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doi:10.2204/iodp.proc.330.103.2012

Microbiology

The goals of microbiology sampling at Site U1372 were to collect samples for shipboard cell counts and shore-based molecular biological studies and δ³⁴S and δ¹³C stable isotope analyses and to inoculate media for cultivation of subseafloor microbes. Of particular interest are the differences between microbes in the sediment and those in the underlying basement. Fifteen whole-round samples (5–10 cm long) were collected for microbiological analysis (Figs. F70, F71). In particular, more altered rocks and rocks with indications of high porosity were selected for microbiological analysis. All samples were preserved for shore-based DNA analysis, cell counting, and δ³⁴S and δ¹³C analyses (isotope analysis will be done only on rock samples). Four samples were used to inoculate culturing experiments with as many as 10 different types of cultivation media, and one sample was collected for shipboard analysis of contamination via fluorescent microsphere analysis. Samples ranged from unconsolidated sediment (2) to volcaniclastic breccia (3) to basaltic lava flows (10).

Cell counts

Cell counts were 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. An example of a typical field of view illustrates the difficulty in distinguishing between cells and rock pieces (Fig. F72). Therefore, the presence of microorganisms can neither be confirmed nor denied until shore-based cell counts are performed under more controlled conditions. However, initial indications from one sample (330-U1372A-18R-2, 44–51 cm) revealed likely microorganisms. Tentative cell counts from this sample indicate there may be as many as 7.7 × 10⁶ microbial cells per gram of rock, but these need to be confirmed through shore-based analysis. For comparison, prokaryotic cell counts for seafloor-exposed basalts range from 3 × 10⁶ to 1 × 10⁹ cells per gram of rock (Santelli et al., 2008).

Culturing experiments

Fifty-four vials were inoculated for culturing experiments with 10 different kinds of media (Table T16). Media were monitored for oxygen levels with the redox dye resazurin. The addition of resazurin turns media with saturated oxygen levels blue and low oxygen levels pink; anoxic media remain clear. Several types of media that require anoxic conditions (for methanogens and sulfate reducers) were pink upon arrival at Site U1372, which indicated that these solutions did not remain anoxic. Several attempts to purge the oxygen in these media failed, including bubbling with N₂ gas and adding the reducing agents sodium sulfide and sodium bicarbonate. Therefore, these media types were not inoculated for every sample. Media targeting iron reducers, iron oxidizers, sulfur oxidizers, and oxygenic heterotrophs were inoculated with all four samples used for culturing experiments. Several culture experiments appeared to have growth in them on the basis of turbidity, and cells were observed in an incubation with 1% marine broth when a small sample was taken and stained with SYBR Green I DNA stain (Sample 330-U1372A-18R-2, 44–51 cm).

Contamination testing

Fluorescent microspheres were deployed during drilling for Core 330-U1372A-9R. Upon retrieval of the microbiology (MBIO) whole-round sample (330-U1372A-9R-4, 0–9 cm), four subsamples were taken during standard MBIO sampling for DNA analysis and cell counts. Two were taken from the outside portion of the whole-round sample: one from the center and one in the area between the outside and 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. The slab was divided into five sections with the Feckler saw (Fig. F73) for microsphere counting of specific zones in the recovered basement core. Shipboard counts of fluorescent microspheres revealed a range of values from 0 to nearly 6.3 × 10⁵ microspheres per gram of rock (Table T17). There are ~10⁵–10⁶ microbial cells per gram (or milliliter) of surface seawater, the source of the drilling fluid. On the basis of preliminary cell counts, there may be ~8 × 10⁶ cells per gram of rock. Therefore, contamination may be substantial (as much as 10% of the cells present in the basaltic lava flow samples and nearly 100% of the cells in the drilling fluid). However, closer examination of the counts reveals that the absolute numbers may be an overestimation because, in general, there were no microspheres present in 90% of the microscopic fields observed. Additionally, when microspheres were present, they were often clumped together. As a consequence, the total number of microspheres observed might be high even though contamination was really limited to one location (e.g., a vesicle or vug). Therefore, the actual number of potentially contaminating cells is likely lower than that observed. Additional counts at other Expedition 330 sites will aid in interpretation of the contamination testing of Sample 330-U1372A-9R-4, 0–9 cm.

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