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Abstract Microbes in marine sediments detected and counted by direct observation of membrane-filtered sediment samples stained with acridine orange. This technique can still be applied to high-biomass (> 105 cells/cm3) sedimentary habitats, such as organic-rich sediments collected in shallow areas near the seafloor. However, to further explore the nutrients and energy turnover under extremely low energy flux conditions, or in habitats that are close to the lower limit of the biosphere, technological breakthroughs have been required to increase the detection sensitivity for microbial life at densities of a few cells/cm3 of sediment. These technological developments contributed to increasing fundamental information on microbial life at the fringes of the subseafloor biosphere and led to the discovery of revivable microbes in sediments aged up to 101.5 million years old. More recently, chemical detection methods have revealed the existence of spores in the deep biosphere that are impermeable to conventional DNA stains. Previous applications of molecular biology-based approaches have been limited to relatively higher biomass samples, potentially because the cells surviving in these very low energy flux environments have less integrated genomes. Here, I review the contribution and importance of the technological developments that have been made in the study of microbes from the subseafloor biosphere, recent developments of alternative methods to microscopically detect microbial spores and their application to deep subseafloor sediments, and the challenges associated with applying molecular biological approaches to study low-biomass samples.

Microbes in marine sediments detected and counted by direct observation of membrane-filtered sediment samples stained with acridine orange. This technique can still be applied to high-biomass (> 105 cells/cm3) sedimentary habitats, such as organic-rich sediments collected in shallow areas near the seafloor. However, to further explore the nutrients and energy turnover under extremely low energy flux conditions, or in habitats that are close to the lower limit of the biosphere, technological breakthroughs have been required to increase the detection sensitivity for microbial life at densities of a few cells/cm3 of sediment. These technological developments contributed to increasing fundamental information on microbial life at the fringes of the subseafloor biosphere and led to the discovery of revivable microbes in sediments aged up to 101.5 million years old. More recently, chemical detection methods have revealed the existence of spores in the deep biosphere that are impermeable to conventional DNA stains. Previous applications of molecular biology-based approaches have been limited to relatively higher biomass samples, potentially because the cells surviving in these very low energy flux environments have less integrated genomes. Here, I review the contribution and importance of the technological developments that have been made in the study of microbes from the subseafloor biosphere, recent developments of alternative methods to microscopically detect microbial spores and their application to deep subseafloor sediments, and the challenges associated with applying molecular biological approaches to study low-biomass samples. [ABSTRACT FROM AUTHOR]

In recent years, research funding agencies, universities, and governments have become increasingly concerned with promoting the reuse of research datasets. Enabling researchers to evaluate the trustworthiness and fitness-for-use of research datasets produced by others is critical for facilitating the reuse of these datasets. Understanding how researchers make these evaluations is crucial for developing digital infrastructure and tools, such as data repositories and metadata schema, in a way that better supports researchers in making these evaluations. Physical samples such as rocks are critical for generating datasets in many scientific domains. Often, samples are collected on field expeditions conducted by large infrastructural projects. These projects comprise many human and non-human components that affect the quality and integrity of samples. However, little is known about whether and how prospective dataset users evaluate the samples' trustworthiness and sample collection processes underlying these datasets. Researchers‘strategies for evaluating sample trustworthiness are explored through a longitudinal qualitative case study (ethnographic observation, interviews (n = 66), and document analysis) of subseafloor biosphere research, an earth sciences domain. Domain researchers use rock samples collected on research cruises conducted by the International Ocean Discovery Program (IODP). Subseafloor biosphere researchers are primarily concerned about samples being compromised by microbiological contamination. Researchers vary regarding the components of IODP infrastructure they consider when evaluating sample trustworthiness. These components include methods to process samples, people handling samples, IODP policies and procedures, and IODP organizational politics. Researchers‘strategies vary according to their disciplinary background, with microbiologists employing more fine-grained judgments about methods; whether researchers have participated in IODP expeditions, with those who have employing more fine-grained judgments about people involved; and whether researchers have ever been involved in organizing cruises or serving on IODP committees, with those who have employing more fine-grained judgments about many aspects of cruises. Researchers who make less complex decisions may be prone to erroneously trusting contaminated samples; researchers who make more complex decisions may be prone to erroneously discarding uncontaminated samples. The paper concludes by considering implications for the design of digital infrastructures to support researchers in evaluating sample trustworthiness.

Diagenetic precipitates, such as dolomite, and the chemistry of residual deeply buried porewater often represent the only traces of past biogeochemical activity in marine sediments. A 600 m thick sedimentary section, recently drilled at Integrated Ocean Drilling Program (IODP) Site U1341 on Bowers Ridge (southern Bering Sea), provides insight into such a 4.3 Ma old paleo-diagenetic archive. Hard-lithified calcite–dolomite layers, and laminae of disseminated carbonate, were recovered in diatom-rich sediments over a depth range of 400 m. Carbon isotope values of the diagenetic carbonates between −16.6 and −14.4‰ (VPDB) and strontium isotope ratios of dolomites close to past seawater values suggest carbonate precipitation induced by the production of dissolved inorganic carbon (DIC) during elevated rates of organic carbon mineralization, primarily via sulfate reduction, at shallow sediment depth below the paleo-seafloor. Diagenetic carbonates at 280–440 m below seafloor were likely also produced by the intermittent onset of sulfate reduction coupled to the anaerobic oxidation of methane (AOM) at sulfate–methane transition zones (SMTZ). These microbially mediated processes do not occur in the sediment at this site at present but were likely connected to the presence of a methanogenic zone at 2.58–2.51 Ma. A minimum in sulfate concentrations in modern porewaters and low sedimentary Ba/Al ratios resulting from former sulfate depletion are reminiscent of the presence of this large methanogenic zone. The minimum in sulfate concentrations is reflected in a minimum in magnesium concentrations, less radiogenic strontium and isotopically light calcium in the porewater. It is proposed that magnesium was removed from the porewater during carbonate precipitation and volcanic ash alteration which occurred in the former methanogenic zone and also released strontium with a less radiogenic isotope ratio and isotopically light calcium into the porewater. The isotopic composition of porewater calcium was also influenced by ammonium–calcium exchange on clay minerals and carbonate recrystallization. Our study elucidates the response of porewater element concentrations and isotopic profiles interlinked with the formation of diagenetic carbonates to changes in the deposition of organic carbon in sediments of deeper water sites (>2000 m water depth) over prolonged timescales. It shows that variations in biogeochemical processes in response to changes in oceanographic conditions and a dynamic subseafloor biogeochemical zonation have to also be taken into account at these deep water sites for a global assessment of organic carbon burial fluxes and remineralization. [ABSTRACT FROM AUTHOR]

During the Integrated Ocean Drilling Program (IODP) Expedition 331 at the Iheya North hydrothermal system in the Mid-Okinawa Trough by the D/V Chikyu, we conducted microbiological contamination tests of the drilling and coring operations. The contamination from the drilling mud fluids was assessed using both perfluorocarbon tracers (PFT) and fluorescent microsphere beads. PFT infiltration was detected from the periphery of almost all whole round cores. By contrast, fluorescent microspheres were not detected in hydrothermally active core samples, possibly due to thermal decomposition of the microspheres under high-temperature conditions. Microbial contamination from drilling mud fluids to the core interior subsamples was further characterized by molecular-based evaluation. The microbial 16S rRNA gene phylotype compositions in the drilling mud fluids were mainly composed of sequences of Beta- and Gammaproteobacteria, and Bacteroidetes and not archaeal sequences. The phylotypes that displayed more than 97% similarity to the sequences obtained from the drilling mud fluids were defined as possible contaminants in this study and were detected as minor components of the bacterial phylotype compositions in 13 of 37 core samples. The degree of microbiological contamination was consistent with that determined by the PFT and/or microsphere assessments. This study suggests a constructive approach for evaluation and eliminating microbial contamination during riser-less drilling and coring operations by the D/V Chikyu.

In recent years, research funding agencies, universities, and governments have become increasingly concerned with promoting the reuse of research datasets. Enabling researchers to evaluate the trustworthiness and fitness-for-use of research datasets produced by others is critical for facilitating the reuse of these datasets. Understanding how researchers make these evaluations is crucial for developing digital infrastructure and tools, such as data repositories and metadata schema, in a way that better supports researchers in making these evaluations. Physical samples such as rocks are critical for generating datasets in many scientific domains. Often, samples are collected on field expeditions conducted by large infrastructural projects. These projects comprise many human and non-human components that affect the quality and integrity of samples. However, little is known about whether and how prospective dataset users evaluate the samples' trustworthiness and sample collection processes underlying these datasets. Researchers'strategies for evaluating sample trustworthiness are explored through a longitudinal qualitative case study (ethnographic observation, interviews ( n = 66), and document analysis) of subseafloor biosphere research, an earth sciences domain. Domain researchers use rock samples collected on research cruises conducted by the International Ocean Discovery Program (IODP). Subseafloor biosphere researchers are primarily concerned about samples being compromised by microbiological contamination. Researchers vary regarding the components of IODP infrastructure they consider when evaluating sample trustworthiness. These components include methods to process samples, people handling samples, IODP policies and procedures, and IODP organizational politics. Researchers'strategies vary according to their disciplinary background, with microbiologists employing more fine-grained judgments about methods; whether researchers have participated in IODP expeditions, with those who have employing more fine-grained judgments about people involved; and whether researchers have ever been involved in organizing cruises or serving on IODP committees, with those who have employing more fine-grained judgments about many aspects of cruises. Researchers who make less complex decisions may be prone to erroneously trusting contaminated samples; researchers who make more complex decisions may be prone to erroneously discarding uncontaminated samples. The paper concludes by considering implications for the design of digital infrastructures to support researchers in evaluating sample trustworthiness., Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Darch.)

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

The microbial community structure of five geographically distinct hydrothermal vents located within the Axial Seamount caldera, Juan de Fuca Ridge, was examined over 6 years following the 1998 diking eruptive event. Terminal restriction fragment length polymorphism (TRFLP) and 16S rRNA gene sequence analyses were used to determine the bacterial and archaeal diversity, and the statistical softwareprimer v6 was used to compare vent microbiology, temperature and fluid chemistry. Statistical analysis of vent fluid temperature and composition shows that there are significant differences between vents in any year, but that the fluid composition changes over time such that no vent maintains a chemical composition completely distinct from the others. In contrast, the subseafloor microbial communities associated with individual vents changed from year to year, but each location maintained a distinct community structure (based on TRFLP and 16S rRNA gene sequence analyses) that was significantly different from all other vents included in this study. Epsilonproteobacterial microdiversity is shown to be important in distinguishing vent communities, while archaeal microdiversity is less variable between sites. We propose that persistent venting at diffuse flow vents over time creates the potential to isolate and stabilize diverse microbial community structures between vents. [ABSTRACT FROM AUTHOR]

The subseafloor microbial communities in the turbidite depositional basins Brazos-Trinity Basin IV (BT Basin) and the Mars-Ursa Basin (Ursa Basin) on the Gulf of Mexico continental slope (IODP holes U1319A, U1320A, U1322B and U1324B) were investigated by PCR-dependent molecular analyses targeted to the small subunit (SSU) rRNA genes, dsrA and mcrA, and hydrogenase activity measurements. Biomass at both basins was very low, with the maximum cell or the SSU rRNA gene copy number <1 × 107 cells mL−1 or copies g−1 sediments, respectively. Hydrogenase activity correlated with biomass estimated by SSU rRNA gene copy number when all data sets were combined. We detected differences in the SSU rRNA gene community structures and SSU rRNA gene copy numbers between the basin-fill and basement sediments in the BT Basin. Examination of microbial communities and hydrogenase activity in the context of geochemical and geophysical parameters and sediment depositional environments revealed that differences in microbial community composition between the basin-fill and basement sediments in the BT Basin were associated with sedimentation regimes tied to the sea-level change. This may also explain the distributions of relatively similar archaeal communities in the Ursa Basin sediments and basement sediments in the BT Basin. [ABSTRACT FROM AUTHOR]

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

Novel alkaliphilic, mesophilic bacteria were isolated from subseafloor alkaline serpentine mud from the Ocean Drilling Program (ODP) Hole 1200D at a serpentine mud volcano, South Chamorro Seamount in the Mariana Forearc. The cells of type strain ODP1200D-1.5T were motile rods with a single polar flagellum. Growth was observed between 10 and 45-50°C (optimum temperature: 30-35°C, 45-min doubling time), between pH 6.5 and 10.8-11.4 (optimum: pH 8.5-9.0), and between NaCl concentrations of 0 and 21% (w/v) (optimum NaCl concentration: 2.5-3.5%). The isolate was a facultatively anaerobic heterotroph utilizing various complex substrates, hydrocarbons, carbohydrates, organic acids, and amino acids. Nitrate or fumarate could serve as an electron acceptor to support growth under anaerobic conditions. The G+C content of the genomic DNA was 57.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genusMarinobacterand was the most closely related toM. aquaeoleistrain VT8T andM. hydrocarbonoclasticusstrain SP.17T, while DNA-DNA hybridization demonstrated that the new isolate could be genetically differentiated from the previously described species ofMarinobacter. Based on the physiological and molecular properties of the new isolate, we propose the nameMarinobacter alkaliphilussp. nov., type strain: ODP1200D-1.5T (JCM12291T and ATCC BAA-889T). [ABSTRACT FROM AUTHOR]

The bacterial diversity in a diffuse flow hydrothermal vent habitat at Axial Volcano, Juan de Fuca Ridge was examined shortly after an eruptive event in 1998 and again in 1999 and 2000 using PCR-amplified 16S rRNA gene sequence analyses. While considerable overlap with deep-sea background seawater was found within the α- and γ-proteobacteria, unique subseafloor phylotypes were distinguishable. These included diverse members of the ∊-proteobacteria, high temperature groups such as Desulfurobacterium, Gram-positive bacteria, and members of novel candidate divisions WS6 and ABY1. Phylotype richness was highest in the particle-attached populations from all three sampling periods, and diversity appeared to increase over that time, particularly among the ∊-proteobacteria. A preliminary model of the subseafloor is presented that relates microbial diversity to temperature, chemical characteristics of diffuse flow fluids and the degree of mixing with seawater. [Copyright &y& Elsevier]

During the Integrated Ocean Drilling Program (IODP) Expedition 331 at the Iheya North hydrothermal system in the Mid-Okinawa Trough by the D/V Chikyu, we conducted microbiological contamination tests of the drilling and coring operations. The contamination from the drilling mud fluids was assessed using both perfluorocarbon tracers (PFT) and fluorescent microsphere beads. PFT infiltration was detected from the periphery of almost all whole round cores (WRCs). By contrast, fluorescent microspheres were not detected in hydrothermally active core samples, possibly due to thermal decomposition of the microspheres under high-temperature conditions. Microbial contamination from drilling mud fluids to the core interior subsamples was further characterized by molecular-based evaluation. The microbial 16S rRNA gene phylotype compositions in the drilling mud fluids were mainly composed of sequences of Beta- and Gammaproteobacteria, and Bacteroidetes and not archaeal sequences. The phylotypes that displayed more than 97% similarity to the sequences obtained from the drilling mud fluids were defined as possible contaminants in this study and were detected as minor components of the bacterial phylotype compositions in 13 of 37 core samples. The degree of microbiological contamination was consistent with that determined by the PFT and/or microsphere assessments. This study suggests a constructive approach for evaluation and eliminating microbial contamination during riser-less drilling and coring operations by the D/V Chikyu.