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doi:10.2204/iodp.proc.322.102.2010

Microbiology

Upon extraction from the seafloor, sediment cores begin to equilibrate to surface environmental conditions. Microbes sensitive to changes in temperature, pressure, redox potential, and other environmental parameters can be significantly impacted in their activity, growth, cell structure, and/or cellular composition (cf. Ehrlich and Newman, 2009). Intra- and extracellular biomolecules such as deoxyribonucleic acid, ribonucleic acid (RNA), intact polar lipids, and various enzymes can be altered or damaged such that an accurate assessment of in situ microbial community structure and/or activity is impeded. Furthermore, there exists the potential for introducing nonindigenous microbial and/or biomolecular contamination from the surface environment, an issue of possibly greater relevance for sediments with lower biomass. Thus, careful attention must be paid when possible to time-sensitive and aseptic shipboard handling, processing, and storage of microbiology samples.

Whole-round core sampling and handling

Multiple studies using microbiological, molecular biological, and biogeochemical approaches will be performed on shore using whole-round cores and subsampled sediments. Shipboard protocols were limited to the appropriate sampling, processing, and storage of subseafloor sediment whole-round cores and basalt core fragments. Sample processing was usually completed within 30 min from the time of core recovery on deck, and samples were held at 2°–8°C during most of this period.

Upon receiving sediment cores from the drilling rig to the core cutting area, an interval was selected for adjacently located whole-round core sampling for interstitial water (variable in length depending on expected interstitial water volume recovery), microbiology (15 cm), and organic geochemistry (10 cm) analyses. The sample collocation maximizes the contextual data from adjacent specimens. From Hole C0011B, microbiology whole-round cores were obtained at roughly 50 m intervals. Sampling at higher spatial resolutions (every 10–30 m) was possible for Hole C0012A.

After removal from the core cutting area, the whole-round core (still containing the microbiology whole-round core at this stage) was sent directly to the X-ray CT imaging laboratory where it was either accepted or rejected for interstitial water microbiology/organic geochemistry whole-round core sampling, based on the presence or absence of features important to structural and sedimentological studies. If accepted, this composite whole-round core was taken to the QA/QC laboratory, where 10 and 5 cm long whole-round cores were cut for onshore biomolecular extractions and cultivation/staining/cell counting analyses, respectively. All sediment cores were cut with sterilized cutting tools, and microbiology whole-round cores were quickly covered with end caps that were irradiated for >45 min (at ~60 cm distance) with UV-C light and sealed with plastic tape.

Sealed 10 cm whole-round cores were placed into zip freezer bags for immediate transfer to the shipboard –80°C freezer for the duration of Expedition 322, at which time they were transferred on dry ice to KCC. The 5 cm whole-round cores were brought directly from the QA/QC laboratory into an anaerobic chamber (Coy Laboratory Products) in the adjacent microbiology laboratory and sampled for roughly 5–10 min at 2°–8°C under a 98:2 N₂:H₂ atmosphere for fluorescence in situ hybridization (FISH) studies (see "Sample processing for cell detection by epifluorescence microscopy"). The samples were then sealed in their core liners as described above and placed into oxygen-impermeable vinyl bags with inserted AnaeroPack oxygen-removing CO₂-generating paper sachets (Mitsubishi Gas Chemical Co., Inc.). Sealed anaerobic whole-round cores were transferred into storage at 3°–4°C for the duration of Expedition 322 until they could be transferred at 2°–8°C to KCC and/or Expedition 322 microbiology science party home institutions. These whole-round cores will be used for multiple biological studies (e.g., cultivation, activity measurements using stable and radiogenic isotopes, and extracellular enzymes).

When the core was heavily fragmented or the ratio of core recovery to advance was low (<0.3), microbiology whole-round cores were not possible to obtain. For most of these cases, however, small "chips" (≥2 cm³ in diameter) were obtained from interstitial water whole-round cores that were trimmed of drilling mud contamination in an N₂-flushed glove bag. These chips were washed with sterile RNA-ase (RNA Later) and frozen at –80°C.

Sample processing for cell detection by epifluorescence microscopy

Epifluorescent cell staining and enumeration (Morono et al., 2009), FISH (Amann and Fuchs, 2008), and catalyzed reporter deposition–fluorescence in situ hybridization (CARD-FISH) (Schippers et al., 2005) staining are the primary tools used for species-specific visualization of living microbes. 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. The 5 cm whole-round core for cultivation and FISH work was placed into a gel ice pack mold preformed to wrap snugly around its circumference and held in place by rubber bands (Fig. F18). Whole-round cores were subsampled in their centers using an autoclaved metal coring device tapped gently into the sediment with a small hammer inside the anaerobic chamber. Sediment subcores were ejected into small polypropylene tubes, homogenized manually with a sterile metal spatula, fixed with sterile 2% paraformaldehyde in PBS solution (Invitrogen, pH adjusted to 7.6) for 8–12 h at 3°–4°C, washed by centrifugation/decanting/resuspension 2–3 times with sterile PBS, and finally stored at –20°C in a 10 mL 1:1 mixture of cold PBS:ethanol (99.9% purity). Centrifuge conditions for these washing steps were 9000 × g at 4°C for 10 min.

Routine microbiology samples

As per the STP recommendation 0908-09, Expedition 322 attempted the implementation of routine microbiological sampling for community archival purposes. However, significant inconsistencies in both core recovery and core quality rendered it necessary to relocate multiple planned whole-round core samples for both science party and archival purposes. Prioritization was given to shipboard and shore-based Expedition 322 science party members, and only two dedicated whole-round cores for the IODP community archive could be obtained. Subsamples from these whole-round cores will be deposited at KCC (Masui et al., 2009) into the community archive for postexpedition science requests.

Where dedicated archival whole-round cores could be taken from recovered cores, they were taken adjacent to interstitial water whole-round cores. Although the STP recommendation mentions only the shipboard storage of archival whole-round cores at –80°C, we attempted to expediently subsample these for future FISH-based studies. The purpose of this implementation was to review the feasibility of sample processing as a routine task for the onboard microbiologist and investigate the temporal change in the quality of preserved samples. FISH subsamples from archival whole-round cores were taken as described above.

Evaluation of potential contamination

As a control sample for contamination, freshly prepared drilling mud (seawater gel) was obtained during Expedition 322. Seawater gel is a seawater-base bentonite mud (pH 12.3) used for drilling and coring throughout the entire section and contains 0.5 m³ seawater, 0.5 m³ drill water, 60.0 kg bentonite, 2.0 kg caustic soda, and 2.0 kg lime. The density of this drilling mud is 1.05 g/cm³. Subsamples of the seawater gel were stored at 4° and –80°C for culturing and molecular analysis, respectively. Also, natural chemical tracers such as sulfate in interstitial water were measured (see "Inorganic geochemistry"). Because of drilling constraints, we were not able to perform other conventional tracer tests such as monitoring the infiltration of micrometer-sized fluorescent spheres or perfluorocarbon tracer (Smith et al., 2000).

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