Sequences of reverse-transcribed dsrAB and mcrA mRNA, as well as bacterial and archaeal 16S rRNA, were analyzed in conjunction with DNA-based V6-tag sequencing, porewater concentrations of methane and sulfate, radiotracer measurements of sulfate reduction and methane oxidation rates, and stable carbon isotopic values of methane to describe the spatial structure and activity patterns of sediment microorganisms with respect to Beggiatoa spp. mat location and hydrocarbon seep geochemistry at MC118. Results Geochemistry Steep sulfate and methane gradients were observed directly under the mat as well as at its edge (Fig. 2a–c). Sulfate was depleted to a relatively constant background concentration at 5 cmbsf and methane increased immediately below the seafloor. The decrease in radiotracer-measured sulfate reduction rates mirrored the sulfate concentrations. Although precautions were taken to minimize sulfide oxidation during sediment processing, the finite background concentration below 5 cm (0.760.3 mM) may be a sampling artifact; hence, measured sulfate reduction rates below 5 cm may overestimate in situ rates. The measured sulfate reduction rates cannot account for the shallow sulfate depletion depth: a 1-D, steady-state, reaction-transport model for sulfate using measured rates predicts that sulfate penetrates to .15 cm Patterns of CH4-Seep RNA (Figure S1). This disparity could be due to upward advection of pore fluids at MC118, although we lack porewater chloride data to test this possibility. Lateral fluid flow is unlikely, given that sediments were compacted, but this is always a possibility [36]. Similar sulfate reduction rates were measured at the surface of another seep that has a similarly steep sulfate gradient [37]. Methane oxidation rates were much lower than sulfate reduction rates and also decrease with depth and sulfate concentration (Fig. 2e–g). Since methane concentrations were measured shipboard at 1 atm, any values above ,1.2 mM (methane saturation at sea level) most likely underestimate methane concentrations at in situ pressure. Methane has previously been shown to be both thermogenic and biogenic at MC118 [3]. The upcore 13C-depletion trends in the Mat-A and Edge cores also indicate methanogenesis (Fig. 2i–k) [38]. The 13C-enrichment of methane in the sediments of Mat-B suggests methane oxidation, since methane-oxidizing microorgan- isms have a kinetic preference for the lighter isotope as it diffuses upwards through the sediments [39]. Outgassing is unlikely to alter the methane d13C profiles since this process has negligible isotopic fractionation [40]. Interpreting the relative locations of net methane oxidation or methanogenesis in the Mat-A and Edge cores is not possible, since 13C-enrichment was observed only in a single point at the surface for each core, and could reflect aerobic methane oxidation. Figure 2. Geochemical measurements. Porewater measurements of A–D) sulfate concentrations (filled diamonds) and duplicate sulfate reduction rates (open squares), E–G) methane concentrations (filled diamonds) and duplicate methane oxidation rates (open squares), H) methane concentrations outside the mat with a smaller scale than the two other cores, and I–K) d13C values for methane. Cores are from within the mat (A, B, E, F, I, and J), at its edge (C, G, and K), and outside the mat (D and H). Methane concentrations above ,1.2 mM are lower limit estimations, since methane outgases to this value at normal atmospheric pressure. Only Mat-B, Edge, and Outside cores were used for microbiological analysis. doi:10.1371/journal.pone.0008738.g002 PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8738