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The goals of microbiology sampling at Site U1377 were to collect samples for shore-based cell counts, molecular biological analyses, and δ34S and δ13C stable isotope analyses; to inoculate media for cultivation of subseafloor microbes; and to set up stable isotope addition bioassays whereby the rate of incorporation of compounds labeled with 14C, 15N, and 34S can be measured. Such bioassays will allow calculation of metabolic rates of subseafloor microbes at Hadar Guyot. Two whole-round samples (8–11 cm long) were collected for microbiological analysis: a moderately olivine-phyric trachybasalt from 46.84 mbsf in Hole U1377A and an aphyric trachybasalt from 18.1 mbsf in Hole U1377B (Fig. F31). Both samples were preserved for shore-based DNA analysis, cell counting, and δ34S and δ13C analyses. Sample 330-U1377B-4R-1, 0–8 cm, was used to inoculate 18 culturing experiments with nine different types of cultivation media (Table T12). Both samples were used to set up stable isotope addition bioassays to determine rates of carbon, nitrogen, and sulfur utilization by subsurface microbes at Hadar Guyot (Table T13). A sample of drill fluid was collected for analysis of the microbial community for comparison with microbial communities in the subsurface rocks collected and for detection of possible contamination.

Culturing experiments

Sample 330-U1377B-4R-1, 0–8 cm, was used to inoculate 18 culturing experiments with nine different types of cultivation media targeting autotrophic sulfur oxidizers, heterotrophic sulfur oxidizers, autotrophic iron oxidizers, autotrophic iron reducers, heterotrophic iron reducers, and nonspecific heterotrophs (Table T12; for details on media recipes, see Table T14 in the “Methods” chapter [Expedition 330 Scientists, 2012a]). Visual observation of turbidity to assess growth was not carried out because the experiments were inoculated shortly before the end of the cruise and there was not enough time for detectable growth to occur. All experiments will be analyzed during shore-based research.

Stable isotope addition bioassays

Both samples collected were used to initiate stable isotope addition bioassays to study rates of carbon, nitrogen, and sulfur cycling by subsurface microbes at Hadar Guyot (Table T13). Enhanced extraction (see “Microbiology” in the “Methods” chapter [Expedition 330 Scientists, 2012a]) was carried out on both samples.

For both samples, 2.71 mM 13C bicarbonate, 300 µM 34S elemental sulfur, and 0.5 µM 15N ammonia were added to the bioassays (Table T13). Both bioassays included “killed” control vials. These vials were treated in the same way as the other experimental vials, with the exception that after the rock chips were added the vials were combusted at >400°C for 3 h to kill all microbes. After the vials were cooled to room temperature, basic seawater media (see “Microbiology” in the “Methods” chapter [Expedition 330 Scientists, 2012a]) and stable isotopes were added to the vials, and from that point on the killed vials were treated like the other (“live”) vials. This kill treatment acts as a negative control and provides a baseline stable isotope reading for the rocks in the experiment. One vial will be terminated at t2 (2 months; Sample 330-U1377A-6R-3, 56–66 cm, only) and one will be terminated at t3 (6 months; both samples).

As with the stable isotope addition bioassays performed at other sites, stable isotopes and rock chips were added to 125 mL serum vials, followed by 100 mL of basic seawater media. The vials were then placed in a 4°C incubator in the dark. At time points of 2 weeks (t1), 2 months (t2), and 6 months (t3), the incubation in one or more vials (depending on number of vials per condition) will be terminated, and the rocks will be collected to measure incorporation of labeled carbon, nitrogen, and sulfur.

Contamination testing

Drill fluid was collected from a tap located on the drill rig floor, and 2 L of the fluid was filtered onto a 0.2 µm polycarbonate filter. The filter will be analyzed for microbial community composition during shore-based research. Drill fluid is composed primarily of surface seawater that is circulated down through the borehole, and it is unlikely that microbes resident in surface seawater would also be present in the deep subsurface of a seamount. If microbes detected in the drill fluid are also detected in one or more samples, we will know that these samples were most likely contaminated. The Site U1377 drill fluid sample will complement the drill fluid sample collected at Site U1374 (see the “Site U1374” chapter [Expedition 330 Scientists, 2012b]) for the same type of analysis.