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Phylogenetic tree of the domains Archaea and Bacteria based on 16S rRNA gene sequences. Red and blue lines respectively represent branches of archaea and bacteria that have been frequently detected in deep subseafloor sediments. The scale bar indicates the expected number of changes per nucleotide positions. Sequence data were analyzed with the ARB software package (Ludwig et al., 2004).
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Deep drilling of marine sediments and igneous crust offers a unique opportunity to explore how life persists and evolves in the Earth’s deepest subsurface ecosystems. Resource availability deep beneath the seafloor may impose constraints on microbial growth and dispersal patterns that differ greatly from those in the surface world. Processes that m...
Context in source publication
Context 1
... understand subseafloor diversity, it will be necessary to conduct surveys of appropriate phylogenetic (16S rRNA genes ( Fig. 2) and 18S rRNA genes) and functional gene markers. A variety of methods exist for such microbial diver- sity analyses (denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymor- phism (T-RFLP), clone libraries, and tag sequencing); indi- vidual investigators should choose the method that is most ...
Citations
... Sub-sea floor sediments encompasses high abundant of microorganisms, estimated by indirect chemical evidences or measured by microbial cell counting (Cragg et al. 1990; Thierstein and Störrlein 1991; Parkes et al. 2000). The microorganisms in sub-sea floor sediments survive under conditions of high pressure and very low temperatures, combined with low concentrations of nutrients and a lack of light (D'Hondt et al. 2007; Fry et al. 2008 ). Both cultured and uncultured molecular techniques have detected high diversity of microbes in sub-seafloor habitats (Reed et al. 2002; Inagaki et al. 2003 Inagaki et al. , 2006 Teske 2006; Webster et al. 2006). ...
... However, little is known about the microbial population size, community composition, diversity, distribution and metabolism (Schippers et al. 2005; Fry et al. 2008; D'Hondt et al. 2007; Batzke et al. 2007). To better understand bacterial community structure and function in sub-sea floor habitats, 16S rRNA genes have been commonly used to identify bacterial diversity (D'Hondt et al. 2007). For example, during the Ocean Drilling Program (ODP) cruises, researchers analyzed the microbial community composition of sediment samples from several marginal seas, and it turned out that Alpha-, Gamma-, Delta-proteobacteria and JS1 dominated at bacterial community in the Japan Sea (Rochelle et al. 1994). ...
We explored the bacterial diversity and vertical distribution along a sediment core (MD05-2896) from the coral reefs of the Nansha carbonate platform in the South China Sea. Bacterial diversity is determined by 16S rRNA molecular survey from twelve subsamples A, obtained via cloning, sequencing and phylogenetic analyses. We estimated the species richness by parametric and nonparametric models, which identified 326 ± 40 (SE) bacteria species. The dominant bacterial groups included Planctomycetes, Deltaproteobacteria, and candidate division OP3, which constituting 23.7, 10.4, and 9.5 % of bacterial 16S rRNAclone libraries, respectively. The observed stratification of bacterial communities was correlated with C/N ratio. This study improves our understanding of the species-environment relationship in the sub-sea floor sediment.
... Microbial populations in subseafloor sediments on the global continental margins account for one tenth to one third of the total biomass on Earth (Whitman et al., 1998; Parkes et al., 2000; Lipp et al., 2008). Although microbial populations have been detected ubiquitously in the marine subseafloor environment, the diversity , activity, metabolic processes, and interactions with geochemistry are still largely unknown (D'Hondt et al., 2007; Bach et al., 2010). Microbial processes in the marine subsurface are potentially significant to global carbon and nutrient cycles (Whiticar, 1999; D'Hondt et al., 2002) and provide relevant analogs to the emerging astrobiology field (Gold, 1992; Chapelle et al., 2002). ...
A remarkable number of microbial cells have been enumerated within subseafloor sediments, suggesting a biological impact on geochemical processes in the subseafloor habitat. However, the metabolically active fraction of these populations is largely uncharacterized. In this study, an RNA-based molecular approach was used to determine the diversity and community structure of metabolically active bacterial populations in the upper sedimentary formation of the Nankai Trough seismogenic zone. Samples used in this study were collected from the slope apron sediment overlying the accretionary prism at Site C0004 during the Integrated Ocean Drilling Program Expedition 316. The sediments represented microbial habitats above, within, and below the sulfate–methane transition zone (SMTZ), which was observed approximately 20 m below the seafloor (mbsf). Small subunit ribosomal RNA were extracted, quantified, amplified, and sequenced using high-throughput 454 pyrosequencing, indicating the occurrence of metabolically active bacterial populations to a depth of 57 mbsf. Transcript abundance and bacterial diversity decreased with increasing depth. The two communities below the SMTZ were similar at the phylum level, however only a 24% overlap was observed at the genus level. Active bacterial community composition was not confined to geochemically predicted redox stratification despite the deepest sample being more than 50 m below the oxic/anoxic interface. Genus-level classification suggested that the metabolically active subseafloor bacterial populations had similarities to previously cultured organisms. This allowed predictions of physiological potential, expanding understanding of the subseafloor microbial ecosystem. Unique community structures suggest very diverse active populations compared to previous DNA-based diversity estimates, providing more support for enhancing community characterizations using more advanced sequencing techniques.
... Hence, such efforts to enumerate AO-stained cells from the subseafloor on photographic images have been difficult, and a verification of counts by other methods has been impossible. In addition, providing mean statistical values from low biomass sedimentary habitats has been complicated by physical and time limitations, yet these habitats are considered critical for understanding the Earth's biosphere close to the limits of habitable zones (Hoehler, 2004; D'Hondt et al., 2007). ...
The marine subsurface environment is considered the potentially largest ecosystem on Earth, harboring one-tenth of all living biota (Whitman et al., 1998) and comprising diverse microbial components (Inagaki et al., 2003, 2006; Teske, 2006; Inagaki and Nakagawa, 2008). In deep marine sediments, the discrimination of life is significantly more difficult than in surface sediments and terrestrial soils because buried cells generally have extremely low metabolic activities (D’Hondt et al., 2002, 2004), and a highly consolidated sediment matrix produces auto-fluorescence fromdiatomaceous spicules and other mineral particles (Kallmeyer et al., 2008). The cell abundance in marine subsurface sediments has conventionally been evaluated by acridine orange direct count (AODC; Cragg et al., 1995; Parkes et al., 2000) down to 1613 meters below the seafloor (mbsf) (Roussel et al., 2008). Since the cell-derived AOsignals often fade out in a short exposure time, recognizing and counting cells require special training. Hence, such efforts to enumerate AO-stained cells from the subseafloor on photographic images have been difficult, and a verification of counts by other methods has been impossible. In addition, providing mean statistical values from low biomass sedimentary habitats has been complicated byphysical and time limitations, yet these habitats are considered critical for understanding the Earth’s biosphere close to the limits of habitable zones (Hoehler, 2004; D’Hondt et al., 2007).
... Hence, such efforts to enumerate AO-stained cells from the subseafloor on photographic images have been difficult, and a verification of counts by other methods has been impossible. In addition, providing mean statistical values from low biomass sedimentary habitats has been complicated by physical and time limitations, yet these habitats are considered critical for understanding the Earth's biosphere close to the limits of habitable zones (Hoehler, 2004;D'Hondt et al., 2007). ...
No abstract available.
doi:10.2204/iodp.sd.9.05.2010
... Subseafloor microbes play important roles in bio- geochemical cycling of carbon, nitrogen, sulfur, metals, and other elements on geologic timescales; however, their growth and metabolic characteristics remain largely unknown because most subseafloor microbes are phylogenetically distinct from known isolates and resistant to culturing in laboratories. Given the significance of this region as potential habitats of subseafloor life, cored materials (or portions thereof) should be frozen in long-term storage to provide opportunities for future molecular analyses arising from rapid biotechnological developments (D'Hondt et al., 2007). Here we report a semi-aseptic subsampling technique for frozen core samples using an electric saw system in a clean booth. ...
No abstract available.
doi:10.2204/iodp.sd.8.05.2009
... Hence, such efforts to enumerate AO-stained cells from the subseafloor on photo-images have been difficult and verification of findings by others has been impossible . In addition, to provide mean statistical values from low biomass sedimentary habitats has been complicated by human efforts and time limitations, yet these habitats are considered critical for understanding the Earth's biosphere close to the limits of habitable zones (Hoehler 2004; D'Hondt et al., 2007). ...
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.
... Long-term storage under these conditions causes significant contamination through growth of microbes and also damages the small amounts of fragile biomolecules in the cores. Over the years there has been a number of task forces formed to make recommendations to IODP concerning sampling and archival of microbiological material (D'Hondt et al., 2007). Recently a technique using a diamond-tip band saw system was developed that enables subsampling of requested frozen core material without melting . ...
Drilling and coring methods developed for sampling deep subsurface terrestrial environments have led to the discovery of active, diverse, indigenous microbial communities in a variety of subsurface habitats, including oil and natural gas reservoirs. The primary drilling and coring methods are hollow-stem augering, direct-push coring, cable-tool drilling, and rotary drilling. Rotary drilling is required for depths >300 m and for hard rock environments. The potential for chemical and microbiological contamination during drilling, coring, and sample handling is great, and so obtaining subsurface samples that are truly representative of the subsurface and that are suitable for geochemical and microbiological analyses requires specialized techniques. Solute and particulate tracers are used to quantify chemical and microbiological contamination, respectively. Cores are dissected to remove inner subcore material, in which tracer concentrations should be orders of magnitude lower than in the surrounding material. Samples are generally processed in an anaerobic chamber to avoid exposure of redox-sensitive chemical species and strictly anaerobic microbes to O2. Once drilled, boreholes can be further used to collect groundwater microbes, monitor subsurface chemistry and microbial processes, and enrich for microorganisms. While the methods described here have been successfully used in a variety of subsurface environments, including deep marine sediments, other approaches have also been used, e.g., sampling in deep mines, and still others are being developed.
Coring methods developed for sampling deep subsurface terrestrial environments have led to the discovery of active, diverse, indigenous subterranean microorganisms. However, coring has rarely been used for microbiological sampling of deep hydrocarbon reservoirs. The primary drilling and coring methods are hollow-stem augering, cable-tool drilling, and rotary drilling. Rotary drilling is required for depths >300 m and for hard rock environments. The potential for chemical and microbiological contamination during drilling, coring, and sample handling is great, and so obtaining subsurface samples that are truly representative of the subsurface and that are suitable for geochemical and microbiological analyses requires specialized techniques. Solute and particulate tracers are used to quantify chemical and microbiological contamination, respectively. Cores are dissected to remove inner subcore material, in which tracer concentrations should be orders of magnitude lower than in the surrounding material. Samples are generally processed in an anaerobic chamber to avoid exposure of redox-sensitive chemical species and strictly anaerobic microbes to O2. While these established methods have been successfully used in a variety of subsurface environments, including deep marine sediments, other approaches have also been used, e.g., sampling in deep mines, and still others are being developed.
