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doi:10.2204/iodp.proc.331.107.2011

Microbiology

Total prokaryotic cell counts

The abundance of microbial cells in subseafloor sediments at Site C0017 was evaluated by fluorescent microscopy using SYBR Green I as a fluorochrome dye. Maximum cell numbers were observed at 6.36 mbsf (3.2 × 10⁷ cells/mL), and cell numbers range from 2.4 × 10⁷ to 3.2 × 10⁷ cells/mL at depths above 10.8 mbsf. Microbial cell counts irregularly decrease with depth (Fig. F15; Table T10). Below 50 mbsf, cell counts decrease to less than the limit of detection of ~1 × 10⁶ to 4 × 10⁶ cells/mL.

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 cores from a high-temperature zone below 94.7 mbsf. No growth of thermophiles was observed on board.

Contamination tests

Fluorescent microspheres and perfluorocarbon tracer (PFT) were used to test for contamination (Tables T11, T12). The PFT test suggests that even in sediment layers containing large amounts of pumice, contamination by drilling fluid was quite low. This was the case even at the outer surface of the core and even with larger grain sizes, which have the greatest risk of contamination. We found evidence for contamination by drilling fluid in the interior of cores obtained from Hole C0017D by the ESCS, but detected PFT concentrations were much lower than those in the drilling mud fluid collected from the onboard tank and that adhering to the massive sulfide of Site C0016 (Table T12; see Table T8 in Expedition 331 Scientists, 2011b, and Table T6 in Expedition 331 Scientists, 2011f).

Cultivation of iron-oxidizing bacteria

The depth interval from ~26 to 35 mbsf at Site C0017 probably represents a zone of recharge flow in this hydrothermal system, as suggested by the temperature and pore water chemistry profiles (see “Petrology” and “Geochemistry”). It looks to be heavily oxidized, by either biotic or abiotic means. The interaction of infiltrating cold seawater with the surrounding reduced layers would produce a redox boundary that can be utilized by many microorganisms, including iron-oxidizing bacteria. For this reason we targeted this oxidized interval for FeOB cultivation experiments (Table T13). Samples 331-C0017C-1H-7, 1 cm, and 1H-7, 2 cm, are two subsamples from the same whole round, but the latter is more rust-colored and appears to be more oxidized.

After 4.5 days of growth, samples from the top of this depth interval, in a section of iron oxide–stained pumice fragments at 26.26–28.45 mbsf, showed significant growth in artificial seawater (ASW) media A (microaerophilic). The preference of these microbes for a microaerophilic environment agrees with the identification of this layer as a zone of recharge flow (oxygenated seawater). Above this layer, outside of the oxidized zone, little to no growth of FeOB was observed. Below this layer, where the sediments transition from yellow-to-brown clay, silt, and sand (see “Petrology”), there was also no growth, suggesting a FeOB preference for growth where there is less fine-grained material.

Observations of putative FeOB cellular morphology were similar to those from other sites from Expedition 331. These include ~1.5 µm rod-shaped cells associated with small particles (Fig. F16A, F16B, F16E, F16F) in addition to large aggregates of cells colonizing larger particles (possibly iron oxides; Fig. F16G, F16H). Of particular importance are the two cells localized on the end of a stalklike structure (Fig. F16C, F16D). Though difficult to see because of the angle of observation, moving up and down through the plane of focus showed that these cells were on opposite sides of a stalk jutting out of the top of the larger particle. This is the first observation of a putative bifurcated iron oxide twisted stalk with attached cells in any of the incubations viewed during the expedition.

Contamination tests from Site C0017 (Tables T11, T12) reveal that all samples that showed growth also exhibited some level of contamination. For Section 331-C0017C-1H-7, contamination was below detection after counting 100 fields, though a single bead was seen upon cursory examination. This may be due to a cracked core liner for that section allowing washout to have occurred. Section 331-C0017C-1H-6 was taken after this core was split and was not tested for contamination, though it came from an intact internal location within the core. Overall, caution must still be exercised when considering whether these Site C0017 enrichments represent FeOB originating from in situ locations.

In an attempt to test whether biotic iron oxidation was occurring in situ at depth, the original sample used to inoculate the enrichment for Section 331-C0017C-1H-7 was dried and imaged using the scanning electron microscope (SEM) and analyzed with energy dispersive spectrometry (EDS) to test for the presence of putative iron oxide filaments of biotic origin. As the doubling time for FeOB is ~12 h (Emerson et al., 2007), these filaments would not have had time to form as the core was brought on deck. SEM results show a number of patches of iron and iron oxide filaments, many located within protected cavities of pumice fragments. Photos were taken of filaments within these cavities (Fig. F17A, F17B), which may explain the lack of a significant oxygen peak in the EDS spectrum. Figure F17C is the only EDS spectrum that has a significant oxygen peak; however, because of the low ratio of oxygen to iron, an iron oxyhydroxide (FeOOH) filament is improbable as compared to other marine FeOB (Emerson and Moyer, 2002; Emerson et al., 2007). Though it is possible that all three of these iron/iron oxide filament patches are of biotic origin, this evidence is not conclusive. Further testing must be conducted onshore.

Conclusions

Site C0017 is located in a probable seawater recharge area in the Iheya North field. Significant impacts of hydrothermal fluids were not observed in geochemical profiles and sediment alteration, except for localized oxidation (see “Geochemistry” and “Lithostratigraphy”). The temperature at the bottom of the deepest Hole C0017D at 151 mbsf is 90°C. We have thus had an opportunity to observe subseafloor microbial communities likely supported by buried organic matter, over a considerable range of temperature.

Relatively low microbial abundance compared with previous Ocean Drilling Program/IODP sites on continental margins (Parkes et al., 1994, 2000; D’Hondt et al., 2004) was seen at Site C0017 (as many as 3.2 × 10⁷ cells/mL), as was also the case at Site C0015 (as many as 1.2 × 10⁷ cells/mL). Cell abundance at Site C0017 may also be depressed as a result of the generally low levels of organic carbon available in the subseafloor sediments around the Iheya North field (see “Microbiology” and “Geochemistry,” both in Expedition 331 Scientists, 2011e). At Site C0017, effects of lithology on cell abundance were again observed, as at Site C0015, where a relatively abundant microbial biomass was observed in samples with pumiceous sands and gravels. (Compare, for example, cell counts in the pumiceous sample from 10.8 mbsf in Hole C0017B with that in the hemipelagic mud sample from 14.8 mbsf) (Table T10). As is usually the case (Parkes et al., 1994, 2000), the subseafloor microbial abundance generally decreases with depth (Fig. F15). Lithostratigraphy at Site C0017 consists of diverse units such as hemipelagic mud and clay, pumiceous sediment, and volcaniclastic-pumiceous mixed sand (see “Lithostratigraphy”). Cell abundances are strongly influenced by this lithological variation and its effect on porosity and pore space size, as seen in previous studies (Schmidt et al., 1998; Zhang et al., 1998; Parkes et al., 2000; Inagaki et al., 2003; Rebata-Landa and Santamarina, 2006), as well as by geochemical constraints imposed by seawater recharge.

Though contamination cannot be ruled out, enrichment experiments represent the deepest successful FeOB enrichments to date in a marine environment and could lend insight into the colonization of low-temperature hydrothermal systems via recharge zone flow.

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