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doi:10.2204/iodp.proc.304305.103.2006

Microbiology

Lost City, an extensive hydrothermal field, was recently discovered near the MAR at 30°N (Kelley et al., 2001). This example of an ultramafic hosted hydrothermal system is postulated to be driven by the heat of the exothermic serpentinization reaction (Kelley et al., 2001), as opposed to the cooling of basaltic material, which drives the hydrothermal flow in younger basaltic crust (Fornari and Embley, 1995). Serpentinization reactions produce significant volumes of hydrogen, methane, and, possibly, low molecular weight organic compounds (Allen et al., 1998; Shock and Schulte, 1998; Berndt et al., 1996; Janecky and Seyfried, 1986; Neal and Stanger, 1983). At the Lost City site, an analysis of the microbial communities revealed the predominance of a single group of methane-metabolizing Archaea (Schrenk et al., 2004). The results of this study suggest that a microbial niche may exist for hydrogen-utilizing methanogens and that the high level of methane (as compared to seawater) measured at Lost City may be produced as a direct result of microbial metabolism. In addition to the microbial niche potentially occupied by methanogens, the hydrogen produced from serpentinization reactions and the iron present in ultramafic rocks may also support other microbial populations, including iron-reducing bacteria.

To further elucidate the microbial communities present in a system possibly similar to the Lost City, a microbiological analysis of the rocks cored at Atlantis Massif, 30°N, was undertaken during Expeditions 304 and 305. A culture collection of anaerobic sediment and endolithic microbes was established and monitored throughout both expeditions. Additionally, core samples from Hole U1309D were frozen at –80°C for shore-based molecular analysis. Unfortunately, only minor amounts of ultramafic rocks were recovered and Hole U1309D is lithologically defined as a mafic system. Even though only a small proportion of the rocks recovered during Expeditions 304 and 305 had an ultramafic protolith, fluids circulating through the massif potentially reacted with substantial volumes of ultramafic rock. Thus, a comparison of microbial communities present at the Lost City hydrothermal vent field and Site U1309 is warranted. Regardless of the lithology, microbiological analyses of Expedition 304 and 305 samples will provide an unprecedented body of knowledge of the deep subsurface biosphere from the seafloor to 1400 mbsf.

Sample information

Carbonate sediment was sampled from 0.45 mbsf in Hole U1309A (Sample 304-U1309A-1R-2, 45–58 cm). Three types of igneous rocks (Samples 304-U1309B-2R-1, 18–26 cm, basalt; 12R-2, 34–43 cm, diabase; and 16R-2, 0–8 cm, gabbro) were sampled in Hole U1309B from the depths of 15.8, 61.9, and 80.4 mbsf, respectively. In Hole U1309D, five types of igneous rocks were sampled. Serpentinized peridotites (Samples 304-U1309D-10R-1, 107–112 cm, and 58R-1, 67–73 cm) were sampled from 60.20 and 300.40 mbsf, respectively. Olivine gabbros (Samples 305-U1309D-80R-1, 18–28 cm, 90R-1, 30–36 cm, 184R-1, 78–85 cm, and 250R-1, 0–11 cm) were sampled from depths of 401.48, 448.90, 895.78, and 1201.50 mbsf, respectively. Olivine-bearing gabbros (Samples 305-U1309D-82R-1, 27–39 cm, 102R-2, 67–78 cm, 122R-2, 76–89 cm, 142R-3, 0–13 cm, and 290R-3, 136–145 cm) were sampled from depths of 410.47, 508.31, 604.24, 701.05, and 1391.01 mbsf, respectively. Olivine-rich troctolites (Samples 305-U1309D-100R-1, 80–89 cm, and 235R, 52–63 cm) were sampled from depths of 497.40 and 1131.28 mbsf, respectively. Gabbros (Samples 304-U1309D-12R-3, 46–54 cm, 28R-4, 43–56 cm, 37R-2, 94–102 cm, 53R-1, 100–111 cm, 68R-1, 88–101 cm, and 78R-1, 82–90 cm, and 305-U1309D-133R-3, 122–132 cm, 164R-1, 60–74 cm, 208R-4, 0–12 cm, and 273R-1, 116–132 cm) were collected at depths of 69.80, 160.93, 201.90, 276.40, 348.40, 396.50, 658.73, 799.60, 1004.79, and 1313.06 mbsf, respectively.

In addition to rock samples, 5 mL of seawater was collected using a sterile WSTP from 5 m above the seafloor (see “Appendix A”) and at 390 and 1215 mbsf (see “Appendix B”). Water samples will serve as a control for molecular analysis to constrain the degree of contamination of core samples by microorganisms from the surrounding seawater.

In situ cell densities

Interior portions of core samples were stained with 4′,6-diamidino-2′-phenylindole-dihydrochloride (DAPI) (Expedition 304) and acridine orange (Expedition 305) and viewed using bright-field illumination and epifluorescence microscopy to determine in situ cell densities. The cell densities in carbonate sediment were determined. The density in the exterior and interior of carbonate cores were 1.1 × 10⁴ ± 0.9 × 10⁴ cells/mL and 1.2 × 10⁴ ± 1.0 × 10⁴ cells/mL, respectively. Because of the overwhelming autofluorescence of rock material, it was not possible to quantify cell densities of igneous rock samples. The question as to whether microbial communities are present in rock samples will be addressed by shore-based molecular analysis of core sections frozen at –80°C.

Cultivation experiments

Expedition 304

Cultures that were established during Expedition 304 were monitored for cell growth using phase-contrast and epifluorescent microscopy (Table T24). A subsample extracted from the interior of the carbonate ooze was used as the innoculum in enrichment experiments to bring into culture methanogenic and sulfate-reducing microbes at 5°C. No positive growth was observed in these cultures. Portions from the interiors of basalt, gabbro, and serpentinized peridotite core samples were used as innoculum to bring into culture methanogens, sulfate-reducing microorganisms, and organic matter–utilizing microbes. After 2 days of incubation at 65°C, fluorescing particles were observed in a culture bottle containing methanogen enrichment media (Section 304-U1309B-16R-2). These particles radiated green light under the blue filter of wavelength 450–490 nm (Fig. F279). A methanogen is able to radiate green light because the absorption spectrum of methyl-coenzyme M reductase, which is the terminal enzyme of methanogenesis (Thauer, 1998). After 5 days of incubation at 85°C, fluorescence of cell-like particles from Section 304-U1309B-16R-2, stained with DAPI, were observed in a culture containing methanogen enrichment media (Fig. F279C).

Molecular analysis is required to confirm that the observed fluorescence is caused by the presence of microbes and not by the presence of minerals that fluoresce when exposed to ultraviolet light. Two gabbro incubations at 37°C (Sections 304-U1309B-16R-2 and 12R-3) resulted in growth of organic matter–utilizing microorganisms after 6 and 7 days, respectively (Fig. F279D; Table T24).

Expedition 305

In an effort to establish a culture collection of endolithic microbes from Site U1309, four different types of media (~300 cultures established), as well as agar plates, were inoculated with interior portions of core samples. Growth was observed in samples from Sections 305-U1309D-80R-1 (Fig. F280) and 82R-1 on marine agar 2216. To begin the process of isolation, a portion of each of the two colonies was transferred from the original plate to a second marine agar 2216 plate. Subsequently, an additional transfer was made from the second plate to a third marine agar 2216 plate. Additionally, using a sterile innoculating loop, tubes of each of the four types of liquid media discussed above were innoculated with a small portion of the viable colony and incubated at 30°C. Growth in liquid media and on marine agar 2216 plates was monitored.

Liquid cultures were monitored for growth via increased turbidity. Cultures that appeared more turbid with time were subsampled, stained with acridine orange, and observed using epifluorescence microscopy (Fig. F281). Several cultures contain fluorescing spheres and small rods, both on and off the rock surfaces; however, because of the autofluorescence of rock particles, the presence of microorganisms will not be confirmed until a shore-based molecular analysis of cultures is undertaken.

Contamination experiments

Expedition 304

The extent of contamination of the carbonate sediment sample was determined by presence/absence of fluorescent microspheres. One microsphere was detected from the exterior of the core sample. No microspheres were detected in the interior of the sample. This result suggests that the exterior of the carbonate sediment sample was potentially contaminated by microorganisms from ambient seawater during the drilling process. Perfluorocarbon tracer (PFT) was not used as an indicator of contamination for the sediment sample. A contamination test of igneous rocks was conducted both with bacterial-sized fluorescent microspheres and PFT. A perfluorocarbon analysis with a gas chromatograph (Hewlett Packard 8059) revealed that all subsamples had PFT concentrations that were below detection limits. A maintenance inspection on the PFT system during the Expedition 305 transit to Site U1309 revealed that both the primary and secondary PFT pumps had suffered a failure (for different reasons). The storage tank for PFTs was depleted, but it cannot be determined if this was caused by use or evaporation through the damaged systems. This is the first occurrence of a system failure, and it cannot be determined whether PFTs were introduced into the drill water system during Expedition 304. However, because no PFTs were detected in any of the samples collected, we presume that PFTs were not incorporated into the drill water circulation system during Expedition 304.

Fluorescent microspheres were detected in seawater used for rinsing the surface of the samples. No microspheres, however, were detected from the interior of igneous rock samples. This result suggests that interior rock samples were not contaminated by microorganisms from surface seawater used for circulation during the drilling.

Samples from Sections 304-U1309D-12R-3 and 58R-1 were not initially intended for microbiological sampling; therefore, neither PFT tracer nor microspheres was used as a contamination indicator.

Expedition 305

To determine the extent of contamination in core samples, fluorescent microspheres were deployed in the core catcher for samples intended for microbiological sampling. The exterior of each core sample was rinsed with nanopure water. This water sample was collected, filtered, and viewed using epifluorescence microscopy. All core samples had detectable levels of microspheres on the exterior. Interior portions of each core were also viewed using epifluorescence, and it was determined that no microspheres were present from the interior subsamples. This initial contamination assay indicates that if seawater penetrated rock samples, microorganisms >0.5 µm were excluded. PFTs were not used during this expedition because of an inability to process samples.

In addition to microspheres, deoxyribonucleic acid will be extracted from water samples collected in Hole U1309D at 390 and 1215 mbsf and used to conduct a comparison of the microbes present in seawater versus those found on the interior of core samples. This comparison will serve as the ultimate control in determining the extent of contamination of rock samples by seawater microorganisms.

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