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doi:10.2204/iodp.proc.311.105.2006

Microbiology

Site U1327, located near the center of the transect of sites cored across the northern Cascadia accretionary prism, was the second site sampled for microbiology. At this site, the base of the GHSZ was estimated at ~223 mbsf. This site is located near Site 889. The goals of the microbiological sampling program and study were to identify the subsurface spatial distribution of optimal microbial activity and growth of methanogens and to elucidate how methanogenesis contributes to the formation of gas hydrate in the northern Cascadia margin. Within this subsurface zone of optimal microbial activity, measurements of methane production rate and organic compounds used for methane production should correlate with the biomass determined from direct cell counts and microbial community structures. It is also a goal of this effort to cultivate and characterize the anaerobic methane oxidizing and other unique communities at the SMI and in gas hydrate–associated sediment. This expedition provides the opportunity to culture novel piezophilic, aerobic (heterotrophic), and anaerobic (sulfate reducing) prokaryotes. Most of the shipboard effort was devoted to developing and implementing a sampling scheme to meet the goals of the program and to begin the cultivation of the high pressure–adapted microorganisms.

Microbiological sampling

Sampling from the mudline (interval 311-U1327C-1H-1, 0–3 cm) to the deepest core (interval 35X-2, 5–15 cm; 297.05 mbsf) in Hole U1327C targeted microorganisms for aerobic and anaerobic high-pressure culturing. Sampling in the upper sediment layers of Holes U1327D and U1327E focused on the SMI, where methane consumption is expected to occur. Anaerobic oxidation of methane using sulfate as the electron acceptor has been the focus of several recent studies (Boetius et al., 2000; Orphan et al., 2002; Michaelis et al., 2002; Zhang et al., 2002) and is a major focus for the microbiology program of Expedition 311. Hole U1327C was completed as a continuous, deep hole, and once the sulfate and methane data had been examined, the SMI was targeted for intensive, coupled microbiological and geochemical sampling in both Holes U1327D and U1327E (see "Interstitial water geochemistry" and "Organic geochemistry"). Samples from Hole U1327D were collected from the mudline to ~15 mbsf; however, the SMI region was in a coring gap and a second attempt in Hole U1327E successfully recovered the SMI between 8.2 and 9.6 mbsf (see "Interstitial water geochemistry" and "Organic geochemistry"). Samples collected from the SMI and 1–5 m above and below the SMI were divided into subsamples and stored at –80°C (for deoxyribonucleic acid [DNA] analysis) or 4°C under nitrogen gas (for culturing and activity analyses).

Methanogenesis can exist in most anaerobic environments, but it becomes the major process when other electron donors such as nitrate, Fe(III), and sulfate are depleted. We sampled regularly downhole to below the predicted depth of the BSR to quantify methanogenesis in the sediment. Most of the methane bound within marine gas hydrate is of biogenic origin (Appenzeller, 1991), which implies that the sediment associated with gas hydrate may consist of microbial communities dominated by methanogens. The Hole U1327A LWD data (see "Downhole logging") indicated that gas hydrate might be widely distributed above the BSR. Therefore, methanogens closely associated with gas hydrate were expected to be present in this deep sediment. We started an enrichment culture experiment for methanogens with these sediment samples (see "Enrichment cultures" in the "Methods" chapter). Across this gas hydrate–bearing zone, Samples 311-U1327C-16X-4, 60–75 cm (128.2 mbsf); 22X-3, 115–130 cm (184.1 mbsf); and 28X-4, 40–55 cm (232 mbsf), were used for methanogen enrichment culture studies. In postcruise experiments, we will examine the correlation between among the rates of methanogenesis and a range of other sediment properties such as direct cell counts, phylogenetic analysis, geochemical analyses of methane gas concentrations, and IW chemistry. Samples were prepared for shore-based experiments in which sediments will be heated to in situ temperatures for measuring total methane production and estimating organic compounds utilized in methanogenesis.

Gas hydrate was inferred to exist in the cores recovered from this site, and whole-round core samples spatially associated with IR thermal anomalies were collected (see "Physical properties"). The emphasis in sample processing was to work as quickly as possible without compromising microbiological integrity of the samples by minimizing warming of cores above in situ formation temperatures and minimizing exposure to oxygen. Subsamples for DNA analysis were removed immediately from the center of the whole-round core and stored at –80°C. All other cores were kept at 4°C and left undisturbed in core liners until processed. Processing of samples from Hole U1327C was conducted in the hold deck reefer on the JOIDES Resolution.

Contamination tests

Perfluorocarbon tracer

Samples for perfluorocarbon tracer (PFT) and fluorescent microsphere analyses were conducted on the whole-round cores in the reefer. Each of the analyzed cores had ~5 cm3 subsamples taken from outer and inner portions for gas chromatograph analysis as described in "Microbiology" in the "Methods" chapter. We found that the contamination level of the samples at this site was less than that at Site U1329 (Table T9); however, we may still be detecting apparent PFT contamination in samples processed in the reefer from PFT-contaminated air.

Fluorescent microspheres

Comparison of paired samples collected from the edges and centers of cores for fluorescent microsphere penetration are summarized in Table T9. Microscopic analysis of the outer portion of the core showed detectable numbers of microspheres of 104 microspheres/g of sediment, whereas numbers were generally below the detection limit of 100 microspheres/g in samples taken from core interiors.

Shipboard analysis

Samples were taken from the top (interval 311-U1327C-1H-1, 0–10 cm) and bottom (interval 35X-2, 5–15 cm; 297.05 mbsf) of the sedimentary section for inoculation of enrichment cultures targeting high pressure–adapted heterotrophic and sulfate-reducing microorganisms. Samples were maintained at low temperature, and dilution series were inoculated to culture for microorganisms at 55.1 MPa and 4°C. Cultures for sulfate reducers were prepared in the anaerobic chamber and were fed formate, acetate, or lactate as a carbon source.