Sediment samples from Holes 1149A, 1149B, and 1149C at Site 1149 were collected to characterize the microbial community inhabiting this environment. Whole rounds, cut on the catwalk, were used for both microbiological analyses and contamination tests. Surface seawater was collected to enumerate and characterize background levels of microbes possibly introduced during drilling. Interstitial water from the whole rounds was collected after the microbiological samples and analyzed for major elements, iron, manganese, and nutrients (see "Interstitial Water Chemistry and Headspace Gas"). These analyses will allow interpretation of the microbiological data in a geochemical context.
A list of samples taken at Site 1149 for microbiological analyses is shown in Table T21. The analyses include (1) adenosine triphosphate (ATP) analysis to estimate total biomass, (2) most probable number (MPN) cultivation at 1 atm, (3) enumeration of cells by epifluorescence microscopy, (4) DNA extraction and community characterization, and (5) maintenance at in situ pressure. Shipboard work included ATP analysis, initiation of MPN cultures in anaerobic media, and preservation of samples for shore-based analyses (see "Microbiology" in the "Explanatory Notes" chapter for procedures).
Tracer tests were carried out as a part of Leg 185 microbiology studies to investigate if the drilling and sample-handling processes contaminate cores. At Site 1149, tests were carried out using perfluorocarbon (PFT) and fluorescent microspheres in four cores (Tables T22, T23). Methods used for these tests can be found in "Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring" (Smith et al., 2000). The PFT was present on the exterior of the sediment samples in all cases. The interior of the unconsolidated sediments sampled with the APC remained uncontaminated in most cases (Table T22), whereas consolidated sediments sampled with the RCB contained traces of the PFT (Fig. F77; see also Fig. F75 in the "Site 801" chapter). Fluorescent microspheres were not observed in the interior of any sediment sample, and they were not abundant on the outside of the cores (Table T23; see also Table T16 in the "Site 801" chapter).
One igneous sample was collected for microbial analyses from Site 1149. The sample was a carbonate and clay vein, Sample 185-1149B-32R-1 (Piece 9, 83-86 cm). Interior parts of the vein were isolated in the anaerobic chamber with sterile techniques. The sample was used to inoculate anaerobic cultures, preserved for enumeration of total cells, for DNA extraction and community analysis, scanning electron microscopy, and in situ hybridization.
Core 185-1149B-32R was cored with microspheres added to the drilling fluid (see "Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring" [Smith et al., 2000]). Sample 185-1149B-32R-1 (Piece 2, 22-42 cm) was washed, and microspheres were observed in the wash water, indicating that spheres were delivered to the core. The crushed rock for the microbiology sample was filtered onto a polycarbonate filter and observed with epifluorescence microscopy. No microspheres were observed. This indicates that the microbiology sample was isolated without contamination from the outside of the core.
Two additional samples were maintained at in situ pressure for shore-based culturing. Sample 185-1149B-29R-1, 148-149 cm, is a mixture of basalt and chalk from the sediment/basement interface. Sample 185-1149C-10R-2, 0-4 cm, consists of a carbonate vein with alteration halos plus the host basalt.
A sample of the water in the drill string was collected when the pipe was opened to retrieve Core 185-1149C-10R. This sample contained 4.4 × 105 (±1.3 × 105) cells/mL. A surface-water sample was collected from the bow of the ship at the location of Hole 1149D. This sample contained 5.8 × 105 (±1.0 × 105) cells/mL. These numbers are typical for surface waters and not significantly different from each other.
A sample of sepiolite mud that is added to the drilling fluid to flush the borehole was also collected for microbial analyses. Total cells could not be enumerated because of the high background fluorescence of the sample. All three of the above samples were inoculated into anaerobic growth media and incubated at 25°C. Samples were filtered and/or frozen for DNA extraction and community analyses. These samples will aid in evaluation of microbes that may have been introduced as contaminants. Evaluation of cultures and DNA extraction will be shore-based analyses.
The majority of the microbiological data will not be available until after significant sample analysis has been completed at shore-based facilities. The results of the contamination experiments are very encouraging and suggest that these samples will be extremely useful in characterizing the microbial community in deep sediments. The particulate tracers were not found in the interior of any of the samples taken for microbiological analysis. The chemical tracer was either absent or at extremely low levels (nannoliters of seawater per gram of sediment) (Fig. F77; see also Fig. F75 in the "Site 801" chapter). Coordinated effort in analyzing the microbiological data along with the chemistry data should result in a more coherent view of microbiological processes in deep Pacific sediments.