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The goals of microbiology sampling at Site U1374 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. These bioassays will allow for calculation of metabolic rates of subseafloor microbes at Rigil Guyot. Twenty-nine whole-round samples (5–13 cm long) were collected for microbiological analysis (Figs. F95, F96). The samples were unconsolidated sediment (1), volcanic sandstone (2), volcanic or sedimentary basalt breccia (23), and aphyric basaltic lava flows (3). All samples were preserved for shore-based DNA analysis, cell counting, and δ34S and δ13C analyses. Eleven samples were used to inoculate culturing experiments with as many as seven different types of cultivation media (Table T18). Five samples were used to set up stable isotope addition bioassays to determine rates of carbon and nitrogen utilization by subsurface microbes at Rigil Guyot (Table T19). Two cores were seeded with fluorescent microspheres, and samples from these cores were collected for shipboard analysis of contamination via fluorescent microsphere counts (Table T20).

Cell counts

Performing shipboard cell counts on rock samples is difficult to nearly impossible because of the combination of autofluorescence from rock particles and the difficulty of focusing using a 100× objective paired with a 10× eyepiece (1000× total magnification) on a moving ship. Therefore, cell counts were not attempted on samples from Site U1374.

Culturing experiments

Eleven samples were used to inoculate culturing experiments with as many as seven 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 T18; for details on media recipes, see Table T14 in the “Methods” chapter [Expedition 330 Scientists, 2012a]). On the basis of visual observation of turbidity, several vials had obvious growth in them. Media targeting sulfur-oxidizing bacteria and general heterotrophic bacteria appear to have yielded growth in multiple samples; however, media targeting methanogens and sulfate-reducing bacteria were not successful, probably because of the continued difficulty in maintaining anoxic conditions, which are required for the growth of both groups of bacteria. The deepest depth from which positive results were obtained is 400 mbsf (Sample 330-U1374A-58R-6, 50–62 cm). All preliminary results will be verified during shore-based research.

Stable isotope addition bioassays

Five samples were used to initiate stable isotope addition bioassays to study rates of carbon, nitrogen, and sulfur cycling by subsurface microbes at Rigil Guyot (Table T19). A more effective method for obtaining large amounts of uncontaminated rock sample for these bioassays (“enhanced extraction”) was developed during processing of Sample 330-U1374A-15R-2, 79–89 cm (see “Microbiology” in the “Methods” chapter [Expedition 330 Scientists, 2012a]). Enhanced extraction was carried out on all samples from Hole U1374A used to initiate stable isotope analyses.

Depending on the volume of rock obtained, one or two experiments were executed with two sets of conditions. The first experiment was addition of 13C bicarbonate and 34S elemental sulfur. This experiment was conducted with all samples. Beginning with Sample 330-U1374A-21R-2, 111–119 cm, 15N compounds were also added to all samples. To bioassays with 13C carbonate, 15N ammonia was added. When large volumes of sample were available for stable isotope addition bioassays, an additional experiment with 13C glucose, 13C acetate, 34S elemental sulfur, and 15N nitrate was conducted. This combination of stable isotope additions aimed to quantify C, N, and S cycling specifically among heterotrophic microbes. Because quantification of elemental cycling by heterotrophs was a secondary goal of the stable isotope addition bioassays, addition of 13C glucose, 13C acetate, 34S elemental sulfur, and 15N nitrate was not always done (Table T19). Additionally, the amount of 13C glucose available was depleted after adding it to two samples (330-U1374A-21R-2, 111–119 cm, and 24R-3, 90–101 cm) and it was therefore not used further.

Incubations from Samples 330-U1374A-31R-3, 142–150 cm, and 71R-3, 47–55 cm, included two “killed” control vials. These vials were treated in the same way as the other experimental vials, with the exception that after adding the rock chips, 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 “Site U1373” chapter [Expedition 330 Scientists, 2012c]) and stable isotopes were added to the vials, and they were then treated as the other experiments. This 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) and the other will be terminated at t3 (6 months).

As with the stable isotope addition bioassay performed at Site U1373, 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, 2 months, and 6 months, 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

Fluorescent microspheres were deployed during drilling for Cores 330-U1374A-8R and 45R. As the core arrived on the rig floor, a rig floor worker collected the drill fluid draining out of the core liner with a beaker (Fig. F97). Analysis of this drill fluid should provide the concentration of microspheres delivered to the core. Water samples for microsphere counts were also collected from each of the three separate sterile seawater rinses that every microbiology whole-round sample is subjected to before subsampling. For each wash, 50 mL of sterile seawater was used. After the microbiology (MBIO) whole round was washed, subsamples were taken during standard microbiology sampling and preserved the same way as cell count samples. The subsamples were analyzed via fluorescent microscopy to quantify microspheres in the rocks. Additionally, following MBIO sterile processing, a ~1 cm thick whole-round slab was cut from the remainder of the whole-round sample with the Feckler saw. This slab was divided into five sections with the Feckler saw (Fig. F97) for microsphere counting of specific areas. Subsamples from these sections were taken specifically from the middle of the section (area shown between dashed lines in Fig. F97).

Shipboard counts of fluorescent microspheres revealed that the microspheres were indeed delivered within the core liner, as indicated by their presence in the drill fluid (Table T20). Microsphere counts of the rinse water are lower than those in the drill fluid and are zero by the third wash. This indicates that few, if any, microspheres were left on the surface of the whole round when the microbiologist began processing samples. The absence of any microspheres on or inside the whole-round samples indicates that the potential for microbial contamination was extremely low during drilling of Hole U1374A.

As an additional test for microbial contamination, drill fluid was collected from a tap located on the drill rig floor. One liter of the fluid was filtered on a 0.2 µm polycarbonate filter. One liter was also frozen unfiltered. 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. It is unlikely that microbes resident in surface seawater would also be present in the deep subsurface of a seamount. Therefore, if microbes detected in the drill fluids were also detected in one or more samples, we would know that the samples were most likely contaminated. An example of obvious drill fluid contamination would be the presence of cyanobacteria, which require sunlight for survival.