The Oman Drilling Project established an “Active Alteration” multi‐borehole observatory in peridotites undergoing low‐temperature serpentinization in the Samail Ophiolite. The highly serpentinized ...rocks are in contact with strongly reducing fluids. Distinct hydrological regimes, governed by differences in rock porosity and fracture density, give rise to steep redox (Eh +200 to −750 mV) and pH (pH range 8.5–11.2) gradients within the 300–400 m deep boreholes. The serpentinites and fluids host an active subsurface ecosystem. Microbial cell abundances in serpentinite vary at least six orders of magnitude, from ≤3.5 × 101 to 2.9 × 107 cells/g. Low levels of biological sulfate reduction (2–1,000 fmol/cm3/day) can be detected in rock cores, particularly in rocks in contact with reduced groundwaters with pH < 10.5. Thermodesulfovibrio is the predominant sulfate reducer identified via metagenomic sequencing of adjacent groundwater communities. We infer that transport and reaction of microbially generated sulfide with the serpentine and brucite assemblages gives rise to optical darkening and sulfide overprinting, including the formation of tochilinite‐vallerite group minerals, potentially serving as an indicator that this system is inhabited by microbial life. Olivine mesh‐cores replaced with ferroan brucite and minor awaruite, abundant veins containing hydroandradite garnet and polyhedral serpentine, and late‐stage carbonate veins are suggested as targets for future spatially resolved life‐detection investigations. The high‐quality whole‐round core samples that have been preserved can be further probed to define how life distributes itself and functions within a system where chemical disequilibria are sustained by low‐temperature water/rock interaction, and how biosignatures of in situ microbial activity are generated.
Plain Language Summary
Ultramafic rocks undergoing water/rock interaction, and storing fluids that are far from chemical equilibrium, may be one of the most common habitats in our solar system. Through the Oman Drilling Project we collected >1 km of intact serpentinite in contact with groundwaters. These cores capture parts of the rock‐hosted biosphere and show how cells are distributed within serpentinites that vary in their mineralogical, physical and chemical properties. The cores are also biologically active, enabling us to detect specific metabolisms, such as when microorganisms combine hydrogen as reductant and sulfate as an oxidant to fuel their metabolism. Although the distribution of microbial cells in the rock cores is very heterogeneous, there are many intervals where the abundance of cells constitutes robust biomass. In the deeper cores, slow, albeit detectable, microbial sulfate reduction proceeds. We suggest that this pervasive biological activity releases byproducts such as sulfide that can react with the serpentinite and change the optical and chemical properties of the rocks. The feedbacks between the rock alteration and microbial activity produce markers that enable us to focus our search for rock‐hosted life and any specific biosignatures it may produce on Earth and perhaps on other planetary bodies.
Key Points
Highly serpentinized subsurface rocks exhibit steep redox gradients and host microbial cell abundances that vary >6 orders of magnitude
Low rates of microbial sulfate reduction in rock cores are inferred to result in optical darkening and sulfide overprinting of the mineralogy
Widespread andradite garnet, abundant ferroan brucite, and rare carbonate are targets for future spatially resolved life‐detection efforts
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.
Abstract
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.
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
m
2
and the pore size ranging from < 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 (< 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.
Abstract
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 (103–105 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.
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.
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.
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.
Summary
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) °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.
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