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doi:10.2204/iodp.proc.311.106.2006

Microbiology

Site U1328 is an active cold vent located off the main transect of sites established by Expedition 311 across the northern Cascadia accretionary prism. It was the third site sampled for microbiology on this expedition. We anticipated a shallower SMI and a GHSZ that extended to a depth of ~220 mbsf. Efforts continued for obtaining live anaerobic methane oxidizers, unknown gas hydrate–associated microbial communities, high pressure adapted microorganisms, and methanogens. With the challenge of biological contamination from drilling, we also conducted tests assess the level of microbial contamination from drilling slurry.

Microbiological sampling

Sampling from the mudline in Hole U1328B (Core 311-U1328B-1H-1, 0–3 cm) and the deepest core (Core 311-U1328C-27X-3, 55–70 cm; 297.0 mbsf) in Hole U1328C targeted microorganisms for aerobic and anaerobic high-pressure culturing. Sampling in the upper sedimentary section of Holes U1328D and U1328E targeted the SMI, where methane consumption is expected to exist. The upper part of Hole U1328B had limited recovery because of the difficulty penetrating hard layers near the seafloor with the APC and delays in core retrieval because of dangerous levels of hydrogen sulfide. Hole U1328C resumed coring where Hole U1328B ended (56 mbsf) and completed the targeted sedimentary section. The SMI was targeted for intensive, coupled microbiological and geochemical sampling in both Holes U1328D and U1328E (see "Interstitial water geochemistry" and "Organic geochemistry"). The SMI, however, was difficult to physically and chemically examine because of the challenges in recovering sediment that was highly disturbed by free gas and gas hydrate within the first several meters below the seafloor. The recovered cores were contaminated with seawater and appeared soupy in texture. Sediment quality was poor for geochemical analysis in both Holes U1328D and U1328E (see "Interstitial water geochemistry" and "Organic geochemistry"). Because LWD resistivity data and physical evidence (i.e., gas hydrate in the core catcher) indicated that gas hydrate was present within several meters below the seafloor, one 10 cm whole-round core from the uppermost 7 m was retained for microbiological study of gas hydrate–associated sediment.

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 depth of the predicted BSR to quantify methanogenesis in these sediments (see "Microbiology" in the "Methods" chapter).

Contamination tests

Perfluorocarbon tracer

Samples for perfluorocarbon tracer (PFT) and microsphere analyses were taken immediately from the ends of whole-round cut sections on the catwalk. Because the freshly cut sediment surface was potentially contaminated by PFT smeared along the core liner, the sediment surface at the sampling spots was scraped away using a clean spatula before sampling with a syringe. Subsamples (~5 cm³) were taken from outer and inner layers from each core for gas chromatograph analysis as described in "Microbiology" in the "Methods" chapter. Samples were analyzed as described and the raw data are presented in Table T12. We also measured the catwalk air to confirm background PFT levels. When we processed the first APC core (Core 311-U1328B-1H-3), catwalk air contained PFT at 6.5 pg PFT/mL air. In the other cases, however, we found that PFT concentrations were below the detection level in catwalk air samples. Therefore, we could avoid false PFT contamination during the sampling process by taking subsamples for PFT measurements on the catwalk instead of in the Hold Deck reefer. Sampling the cores on the catwalk eliminated most of the background PFT contamination in measurements from Site U1328.

Fluorescent microspheres

Comparison of paired samples collected from the edges and centers of cores to assess fluorescent microsphere penetration is summarized in Table T12. Microscopic analysis of the outer portion of the APC cores showed detectable numbers of microspheres at 10⁴ per gram of sediment, whereas microspheres were generally below the detection limit of 100 microspheres/g in samples taken from core interiors. Biscuit and sand layer samples obtained from XCB cores have a high possibility of contamination because of the nature of XCB coring or the porous structure of sand layers. In this hole, the quality of these characteristic samples was evaluated in detail. The samples were collected from the split cores because it is easy to identify the specific structures (Fig. F36). Numerous microspheres (10³) were observed in the interior of one sand layer (Section 311-U1328C-16X-3), indicating contamination from drill fluid (Table T12). We also took samples from the inner and outer parts of biscuit and nonbiscuit (slurry) intervals at two different depths (Fig F36), but never observed microspheres in the inner portions of biscuits (Table T12). This observation suggests that the interiors of biscuits were biologically "clean."

Shipboard analysis

Samples were taken from the top (Sample 311-U1328B-1H-1, 0 cm) and bottom (Sample 311-U1328C-27X-3, 55–70 cm; 297.0 mbsf) of the sedimentary section for inoculation of enrichment cultures targeting high-pressure adapted heterotrophs and sulfate-reducing bacteria. Samples were maintained at low temperature, and dilution series were inoculated to culture for microorganisms at 55.1 MPa and 4°C. Only the sulfate reducers required preparation in the anaerobic chamber and were fed formate, acetate, or lactate as a carbon source. Samples from Hole U1328D were collected from the mudline to ~15 mbsf.

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