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.
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 (µXCT) and scanning electron microscopy (SEM). The µXCT 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.
X-ray computed tomography (XCT) can be used to identify lithologies and deformation structures within geological core, with the potential for the identification processes to be applied automatically. ...However, because of drilling disturbance and other artifacts, the use of large XCT-datasets in automated processes requires methods of quality control that can be applied systematically. We propose a new systematic method for quality control of XCT data that applies numerical measures to CT slices, and from this obtains data reflective of core quality. Because the measures are numerical they can be applied quickly and consistently between different sections and cores. This quality control processing protocol produces downhole radiodensity profiles from mean CT-values that can be used for geological interpretation. The application of this quality control protocols was applied to XCT data from International Ocean Discovery Program (IODP) Expedition 370 Site C0023 located at the toe of the Nankai accretionary complex. The evaluation of core quality based on this protocol was found to be a good fit to standard-evaluations based on the visual description of core, and could be used to select samples free from drilling disturbance or contamination. The quality-controlled downhole mean CT-value profile has features that can be used to identify lithologies within a formation, the presence and type of deformation structures and to distinguish formations.
“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 ~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 10⁷ 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.
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 104 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.
Geological CO2 sequestration in unmineable subsurface oil/gas fields and coal formations has been proposed as a means of reducing anthropogenic greenhouse gasses in the atmosphere. However, the ...feasibility of injecting CO2 into subsurface depends upon a variety of geological and economic conditions, and the ecological consequences are largely unpredictable. In this study, we developed a new flow-through-type reactor system to examine potential geophysical, geochemical and microbiological impacts associated with CO2 injection by simulating in-situ pressure (0-100 MPa) and temperature (0-70°C) conditions. Using the reactor system, anaerobic artificial fluid and CO2 (flow rate: 0.002 and 0.00001 ml/min, respectively) were continuously supplemented into a column comprised of bituminous coal and sand under a pore pressure of 40 MPa (confined pressure: 41 MPa) at 40°C for 56 days. 16S rRNA gene analysis of the bacterial components showed distinct spatial separation of the predominant taxa in the coal and sand over the course of the experiment. Cultivation experiments using sub-sampled fluids revealed that some microbes survived, or were metabolically active, under CO2-rich conditions. However, no methanogens were activated during the experiment, even though hydrogenotrophic and methylotrophic methanogens were obtained from conventional batch-type cultivation at 20°C. During the reactor experiment, the acetate and methanol concentration in the fluids increased while the δ(13)Cacetate, H2 and CO2 concentrations decreased, indicating the occurrence of homo-acetogenesis. 16S rRNA genes of homo-acetogenic spore-forming bacteria related to the genus Sporomusa were consistently detected from the sandstone after the reactor experiment. Our results suggest that the injection of CO2 into a natural coal-sand formation preferentially stimulates homo-acetogenesis rather than methanogenesis, and that this process is accompanied by biogenic CO2 conversion to acetate.
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.
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 (δ13C) of acetate in pore water of two core samples stored at near in-situ temperatures (4°C) within 3 hours and at room temperatures (25°C) for 19 hours after sample recovery. Acetate concentrations at 4°C were less than 3 μM throughout the sediment column, whereas they increased up to 13 μM in the samples stored at 25°C and corresponding δ13C values of acetate were enriched up to 8.7‰ 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 pore-water geochemical analysis.
Abstract Guaymas Basin, located off the Gulf of California, is a hydrothermally active marginal basin. Due to steep geothermal gradients and localized heating by sill intrusions, microbial substrates ...like short-chain fatty acids and hydrocarbons are abiotically produced from sedimentary organic matter at comparatively shallow depths. We analyzed the effect of hydrocarbons on uptake of hydrocarbons by microorganisms via nanoscale secondary ion mass spectrometry (NanoSIMS) and microbial sulfate reduction rates (SRR), using samples from two drill sites sampled by IODP Exp. 385 (U1545C and U1546D). These sites are in close proximity of each other (ca. 1 km) and have very similar sedimentology. Site U1546D experienced the intrusion of a sill that has since then thermally equilibrated with the surrounding sediment. Both sites currently have an identical geothermal gradient, despite their different thermal history. The localized heating by the sill led to thermal cracking of sedimentary organic matter and formation of potentially bioavailable organic substrates. There were low levels of hydrocarbon and nitrogen uptake in some samples from both sites, mostly in surficial samples. Hydrocarbon and methane additions stimulated SRR in near-seafloor samples from Site U1545C, while samples from Site U1546D reacted positively only on methane. Our data indicate the potential of microorganisms to metabolize hydrocarbons even in the deep subsurface of Guaymas Basin.
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.
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