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doi:10.2204/iodp.proc.331.105.2011

Microbiology

Total prokaryotic cell counts

The abundance of microbial cells in subseafloor sediments at Site C0015 was evaluated by fluorescent microscopy using SYBR Green I as a fluorochrome dye. The maximum cell abundance was observed at 0.3 mbsf (1.2 × 10⁷ cells/mL). The microbial cell count decreased with depth and was below our detection limit of ~1–4 × 10⁶ cells/mL at 5.6 mbsf. However, the cell count increased again to 8.8 × 10⁶ cells/mL sediment at 6.9 mbsf and 4.8 × 10⁶ cells/mL at 8.8 mbsf (Fig. F14; Table T10).

Contamination tests

Fluorescent microspheres and perfluorocarbon tracer (PFT) were not detected in most of the core samples (Tables T11, T12), indicating that the cores were mostly uncontaminated. Exceptions were samples from 6.9 and 8.8 mbsf, which contained detectable amounts of both tracers and so appear to be contaminated with drilling fluid.

Cultivation of iron-oxidizing bacteria

Onboard cultivation/enrichment experiments for iron-oxidizing bacteria (FeOB) showed growth after 5–6 days of incubation at room temperature (~21°C) for most of the samples inoculated (Table T13). Observation of petri plate coloration and turbidity after incubation (Fig. F15), though not effective for assessment of growth, did confirm the effectiveness of the GasPak in producing an anaerobic environment. Assessment of relative cell growth using epifluorescence microscopy yielded a variety of cellular morphologies and possible colonization strategies (Fig. F16). In Section 331-C0015B-1H-1, artificial seawater (ASW) media A, a long (~4 µm) rod-shaped bacterium can be seen contained within a twisted filament along with other, shorter rods at ~2.5 µm. Section 331-C0015C-1H-3, ASW media B, also contained short rods that are often associated with larger particles. In Section 331-C0015C-1H-3, ASW media A, rod-shaped bacteria colonize the surface of a large, dense particle. This observation could lend some insight into the colonization and weathering of the particle in Figure F17A.

Though no twisted iron oxide stalks, such as those created by Mariprofundus ferrooxydans, were observed in these enrichment experiments, SEM imaging revealed several possible sheathlike structures composed of iron oxides (Fig. F17). Imaging of original samples from Sample 331-C0015C-1H-3, 84–85 cm (see “Biostratigraphy”), showed extensive iron oxide filaments of putative biological origin. Also, several putative bifurcated iron oxide twisted stalks were observed under epifluorescence microscopy, though these structures were not associated with cells (Fig. F18).

Conclusions

As was the case at Sites C0013 and C0014, cell abundances at Site C0015 were significantly lower than those found in previous Ocean Drilling Program/IODP sites on continental margins (Parkes et al., 1994, 2000; D’Hondt et al., 2004), despite the location of the Iheya North field in a backarc basin associated with a continental margin. The generally low microbial abundance in the subseafloor at the Iheya North field may be associated with low primary production in the surface ocean and/or low influx of terrigenous organic matter in the middle Okinawa Trough, as previously suggested for the Brazos-Trinity Basin IV and the Mars-Ursa Basin in the Gulf of Mexico continental margin drilled by IODP Expedition 308 (Holes U1319A, U1320A, U1322B, and U1324B) (Nunoura et al., 2009).

In the deeper sediments at Site C0015 the cell count increased again. Although depth profiles of interstitial water chemistry and dissolved gas concentrations (see “Geochemistry”) are relatively invariant at Site C0015, there are lithological differences between the surficial and the deeper sediment. Layers with low cell abundances at 3.4 and 5.6 mbsf consist of pelagic clay, whereas those with higher cell counts at 6.9 and 8.8 mbsf occur in sandy layers (see “Lithostratigraphy”). The depth profile of cell abundance at Site C0015 may thus be explained by the physical properties of the sediments such as porosity, as has been pointed out in previous studies (Schmidt et al., 1998; Zhang et al., 1998; Parkes et al., 2000; Inagaki et al., 2003; Rebata-Landa and Santamarina, 2006).

Samples from Site C0015 represent the deepest relatively pristine inoculum that showed positive growth for putative FeOB for any site during Expedition 331. Contamination was below our detection limit. With increasing depth, relative growth shifted from a preference toward a microaerophilic environment to a more anaerobic environment. The detection of FeOB at this “cooler” site is consistent with previous detection of FeOB in other low-temperature hydrothermal vent systems (Rassa et al., 2009).

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