© 2020, The Author(s). Sparse microbial populations persist from seafloor to basement in the slowly accumulating oxic sediment of the oligotrophic South Pacific Gyre (SPG). The physiological status of these communities, including their substrate metabolism, is previously unconstrained. Here we show that diverse aerobic members of communities in SPG sediments (4.3‒101.5 Ma) are capable of readily incorporating carbon and nitrogen substrates and dividing. Most of the 6986 individual cells analyzed with nanometer-scale secondary ion mass spectrometry (NanoSIMS) actively incorporated isotope-labeled substrates. Many cells responded rapidly to incubation conditions, increasing total numbers by 4 orders of magnitude and taking up labeled carbon and nitrogen within 68 days after incubation. The response was generally faster (on average, 3.09 times) for nitrogen incorporation than for carbon incorporation. In contrast, anaerobic microbes were only minimally revived from this oxic sediment. Our results suggest that microbial communities widely distributed in organic-poor abyssal sediment consist mainly of aerobes that retain their metabolic potential under extremely low-energy conditions for up to 101.5 Ma.

© 2019, The Author(s). Ferromanganese minerals are widely distributed in subseafloor sediments and on the seafloor in oceanic abyssal plains. Assessing their input, formation and preservation is important for understanding the global marine manganese cycle and associated trace elements. However, the extent of ferromanganese minerals buried in subseafloor sediments remains unclear. Here we show that abundant (10 8 –10 9 particles cm −3 ) micrometer-scale ferromanganese mineral particles (Mn-microparticles) are found in the oxic pelagic clays of the South Pacific Gyre (SPG) from the seafloor to the ~100 million-year-old sediments above the basement. Three-dimensional micro-texture, and major and trace element compositional analyses revealed that these Mn-microparticles consist of poorly crystalline ferromanganese oxides precipitating from bottom water. Based on our findings, we extrapolate that 1.5–8.8 × 10 28 Mn-microparticles, accounting for 1.28–7.62 Tt of manganese, are globally present in oxic subseafloor sediments. This estimate is at least two orders of magnitude larger than the manganese budget for nodules and crusts on the seafloor. Subseafloor Mn-microparticles thus contribute significantly to the global manganese budget.

Marine sediments host an unexpectedly large microbial biosphere, suggesting unique microbial mechanisms for surviving burial and slow metabolic turnover. Although dormancy is generally considered an important survival strategy, its specific role in subsurface sediments remains unclear. We quantified dormant bacterial endospores in 331 marine sediment samples from diverse depositional types and geographical origins. The abundance of endospores relative to vegetative cells increased with burial depth and endospores became dominant below 25 m, with an estimated population of 2.5 x 10(28) to 1.9 x 10(29) endospores in the uppermost kilometer of sediment and a corresponding biomass carbon of 4.6 to 35 Pg surpassing that of vegetative cells. Our data further identify distinct endospore subgroups with divergent resistance to burial and aging. Endospores may shape the deep biosphere by providing a core population for colonization of new habitats and/or through low-frequency germination to sustain slow growth in this environment.

© 2019, The Author(s), under exclusive licence to Springer Nature Limited. Nearly half of the global seafloor is overlain by sediment oxygenated to the basement. Yet, despite the availability of oxygen to fuel aerobic respiration, organic carbon persists over million-year timescales. Identifying the controls on organic carbon preservation requires an improved understanding of the composition and distribution of organic carbon within deep oligotrophic marine sediments. Here we show that organic carbon in sediment from the oligotrophic North Atlantic and South Pacific is low (<0.1%), yet stable to depths of 25 m and ages of 24 million years. This organic carbon is not bound in biomass and has a low carbon/nitrogen ratio. X-ray imaging and spectroscopic analyses reveal that the chemical composition of this old, deep organic carbon is dominated (40–60%) by amide and carboxylic carbon with a proteinaceous nature. We posit that organic carbon persists in oxic oligotrophic sediment through a combination of protective processes that involve adsorption to mineral surfaces and physical inaccessibility to the heterotrophic community. We estimate that up to 1.6 × 1022 g of organic carbon are sequestered on million-year timescales in oxic pelagic sediment, which exceeds current estimates of the total global sediment organic carbon and constitutes an important, previously overlooked carbon reservoir.

© 2019, Springer Nature Limited. The surface of Mars has been well mapped and characterized, yet the subsurface — the most likely place to find signs of extant or extinct life and a repository of useful resources for human exploration — remains unexplored. In the near future this is set to change.

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.

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 degrees C). The 30-month SIP incubations were amended with deuterated water as a passive tracer for growth and different combinations of C-13- or N-15-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 life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ∼40° to 60°C sediment associated with lignite coal beds at ∼1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ∼10⁴ cells cm^-3. Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

The depth of oxygen penetration into marine sediments differs considerably from one region to another1,2. In areas with high rates of microbial respiration, O-2 penetrates only millimetres to centimetres into the sediments(3), but active anaerobic microbial communities are present in sediments hundreds of metres or more below the sea floor(4-7). In areas with low sedimentary respiration, O-2 penetratesmuchdeeper(8-12) but the depth to which microbial communities persist was previously unknown(9,10,13). The sediments underlying the South Pacific Gyre exhibit extremely low areal rates of respiration(9). Here we show that, in this region, microbial cells and aerobic respiration persist through the entire sediment sequence to depths of at least 75 metres below sea floor. Based on the Redfield stoichiometry of dissolved O-2 and nitrate, we suggest that net aerobic respiration in these sediments is coupled to oxidation of marine organic matter. We identify a relationship of O-2 penetration depth to sedimentation rate and sediment thickness. Extrapolating this relationship, we suggest that oxygen and aerobic communities may occur throughout the entire sediment sequence in 15-44% of the Pacific and 9-37% of the global sea floor. Subduction of the sediment and basalt from these regions is a source of oxidized material to the mantle.

Sediment-covered basalt on the flanks of mid-ocean ridges constitutes most of Earth's oceanic crust, but the composition and metabolic function of its microbial ecosystem are largely unknown. By drilling into 3.5-million-year-old subseafloor basalt, we demonstrated the presence of methane-and sulfur-cycling microbes on the eastern flank of the Juan de Fuca Ridge. Depth horizons with functional genes indicative of methane-cycling and sulfate-reducing microorganisms are enriched in solid-phase sulfur and total organic carbon, host delta C-13- and delta S-34-isotopic values with a biological imprint, and show clear signs of microbial activity when incubated in the laboratory. Downcore changes in carbon and sulfur cycling show discrete geochemical intervals with chemoautotrophic delta C-13 signatures locally attenuated by heterotrophic metabolism.

A recent study provides new constraints on the size and distribution of microbial biomass beneath the ocean floor, but key factors remain uncertain.

Remarkable numbers of microbial cells have been observed in global shallow to deep subseafloor sediments. Accumulating evidence indicates that deep and ancient sediments harbor living microbial life, where the flux of nutrients and energy are extremely low. However, their physiology and energy requirements remain largely unknown. We used stable isotope tracer incubation and nanometer-scale secondary ion MS to investigate the dynamics of carbon and nitrogen assimilation activities in individual microbial cells from 219-m-deep lower Pleistocene (460,000 y old) sediments from the northwestern Pacific off the Shimokita Peninsula of Japan. Sediment samples were incubated in vitro with (13)C- and/or (15)N-labeled glucose, pyruvate, acetate, bicarbonate, methane, ammonium, and amino acids. Significant incorporation of (13)C and/or (15)N and growth occurred in response to glucose, pyruvate, and amino acids (∼76% of total cells), whereas acetate and bicarbonate were incorporated without fostering growth. Among those substrates, a maximum substrate assimilation rate was observed at 67 × 10(-18) mol/cell per d with bicarbonate. Neither carbon assimilation nor growth was evident in response to methane. The atomic ratios between nitrogen incorporated from ammonium and the total cellular nitrogen consistently exceeded the ratios of carbon, suggesting that subseafloor microbes preferentially require nitrogen assimilation for the recovery in vitro. Our results showed that the most deeply buried subseafloor sedimentary microbes maintain potentials for metabolic activities and that growth is generally limited by energy but not by the availability of C and N compounds.

Deep-sea sediments harbour a vast biosphere. Archaea-one of the three domains of life(1)-are prevalent in marine environments(2-5), and comprise a significant fraction of the biomass in marine sediments(6). Archaeal membranes are well characterized, and are comprised of a glycerol backbone and a nonpolar isoprenoid chain. However, the ecology of sedimentary archaea remains elusive, because it is difficult to grow them in the laboratory. Here, we trace the fate of C-13-labelled glucose added to marine sediments in Sagami Bay, Japan, to determine the in situ mechanisms of membrane synthesis. Following the addition of labelled glucose to sediment samples collected in the region, we placed the cores on the sea floor and sampled them after 9 and 405 days. We found that the C-13 was incorporated into the glycerol backbone of archaeal membranes; C-13 was apparent after 9 days of incubation, but most pronounced after 405 days. However, the isoprenoid chain of the membranes remained unlabelled. On the basis of the differential uptake of C-13, we suggest that the glycerol unit is synthesized de novo, whereas the isoprenoid unit is synthesized from relic archaeal membranes and detritus, because of the prevalence of these compounds in marine sediments. We therefore suggest that some benthic archaea build their membranes by recycling sedimentary organic compounds.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells(1,2). Extrapolation of direct counts of stained microbial cells to the total volume of habitable marine subsurface sediments suggests that between 56 Pg ( ref. 1) and 303 Pg ( ref. 3) of cellular carbon could be stored in this largely unexplored habitat. From recent studies using various cultureindependent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem(4-7). Here we show that in subsurface sediments buried deeper than 1 m in a wide range of oceanographic settings at least 87% of intact polar membrane lipids, biomarkers for the presence of live cells(7,8), are attributable to archaeal membranes, suggesting that Archaea constitute a major fraction of the biomass. Results obtained from modified quantitative polymerase chain reaction and slot- blot hybridization protocols support the lipid- based evidence and indicate that these techniques have previously underestimated archaeal biomass. The lipid concentrations are proportional to those of total organic carbon. On the basis of this relationship, we derived an independent estimate of amounts of cellular carbon in the global marine subsurface biosphere. Our estimate of 90 Pg of cellular carbon is consistent, within an order of magnitude, with previous estimates, and underscores the importance of marine subsurface habitats for global biomass budgets.

Increasing levels Of CO2 in the atmosphere are expected to cause climatic change with negative effects on the earth's ecosystems and human society. Consequently, a variety of CO2 disposal options are discussed, including injection into the deep ocean. Because the dissolution Of CO2 in seawater will decrease ambient pH considerably, negative consequences for deep-water ecosystems have been predicted. Hence, ecosystems associated with natural Co-2 reservoirs in the deep sea, and the dynamics of gaseous, liquid, and solid CO2 in such environments, are of great interest to science and society. We report here a biogeochemical and microbiological characterization of a microbial community inhabiting deep-sea sediments overlying a natural CO2 lake at the Yonaguni Knoll IV hydrothermal field, southern Okinawa Trough. We found high abundances (&gt; 10(9) CM-3) of microbial cells in sediment pavements above the CO2 lake, decreasing to strikingly low cell numbers (107 CM-3) at the liquid CO2/CO2-hydrate interface. The key groups in these sediments were as follows: (i) the anaerobic methanotrophic archaea ANME-2c and the Eel-2 group of Deltaproteobacteria and (ii) sulfur-metabolizing chemolithotrophs within the Gamma- and Epsilonproteobacteria. The detection of functional genes related to one-carbon assimilation and the presence of highly C-13-depleted archaeal and bacterial lipid biomarkers suggest that microorganisms assimilating CO2 and/or CH4 dominate the liquid CO2 and CO2-hydrate-bearing sediments. Clearly, the Yonaguni Knoll is an exceptional natural laboratory for the study of consequences of CO2 disposal as well as of natural CO2 reservoirs as potential microbial habitats on early Earth and other celestial bodies.

The deep subseafloor biosphere is among the least-understood habitats on Earth, even though the huge microbial biomass therein plays an important role for potential long-term controls on global biogeochemical cycles. We report here the vertical and geographical distribution of microbes and their phylogenetic diversities in deeply buried marine sediments of the Pacific Ocean Margins. During the Ocean Drilling Program Legs 201 and 204, we obtained sediment cores from the Peru and Cascadia Margins that varied with respect to the presence of dissolved methane and methane hydrate. To examine differences in prokaryotic distribution patterns in sediments with or without methane hydrates, we studied &gt; 2,800 clones possessing partial sequences (400-500 bp) of the 16S rRNA gene and 348 representative clone sequences (approximate to 1 kbp) from the two geographically separated subseafloor environments. Archaea of the uncultivated Deep-Sea Archaeal Group were consistently the dominant phylotype in sediments associated with methane hydrate. Sediment cores lacking methane hydrates displayed few or no Deep-Sea Archaeal Group phylotypes. Bacterial communities in the methane hydrate-bearing sediments were dominated by members of the JS1 group, Planctomycetes, and Chloroflexi. Results from cluster and principal component analyses, which include previously reported data from the West and East Pacific Margins, suggest that, For these locations in the Pacific Ocean, prokaryotic communities from methane hydrate-bearing sediment cores are distinct from those in hydrate-free cores. The recognition of which microbial groups prevail under distinctive subseafloor environments is a significant step toward determining the role these communities play in Earth's essential biogeochemical processes.

Diverse microbial communities and numerous energy-yielding activities occur in deeply buried sediments of the eastern Pacific Ocean. Distributions of metabolic activities often deviate from the standard model. Rates of activities, cell concentrations, and populations of cultured bacteria vary consistently from one subseafloor environment to another. Net rates of major activities principally rely on electron acceptors and electron donors from the photosynthetic surface world. At open-ocean sites, nitrate and oxygen are supplied to the deepest sedimentary communities through the underlying basaltic aquifer. In turn, these sedimentary communities may supply dissolved electron donors and nutrients to the underlying crustal biosphere.

A novel mesophilic sulfur- and thiosulfate-oxidizing bacterium, strain 42BKT(T), was isolated from the gas-bubbling sediment at the Iheya North hydrothermal system in the mid-Okinawa Trough, Japan. The isolate was a Gram-negative, non-motile and coccoid to oval-shaped bacterium. Growth was observed at 10-40degreesC (optimum 28-30degreesC) and in the pH range 5.0-9.0 (optimum 6.5-7.0). Strain 42BKT(T) grew chemolithoautotrophically with elemental sulfur or thiosulfate as a sole electron donor and oxygen (optimum 5% in gas phase) or nitrate as an electron acceptor. The G+C content of the genomic DNA was 48.0 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the isolate belonged to the previously uncultivated Group F within the epsilon-Proteobacteria, which includes phylotypes of vent epibiont and environmental sequences from global deep-sea cold seep and hydrothermal vent fields. On the basis of the physiological and molecular characteristics of this isolate, the type species of a novel genus, Sulfurovum lithotrophicum gen. nov., sp. nov., is proposed. The type strain is 42BKTT ( = ATCC BAA-797(T) = JCM 12117(T)).

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated by performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, which extended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing fine pumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA gene fragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger than those in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences in the microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblages in the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the most predominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of 16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ash layers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominated the pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed that there was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some grain-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands of years to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, which are capable of harboring microbial communities that are very distinct from the communities in the more abundant pelagic clays.

A novel mesophilic, sulfur- and thiosulfate-oxidizing bacterium, strain OK10(T), was isolated from deep-sea sediments at the Hatoma Knoll in the Mid-Okinawa Trough hydrothermal field. Cells of strain OK10(T) were short rods, each being motile by means of a single polar flagellum. The isolate grew at 10-40 degreesC (optimum 25 degreesC) and pH 4(.)5-9(.)0 (optimum pH 6(.)5). It grew chemolithoautotrophically with elemental sulfur, sulfide and thiosulfate as sole electron donors and oxygen as electron acceptor. Molecular hydrogen did not support growth. The G + C content of the genomic DNA of strain OK10(T) was 35(.)2 mol%. Phylogenetic analysis, based on 16S rRNA gene sequences, indicated that the isolate belonged to the epsilon-Proteobacteria. On the basis of its physiological and molecular characteristics, strain OK10(T) (=ATCC BAA-671(T)=JCM 11897(T)) represents the sole species of a new genus, Sulfurimonas, for which the name Sulfurimonas autotrophica is proposed.

ABSTRACT Deep-sea and subseafloor sedimentary environments host heterotrophic microbial communities that contribute to Earth’s carbon cycling. However, the potential metabolic functions of individual microorganisms and their biogeographical distributions in hadal ocean sediments remain largely unexplored. In this study, we conducted single-cell genome sequencing on sediment samples collected from six sites (7,445–8,023 m water depth) along an approximately 500 km transect of the Japan Trench during the International Ocean Discovery Program Expedition 386. A total of 1,886 single-cell amplified genomes (SAGs) were obtained, offering comprehensive genetic insights into sedimentary microbial communities in surface sediments (&lt;1 m depth) above the sulfate-methane transition zone along the Japan Trench. Our genome data set included 269 SAGs from Atribacterota JS1, the predominant bacterial clade in these hadal environments. Phylogenetic analysis classified SAGs into nine distinct phylotypes, whereas metagenome-assembled genomes were categorized into only two phylotypes, advancing JS1 diversity coverage through a single cell-based approach. Comparative genomic analysis of JS1 lineages from different habitats revealed frequent detection of genes related to organic carbon utilization, such as extracellular enzymes like clostripain and α-amylase, and ABC transporters of oligopeptide from Japan Trench members. Furthermore, specific JS1 phylotypes exhibited a strong correlation with in situ methane concentrations and contained genes involved in glycine betaine metabolism. These findings suggest that the phylogenomically diverse and novel Atribacterota JS1 is widely distributed in Japan Trench sediment, playing crucial roles in carbon cycling within the hadal sedimentary biosphere. IMPORTANCE The Japan Trench represents tectonically active hadal environments associated with Pacific plate subduction beneath the northeastern Japan arc. This study, for the first time, documented a large-scale single-cell and metagenomic survey along an approximately 500 km transect of the Japan Trench, obtaining high-quality genomic information on hadal sedimentary microbial communities. Single-cell genomics revealed the predominance of diverse JS1 lineages not recoverable through conventional metagenomic binning. Their metabolic potential includes genes related to the degradation of organic matter, which contributes to methanogenesis in the deeper layers. Our findings enhance understanding of sedimentary microbial communities at water depths exceeding 7,000 m and provide new insights into the ecological role of biogeochemical carbon cycling in the hadal sedimentary biosphere.

Abstract Dissimilatory iron reduction (DIR) is suggested to be one of the earliest forms of microbial respiration. It plays an important role in the biogeochemical cycling of iron in modern and ancient sediments. Since microbial iron cycling is typically accompanied by iron isotope fractionation, stable iron isotopes are used as tracer for biological activity. Here we present iron isotope data for dissolved and sequentially extracted sedimentary iron pools from deep and hot subseafloor sediments retrieved in the Nankai Trough off Japan. Dissolved iron (Fe(II)_aq) is isotopically light throughout the ferruginous sediment interval but some samples have exceptionally light isotope values. Such light values have never been reported in natural marine environments and cannot be solely attributed to DIR. We show that the light isotope values are best explained by a Rayleigh distillation model where Fe(II)_aq is continuously removed from the pore water by adsorption onto iron (oxyhydr)oxide surfaces. While the microbially mediated Fe(II)_aq release has ceased due to an increase in temperature beyond the threshold of mesophilic microorganisms, the abiotic adsorptive Fe(II)_aq removal continued, leading to uniquely light isotope values. These findings have important implications for the interpretation of dissolved iron isotope data especially in deep subseafloor sediments.

Mud volcanoes are topographic features through which over-pressurized sediments are intruded upward and erupted, transporting fluids and sediments from the deep subsurface to the surface/seafloor. To understand material and biogeochemical cycling through mud volcanoes, we investigated the origins of sediments and fluids erupted by four submarine mud volcanoes off Tanegashima Island, along the northern Ryukyu Trench: MV#1–3 and MV#14. We estimated the ages of the source sequences of the mud volcano sediments to be middle to late Miocene based on nannofossils and ¹⁰Be ages. The sediments were characteristically enriched in illite. The similar mineral compositions and vitrinite reflectance values (0.42%–0.45%) of sediments erupted by the mud volcanoes strongly suggest that each is rooted in the same source sequence. Pore waters had Cl⁻ concentrations ∼40% that of seawater and were proportionally enriched in ¹⁸O and depleted in D, indicating the addition of freshwater from the dehydration of clay minerals. However, the smectite and illite contents (&amp;lt;40%) in the clay size fraction ruled out in situ smectite dewatering as the cause of the pore water Cl⁻ dilution. Thus, fluids derived from clay dewatering must have originated from deeper than the source sequence of the mud volcano sediments. Vertical Cl⁻ profiles indicate that the upward fluid advection rate and eruption frequency decrease from MV#3 to MV#2, MV#1, and MV#14 (MV#14 being dormant). At the active mud volcanoes, the C₁/C₂ ratios (&amp;lt;100) and methane δ¹³C values (−56‰ to −42‰) indicate that hydrocarbon gases are mostly derived from the thermal decomposition of organic matter in deep sediments where the in situ temperature exceeds 80°C. At the dormant MV#14, high C₁/C₂ ratios (700–4,000) and low methane δ¹³C values (ca. −75‰) suggest the limited supply of thermogenic methane and the subsequent shallow methanogenesis. Because vitrinite reflectance values indicate that the source sediments are too immature to produce thermogenic hydrocarbons, the hydrocarbon gases, like the fluids derived from clay dewatering, were probably supplied from deeper than the source strata. The supply of deep fluids into the source sequence might be a universal phenomenon in subduction zones, and may play an important role in mud volcanism and associated biogeochemical cycling.

BACKGROUND: Marine deep subsurface sediments were once thought to be devoid of eukaryotic life, but advances in molecular technology have unlocked the presence and activity of well-known closely related terrestrial and marine fungi. Commonly detected fungi in deep marine sediment environments includes Penicillium, Aspergillus, Cladosporium, Fusarium, and Schizophyllum, which could have important implications in carbon and nitrogen cycling in this isolated environment. In order to determine the diversity and unknown metabolic capabilities of fungi in deep-sea sediments, their genomes need to be fully analyzed. In this study, two Penicillium species were isolated from South Pacific Gyre sediment enrichments during Integrated Ocean Drilling Program Expedition 329. The inner gyre has very limited productivity, organic carbon, and nutrients. RESULTS: Here, we present high-quality genomes of two proposed novel Penicillium species using Illumina HiSeq and PacBio sequencing technologies. Single-copy homologues within the genomes were compared to other closely related genomes using OrthoMCL and maximum-likelihood estimation, which showed that these genomes were novel species within the genus Penicillium. We propose to name isolate SPG-F1 as Penicillium pacificasedimenti sp. nov. and SPG-F15 as Penicillium pacificagyrus sp. nov. The resulting genome sizes were 32.6 Mbp and 36.4 Mbp, respectively, and both genomes were greater than 98% complete as determined by the presence of complete single-copy orthologs. The transposable elements for each genome were 4.87% for P. pacificasedimenti and 10.68% for P. pacificagyrus. A total of 12,271 genes were predicted in the P. pacificasedimenti genome and 12,568 genes in P. pacificagyrus. Both isolates contained genes known to be involved in the degradation of recalcitrant carbon, amino acids, and lignin-derived carbon. CONCLUSIONS: Our results provide the first constructed genomes of novel Penicillium isolates from deep marine sediments, which will be useful for future studies of marine subsurface fungal diversity and function. Furthermore, these genomes shed light on the potential impact fungi in marine sediments and the subseafloor could have on global carbon and nitrogen biogeochemical cycles and how they may be persisting in the most energy-limited sedimentary biosphere.

Here, we demonstrate that wood-rot fungi produce methane anaerobically without the involvement of methanogenic archaea via a new, halomethane-dependent pathway. These findings of an anaerobic fungal methane formation pathway open another avenue in methane research and will further assist with current efforts in the identification of the processes involved and their ecological implications.

Pore pressure plays a key role in the generation of earthquakes in subduction zones. However, quantitative constraints for its determination are quite limited. Here, we estimate the subsurface pore pressure by analyzing the transient upwelling flow of drilling mud from borehole C0023A of the International Ocean Discovery Program (IODP) Expedition 370, in the Nankai Trough off Cape Muroto. This upward flow provided the first direct evidence of an overpressured aquifer in the underthrust sediments off Cape Muroto. To estimate the pre-drilling pore pressure in the overpressured aquifer around a depth of 950–1,050 m below sea floor, we examined the measured porosities of core samples retrieved from nearby IODP wells; we then proceeded to explain the observed time evolution of the flow rate of the upwelling flow by modeling various sized aquifers through solving a radial diffusion equation. It was observed that for a permeability of 10−13 m2, the aquifer possessed an initial excess pore pressure of ∼5–10 MPa above the hydrostatic pressure, with a lateral dimension of several hundred meters and thickness of several tens of meters. The overpressure estimates from the porosity-depth profile at Site C0023 differ from those at other drill sites in the region, suggesting the possible existence of multiple overpressured aquifers with a patchy distribution in the underthrust sediments of the Nankai Trough. As pore pressure is relevant in maintaining fault stability, the overpressured aquifers may be the source of slow earthquakes that have been observed around the drilling site.

Abstract. For more than half a century, exploring a complete sequence of the oceanic crust from the seafloor through the Mohorovičić discontinuity (Moho) and into the uppermost mantle has been one of the most challenging missions of scientific ocean drilling. Such a scientific and technological achievement would provide humankind with profound insights into the largest realm of our planet and expand our fundamental understanding of Earth's deep interior and its geodynamic behavior. The formation of new oceanic crust at mid-ocean ridges and its subsequent aging over millions of years, leading to subduction, arc volcanism, and recycling of some components into the mantle, comprise the dominant geological cycle of matter and energy on Earth. Although previous scientific ocean drilling has cored some drill holes into old (&gt; 110 Ma) and young (&lt; 20 Ma) ocean crust, our sampling remains relatively shallow (&lt; 2 km into intact crust) and unrepresentative of average oceanic crust. To date, no hole penetrates more than 100 m into intact average-aged oceanic crust that records the long-term history of seawater–basalt exchange (60 to 90 Myr). In addition, the nature, extent, and evolution of the deep subseafloor biosphere within oceanic crust remains poorly unknown. To address these fundamentally significant scientific issues, an international workshop “Exploring Deep Oceanic Crust off Hawai`i” brought together 106 scientists and engineers from 16 countries that represented the entire spectrum of disciplines, including petrologists, geophysicists, geochemists, microbiologists, geodynamic modelers, and drilling/logging engineers. The aim of the workshop was to develop a full International Ocean Discovery Program (IODP) proposal to drill a 2.5 km deep hole into oceanic crust on the North Arch off Hawai`i with the drilling research vessel Chikyu. This drill hole would provide samples down to cumulate gabbros of mature (∼ 80 Ma) oceanic crust formed at a half spreading rate of ∼ 3.5 cm a−1. A Moho reflection has been observed at ∼ 5.5 km below the seafloor at this site, and the workshop concluded that the proposed 2.5 km deep scientific drilling on the North Arch off Hawai`i would provide an essential “pilot hole” to inform the design of future mantle drilling.

© 2019 The Authors. Basin Research © 2019 John Wiley & Sons Ltd, European Association of Geoscientists & Engineers and International Association of Sedimentologists Basin models can simulate geological, geochemical and geophysical processes and potentially also the deep biosphere, starting from a burial curve, assuming a thermal history and utilizing other experimentally obtained data. Here, we apply basin modelling techniques to model cell abundances within the deep coalbed biosphere off Shimokita Peninsula, Japan, drilled during Integrated Ocean Drilling Program Expedition 337. Two approaches were used to simulate the deep coalbed biosphere: (a) In the first approach, the deep biosphere was modelled using a material balance approach that treats the deep biosphere as a carbon reservoir, in which fluxes are governed by temperature-controlled metabolic processes that retain carbon via cell-growth and cell-repair and pass it back via cell-damaging reactions. (b) In the second approach, the deep biosphere was modelled as a microbial community with a temperature-controlled growth ratio and carrying capacity (a limit on the size of the deep biosphere) modulated by diagenetic-processes. In all cases, the biosphere in the coalbeds and adjacent habitat are best modelled as a carbon-limited community undergoing starvation because labile sedimentary organic matter is no longer present and petroleum generation is yet to occur. This state of starvation was represented by the conversion of organic carbon to authigenic carbonate and the formation of kerogen. The potential for the biosphere to be stimulated by the generation of carbon-dioxide from the coal during its transition from brown to sub-bituminous coal was evaluated and a net thickness of 20 m of lignite was found sufficient to support an order of magnitude greater number of cells within a low-total organic carbon (TOC) horizon. By comparison, the stimulation of microbial populations in a coalbed or high-TOC horizon would be harder to detect because the increase in population size would be proportionally very small.

Abstract. The Chikyu Shallow Core Program (SCORE) has been started to provide more opportunities for the scientific ocean drilling of shallow boreholes (up to 100 m) during a short-term expedition. The proposal flow is a simplified version of that of the International Ocean Discovery Program (IODP). Although there are several limitations for a SCORE project, the opportunity to retrieve 100 m of continuous core samples will be of great interest for the scientific ocean drilling community in multiple disciplines. The first expedition of the SCORE program was implemented off Cape Erimo, Hokkaido, northern Japan. The target of the drilling was to investigate the impact of submarine mass transport on the subseafloor sedimentary biosphere. In the preliminary observation of the core samples, including X-ray computed tomography (CT) scan image analysis, chaotic and inclined beds were found and interpreted as mass transport deposit (MTD) units.

© 2020, The Author(s). The upper oceanic crust is mainly composed of basaltic lava that constitutes one of the largest habitable zones on Earth. However, the nature of deep microbial life in oceanic crust remains poorly understood, especially where old cold basaltic rock interacts with seawater beneath sediment. Here we show that microbial cells are densely concentrated in Fe-rich smectite on fracture surfaces and veins in 33.5- and 104-million-year-old (Ma) subseafloor basaltic rock. The Fe-rich smectite is locally enriched in organic carbon. Nanoscale solid characterizations reveal the organic carbon to be microbial cells within the Fe-rich smectite, with cell densities locally exceeding 1010 cells/cm3. Dominance of heterotrophic bacteria indicated by analyses of DNA sequences and lipids supports the importance of organic matter as carbon and energy sources in subseafloor basalt. Given the prominence of basaltic lava on Earth and Mars, microbial life could be habitable where subsurface basaltic rocks interact with liquid water.

© 2019 The Authors Drilling during International Ocean Discovery Program (IODP) Expedition 370 at Site C0023 encountered instances of hydrothermal mineralization from 775 to 1121 m below seafloor. Fluid inclusion homogenization temperatures measured on barite veins within this interval indicate precipitation from fluids with temperatures up to 220 °C, and salinities ten times higher than interstitial water (16–25 c.f. 2.8–3.6 wt % NaCl). Patches of stratabound mineralization (rhodochrosite, calcite, barite and anhydride) are largely confined to the vicinity of veins and have vertical thicknesses and extents that can be explained by precipitation within the thermal aureoles of veins. Thermal maturities measured on petroleum biomarkers in underthrust sediments record a rise from pre-oil window to early oil window thermal maturities. Basin models show that increases in thermal maturity can be explained by burial metamorphism, and kinetic-based calculations suggest that hot fluids would only have had a minimal effect on hydrocarbon generation. However, the movement of hot fluids still has geochemical implications and creates a complex thermal history where both short-duration, localized heating within fracture zones at hot temperatures (~200 °C for less than 1 yr) and heating during burial over long durations (80–110 °C for 0.5–2 Myrs) need to be considered.

© 2019, The Author(s). 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 13 C-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.

National Gas Hydrate Program Expedition 02 was conducted in 2015 by D/V Chikyu in the western Bay of Bengal, India. Analyses of interstitial water in sediment from four drilling sites along the eastern and western transect in the Krishna–Godavari Basin show that dissolved chloride (Cl−) concentrations decreased with depth from seawater values (>550 mM) near the sediment surface to 300–400 mM at 350–400 m below seafloor (mbsf), suggesting the upward advection of low-Cl– fluid. Excursions of Cl− concentrations toward low values in gas hydrate zones were attributed to the presence of fresh water released by dissociation of gas hydrate during core recovery. At Sites in the western transect, Cl− concentrations below the gas hydrate reservoir (around 280 mbsf) were ca. 60 mM and ca. 45 mM lower than those from above the reservoir, and δ18O and δD values were 1–2‰ and 0.7–1.3‰ higher than those from above the reservoir, respectively. These results suggest that a fraction of migrating low-Cl– fluid is trapped in the gas hydrate zone, causing decreases of δ18O and δD in the residual water due to isotopic fractionation during gas hydrate formation. From mass-balance calculations, we estimated that 10–20% of the advected fluid is trapped at the gas hydrate zone. At all sites, the δ18O and δD values below the gas hydrate zone, representing relatively unaltered low-Cl– fluid, were 0.5–1‰ higher and ca. 13–15‰ lower than those of seawater, respectively. This trend is consistent with water derived from the dehydration of clay minerals, which generally occurs in deeply buried sediments. The contribution of clay mineral dehydration is relatively large at the western transect compared to that in the eastern transect. Our results imply that fluid advection contributes to the accumulation of gas hydrate in the study area.

© 2019, Springer Nature Limited. In the version of the Article originally published, incorrect units were used in the sediment thickness grid used in the integration of sedimentary organic carbon content. This mistake resulted in an overestimate of the oxic sedimentary organic carbon reservoir by three orders of magnitude.

The occurrence of microbially induced smectite-to-illite (S-I) reaction has challenged both the notions of solely inorganic chemical control for this reaction and the conventional concept of a semiquantitative illite geothermometer for the reconstruction of the thermal and tectonic histories of sedimentary basins. Here, we present evidence for a naturally occurring microbially induced S-I transition, via biotic reduction of phyllosilicate structural Fe(III), in mudstones buried at the Nankai Trough, offshore Japan (International Ocean Discovery Program Site C0023). Biotic S-I reaction is a consequence of a bacterial survival and growth strategy at diagenetic temperatures up to 80 degrees C within the Nankai Trough mudstones. These results have considerable implications for petroleum exploration, modification of fault behavior, and the understanding of microbial communities in the deep biosphere.

Here, we report genome sequences of two Penicillium isolates from below the seafloor of the oligotrophic South Pacific Gyre. These genomes are the first reported for fungi from deeply buried marine sediment. Both genomes will provide valuable information regarding the role of fungi and carbon cycling in the energy-limited subsurface biosphere.

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.

Viruses are the most abundant biological entities on Earth and perform essential ecological functions in aquatic environments by mediating biogeochemical cycling and lateral gene transfer. Cellular life as well as viruses have been found in deep subseafloor sediment. However, the study of deep sediment viruses has been hampered by the complexities involved in efficiently extracting viruses from a sediment matrix. Here, we developed a new method for the extraction of viruses from sediment based on density separation using a Nycodenz density step gradient. The density separation method resulted in up to 2 orders of magnitude greater recovery of viruses from diverse subseafloor sediments compared to conventional methods. The density separation method also showed more consistent performance between samples of different sediment lithology, whereas conventional virus extraction methods were highly inconsistent. Using this new method, we show that previously published virus counts have underestimated viral abundances by up to 2 orders of magnitude. These improvements suggest that the carbon contained within viral biomass in the subseafloor environment may potentially be revised upward to 0.8-3.7 Gt from current estimates of 0.2 Gt. The vastly improved recovery of viruses indicate that viruses represent a far larger pool of organic carbon in subseafloor environments than previously estimated.

© 2018, The Author(s). To understand the ability of microbial life to inhabit a deep subseafloor coalbed sedimentary basin, the correlation between fluid transport properties and the abundance of microbial cells was investigated based on core samples collected down to about 2.5 km below the seafloor during the Integrated Ocean Drilling Program Expedition 337 off the Shimokita Peninsula, Japan. The overall depth profiles for porosity and permeability exhibited a decreasing trend with increasing depth. However, at depths greater than 1.2 km beneath the seafloor, the transport characteristics of the sediments were highly variable, with the permeability ranging from 10−16 to 10−22 m2 and the pore size ranging from &lt; 0.01 to 100 μm. This is mainly attributed to the diversity of the lithology, which exhibits a range of pore sizes and pore geometries. Fracture channels in coal seams had the highest permeability, while shale deposits had the smallest pore size and lowest permeability. A positive correlation between permeability and pore size was confirmed by the Kozeny-Carman equation. Cell abundance at shallower depths was positively correlated with porosity and permeability, and was less strongly correlated with pore size. These findings suggest that one of the factors affecting the decrease in microbial cell abundance with increasing depth was a reduction in nutrient and water supply to indigenous microbial communities as a result of a decrease in porosity and permeability due to sediment compaction. Anomalous regions with relatively high cell concentrations in coal-bearing units could be explained by the higher permeability and larger pore size for these units compared to the surrounding sediments. Nutrient transport through permeable cleats in coal layers might occur upwards toward the upper permeable sandstone layers, which are well suited for sustaining sizable microbial populations. Conversely, impermeable shale and siltstone with small pores (&lt; 0.2 μm, which is smaller than microbial cell size) may act as barriers to water and energy-yielding substrates for deep microbial life. We propose that the pore size and permeability govern the threshold for microbial habitability in the deep subseafloor sedimentary biosphere. [Figure not available: see fulltext.].

© 2018, The Author(s). The study investigates the in-situ strength of sediments across a plate boundary décollement using drilling parameters recorded when a 1180-m-deep borehole was established during International Ocean Discovery Program (IODP) Expedition 370, Temperature-Limit of the Deep Biosphere off Muroto (T-Limit). Information of the in-situ strength of the shallow portion in/around a plate boundary fault zone is critical for understanding the development of accretionary prisms and of the décollement itself. Studies using seismic reflection surveys and scientific ocean drillings have recently revealed the existence of high pore pressure zones around frontal accretionary prisms, which may reduce the effective strength of the sediments. A direct measurement of in-situ strength by experiments, however, has not been executed due to the difficulty in estimating in-situ stress conditions. In this study, we derived a depth profile for the in-situ strength of a frontal accretionary prism across a décollement from drilling parameters using the recently established equivalent strength (EST) method. At site C0023, the toe of the accretionary prism area off Cape Muroto, Japan, the EST gradually increases with depth but undergoes a sudden change at ~ 800 mbsf, corresponding to the top of the subducting sediment. At this depth, directly below the décollement zone, the EST decreases from ~ 10 to 2 MPa, with a change in the baseline. This mechanically weak zone in the subducting sediments extends over 250 m (~ 800–1050 mbsf), corresponding to the zone where the fluid influx was discovered, and high-fluid pressure was suggested by previous seismic imaging observations. Although the origin of the fluids or absolute values of the strength remain unclear, our investigations support previous studies suggesting that elevated pore pressure beneath the décollement weakens the subducting sediments. [Figure not available: see fulltext.].

©2018. American Geophysical Union. All Rights Reserved. Magnetic mineral diagenesis is an important process in sediments that is responsible for the partial or total destruction of records of Earth&#039;s magnetic field variations and also plays an important part in iron and sulfur cycling. A rock magnetic study has been carried out at International Ocean Discovery Program Expedition 350 Site U1437 in the Izu Bonin rear arc to investigate magnetic mineral diagenesis in deeply buried sediments from ~775 to ~1,002 m below sea floor (mbsf) with burial temperatures ranging from ~67 to 85 °C. Nonsteady state geochemical environments occur within an unusual deep methanic zone (below 850 mbsf) because of a release of sulfate below the shallow sulfate reduction zone. A drastic decline in magnetic susceptibility and remanence is observed at ~850 mbsf due to a decrease in ferrimagnetic iron oxide contents. Reduction of (titano)-magnetite and pyritization occur at this inferred deep sulfate-methane transition zone. Below ~850 mbsf, lower magnetic mineral contents coincide with further methane release. Geochemical analyses support a change in redox conditions and secondary precipitation of iron sulfides and carbonates in the methane-rich zone. Magnetic mineral alteration is enhanced in zones where methane accumulates underneath low porosity intervals that act as seals. Although geochemical processes due to methane occurrence are likely dominant, more than one mechanism, possibly involving microbial activity, is probably responsible for the observed magnetic mineral assemblage changes.

© Author(s) 2018. An International Ocean Discovery Program (IODP) workshop was held at Sydney University, Australia, from 13 to 16 June 2017 and was attended by 97 scientists from 12 countries. The aim of the workshop was to investigate future drilling opportunities in the eastern Indian Ocean, southwestern Pacific Ocean, and the Indian and Pacific sectors of the Southern Ocean. The overlying regional sedimentary strata are underexplored relative to their Northern Hemisphere counterparts, and thus the role of the Southern Hemisphere in past global environmental change is poorly constrained. A total of 23 proposal ideas were discussed, with ~12 of these deemed mature enough for active proposal development or awaiting scheduled site survey cruises. Of the remaining 11 proposals, key regions were identified where fundamental hypotheses are testable by drilling, but either site surveys are required or hypotheses need further development. Refinements are anticipated based upon regional IODP drilling in 2017/2018, analysis of recently collected site survey data, and the development of site survey proposals. We hope and expect that this workshop will lead to a new phase of scientific ocean drilling in the Australasian region in the early 2020s.

The Challenger Deep is the deepest ocean on Earth. The present study investigated microbial community structures and geochemical cycles associated with the trench bottom sediments of the Challenger Deep, the Mariana Trench. The SSU rRNA gene communities found in trench bottom sediments were dominated by the bacteria Chloroflexi (SAR202 and other lineages), Bacteroidetes, Planctomycetes, "Ca. Marinimicrobia" (SAR406), and Gemmatimonadetes and by the archaeal α subgroup of MGI Thaumarchaeota and "Ca. Woesearchaeota" (Deep-sea Hydrothermal Vent Euryarchaeotic Group 6). The SSU rRNA gene sequencing analysis indicated that the dominant populations of the thaumarchaeal α group in hadal water and sediments were similar to each other at the species or genus level. In addition, the co-occurrence of nitrification and denitrification was revealed by the combination of pore water geochemical analyses and quantitative PCR for nitrifiers.

Experimental contamination by exogenous DNA is a major issue in molecular biological studies for data quality and its management. We herein assessed DNA aerosols for the risk of contamination and tested the capacity of clean air filters to trap and remove DNA aerosols. DNA aerosols were generated by atomizing a DNA solution and introduced into a laminar flow clean air unit. Capture and detection performed upstream and downstream of the clean air unit showed that a significant fraction (>99.96%) of introduced molecules was trapped and removed by the filter. Although DNA aerosols appear to be an avoidable source of exogenous contamination, a clearer understanding and careful experimental procedures are needed in order to perform contamination-free, high-quality molecular biology experiments.

Clay mineralogy is an important characteristic of mud volcano sediments. This study determined the clay mineral compositions of sediment from two submarine mud volcanoes in the Kumano forearc basin, Nankai Trough, by X-ray diffraction analysis. Similar compositions dominated by smectite in the two mud volcanoes indicate that the mud volcanoes in the basin are rooted in the same source sequence. These clay mineral compositions differed from those in Pleistocene basin sediment, suggesting that the mud volcano sediment originated beneath the Pleistocene sediment. The illite content in the illite–smectite mixed layer averaged 32% in the mud volcano sediment, which implies that the sediment experienced temperatures above 60◦C that promoted the smectite-to-illite transformation. However, porewater extracted from the mud volcano sediment had Cl- concentrations roughly half that of seawater and proportional enrichment in18O and depletion in D, indicating that dehydration reactions of clay minerals had previously occurred in a deeply buried sedimentary layer. The smectite and illite contents (&lt 60%) in the clay-size fraction rule out in situ smectite dewatering as the cause of the dilution of Cl- in porewater. Thus, fluids derived from clay dewatering must have originated from deeper than the source of the mud volcano sediment.

We investigated the impact of temperature on the microbial turnover of organic matter (OM) in a hydrothermal vent system in Guaymas Basin, by calculating microbial bio- and necromass turnover times based on the culture-independent D:L-amino acid model. Sediments were recovered from two stations near hydrothermal mounds (&lt 74°C) and from one cold station (&lt 9°C). Cell abundance at the two hydrothermal stations dropped from 108 to 106 cells cm-3 within ~5 m of sediment depth resulting in a 100-fold lower cell number at this depth than at the cold site where numbers remained constant at 108 cells cm-3 throughout the recovered sediment. There were strong indications that the drop in cell abundance was controlled by decreasing OM quality. The quality of the sedimentary OM was determined by the diagenetic indicators %TAAC (percentage of total organic carbon present as amino acid carbon), %TAAN (percentage of total nitrogen present as amino acid nitrogen), aspartic acid:β-alanine ratios, and glutamic acid:γ-amino butyric acid ratios. All parameters indicated that the OM became progressively degraded with increasing sediment depth, and the OM in the hydrothermal sediment was more degraded than in the uniformly cold sediment. Nonetheless, the small community of microorganisms in the hydrothermal sediment demonstrated short turnover times. The modeled turnover times of microbial bio- and necromass in the hydrothermal sediments were notably faster (biomass: days to months necromass: up to a few hundred years) than in the cold sediments (biomass: tens of years necromass: thousands of years), suggesting that temperature has a significant influence on the microbial turnover rates. We suggest that short biomass turnover times are necessary for maintance of essential cell funtions and to overcome potential damage caused by the increased temperature. The reduced OM quality at the hyrothemal sites might thus only allow for a small population size of microorganisms.

Widespread diagenesis of clay minerals occurs in deeply buried marine sediments under high-temperature and high-pressure conditions. For example, the smectite-to-illite (S-I) transformation has been often observed in sediments at in situ temperatures above similar to 60 degrees C. However, it remains largely unknown whether such diagenetic processes naturally occur in relatively shallow and low-temperature sediments and, if so, what the consequences are of any related chemical reactions to the geochemical characteristics in the deep biosphere. We evaluated the possibility of naturally occurring S-I transformation at temperatures below 40 degrees C in continental slope sediments of the Bering Sea by examining porewater chemistry, clay mineralogy, and chemical composition of clay minerals measured to similar to 800 m beneath the seafloor (mbsf) in core samples acquired during Integrated Ocean Drilling Program Expedition 323. In porewater from these cores, chloride concentrations decreased with increasing depth from 560 mM near the seafloor to 500 mM at similar to 800 mbsf; delta O-18 increased from 0 to 1.5 parts per thousand and delta D decreased from -1 to -9%0. These trends are consistent with the addition of water derived from S-I transformation. The discrete low Cl- spikes observed between similar to 200 and similar to 450 mbsf could be attributed to the dissociation of methane hydrate. X-ray diffraction analysis of the clay-size fraction (<21.1 mu m) showed an increase of illite content in the I/S mixed layer with increasing depth to 150 mbsf. This increase may imply the occurrence of S-I transformation. The decrease of Fe3+/Fe2+ in the clay-size fraction with increasing depth strongly suggests microbial reduction of Fe(III) in clay minerals with burial, which also has the potential to promote the S-I transformation. Our results imply the significant ecological roles on the diagenesis of siliciclastic clay minerals underlying the high-productivity surface seawater at continental margins.

Deep-sea mud extremely enriched in rare-earth elements and yttrium (together called REY) has been discovered around Minamitorishima Island, in the western North Pacific. The REY-rich mud was previously observed to contain abundant phillipsite and biogenic calcium phosphate. We analyzed the chemical compositions of porewater in a sediment core containing REY-rich mud and observed a decrease of dissolved phosphate (PO43-) from similar to 1 mu M to similar to 0.5 mu M around the REY-rich mud layer. The presence of dissolved nitrate + nitrite throughout the cored depth indicated oxic sediment conditions. In the REY-rich mud, PO43- was presumed to be adsorbed onto the surfaces of minerals such as phillipsite. Our results support that oxic pelagic clay containing phillipsite can be a sink for PO43-. Concentrations of REY in porewater were below the detection limit.

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 degrees 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.

Water from The Cedars springs that discharge from serpentinized ultramafic rocks feature highly basic (pH=similar to 12), highly reducing (E-h&lt;-550 mV) conditions with low ionic concentrations. These conditions make the springs exceptionally challenging for life. Here, we report the metagenomic data and recovered draft genomes from two different springs, GPS1 and BS5. GPS1, which was fed solely by a deep groundwater source within the serpentinizing system, was dominated by several bacterial taxa from the phyla OD1 ('Parcubacteria') and Chloroflexi. Members of the GPS1 community had, for the most part, the smallest genomes reported for their respective taxa, and encoded only archaeal (A-type) ATP synthases or no ATP synthases at all. Furthermore, none of the members encoded respiration-related genes and some of the members also did not encode key biosynthesis-related genes. In contrast, BS5, fed by shallow water, appears to have a community driven by hydrogen metabolism and was dominated by a diverse group of Proteobacteria similar to those seen in many terrestrial serpentinization sites. Our findings indicated that the harsh ultrabasic geological setting supported unexpectedly diverse microbial metabolic strategies and that the deep-water-fed springs supported a community that was remarkable in its unusual metagenomic and genomic constitution.

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutS, which recognizes Holliday junction to promote homologous recombination, has been identified in archaea. Hence, the whole molecular mechanism of archaeal homologous recombination has not yet been revealed. In this study, to identify the archaeal functional homolog of MutS gamma, we focused on a functionally uncharacterized MutS homolog, MutS5, from a hyperthermophilic archaeon Pyrococcus horikoshii (phMutS5). Archaeal MutS5 has a Walker ATPase motif-containing amino acid sequence that shows similarity to the ATPase domain of MutS gamma. It is known that the ATPase domain of MutS homologs is also a dimerization domain. Chemical cross-linking revealed that purified phMutS5 has an ability to dimerize in solution. phMutS5 bound to Holliday junction with a higher affinity than to other branched and linear DNAs, which resembles the DNA-binding specificities of MutS gamma and bacterial MutS2, a Holliday junction-resolving MutS gamma homolog. However, phMutS5 has no nuclease activity against branched DNA unlike MutS2. The ATPase activity of phMutS5 was significantly stimulated by the presence of Holliday junction similarly to MutS gamma. Furthermore, site-directed mutagenesis revealed that the ATPase activity is dependent on the Walker ATPase motif of the protein. These results suggest that archaeal MutS5 should stabilize the Holliday junction and play a role in homologous recombination, which is analogous to the function of eukaryotic MutS gamma.

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 x 10(4) 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.

Carbon monoxide (CO) is the simplest oxocarbon generated by the decomposition of organic compounds, and it is expected to be in marine sediments in substantial amounts. However, the availability of CO in the deep subseafloor sedimentary biosphere is largely unknown even though anaerobic oxidation of CO is a thermodynamically favourable reaction that possibly occurs with sulphate reduction, methanogenesis, acetogenesis and hydrogenesis. In this study, we surveyed for the first time the distribution of the CO dehydrogenase gene (cooS), which encodes the catalytic beta subunit of anaerobic CO dehydrogenase (CODH), in subseafloor sediment-core samples from the eastern flank of the Juan de Fuca Ridge, Mars-Ursa Basin, Kumano Basin, and off the Shimokita Peninsula, Japan, during Integrated Ocean Drilling Program (IODP) Expeditions 301, 308 and 315 and the D/V Chikyu shakedown cruise CK06-06, respectively. Our results show the occurrence of diverse cooS genes from the seafloor down to about 390m below the seafloor, suggesting that microbial communities have metabolic functions to utilize CO in anoxic microbial ecosystems beneath the ocean floor, and that the microbial community potentially responsible for anaerobic CO oxidation differs in accordance with possible energy-yielding metabolic reactions in the deep subseafloor sedimentary biosphere. Significance and Impact of the StudyLittle is known about the microbial community associated with carbon monoxide (CO) in the deep subseafloor. This study is the first survey of a functional gene encoding anaerobic carbon monoxide dehydrogenase (CODH). The widespread occurrence of previously undiscovered CO dehydrogenase genes (cooS) suggests that diverse micro-organisms are capable of anaerobic oxidation of CO in the deep subseafloor sedimentary biosphere.

The factors controlling the relative abundances of Archaea and Bacteria in marine sediments are poorly understood. We determined depth distributions of archaeal and bacterial 16S rRNA genes by quantitative PCR at eight stations in Aarhus Bay, Denmark. Bacterial outnumber archaeal genes 10-60-fold in uppermost sediments that are irrigated and mixed by macrofauna. This bioturbation is indicated by visual observations of sediment color and faunal tracks, by porewater profiles of dissolved inorganic carbon and sulfate, and by distributions of unsupported Pb-210 and Cs-137. Below the depth of bioturbation, the relative abundances of archaeal genes increase, accounting for one third of 16S rRNA genes in the sulfate zone, and half of 16S rRNA genes in the sulfate-methane transition zone and methane zone. Phylogenetic analyses reveal a strong shift in bacterial and archaeal community structure from bioturbated sediments to underlying layers. Stable isotopic analyses on organic matter and porewater geochemical gradients suggest that macrofauna mediate bacterial dominance and affect microbial community structure in bioturbated sediment by introducing fresh organic matter and high-energy electron acceptors from overlying seawater. Below the zone of bioturbation, organic matter content and the presence of sulfate exert key influences on bacterial and archaeal abundances and overall microbial community structure.

Rich animal and microbial communities have been found at deep-sea hydrothermal vents. Although the biogeography of vent macrofauna is well understood, the corresponding knowledge about vent microbial biogeography is lacking. Here, we apply the multilocus sequence analysis (MLSA) to assess the genetic variation of 109 Sulfurimonas strains with &gt;= 98% 16S rRNA gene sequence similarity, which were isolated from four different geographical regions (Okinawa Trough (OT), Mariana Volcanic Arc and Trough (MVAT), Central Indian Ridge (CIR) and Mid-Atlantic Ridge (MAR)). Sequence typing based on 11 protein-coding genes revealed high genetic variation, including some allele types that are widespread within regions, resulting in 102 nucleotide sequence types (STs). This genetic variation was predominantly due to mutation rather than recombination. Phylogenetic analysis of the 11 concatenated genes showed a clear geographical isolation corresponding to the hydrothermal regions they originated from, suggesting limited dispersal. Genetic differentiation among Sulfurimonas populations was primarily influenced by geographical distance rather than gas composition of vent fluid or habitat, although in situ environmental conditions of each microhabitat could not be examined. Nevertheless, Sulfurimonas may possess a higher dispersal capability compared with deep-sea hydrothermal vent thermophiles. This is the first report on MLSA of deep-sea hydrothermal vent Epsilonproteobacteria, which is indicative of allopatric speciation.

© 2017 Hoshino, Inagaki. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. There are errors in the fourth and fifth sentences of the Quantification by qSeq subsection of the Materials and Methods section. The correct sentences are: The SPE conditions consisted of initial denaturation at 98°C for 2 min, cooling to 55°C at 0.3°C/s, and elongation at 68°C for 10 min. Subsequently, excess primers were digested by addition of 4μl of Exonuclease I (5 U/μl, TaKaRa Bio) and incubation at 37°C for 120 min, followed by inactivation of the enzyme by incubation at 80°C for 30 min.

Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down to similar to 2.5 km below the seafloor (kmbsf). However, very little is known about the pressure-adapted and/or pressure-loving microorganisms, the so called piezophiles, in the deep subseafloor biosphere, despite that pressure directly affects microbial physiology, metabolism, and biogeochemical processes of carbon and other elements in situ. In this study, we studied taxonomic compositions of microbial communities in high-pressure incubated sediment, obtained during the Integrated Ocean Drilling Program (IODP) Expedition 337 off the Shimokita Peninsula, Japan. Analysis of 16S rRNA gene-tagged sequences showed that members of spore-forming bacteria within Firmicutes and Actinobacteria were predominantly detected in all enrichment cultures from similar to 1.5 to 2.4 km-deep sediment samples, followed by members of Proteobacteria, Acidobacteria, and Bacteroidetes according to the sequence frequency. To further study the physiology of the deep subseafloor sedimentary piezophilic bacteria, we isolated and characterized two bacterial strains, 19R1-5 and 29R7-12, from 1.9 and 2.4 km-deep sediment samples, respectively. The isolates were both low G+C content, gram-positive, endospore-forming and facultative anaerobic piezophilic bacteria, closely related to Virgibacillus pantothenticus and Bacillus subtilis within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42 degrees C for strain 19R1-5, and 10 MPa and 43 degrees C for strain 29R7-12. Bacterial (endo)spores were observed in both the enrichment and pure cultures examined, suggesting that these piezophilic members were derived from microbial communities buried in the similar to 20 million-year-old coal-bearing sediments after the long-term survival as spores and that the deep biosphere may host more abundant gram-positive spore-forming bacteria and their spores than hitherto recognized.

Although subseafloor sediments are known to harbour a vast number of microbial cells, the distribution, diversity, and origins of fungal populations remain largely unexplored. In this study, we cultivated fungi from 34 of 47 deep coal-associated sediment samples collected at depths ranging from 1289 to 2457 m below the seafloor (mbsf) off the Shimokita Peninsula, Japan (1118 m water depth). We obtained a total of 69 fungal isolates under strict contamination controls, representing 61 Ascomycota (14 genera, 23 species) and 8 Basidiomycota (4 genera, 4 species). Penicillium and Aspergillus relatives were the most dominant genera within the Ascomycetes, followed by the members of genera Cladosporium, Hamigera, Chaetomium, Eutypella, Acremonium, Aureobasidium, Candida, Eurotium, Exophiala, Nigrospora, Bionectria and Pseudocercosporella. Four Basidiomycota species were identified as genera Schizophyllum, Irpex, Bjerkandera and Termitomyces. Among these isolates, Cladosporium sphaerospermum and Aspergillus sydowii relatives were isolated from a thin lignite coal-sandstone formation at 2457 mbsf. Our results indicate that these cultivable fungal populations are indigenous, originating from past terrigenous environments, which have persisted, possibly as spores, through similar to 20 million years of depositional history.

Next-generation sequencing (NGS) is a powerful tool for analyzing environmental DNA and provides the comprehensive molecular view of microbial communities. For obtaining the copy number of particular sequences in the NGS library, however, additional quantitative analysis as quantitative PCR (qPCR) or digital PCR (dPCR) is required. Furthermore, number of sequences in a sequence library does not always reflect the original copy number of a target gene because of biases caused by PCR amplification, making it difficult to convert the proportion of particular sequences in the NGS library to the copy number using the mass of input DNA. To address this issue, we applied stochastic labeling approach with random-tag sequences and developed a NGS-based quantification protocol, which enables simultaneous sequencing and quantification of the targeted DNA. This quantitative sequencing (qSeq) is initiated from single-primer extension (SPE) using a primer with random tag adjacent to the 5' end of target-specific sequence. During SPE, each DNA molecule is stochastically labeled with the random tag. Subsequently, first-round PCR is conducted, specifically targeting the SPE product, followed by second-round PCR to index for NGS. The number of random tags is only determined during the SPE step and is therefore not affected by the two rounds of PCR that may introduce amplification biases. In the case of 16S rRNA genes, after NGS sequencing and taxonomic classification, the absolute number of target phylotypes 16S rRNA gene can be estimated by Poisson statistics by counting random tags incorporated at the end of sequence. To test the feasibility of this approach, the 16S rRNA gene of Sulfolobus tokodaii was subjected to qSeq, which resulted in accurate quantification of 5.0 x 10(3) to 5.0 x 10(4) copies of the 16S rRNA gene. Furthermore, qSeq was applied to mock microbial communities and environmental samples, and the results were comparable to those obtained using digital PCR and relative abundance based on a standard sequence library. We demonstrated that the qSeq protocol proposed here is advantageous for providing less-biased absolute copy numbers of each target DNA with NGS sequencing at one time. By this new experiment scheme in microbial ecology, microbial community compositions can be explored in more quantitative manner, thus expanding our knowledge of microbial ecosystems in natural environments.

Sulfate reduction is the predominant anaerobic microbial process of organic matter mineralization in marine sediments, with recent studies revealing that sulfate reduction not only occurs in sulfate-rich sediments, but even extends to deeper, methanogenic sediments at very low background concentrations of sulfate. Using samples retrieved off the Shimokita Peninsula, Japan, during the Integrated Ocean Drilling Program (IODP) Expedition 337, we measured potential sulfate reduction rates by slurry incubations with S-35-labeled sulfate in deep methanogenic sediments between 1276.75 and 2456.75 meters below the seafloor. Potential sulfate reduction rates were generally extremely low (mostly below 0.1 pmol cm(-3) d(-1)) but showed elevated values (up to 1.8 pmol cm-3 d-1) in a coal bearing interval (Unit III). A measured increase in hydrogenase activity in the coal-bearing horizons coincided with this local increase in potential sulfate reduction rates. This paired enzymatic response suggests that hydrogen is a potentially important electron donor for sulfate reduction in the deep coalbed biosphere. By contrast, no stimulation of sulfate reduction rates was observed in treatments where methane was added as an electron donor. In the deep coalbeds, small amounts of sulfate might be provided by a cryptic sulfur cycle. The isotopically very heavy pyrites (delta S-34 = +43 parts per thousand) found in this horizon is consistent with its formation via microbial sulfate reduction that has been continuously utilizing a small, increasingly S-34-enriched sulfate reservoir over geologic time scales. Although our results do not represent in-situ activity, and the sulfate reducers might only have persisted in a dormant, spore-like state, our findings show that organisms capable of sulfate reduction have survived in deep methanogenic sediments over more than 20 Ma. This highlights the ability of sulfate-reducers to persist over geological timespans even in sulfate-depleted environments. Our study moreover represents the deepest evidence of a potential for sulfate reduction in marine sediments to date.

During Integrated Ocean Drilling Program (IODP) Expedition 329, a deep-sea ferromanganese nodule and surrounding sediment were collected from the South Pacific Gyre, the most oligotrophic oceanic environment on earth. Using a combination of cryo-sectioning and fluorescence-based cell counting techniques, we determined that the microbial cell density at the very surface of the nodule was similar to 10(8) cells cm(-3), three orders of magnitude higher than that in the surrounding sediment. Analysis of bacterial and archaeal 16S rRNA gene sequences (similar to 1400 bp) indicated that the taxonomic composition of the nodule-associated community differed markedly from that of the sediment-associated community. Members of Marine Group I (MGI) Thaumarchaeota are potentially crucial for sustaining the high cell density because both ammonia and Cu were available on the nodule surface, making it suitable for ammonia-oxidizing chemolithoautotrophy mediated by copper enzymes. Combined cryo-sectioning and synchrotron analysis of the nodule surface revealed both hexagonal birnessite resembling delta-MnO2 and triclinic birnessite, minerals characteristic of biogenic oxide and its secondary product, respectively. Regardless of these possible biogenic features, only one gene sequence exhibited some similarity to previously identified manganese-oxidizing bacteria. On the other hand, MGI Thaumarchaeota were assumed as potential candidate of manganese oxidizers because they have multi-copper oxidase that is utilized by most known manganese oxidizers. Therefore, this archaeal group is considered to play a significant ecological role as a primary producer in biogeochemical elemental cycles in the ultra-oligotrophic abyssal plain. (C) 2016 Elsevier B.V. All rights reserved.

© 2016 Author(s). The Integrated Ocean Drilling Program (IODP) Expedition 337 was the first expedition dedicated to subseafloor microbiology that used riser-drilling technology with the drilling vessel Chikyu. The drilling Site C0020 is located in a forearc basin formed by the subduction of the Pacific Plate off the Shimokita Peninsula, Japan, at a water depth of 1180 m. Primary scientific objectives during Expedition 337 were to study the relationship between the deep microbial biosphere and a series of ~2 km deep subseafloor coalbeds and to explore the limits of life in the deepest horizons ever probed by scientific ocean drilling. To address these scientific objectives, we penetrated a 2.466 km deep sedimentary sequence with a series of lignite layers buried around 2 km below the seafloor. The cored sediments, as well as cuttings and logging data, showed a record of dynamically changing depositional environments in the former forearc basin off the Shimokita Peninsula during the late Oligocene and Miocene, ranging from warm-temperate coastal backswamps to a cool water continental shelf. The occurrence of small microbial populations and their methanogenic activity were confirmed down to the bottom of the hole by microbiological and biogeochemical analyses. The factors controlling the size and viability of ultra-deep microbial communities in those warm sedimentary habitats could be the increase in demand of energy and water expended on the enzymatic repair of biomolecules as a function of the burial depth. Expedition 337 provided a test ground for the use of riser-drilling technology to address geobiological and biogeochemical objectives and was therefore a crucial step toward the next phase of deep scientific ocean drilling.

Recovering high quality genomic DNA from environmental samples is a crucial primary step to understand the genetic, metabolic, and evolutionary characteristics of microbial communities through molecular ecological approaches. However, it is often challenging because of the difficulty of effective cell lysis without fragmenting the genomic DNA. This work aims to improve the previous SDS-based DNA extraction methods for high biomass seafloor samples, such as pelagic sediments and metal sulfide chimney, to obtain high quality and high molecular weight of the genomic DNA applicable for the subsequent molecular ecological analyses. In this regard, we standardized a modified SDS-based DNA extraction method (M-SDS), and its performance was then compared to those extracted by a recently developed hot-alkaline DNA extraction method (HA) and a commercial DNA extraction kit. Consequently, the M-SDS method resulted in higher DNA yield and cell lysis efficiency, lower DNA shearing, and higher diversity scores than other two methods, providing a comprehensive DNA assemblage of the microbial community on the seafloor depositional environment.

Subseafloor pelagic sediments with high concentrations of organic matter form habitats for diverse microorganisms. Here, we determined depth profiles of genes for SSU rRNA, mcrA, dsrA and amoA from just beneath the seafloor to 363.3 m below the seafloor (mbsf) using core samples obtained from the forearc basin off the Shimokita Peninsula. The molecular profiles were combined with data on lithostratigraphy, depositional age, sedimentation rate and pore-water chemistry. The SSU rRNA gene tag structure and diversity changed at around the sulfate-methane transition zone (SMTZ), whereas the profiles varied further with depth below the SMTZ, probably in connection with the variation in pore-water chemistry. The depth profiles of diversity and abundance of dsrA, a key gene for sulfate reduction, suggested the possible niche separations of sulfate-reducing populations, even below the SMTZ. The diversity and abundance patterns of mcrA, a key gene for methanogenesis/anaerobic methanotrophy, suggested a stratified distribution and separation of anaerobic methanotrophy and hydrogenotrophic or methylotrophic methanogensis below the SMTZ. This study provides novel insights into the relationships between the composition and function of microbial communities and the chemical environment in the nutrient-rich continental margin subseafloor sediments, which may result in niche separation and variability in subseafloor microbial populations.

Heat transport properties are significant parameters that affect geothermal processes in deep sedimentary basins. We measured the thermal properties of deep sediment core samples to a depth of approximately 2500 m below sea floor (mbsf), whereas most previous studies have only presented data &lt;800 mbsf for marine sediments, taken from the basin off Shimokita, Japan during Integrated Ocean Drilling Program (IODP) Expedition 337. Thermal conductivity and thermal diffusivity increased with depth, and heat capacity decreased with depth, although the data were highly scattered at greater depths. The increase in thermal conductivity is explained primarily by the porosity reduction of sediment resulting from consolidation during sedimentation. The high variations in thermal conductivity at the similar depth are reflected by various lithological rocks formed at the same core section. Coal exhibits the lowest thermal conductivity of 0.4 Wm(-1) K-1, and calcite-cemented sandstoneisiltstone exhibits the highest conductivity of approximately 3 Wm(-1) K-1. The other thermal properties are also influenced by the porosity and lithological contrast. The correlation between thermal conductivity and porosity is explained by the mixed-law model of the geometric mean. The one-dimensional temperature-depth profile at site C0020 estimated from measured thermal conductivity reveals a regression of the thermal gradient with depth, and the surface heat flow of 30 mWm(-2), which is consistent with the heat flow data near this site, was evaluated. The obtained results suggest that the linear geothermal gradient estimated from the average thermal conductivity value at shallow sediment materials could overestimate the temperature at greater depths and underestimate the heat flow. (C) 2016 Elsevier Ltd. All rights reserved.

There are very few natural environments where life is absent in the surface biosphere on Earth. Over the past decade, explorations through scientific ocean drilling revealed the presence of vast microbial ecosystems in marine subsurface environments, even down to ~2.5 km deep below the ocean floor [1]. Molecular ecological studies demonstrated that microbial communities in subseafloor sediments consist mostly of unknown species that have never isolated from the Earth’s surface biosphere [2]. Activity of subseafloor microbial life is thought to be extraordinary slow due to the limited supply of nutrient and energy substrates from the seawater and/or oceanic crust [3,4]. To date, metabolic functions and ecological roles of deep subseafloor life and the biosphere are still largely unknown [5].

Previous studies of microbial communities in deep-sea hydrothermal ferric deposits have demonstrated that members of Zetaproteobacteria play significant ecological roles in biogeochemical iron-cycling. However, the ecophysiological characteristics and interaction between other microbial members in the habitat still remain largely unknown. In this study, we investigated microbial communities in a core sample obtained from shallow hydrothermal iron-oxyhydroxide deposits at Nagahama Bay of Satsuma Iwo-Jima, Japan. Scanning electron microscopic observation showed numerous helical stalk structures, suggesting the occurrence of iron-oxidizing bacteria. Analysis of 16S rRNA gene sequences indicated the co-occurrence of iron-oxidizing Zetaproteobacteria and iron-reducing bacteria such as the genera Deferrisoma and Desulfobulbus with strong correlations on the sequence abundance. CARD FISH indicated that the numbers of Zetaproteobacteria were not always consistent to the frequency of stalk structures. In the stalk-abundant layers with relatively small numbers of Zetaproteobacteria cells, accumulation of polyphosphate was observed inside Zetaproteobacteria cells, whereas no polyphosphate grains were observed in the topmost layers with fewer stalks and abundant Zetaproteobacteria cells. These results suggest that Zetaproteobacteria store intracellular polyphosphates during active iron oxidation that contributes to the mineralogical growth and biogeochemical iron cycling.

We report detailed lithological and chemical characteristics of deep-sea sediments, including rare-earth elements and yttrium-rich mud (REY-rich mud), in the Japanese Exclusive Economic Zone (EEZ) around Minamitorishima Island. Three research cruises obtained fourteen sediment cores collected by piston coring. Based on the visual descriptions and geochemical analysis of the sediment cores, we confirm the presence of REY-rich mud containing more than 400 ppm total REY (Sigma REY) in the southern and northwestern areas of the Minamitorishima EEZ. The REY-rich mud layers are characterized by abundant grains of phillipsite, biogenic calcium phosphate, and manganese oxides, and are widely distributed in relatively shallow depths beneath the seafloor. In contrast, relatively thick, non-REY-rich mud lies near the seafloor in the northern areas of the EEZ. In the three cores from the southern part of the EEZ, we also confirm the presence of highly/extremely REY-rich mud layers. Further accumulation of geochemical data from the sediments will be required to constrain the extent of the highly/extremely REY-rich mud layers.

We have discovered deep-sea mud that is extremely enriched in rare-earth elements and yttrium (together called REY) in the Japanese Exclusive Economic Zone around Minamitorishima Island, in the western North Pacific Ocean. The maximum total REY concentration reaches approximately 7000 ppm, which is much higher than that reported for conventional REY deposits on land and other known potential REY resources in the ocean. The extremely REY-rich mud is characterized by abundant phillipsite and biogenic calcium phosphate. In addition, the stratigraphic layer with the highest REY concentration occurs just similar to 3 m beneath the seafloor. The shallow burial of these strata together with the high REY content, especially those of heavy rare-earth elements, suggest that the newly discovered extremely REY-rich mud may be a promising REY resource.

Over the past few decades, the subseafloor biosphere has been explored by scientific ocean drilling to depths of about 2.5 km below the seafloor. Although organic-rich anaerobic sedimentary habitats in the ocean margins harbor large numbers of microbial cells, microbial populations in ultraoligotrophic aerobic sedimentary habitats in the open ocean gyres are several orders of magnitude less abundant. Despite advances in cultivation-independent molecular ecological techniques, exploring the low-biomass environment remains technologically challenging, especially in the deep subseafloor biosphere. Reviewing the historical background of deep-biosphere analytical methods, the importance of obtaining clean samples and tracing contamination, as well as methods for detecting microbial life, technological aspects of molecular microbiology, and detecting subseafloor metabolic activity will be discussed.

The proper pre-drilling preparation, on-site acquisition and post-drilling preservation of high-quality subsurface samples are crucial to ensure significant progress in the scientifically and societally important areas of subsurface biosphere and paleoclimate research. Two of the four research themes of IODP and ICDP and one of the four research areas of the Deep Carbon Observatory (DCO) focus on the subsurface biosphere. Increasing understanding of paleoclimate is a central goal of IODP and incorporated within the scope of the IMPRESS program, the successor of the IMAGES program. Therefore, the goal of our IODP-ICDP-DCO-JDESC- MagellanPlus-sponsored workshop was to help advance deep biosphere and paleoclimate research by identifying needed improvements in scientific drilling planning and available technology, sample collection and initial analysis, and long-term storage of subsurface samples and data. Success in these areas will (a) avoid biological and other contamination during drilling, sampling, storage and shipboard/shore-based experiments (b) build a repository and database of high-quality subsurface samples for microbiological and paleoclimate research available for the scientific community world-wide over the next decades and (c) standardize, as much as possible, microbiological and paleoclimate drilling, sampling and storage workflows to allow results and data to be comparable across both space and time. A result of this workshop is the development and suggested implementation of new advanced methods and technologies to collect high-quality samples and data for the deep biosphere and paleoclimate scientific communities to optimize expected substantial progress in these fields. The members of this workshop will enhance communication within the scientific drilling community by crafting a handbook focused on pre-drilling, drilling and post-drilling operations.

The study of environmental samples requires a preservation system that stabilizes the sample structure, including cells and biomolecules. To address this fundamental issue, we tested the cell alive system (CAS)-freezing technique for subseafloor sediment core samples. In the CAS-freezing technique, an alternating magnetic field is applied during the freezing process to produce vibration of water molecules and achieve a stable, super-cooled liquid phase. Upon further cooling, the temperature decreases further, achieving a uniform freezing of sample with minimal ice crystal formation. In this study, samples were preserved using the CAS and conventional freezing techniques at 4, -20, -80 and -196 (liquid nitrogen) degrees C. After 6 months of storage, microbial cell counts by conventional freezing significantly decreased (down to 10.7% of initial), whereas that by CAS-freezing resulted in minimal. When Escherichia coli cells were tested under the same freezing conditions and storage for 2.5 months, CAS-frozen E.coli cells showed higher viability than the other conditions. In addition, an alternating magnetic field does not impact on the direction of remanent magnetization in sediment core samples, although slight partial demagnetization in intensity due to freezing was observed. Consequently, our data indicate that the CAS technique is highly useful for the preservation of environmental samples.

Spore formation is considered to be one of the microbial strategies for long-term survival in subseafloor sedimentary habitats. However, our knowledge of the genetic and physiological characteristics of subseafloor microbes is limited. Here, we studied the distribution and frequency of genes that are related to endospore formation in 10 subseafloor sedimentary metagenomes from Site C9001 off Japan and Site 1229 off Peru. None or very low frequencies of endospore-specific genes (e.g. dpaA, dpaB, sspA, spo0A, spoIIGA, spoIIM, spoIIIAB, spoIVA, spoIVB, yabP, yunB, spoVM) were observed in the subseafloor metagenomes. Based on the number of universally conserved single copy genes, the frequency ratio of putative endospore-formers was estimated to be &lt;10%, which is consistent with the frequency of Clostridia-derived genomes (2-4%) but is lower than previous estimates based on the concentration of dipicolinic acid. Conceivable explanations for this discrepancy are as follows: the efficiency of lysis and DNA extraction of subseafloor endospore cells may have been lower than those of vegetative cells, conversion factor of dipicolinic acid content per cell may differ, and/or sporulation-related genes and other functional strategies for long-term survival in the deep subseafloor biosphere are evolutionarily distinct from known spore-forming gene repertoires.

Markedly diverse sequences of the adenosine-5'-phosphosulfate reductase alpha subunit gene (aprA), which encodes a key enzyme in microbial sulfate reduction and sulfur oxidation, were detected in subseafloor sediments on the northwestern Pacific off Japan. The aprA gene sequences were grouped into 135 operational taxonomic units (90% sequence identity), including genes related to putative sulfur-oxidizing bacteria predominantly detected in sulfate-depleted deep sediments. Our results suggest that microbial ecosystems in the subseafloor biosphere have phylogenetically diverse genetic potentials to mediate cryptic sulfur cycles in sediments, even where sulfate is rarely present.

© 2015 Elsevier B.V. To investigate fluid regimes in the Nankai accretionary wedge, we estimated the temperatures of lithium (Li) reservoirs from the Li isotope ratio (7Li/6Li) in fluids recovered from Kumano mud volcano No. 5 (KMV#5) in the Nankai forearc basin. The 7Li/6Li ratios in the KMV#5 fluids are among the lightest reported from submarine mud volcanoes. The estimated Li reservoir temperatures (310 °C at maximum) were significantly higher than the smectite-to-illite conversion temperature (60-150 °C), whereas the temperature of fluid from the deeper Nankai décollement was estimated to be 150 °C at maximum. Data obtained from previous conductivity surveys across the trench suggest that there are two fluid reservoirs associated with distinct dehydration processes in the Nankai subduction zone. From these results, we inferred that the discontinuous mud diapirism in the Kumano forearc basin results from the periodic injection of deep-seated fluid accumulated in the corner of the serpentinized forearc mantle wedge that travels upward via a thrust fault. The intermittent upwelling of deep-seated fluid from the Nankai accretionary wedge due to periodic breaches of a low-permeability barrier above the fluid reservoir may be associated with seismogenic fault activity.

Increasing anthropogenic CO₂ in the atmosphere causes global warming and subsequent environmental changes, which may lead to an increase in natural disasters jeopardizing human society. Prompt technological development for CO₂ capture and sequestration is required in the international community. In this study, we performed CO₂ emission and shallow-type methane hydrate decomposition experiments at the Joetsu Knoll, offshore Joetsu, Niigata, Japan, as pilot studies to test feasibility of CO₂ sequestration and methane recovery using methane-CO₂ replacement in shallow-type methane hydrates. An isobaric cylinder pump and probe with a built-in heater ("Heat sonde") were developed to inject CO₂ in deep-sea, high-pressure conditions. Before injecting CO₂ into a methane hydrate located in deep-sea sediments, we attempted CO₂ emission directly into deep-seafloor. In the experiment, liquid CO₂ was emitted at the head of Heat sonde, however, the isobaric cylinder pump became clogged during operation. The result reveals that precipitates of CO₂ hydrate, which are generated during mixing of inflow seawater and outflow liquid CO₂, blocked flow lines of the isobaric cylinder pump and Heat sonde. This suggests that our developed instruments must be improved for future work. We also observed the collapse of an exposed methane hydrate layer at the seafloor upon contact with the Heat sonde in our experiment.

Barnacles of the genus Neoverruca are abundant near deep-sea hydrothermal vents of the northwestern Pacific Ocean, and are useful for understanding processes of population formation and maintenance of deep-sea vent faunas. Using next-generation sequencing, we isolated 12 polymorphic microsatellite loci from Neoverruca sp., collected in the Okinawa Trough. These microsatellite loci revealed 2-19 alleles per locus. The expected and observed heterozygosities ranged from 0.286 to 1.000 and 0.349 to 0.935, respectively. Cross-species amplification showed that 9 of the 12 loci were successfully amplified for Neoverruca brachylepadoformis in the Mariana Trough. A pairwise FST value calculated using nine loci showed significant genetic differentiation between the two species. Consequently, the microsatellite markers we developed will be useful for further population genetic studies to elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.

Barnacles of the genus Neoverruca are abundant near deep-sea hydrothermal vents of the northwestern Pacific Ocean, and are useful for understanding processes of population formation and maintenance of deep-sea vent faunas. Using next-generation sequencing, we isolated 12 polymorphic microsatellite loci from Neoverruca sp., collected in the Okinawa Trough. These microsatellite loci revealed 2-19 alleles per locus. The expected and observed heterozygosities ranged from 0.286 to 1.000 and 0.349 to 0.935, respectively. Cross-species amplification showed that 9 of the 12 loci were successfully amplified for Neoverruca brachylepadoformis in the Mariana Trough. A pairwise F-ST value calculated using nine loci showed significant genetic differentiation between the two species. Consequently, the microsatellite markers we developed will be useful for further population genetic studies to elucidate genetic diversity, differentiation, classification, and evolutionary processes in the genus Neoverruca.

Marine sediments are composed of various mineral species and are characterized by micro-scale grain fabric. The arrangements and interactions of component particles place critical constraints on the physical, chemical, and biological processes that occur in subseafloor environments. However, the observation of nearly intact microstructures is difficult, especially in soft, muddy sediments because of their high water content and the presence of organic molecules. In this study, a modified version of the resin-embedding method generally used for biological samples was applied to marine sediment sample preparation. The new method was compared with the conventional t-butyl alcohol freeze-drying method using microfocus X-ray computed tomography (mu XCT) and scanning electron microscopy (SEM). The mu XCT and SEM results showed that all t-butyl alcohol freeze-dried sediment samples contained microstructural disturbances (e.g., cracks). In contrast, no cracks were observed in the samples prepared using the new resin-embedding method, and the microstructural arrangement of the sediment particles were clearly visible. In addition, the porosity visible from SEM images of the resin-embedded samples was similar to that measured using the moisture and density method, providing additional evidence that the microstructures of the resin-embedded samples were well preserved. The resin-embedding method allowed observation of the limited contact of the fine particles in clayey sediments, the clay microaggregates throughout the continental margin to pelagic sediments, and the presence of organic materials in environmental sediments. This modified biological resin-embedding method is suitable for the detailed observation and characterization of fine-grained marine sediment microstructure.

The linkage of microbial phylogenetic and metabolic analyses by combining ion imaging analysis with nano-scale secondary ion mass spectrometry (NanoSIMS) has become a powerful means of exploring the metabolic functions of environmental microorganisms. Phylogenetic identification using NanoSIMS typically involves probing by horseradish peroxidase-mediated deposition of halogenated fluorescent tyramides, which permits highly sensitive detection of specific microbial cells. However, the methods require permeabilization of target microbial cells and inactivation of endogenous peroxidase activity, and the use of halogens as the target atom is limited because of heavy background signals due to the presence of halogenated minerals in soil and sediment samples. Here, we present "Gold-ISH," a non-halogen phylogenetic probing method in which oligonucleotide probes are directly labeled with Undecagold, an ultra-small gold nanoparticle. Undecagold-labeled probes were generated using a thiol-maleimide chemical coupling reaction and they were purified by polyacrylamide gel electrophoresis. The method was optimized with a mixture of axenic (13)C-labeled Escherichia coli and Methanococcus maripaludis cells and applied to investigate sulfate-reducing bacteria in an anaerobic sludge sample. Clear gold-derived target signals were detected in microbial cells using NanoSIMS ion imaging. It was concluded that Gold-ISH can be a useful approach for metabolic studies of naturally occurring microbial ecosystems using NanoSIMS.

Vesicomyidae clams harbor sulfide-oxidizing endosymbionts and are typical members of cold seep communities where active venting of fluids and gases takes place. We investigated the central biogeochemical processes that supported a vesicomyid clam colony as part of a locally restricted seep community in the Japan Trench at 5346m water depth, one of the deepest seep settings studied to date. An integrated approach of biogeochemical and molecular ecological techniques was used combining in situ and ex situ measurements. In sediment of the clam colony, low sulfate reduction rates (maximum 128nmolmL(-1)day(-1)) were coupled to the anaerobic oxidation of methane. They were observed over a depth range of 15cm, caused by active transport of sulfate due to bioturbation of the vesicomyid clams. A distinct separation between the seep and the surrounding seafloor was shown by steep horizontal geochemical gradients and pronounced microbial community shifts. The sediment below the clam colony was dominated by anaerobic methanotrophic archaea (ANME-2c) and sulfate-reducing Desulfobulbaceae (SEEP-SRB-3, SEEP-SRB-4). Aerobic methanotrophic bacteria were not detected in the sediment, and the oxidation of sulfide seemed to be carried out chemolithoautotrophically by Sulfurovum species. Thus, major redox processes were mediated by distinct subgroups of seep-related microorganisms that might have been selected by this specific abyssal seep environment. Fluid flow and microbial activity were low but sufficient to support the clam community over decades and to build up high biomasses. Hence, the clams and their microbial communities adapted successfully to a low-energy regime and may represent widespread chemosynthetic communities in the Japan Trench. In this regard, they contributed to the restricted deep-sea trench biodiversity as well as to the organic carbon availability, also for non-seep organisms, in such oligotrophic benthic environment of the dark deep ocean.

We studied the relationship between viral particle and microbial cell abundances in marine subsurface sediments from three geographically distinct locations in the continental margins (offshore of the Shimokita Peninsula of Japan, the Cascadia Margin off Oregon, and the Gulf of Mexico) and found depth variations in viral abundances among these sites. Viruses in sediments obtained offshore of the Shimokita and in the Cascadia Margin generally decreased with increasing depth, whereas those in sediments from the Gulf of Mexico were relatively constant throughout the investigated depths. In addition, the abundance ratios of viruses to microbial cells notably varied among the sites, ranging between 10(-3) and 10(1). The subseafloor viral abundance offshore of the Shimokita showed a positive relationship with the microbial cell abundance and the sediment porosity. In contrast, no statistically significant relationship was observed in the Cascadia Margin and the Gulf of Mexico sites, presumably due to the long-term preservation of viruses from enzymatic degradation within the low-porosity sediments. Our observations indicate that viral abundance in the marine subsurface sedimentary environment is regulated not only by in situ production but also by the balance of preservation and decay, which is associated with the regional sedimentation processes in the geological settings.

Marine subsurface sediments on the Pacific margin harbor diverse microbial communities even at depths of several hundreds meters below the seafloor (mbsf) or more. Previous PCR-based molecular analysis showed the presence of diverse reductive dehalogenase gene (rdhA) homologs in marine subsurface sediment, suggesting that anaerobic respiration of organohalides is one of the possible energy-yielding pathways in the organic-rich sedimentary habitat. However, primer-independent molecular characterization of rdhA has remained to be demonstrated. Here, we studied the diversity and frequency of rdhA homologs by metagenomic analysis of five different depth horizons (0.8, 5.1, 18.6, 48.5, and 107.0 mbsf) at Site C9001 off the Shimokita Peninsula of Japan. From all metagenomic pools, remarkably diverse rdhA-homologous sequences, some of which are affiliated with novel clusters, were observed with high frequency. As a comparison, we also examined frequency of dissimilatory sulfite reductase genes (dsrAB), key functional genes for microbial sulfate reduction. The dsrAB were also widely observed in the metagenomic pools whereas the frequency of dsrAB genes was generally smaller than that of rdhA-homologous genes. The phylogenetic composition of rdhA-homologous genes was similar among the five depth horizons. Our metagenomic data revealed that subseafloor rdhA homologs are more diverse than previously identified from PCR-based molecular studies. Spatial distribution of similar rdhA homologs across wide depositional ages indicates that the heterotrophic metabolic processes mediated by the genes can be ecologically important functioning in the organic-rich subseafloor sedimentary biosphere.

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98 degrees C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., similar to 1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.

A novel, anaerobic filamentous bacterium, strain MO-CFX1(T), was isolated from a methanogenic community, which was originally established from subseafloor sediments collected from off the Shimokita Peninsula, Japan. Cells were non-spore-forming, non-motile, Gram-stain-negative and filamentous. The filaments were longer than 10 mu m and 130-150 nm in width. Growth of the strain was observed at 10-37 degrees C (optimum 25-30 degrees C), at pH 5.5-8.5 (optimum pH 7.0) and in 0-50 g NaCl I-1 (optimum 15 g NaCl I-1). The strain was able to grow with a number of carbohydrates in the presence of yeast extract. The major cellular fatty acids were monounsaturated C-18:1 omega 9, C-16:1 omega 7 and saturated C-18:0 and C-16:0. The intact polar lipids of the strain were dominated by diacylglyceride and sphingolipid core lipid structures with monoglycosidic, mixed phosphomonoglycosidic and fatty-acid-modified monoglycosidic polar head groups. The G+C content of the genomic DNA was 52.4 mol%. Based on the comparative 165 rRNA gene sequence analysis, strain MO-CFX1(T) was affiliated with the class Anaerolineae within the phylum Chloroflexi and was most closely related to Leptolinea tardivitalis YMTK-2(T) (sequence identity of 91.0%). Based on phenotypic and genetic properties of the novel isolate, we propose a novel species representing a new genus Pelolinea submarina gen. nov., sp. nov., for strain MO-CFX1(T) (=JCM 17238(T),=KCTC 5975(T)). This is the first formal description, to our knowledge, of an isolate of the phylum Chloroflexi from the deep-sea sedimentary environment.

Two active hydrothermal fields, Jade and Hakurei fields have been discovered within the Izena Hole, a rectangular 6 km x 3 km shape depression located in the middle Okinawa Trough back arc basin. In both fields, intense hydrothermal activity is represented by venting of high-temperature fluid (&gt;300 degrees C) and occurrence of sulfide/sulfate ore deposits. We collected hydrothermal fluids during dive expeditions of ROV Hyper Dolphin conducted in 2003, 2010 and 2011, in order to analyze both elemental and gas species. The geochemistry of high temperature hydrothermal fluids collected from the Jade and Hakurei fields is very similar to each other with exceptions in minor gas composition. Little temporal variation was observed in geochemistry of the high-temperature hydrothermal fluid of the Jade field over two decades, since a previous study carried out in 1989. These results suggest that these fluids are derived from a common fluid reservoir where fluid chemistry is basically controlled by fluid-mineral equilibria and gas species are dominantly contributed from the same magma. Venting of low temperature fluid (about 104 degrees C) was discovered in the distal part of the Jade field, which was named as the Biwako vent. Chemical composition of the Biwako vent fluid was distinctive from that of the high temperature fluid in the proximal part of the Jade field, and could not be explained by simple dilution or cooling. This intra-field chemical diversity could be caused by phase separation and segregation during fluid upwelling, based on relationships in concentrations of Cl and major cations. On the other hand, the chemical diversity recognized in minor gas composition between the Jade and Hakurei fields is in accordance with results from previous plume survey. Difference in concentrations of minor gases such as H-2, is attributed to contribution from thermal degradation of organic matter in the sediment, during fluid,upwelling.

Retrieving core samples without releasing the in situ hydrostatic pressure during core recovery is one of the many technical challenges in scientific drilling. We report here a newly developed hybrid pressurecoring system for the use on the drilling vessel Chikyu and its successful use during expeditions 906 and 802 in the Nankai Trough of Japan. The system is gas-tight and hence enables researchers to study in situ geophysical and geochemical characteristics of sediments containing gaseous components, such as methane hydrates that cannot be reliably recovered with nonpressure coring systems. In addition, pressure coring is a powerful tool, not only for scientific but also for hydrocarbon resources research. © Author(s) 2014.

The scientific drilling of marine subsurface environments has offered unique opportunities to explore how life persists and evolves in the Earth's interior. There are very few natural environments on the surface of the earth where life is absent however, there are predicted constraints and limits to life associated with some deep subseafloor environments. The deep subseafloor biosphere is vast in scale and differs both physically and chemically from the Earth's surface biosphere. The processes that mediate genetic and metabolic evolution of life in the subseafloor may be very different from those well-studied systems on the Earth's surface. Research on the deep subseafloor biosphere is anticipated to have the potential to impact major scientific and societal questions such as energy creation, climate change, and the process of microbial evolution on Earth over geologic time.

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 degrees 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 degrees 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 degrees C. During the reactor experiment, the acetate and methanol concentration in the fluids increased while the delta C-13(acetate), H-2 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.

Development of an improved technique for separating microbial cells from marine sediments and standardization of a high-throughput and discriminative cell enumeration method were conducted. We separated microbial cells from various types of marine sediment and then recovered the cells using multilayer density gradients of sodium polytungstate and/or Nycodenz, resulting in a notably higher percent recovery of cells than previous methods. The efficiency of cell extraction generally depends on the sediment depth; using the new technique we developed, more than 80% of the total cells were recovered from shallow sediment samples (down to 100 meters in depth), whereas approximate to 50% of cells were recovered from deep samples (100-365m in depth). The separated cells could be rapidly enumerated using flow cytometry (FCM). The data were in good agreement with those obtained from manual microscopic direct counts over the range 10(4)-10(8)cellscm(-3). We also demonstrated that sedimentary microbial cells can be efficiently collected using a cell sorter. The combined use of our new cell separation and FCM/cell sorting techniques facilitates high-throughput and precise enumeration of microbial cells in sediments and is amenable to various types of single-cell analyses, thereby enhancing our understanding of microbial life in the largely uncharacterized deep subseafloor biosphere.

To obtain obtain a better understanding of metabolically active microbial communities, we tested a molecular ecological approach using poly(A) tailing of environmental 16S rRNA, followed by full-length complementary DNA (cDNA) synthesis and sequencing to eliminate potential biases caused by mismatching of polymerase chain reaction (PCR) primer sequences. The RNA pool tested was extracted from marine sediments of the Yonaguni Knoll IV hydrothermal field in the southern Okinawa Trough. The sequences obtained using the poly(A) tailing method were compared statistically and phylogenetically with those obtained using conventional reverse transcription-PCR (RT-PCR) with published domain-specific primers. Both methods indicated that Deltaproteobacteria are predominant in sediment (&gt;85% of the total sequence read). The poly(A) tailing method indicated that Desulfobacterales were the predominant Deltaproteobacteria, while most of the sequences in libraries constructed using RT-PCR were derived from Desulfuromonadales. This discrepancy may have been due to low coverage of Desulfobacterales by the primers used. A comparison of library diversity indices indicated that the poly(A) tailing method retrieves more phylogenetically diverse sequences from the environment. The four archaeal 16S rRNA sequences that were obtained using the poly(A) tailing method formed deeply branching lineages that were related to Candidatus "Parvarchaeum" and the ancient archaeal group. These results clearly demonstrate that poly(A) tailing followed by cDNA sequencing is a powerful and less biased molecular ecological approach for the study of metabolically active microbial communities.

A facultatively anaerobic organoheterotroph, designated JAM-BA0302(T), was isolated from a deep subseafloor sediment at a depth of 247.1 m below the seafloor off the Shimokita Peninsula of Japan in the north-western Pacific Ocean (Site 09001, water depth 1180 m). Cells of strain JAM-BA0302(T) showed gliding motility and were thin, long rods with peritrichous fimbriae-like structures. Growth occurred at 4-37 degrees C (optimum 30 degrees C; doubling time 8 h), at pH 5.4-8.3 (optimum pH 7.5) and with 5-60 g NaCl l(-1) (optimum 20-25 g l(-1)). The isolate utilized proteinaceous substrates such as yeast extract, tryptone, casein and Casamino acids with O-2 respiration or fermentation. Strain JAM-BA0302(T) was a piezotolerant bacterium that could grow at pressures as high as 25 MPa under aerobic conditions and 10 MPa under anaerobic conditions. The G+C content of the genomic DNA was 43.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JAM-BA0302(T) was most closely related to yet-undescribed strains recently isolated from various marine sedimentary environments (&gt;99.6% 16S rRNA gene sequence similarity) and was moderately related to Sunxiuqinia elliptica DOHS-4(T), isolated from a sea cucumber farm sediment (95.5% 16S rRNA gene sequence similarity) within the Bacteroidetes. The phylogenetic analysis suggested that the isolate should belong to the genus Sunxiuqinia. However, low DNA-DNA relatedness (&lt;11%) and many physiological and molecular properties differentiated the isolate from those previously described. We propose here a novel species of the genus Sunxiuqinia, with the name Sunxiuqinia faeciviva sp. nov. The type strain is JAM-BA0302(T) (=JCM 15547(T) =NCIMB 14481(T)).

Halogenated organic matter buried in marine subsurface sediment may serve as a source of electron acceptors for anaerobic respiration of subseafloor microbes. Detection of a diverse array of reductive dehalogenase-homologous (rdhA) genes suggests that subseafloor organohalide-respiring microbial communities may play significant ecological roles in the biogeochemical carbon and halogen cycle in the subseafloor biosphere. We report here the spatial distribution of dehalogenation activity in the Nankai Trough plate-subduction zone of the northwest Pacific off the Kii Peninsula of Japan. Incubation experiments with slurries of sediment collected at various depths and locations showed that degradation of several organohalides tested only occurred in the shallow sedimentary basin, down to 4.7 metres below the seafloor, despite detection of rdhA in the deeper sediments. We studied the phylogenetic diversity of the metabolically active microbes in positive enrichment cultures by extracting RNA, and found that Desulfuromonadales bacteria predominate. In addition, for the isolation of genes involved in the dehalogenation reaction, we performedasubstrate-induced gene expression screeningonDNA extracted from the enrichment cultures. Diverse DNA fragments were obtained and some of them showed best BLAST hit to known organohalide respirers such as Dehalococcoides, whereas no functionally known dehalogenation-related genes such as rdhA were found, indicating the need to improve the molecular approach to assess functional genes for organohalide respiration. © 2013 The Author(s) Published by the Royal Society. All rights reserved.

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 (13-30cm 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. The ISME Journal (2013) 7, 555-567; doi:10.1038/ismej.2012.124; published online 25 October 2012

Surface seawater in the South Pacific Gyre (SPG) is one of the cleanest oceanic environments on earth, and the photosynthetic primary production is extremely low. Despite the ecological significance of the largest aquatic desert on our planet, microbial community composition in the ultra-oligotrophic seawater remain largely unknown. In this study, we collected surface seawater along a southern transect of the SPG during the Integrated Ocean Drilling Program (IODP) Expedition 329. Samples from four distinct sites (Sites U1368, U1369, U1370 and U1371) were examined, representing similar to 5400 kilometers of transect line from the gyre heart to the edge area. Real-time PCR analysis showed 16S rRNA gene abundance in the gyre seawater, ranging from 5.96x10(5) to 2.55x10(6) copies ml(-1) for Bacteria and 1.17x10(3) to 1.90x10(4) copies ml(-1) for Archaea. The results obtained by statistic analyses of 16S rRNA gene clone libraries revealed the community composition in the southern SPG area: diversity richness estimators in the gyre center (Sites U1368 & U1369) are generally lower than those at sites in the gyre edge (Sites U1370 & U1371) and their community structures are clearly distinguishable. Phylogenetic analysis showed the predominance of Proteobacteria (especially Alphaproteobacteria) and Cyanobacteria in bacterial 16S rRNA gene clone libraries, whereas phylotypes of Betaproteobacteria were only detected in the central gyre. Archaeal 16S rRNA genes in the clone libraries were predominated by the sequences of Marine Group II within the Euryarchaeota, and the Crenarchaeota sequences were rarely detected, which is consistent with the real-time PCR data (only 9.9 to 22.1 copies ml(-1)). We also performed cultivation of heterotrophic microbes onboard, resulting in 18.9% of phylogenetically distinct bacterial isolates at least at the species level. Our results suggest that the distribution and diversity of microbial communities in the SPG surface seawater are closely related to the ultra-oligotrophic oceanographic features in the Pacific Ocean.

© 2013 JFES. We conducted coring, well logging and formation fluid sampling during the Integrated Ocean Drilling Program (IODP) Expedition 337 &quot;Deep coalbed biosphere off Shimokita&quot; on September 2012 in off-shore Shimokita, Japan. The purpose of this experiment is basic scientific research for deep biosphere and carbon cycle system in the coal bed methane. Wireline logs, cores, cuttings, and mud gas were taken for scientific study. Measurement properties of the well logging were natural gamma-ray, resistivity, density, neutron porosity, sonic velocity, borehole resistivity image, NMR log, pressure, temperature, permeability, and zero-offset VSP. Our strategy for formation fluid sampling was follows: 1) Geological and lithological interpretation with standard logs, 2) accuate identification of target layers with borehole resistivity image, 3) screening of target points with pearmeavility estimation by NMR log, 4) final screening by permeability with MDT (modular dynamic tester) pretest, 5) fluid sampling with MDT. We prepared MDT with Quicksilver probe and in-situ fluid analyzer to take low contamination fluid, special coating single-phase sampling bottles to prevent chemical interaction and to keep in-situ fluid pressure, a more powerful pump module for low permeability, stronger wireline cable to prevent to stack heavy tool stack. We obtained six 250ml full sample bottles from 2456-3646m with water depth 1180m successfully. Our sampling targets were sand layers nearby coal beds. It was very challenging to obtain fluid sampling under the conditions of low permeability formations, open hole, water base mud, and limited operation time. To complete our mission, we used Schlumberger&#039;s MDT, quicksilver probe, DURSAN coating shingle phase bottles, and IFA (In-site Fluid Analyzer) to maximize operation efficiency and to minimize contamination. Sampled formation fluid was shared with scientists, and advanced analysis is being carried out in their shore-base laboratories.

Microbial activity in marine sediment plays an important biogeochemical role in cycling of carbon and other elements. Acetate is a key intermediate of various microbial metabolic pathways. In this study, we measured concentrations and stable carbon isotopic compositions (delta C-13) of acetate in pore water of two core samples stored at near in-situ temperatures (4 degrees C) within 3 hours and at room temperatures (25 degrees C) for 19 hours after sample recovery. Acetate concentrations at 4 degrees C were less than 3 mu M throughout the sediment column, whereas they increased up to 13 mu M in the samples stored at 25 degrees C and corresponding delta C-13 values of acetate were enriched up to 8.7 parts per thousand relative to those of total organic carbon. Our results indicate that acetate-mediated microbial activity is rapidly changed with temperature increase and possible air contamination, and also suggest that sample processing at near in-situ temperatures in the short-term is required for accurate porewater geochemical analysis.

Integrated Ocean Drilling Program (IODP) Expedition 329 made major strides toward fulfilling its objectives. Shipboard studies documented (1) fundamental aspects of habitability and life in this very low activity subseafloor sedimentary ecosystem and (2) first-order patterns of habitability within the igneous basement. A broad range of postexpedition studies will complete the expedition objectives. Throughout the South Pacific Gyre (SPG Sites U1365- U1370), dissolved oxygen and nitrate are present throughout the entire sediment sequence, and sedimentary microbial cell counts are lower than at all previously drilled IODP/ Ocean Drilling Program (ODP)/Deep Sea Drilling Program (DSDP) sites. In contrast, at Site U1371 in the upwelling zone just south of the gyre, detectable oxygen and nitrate are limited to the top and bottom of the sediment column, manganese reduction is a prominent electron-accepting process, and cell concentrations are higher than at the same depths in the SPG sites throughout the sediment column. Geographic variation in subseafloor profiles of dissolved and solid-phase chemicals are consistent with the magnitude of organic-fueled subseafloor respiration declining from outside the gyre to the gyre center. Chemical profiles in the sedimentary pore water and secondary mineral distributions in the basaltic basement indicate that basement alteration continues on the timescale of formation fluid replacement, even at the sites with the oldest basement (84-120 Ma at Sites U1365 and U1366).

This study focused on biogeochemical processes and microbial activity in sediments of a natural deep-sea CO2 seepage area (Yonaguni Knoll IV hydrothermal system, Japan). The aim was to assess the influence of the geochemical conditions occurring in highly acidic and CO2 saturated sediments on sulfate reduction (SR) and anaerobic methane oxidation (AOM). Porewater chemistry was investigated from retrieved sediment cores and in situ by microsensor profiling. The sites sampled around a sediment-hosted hydrothermal CO2 vent were very heterogeneous in porewater chemistry, indicating a complex leakage pattern. Near the vents, droplets of liquid CO2 were observed emanating from the sediments, and the pH reached approximately 4.5 in a sediment depth &gt 6 cm, as determined in situ by microsensors. Methane and sulfate co-occurred in most sediment samples from the vicinity of the vents down to a depth of 3 m. However, SR and AOM were restricted to the upper 7-15 cm below seafloor, although neither temperature, low pH, nor the availability of methane and sulfate could be limiting microbial activity. We argue that the extremely high subsurface concentrations of dissolved CO2 (1000-1700 mM), which disrupt the cellular pH homeostasis, and lead to end-product inhibition. This limits life to the surface sediment horizons above the liquid CO2 phase, where less extreme conditions prevail. Our results may have to be taken into consideration in assessing the consequences of deep-sea CO2 sequestration on benthic element cycling and on the local ecosystem state. © Author(s) 2013. CC Attribution 3.0 License.

In this study, we tested a radical gas-based decontamination technique to prevent possible DNA contamination by the air and/or equipment used in molecular experiments. We prepared 10(4) molecules of model DNA contaminant and placed the dried DNA into test tubes, which were then exposed to radical gas. Quantitative PCR analysis showed that, even after a short exposure time of 30 minutes, 99.54% of the model DNA contaminant was effectively decomposed to undetectable levels. Our results demonstrate that the radical gas-based treatment is a useful method for eliminating potential DNA contaminant in ultra-sensitive molecular experiments.

In order to study bacterial activity, and turnover times of bacterial necromass and biomass in marine sediment, two stations from the Aarhus Bay, Denmark were analyzed. Sediment cores were up to 11 m deep and covered a timescale from the present to similar to 11,000 years ago. Sediment was analyzed for total hydrolysable amino acids (THAA), total hydrolysable amino sugars, the bacterial endospore marker dipicolinic acid (DPA), and amino acid enantiomers (L-and D-form) of aspartic acid. Turnover times of bacterial necromass and vegetative cells, as well as carbon oxidation rates were estimated by use of the D: L-amino acid racemization model. Diagenetic indicators were applied to evaluate the diagenetic state of the sedimentary organic matter. The contribution of amino acids to total organic carbon, and the ratio between the amino acids aspartic acid and glutamic acid, and their respective non protein degradation products, beta-alanine and gamma-amino butyric acid, all indicated increasing degradation state of the organic matter with sediment depth and age. Quantification of DPA showed that endo-spores were abundant, and increased with depth relative to vegetative cells. Most of the amino acids (97%) could be ascribed to microbial necromass, i.e. the remains of dead bacterial cells. Model estimates showed that the turnover times of microbial necromass were in the range of 0.5-1 x 10(5) years, while turnover times of vegetative cells were in the range of tens to hundreds of years. The turnover time of the TOC pool increased with depth in the sediment, indicating that the TOC pool became progressively more refractory and unavailable to microorganisms with depth and age of the organic matter. (C) 2012 Elsevier Ltd. All rights reserved.

A novel chemolithoautotrophic hydrogen-oxidizing and sulfur-reducing bacterium, strain 496Chim(T), was isolated from a deep-sea hydrothermal vent chimney collected from the hydrothermal field at the summit of Nikko Seamount field, in the Mariana Arc. Cells were rods or curved rods, motile by means of a single polar flagellum. Growth was observed between 15 and 45 A degrees C (optimum 37 A degrees C; doubling time, 2.1 h) and between pH 5.3 and 8.0 (optimum pH 6.0). The isolate was a strictly anaerobic, obligate chemolithoautotroph capable of growth using molecular hydrogen as the sole energy source, carbon dioxide as the sole carbon source, ammonium or nitrate as the sole nitrogen source, and elemental sulfur as the electron acceptor. The G+C content of genomic DNA was 35 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the new isolate belonged to the class Epsilonproteobacteria, but the isolate was distantly related to the previously described Epsilonproteobacteria species potentially at the genus level (&lt; 90 %). On the basis of its physiological and molecular characteristics, strain 496Chim(T) (=DSM 22050(Icurrency sign) = JCM 15747(Icurrency sign) = NBRC 105224(Icurrency sign)) represents the sole species of a new genus, Thiofractor, for which the name Thiofractor thiocaminus is proposed.

Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using X DNA and humic acids, underestimation of gimel DNA at 1/4400 of the theoretical value was observed with 6.58 ng mu L-1 humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34 ng mu L-1. The inhibitory effect of paddy field soil extract on quantification of the archaeal 165 rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04 x 10(3) copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64 x 102 copies/gsediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats. (c) 2012 Elsevier GmbH. All rights reserved.

The Integrated Ocean Drilling Program site U1365 drilled into the basement of the southwest Pacific crust formed from the mid-Cretaceous Osbourn Trough that rifted apart the Manihiki and Hikurangi Plateaus (the Greater Manihiki). The basalt geochemistry at this site is crucial for understanding the magmatic processes and mantle source of the mid-Cretaceous Osbourn Trough. The recovered fresh basalts were low-K tholeiitic normal (N) and depleted (D) mid-ocean ridge basalt (MORB). Their trace element and Sr-Nd isotope compositions indicate a Pacific-type mantle source rather than any significant influences from the nearby Louisville Seamount Chain or from the Greater Manihiki Plateau. Despite the presence of a plume head underneath the Osbourn Trough at its initial stage, the insignificance of a plume head could be explained by the long-distance (&gt;1000 km) southward migration of the Osbourn Trough. Lavas at site U1365 vary from low-MgO (&lt;6.9 wt.%) N-MORB at the bottom to high-MgO (8 wt.% to 9.5 wt.%) D-MORB and, then, to medium-MgO (7.3 wt.% to 8.2 wt.%) N-MORB according to their eruption sequences, which was accompanied by magma mixing in the magma reservoir. The D-MORB group lavas have higher melting degrees than those of N-MORB group based on their concentrations of TiO2, Na2O and CaO corrected for crystallization relative to MgO = 7.8 wt.%. The major element compositions of the high-MgO D-MORB lavas were consistent with partial melting in the spinel-peridotite zone over a pressure interval from similar to 3.1 GPa to 2 GPa in the mantle. The significant overlap of N-MORB and D-MORB in Sr-Nd isotopes suggests that chemical differences between the two groups were derived from the mantle melting processes. Based on comparison with lavas from the East Pacific Rise where a positive correlation of mantle melting degree vs. spreading rate is shown, we suggest that the Osbourn Trough might have a full spreading rate of similar to 140 mm/yr. Thus, the slow ridge-like axial morphology of the Osbourn Trough should be a character of an extinct fast ridge. (C) 2012 Elsevier B.V. All rights reserved.

The degradation of organic carbon in subseafloor sediments on continental margins contributes to the largest reservoir of methane on Earth. Sediments in the Andaman Sea are composed of 1% marine-derived organic carbon and biogenic methane is present. Our objective was to determine microbial abundance and diversity in sediments that transition the gas hydrate occurrence zone (GHOZ) in the Andaman Sea. Microscopic cell enumeration revealed that most sediment layers harbored relatively low microbial abundance (103105cellscm-3). Archaea were never detected despite the use of both DNA- and lipid-based methods. Statistical analysis of terminal restriction fragment length polymorphisms revealed distinct microbial communities from above, within, and below the GHOZ, and GHOZ samples were correlated with a decrease in organic carbon. Primer-tagged pyrosequences of bacterial 16S rRNA genes showed that members of the phylum Firmicutes are predominant in all zones. Compared with other seafloor settings that contain biogenic methane, this deep subseafloor habitat has a unique microbial community and the low cell abundance detected can help to refine global subseafloor microbial abundance.

A novel, facultatively anaerobic bacterium (strain JAM-BA0501(T)) was isolated from a deep subseafloor sediment sample at a depth of 247 m below seafloor off the Shimokita Peninsula of Japan in the north-western Pacific Ocean (Site C9001, 1180 m water depth). Cells of strain JAM-BA0501(T) were Gram-negative, filamentous, non-spore-forming and motile on solid medium by gliding. Phylogenetic analysis based on the 16S rRNA gene sequence of strain JAM-BA0501(T) indicated a distant relationship to strains representing genera within the order Bacteroidales, such as Alkaliflexus imshenetskii Z-7010(T) (91.1 % similarity), Marinilabilia salmonicolor ATCC 19041(T) (86.2 %) and Anaerophaga thermohalophila Fru22(T) (89.3 %). The new isolate produced isoprenoid quinones with menaquinone MK-7 as the major component, and the predominant fatty acids were iso-C-15:0 and anteiso-C-15:0. The DNA G + C content of the isolate was 42.9 mol%. Based on its taxonomic distinctiveness, strain JAM-BA0501(T) is considered to represent a novel species of a new genus within the family Marinilabiliaceae, for which the name Geofilum rubicundum gen. nov., sp. nov. is proposed. The type strain of Geofilum rubicundum is JAM-BA0501(T) (=JCM 15548(T) =NCIMB 14482(T)).

The influence of hydrostatic pressure on microbial sulfate reduction (SR) was studied using sediments obtained at cold seep sites from 5500 to 6200 m water depth of the Japan Trench. Sediment samples were stored under anoxic conditions for 17 months in slurries at 4 degrees C and at in situ pressure (50 MPa), at atmospheric pressure (0.1 MPa), or under methanic conditions with a methane partial pressure of 0.2 MPa. Samples without methane amendment stored at in situ pressure retained higher levels of sulfate reducing activity than samples stored at 0.1 MPa. Piezophilic SR showed distinct substrate specificity after hydrogen and acetate addition. SR activity in samples stored under methanic conditions was one order of magnitude higher than in non amended samples. Methanic samples stored under low hydrostatic pressure exhibited no increased SR activity at high pressure even with the amendment of methane. These new insights into the effects of pressure on substrate specific sulfate reducing activity in anaerobic environmental samples indicate that hydrostatic pressure must be considered to be a relevant parameter in ecological studies of anaerobic deep-sea microbial processes and long-term storage of environmental samples.

A remarkable number of microbial cells have been enumerated within subseafloor sediments, suggesting a biological impact on geochemical processes in the subseafloor habitat. However, the metabolically active fraction of these populations is largely uncharacterized. In this study, an RNA-based molecular approach was used to determine the diversity and community structure of metabolically active bacterial populations in the upper sedimentary formation of the Nankai Trough seismogenic zone. Samples used in this study were collected from the slope apron sediment overlying the accretionary prism at Site C0004 during the Integrated Ocean Drilling Program Expedition 316. The sediments represented microbial habitats above, within, and below the sulfate methane transition zone (SMTZ), which was observed approximately 20 m below the seafloor (mbsf). Small subunit ribosomal RNA were extracted, quantified, amplified, and sequenced using high-throughput 454 pyrosequencing, indicating the occurrence of metabolically active bacterial populations to a depth of 57 mbsf. Transcript abundance and bacterial diversity decreased with increasing depth. The two communities below the SMTZ were similar at the phylum level, however only a 24% overlap was observed at the genus level. Active bacterial community composition was not confined to geochemically predicted redox stratification despite the deepest sample being more than 50 m below the oxic/anoxic interface. Genus-level classification suggested that the metabolically active subseafloor bacterial populations had similarities to previously cultured organisms. This allowed predictions of physiological potential, expanding understanding of the subseafloor microbial ecosystem. Unique community structures suggest very diverse active populations compared to previous DNA-based diversity estimates, providing more support for enhancing community characterizations using more advanced sequencing techniques.

Microbial communities that thrive in subterranean consolidated sediments are largely unknown owing to the difficulty of extracting DNA. As this difficulty is often attributed to DNA binding onto the silica-bearing sediment matrix, we developed a DNA extraction method for consolidated sediment from the deep subsurface in which silica minerals were dissolved by being heated under alkaline conditions. NaOH concentrations (0.07 and 0.33 N), incubation temperatures (65 and 94 degrees C) and incubation times (30-90 min) before neutralization were evaluated based on the copy number of extracted prokaryotic DNA. Prokaryotic DNA was detected by quantitative PCR analysis after heating the sediment sample at 94 degrees C in 0.33 N NaOH solution for 50-80 min. Results of 16S rRNA gene sequence analysis of the extracted DNA were all consistent with regard to the dominant occurrence of the metallophilic bacterium, Cupriavidus metallidurans, and Pseudomonas spp. Mineralogical analysis revealed that the dissolution of a silica mineral (opal-CT) during alkaline treatment was maximized at 94 degrees C in 0.33 N NaOH solution for 50 min, which may have resulted in the release of DNA into solution. Because the optimized protocol for DNA extraction is applicable to subterranean consolidated sediments from a different locality, the method developed here has the potential to expand our understanding of the microbial community structure of the deep biosphere.

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 degrees C. 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. The ISME Journal (2011) 5, 1913-1925; doi: 10.1038/ismej.2011.64; published online 9 June 2011

A novel methane-oxidizing bacterium, strain HTM55(T), was isolated from subsurface hot aquifer water from a Japanese gold mine. Strain HTM55(T) was a Gram-negative, aerobic, motile, coccoid bacterium with a single polar flagellum and the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain HTM55(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 37-65 degrees C (optimum 55-60 degrees C). The isolate grew at pH 5.2-7.5 (optimum 5.8-6.3) and with 0-1% NaCl (optimum 0-0.3%). The ribulose monophosphate pathway was operative for carbon assimilation. The DNA G+C content was 54.4 mol% and the major fatty acids were C-16:0, (52.0%) and C-18:1 omega 7c (34.8%). Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HTM55(T) was closely related to Methylothermus thermalis MYHTT (99.2% 16S rRNA gene sequence similarity), which is within the class Gammaproteobacteria. However, DNA DNA relatedness between strain HTM55(T) and Methylothermus thermalis MYHTT was On the basis of distinct phylogenetic, chemotaxonomic and physiological characteristics, strain HTM55(T) represents a novel species of the genus Methylothermus, for which the name Methylothermus subterraneus sp. nov. is proposed. The type strain is HTM55(T) (=JCM 13664(T)=DSM 19750(T)).

We obtained 78 heat flow measurements during 2002-2008 in the area of the Iheya-North hydrothermal field in the middle Okinawa Trough, in order to clarify the spatial extent of the hydrothermal circulation system. Within a small basin surrounded by knolls, three distinct zones are identified with different heat flow values, which we termed the high-, moderate-, and low-heat-flow zones. In the high-heat-flow zone located near the western edge of the basin, extremely high and widely scattered heat flow values (0.01-100 Wm^-2) were measured within ∼500 m of the active hydrothermal mounds, venting black smoker fluid of maximum 311 °C. With increasing distance east of the high-heat-flow zone, heat flow gradually decreases from 1.0 to ∼0.1 Wm^-2 in a region where surface sediment is dominated by clay and a high-resolution bathymetry indicates a smooth seafloor surface. We term this area the moderate-heat-flow zone. Further to the east (∼2 km from the high-heat-flow zone), the seafloor consists of coarser sediment with a rugged surface, and heat flow is very low (<0.1 Wm^-2), as designated the low-heat-flow zone. We suggest that such anomalously low heat flow can be explained by the recharge of seawater into the formation, and that hydrothermal vents or diffuse flow in the high-heat-flow zone can drive this kilometer-scale hydrothermal circulation within the Iheya-North knoll complex, if the sediment below the moderate-heat-flow zone is impermeable enough to prevent vertical fluid migration but is permeable enough to encourage horizontal flow. Although some geological data such as piston core samples, surface morphology and seismic data would support this inference, this hypothesis needs to be tested through more direct evidence and by numerical simulation studies.

Subseafloor sedimentary environments harbor remarkably diverse microbial communities. However, it remains unknown if the deeply buried fossils in these sediments play ecological roles in deep microbial habitats, or whether the microbial communities inhabiting such fossils differ from those in the surrounding sediment matrix. Here we compare the community structures of subseafloor microbes in cold-water coral carbonates (Madrepora oculata and Lophelia pertusa) and the clay matrix. Samples were obtained from the Challenger Mound in the Porcupine Seabight at Site U1317 Hole A during the Integrated Ocean Drilling Program Expedition 307. DNA was extracted from coral fossils and the surrounding sedimentary matrix at 4, 20, and 105 m below the seafloor. 16S rRNA genes of Bacteria and Archaea were amplified by PCR, and a total of 213,792 16S rRNA gene-tagged sequences were analyzed. At the phylum level, dominant microbial components in both habitats consisted of Proteobacteria, Firmicutes, Nitrospirae, Chloroflexi, and Miscellaneous Crenarchaeota Group (MCG) at all three of the depths examined. However, at the genus and/or species level (similarity threshold 97.0%), the community compositions were found to be very different, with 69-75 and 46-57% of bacterial and archaeal phylotypes not overlapping in coral fossils and the clay matrix, respectively. Species richness analysis revealed that bacterial communities were generally more diverse than archaea, and that the diversity scores of coral fossils were lower than those in sediment matrix. However, the evenness of microbial communities was not significantly different in all the samples examined. No eukaryotic DNA sequences, such as 18S rRNA genes, were obtained from the corals. The findings suggested that, even at the same or similar depths, the sedimentological characteristics of a habitat are important factors affecting microbial diversity and community structure in deep subseafloor sedimentary habitats.

In this study, we investigated the diversity and spatial distribution of anaerobic methanotrophic archaea (ANMEs) in sediments of a gas hydrate field off Joetsu in the Japan Sea. Distribution of ANMEs in sediments was identified by targeting the gene for methyl coenzyme M reductase alpha subunit (mcrA), a phylogenetically conserved gene that occurs uniquely in methanotrophic and methanogenic archaea, in addition to 16S rRNA genes. Quantitative PCR analyses of mcrA genes in 14 piston core samples suggested that members of ANME-1 group would dominate AOM communities in sulfate-depleted sediments, even below the sulfate-methane interface, while ANME-2 archaea would prefer to populate in shallower sediments containing comparatively higher sulfate concentrations. These results suggest that, although the potential electron acceptors in sulfate-depleted habitats remain elusive, the niche separation of ANME-1 and -2 may be controlled by in situ concentration of sulfate and the availability in sediments.

A novel thermophilic and lithoautotrophic sulfate-reducing archaeon was isolated from black rust formed on the steel surface of a borehole observatory (CORK 1026B) retrieved during IODP Expedition 301 on the eastern flank of Juan de Fuca Ridge, eastern Pacific Ocean. Cells of the strain were lobe-shaped or triangular. The optimum temperature, pH and NaCl concentration for growth were 75 degrees C, pH 7 and 2% (w/v), respectively. The isolate was strictly anaerobic, growing lithoautotrophically on H-2 and CO2 using sulfate, sulfite or thiosulfate as electron acceptors. Lactate and pyruvate could serve as alternative energy and carbon sources. The G+C content of the genomic DNA was 42 mol%. Phylogenetic analyses of the 16S rRNA gene indicated that the isolate was closely related to members of the family Archaeoglobaceae, with sequence similarities of 90.3-94.4%. Physiological and molecular properties showed that the isolate represents a novel species of the genus Archaeoglobus. The name Archaeoglobus sulfaticallidus sp. nov. is proposed; the type strain is PM70-1(T) (=DSM 19444(T)=JCM 14716(T)).

A variety of archaeal lineages have been identified using culture-independent molecular phylogenetic surveys of microbial habitats occurring in deep-sea hydrothermal environments such as chimney structures, sediments, vent emissions, and chemosynthetic macrofauna. With the exception of a few taxa, most of these archaea have not yet been cultivated, and their physiological and metabolic traits remain unclear. In this study, phylogenetic diversity and distribution profiles of the archaeal genes encoding small subunit (SSU) rRNA, methyl coenzyme A (CoA) reductase subunit A, and the ammonia monooxygenase large subunit were characterized in hydrothermally influenced sediments at the Yonaguni Knoll IV hydrothermal field in the Southern Okinawa Trough. Sediment cores were collected at distances of 0.5, 2, or 5 m from a vent emission (90 degrees C). A moderate temperature gradient extends both horizontally and vertically (5 to 69 degrees C), indicating the existence of moderate mixing between the hydrothermal fluid and the ambient sediment pore water. The mixing of reductive hot hydrothermal fluid and cold ambient sediment pore water establishes a wide spectrum of physical and chemical conditions in the microbial habitats that were investigated. Under these different physico-chemical conditions, variability in archaeal phylotype composition was observed. The relationship between the physical and chemical parameters and the archaeal phylotype composition provides important insight into the eco-physiological requirements of uncultivated archaeal lineages in deep-sea hydrothermal vent environments, giving clues for approximating culture conditions to be used in future culturing efforts.

In deep subsurface sediments of the Juan de Fuca Ridge Flank, porewater acetate that is depleted in 13C relative to sedimentary organic matter indicates an acetogenic component to total acetate production. Thermodynamic calculations indicate common fermentation products or lignin monomers as potential substrates for acetogenesis. The classic autotrophic reaction may contribute as well, provided that dihydrogen (H2) concentrations are not drawn down to the thermodynamic thresholds of the energetically more favorable processes of sulfate reduction and methanogenesis. A high diversity of novel formyl tetrahydrofolate synthetase (fhs) genes throughout the upper half of the sediment column indicates the genetic potential for acetogenesis. Our results suggest that a substantial fraction of the acetate produced in marine sediment porewaters may derive from acetogenesis, in addition to the conventionally invoked sources fermentation and sulfate reduction.

Integrated Ocean Drilling Program (IODP) Expedition 316 Sites C0006 and C0007 examined the deformation front of the Nankai accretionary prism offshore the Kii Peninsula, Japan. In the drilling area, the frontal thrust shows unusual behavior as compared to other regions of the Nankai Trough. Drilling results, integrated with observations from seismic reflection profiles, suggest that the frontal thrust has been active since similar to 0.78-0.436 Ma and accommodated similar to 13 to 34% of the estimated plate convergence during that time. The remainder has likely been distributed among out-of-sequence thrusts further landward and/or accommodated through diffuse shortening. Unlike results of previous drilling on the Nankai margin, porosity data provide no indication of undercompaction beneath thrust faults. Furthermore, pore water geochemistry data lack clear indicators of fluid flow from depth. These differences may be related to coarser material with higher permeability or more complex patterns of faulting that could potentially provide more avenues for fluid escape. In turn, fluid pressures may affect deformation. Well-drained, sand-rich material under the frontal thrust could have increased fault strength and helped to maintain a large taper angle near the toe. Recent resumption of normal frontal imbrication is inferred from seismic reflection data. Associated decollement propagation into weaker sediments at depth may help explain evidence for recent slope failures within the frontal thrust region. This evidence consists of seafloor bathymetry, normal faults documented in cores, and low porosities in near surface sediments that suggest removal of overlying material. Overall, results provide insight into the complex interactions between incoming materials, deformation, and fluids in the frontal thrust region.

Halogenated organic compounds serve as terminal electron acceptors for anaerobic respiration in a diverse range of microorganisms. Here, we report on the widespread distribution and diversity of reductive dehalogenase homologous (rdhA) genes in marine subsurface sediments. A total of 32 putative rdhA phylotypes were detected in sediments from the southeast Pacific off Peru, the eastern equatorial Pacific, the Juan de Fuca Ridge flank off Oregon, and the northwest Pacific off Japan, collected at a maximum depth of 358 m below the seafloor. In addition, significant dehalogenation activity involving 2,4,6-tribromophenol and trichloroethene was observed in sediment slurry from the Nankai Trough Forearc Basin. These results suggest that dehalorespiration is an important energy-yielding pathway in the subseafloor microbial ecosystem.

Detection and enumeration of microbial life in natural environments provide fundamental information about the extent of the biosphere on Earth. However, it has long been difficult to evaluate the abundance of microbial cells in sedimentary habitats because non-specific binding of fluorescent dye and/or auto-fluorescence from sediment particles strongly hampers the recognition of cell-derived signals. Here, we show a highly efficient and discriminative detection and enumeration technique for microbial cells in sediments using hydrofluoric acid (HF) treatment and automated fluorescent image analysis. Washing of sediment slurries with HF significantly reduced non-biological fluorescent signals such as amorphous silica and enhanced the efficiency of cell detachment from the particles. We found that cell-derived SYBR Green I signals can be distinguished from non-biological backgrounds by dividing green fluorescence (band-pass filter: 528/38nm (center-wavelength/bandwidth)) by red (617/73 nm) per image. A newly developed automated microscope system could take a wide range of high-resolution image in a short time, and subsequently enumerate the accurate number of cell-derived signals by the calculation of green to red fluorescence signals per image. Using our technique, we evaluated the microbial population in deep marine sediments offshore Peru and Japan down to 365 m below the seafloor, which provided objective digital images as evidence for the quantification of the prevailing microbial life. Our method is hence useful to explore the extent of sub-seafloor life in the future scientific drilling, and moreover widely applicable in the study of microbial ecology. The ISME Journal (2009) 3, 503-511; doi:10.1038/ismej.2009.1; published online 12 February 2009

The effects of silicic acid on the growth of Thermus thermophilus TMY, an extreme thermophile isolated from a siliceous deposit formed from geothermal water at a geothermal power plant in Japan, were examined at 75 degrees C. At concentrations higher than the solubility of amorphous silica (400 to 700 ppm SiO(2)), a silica-induced protein (Sip) was isolated from the cell envelope fraction of log-phase TMY cells grown in the presence of supersaturated silicic acid. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the molecular mass and pI of Sip to be about 35 kDa and 9.5, respectively. Induction of Sip expression occurred within 1 h after the addition of a supersaturating concentration of silicic acid to TM broth. Expression of Sip-like proteins was also observed in other thermophiles, including T. thermophilus HB8 and Thermus aquaticus YT-1. The amino acid sequence of Sip was similar to that of the predicted solute-binding protein of the Fe(3+) ABC transporter in T. thermophilus HB8 (locus tag, TTHA1628; GenBank accession no. NC_006461; GeneID, 3169376). The sip gene (987-bp) product showed 87% identity with the TTHA1628 product and the presumed Fe(3+)-binding protein of T. thermophilus HB27 (locus tag TTC1264; GenBank accession no. NC_005835; GeneID, 2774619). Within the genome, sip is situated as a component of the Fbp-type ABC transporter operon, which contains a palindromic structure immediately downstream of sip. This structure is conserved in other T. thermophilus genomes and may function as a terminator that causes definitive Sip expression in response to silica stress.

Iodine concentration and radioisotopic composition (129I/I) were measured in the pore waters from the gas hydrate occurrence in the forearc basin offshore Shimokita Peninsula, north-eastern Japan, to determine the source formation of I and accompanying hydrocarbons. Iodine concentrations correlate well with the alkalinity and SO4 patterns, reflecting degradation stages of I-rich buried organic matter, rapidly increasing in the sulfate reduction interval, and becoming constant below 250 meters below the seafloor with an upwelling flux of 1.5 x 10-11 mu mol cm-2 year-1. The 129I/I ratios of 300 x 10-15-400 x 10-15 in deep pore waters suggest ages for iodine and hydrocarbon sources as old as 40 Ma. These ages correlate well with the coaly source formations of the Eocene age thought to be responsible for the conventional natural gas deposits underlying the gas hydrate stability zone. Similar profiles are observed in 129I/I ratios of pore waters in the gas hydrate stability zone from the forearc basin in the eastern Nankai Trough, offshore central Japan, where pore waters are enriched in I and reach ages as old as similar to 50 Ma through the sediment column. At the outer ridge site along the trough, on the other hand, relatively younger I are more frequently delivered probably through thrusts/faults associated with subduction. The nature of source formations of I and hydrocarbons in the offshore Shimokita Peninsula has a more terrestrial contribution compared with those in the Nankai Trough, but these formations are also considerably older than the host sediments, suggesting long-term transport of I and hydrocarbons for the accumulation of gas hydrates in both locations.

"A meta-enzyme approach" is proposed as an ecological enzymatic method to explore the potential functions of microbial communities in extreme environments such as the deep marine subsurface. We evaluated a variety of extra-cellular enzyme activities of sediment slurries and isolates from a deep subseafloor sediment core. Using the new deep-sea drilling vessel "Chikyu", we obtained 365 m of core sediments that contained similar to 2% organic matter and considerable amounts of methane from offshore the Shimokita Peninsula in Japan at a water depth of 1,180 m. In the extra-sediment fraction of the slurry samples, phosphatase, esterase, and catalase activities were detected consistently throughout the core sediments down to the deepest slurry sample from 342.5 m below seafloor (mbsf). Detectable enzyme activities predicted the existence of a sizable population of viable aerobic microorganisms even in deep subseafloor habitats. The subsequent quantitative cultivation using solid media represented remarkably high numbers of aerobic, heterotrophic microbial populations (e.g., maximally 4.4 x 10(7) cells cm(-3) at 342.5 mbsf). Analysis of 16S rRNA gene sequences revealed that the predominant cultivated microbial components were affiliated with the genera Bacillus, Shewanella, Pseudoalteromonas, Halomonas, Pseudomonas, Paracoccus, Rhodococcus, Microbacterium, and Flexibacteracea. Many of the predominant and scarce isolates produced a variety of extra-cellular enzymes such as proteases, amylases, lipases, chitinases, phosphatases, and deoxyribonucleases. Our results indicate that microbes in the deep subseafloor environment off Shimokita are metabolically active and that the cultivable populations may have a great potential in biotechnology.

Integrated Ocean Drilling Program (IODP) Expedition 316 is part of the Nankai Trough Seismogenic Zone Experiment (NanTroSEIZE) complex drilling project (CDP). This coordinated, multiplatform, and multiexpedition drilling project is designed to investigate fault mechanics and seismogenesis along subduction megathrusts through direct sampling, in situ measurements, and long-term monitoring in conjunction with allied laboratory and numerical modeling studies. Expedition 316 was designed to evaluate the deformation, inferred depth of detachment, structural partitioning, fault zone physical characteristics, and fluid flow at the frontal thrust and at the shallow portion of the megasplay system. To accomplish these objectives, drilling was conducted at two sites in the megasplay region, one within the fault zone and one in the slope basin seaward of the megasplay. Two sites were also drilled within the frontal thrust region. During Expedition 314, two of these sites were characterized using logging while drilling. Site C0004 is located along the slope of the accretionary prism landward of the inferred intersection of the megasplay fault zone with the seafloor. Drilling at this site examined the youngest sediments on the slope overlying the accretionary prism; these sediments consist of slowly deposited marine sediments and redeposited material from upslope. This redeposited material provides information about past slope failures, which may be related to past megasplay movement, earthquakes, and tsunamigenesis. The accretionary prism was sampled and the megasplay fault zone was successfully drilled. Structural observations of core material from the fault zone and two age reversals suggested by nannofossils indicate a complex history of deformation. The sediments under the fault zone were sampled to understand their deformation, consolidation, and fluid flow history. Drilling at Site C0008 targeted the slope basin seaward of the megasplay fault. This basin records the history of fault movement. In addition, sediment layers within this basin provide a reference for sediment underthrusting Site C0004. Drilling at Sites C0006 and C0007 allowed examination of the frontal thrust region. At Site C0006, several fault zones within the prism were penetrated before drilling was stopped because of poor conditions. The plate boundary frontal thrust was successfully drilled and thrust fault material ranging from breccia to fault gouge was successfully recovered at Site C0007.

A previous study interpreted extremely C-13-depleted excursions of planktonic and benthic foraminifera in last glacial sediments (17,500 to 25,400 cal years B. P.) of the core retrieved from off Shimokita Peninsula and off Hokkaido, Japan, as evidence for periodic releases of methane, arising from the dissociation of methane hydrate. To better understand the formation process of the C-13-depleted excursions, we conducted high-resolution natural radiocarbon measurements and biogeochemical analyses. We found highly depleted 13 C excursions ranging from -10.2% to -1.6% and -6.8% to -1.6% in planktonic and benthic foraminifera, respectively. Most of the foraminiferal tests in these horizons were brown, most likely as a result of postdepositional alteration, reflecting the formation of authigenic carbonate on the surface of tests. These alterations were also supported by high levels of Mg-calcite and the acid-leaching test for anomalous foraminifera. To evaluate the carbon sources in the altered foraminifera tests, we quantified the relative contributions of C-14-free methane-derived carbon sources to the formation of authigenic carbonates in foraminifera with depleted 13 C excursions using a coupled mass balance isotopic model (C-14/C and C-13/C-12). The radiocarbon ages of both planktonic and benthic C-13-depleted foraminifera were approximately 600 to 2000 years older than those of normal tests from nearby horizons. The relative contributions of authigenic carbonates derived from the methane oxidizing process reached to -22 wt% for planktonic foraminifera and -15 wt% for benthic foraminifera. The delta C-13 values of methane calculated from the mass balance model were between -29% and -68% for planktonic foraminifera and between -40% and -108% for benthic foraminifera, consistent with delta C-13 values reported for thermogenic and abiogenic methane in global methane hydrate reservoirs. These data consistently suggest that methane-related drastic environmental change occurred in the horizons that included delta C-13 anomalies. This study provides important information for interpreting geological records of the methane hydrate instability associated with climate.

Marine subsurface sediments that cover more than two-thirds of the Earth harbor remarkable numbers of microbial cells. Subseafloor microbial activities may affect global biogeochemical cycles; however, our knowledge of the deep-subseafloor biosphere remains very limited. Recent molecular ecological studies have shown that subseafloor microbial communities are predominantly composed of yet -uncultivated, -uncharacterized bacteria and archaea with great phylogenetic diversity. Some phylogenetic groups are commonly detected in global marine sediments regardless of location and depth, and environmental factors such as sedimentological, geochemical, and geophysical characteristics probably control their activities, biomass, and community structures. This chapter reviews emerging patterns of microbial diversity in deeply buried marine sediments and discusses the potential ecological roles and distribution of microbial communities.

The importance of crustal fluid chemical composition in driving the marine deep subseafloor biosphere was examined in northeast Pacific ridge-flank sediments. At IODP Site U1301, sulfate from crustal fluids diffuses into overlying sediments, forming a transition zone where sulfate meets in situ-produced methane. Enhanced cell counts and metabolic activity suggest that sulfate stimulates microbial respiration, specifically anaerobic methane oxidation coupled to sulfate reduction. Cell counts and activity are also elevated in basement-near layers. Owing to the worldwide expansion of the crustal aquifer, we postulate that crustal fluids may fuel the marine deep subseafloor biosphere on a global scale.

Aims: To identify an extreme thermophile, strain TMY, isolated from silica scale from the geothermal electric power plant and to examine microdiversity of Thermus thermophilus strains. Materials and Results: The isolated strain TMY was identified by morphological, biochemical and physiological tests. Phylogenetic comparison of the strain and other Thermus strains with 16S rDNA analysis, RAPD and ERIC-PCR fingerprinting were performed. Strain TMY was closely related to strain which was isolated from a hot spring in New Zealand and shown to belong to the Japanese Thermus cluster. However, there were considerable genetic differences between strain TMY and other Thermus species using DNA fingerprinting. Conclusions: Based on morphological, physiological and genetic properties, strain TMY could be a strain of T. thermophilus. The distinct properties of strain TMY suggest that microdiversity of T. thermophilus strains should be considered. Significance and Impact of the Study: The results of this study have demonstrated genetic diversity within T. thermophilus strains, which were previously masked by an almost identical 16S rDNA sequence. RAPD and ERIC-PCR could be potential methods for distinguishing between Thermus strains.

Quality assurance and control (QA/QC) is significant for the scientific drilling in order to accurately characterize physical, geochemical, and biological properties in the cored deep subseafloor materials. To explore the deep subseafloor life and its biosphere, identification and control of microbial contamination in drilling cores is critical for highly sensitive molecular analyses as well as cultivations, especially for the evaluation of low biomass and/or extremely harsh deep environments. Here we report some microbiological characteristics of circulation mud fluids before and after the first riser drilling operation by the newly constructed deep-earth research vessel Chikyu. During the Chikyu shakedown expedition CK06-06 in 2006, we used the riser system for drilling 547 to 647 meter below the seafloor into the sediments offshore the Shimokita Peninsula of Japan. Cultivation experiments showed that no microbial growth was observed in the precirculation mud fluid, while 4 105 colonies per 1 ml were observed in the postcirculation mud fluid; all cultured bacterial isolates were found to be Halomonas. Using culture-independent molecular analysis, 16S rRNA gene sequences of Xanthomonas, which is used for industrial production of the mud fluid viscosifier xanthan gum, were predominantly detected in the precirculation mud fluid, while Halomonas sequences consistently dominated the clone library constructed from the postcirculation mud fluid. Archaeal 16S rRNA genes were amplified only from the postcirculation mud fluid; these archaeal clone sequences were affiliated to the Marine Crenarchaeota Group I (MGI), Marine Euryarchaeota Group II (MGII), Miscellaneous Crenarchaeotic Group (MCG), South African Gold Mine Euryarchaeotic Group (SAGMEG), Soil Group, and Methanococcus aeolicus. These results suggest that Halomonas contaminated and grew in the tank of circulation mud fluids, and other indigenous deep subseafloor microbial components, especially deep subsurface archaea, were also mixed into the post-circulation mud fluid.

The prokaryotic communities in deep subseafloor sediment collected during Ocean Drilling Program (ODP) Leg 204 from the South Hydrate Ridge (SHR) on the Cascadia Margin were analyzed by 16S rRNA gene clone sequencing and a fluorescent quantitative PCR technique. The microbial communities came from sites with contrasting geological characteristics on the SHR: sites 1244 and 1245 (located on the flank of the ridge, hydrate-rich sediment) and site 1251 (located on the slope basin of SHR, hydrate-poor sediment). The overall copy numbers of the 16S rRNA gene, and the proportion of archaeal 16S rRNA gene in all 16S rRNA gene community in sediment were larger on the slope basin than on the flank of the SHR. Archaeal community structure around the sulfate-methane transition zone at site 125 1 (4.5 mbsf) was intensively investigated using two different PCR primer sets. A relatively abundant distribution of the 16S rRNA gene sequences related to mesophilic methanogen of the genus Methanoculleus was identified at a depth of 43.2 mbsf, and suggested that the methanogens occur in relatively shallow zones of sediment. This study demonstrated that the subseafloor microbial communities shown by 16S rRNA gene clone analyses were not directly associated with subseafloor methane hydrate deposits.

Since their discovery in the late 1970s, deep-sea Hydrothermal systems have been considered as likely candidates for the origin and early evolution of life on Earth. However, while subsequent investigations have revealed a great diversity of modern deep-sea hydrothermal ecosystems, they have done little to shed light on the issues of the origin and early evolution of life, metabolism, cells, or communities. Phylogenetic, biochemical and geochemical clues all seem to point to the early evolution of hydrogenotrophic chemolithoautotrophy such as methanogenesis and sulfur-reduction, thus pinpointing the availability of hydrogen as one of the key elements needed for the early evolution of earthly life. Hydrogen-driven, photosynthesis-independent communities are very rare on the contemporary Earth, however, being unambiguously found only in subsurface environments of H2-dominated hydrothermal systems. Such systems have been termed hyperthermophilic subsurface lithoautotrophic microbial ecosystems (HyperSLiMEs) (Takai et al., 2004; Nealson et al., 2005). The supply of abundant hydrogen and available inorganic carbon sources to power such communities is most likely coupled to hydrothermal serpentinization of ultramafic rocks and input of magmatic volatiles, both of which are related to specific geological settings. We propose here, on the basis of findings in the modern Earth and implications for the deep-sea hydrothermal systems in the Archean Earth, that "Ultramafics-Hydrothermalism-Hydrogenesis-HyperSLiME", a linkage we refer to as Ultra H3, provided a suitable habitat for the early microbial ecosystem on the Archean Earth. © by the Palaeontological Society of Japan.

During Integrated Ocean Drilling Program Expedition 301, we obtained a sample of black rust from a circulation obviation retrofit kit (CORK) observatory at a borehole on the eastern flank of Juan de Fuca Ridge. Due to overpressure, the CORK had failed to seal the borehole. Hot fluids from oceanic crust had discharged to the overlying bottom seawater and resulted in the formation of black rust analogous to a hydrothermal chimney deposit. Both culture-dependent and culture-independent analyses indicated that the black-rust-associated community differed from communities reported from other microbial habitats, including hydrothermal vents at seafloor spreading centers, while it shared phylotypes with communities previously detected in crustal fluids from the same borehole. The most frequently retrieved sequences of bacterial and archaeal 16S rRNA genes were related to the genera Ammonifex and Methanothermococcus, respectively. Most phylotypes, including phylotypes previously detected in crustal fluids, were isolated in pure culture, and their metabolic traits were determined. Quantification of the dissimilatory sulfite reductase (dsrAB) genes, together with stable sulfur isotopic and electron microscopic analyses, strongly suggested the prevalence of sulfate reduction, potentially by the Ammonifex group of bacteria. Stable carbon isotopic analyses suggested that the bulk of the microbial community was trophically reliant upon photosynthesis-derived organic matter. This report provides important insights into the phylogenetic, physiological, and trophic characteristics of subseafloor microbial ecosystems in warm ridge flank crusts.

Perfluorocarbon tracers (PFTs) are used during cruises of the Ocean Drilling Program (ODP) and Integrated Ocean Program (IODP) to measure sample contamination with drilling fluid. Drilling fluid is supplied with a constant PFT concentration that can then be detected and quantified in sediment and basalt core samples. During IODP Expedition 301, we used washing (2x) and flaming to effectively remove PFT from the exterior of basalt rocks. Near-complete removal from the exterior allowed us to demonstrate that the interior of basalts was only minutely, if at all, contaminated with drilling fluid. We examined horizontal and vertical trends in sediment core contamination. Contamination decreased greatly between the core exterior to halfway along the core radius, and slightly from halfway to the center of cores, and was generally very low in halfway and center portions. Clay cores were, on average, more contaminated than cores with fine sand. Contamination was typically highest in the two uppermost sections (sections 1 and 2) and lower below (sections 3-5). There was no relationship between depth of core origin and contamination. To determine mechanisms of contamination in halfway and interior parts of cores, we estimated the diffusive flux of PFT from the core liner towards the core center. Based on conservative estimates, we concluded that diffusion did not account for any of the PFT measured in halfway and interior parts of cores in this study. Any measurable PFT concentrations in halfway and center parts of cores were caused by advection.

Culture-dependent and independent methods were used to evaluate the microbial communities in cores collected at depths up to 200 m in oil-bearing and oil-free strata near the Sagara oil reservoir near Shizuoka, Japan. Direct microbial counts revealed much higher numbers (2.2 x 10(4)-7.9 x 10(6)/g) of microbes in the oil-bearing strata than in the oil-free zones, where counts were uniform at approximately 1.0 x 10(4)/g. Molecular taxonomic analyses via 16S rRNA gene sequence comparisons showed that the oil-free strata were dominated by members of the gamma-Proteobacteria including Pseudomonas, Stenotrophomonas and Sphingomonas, whereas the oil-bearing strata were dominated by a single species closely related to Pseudomonas stutzeri. All archaeal clones were phylogenetically affiliated with the uncultured soil group in Crenarchaeota with the exception of a single phylotype that belonged to the genus Thermococcus. Culture-dependent analysis was carried out by most-probable-number culturing as well as direct plating to determine viable cell counts, using both complex organic substrates or native oil and autotrophic media. Both culture-dependent and independent methods revealed the abundant cultivable member was the aerobic oil-degrading bacterium Pseudomonas stutzeri and neither autotrophs nor anaerobic heterotrophs could be detected in the oil-bearing strata.

A novel mesophilic bacterium, strain GO25(T), was isolated from a nest of hydrothermal vent polychaetes, Paralvinella sp., at the Iheya North field in the Mid-Okinawa Trough. Cells were motile short rods with a single polar flagellum. Growth was observed between 4 and 35 degrees C (optimum 30 degrees C; 13-16 h doubling time) and between pH 5(.)4 and 8(.)6 (optimum pH 6(.)1). The isolate was a facultatively anaerobic chemolithoautotroph capable of growth using molecular hydrogen, elemental sulfur or thiosulfate as the sole energy source, carbon dioxide as the sole carbon source, ammonium or nitrate as the sole nitrogen source and elemental sulfur, thiosulfate or yeast extract as the sole sulfur source. Strain GO25(T) represents the first deep-sea epsilonproteobacterium capable of growth by both hydrogen and sulfur oxidation. Nitrate or molecular oxygen (up to 10 % partial pressure) could serve as the sole electron acceptor to support growth. Metabolic products of nitrate reduction shifted in response to the electron donor provided. The G + C content of genomic DNA was 37(.)6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurimonas and was most closely related to Sulfurimonas autotrophica OK10(T) (96.3 % sequence similarity). DNA-DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurimonas autotrophica OK10(T). On the basis of the physiological and molecular properties of the novel isolate, the name Sulfurimonas paralvinellae sp. nov. is proposed, with strain GO25(T) (=JCM 13212(T) = DSM 17229(T)) as the type strain. Thiomicrospira denitrificans DSM 1251(T) (=ATCC 33889(T)) is phylogenetically associated with Sulfurimonas autotrophica OK10(T) and Suffurimonas paralvinellae GO25(T). Based on the phylogenetic relationship between Thiomicrospira denitriticans DSM 1251(T), Sulfurimonas autotrophica OK10(T) and Suffurimonas paralvinellae GO25T, we propose the reclassification of Thiomicrospira denitrificans as Suffurimonas denitrificans comb. nov. (type strain DSM 1251(T) = ATCC 33889(T)). In addition, an emended description of the genus Sulfurimonas is proposed.

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier (C-13-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.

We propose a hypothesis that Ultra-H3 (Ultramafic rock, Hydrothermal activity, Hydrogen, and Hyper SLiME) linkage was responsible for the development of primitive life on the early Earth. In order to establish the Ultra-H3 linkage hypothesis, we plan experimental investigations of hydrothermal reactions among hydrothermal solution, ultramafic rocks, and microorganisms using newly designed hydrothermal apparatus with both flexible reaction-cell and fluid-flow systems. The hydrothermal experiments will be performed to resolve the following issues: 1) relationships between HyperSLiME activity and hydrogen concentration of hydrothermal fluid, 2) hydrothermal hydrogen-generation under the specific condition of the early Earth (e.g., low-O2, low-SO4, high-CO2, low-pH, and komatiitic rocks), and 3) probability of formation of organic compounds such as amino acids that are essential to cellular life during the hydrothermal reaction.

Recent advances in molecular ecological techniques have led to the revelation that sedimentary environments contain molecular signals in the form of DNA sequences: signals that tend to be consistent with the in situ geochemical environment. However, as one moves to more ancient environments, the correlation of molecular signals with the in situ environment becomes more difficult. We have called the pool of molecular information that can be obtained from such ancient environments the 'Paleome'. This concept is controversial, in part because it demands that the molecular sequences be stable for long periods of time, and in part because the interpretation of such sequences in a geochemical context is often difficult, even for contemporary samples. We review results from a variety of environments, discussing the potential explanations for each, and focusing on the relationship of the molecular signals to the biological history of the sample. In almost every case, while molecular signals were abundant, viable cells were not cultivated. We posit here that the paleome is the genetic record of the biological past, a record that has been preserved on geologic timescales: a record that may well provide us with insights into both the paleoenvironment and the co-evolution of Earth and its biota.

Within a phylum Crenarchaeota, only some members of the hyperthermophilic class Thermoprotei, have been cultivated and characterized. In this study, we have constructed a metagenomic library from a microbial mat formation in a subsurface hot water stream of the Hishikari gold mine, Japan, and sequenced genome fragments of two different phylo-groups of uncultivated thermophilic Crenarchaeota : (i) hot water crenarchaeotic group (HWCG) I (41.2 kb), and (ii) HWCG III (49.3 kb). The genome fragment of HWCG I contained a 16S rRNA gene, two tRNA genes and 35 genes encoding proteins but no 23S rRNA gene. Among the genes encoding proteins, several genes for putative aerobic-type carbon monoxide dehydrogenase represented a potential clue with regard to the yet unknown metabolism of HWCG I Archaea. The genome fragment of HWCG III contained a 16S/23S rRNA operon and 44 genes encoding proteins. In the 23S rRNA gene, we detected a homing-endonuclease encoding a group I intron similar to those detected in hyperthermophilic Crenarchaeota and Bacteria, as well as eukaryotic organelles. The reconstructed phylogenetic tree based on the 23S rRNA gene sequence reinforced the intermediate phylogenetic affiliation of HWCG III bridging the hyperthermophilic and non-thermophilic uncultivated Crenarchaeota

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities-ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase-while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and 11 RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5'-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.

Epsilon-Proteobacteria is increasingly recognized as an ecologically significant group of bacteria, particularly in deep-sea hydrothermal environments. In this study, we studied the spatial distribution, diversity and physiological characteristics of the epsilon-Proteobacteria in various microbial habitats in the vicinity of a deep-sea hydrothermal vent occurring in the Iheya North field in the Mid-Okinawa Trough, by using culture-dependent and -independent approaches. The habitats studied were inside and outside hydrothermal plume, and annelid polychaete tubes. In addition, we deployed colonization devices near the vent emission. The polychaete tubes harboured physiologically and phylogenetically diverse microbial community. The in situ samplers were predominantly colonized by epsilon-Proteobacteria. Energy metabolism of epsilon-Proteobacteria isolates was highly versatile. Tree topology generated from the metabolic traits was significantly different (P = 0.000) from that of 16S rRNA tree, indicating current 16S rRNA gene-based analyses do not provide sufficient information to infer the physiological characteristics of epsilon-Proteobacteria. Nevertheless, culturability of epsilon-Proteobacteria in various microbial habitats differed among the phylogenetic subgroups. Members of Sulfurimonas were characterized by the robust culturability, and the other phylogenetic subgroups appeared to lose culturability in seawater, probably because of the sensitivity to oxygen. These results provide new insight into the ecophysiological characteristics of the deep-sea hydrothermal vent epsilon-Proteobacteria, which has never been assessed by comparative analysis of the 16S rRNA genes.

One of the keys to success of many anaerobic ecosystems is the process of syntrophic intercellular hydrogen transfer. This process facilitates the overall reaction by end-product removal, taking advantage of a wide variety of organisms that are able to use hydrogen directly as an energy source by uptake hydrogenases. Thus, the issue is not whether there are hydrogen-driven processes or communities but whether there are hydrogen-driven communities that exist and persist independently of the products of photosynthesis (so called subsurface lithoautotrophic microbial ecosystems, or SLiMEs). It is the proof of long-term independence from photosynthesis and its products that is the most difficult issue to establish, and perhaps the most important one with regard to searching for SLiMEs both on and off our planet. Although the evidence is not yet unequivocal, a growing body of evidence supports the existence of SLiME-like communities: if they exist, the implications are immense with regard to understanding subsurface environments on Earth, looking for present day analogs of early Earth and the search for life in other worlds.

Phase-separation and -segregation (boiling/distillation of subseafloor hydrothermal fluids) represent the primary mechanisms causing intra-field variations in vent fluid compositions. To determine whether this geochemical process affects the formation of microbial communities, we examined the microbial communities at three different vent sites located within a few tens meters of one another. In addition to chimney structures, colonization devices capturing subseafloor communities entrained by the vent fluids were studied, using culture-dependent and -independent methods. Microbiological analyses demonstrated the occurrence of distinctive microbial communities in each of the hydrothermal niches. Within a chimney structure, there was a transition from a mixed community of mesophiles and thermophiles in the exterior parts to thermophiles in the interior. Beside the transition within a chimney structure, intra-field variations in microbial communities in vent fluids were apparent. Geochemical analysis demonstrated that different vent fluids have distinctive end-member compositions as a consequence of subscafloor phase-separation and -segregation, which were designated gas-depleted, normal and gas-enriched fluids. In comparison to gas-depleted and normal fluids, gas-enriched fluids harbored more abundant chemolithoautotrophs with gaseous component-dependent energy metabolism, such as hydrogenotrophic methanogenesis. Subseafloor phase-separation and -segregation may play a key role in supplying energy and carbon sources to vent-associated chemolithoautotrophs and subvent microbial communities. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Change of bacterial community occurring along a hot water stream in the Hishikari gold mine, Japan, was investigated by applying a combination of various culture-independent techniques. The stream, which is derived from a subsurface anaerobic aquifer containing plentiful CO2, CH4, H-2, and NH4+, emerges in a mine tunnel 320 m below the surface providing nutrients for a lush microbial community that extends to a distance of approximately 7 m in the absence of sunlight-irradiation. Over this distance, the temperature decreases from 69 degrees C to 55 degrees C, and the oxidation-reduction potential increases from - 130 mV to + 59 mV. In the hot upper reaches of the stream, the dominant phylotypes were: 1) a deeply branching lineage of thermophilic methane-oxidizing gamma-Proteobacteria, and 2) a thermophilic hydrogen- and sulfur-oxidizing Sulfurihydrogenibium sp. In contrast, the prevailing phylotypes in the middle and lower parts of the stream were closely related to ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira spp.. Changes in the microbial metabolic potential estimated by competitive PCR analysis of genes encoding the enzymes, particulate methane monooxygenase (pmoA), ammonia monooxygenase (amoA), and putative nitrite oxidoreductase ( norB), also substantiated the community shift indicated by 16S rRNA gene analysis. The diversity of putative norB lineages was assessed for the first time in the hot water environment. Estimation of dominant phylotypes by whole-cell fluorescent in situ hybridization and changes in inorganic nitrogen compounds such as decreasing ammonium and increasing nitrite and nitrate in the mat-interstitial water along the stream were consistent with the observed transition of the bacterial community structure in the stream.

Molecular genetic methods were used to analyze the remnants of microbial ecosystems contained within an ancient oceanic microbial habitat that was recovered from a continental drilled core of black shale similar to 100 million years in age. Bacterial ribosomal RNA genes were vertically amplified from the six different depths of a black shale core associated with a phosphate-rich stratum, defined as one of the mid-Cretaceous oceanic anoxic events (OAEs). Although the black shale core was recovered from a terrestrial coring effort, the recovered 16S rRNA gene sequences showed affinity to microbial communities previously seen in deep-sea sedimentary environments (i.e., the microbial assemblage was easily recognizable as a marine community). In particular, a number of 16S rRNA gene clones of oceanic sulfate-reducing bacteria within the delta-Proteobacteria predominated at the OAE layer. The recovered bacterial DNA signatures are consistent with the interpretation that the sequences are derived from the past microbial communities buried in either sea-bottom or subseafloor environments during the sedimentation process and, after ceasing growth, preserved until the present.

Two novel denitrifying bacteria, designated strains MI55-1(T) and E9I37-1(T), were isolated from deep-sea hydrothermal vent chimney structures at the Iheya North hydrothermal field in the Mid-Okinawa Trough, Japan. Both isolates were strict chemolithoautotrophs growing by respiratory nitrate reduction with H-2, forming N-2 as a metabolic product. Oxygen (at low concentrations) could serve as an alternative electron acceptor for growth of the isolates. Growth of strain MI55-1(T) was observed at temperatures between 40 and 57 degrees C (optimum, 55 degrees C; doubling time, 2 h), at pH values between 5.4 and 6.9 (optimum, pH 6.4) and in the presence of between 1.5 and 4.0% (w/v) NaCl (optimum, 2.5%). Growth of strain E9I37-1(T) was observed at temperatures between 28 and 40 degrees C (optimum, 37 degrees C; doubling time, 2.5 h), at pH values between 5.6 and 7.6 (optimum, pH 7.0) and in the presence of between 1.5 and 3.5% (w/v) NaCl (optimum, 3.0%). The G+C contents of the genomic DNA of strains MI55-1(T) and E9I37-1(T) were 29.6 and 35.5 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strains MI55-1(T) and E9I37-1(T) belonged to groups A and F of the epsilon-Proteobacteria, but that they had distant phylogenetic relationships with any species, within the phylogenetic groups, that had validly published names (sequence similarities were less than 91%). On the basis of the physiological and molecular characteristics of the novel isolates, it is proposed that they should each be classified in a novel genus: Nitratiruptor tergarcus gen. nov., sp. nov., with MI55-1(T) (= JCM 12459(T) = DSM 16512(T)) as the type strain, and Nitratifractor salsuginis gen. nov., sp. nov., with E9I37-1(T) (= JCM 12458(T) = DSM 16511(T)) as the type strain.

A novel mesophilic, hydrogen-oxidizing, sulfur-reducing bacterium, designated strain BKB25Ts-Y-T, was isolated from hydrothermal sediments at Iheya North in the Mid-Okinawa Trough, Japan. Cells were Gram-negative, motile rods (1(.)8-2(.)1 mu m long and 0(.)5-0(.)7 mu m wide). The isolate was a strictly anaerobic chemolithoautotroph capable of using molecular hydrogen as the sole energy source and carbon dioxide as the sole carbon source. Elemental sulfur and nitrate served as electron acceptors, respectively yielding hydrogen sulfide and ammonium. Growth was observed at 20-42 degrees C (optimum 32 degrees C; 3 h doubling time), pH 5.0-6.5 (optimum 6.0) and in the presence of 2.0-4.0 % NaCl (optimum 2.5 %) via respiratory So reduction with H-2. The G + C content of the genomic DNA was 37.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate represented the first strain for which taxonomic properties have been characterized within the previously uncultivated epsilon-Proteobacteria Group G. On the basis of the physiological and molecular properties of the novel isolate, the genus name Thioreductor gen. nov. is proposed, with Thioreductor micantisoli sp. nov. as the type species. The type strain is BKB25Ts-Y-T (=JCM 12457(T) =DSM 16661(T)).

A novel, thermophilic, obligately chemolithoautotrophic, sulfur/thiosulfate-oxidizing bacterium was isolated from subsurface geothermal aquifer water (temperature approximately 70 degreesC) in the Hishikari gold mine, Japan. Cells of the isolate, designated strain C55(T), were motile, straight rods with a single polar flagellum. Growth was observed at temperatures between 35 and 62 degreesC (optimum 50-55 degreesC; 60 min doubling time) and pH between 5(.)2 and 7(.)7 (optimum pH 6(.)5-7(.)0). High growth rate of strain C55(T) was observed on either thiosulfate or elemental sulfur as a sole energy source, with molecular oxygen as the only electron acceptor. None of the organic compounds tested supported or stimulated growth of strain C55(T). The G+C content of the genomic DNA was 66(.)9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C55(T) was affiliated to the beta-Proteobacteria, but was distantly related to recognized genera. On the basis of its physiological and molecular properties, strain C55(T) (= JCM 12421(T) = DSM 16629(T) = ATCC BAA-941(T)) is proposed as the type strain of Thiobacter subterraneus gen. nov., sp. nov.

The population histories of neoverrucid barnacles occurring at 2 different hydrothermal vent systems around Japan, namely the Izu-Ogasawara Arc and the Okinawa Trough, were investigated based on the sequence variation in a 661 bp fragment of the mitochondrial COI gene. Compared with other barnacles, the investigated neoverrucids showed low levels of genetic diversity. The mismatch analysis suggested that these neoverrucids have experienced extinction-recolonization events associated with vent activity, and this may have led to the low levels of genetic diversity in neoverrucids around Japan. No haplotypes were shared between the Izu-Ogasawara Arc and the Okinawa Trough and no significant genetic differences were detected among the populations within each of these 2 regions. These results suggest that neoverrucid larvae only disperse within each area and are unable to migrate between the 2 areas. The haplotypes of the Okinawa Trough formed a paraphyletic cluster, with the exclusion of a monophyletic cluster from the Izu-Ogasawara Arc. Furthermore, the population expansion date of the Okinawa Trough, as estimated from the mismatch analysis, was earlier than that of the Izu-Ogasawara Arc. These results suggested that the neoverrucid population in the Okinawa Trough was a source of neoverrucids inhabiting the Izu-Ogasawara Arc.

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (similar to34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA) soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C, metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.

A novel extremely thermophilic, hydrogen- and sulfur-oxidizing bacterium, designated strain IBSK3(T), was isolated from a coastal hot spring in lbusuki, Kagoshima Prefecture, Japan. The cells were motile, straight to slightly curved rods (1(.)2-3(.)0 mum long and 0-3-0-4 mum wide), Strain IBSK3T was an obligate chemolithoautotroph growing by respiratory nitrate reduction with H-2, forming N2O as an end product. Low concentrations of O-2 (0-4-7-7 %, v/v; optimum 2-0 %, v/v) could serve as an alternative electron acceptor to growth. In addition, strain IBSK3T was able to utilize elemental sulfur as a sole electron donor with either nitrate or low concentrations Of 02 as an electron acceptor. Growth was observed between 55 and 77-5 degreesC (optimum 75 degreesC; 2 h doubling time), pH 5-5 and 8-3 (optimum pH 6-5-7-0)(1) and in the presence of 0(.)5 and 4(.)0% NaCl (optimum 2-0 %). The G +C content of the genomic DNA was 49(.)2 mol%. On the basis of 16S rRNA gene sequence analysis, strain IBSK3T belonged to the family Aquificaceae, but it only demonstrated a distant phylogenetic relationship with any recognized species within the family (sequence similarity was less than 92 %). On the basis of the physiological and molecular characteristics of the novel isolate, a new genus and novel species are proposed: the type strain of Hydrogenivirga caldilitoris gen. nov., sp. nov. is IBSK3(T) (=jCM 12173(T) =ATCC BAA-821(T)).

Distribution profiles of marine crenarchaeota group I in the vicinity of deep-sea hydrothermal systems were mapped with culture-independent molecular techniques. Planktonic samples were obtained from the waters surrounding two geographically and geologically distinct hydrothermal systems, and the abundance of marine crenarchaeota group I was examined by 16S ribosomal DNA clone analysis, quantitative PCR, and whole-cell fluorescence in situ hybridization. A much higher proportion of marine crenarchaeota group I within the microbial community was detected in deep-sea hydrothermal environments than in normal deep and surface seawaters. The highest proportion was always obtained from the ambient seawater adjacent to hydrothermal emissions and chimneys but not from the hydrothermal plumes. These profiles were markedly different from the profiles of epsilon-Proteobacteria, which are abundant in the low temperatures of deep-sea hydrothermal environments.

The phylogenetic diversity of sulfate-reducing prokaryotes occurring in active deep-sea hydrothermal vent chimney structures was characterized based on the deduced amino acid sequence analysis of the polymerase chain reaction-amplified dissimilatory sulfite reductase (DSR) gene. The DSR genes were successfully amplified from microbial assemblages of the chimney structures, derived from three geographically and geologically distinct deep-sea hydrothermal systems in the Central Indian Ridge (CIR), in the Izu-Bonin Arc (IBA). and the Okinawa Trough (OT), respectively. Phylogenetic analysis revealed seven major phylogenetic groups. More than half of the clones from the CIR chimney structure were related to DSR amino acid sequences of the hyperthermophilic archaeal members of the genus Archaeoglobus, and those of environmental DSR clones within the class Thermodesulfobacteria. From the OT chimney structure, a different group was obtained, which comprised a novel, deep lineage associated with the DSRs of the thermophilic sulfate-reducing bacterium Thermodesulfovibrio. Most of the DSR clones from the IBA chimney structure were phylogenetically associated with the delta-proteobacterial sulfate-reducing bacteria represented by the genus Desulfobulbus. Sequence analysis of DSR clones demonstrated a diverse sulfate-reducing prokaryotic community in the active deep-sea hydrothermal chimney structures. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Massive chimney structures, which arc characteristic of many hydrothermally active zones, harbor diverse Microbial communities containing both thermophilic and hyperthermophilic microbes. However, vent chimneys ultimately become hydrothermally inactive, and the changes that occur in the microbial communities upon becoming inactive have not been documented. We thus collected inactive chimneys from two geologically and geographically distinct hydrothermal fields, Iheya North in the western Pacific Ocean and the Kalrei field in the Indian Ocean. The chimneys displayed easily distinguishable strata, which were analyzed with regard to both mineralogical and microbiological properties. X-ray diffraction pattern and energy-dispersive spectroscopic analyses revealed that the main mineral components of the chimney substructures from Iheya North and the Kairei field were barite (BaSO4) and chalcopyrite (CuFeS2), respectively. Microbial cell densities in the substructures determined by DAPI counting ranged from 1.7 X 10(7) cells g(-1) to 3.0 X 10(8) cells g(-1). The proportions of archaeal rDNA in the whole microbial rDNA assemblages in all substructures were, at most, a few percent as determined by quantitative fluorogenic PCR. The microbial rDNA clone analysis and whole-cell fluorescence in Situ hybridization revealed a community that was decidedly different from any communities previously reported ill active chimneys. Curiously, both samples revealed the abundant presence of a group of Bacteria related to a magnetosome-bearing bacterium, "Magnetobacterium bavaricum" of the Nitrospirae division. These results suggest that inactive chimneys provide a distinct microbial habitat.

Recent microbiological surveys of terrestrial and oceanic subsurface biospheres have revealed that sizable microbial populations are present in global subsurface environments. However, little is known about the community structure, genetic diversity, and distribution pattern of subsurface bacteria and archaea since these surveys are mainly dependent on microscopic observations and conventional cultivation techniques. Culture-independent, molecular phylogenetic techniques are now utilized to explore microbial communities in various subsurface environments such as underground mines, subterrestrial rocks, continental and ocean oil reservoirs, subseafloor sediments and subvent microbial ecosystems. It has become apparent that unique archaeal components are commonly present in these subsurface microbial habitats. The most frequently recovered genetic signatures are of members of the hyperthermophiles Thermococcus. Their unexpected ubiquity even in non-extreme subsurface environments may represent the great biomass potential of probably dormant extremophilic archaea in the global subsurface biosphere. Archaeal populations in deep-sea hydrothermal vents and subvent environments might serve as sources of dormant extremophiles. It therefore appears likely that global and local ocean hydrothermal activities have had a persistent and significant impact on the formation of subsurface microbial communities and the distribution of subsurface microorganisms.

Distribution and phylogenetic diversity of microbial communities in hot, deep underground environments in the Hishikari epithermal gold mine, southern part of Kyushu, Japan, were evaluated using molecular phylogenetic analyses. Samples included drilled cores such as andesitic volcanic rock (0.95-1.78 Ma) and the oceanic sedimentary basement rock of Shimanto-Supergroup (100 Ma), as well as geothermal hot aquifer waters directly collected from two different sites: AW-site (71.5degreesC, pH 6.19) and XW-site (85.0degreesC, pH 6.80) at a depth of 350 mbls (meters below land surface). Based on PCR-amplified 16S rRNA gene clone analysis, the microbial communities in the drilled cores and the hot aquifer water from the XW-site consisted largely of the 16S rRNA gene sequences, closely related to the sequences often found in marine environments, while the aquifer water from the AW-site contained 16S rRNA gene sequences representing members of Aquificales, thermophilic methanotrophs within the gamma-subdivision of the Proteobacteria and uncultivated strains within the beta-subdivision of Proteobacteria. The cultivable microbial community detected by enrichment cultivation analysis largely matched that detected by the culture-independent molecular analysis.

A combined use of molecular ecological techniques and geochemical surveys revealed that thermophilic or hyperthermophilic microorganisms living in geothermal environments are likely to be implicated in the formation of biogenic siliceous deposits. Electron microscopic observations indicated that numerous microorganism-like fabrics were preserved in naturally occurring siliceous deposits such as siliceous sinter, geyserite, and silica scale, which suggests microbial contribution to silica precipitation. Molecular phylogenetic analyses suggested that extreme thermophilic bacteria within the genera Thermus and Hydrogenobacter are predominant components among the indigenous microbial community in siliceous deposits formed in pipes and equipment of Japanese geothermal power plants. These bacteria seem to actively contribute to the rapid formation of huge siliceous deposits. Additionally, in vitro examination suggested that Thermus cells induced the precipitation of supersaturated amorphous silica during the exponential growth phase, concomitant with the production of a specific cell envelope protein. Dissolved silica in geothermal hot water may be a significant component in the maintenance of position and survival of microorganisms in limited niches.

We report the successful cultivation and partial characterization of novel members of epsilon-Proteobacteria, which have long been recognized solely as genetic signatures of small subunit ribosomal RNA genes (rDNA) from a variety of habitats occurring in deep-sea hydrothermal fields. A newly designed microhabitat designated 'in situ colonization system' was used for enrichment. Based on phylogenetic analysis of the rDNA of the isolates, most of these represent the first cultivated members harboring previously uncultivated phylotypes classified into the Uncultivated epsilon-Proteobacteria Groups A, B, F and G, as well as some novel members of Group D. Preliminary characterization of the isolates indicates that all are mesophilic or thermophilic chemolithoautotrophs using H-2 or reduced sulfur compounds (elemental sulfur or thiosulfate) as an electron donor and O-2, nitrate or elemental sulfur as an electron acceptor. The successful cultivation will enable the subsequent characterization of physiological properties and ecological impacts of a diversity of epsilon-Proteobacteria in the deep-sea hydrothermal environments. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

Microbial community structure in the depth profile of a deep-sea sedimentary rock collected from the Sanriku Escarpment in the Japan Trench at a depth of 6337 m were analyzed using enrichment culture methods and culture-independent molecular phylogenetic techniques. The rock was subsampled at four depths (S1 to S4; from the surface to the inside), and carbon concentrations and colony-forming units (CFU) were determined under several culture conditions. Terminal-restriction fragment length polymorphism(T-RFLP) analysis of PCR-amplified 16S rRNA gene (rDNA) sequences indicated that a shift in bacterial and archaeal ribotype structures occurred in the sections at different depths from the surface. rDNA clone analysis revealed a significant change in microbial rDNA community structure. Bacterial community rDNA in sections S1 to S3 consisted of typical marine bacteria mainly members of the alpha and gamma-subclass of Proteobacteria, while the inner most section, S4, contained rDNA signatures for the beta-subclass of Proteobacteria and the High G(+) C Gram-Positive Group. Major archaeal rDNA clones shifted from Marine Group I (S1) to Thermococcales(S2-S4). The changes in bacterial and archaeal rDNA community structure indicated the possible infiltration of seawater and microorganisms into the rock and strongly suggested the isolation of endolithic microbial communities over the geological history of the rock.

Culture-dependent and -independent techniques were combined to characterize the physiological properties and the ecological impacts of culture-resistant phylotypes of thermophiles within the order Aquificales from a subsurface hot aquifer of a Japanese gold mine. Thermophilic bacteria phylogenetically associated with previously uncultured phylotypes of Aquificales were successfully isolated. 16S ribosomal DNA clone analysis of the entire microbial DNA assemblage and fluorescence in situ whole-cell hybridization analysis indicated that the isolates dominated the microbial population in the subsurface aquifer. The isolates were facultatively anaerobic, hydrogen- or sulfur/thiosulfate-oxidizing, thermophilic chemolithoautotrophs utilizing molecular oxygen, nitrate, ferric iron, arsenate, selenate, and selenite as electron acceptors. Their versatile energy-generating systems may reflect the geochemical conditions of their habitat in the geothermally active subsurface gold mine.

A depth profile of naturally occurring bacterial community structures associated with the deep-sea cold seep push-core sediment in the Japan Trench at a depth of 5343 m were evaluated using molecular phylogenetic analyses of RNA reverse transcription-PCR (RT-PCR) amplified 16S crDNA fragments. A total of 137 clones of bacterial crDNA (complimentary rDNA) phylotypes (phylogenetic types) obtained at three different depths (2-4, 8-10 and 14-16 cm) were identified in partial crDNA sequencings, crDNA phylotypes from the cold seep sediment were dominantly composed of δ- and ε-Proteobacteria (36% and 42% respectively). Phylotype analysis of crDNA clone libraries and terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the majority of bacterial components shifted from δ-Proteobacteria to ε-Proteobacteria with increasing depth. Among the δ-proteobacterial crDNA clones, the sequences related to the genus Desulfosarcina were dominant. Almost all sequences of crDNA belonging to ε-Proteobacteria were affiliated with the same cluster (ε-CSG: ε-proteobacterial cold seep group), and were closely related with rDNA sequences from deep-sea hydrothermal vent environments.

A record of the history of the Earth is hidden in the Earth's crust, like the annual rings of an old tree. From very limited records retrieved from deep underground, one can infer the geographical, geological, and biological events that occurred throughout Earth's history. Here we report the discovery of vertically shifted community structures of Archaea in a typical oceanic subseafloor core sample (1410 cm long) recovered from the West Philippine Basin at a depth of 5719 m. Beneath a surface community of ubiquitous deep-sea archaea (marine crenarchaeotic group I; MGI), an unusual archaeal community consisting of extremophilic archaea, such as extreme halophiles and hyperthermophiles, was present. These organisms could not be cultivated, and may be microbial relicts more than 2 million years old. Our discovery of archaeal rDNA in this core sample, probably associated with the past terrestrial volcanic and submarine hydrothermal activities surrounding the West Philippine Basin, serves as potential geomicrobiological evidence reflecting novel records of geologic thermal events in the Pleistocene period concealed in the deep-sea subseafloor.

Archaeal community structures in microhabitats in a deep-sea hydrothermal vent chimney structure were evaluated through the combined use of culture-independent molecular analyses and enrichment culture methods. A black smoker chimney was obtained from the PACMANUS site in the Manus Basin near Papua New Guinea, and subsamples were obtained from vertical and horizontal sections. The elemental composition of the chimney was analyzed in different subsamples by scanning electron microscopy and energy-dispersive X-ray spectroscopy, indicating that zinc and sulfur were major components while an increased amount of elemental oxygen in exterior materials represented the presence of oxidized materials on the outer surface of the chimney. Terminal restriction fragment length polymorphism analysis revealed that a shift in archaeal ribotype structure occurred in the chimney structure. Through sequencing of ribosomal DNA (rDNA) clones from archaeal rDNA clone libraries, it was demonstrated that the archaeal communities in the chimney structure consisted for the most part of hyperthermophilic members and extreme halopbiles and that the distribution of such extremophiles in different microhabitats of the chimney varied. The results of the culture-dependent analysis supported in part the view that changes in archaeal community structures in these microhabitats are associated with the geochemical and physical dynamics in the black smoker chimney.

At Steep Cone hot spring, Lower Geyser Basin, Yellowstone National Park, a mound of siliceous deposits called “siliceous sinter” forms from discharged boiling geothermal water that is supersaturated with amorphous silica. Electron-probe microanalyzer (EPMA) observations suggested that silica is deposited on the surface of the cells and that microorganisms affect the formation of the siliceous sinter. EPMA signals and backscattered electron images of sinter samples taken at the inner wall of the boiling pool revealed framboidal pyrite structures associated with a network of silicified microbial structures. The structure of bacterial community in a beige-colored sinter sediment (75.8°C, pH 8.6) was studied by molecular clone type phylogenetic analysis of PCR-mediated 16S rDNA fragments. The bacterial rDNA clones found indicated the presence of a complex community in the sinter sediment. Sequences closely related to the genera Thermus and Saccharomonospora were dominated evidence of indigenous microbial components. © 2001, Japanese Society of Microbial Ecology &amp The Japanese Society of Soil Microbiology. All rights reserved.

Silica scale, one of the major problems for geothermal power development, was formed on the copper test plates in geothermal hot water (85+/-2 degrees C, pH 7.2). Numerous bacterial shaped structures were observed and mixed bacterial population of genomic DNA was stably extracted from the silica scale. The amount of silica scale and extracted DNA increased exponentially with the time of incubation in the geothermal hot water. The molecular phyogenetic survey of extremely thermophilic bacterial diversities in silica scale was carried out by using the PCR-mediated small subunit rRNA gene (rDNA) sequencing. The community was composed mainly of three phylogenetic types in domain Bacteria. Cluster I was affiliated with the Aquificales and cluster II was closely related to the genus Thermus. Cluster III was homologus with Gram-positive anaerobic thermophilic bacteria. These bacterial communities may possibly contribute to the rapid aggregation of silica.

The bio-deposition of amorphous silica, which occurred in vitro by exposure to the extremely thermophilic bacterium Thermus spp. began from the latter part of the exponential phase of growth of the bacteria. The concentration with which the deposition occurred exceeded the solubility of amorphous silica of neutral pH at the temperature 60-85°C. Our observations suggest that Thermus spp. promotes the formation of siliceous minerals in a geothermal environment. © 1998 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

Numerous rod-shaped bacterial cells were present in amorphous silica which had formed on the copper plate placed in geothermal hot water, under conditions of pH 7.2 and 85 +/- 2 degrees C. Bulk genomic DNA in the siliceous deposit was-extracted, using lysozymes and the freeze-thaw method. The Volume of siliceous deposit formed on one copper plate and the amount of genomic DNA extracted from siliceous deposits exponentially increased with the time of incubation. The phylogenetic diversity in these DNA extracts was investigated by cloning and sequencing of partial 16S rRNA genes obtained by PCR. The bacterial community was composed mainly of three phylogenetic types in domain Bacteria. Cluster I (8 clones) was affiliated with the Aquificales and cluster II (15 clones) was closely related to the genus Thermus. Cluster III (2 clones), the sequences of which were homologous with Gram-positive anaerobic thermophilic bacteria, was also detected. These extremely thermophilic bacteria may possibly contribute to the rapid aggregation of amorphous silica.