Phylogenetically diverse microorganisms have been observed in marine subsurface sediments down 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 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 ~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
and
within the phylum Firmicutes, respectively. The optimal pressure and temperature conditions for growth were 20 MPa and 42°C for strain 19R1-5, and 10 MPa and 43°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 ~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.
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
C-labeled carbon sources (glucose, acetate, bicarbonate, methanol, or methane), and
N-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
C and
N incorporation in individual cells, while isotope ratio monitoring-gas chromatography-mass spectrometry (IRM-GC-MS) was used to measure
CO
, and
CH
as metabolic byproducts. Glucose, acetate, and methanol were all assimilated by microorganisms under anoxic conditions.
CO
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
. The primary genera detected in the incubations,
and
, 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.
In this announcement, we present the set of putative terpene synthase (TS) gene fragments detected in a subseafloor sediment sample collected off Shimokita Peninsula, Japan. This data set contains ...sequences with 72 to 100% identity to TS from actinobacteria and cyanobacteria.
Serpentinitic systems are potential habitats for microbial life due to frequently high concentrations of microbial energy substrates, such as hydrogen (H
), methane (CH
), and short-chain organic ...acids (SCOAs). Yet, many serpentinitic systems are also physiologically challenging environments due to highly alkaline conditions (pH > 10) and elevated temperatures (>80°C). To elucidate the possibility of microbial life in deep serpentinitic crustal environments, International Ocean Discovery Program (IODP) Expedition 366 drilled into the Yinazao, Fantangisña, and Asùt Tesoru serpentinite mud volcanoes on the Mariana Forearc. These mud volcanoes differ in temperature (80, 150, 250°C, respectively) of the underlying subducting slab, and in the porewater pH (11.0, 11.2, 12.5, respectively) of the serpentinite mud. Increases in formate and acetate concentrations across the three mud volcanoes, which are positively correlated with temperature in the subducting slab and coincide with strong increases in H
concentrations, indicate a serpentinization-related origin. Thermodynamic calculations suggest that formate is produced by equilibrium reactions with dissolved inorganic carbon (DIC) + H
, and that equilibration continues during fluid ascent at temperatures below 80°C. By contrast, the mechanism(s) of acetate production are not clear. Besides formate, acetate, and H
data, we present concentrations of other SCOAs, methane, carbon monoxide, and sulfate, δ
C-data on bulk carbon pools, and microbial cell counts. Even though calculations indicate a wide range of microbial catabolic reactions to be thermodynamically favorable, concentration profiles of potential energy substrates, and very low cell numbers suggest that microbial life is scarce or absent. We discuss the potential roles of temperature, pH, pressure, and dispersal in limiting the occurrence of microbial life in deep serpentinitic environments.
Abstract
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 (13–30 cm 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.
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 (<74°C) and from one cold station (<9°C). Cell abundance at the two hydrothermal stations dropped from 10
to 10
cells cm
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 10
cells cm
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 %T
C (percentage of total organic carbon present as amino acid carbon), %T
N (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.
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.
•Thermal histories for the underthrust domain of the Nankai Accretionary Complex, IODP 370 "Temperature Limits of the Deep Biosphere".•Hydrothermal and burial metamorphism at IODP Site C0023 with basin models.•Fluid inclusion homogenisation temperatures significantly hotter than present-day formation temperatures.•First direct evidence for anomalously hot fluids in underthrust sediments of Nankai Accretionary Complex.•Biomarkers in underthrust sediments record rise from pre-oil window to early oil window thermal maturities.
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.
Abstract
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 101. 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.
The comparative study of viral abundances across the three subseafloor sediments demonstrates that the viruses are regulated by production, preservation and decay.
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 since it enables one to observe specimens using both SEM and optical microscopes.
PDF
