The fascinating possibility that microbial activity may inhabit oceanic crust as old and as deep as that at Site 801 provided the motivation for sampling the subseafloor in the search for extremophilic life. Water, sediment, and rock samples were collected to determine cell abundance and community composition. Samples were examined microscopically and used to establish cultures and to extract and characterize DNA. Sample protocols were established and tracer tests conducted to determine the extent of microbial contamination potentially introduced during drilling and sample preparation.
Water samples were collected to determine background microbial populations potentially introduced in the drilling process. Water samples were collected from the drill pipe and from the sea surface (from the z-boat) upwind of the Resolution. Undisturbed water in Hole 801C may be an excellent in situ microbial culture and was thus sampled using the water-sampling temperature probe (WSTP) prior to Leg 185 drilling; ~300 mL of water was recovered from a depth of 540 m in the hole.
A series of samples of different rock types was collected for culturing, total cell counts, and DNA and adenosine triphosphate (ATP) analyses. Figure F22 shows an example of a bacterial population from surface seawater with fluorescent microsphere traces. Samples of basement rocks, including glass samples, were (1) taken and transported to an anaerobic chamber where they were cracked open to obtain interior pieces used to inoculate cultures and (2) used for shore-based microscopic analyses and DNA extraction. Cultures have also been started at in situ pressure (620-650 atm), and some samples have been stored under pressure for shore-based analyses.
Hole 801C and, to a lesser extent, volcanic rocks from Site 1149 preserve small amounts (several grams to a few grains) of fresh basaltic glass. These samples were studied microscopically for traces of microbial activity. Several samples preserve filament-like textures that are identical to textures attributed to microbes in deep-sea basalt from young oceanic pillow basalt (see Fig. F23 and Thorseth et al., 1992; Fisk et al., 1998; Furnes and Staudigel, 1999). Whether these textures are trace fossils of past microbial activity or signs of extant microbes living in this high-pressure seafloor environment remains a fascinating subject of shore-based study.
Great strides in the study of the deep biosphere have been made using samples collected during previous drilling (e.g., Oremland et al., 1982; Whelan et al., 1986; Thorseth et al., 1992; Parkes et al., 1994; Griffin et al., 1997; Fisk et al., 1998). The drilling capabilities and extensive areas of operation of the Resolution give unparalleled access to samples from this environment. Critical to conducting research in the deep biosphere is the capability to core and retrieve samples without contaminating the samples with nonindigenous microbes. During Leg 185, we conducted the first extensive experiments on the Resolution to explicitly assess the suitability of this platform for sampling the deep biosphere.
Two types of tests were conducted to quantify the potential contamination. Particulate tracers (0.5-µm-diameter fluorescent microspheres) were introduced into the core barrel at a concentration of ~1010 spheres/mL and quantified in recovered cores using epifluorescence microscopy. In addition, a chemical tracer (perfluoro[methylcyclohexane]) was added to the drilling fluid (surface seawater) to produce a 1-ppm solution. Gas chromatography was used to quantify the tracer in recovered cores. Cores were collected with the APC in unconsolidated sediments at Hole 1149A and with the DCB and RCB in sedimentary and igneous rock at Sites 801 and 1149.
Detection of the tracers on the exterior of the cores confirmed successful delivery. Contamination was low in the unconsolidated sediment cores using the APC. Spheres were never detected in the interiors of these cores (N = 24). The average concentration of the perfluorocarbon (N = 12) was equivalent to 0.03 µL of drilling fluid per gram of sediment. Contamination was also low in the igneous rock samples cored with the RCB. Spheres were not found in the interiors of igneous rock samples that were crushed (N = 4) but appeared in the interiors of 64% of the thin sections examined (N = 12). This suggests that the samples were contaminated during sectioning rather than drilling. Drilling fluid in the igneous rock samples averaged 0.01 µL/g rock. Based on the abundance of bacteria in the surface seawater (4.2 × 108 cells/L), the potential contamination of both sample types is on the order of 1-10 cells/g of cored material.
The tracer techniques adapted for use on the Resolution are a sensitive means of evaluating microbial contamination in deep-sea cores and should be deployed in the future when microbiological samples are collected. The perfluorocarbon tracer is also useful for evaluating the incursion of drilling fluid into pore-water samples. The methods used in these tests are described in detail in "Methods for Quantifying Potential Microbial Contamination during Deep Ocean Coring" (Smith et al., 2000) so that they may be routinely employed when coring for samples to be used in microbiological research in future ODP legs.