A key contribution to the Leg 204 investigations was the characterization of various components of the carbon cycle, which allows inferences on microbial processes and activity rates in this region. In areas of active methane venting near the summit of SHR, microbiological (e.g., Boetius et al., 2000; Knittel et al., 2005) and isotopic (Torres et al., 2003; Torres and Rugh, this volume) data indicate that high anaerobic methane oxidation rates near the seafloor are supported by rapid methane flux. In contrast, at the sites drilled in slope basin and on the saddle north of the southern summit, anaerobic oxidation of methane is minor or absent, as indicated by the distribution of metabolites in the pore fluids and isotopic composition of the dissolved inorganic carbon (Claypool et al., this volume; Torres and Rugh, this volume; Borowski, this volume). At these sites, sulfate is consumed primarily by oxidation of organic matter, and sulfate reduction rates estimated from curve fitting of concentration gradients are in the range of 2–4 mmol/m3/yr, with integrated net rates of 20–50 mmol/m2/yr. Microbial methane production rates derived from the geochemical data are ~1.5 mmol/m3/yr in sediments just beneath the sulfate reduction zone and decrease to <0.1 mmol/m3/yr at depths greater than 100 mbsf. These rates overlap with, but are generally lower than, those obtained in laboratory incubations of samples from NHR, where methane was produced at ~0.9–9.0 mmol/m3/yr (Cragg et al., 1996).
Using a method that involved enumeration of methanogens in nearly 50 Leg 204 samples combined with determinations of minimal rates of methane production obtained from lab reactors, Colwell et al. (2004) estimated that most of the samples measured (~75%) exhibited rates of <0.0016 mmol/m3/yr and rare samples had rates estimated as high as 70 mmol/m3/yr. Centimeter-scale investigations of the geological or geochemical conditions that determine how rapidly methanogens make methane in Hydrate Ridge sediments were not attempted as a part of this research; however, such studies would advance our understanding of the abiotic controls that determine how methanogenic rates can apparently range over several orders of magnitude in closely spaced samples. Other research in deep marine sediments has found that geological and geochemical characteristics determine the rates for key biogeochemical processes (D'Hondt et al., 2004; Parkes et al., 2005). A study of the distribution of acetate and hydrogen, two key methanogenic electron donors, performed on Leg 204 sediments indicated that these sources of energy are present in relatively high concentrations (3.17–2515 M) and show intriguing peaks at some depths in the sediments (Lorenson et al., this volume); for example, acetate maxima and localized high acetate concentrations occurred at the BSR at all sites and frequently corresponded with areas of gas hydrate accumulation.
In order to evaluate whether the presence of gas hydrates has bearing on the biogeographical distribution and phylogenetic diversity of microbial communities present in seafloor sediments, microbial deoxyribonucleic acid (DNA) was extracted from samples collected as a part of Leg 204 and then amplified using universal primers that target 16S ribosomal ribonucleic acid (rRNA) genes. The resulting Leg 204 clone libraries were used together with similar libraries constructed from Leg 201 samples. The analysis of this extraordinarily large collection of clone sequences demonstrated that for the tested sediments the presence of hydrates corresponded to microbial communities that were statistically distinct from the communities that were present in sediments lacking hydrates (Inagaki et al., 2006). Notably, the as-yet-uncultivated Archaea of the Deep Sea Archaeal Group, and the Bacteria of the JS1 Group, Planctomycetes, and the Chloroflexi, were important members of hydrate-bearing sediments, but they were less apparent in nonhydrate-bearing sediments. As in a number of prior investigations that sought the presence of methanogenic Archaea in hydrate-bearing sediments, these methane-producing cells were not detected in the clone libraries. However, when the methanogenic functional gene for methyl coenzyme M reductase (mcr) was targeted in quantitative polymerase chain reaction experiments, ~25% of the Leg 204 samples showed evidence of detectable levels of this gene, suggesting the presence of these cells, albeit at low levels (Colwell et al., 2004). These studies augment past investigations of the Cascadia margin sediments, which found that methanogen diversity was low, with all of the detected sequences associated with the Methanosarcinales and Methanobacteriales groups (Marchesi et al., 2001).
To further address the metabolic capability of microorganisms in this methane-rich deep biosphere environment, the stable carbon isotopic compositions of intact polar lipids, derived from live cells in the samples, were correlated with microbial diversity and activity measurements in discrete samples (B. Orcutt, pers. comm., 2006; Orcutt, 2006). In near-surface samples from the summit of SHR, where massive gas hydrate is supported by upward methane advection, highly depleted derivatives of archaeal and bacterial lipids indicate the incorporation of methane-derived carbon into microbial biomass, presumably by members of the ANME-1 clade. Samples collected below zones of massive hydrate at the summit sites contain both Archaea and Bacteria in varying proportions as evidenced by lipid biomarker and 16S rDNA based tools. Archaea of the Deep Sea Archaeal Group of Crenarchaea were detected in all subsurface samples analyzed, and the ANME-1 group of Euryarchaea, together with sulfate-reducing bacteria, were detected in the deep subsurface (54 mbsf) for the first time. However, archaeal and bacterial lipids derived from these samples show that these microorganisms are not substantially depleted in 13C, as known from other microbial habitats dominated by anaerobic oxidation of methane. It is possible that environmental factors such as sulfate depletion limit the distribution, activity, and growth of anaerobic methanotrophs in the deep subsurface. In combination with previous investigations (Biddle et al., 2006), these results may also indicate that the low diversity of Archaea in the deep biosphere cycle methane in a manner that is biochemically different from methane consumers in the near surface, likely due to the constraint of energy limitation in the deep biosphere.