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The goals of microbiology sampling at Site U1373 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 and 34S stable isotopes can be measured. Such bioassays then allow for calculation of metabolic rates of subseafloor microbes. Five whole-round samples (5–10 cm long) were collected for microbiological analysis (Figs. F61, F62). The samples were sedimentary conglomerate (one), aphyric basalt breccia (one), and aphyric basalt lava flows (three). In the thick lava flow at the bottom of Hole U1373A, samples with visible veins were preferably taken for microbiological analyses. Four of the five samples were preserved for shore-based DNA analysis, cell counting, and δ34S and δ13C analyses. Sample 330-U1373A-10R-2, 114–123 cm, contained no veins or features that would allow fluid flow, and after several failed attempts to crack open the core with a hammer and chisel, it was determined that this rather massive basalt was likely to have little or no microbial life. Therefore, a subsample was taken for cell counts only. Sample 330-U1373A-2R-1, 120–133 cm, was used to inoculate culturing experiments with six different types of cultivation media; Sample 330-U1373A-11R-2, 122–133 cm, was collected for shipboard analysis of contamination via fluorescent microsphere analysis; and Sample 330-U1373A-13R-3, 118–128 cm, was used to set up a stable isotope addition bioassay.

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 U1373.

Culturing experiments

Twelve vials were inoculated for culturing experiments with media targeting autotrophic sulfur oxidizers, heterotrophic sulfur oxidizers, autotrophic iron oxidizers, autotrophic iron reducers, heterotrophic iron reducers, and nonspecific heterotrophs (for details on media recipes, see Table T14 in the “Methods” chapter [Expedition 330 Scientists, 2012a]). Media were monitored for oxygen levels with the redox dye resazurin. The addition of resazurin turns media saturated in oxygen blue and media with low oxygen levels pink; anoxic media remain clear. Several types of media that require anoxic conditions (for methanogens and sulfate reducers) remained pink (instead of clear) despite continued efforts to generate anoxic conditions, and therefore these were not used for Hole U1373A samples because the targeted microbes would be poisoned by the presence of oxygen. Some culture experiments for autotrophic and heterotrophic sulfur oxidizers and nonspecific heterotrophs appeared to show growth on the basis of turbidity, but this requires verification by shore-based research.

Contamination testing

Fluorescent microspheres were deployed during drilling for Core 330-U1373A-11R. As the core arrived on the rig floor, a core laboratory technician collected the drill fluid draining from the core liner with a beaker. Analysis of this drill fluid provides the concentration of microspheres delivered to a core. Unfortunately, during retrieval of Core 330-U1373A-11R, there was difficulty removing the core catcher, and nearly all of the drill fluid was lost. Therefore, only a few milliliters of drill fluid was recovered.

Water samples for microsphere counts were also collected from each of the three separate sterile seawater rinses that every microbiology (MBIO) whole-round sample was subjected to before subsampling. For each wash, 100 mL of sterile seawater was used. After MBIO whole-round Sample 330-U1372A-11R-2, 122–133 cm, was washed, four subsamples were taken during standard MBIO sampling for DNA and cell counts and were preserved in the same way as cell count samples. Two subsamples were taken from the outside portion of the whole-round sample and two were taken from the center. 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 (Fig. F63) with the Feckler saw for microsphere counting of specific zones. Subsamples from these sections were taken specifically from the middle of the sections (area shown between dashed lines in Fig. F63).

Shipboard counts of fluorescent microspheres revealed only one microsphere in the drill fluid and one microsphere in Section 1 of the slabbed whole round; all other observed microscope fields, including those from the three sterile seawater rinses, the five sections of cut rock, and the four rock samples collected during standard MBIO sampling, were devoid of microspheres. The general absence of any microspheres, especially in the drill fluid, indicates three possibilities. First, it is possible that the microspheres were not delivered or that they remained very near the spot of delivery. However, we can rule out a lack of delivery, because the broken bag from which the microspheres were deployed was recovered within the core liner (Fig. F63). Second, it is possible that the microspheres did not circulate sufficiently in the core. A third possibility, one that is more likely, is that because of the relative impermeability of the lava flow collected in Core 330-U1373A-11R, the microspheres could not penetrate into the massive basalt and were flushed out of the core barrel as the core ascended the water column on the way to the rig floor. This indicates that the chance for microbial contamination in this core is low and that the MBIO samples collected were pristine.

Stable isotope addition bioassays

Sample 330-U1373A-13R-3, 118–128 cm, was used to initiate a stable isotope addition bioassay. The purpose of these experiments is to determine metabolic rates of microbes living in the subseafloor basement environment. Recent work has illustrated the presence of microbial cells in basement habitats (Cowen et al., 2003; Mason et al., 2010; Santelli et al., 2010; Orcutt et al., 2011), but there are as yet no physiological data or metabolic rate estimates available for these microbes. The stable isotope addition bioassays performed during Expedition 330 aimed to constrain the rates of carbon cycling in the basement rocks of the Louisville Seamounts.

Approximately 15 cm3 of rock was collected from veins in Section 330-U1373A-13R-3 and split into three separate 125 mL precombusted glass serum vials. To these vials, 100 mL of sterile seawater (Sigma-Aldrich; St. Louis, MO) modified with 50 µM sodium nitrate, 3 µM potassium phosphate, and 0.5 µM ammonium chloride was added as base media. We herein refer to this as the basic seawater media. In each vial containing ~5 cm3 of rocks and 100 mL of basic seawater media, 2.71 µM 13C-labeled sodium bicarbonate and 0.30 µM 34S elemental sulfur was added. 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 of these three vials will be terminated, and the rocks will be collected for onshore measurement of the incorporation of labeled bicarbonate and sulfur.