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doi:10.2204/iodp.proc.331.104.2011

Microbiology

Total prokaryotic cell counts

The abundance of microbial cells in the subseafloor at Site C0014 was evaluated by fluorescent microscopy using SYBR Green I as a fluorochrome dye. Commonly among the holes at Site C0014, the highest cell abundances were found in sediments near the seafloor. The maximal abundances in Holes C0014B and C0014G are 1.8 × 10⁷ and 3.3 × 10⁷ cells/mL sediment (at 0.33 and 0.27 mbsf), respectively, whereas that in Hole C0014D is 4.7 × 10⁸ cells/mL sediment (at 0.23 mbsf) (Fig. F29; Table T13). Cell numbers generally decrease with increasing depth, although there are secondary peaks at 14.3, 8.6, and 22.1 mbsf in Holes C0014B, C0014D, and C0014E, respectively. At depths below 17.2 mbsf in Hole C0014B, 11.4 mbsf in Hole C0014D, and 4.1 mbsf in Hole C0014G, microbial cell numbers decrease to below our detection limit of ~1–4 × 10⁶ cells/mL. Maximum cell abundances in Holes C0014B and C0014G are similar to those at Sites C0015 and C0017, but Hole C0014D yielded a significantly higher maximum than any other hole drilled on Expedition 331.

Cultivation of thermophiles

Growth of Thermococcales (e.g., Thermococcus) at 80°C and Aquificales (e.g., Persephonella) and thermophilic Epsilonproteobacteria (e.g., Nitratiruptor) at 55°C was examined for all core samples taken at Site C0014. No growth of any thermophilic microorganism tested for was observed.

Cultivation of iron-oxidizing bacteria

Selected samples from Site C0014 were incubated in artificial seawater (ASW) media A and B for depths from 0.23 to 42.45 mbsf (Table T14). Growth was observed only within the uppermost 1.93 m. It is likely that extreme temperatures at depth at Site C0014 make the habitat inhospitable for mesophilic iron oxidizers, as was also inferred for Site C0013. Of the samples that grew, observed cellular morphologies are similar to what was seen at Site C0015. From Section 331-C0014B-1H-1, rod-shaped cells ~2 µm long were observed aggregated on large particles (Fig. F30A, F30B). Figure F30C and F30D shows a sheath-like structure containing two rod-shaped particle-associated cells. Though found only in shallow sediments, these Site C0014 enrichments may prove useful for comparisons with Site C0015 iron oxidizers, both shallow and deep.

Contamination tests

Fluorescent microspheres were used to test for contamination in all cores from Site C0014 obtained with the HPCS (Table T15). Perfluorocarbon tracer (PFT) was also used as a contamination tracer in all cores from this site (Table T16). Microspheres were not observed in most of the core samples, even in exterior parts of the cores. As at Site C0013, the plastic microspheres may have melted at greater depths, as temperature exceeded 120°C at 38 mbsf and deeper in Hole C0014G.

PFT was not detected from the interior of more than half of the core samples obtained by the HPCS. Conversely, it was detected in the interiors of most samples taken by the ESCS or EPSC, although at concentrations much lower than in the drilling mud (Table T16). PFT was also detected in cores exposed to high temperatures. Although PFT is a highly volatile chemical, our results suggest that it may be used as a tracer for contamination even at high temperatures.

Relatively high numbers of microspheres and high concentrations of PFT were observed in samples containing pumice, for example at 6.67 mbsf in Hole C0014D and at 7.84 mbsf in Hole C0014G. PFT concentrations in such samples, however, were at least an order of magnitude lower than concentrations in the drilling fluid. Permeable layers are often highly disturbed during recovery, making the external contamination unavoidable.

Conclusion

Site C0014 is located at a Calyptogena colony site in the Iheya North hydrothermal field. Also observed at this site are patches of black-colored sediments and white bacterial or sulfur mats. Calyptogena clams require H₂S as an energy source (Cavanaugh et al., 2006). Consequently, the transition from a psychrophilic microbial community at and near the seafloor that is not obviously dependent on hydrothermal input to a deeper one that is thermophilic and highly dependent on hydrothermal input was the major focus for microbiology at Site C0014. Unfortunately, our shipboard cell counts and cultivation experiments did not detect a deeper (hyper)thermophilic community, even though there is a potentially habitable zone at <120°C that extends as deep as 38 mbsf, over which sulfate decreases to nearly zero (by 12–31 mbsf in all holes) and methane is generated biogenically. Growth of mesophilic FeOB was detected within the uppermost 2 m only. It appears that functionally active, metabolically diverse microbial communities may be limited to the shallower zones of the subseafloor at Site C0014. As was the case at Site C0013, it is likely that temperatures over the key depth interval of ~15–38 mbsf exceeded the limits for life recently enough that microbial communities are largely absent. This conclusion is consistent with the presence of illite at ~10–25 mbsf and Mg chlorite at 25 mbsf to the maximum depth drilled at Site C0014.

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