Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota.

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45-75 °C (optimum 70-75 °C) and pH 6.5-8.5 (optimum pH 7.5-8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Impact of Concurrent aerobic-anaerobic Methanotrophy on Methane Emission from Marine Sediments in Gas Hydrate Area.

Microbial methane oxidation has a significant impact on the methane flux from marine gas hydrate areas. However, the environmental fate of methane remains poorly constrained. We quantified the relative contributions of aerobic and anaerobic methanotrophs to methane consumption in sediments of the gas hydrate-bearing Sakata Knoll, Japan, by in situ geochemical and microbiological analyses coupled with 13C-tracer incubation experiments. The anaerobic ANME-1 and ANME-2 species contributed to the oxidation of 33.2 and 1.4% methane fluxes at 0-10 and 10-22 cm below the seafloor (bsf), respectively. Although the aerobic Methylococcaceae species consumed only 0.9% methane flux in the oxygen depleted 0.0-0.5 cmbsf zone, their metabolic activity was sustained down to 6 cmbsf (based on rRNA and lipid biosyntheses), increasing their contribution to 10.3%. Our study emphasizes that the co-occurrence of aerobic and anaerobic methanotrophy at the redox transition zone is an important determinant of methane flux.

Methane diffusion affects characteristics of benthic communities in and around microbial mat-covered sediments in the northeastern Japan sea.

We investigated relationships between features of benthic macrofaunal communities and geochemical parameters in and around microbial mat-covered sediments associated with a methane seepage on Sakata Knoll in the eastern Japan Sea. A depression on top of the knoll corresponds to a gas-hydrate-bearing area with seepage of methane-rich fluid, and microbial mats cover the seafloor sediments. Sediment cores were collected at three sites for this study: one within a microbial mat, a second a few meters outside of the microbial mat, and a third from a reference site outside the gas-hydrate-bearing areas. Morphological analysis showed that the site inside the microbial mat had higher macrofaunal density and biomass compared with the other sites. 18S rRNA gene analysis showed that annelids were dominant in the surface sediment inside the microbial mat with the possible occurrence of microbial anaerobic oxidation of methane (AOM), whereas in the surface sediments outside the microbial mat and at the reference site the predominant species belonged to phylum Cercozoa. Morphological analysis also showed that the surface sediment inside the microbial mat noticeably favored annelids, with dorvilleid Ophryotrocha sp. and ampharetid Neosabellides sp. identified as major constituents. Statistical analysis showed that sulfidic sediment conditions with concentrations of H2S up to 121 µM resulting from AOM likely resulted in the predominance of annelids with tolerance to sulfide. Both the 18S rRNA genes and macrofaunal characteristics showed that benthic biodiversity among the three sites was greatest outside the microbial mat. The site outside the microbial mat may represent geochemical transition conditions, including a lower rate of upward methane gas-flow compared with the site inside the microbial mat. The high biodiversity there might result from the presence of species specifically suited to the transition zone as well as species also found in photosynthesis-based communities of the background environment.

Cultivation and biogeochemical analyses reveal insights into methanogenesis in deep subseafloor sediment at a biogenic gas hydrate site.

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.

Acetate excretion by a methanotroph, Methylocaldum marinum S8, under aerobic conditions.

Methane-oxidizing bacteria (methanotrophs) often coexist with methylotrophs that utilize methanol excreted by methanotrophs. Recently, we found that a facultative methylotroph, Methyloceanibacter caenitepidi Gela4T, possibly utilizes acetate rather than methanol in the coculture with a methanotroph, Methylocaldum marinum S8. Here, we examined the effects of oxygen concentrations on growth of and acetate excretion by M. marinum S8 in pure culture and the coculture with M. caenitepidi Gela4T. M. marinum S8 excreted acetate during the exponential growth phase not only under microaerobic (O2 concentrations of 3.5%-6%) but also under aerobic (O2 concentrations of 20%-31%) conditions. RNA-Seq analyses of M. marinum S8 cells grown under aerobic conditions suggested that phosphoketolase and acetate kinase were candidate genes involved in acetate production. Nonmethylotrophic bacteria, Cupriavidus necator NBRC 102504, could grow when cocultured with M. marinum S8, also supporting the existence of methanol-independent cross-feeding from M. marinum S8 under aerobic conditions.

Correction: Possible cross-feeding pathway of facultative methylotroph Methyloceanibacter caenitepidi Gela4 on methanotroph Methylocaldum marinum S8.

[This corrects the article DOI: 10.1371/journal.pone.0213535.].

Isolation of a member of the candidate phylum 'Atribacteria' reveals a unique cell membrane structure.

A key feature that differentiates prokaryotic cells from eukaryotes is the absence of an intracellular membrane surrounding the chromosomal DNA. Here, we isolate a member of the ubiquitous, yet-to-be-cultivated phylum 'Candidatus Atribacteria' (also known as OP9) that has an intracytoplasmic membrane apparently surrounding the nucleoid. The isolate, RT761, is a subsurface-derived anaerobic bacterium that appears to have three lipid membrane-like layers, as shown by cryo-electron tomography. Our observations are consistent with a classical gram-negative structure with an additional intracytoplasmic membrane. However, further studies are needed to provide conclusive evidence for this unique intracellular structure. The RT761 genome encodes proteins with features that might be related to the complex cellular structure, including: N-terminal extensions in proteins involved in important processes (such as cell-division protein FtsZ); one of the highest percentages of transmembrane proteins among gram-negative bacteria; and predicted Sec-secreted proteins with unique signal peptides. Physiologically, RT761 primarily produces hydrogen for electron disposal during sugar degradation, and co-cultivation with a hydrogen-scavenging methanogen improves growth. We propose RT761 as a new species, Atribacter laminatus gen. nov. sp. nov. and a new phylum, Atribacterota phy. nov.

Possible cross-feeding pathway of facultative methylotroph Methyloceanibacter caenitepidi Gela4 on methanotroph Methylocaldum marinum S8.

Non-methanotrophic bacteria such as methylotrophs often coexist with methane-oxidizing bacteria (methanotrophs) by cross-feeding on methane-derived carbon. Methanol has long been considered a major compound that mediates cross-feeding of methane-derived carbon. Despite the potential importance of cross-feeding in the global carbon cycle, only a few studies have actually explored metabolic responses of a bacteria when cross-feeding on a methanotroph. Recently, we isolated a novel facultative methylotroph, Methyloceanibacter caenitepidi Gela4, which grows syntrophically with the methanotroph, Methylocaldum marinum S8. To assess the potential metabolic pathways in M. caenitepidi Gela4 co-cultured with M. marinum S8, we conducted genomic analyses of the two strains, as well as RNA-Seq and chemical analyses of M. caenitepidi Gela4, both in pure culture with methanol and in co-culture with methanotrophs. Genes involved in the serine pathway were downregulated in M. caenitepidi Gela4 under co-culture conditions, and methanol was below the detection limit (< 310 nM) in both pure culture of M. marinum S8 and co-culture. In contrast, genes involved in the tricarboxylic acid cycle, as well as acetyl-CoA synthetase, were upregulated in M. caenitepidi Gela4 under co-culture conditions. Notably, a pure culture of M. marinum S8 produced acetate (< 16 µM) during growth. These results suggested that an organic compound other than methanol, possibly acetate, might be the major carbon source for M. caenitepidi Gela4 cross-fed by M. marinum S8. Co-culture of M. caenitepidi Gela4 and M. marinum S8 may represent a model system to further study methanol-independent cross-feeding from methanotrophs to non-methanotrophic bacteria.

Microbial community structure in deep natural gas-bearing aquifers subjected to sulfate-containing fluid injection.

In the natural gas field located in central Japan, high concentrations of natural gases and iodide ions are dissolved in formation water and commercially produced in deep aquifers. In the iodine recovery process, the produced formation water is amended with sulfate, and this fluid is injected into gas-bearing aquifers, which may lead to infrastructure corrosion by hydrogen sulfide. In this study, we examined the microbial community in aquifers subjected to sulfate-containing fluid injection. Formation water samples were collected from production wells located at different distances from the injection wells. The chemical analysis showed that the injection fluid contained oxygen, nitrate, nitrite and sulfate, in contrast to the formation water, which had previously been shown to be depleted in these components. Sulfur isotopic analysis indicated that sulfate derived from the injection fluid was present in the sample collected from near the injection wells. Quantitative and sequencing analysis of dissimilatory sulfite reductase and 16S rRNA genes revealed that sulfate-reducing bacteria (SRB), sulfur-oxidizing bacteria, and anaerobic methanotrophic archaea (ANME) in the wells located near injection wells were more abundant than those in wells located far from the injection wells, suggesting that fluid injection stimulated these microorganisms through the addition of oxygen, nitrate, nitrite and sulfate to the methane-rich aquifers. The predominant taxa were assigned to the ANME-2 group, its sulfate-reducing partner SEEP-SRB1 cluster and sulfur-oxidizing Epsilonproteobacteria. These results provide important insights for future studies to support the development of natural gas and iodine resources in Japan.

Methane production from coal by a single methanogen.

Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface-derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this "methoxydotrophic" mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl-coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.

Changes in microbial communities associated with gas hydrates in subseafloor sediments from the Nankai Trough.

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.

Physicochemical impacts associated with natural gas development on methanogenesis in deep sand aquifers.

The Minami-Kanto gas field, where gases are dissolved in formation water, is a potential analogue for a marine gas hydrate area because both areas are characterized by the accumulation of microbial methane in marine turbidite sand layers interbedded with mud layers. This study examined the physicochemical impacts associated with natural gas production and well drilling on the methanogenic activity and composition in this gas field. Twenty-four gas-associated formation water samples were collected from confined sand aquifers through production wells. The stable isotopic compositions of methane in the gases indicated their origin to be biogenic via the carbonate reduction pathway. Consistent with this classification, methanogenic activity measurements using radiotracers, culturing experiments and molecular analysis of formation water samples indicated the predominance of hydrogenotrophic methanogenesis. The cultivation of water samples amended only with methanogenic substrates resulted in significant increases in microbial cells along with high-yield methane production, indicating the restricted availability of substrates in the aquifers. Hydrogenotrophic methanogenic activity increased with increasing natural gas production from the corresponding wells, suggesting that the flux of substrates from organic-rich mudstones to adjacent sand aquifers is enhanced by the decrease in fluid pressure in sand layers associated with natural gas/water production. The transient predominance of methylotrophic methanogens, observed for a few years after well drilling, also suggested the stimulation of the methanogens by the exposure of unutilized organic matter through well drilling. These results provide an insight into the physicochemical impacts on the methanogenic activity in biogenic gas deposits including marine gas hydrates.

A long-term cultivation of an anaerobic methane-oxidizing microbial community from deep-sea methane-seep sediment using a continuous-flow bioreactor.

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.

Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus.

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed.

Novel integrons and gene cassettes from a Cascadian submarine gas-hydrate-bearing core.

To determine whether integrons are present in a submarine gas hydrate community, metagenomic DNA was extracted from a gas-hydrate-bearing core, 150 m below the seafloor, from the Cascadian Margin. Integrons and gene cassettes were recovered by PCR from metagenomic DNA and sequenced. Thirty-seven integron integrase phylotypes were identified. The phylotypes were diverse and included members with homology to integrases from Methylomonas methanica, Desulfuromonas acetoxidans, Thermodesulfatator indicus, and marine uncultured bacteria. The gene cassette composition, 153 gene cassettes, was dominated by two types of encoded putative proteins. The first of these was predicted oxidoreductases, such as iron/sulfur cluster-binding proteins. A second type was alkyl transferases. Some cassette proteins showed homologies with those from methane-related archaea. These observations suggest that integrons may assist in the adaptation of microbial communities in this environment.

A distinct freshwater-adapted subgroup of ANME-1 dominates active archaeal communities in terrestrial subsurfaces in Japan.

Anaerobic methane-oxidizing archaea (ANME) are known to play an important role in methane flux, especially in marine sediments. The 16S rRNA genes of ANME have been detected in terrestrial freshwater subsurfaces. However, it is unclear whether ANME are actively involved in methane oxidation in these environments. To address this issue, Holocene sediments in the subsurface of the Kanto Plain in Japan were collected for biogeochemical and molecular analysis. The potential activity of the anaerobic oxidation of methane (AOM) (0.38-3.54 nmol cm⁻³ day⁻¹) was detected in sediment slurry incubation experiments with a (13) CH(4) tracer. Higher AOM activity was observed in low-salinity treatment compared with high-salinity condition (20‰), which supports the adaptation of ANME in freshwater habitats. The 16S rRNA sequence analysis clearly revealed the presence of a distinct subgroup of ANME-1, designated ANME-1a-FW. Phylogenetic analysis of the mcrA genes also implied the presence of the distinct subgroup in ANME-1. ANME-1a-FW was found to be the most dominant active group in the archaeal communities on the basis of 16S rRNA analysis (75.0-93.8% of total archaeal 16S rRNA clones). Sulfate-reducing bacteria previously known as the syntrophic bacterial partners of ANME-1 was not detected. Our results showed that ANME-1a-FW is adapted to freshwater habitats and is responsible for AOM in terrestrial freshwater subsurface environments.

Evidence for syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the high-temperature petroleum reservoir of Yabase oil field (Japan).

The methanogenic communities and pathways in a high-temperature petroleum reservoir were investigated through incubations of the production water and crude oil, combined with radiotracer experiments and molecular biological analyses. The incubations were conducted without any substrate amendment and under high-temperature and pressurized conditions that mimicked the in situ environment (55°C, 5 MPa). Changes in methane and acetate concentrations during the incubations indicated stoichiometric production of methane from acetate. Rates of hydrogenotrophic methanogenesis measured using [(14)C]-bicarbonate were 42-68 times those of acetoclastic methanogenesis measured using [2-(14) C]-acetate, implying the dominance of methane production by syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis in the environment. 16S rRNA gene sequence analyses of the incubated production water showed bacterial communities dominated by the genus Thermacetogenium, known as a thermophilic syntrophic acetate-oxidizing bacterium, and archaeal communities dominated by thermophilic hydrogenotrophic methanogens belonging to the genus Methanothermobacter. Furthermore, group-specific real-time PCR assays revealed that 16S rRNA gene copy numbers of the hydrogenotrophic methanogens affiliated with the order Methanobacteriales were almost identical to those of archaeal 16S rRNA genes. This study demonstrates that syntrophic acetate oxidation is the main methanogenic pathway in a high-temperature petroleum reservoir.

Microbial diversity and methanogenic potential in a high temperature natural gas field in Japan.

Microbial diversity and methanogenic potential in formation water samples from a dissolved-in-water type gas field were investigated by using 16S rRNA gene libraries and culture-based methods. Two formation water samples (of 46 and 53 degrees C in temperature) were obtained from a depth of 700 to 800 m. Coenzyme F(420)-autofluorescence indicated that 10(3)-10(4) cells per ml of active methanogens were present, accounting for at least 10% of the total cell count. The 16S rRNA gene sequence analysis indicated that the diversity of Archaea and Bacteria of the two samples was quite limited; i.e., the archaeal libraries were dominated by the sequences related to Methanobacterium formicicum and Methanothermobacter thermautotrophicus, and the bacterial libraries were dominated by the sequences related to Hydrogenophilus and Deferribacter. Of the methanogenic substrates tested using the formation water-based medium, only H(2)-CO(2) gave rise to methane formation. Those dominant archaeal and bacterial genera have the potential to use hydrogen for growth at the in situ temperatures, suggesting that the formation water of the Pliocene strata in the gas field has been provided with hydrogen, probably from underneath the strata, and thus on-going active methanogenesis has been occurring to date.