conditions of <10 2 Pa-H 2 and two pure cultures of hydrogenotrophic methanogens under conditions of~10 5 Pa-H 2 were tested. Carbon isotope fractionation between CH 4 and CO 2 during hydrogenotrophic methanogenesis was correlated with pH 2 , as indicated in previous studies. The hydrogen isotope ratio of CH 4 produced during rapid growth of the thermophilic methanogen Methanothermococcus okinawensis under high pH 2 conditions (~10 5 Pa) was affected by the isotopic composition of H 2 , as concluded in a previous study of Methanothermobacter thermautotrophicus. This "δD H 2 effect" is a possible cause of the diversity of previously reported values for hydrogen isotope fractionation between CH 4 and H 2 O examined in H 2 -enriched culture experiments. Hydrogen isotope fractionation between CH 4 and H 2 O, defined by (1000 + δD CH 4 )/(1000 + δD H 2 O ), during hydrogenotrophic methanogenesis of the H 2 -syntrophic cocultures was in the range 0.67-0.69. The hydrogen isotope fractionation of our H 2 -syntrophic dataset overlaps with those obtained not only from low-pH 2 experiments reported so far but also from natural samples of "young" methane reservoirs (0.66-0.74). Conversely, such hydrogen isotope fractionation is not consistent with that of "aged" methane in geological samples (≥0.79), which has been regarded as methane produced via hydrogenotrophic methanogenesis from the carbon isotope fractionation. As a possible process inducing the inconsistency in hydrogen isotope signatures between experiments and geological samples, we hypothesize that the hydrogen isotope signature of CH 4 imprinted at the time of methanogenesis, as in the experiments and natural young methane, may be altered by diagenetic hydrogen isotope exchange between extracellular CH 4 and H 2 O through reversible reactions of the microbial methanogenic pathway in methanogenic region and/or geological methane reservoirs.

Anaerobic oxidation of methane (AOM) in marine sediments is an important global methane sink, but the physiological characteristics of AOM-associated microorganisms remain poorly understood. Here we report the cultivation of an AOM microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor with polyurethane sponges, called the down-flow hanging sponge (DHS) bioreactor. We anaerobically incubated deep-sea methane-seep sediment collected from the Nankai Trough, Japan, for 2,013 days in the bioreactor at 10°C. Following incubation, an active AOM activity was confirmed by a tracer experiment using 13C-labeled methane. Phylogenetic analyses demonstrated that phylogenetically diverse Archaea and Bacteria grew in the bioreactor. After 2,013 days of incubation, the predominant archaeal components were anaerobic methanotroph (ANME)-2a, Deep-Sea Archaeal Group, and Marine Benthic Group-D, and Gammaproteobacteria was the dominant bacterial lineage. Fluorescence in situ hybridization analysis showed that ANME-1 and -2a, and most ANME-2c cells occurred without close physical interaction with potential bacterial partners. Our data demonstrate that the DHS bioreactor system is a useful system for cultivating fastidious methane-seep-associated sedimentary microorganisms.