Methyl-compound use and slow growth characterize microbial life in 2-km-deep subseafloor coal and shale beds.

The past decade of scientific ocean drilling has revealed seemingly ubiquitous, slow-growing microbial life within a range of deep biosphere habitats. Integrated Ocean Drilling Program Expedition 337 expanded these studies by successfully coring Miocene-aged coal beds 2 km below the seafloor hypothesized to be "hot spots" for microbial life. To characterize the activity of coal-associated microorganisms from this site, a series of stable isotope probing (SIP) experiments were conducted using intact pieces of coal and overlying shale incubated at in situ temperatures (45 °C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of 13C- or 15N-labeled methanol, methylamine, and ammonium added at low (micromolar) concentrations to investigate methylotrophy in the deep subseafloor biosphere. Although the cell densities were low (50-2,000 cells per cubic centimeter), bulk geochemical measurements and single-cell-targeted nanometer-scale secondary ion mass spectrometry demonstrated active metabolism of methylated substrates by the thermally adapted microbial assemblage, with differing substrate utilization profiles between coal and shale incubations. The conversion of labeled methylamine and methanol was predominantly through heterotrophic processes, with only minor stimulation of methanogenesis. These findings were consistent with in situ and incubation 16S rRNA gene surveys. Microbial growth estimates in the incubations ranged from several months to over 100 y, representing some of the slowest direct measurements of environmental microbial biosynthesis rates. Collectively, these data highlight a small, but viable, deep coal bed biosphere characterized by extremely slow-growing heterotrophs that can utilize a diverse range of carbon and nitrogen substrates.

Microbial community stratification controlled by the subseafloor fluid flow and geothermal gradient at the Iheya North hydrothermal field in the Mid-Okinawa Trough (Integrated Ocean Drilling Program Expedition 331).

The impacts of lithologic structure and geothermal gradient on subseafloor microbial communities were investigated at a marginal site of the Iheya North hydrothermal field in the Mid-Okinawa Trough. Subsurface marine sediments composed of hemipelagic muds and volcaniclastic deposits were recovered through a depth of 151 m below the seafloor at site C0017 during Integrated Ocean Drilling Program Expedition 331. Microbial communities inferred from 16S rRNA gene clone sequencing in low-temperature hemipelagic sediments were mainly composed of members of the Chloroflexi and deep-sea archaeal group. In contrast, 16S rRNA gene sequences of marine group I Thaumarchaeota dominated the microbial phylotype communities in the coarse-grained pumiceous gravels interbedded between the hemipelagic sediments. Based on the physical properties of sediments such as temperature and permeability, the porewater chemistry, and the microbial phylotype compositions, the shift in the physical properties of the sediments is suggested to induce a potential subseafloor recharging flow of oxygenated seawater in the permeable zone, leading to the generation of variable chemical environments and microbial communities in the subseafloor habitats. In addition, the deepest section of sediments under high-temperature conditions (∼90°C) harbored the sequences of an uncultivated archaeal lineage of hot water crenarchaeotic group IV that may be associated with the high-temperature hydrothermal fluid flow. These results indicate that the subseafloor microbial community compositions and functions at the marginal site of the hydrothermal field are highly affected by the complex fluid flow structure, such as recharging seawater and underlying hydrothermal fluids, coupled with the lithologic transition of sediments.

Cultivation of Piezotolerant and Piezophilic Hyperthermophiles with a Newly Developed Constant High Pressure and Temperature Culturing and Monitoring System.

The Earth's microbial biosphere extends from ambient to extreme environments, including deep-sea hydrothermal vents and subseafloor habitats. Despite efforts to understand the physiological adaptations of these microbes, our knowledge is limited due to the technological challenges associated with reproducing in situ high temperature (HT)-high hydrostatic pressure (HHP) conditions and sampling HT-HHP cultures. In the present study, we developed a new high temperature and pressure (HTP) incubation system that enabled the maintenance of HT-HHP conditions while sampling incubation medium and mostly eliminated non-biological reactions, including hydrogen generation or the leakage of small gaseous molecules. The main characteristics of our system are (1) a chamber made of gold with gold-etched lid parts that suppress the majority of non-biological reactions, (2) the exceptional containment of dissolved gas, even small molecules, such as hydrogen, and (3) the sampling capacity of intra-chamber liquid without depressurization and the isobaric transfer of a culture to inoculate new medium. We initially confirmed the retention of dissolved hydrogen in the incubation container at 82°C and 20| |MPa for 9 days. Cultivation tests with an obligate hyperthermophilic piezophile (Pyrococcus yayanosii), hydrogenotrophic hyperthermophile (Archaeoglobus profundus), and heterotrophic hyperthermophile (Pyrococcus horikoshii) were successful based on growth monitoring and chemical ana-lyses. During HTP cultivation, we observed a difference in the duration of the lag phase of P. horikoshii, which indicated the potential effect of a pressure change on the physiology of piezophiles. The present results suggest the importance of a cultivation system designed and developed explicitly for HTP conditions with the capacity for sampling without depressurization of the entire system.

Estimation of the depth of origin of fluids using noble gases in the surface sediments of submarine mud volcanoes off Tanegashima Island.

The helium isotope ratio (3He/4He), concentration ratio of neon-20 to helium-4 (20Ne/4He), argon (Ar), krypton (Kr), and xenon (Xe) concentrations were measured in the porewater of surface sediments of several submarine mud volcanoes. From the 3He/4He values (0.18-0.93RA), the estimated He origin is almost 90% crustal He, with little contribution from mantle-derived He. The determined Ar, Kr, and Xe concentrations lie within the solubility equilibrium range expected for temperatures from 83 °C up to 230 °C and are consistent with the temperature range of the dehydration origin of clay minerals. Considering the geothermal gradient in the investigated region (25 °C/km), these gases are considered to have reached dissolution equilibrium at a depth of about 3.3 km to 9.2 km below the seafloor. As the depth of the plate boundary is 18 km below the seafloor, the noble gas signatures are likely to originate from the crust, not from the plate boundary. This is consistent with the results presented by the He isotope ratios.

Unique H2-utilizing lithotrophy in serpentinite-hosted systems.

Serpentinization of ultramafic rocks provides molecular hydrogen (H2) that can support lithotrophic metabolism of microorganisms, but also poses extremely challenging conditions, including hyperalkalinity and limited electron acceptor availability. Investigation of two serpentinization-active systems reveals that conventional H2-/CO2-dependent homoacetogenesis is thermodynamically unfavorable in situ due to picomolar CO2 levels. Through metagenomics and thermodynamics, we discover unique taxa capable of metabolism adapted to the habitat. This included a novel deep-branching phylum, "Ca. Lithacetigenota", that exclusively inhabits serpentinite-hosted systems and harbors genes encoding alternative modes of H2-utilizing lithotrophy. Rather than CO2, these putative metabolisms utilize reduced carbon compounds detected in situ presumably serpentinization-derived: formate and glycine. The former employs a partial homoacetogenesis pathway and the latter a distinct pathway mediated by a rare selenoprotein-the glycine reductase. A survey of microbiomes shows that glycine reductases are diverse and nearly ubiquitous in serpentinite-hosted environments. "Ca. Lithacetigenota" glycine reductases represent a basal lineage, suggesting that catabolic glycine reduction is an ancient bacterial innovation by Terrabacteria for gaining energy from geogenic H2 even under hyperalkaline, CO2-poor conditions. Unique non-CO2-reducing metabolisms presented here shed light on potential strategies that extremophiles may employ for overcoming a crucial obstacle in serpentinization-associated environments, features potentially relevant to primordial lithotrophy in early Earth.

Temperature limits to deep subseafloor life in the Nankai Trough subduction zone.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

Microbial Metabolism and Community Dynamics in Hydraulic Fracturing Fluids Recovered From Deep Hydrocarbon-Rich Shale.

Hydraulic fracturing is a prominent method of natural gas production that uses injected, high-pressure fluids to fracture low permeability, hydrocarbon rich strata such as shale. Upon completion of a well, the fluid returns to the surface (produced water) and contains natural gas, subsurface constituents, and microorganisms (Barbot et al., 2013; Daly et al., 2016). While the microbial community of the produced fluids has been studied in multiple gas wells, the activity of these microorganisms and their relation to biogeochemical activity is not well understood. In this experiment, we supplemented produced fluid with 13C-labeled carbon sources (glucose, acetate, bicarbonate, methanol, or methane), and 15N-labeled ammonium chloride in order to isotopically trace microbial activity over multiple day in anoxic incubations. Nanoscale secondary ion mass spectrometry (NanoSIMS) was used to generate isotopic images of 13C and 15N incorporation in individual cells, while isotope ratio monitoring-gas chromatography-mass spectrometry (IRM-GC-MS) was used to measure 13CO2, and 13CH4 as metabolic byproducts. Glucose, acetate, and methanol were all assimilated by microorganisms under anoxic conditions. 13CO2 production was only observed with glucose as a substrate indicating that catabolic activity was limited to this condition. The microbial communities observed at 0, 19, and 32 days of incubation did not vary between different carbon sources, were low in diversity, and composed primarily of the class Clostridia. The primary genera detected in the incubations, Halanaerobium and Fusibacter, are known to be adapted to harsh physical and chemical conditions consistent with those that occur in the hydrofracturing environment. This study provides evidence that microorganisms in produced fluid are revivable in laboratory incubations and retained the ability to metabolize added carbon and nitrogen substrates.

Cultivable microbial community in 2-km-deep, 20-million-year-old subseafloor coalbeds through ~1000 days anaerobic bioreactor cultivation.

Recent explorations of scientific ocean drilling have revealed the presence of microbial communities persisting in sediments down to ~2.5 km below the ocean floor. However, our knowledge of these microbial populations in the deep subseafloor sedimentary biosphere remains limited. Here, we present a cultivation experiment of 2-km-deep subseafloor microbial communities in 20-million-year-old lignite coalbeds using a continuous-flow bioreactor operating at 40 °C for 1029 days with lignite particles as the major energy source. Chemical monitoring of effluent samples via fluorescence emission-excitation matrices spectroscopy and stable isotope analyses traced the transformation of coalbed-derived organic matter in the dissolved phase. Hereby, the production of acetate and 13C-depleted methane together with the increase and transformation of high molecular weight humics point to an active lignite-degrading methanogenic community present within the bioreactor. Electron microscopy revealed abundant microbial cells growing on the surface of lignite particles. Small subunit rRNA gene sequence analysis revealed that diverse microorganisms grew in the bioreactor (e.g., phyla Proteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Bacteroidetes, Spirochaetes, Tenericutes, Ignavibacteriae, and SBR1093). These results indicate that activation and adaptive growth of 2-km-deep microbes was successfully accomplished using a continuous-flow bioreactor, which lays the groundwork to explore networks of microbial communities of the deep biosphere and their physiologies.

Deep-biosphere methane production stimulated by geofluids in the Nankai accretionary complex.

Microbial life inhabiting subseafloor sediments plays an important role in Earth's carbon cycle. However, the impact of geodynamic processes on the distributions and carbon-cycling activities of subseafloor life remains poorly constrained. We explore a submarine mud volcano of the Nankai accretionary complex by drilling down to 200 m below the summit. Stable isotopic compositions of water and carbon compounds, including clumped methane isotopologues, suggest that ~90% of methane is microbially produced at 16° to 30°C and 300 to 900 m below seafloor, corresponding to the basin bottom, where fluids in the accretionary prism are supplied via megasplay faults. Radiotracer experiments showed that relatively small microbial populations in deep mud volcano sediments (102 to 103 cells cm-3) include highly active hydrogenotrophic methanogens and acetogens. Our findings indicate that subduction-associated fluid migration has stimulated microbial activity in the mud reservoir and that mud volcanoes may contribute more substantially to the methane budget than previously estimated.

Atribacteria from the Subseafloor Sedimentary Biosphere Disperse to the Hydrosphere through Submarine Mud Volcanoes.

Submarine mud volcanoes (SMVs) are formed by muddy sediments and breccias extruded to the seafloor from a source in the deep subseafloor and are characterized by the discharge of methane and other hydrocarbon gasses and deep-sourced fluids into the overlying seawater. Although SMVs act as a natural pipeline connecting the Earth's surface and subsurface biospheres, the dispersal of deep-biosphere microorganisms and their ecological roles remain largely unknown. In this study, we investigated the microbial communities in sediment and overlying seawater at two SMVs located on the Ryukyu Trench off Tanegashima Island, southern Japan. The microbial communities in mud volcano sediments were generally distinct from those in the overlying seawaters and in the well-stratified Pacific margin sediments collected at the Peru Margin, the Juan de Fuca Ridge flank off Oregon, and offshore of Shimokita Peninsula, northeastern Japan. Nevertheless, in-depth analysis of different taxonomic groups at the sub-species level revealed that the taxon affiliated with Atribacteria, heterotrophic anaerobic bacteria that typically occur in organic-rich anoxic subseafloor sediments, were commonly found not only in SMV sediments but also in the overlying seawater. We designed a new oligonucleotide probe for detecting Atribacteria using the catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). CARD-FISH, digital PCR and sequencing analysis of 16S rRNA genes consistently showed that Atribacteria are abundant in the methane plumes of the two SMVs (0.58 and 1.5 × 104 cells/mL, respectively) but not in surrounding waters, suggesting that microbial cells in subseafloor sediments are dispersed as "deep-biosphere seeds" into the ocean. These findings may have important implications for the microbial transmigration between the deep subseafloor biosphere and the hydrosphere.

Aerobic and Anaerobic Methanotrophic Communities Associated with Methane Hydrates Exposed on the Seafloor: A High-Pressure Sampling and Stable Isotope-Incubation Experiment.

High-pressure (HP) environments represent the largest volumetric majority of habitable space for microorganisms on the planet, including the deep-sea and subsurface biosphere. However, the importance of pressure as an environmental variable affecting deep microbial life and their biogeochemical functions in carbon cycling still remains poorly understood. Here, we designed a new high-volume HP-sediment core sampler that is deployable on the payload of a remotely operated vehicle and can maintain in situ HP conditions throughout multi-month enrichment incubations including daily amendments with liquid media and gases and daily effluent sampling for geochemical or microbiological analysis. Using the HP core device, we incubated sediment and overlying water associated with methane hydrate-exposed on the seafloor of the Joetsu Knoll, Japan, at 10 MPa and 4°C for 45 days in the laboratory. Diversity analyses based on 16S rRNA and methane-related functional genes, as well as carbon isotopic analysis of methane and bicarbonate, indicated the stimulation of both aerobic and anaerobic methanotrophy driven by members of the Methylococcales, and ANME, respectively: i.e., aerobic methanotrophy was observed upon addition of oxygen whereas anaerobic processes subsequently occurred after oxygen consumption. These laboratory-measured rates at 10 MPa were generally in agreement with previously reported rates of methane oxidation in other oceanographic locations.

Defining boundaries for the distribution of microbial communities beneath the sediment-buried, hydrothermally active seafloor.

Subseafloor microbes beneath active hydrothermal vents are thought to live near the upper temperature limit for life on Earth. We drilled and cored the Iheya North hydrothermal field in the Mid-Okinawa Trough, and examined the phylogenetic compositions and the products of metabolic functions of sub-vent microbial communities. We detected microbial cells, metabolic activities and molecular signatures only in the shallow sediments down to 15.8 m below the seafloor at a moderately distant drilling site from the active hydrothermal vents (450 m). At the drilling site, the profiles of methane and sulfate concentrations and the δ13C and δD isotopic compositions of methane suggested the laterally flowing hydrothermal fluids and the in situ microbial anaerobic methane oxidation. In situ measurements during the drilling constrain the current bottom temperature of the microbially habitable zone to ~45 °C. However, in the past, higher temperatures of 106-198 °C were possible at the depth, as estimated from geochemical thermometry on hydrothermally altered clay minerals. The 16S rRNA gene phylotypes found in the deepest habitable zone are related to those of thermophiles, although sequences typical of known hyperthermophilic microbes were absent from the entire core. Overall our results shed new light on the distribution and composition of the boundary microbial community close to the high-temperature limit for habitability in the subseafloor environment of a hydrothermal field.

Biological CO2 conversion to acetate in subsurface coal-sand formation using a high-pressure reactor system.

Geological CO2 sequestration in unmineable subsurface oil/gas fields and coal formations has been proposed as a means of reducing anthropogenic greenhouse gasses in the atmosphere. However, the feasibility of injecting CO2 into subsurface depends upon a variety of geological and economic conditions, and the ecological consequences are largely unpredictable. In this study, we developed a new flow-through-type reactor system to examine potential geophysical, geochemical and microbiological impacts associated with CO2 injection by simulating in-situ pressure (0-100 MPa) and temperature (0-70°C) conditions. Using the reactor system, anaerobic artificial fluid and CO2 (flow rate: 0.002 and 0.00001 ml/min, respectively) were continuously supplemented into a column comprised of bituminous coal and sand under a pore pressure of 40 MPa (confined pressure: 41 MPa) at 40°C for 56 days. 16S rRNA gene analysis of the bacterial components showed distinct spatial separation of the predominant taxa in the coal and sand over the course of the experiment. Cultivation experiments using sub-sampled fluids revealed that some microbes survived, or were metabolically active, under CO2-rich conditions. However, no methanogens were activated during the experiment, even though hydrogenotrophic and methylotrophic methanogens were obtained from conventional batch-type cultivation at 20°C. During the reactor experiment, the acetate and methanol concentration in the fluids increased while the δ(13)Cacetate, H2 and CO2 concentrations decreased, indicating the occurrence of homo-acetogenesis. 16S rRNA genes of homo-acetogenic spore-forming bacteria related to the genus Sporomusa were consistently detected from the sandstone after the reactor experiment. Our results suggest that the injection of CO2 into a natural coal-sand formation preferentially stimulates homo-acetogenesis rather than methanogenesis, and that this process is accompanied by biogenic CO2 conversion to acetate.

A simple method for oxygen-18 determination of milligram quantities of water using NaHCO3 reagent.

This paper presents a modified H(2)O-CO(2) equilibration method for stable oxygen isotopic composition (delta(18)O) analysis of water. This method enables rapid and simple delta(18)O analysis of milligram quantities of water, by employing solid reagent NaHCO(3) as the CO(2) source, a small (0.6 mL) glass vial for the equilibration chamber, and an isotope-monitoring gas chromatography/mass spectrometry (irm-GC/MS) system for delta 18O(CO2) analysis. This method has several advantages, including simple handling for the H(2)O-CO(2) equilibration (without purging and/or evacuation treatments), rapid and easy delta(18)O analysis of equilibrated CO(2), and highly sensitive and highly precise delta(18)O analysis of H(2)O, using samples as small as 10 mg and with a precision of less than +/-0.12 per thousand. The time needed to attain oxygen isotopic equilibration between CO(2) and water is also comparable (17 h for 10 mg H(2)O and 10 h for 100 mg H(2)O) to other previous methods using CO(2) gas for the CO(2) source. The extent of delta(18)O variation of sample water from its initial delta(18)O value due to isotope exchange with added NaHCO(3) is also discussed. It is concluded that the correction needed is negligible (less than 0.1 per thousand ) as long as the oxygen atom ratio (O(NaHCO3)/O(H2O)) is less than 3.3 +/- 10(-3) and provided the delta18O(H2O) determination is made by comparing delta(18)O of CO(2) equilibrated with sample water and that equilibrated with standard water of a moderately close delta(18)O value, less than 30 per thousand difference.