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Biogeochemical processes in subseafloor sediments are closely coupled to global element cycles. To improve the understanding of changes in biogeochemical conditions on geological timescales, we investigate sediment cores from a 1,180 m deep hole in the Nankai Trough offshore Japan (Site C0023) drilled during International Ocean Discovery Program Expedition 370. During its tectonic migration from the Shikoku Basin to the Nankai Trough over the past 15 Ma, Site C0023 has experienced significant changes in depositional, thermal, and geochemical conditions. By combining pore-water, solid-phase, and rock magnetic data, we demonstrate that a transition from organic carbon-starved conditions with predominantly aerobic respiration to an elevated carbon burial environment with increased sedimentation occurred at ∼2.5 Ma. Higher rates of organic carbon burial in consequence of increased nutrient supply and productivity likely stimulated the onset of anaerobic electron-accepting processes during organic carbon degradation. A significant temperature increase by ∼50°C across the sediment column associated with trench-style sedimentation since ∼0.5 Ma could increase the bioavailability of organic matter and enhance biogenic methanogenesis. The resulting shifts in reaction fronts led to diagenetic transformation of iron (oxyhydr)oxides into pyrite in the organic carbon-starved sediments several millions of years after burial. We also show that high amounts of reducible iron(III) which can serve as electron acceptor for microbial iron(III) reduction are preserved and still available as phyllosilicate-bound iron. This is the first study that shows the evolution of long-term variations of (bio-)geochemical processes along tectonic migration of ocean floor, thereby altering the primary sediment composition long after deposition.

Organic matter (OM) in surface soil is largely present within porous mineral-dominant clusters called aggregates. Recent studies have shown the localization of OM within large macroaggregate structure based on synchrotron X-ray micro-computed tomography (μCT) coupled with a vapor-phase, osmium (Os)-staining pretreatment. Here we developed a new approach to identify OM locations within submillimeter-sized aggregates at higher spatial resolution and assessed its applicability using a well-characterized surface volcanic soil, an allophanic Andisol, under no-tillage with leaf compost addition. Water-stable aggregates (500–800 μm in diameter) were embedded in agarose, chemically fixed, and repeatedly stained by osmium tetroxide and thiocarbohydrazide, followed by gradual dehydration with ethanol and resin fixation. Scanning electron microscopy and μCT observation showed no signs of physical alteration during sample preparation. Energy-dispersive X-ray spectroscopy analysis of the microtome-cut surface of the aggregate identified plant detritus highly enriched in Os and much smaller amorphous OM (

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

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 (

Microbial cell counting provides essential information for the study of cell abundance profiles and biogeochemical interactions with the surrounding environments. However, it often requires labor-intensive and time-consuming processes, particularly for subseafloor sediment samples, in which non-cell particles are abundant. We developed a rapid and straightforward method for staining microbial intracellular DNA by SYBR Green I (SYBR-I) to enumerate cells by flow cytometry (FCM). We initially examined the efficiency of microbial cell staining at various dye/sediment ratios (volume ratio of SYBR-I/sediment [vSYBR/vSed]). Non-cell particles in sediment strongly and preferentially adsorbed SYBR-I dye, resulting in the unsuccessful staining of microbial cells when an insufficient ratio (106 cells cm–3). Samples with low cell abundance (

Deep, hot, and more alive than we thought Marine sediments represent a massive microbial ecosystem, but we still do not fully understand what factors shape and limit life underneath the seafloor. Analyzing samples from a subduction zone off the coast of Japan, Heuer et al. found that microbial life, in particular bacterial vegetative cells, decreases as depth and temperature increases down to ∼600 meters below the seafloor, corresponding to temperatures of ∼70°C. Below this limit, endospores are common—a remnant, and a potential reservoir, of bacterial life. Deeper still is a sterile zone, and below 1000 meters is a scalding realm populated by vegetative cells. At such great depths, high concentrations of acetate and sulfate coexist, and there are also signs of hyperthermophilic methanogenesis. These data provide a fascinating window into an extreme and inhospitable environment that nonetheless supports microbial life. Science , this issue p. 1230

Fluorescence in situ hybridization (FISH) is a widely used molecular technique in microbial ecology. However, the non-specific adsorption of fluorescent probes and resulting high intensity of background signals from mineral particles hampers the specific detection of microbial cells in grain-rich environmental samples, such as subseafloor sediments. We herein demonstrated that a new buffer composition containing EDTA efficiently reduced the adsorption of probes without compromising the properties of the FISH-based probing of microbes. The inclusion of a high concentration of EDTA in the buffer in our protocol provides a simple and effective approach for reducing the background in FISH for environmental samples.

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., Yohey Suzuki, Seiya Yamashita et al. discover the presence of bacterial cells in the iron-rich smectite on aged subseafloor basaltic rock using nanoscale solid characterizations. Analysis of their lipid profiles and DNA sequences reveals the dominance of heterotrophic bacteria, suggesting the presence of organic matter resources in the subsea basalt.

South Chamorro Seamount (SCS) is a blueschist-bearing serpentinite mud volcano in the Mariana forearc. Previous scientific drilling conducted at SCS revealed highly alkaline, sulfate-rich formation fluids resulting from slab-derived fluid upwelling combined with serpentinization both beneath and within the seamount. In the present study, a time-series of ROV dives spanning 1000 days was conducted to collect discharging alkaline fluids from the cased Ocean Drilling Program (ODP) Hole 1200C (hereafter the CORK fluid). The CORK fluids were analyzed for chemical compositions (including dissolved gas) and microbial community composition/function. Compared to the ODP porewater, the CORK fluids were generally identical in concentration of major ions, with the exception of significant sulfate depletion and enrichment in sulfide, alkalinity, and methane. Microbiological analyses of the CORK fluids revealed little biomass and functional activity, despite habitable temperature conditions. The post-drilling sulfate depletion is likely attributable to sulfate reduction coupled with oxidation of methane (and hydrogen), probably triggered by the drilling and casing operations. Multiple lines of evidence suggest that abiotic organic synthesis associated with serpentinization is the most plausible source of the abundant methane in the CORK fluid. The SCS formation fluid regime presented here may represent the first example on Earth where abiotic syntheses are conspicuous with little biotic processes, despite a condition with sufficient bioavailable energy potentials and temperatures within the habitable range., Progress in Earth and Planetary Science, 5

AIMS Using substrate-induced gene-expression (SIGEX) screening on subseafloor sediment samples from the Nankai Trough, Japan, we identified gene fragments showing an induction response to metal ions. METHODS AND RESULTS Environmental DNA libraries in Escherichia coli host cells were tested by the addition of metal ions (Ni2+ , Co2+ , Ga3+ or Mo6+ ), followed by cell sorting of clones exhibiting green fluorescence upon co-expression of green fluorescence protein downstream of the inserted gene fragments. One clone displayed Ni2+ -specific induction, three clones displayed Ga3+ -specific induction and three clones displayed an induction response to multiple metal ions. DNA sequence analysis showed that a variety of genes showed induction responses in the screened clones. CONCLUSIONS Using the SIGEX approach, we retrieved gene fragments with no previously identified response to metal ions that exhibited metal-ion-induced expression. This method has the potential to promote exploration of gene function through gene-induction response. SIGNIFICANCE AND IMPACT OF THE STUDY We successfully linked gene-induction response with sequence information for gene fragments of previously unknown function. The SIGEX-based approach exhibited the potential to identify genetic function in unknown gene pools from the deep subseafloor biosphere, as well as novel genetic components for future biotechnological applications.

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.

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

Archaeal DNA recombination mechanism and the related proteins are similar to those in eukaryotes. However, no functional homolog of eukaryotic MutSγ, 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 MutSγ, 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 MutSγ. 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 MutSγ and bacterial MutS2, a Holliday junction-resolving MutS 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 MutSγ. 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 MutSγ.

A scanning electron microscope (SEM) is a very common instrument to observe fossil specimens. Although a low-vacuum SEM is now getting to be a common tool, still a conductive coating is required for observation of specimens at higher magnification. For SEM images of nonconductive fossil specimens, the specimens are usually coated with metals such as gold and platinum. The metal sputter coating method has a problem that the coating reduces the transparency of the fossil specimens when observed under a transmitted optical microscope. Therefore, the metal coating may not be suitable for observation of types in museum research collections. We applied an osmium plasma coating to the SEM preparation of microfossil specimens and found that the osmium coating neither shades transmitted light through the specimens nor masks the internal structures of the specimens. Also this method allowed us to observe specimens at high magnification. The osmium coating has an advantage over the metal sputter coating si...

Summary 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.

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.

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 (10 2 to 10 3 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., Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe, 802

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 (

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

Protocol for cell separation, enumeration, and sorting of low biomass samples for SIP-NanoSIMS preparations.

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 210Pb and 137Cs. 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.

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°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., ∼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.

567 The concentration and carbon isotopic composition (δ13C) of acetate in marine sediment are such recent additions to the pore-water geochemical analyses, following the technological advancement of a sensitive analytical tool (Heuer et al., 2006), which was developed to understand subseafloor biogeochemical processes via acetate. It has been pointed out that acetate in sediment could be sensitive to a temperature increase, resulting in increased acetate concentrations after incubation of sediment at high temperature (Wellsbury et al., 1997). In this brief note, we simulated two possible cases of shipboard sample processing; i) short-term storage of the corer with rubber stoppers in a refrigerator at near in-situ temperature (4°C) until pore water extraction to minimize air contamination and temperature change represents an ordinarily procedure on board, even though a small temperature change from the absolute in-situ temperature to 4°C and air contamination is unavoidable, ii) storage at room temperature for 19 hours represents a worst possible condition. Such a worst case occasionally occur to cut core samples which have to be warmed to room temperature for non-destructive measurement of physical properties using a multi-sensor core logger (MSCL) for 12–17 hours before pore-water extraction (Ijiri et al., 2012). Those two samples have been provided to meaIncrease in acetate concentrations during sediment sample onboard storage: a caution for pore-water geochemical analyses

The discovery of a microbial ecosystem in ocean sediments has evoked interest in life under extreme energy limitation and its role in global element cycling. However, fundamental parameters such as the size and the amount of biomass of sub-seafloor microbial cells are poorly constrained. Here we determined the volume and the carbon content of microbial cells from a marine sediment drill core retrieved by the Integrated Ocean Drilling Program (IODP), Expedition 347, at Landsort Deep, Baltic Sea. To determine their shape and volume, cells were separated from the sediment matrix by multi-layer density centrifugation and visualized via epifluorescence microscopy (FM) and scanning electron microscopy (SEM). Total cell-carbon was calculated from amino acid-carbon, which was analyzed by high-performance liquid chromatography (HPLC) after cells had been purified by fluorescence-activated cell sorting (FACS). The majority of microbial cells in the sediment have coccoid or slightly elongated morphology. From the sediment surface to the deepest investigated sample (∼60 m below the seafloor), the cell volume of both coccoid and elongated cells decreased by an order of magnitude from ∼0.05 to 0.005 μm3. The cell-specific carbon content was 19–31 fg C cell−1, which is at the lower end of previous estimates that were used for global estimates of microbial biomass. The cell-specific carbon density increased with sediment depth from about 200 to 1000 fg C μm−3, suggesting that cells decrease their water content and grow small cell sizes as adaptation to the long-term subsistence at very low energy availability in the deep biosphere. We present for the first time depth-related data on the cell volume and carbon content of sedimentary microbial cells buried down to 60 m below the seafloor. Our data enable estimates of volume- and biomass-specific cellular rates of energy metabolism in the deep biosphere and will improve global estimates of microbial biomass.

Microbial biomolecules, typically from the cell envelope, can provide crucial information about distribution, activity, and adaptations of sub-seafloor microbial communities. However, when cells die these molecules can be preserved in the sediment on timescales that are likely longer than the lifetime of their microbial sources. Here we provide for the first time measurements of the cellular content of biomolecules in sedimentary microbial cells. We separated intact cells from sediment matrices in samples from surficial, deeply buried, organic-rich, and organic-lean marine sediments by density centrifugation. Amino acids, amino sugars, muramic acid, and intact polar lipids were analyzed in both whole sediment and cell extract, and cell separation was optimized and evaluated in terms of purity, separation efficiency, taxonomic resemblance, and compatibility to high-performance liquid chromatography and mass spectrometry for biomolecule analyses. Because cell extracts from density centrifugation still contained considerable amounts of detrital particles and non-cellular biomolecules, we further purified cells from two samples by fluorescence-activated cell sorting (FACS). Cells from these highly purified cell extracts had an average content of amino acids and lipids of 23–28 fg cell-1 and 2.3 fg cell-1, respectively, with an estimated carbon content of 19–24 fg cell-1. In the sediment, the amount of biomolecules associated with vegetative cells was up to 70-fold lower than the total biomolecule content. We find that the cellular content of biomolecules in the marine subsurface is up to four times lower than previous estimates. Our approach will facilitate and improve the use of biomolecules as proxies for microbial abundance in environmental samples and ultimately provide better global estimates of microbial biomass.

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.

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO(2) in the seabed. The emission of CO(2) 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-CO(2) 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 CO(2)-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 CO(2) 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 CO(2) concentration, indicating that microbial activity and community structure are sensitive to CO(2) venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13-30 cm in depth) characterized by high CO(2). 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 CO(2)-seep sedimentary environment; however, CO(2) and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.

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.

In deep subsurface sediments of the Juan de Fuca Ridge Flank, porewater acetate that is depleted in 13 C 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 (H 2 ) 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.

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: 528/38 nm (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.

A technique to distinguish viable and dead cells has long been considered necessary in various fields such as sterilization, toxicity assessment, sanitary evaluation and so on. A bacterial staining method using fluorescent dye is a popular tool, although the weakness of fluorescence intensity and its fading over time constitute notable drawbacks. In the process of esterase-active bacteria staining with carboxyfluorescein diacetate (CFDA), we have reported glutaraldehyde (GTA) affected the discriminative recognition of bacteria due to prevention of fluorescence leakage from the cell. In this study, CFDA was applied to four pure bacterial strains (two Gram-negative strains and two Gram-positive strains) during the exponential growth phase and to activated sludge as an indicator of microbial viability. GTA concentration was also optimized and the effect of GTA addition was compared to the conventional method using ethylenediamine tetraacetic acid (EDTA) and the control without pretreatment. At higher concentrations of GTA, microbial viability decreased because of GTA toxicity. In the case of all conditions where CFDA staining was carried out in the assay of microbial viability, the highest viability was achieved by using of 1 g/L GTA.

A deep sleep in coal beds Deep below the ocean floor, microorganisms from forest soils continue to thrive. Inagaki et al. analyzed the microbial communities in several drill cores off the coast of Japan, some sampling more than 2 km below the seafloor (see the Perspective by Huber). Although cell counts decreased with depth, deep coal beds harbored active communities of methanogenic bacteria. These communities were more similar to those found in forest soils than in other deep marine sediments. Science , this issue p. 420 ; see also p. 376

The depth of oxygen penetration into marine sediments differs considerably from one region to another. In areas with high rates of microbial respiration, O2 penetrates only millimetres to centimetres into the sediments, but active anaerobic microbial communities are present in sediments hundreds of metres or more below the sea floor. In areas with low sedimentary respiration, O2 penetrates much deeper but the depth to which microbial communities persist was previously unknown. The sediments underlying the South Pacific Gyre exhibit extremely low areal rates of respiration. 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 O2 and nitrate, we suggest that net aerobic respiration in these sediments is coupled to oxidation of marine organic matter. We identify a relationship of O2 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.

We have constructed a bacterial library consisting of 97 strains of toluene-degrading bacteria from soil and activated sludge samples for examining their physiological properties in terms of cometabolism of TCE. Large variation of TCE degradation ability was observed in Gram-positive and Gram-negative strains, as well as diverse patterns of availability of aromatics as a growth substrate. No clear correlation was observed between the number of available substrates for growth and TCE degradation ability. However, the growth on some of the aromatics showed positive or negative correlations with TCE degradation ability. Kendall correlation constants (tau) for the growth on cumene, m-xylene, p-xylene, and m-cresol with TCE degradation ability were statistically significant (P

We have previously constructed a bacterial library consisting of toluene-degrading bacteria isolated from soil, activated sludge, and trichloroethylene (TCE) waste. In the present study, the library was subjected to colony hybridization with todC1C2-encoding terminal oxygenase of a multicomponent enzyme that is responsible for the first oxidation of toluene, toluene dioxygenase (TDO). Three strains, A1071, IB5, and B6122, which showed positive signals with todC1C2 by the colony hybridization, were selected on the basis of results of a preliminary degradation assay for detail analyses of degradation kinetics of toluene and TCE. Their possession of the gene homologous to todC1, a large subunit of TDO of Pseudomonas putida F1, was confirmed by dot hybridization analysis using their chromosomal DNA extracts as templates. Results of degradation experiments using resting cells revealed the variety in TCE degradation ability among these strains; A1071 demonstrated degradation kinetics similar to the strain F1 while B6122 could not degrade TCE at all. The interaction between toluene and TCE on their degradation could be simply expressed by competitive inhibition. © 2005 Elsevier B.V. All rights reserved.

Radiation effects on the optical absorption of orthorhombic perovskite-type oxide (Yb-doped SrCeO3) ceramics have been studied at room temperature using 1.8 MeV electrons at a dose rate of 300 Gy/s. The optical absorption in the wavelength range 460 to 800 nm is significantly reduced by annihilation of two absorption bands at approximately 585 and 695 nm as the electron irradiation dose increases up to 7.6 MGy. This is associated with Ce4+ to Ce3+ conversion. Under these irradiation conditions, segregation of SrO to the sample surface was also observed. Further irradiation up to 15 MGy reduced the optical density in the infrared wavelength range between 800–2800 nm due to dissociation of water on the sample surface, activated by radiation damage. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

The rate of trichloroethylene (TCE) degradation by toluene dioxygenase (TDO) in resting cells of Pseudomonas putida F1 gradually decreased and eventually stopped within 1.5 h, as in previous reports. However, the subsequent addition of toluene, which is the principal substrate of TDO, resulted in its immediate degradation without a lag phase. After the consumption of toluene, degradation of TCE restarted at a rate similar to its initial degradation, suggesting that this degradation was mediated by TDO molecules that were present before the cessation of TCE degradation. The addition of benzene and cumene, which are also substrates of TDO, also caused restoration of TCE degradation activity: TCE was degraded simultaneously with cumene, and a larger amount of TCE was degraded after cumene was added than after toluene or benzene was added. But substrates that were expected to supply the cells with NADH or energy did not restore TCE degradation activity. This cycle of pseudoinactivation and restoration of TCE degradation was observed repeatedly without a significant decrease in the number of viable cells, even after six additions of toluene spread over 30 h. The results obtained in this study demonstrate a new type of restoration of TCE degradation that has not been previously reported.

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.

Volume 80, no. 6, p. 1985–1994, 2014. Page 1986, column 1, paragraph 5, lines 7 to 10: “For the PowerMax soil DNA isolation kit, the sediment was vigorously shaken with autoclaved metal beads (5 mm in diameter) using a Shake Master auto (Bio Medical Science, Tokyo, Japan) operated at 1,500 rpm

Summary 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 ∼ 50% of cells were recovered from deep samples (100–365 m 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 104–108 cells cm−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.

Radiation induced optical absorption and radioluminescence impose important limitations on the use of any optical materials under ionizing radiation. Optical transmission of a type of high purity silica glass, KU1 quartz glass, has been studied under radiation (gamma 60Co and 1.8 Mev electrons). The results show that this material maintain their transmission properties, from 400 to 2600 nm under high levels of ionizing radiation and with atomic displacements, during long time. Also radioluminescence produced by electrons and radiation effect on laser damage in this material have been studied.La absorción óptica inducida por radiación así como la emisión de luz (radioluminiscencia) imponen severas limitaciones en el uso de materiales ópticos dentro de un campo de radiación. Para un tipo de sílice de alta pureza, cuarzo KU1, se ha estudiado el efecto de la radiación (gamma 60Co y electrones de 1.8 Mev), en la transmisión óptica. Se ha visto que este material mantiene sus propiedades de transmisión óptica, entre 400 y 2600 nm, en altos niveles de radiación tanto puramente ionizante como con desplazamientos, durante largos periodos de tiempo. También se ha estudiado la radioluminiscencia producida con electrones y el efecto de la radiación en el daño producido por láser en este material.

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 ∼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, β-alanine and γ-amino butyric acid, all indicated increasing degradation state of the organic matter with sediment depth and age. Quantification of DPA showed that endospores 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 × 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.

In this study, we report experimental evidence of the thioautotrophic activity of the epibiotic microbial community associated with the setae of Shinkaia crosnieri, a galatheid crab that is endemic to deep-sea hydrothermal systems in the Okinawa Trough in Japan. Microbial consumption of reduced sulfur compounds under in situ hydrostatic and atmospheric pressure provided evidence of sulfur-oxidizing activity by the epibiotic microbial community; the rate of sulfur oxidation was similar under in situ and decompressed conditions. Results of the microbial consumption of reduced sulfur compounds and tracer experiments using (13)C-labeled bicarbonate in the presence and absence of thiosulfate (used as a thioautotrophic substrate) convincingly demonstrated that the epibiotic microbial community on S. crosnieri drove primary production via an energy metabolism that was coupled with the oxidation of reductive sulfur compounds. A combination of tracer experiments, fluorescence in situ hybridization (FISH) and nano-scale secondary ion mass spectrometry (Nano-SIMS) indicated that the filamentous cells of the genus Sulfurovum belonging to the class Epsilonproteobacteria were thioautotrophs in the epibiotic community of S. crosnieri. In conclusion, our results strongly suggest that thioautotrophic production by Sulfurovum members present as the epibiotic microbial community play a predominant role in a probable nutritional ectosymbiosis with S. crosnieri.

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 (10(3)-10(5) cells cm(-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.

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 °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 °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 °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.

Deep drilling into the marine sea floor has uncovered a vast sedimentary ecosystem of microbial cells. 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 culture-independent techniques, no clear picture has yet emerged as to whether Archaea or Bacteria are more abundant in this extensive ecosystem. 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, 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.