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doi:10.2204/iodp.proc.331.103.2011

Microbiology

Total prokaryotic cell counts

The abundance of microbial cells in the subseafloor at Site C0013 was evaluated by fluorescent microscopy using SYBR Green I as a fluorochrome dye. Total cell biomass at Site C0013 is generally lower than the detection limit of our onboard counting method at this site of 4.4 × 10⁶ cells/mL sediment. Only one sample from 3.14 mbsf in Hole C0013D presented cell numbers greater than the detection limit, and this sample may have been contaminated by drilling fluid (Tables T10, T11). Microbial cell abundance even in the shallowest sediment at Site C0013 was lower than that at Sites C0015 and C0017. This result suggests that most of the core samples at Site C0013 were exposed to a higher temperature range than microbes can survive.

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 from Site C0013. No growth was observed for any of the microorganisms tested.

Cultivation of iron-oxidizing bacteria

Selected samples from Site C0013 were incubated in artificial seawater (ASW) media A and B for depths from 1.09 to 17.04 mbsf. Of these, only Sample 331-C0013B-1T-CC, 6–12 cm, showed significant growth (mainly in ASW media A). Contamination tests were not run for this sample, and the pristine sampling of the core catcher was questioned. In an attempt to answer the question of contamination, another sample (331-C0013F-1H-3, 43–58 cm) from approximately the same depth was inoculated when we returned to drill Hole C0013F. After a 4.5 day incubation period, this sample showed no growth in ASW media A. This result would suggest that the core catcher was indeed contaminated. Nevertheless, subsamples from Section 331-C0013B-1T-CC were stored for future analysis.

Contamination test

Fluorescent microspheres were used to test for contamination in all the cores taken by the HPCS and the first core by the EPCS at Site C0013. Fewer microspheres were observed in the deeper cores from Site C0013 (Table T11). Considering that the plastic core liner often melted at Site C0013, the fluorescent microspheres might also melt in the deeper samples. The perfluorocarbon tracer (PFT) test was conducted only in Hole C0013F (Table T12).

Conclusions

We expected to find microbiological evidence for an active subvent biosphere at Site C0013, but we found neither abundant microbial cells nor detectable growth of (hyper)thermophiles in the onboard measurements and experiments. We were not able to measure temperature in the subseafloor at Site C0013, but there is ample evidence from melting of the plastic core liners, alteration mineralogy and pore water chemistry, and direct observation of hydrothermal fluid emission during the drilling operation that Site C0013 has a steep thermal gradient. It appears that most of the subseafloor environment we sampled at this site has been exposed to temperatures that exceed the habitable and survival limits of microorganisms.

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