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doi:10.2204/iodp.proc.314315316.132.2009

Microbiology and biogeochemistry

Subseafloor environments represent the least explored habitats on Earth with regard to microbial life (D’Hondt et al., 2007). Enumeration of cell abundance by nucleic acid staining methods using acridine orange or SYBR Green I has shown that a large number of microbial cells are present in marine sediments to ~800 meters below seafloor (mbsf) (Parkes et al., 1994, 2000). The metabolic activity of deep microbes is extraordinarily low compared to those in seafloor sediments (D’Hondt et al., 2002, 2004). Nevertheless, this activity plays an important role in global biogeochemical cycling on the geological timescale. One of the subsurface interfaces that has attracted much attention in the past few years is that of the SMT zone where microbial activity is responsible for anaerobic oxidation of methane (e.g., Hinrichs et al., 1999; Parkes et al., 2005; Biddle et al., 2006; Inagaki et al., 2006a). Also, subseafloor microbes are thought to be responsible for methanogenesis, ethanogenesis, and propanogenesis (Hinrichs et al., 2006). Carbon isotopic fractionation through the activity of metabolic enzymes contributes to the accumulation of ¹³C-depleted hydrocarbons in the sediments. Microbial activity is controlled by the availability of electron donors and/or acceptors, which are derived from either the surface via photosynthetic activities or geochemical activities in the underlying crust of the Earth (D’Hondt et al., 2004). Molecular phylogenetic analysis using 16S ribosomal ribonucleic acid (rRNA) gene fragments indicates that subseafloor communities are highly diverse and are often composed of microbial strains lacking closely related cultivated relatives (Inagaki et al., 2003, 2006b). In addition, advanced molecular and lipid analyses suggest that the population of Archaea seems to be more abundant in subseafloor sediments than in the surface world (Lipp et al., 2008). Although the function of yet-uncultivated phylotypes remains largely unknown, significant extracellular enzymatic activities such as phosphatase and liparse have been detected in deep subseafloor sediments (Engelen et al., 2008; Kobayashi et al., 2008). The activity and habitability of life in the deep subseafloor biosphere is highly associated with geological settings in terms of carbon and energy flux.

Given this background, it is hypothesized that microbial abundance and activity are closely associated with energy and fluid flow regimes related to geological structures and plate movements. Expedition 316 provides an unprecedented opportunity to study the active subduction zone that directly connects to the deep seismogenic zone via the megasplay fault system. In addition, no systematic microbiological or biogeochemical studies have been conducted in deep subseafloor accretionary prisms. To understand such geosphere-biosphere interactions, multiple studies using microbiological, molecular biological, and biogeochemical approaches will be performed on shore using whole-round cores and subsampled materials.

Core handling and sampling

Whole-round cores

Because indigenous subsurface living microbes and their biomolecules such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), intact polar lipids (IPLs), and enzymes are highly sensitive to oxygen, temperature, and exposure time, special attention to the processing of microbiology/​biogeochemistry samples was required. After core was recovered in the core cutting area, sections were immediately stored in the refrigerator room adjacent to the QA/QC laboratory. Subsequently, X-ray CT scans were collected and the location of whole-round core sampling was determined according to the image, avoiding specific geological features such as lithologic boundaries and faults. Two types of whole-round cores (7–20 cm in length) were sliced off in the QA/QC laboratory. The first type (frozen whole-round cores) were placed into plastic bags and stored at –80°C. These whole-round cores will be used for molecular (DNA and RNA) analyses as well as structure and carbon isotopic analysis of IPLs. The second type (anaerobically packed whole-round cores) were brought to an anaerobic glove box (Coy Laboratory Products, Inc.) in the microbiology laboratory, placed into an oxygen-impermeable vinyl bag with AnaeroPack (Mitsubishi Gas Chemical Co., Inc.), sealed with a plastic clip, and stored at 4°C. End caps were placed on the whole-round core in the anaerobic glove box; otherwise even small amounts of oxygen (~100 ppm) might contaminate the samples. The anaerobic whole-round core samples will be used for multiple biological studies (e.g., cultivation, activity measurements using radiotracers, and extracellular enzymes).

Cell detection by fluorescent microscopy

Fluorescent in situ hybridization (FISH) and catalyzed reporter deposition (CARD)-FISH techniques are powerful molecular ecological tools for visualization of specific living microbes that contain hybridizable 16S rRNA (Schippers et al., 2005). Because intracellular RNA is easily degraded by enzymatic reaction after cell death, it is necessary to fix the cells as soon as possible after core recovery. During Expedition 316, 3 cm³ minicore samples were taken from the innermost part of the whole-round core in laminar-flow clean bench, fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS, Invitrogen, pH adjusted to 7.6) for 10 h at 4°C, washed twice with PBS, and stored in 3 times volume of PBS:ethanol (1:1) at –20°C. Centrifuge conditions for these washing steps were 9000 × g at 4°C for 10 min. Alternatively, 50 cm³ of soft sediment was taken from the innermost whole-round core by tip-cut syringe, mixed with the same volume of acetone, vigorously shaken by hand for a few minutes, and stored at –20°C (Fukatsu, 1999). The paraformaldehyde-fixed slurry samples were stained with SYBR Green I, which specifically binds double-stranded DNA and produces high fluorescent signals with low background (Lunau et al., 2005). Before staining, the fixed sediment slurry was vortexed and diluted 10 times with 10% (vol/vol) methanol in PBS. The mixture was sonicated at 40 W for 1 min with an ultrasonic homogenizer UH-50 (SMT Co., Ltd), and the sediment particles were removed by centrifuging at 100 × g for 2 min at 4°C. A 5 µL aliquot of the supernatant was mixed with 1 mL PBS and filtered through a black 0.2 µm pore-sized polycarbonate filter. The filter was washed twice with 1 mL PBS, and the ⅛ cut piece was placed on an object glass. A coverslip was mounted on the filter with 8 µL of SYBR Green I staining solution (Lunau et al., 2005). Cells were viewed using an epifluorescence microscope (ZEISS Axioplan 2 imaging microscope), and images were taken with ZEISS AxioCam HRc camera and AxioVision AC software. Total cell abundance was enumerated by the average number of SYBR-stained particles in a microscopic field.

Fractured pieces and rotary core barrel core sampling

Because cores in the fault zone between 270 and 310 m CSF were highly fractured at Site C0004, we picked relatively large pieces (>3 cm in diameter) from the interstitial water whole-round core section after the core surface trimming (see “Inorganic geochemistry”), washed the pieces four times with sterilized PBS (pH 7.6) to remove potential contaminants from the seawater-based drilling mud, and crushed the pieces with a hammer in a sterilized aluminum bag. The PBS-washed powdered sediments were immediately stored at –80°C with or without acetone or were anaerobically stored at 4°C in a glass bottle for cultivation and activity measurements. Bulk sediments without washing were obtained as control samples. Massive pieces of RCB cores were covered with a soft matrix that consists of drilling mud and cuttings. The core surface was washed with either sterilized PBS (pH 7.6) or autoclaved Milli-Q water and processed as described above.

Contamination test

As it was not possible to do perfluorocarbon (PFC) tracer contamination tests shipboard, we monitored the infiltration of 0.5 and 1.0 µm fluorescent microsphere beads according to the previously described protocol (Smith et al., 2000a, 2000b). As a control sample for contamination from drilling mud, freshly prepared drilling mud (seawater gel and kill mud) was obtained during Expedition 316 (Masui et al., 2008). Seawater gel is a seawater-based bentonite mud used for drilling and coring throughout the entire section. Seawater gel (pH 12.3) contains 0.5 m³ seawater, 0.5 m³ drill water, 60.0 kg bentonite, 2.0 kg caustic soda, and 2.0 kg lime. Kill mud is a freshwater-based barite-weighted mud used to suspend or abandon the hole. It was not used before or during Expedition 316 coring. Kill mud (pH 11.3) contains 1 m³ drill water, 60.0 kg bentonite, 1.0 kg caustic soda, 2.0 kg polyanionic cellulose derivatives, and 40.0 kg barite. The density of both drilling muds is 1.05 g/cm³. The subsamples for culturing and molecular analysis were stored at 4°C and –80°C, respectively. Natural chemical tracers such as sulfate in pore water will be carefully checked, especially for highly fractured core samples.

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