Just outside the mat, the sulfate concentrations did not decrease with depth (Fig. 2d). Likewise, the sulfate reduction rates were very low in this core. The methane concentrations were much lower than those of the mat cores, but the curved increase in methane with depth suggests oxidation of methane diffusing upwards from below (Fig. 2h) [41]. Outside the mat, methane concentrations were not high enough to accurately measure d13C and methane oxidation rates were below the detection limit. Sulfate and methane concentration fluxes across the first two depths were compared with total integrated rates of sulfate reduction and methane oxidation as a quality control check for measurements (Table S1). Most flux and rate measurements were in good agreement; only the second measurement of methane oxidation rates in Mat-B appeared to be largely underestimated. Bacterial 16S rRNA and dsrAB Transcripts The dominant sub-mat bacterial 16S rRNA transcripts were present at all sediment depths and position in the mat (center or near the edge of the mat) (Fig. 3a). Mat-A was not included in the molecular biological analysis. The majority of the 16S rRNA bacterial clones came from groups whose closest cultured relatives are SRB (Fig. S2). The clone libraries from sub-mat samples were dominated by phylotypes of the Eel-2 group, a sister group of the Desulfobulbaceae within the Deltaproteobacteria (Fig. 3a). The Eel-2 group has also been found at methane-rich areas off the coast of California [42], in the Black Sea [43], the Gulf of Mexico [15,44], and a deep sea CO2 lake [45]. SEEP-SRB1 and other Patterns of CH4-Seep RNA members of the Desulfobacteraceae also feature prominently, including the subgroup related to Desulfobacterium anilini which can degrade aromatic hydrocarbons. All cultured members of the Desulfobacteraceae oxidize organic carbon compounds complete- ly to CO2. Members of the Desulfobulbaceae are also present; cultured members of this Family oxidize a wide range of carbon molecules incompletely. In particular, cultured members of the genus Desulfocapsa are able to disproportionate elemental sulfur. In the surface sediments outside the mat, bacterial 16S rRNA transcript composition changes abruptly to a diverse assemblage of phylotypes related to aerobic, microaerophilic or nitrate-reducing bacteria (Fig. 3a). Some of these aerobic groups that dominate the transcript libraries at the surface outside the mat, such as Alphaproteobacteria, sulfur-oxidizing Gammaproteobacteria, Acidobacteria, and the Bacteriodetes phylum, are also present at the surface within the mat, but in much smaller clone proportions relative to the SRB (Fig. 3a, Fig. S3). Likewise, SRB 16S rRNA transcripts are also present in surficial sediment outside the mat, but in much lower abundance relative to the aerobic groups. The bacterial community of the deeper sample outside the mat resembles the sub-mat community, and is dominated by SEEP- SRB1 and other members of the Desulfobacteraceae. Many of the aerobic and microaerophilic groups also persist in the deep sample as well, but in lower clone abundance relative to SRB (Fig. 3a, Fig. S3). The only group common to all samples was the Chloroflexi, which are commonly found in deep and shallow subsurface libraries [46]. Although the 0–3 cmbsf sediment samples were Figure 3. Bacterial community structure and stratification. Phylogenetic affiliations of (A) bacterial 16S rRNA transcripts, and (B) dsrAB mRNA transcripts at different depths across the mat transect. Shown are the percent of clones obtained from each group in the color coded bar graph legends. In A), shades of green and blue denote putative sulfate-reducing groups. Numbers in parentheses are the total number of clones analyzed, including full length and short reads. ‘‘Not determined’’ means that no amplification was tried. doi:10.1371/journal.pone.0008738.g003 PLoS ONE | www.plosone.org 4 January 2010 | Volume 5 | Issue 1 | e8738