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doi:10.2204/iodp.proc.311.107.2006

Microbiology

Site U1329 is located on the northeastern end of a transect of sites across the northern Cascadia accretionary prism. 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 at 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 analysis. It is also a goal of this effort to cultivate and characterize the anaerobic methane oxidizing community in sediments where bacterial sulfate reduction and methane oxidation are coupled and high concentrations of gas hydrate exists. This expedition gives the opportunity to explore piezophilic, aerobic (heterotrophs), and anaerobic (sulfate reducers) microorganisms. Most of the shipboard effort was devoted to developing and implementing a sampling program to meet the goals of the program and to begin the cultivation of the high pressure–adapted microorganisms. However, most of the analyses needed to achieve these goals will be performed postcruise.

Microbiological sampling

Site U1329 was the first site sampled for microbiology during Expedition 311, and the samples taken and methods used reflect our learning process. Sampling from the mudline (Core 311-U1329C-1H-1, 0–10 cm) and the deepest core (Core 22X-4, 95–105 cm) in Hole U1329C targeted microorganisms for aerobic and anaerobic high-pressure culturing. Sampling in the upper sediment layers of Holes U1329D and U1329E targeted the SMI, where microbial consumption of methane peaks. 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 U1329C was first 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 U1329D and U1329E (see "Interstitial water geochemistry" and "Organic geochemistry").

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 BSR to quantify methanogenesis in these sediments. In postcruise experiments, we will examine the correlation among the rates of methanogenesis, 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.

The emphasis in sample processing was to work as quickly as possible without compromising microbiological integrity of the samples by maintaining samples at or below in situ formation temperatures and minimizing exposure to oxygen. Cores were kept at 4°C and left undisturbed in core liners until processed. The processing of samples from Hole U1329C was conducted in the Hold Deck reefer on the JOIDES Resolution.

Contamination tests

Perfluorocarbon tracers

Samples for perfluorocarbon tracer (PFT) and microsphere analyses were taken from the whole-round cores in the reefer. Each of the analyzed cores had ~5 cm³ subsamples taken from outer and inner layers 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 T8. We found that the reefer contained high PFT levels (0.15 ng PFT/mL air). This might lead to false positive PFT concentrations for samples processed in the reefer.

Fluorescent microspheres

Comparison of paired samples collected from the edges and centers of cores by fluorescent microsphere penetration are summarized in Table T8. Microscopic analysis of the outer portions of cores showed detectable numbers of microspheres of 10⁴ spheres/g of sediment, whereas microspheres were below the detection limit of 100 spheres/g in samples taken from core interiors.

Shipboard analysis

Samples were taken from the top (Sample 311-U1329C-1H-1, 0–10 cm) and bottom (Sample 22X-4, 95–105 cm; 184.35 mbsf) of Hole U1329C 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. Only the sulfate reducers required preparation in the anaerobic chamber and were fed formate, acetate, or lactate as a carbon source. Because interpretation of the SMI at Site U1329 was unclear, methanogenic enrichments were postponed until the next site (U1327).

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