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The goals of microbiology sampling at Site U1376 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 for calculation of metabolic rates of subseafloor microbes at Burton Guyot. Eleven whole-round samples (5–13 cm long) were collected for microbiological analysis (Figs. F71, F72). The samples were volcanic sandstone (two), limestone (boundstone) (two), volcanic breccia (three), and basaltic lava flows (four). All samples were preserved for shore-based DNA analysis, cell counting, and δ34S and δ13C analyses. Five samples were used to inoculate 70 culturing experiments with as many as 10 different types of cultivation media (Table T15). Two samples were used to set up stable isotope addition bioassays to determine rates of carbon and nitrogen utilization by subsurface microbes at Burton Guyot (Table T16). One core was seeded with fluorescent microspheres, and samples from this core were collected for shipboard analysis of contamination via fluorescent microsphere counts.

Cell counts

Performing shipboard cell counts on rock samples is difficult or 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 U1376.

Culturing experiments

Five samples were used to inoculate 70 culturing experiments with as many as 10 different types of cultivation media targeting autotrophic sulfur oxidizers, heterotrophic sulfur oxidizers, autotrophic iron oxidizers, autotrophic iron reducers, heterotrophic iron reducers, heterotrophic sulfate-reducing bacteria, and nonspecific heterotrophs (Table T15; for details on media recipes, see Table T14 in the “Methods” chapter [Expedition 330 Scientists, 2012a]). On the basis of visual observation of turbidity, 43% of the vials had obvious growth in them. Media targeting sulfur-oxidizing bacteria and general heterotrophic bacteria were the most successful. The highest proportion of inoculated vials with positive growth are from Sample 330-U1376A-2R-3, 0–6 cm, a volcanic sandstone, and Sample 17R-2, 112–120 cm, an aphyric basalt. The deepest depth from which positive results were obtained is 174 mbsf (Sample 330-U1376A-23R-1, 102–114 cm). All preliminary results will be verified during shore-based research.

Stable isotope addition bioassays

Two samples were used to initiate stable isotope addition bioassays to study rates of carbon, nitrogen, and sulfur cycling by subsurface microbes at Burton Guyot (Table T16). 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 T16). Both bioassays include “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 “Site U1373” chapter [Expedition 330 Scientists, 2012c]) and stable isotopes were added to the vials; from that point on the killed vials were treated in the same way as 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-U1376A-7R-3, 59–71 cm, only) and the other will be terminated at t3 (6 months; both samples).

As with the stable isotope addition bioassays performed at Sites U1373 and U1374, 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

Fluorescent microspheres were deployed during drilling for Core 330-U1376A-19R. A small amount of drill fluid was collected from inside the core liner when the microbiology whole-round sample was selected. 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 was subjected to before subsampling. For each wash, 50 mL of sterile seawater was used. After the microbiology whole round was washed, two subsamples were taken from the outside of the whole round and two were collected from the inside of the whole-round sample during standard microbiology sampling. These were preserved the same way as cell count samples and were analyzed via fluorescent microscopy to quantify microspheres in the rocks.

Shipboard counts of fluorescent microspheres revealed 1.2 × 104 microspheres per milliliter of drill fluid. Microsphere counts of the rinse water were negative for the first two rinses, and there was one clump of three microspheres in the third wash. No microspheres were detected on the outside or inside of the whole-round samples. The absence of any microspheres on or inside the whole-round samples indicates that the potential for microbial contamination was low during drilling of Hole U1376A.