Factors structuring bacterial communities
Geological and geochemical conditions of the sedimentary environment play an important role in bacterial community structure and diversity (Kuehl et al., 1996; Edlund et al., 2008). Likewise, the availability of organic carbon and methane at each site likely drives bacterial diversity. With the exception of the PC7 cluster (Fig. 5), there were no distinct separations of communities based on location alone. However, different abiotic factors were attributed as influences on community structure in each core and in each MDS cluster.
The central question of this study was determining the influence of local environmental heterogeneity on bacterial community composition. Local influence was best demonstrated at PC7. The features that delineated the PC7 cluster were minimal Alphaproteobacteria and elevated Betaproteobacteria sequences (Fig. 4b). Samples in the PC7 cluster had a high concentration of Betaproteobacteria (Fig. 5) associated with the order Burkholderiales and genus Achromobacter. Such sequences were 99% similar to two cultured isolates of Achromobacter insolitus (GenBank accession EU221379 and EU520399). In laboratory experiments, these isolates exhibited diazotrophism (Sala et al., 2008) and synthesis of the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which is responsible for hydrolysis of the ethylene precursor ACC into ammonia and α-ketobutyrate. Common to these isolates is the capacity to generate ammonium. The presence of sequences related to the isolates described above may explain the elevated ammonium concentrations in PC7 relative to other cores (Fig. 3e) and suggest a biological pathway for ammonium generation. The geochemical data support a hypothesis of in situ ammonium production in PC7. In PC7, % nitrogen (not shown) averaged 0.05, approximately half that observed elsewhere. The average TOC wt% was reduced compared with the other locations, and C : N ratios were <6.2 compared with an average of 7.6 at other sites. Surface porosity (Fig. 3g) and dissolved silica concentrations were reduced in the upper 100 cm of PC7 relative to other cores (Fig. 3d). Dissolved silica may be used as an indicator of phytodetritus deposition because its concentration in sediment is controlled by the burial and dissolution of silicate (Schink et al., 1974, 1975; Demaster, 1981). The depth profile shape for silica in PC7 was similar to other cores, although the surface concentrations were reduced. This suggests that controls on silica dissolution are similar across transect, but that silicate is diminished at the surface of PC7. Reduced silica, TOC, bulk nitrogen and porosity, and low C : N ratios suggest that resupply of organic matter and nitrogen was reduced in surface sediments at PC7. This was unexpected because basins such as the one PC7 was positioned in often act as sediment traps, which could explain ammonium enrichment. However, seismic data do not suggest substantial sedimentation near PC7 (Pecher et al., 2010). Current flow is channeled down the basin (Carter & Manighetti, 2006) and causes erosion (I. Pecher, pers. commun.), which may remove surface deposition and establish a nitrogen requirement. Because some Achromobacter generate ammonium from nitrate under nitrate-limited conditions (King & Nedwell, 1985), the physical conditions at PC7 may have conferred an advantage to A. insolitus-related taxa and may explain their elevated sequence abundance.
Other studies have demonstrated in situ nitrogen production in methane-rich marine sediments. Dekas et al. (2009) documented diazotrophism in aggregates of ANME-2 and Desulfosarcina/Desulfococcus. Pernthaler et al. (2008) commented on the potential involvement of Burkholderiales in nitrogen biogeochemistry in methane charged marine sediments. Further study may reveal whether diazotrophism, ACC cleavage or other microbial nitrogen cycling partnerships occur in these sediments.
The Main cluster included most samples from PC4, PC17 and PC14. In these cores, Alphaproteobacteria were abundant near the surface and declined with depth (Fig. 4a, c and d). Others report similar depth trends for this group in marine sediments (Webster et al., 2006; Mills et al., 2008). The most abundant Alphaproteobacteria in the Main cluster were 99% related to uncultured isolates from organic-rich surface sediments near the Crozet Island archipelago (GenBank accession FM214379 and FM214399). These phylotypes accounted for 26% of Alphaproteobacteria in this study, were observed in nearly all samples from the Main cluster and were absent from PC7. Rhodobacterales accounted for most Alphaproteobacteria with known phylogeny. The most abundant Rhodobacterales was associated with a Loktanella sp. (GenBank accession FJ196061) isolated from coastal sediments off east Antarctica. The second most abundant Rhodobacterales phylotype was associated with a Rhodobacter sp. isolate (GenBank accession EU979473) from the Columbia River estuarine turbidity maximum. Rhodobacterales are the primary surface sediment colonizers in coastal waters (Dang & Lovell, 2000) and often play a role in the oxidation of newly deposited organic matter. The Bio-Env results implicate silica and TOC as important structuring factors on the Main cluster (Table 3). These results may explain why sequences associated with organic matter utilization were so abundant in surface samples in the Main cluster.
The order Rhizobiales was well represented in samples in the Main cluster and accounted for 9% of Alphaproteobacteria. Rhizobiales related to the Methylocystaceae family were in highest abundance in PC4, limited in PC17 and PC14 and not observed in PC7. The elevated abundance of Methylocystaceae in PC4 compared with the other cores suggests that although Methylocystaceae are methanotrophic, phylotypes in this study may proliferate under conditions of low methane concentration. In addition, anoxic conditions in PC17 and PC7 (indicated by the presence of TDS –Fig. 3b) would inhibit obligately aerobic Methylocystaceae and may explain their reduced sequence abundance in the cores.
The Chloroflexi/GNS featured largely in samples from the Main cluster (Fig. 5). This and other studies report that the location of Chloroflexi/GNS within the sediment column is variable (Inagaki et al., 2003, 2006; Kormas et al., 2003; Heijs et al., 2007; Biddle et al., 2008; Hamdan et al., 2008; Harrison et al., 2009). Chloroflexi/GNS are ubiquitous in methane-rich sediments (Webster et al., 2006). In such sediments from the Chilean margin, Santa Barbara Basin, Mediterranean Sea and Gulf of Mexico, Chloroflexi/GNS are most prevalent below the SMTZ (Reed et al., 2006; Heijs et al., 2007; Hamdan et al., 2008; Harrison et al., 2009). Others have discovered Chloroflexi/GNS to be uniformly distributed in the deep subsurface (>50 m) sediments on the Peru margin (Biddle et al., 2008). Inagaki et al. (2006) noted that on the Peru margin, Chloroflexi/GNS dominate organic-rich methane-bearing sediments that lacked gas hydrates and that this was a key biological factor distinguishing nearby sites that contained gas hydrates from those that did not.
Frequent observations of Chloroflexi/GNS in methane-rich sediments have prompted speculation on their role in methanogenesis (Sekiguchi, 2006). The semi-ubiquitous appearance of Chloroflexi/GNS in studies of marine sediments (Kormas et al., 2003; Biddle et al., 2008) indicates that factors other than methane are correlated with their appearance. In this study, no direct relationship between Chloroflexi/GNS and methane was observed. Methane concentration was generally low in samples with high Chloroflexi/GNS abundance, and the Bio-Env analysis (Table 3) indicated that TOC, not methane, was a structuring factor on the Main cluster.
Chloroflexi/GNS were abundant in sediments from the top of PC7. In PC7 samples that grouped in the Main cluster (Fig. 5), the majority of Chloroflexi/GNS were affiliated with isolates from the Brazos-Trinity Basin of the Gulf of Mexico (Nunoura et al., 2009). The Brazos-Trinity Basin like PC7 is relatively TOC poor, having C : N ratios that are non-Redfield (Gilhooly et al., 2008). The physicochemical and biological parity between these sites suggest bacterial composition to be a highly specific indicator of abiotic conditions in marine sediments. The location where PC7 was obtained was likely eroded by hydraulic forces. Because of this, newly deposited, labile TOC would be limited at the surface. However, erosion would expose buried recalcitrant TOC. Studies demonstrate that recalcitrant TOC is responsible for the long-term survival of microorganisms in the deep subsurface (Fredrickson & Balkwill, 2006) and may explain the observations of Chloroflexi/GNS in this and similar environments (Inagaki et al., 2006; Biddle et al., 2008; Nunoura et al., 2009).
Although most of PC17 was nested in the Main cluster, the robust Spearman Rank correlation coefficients for the core deserve some individual discussion. Silica was the main structuring factor in PC17 (Table 3). A significant increase in the silica concentration was observed below the SMTZ, concomitant with the TDS maximum (Fig. 3d). Studies demonstrate silica dissolution in conjunction with sulfate reduction (SR), AOM and carbonate precipitation (Birnbaum & Wireman, 1984; Jørgensen & Boetius, 2007; Pierre & Fouquet, 2007). Elevated silica concentration near the SMTZ in PC17 was likely a secondary result of metabolic activity. Hence, the high ρ for silica may be the result of covariance of factors rather than direct cause and effect on community structure. This is reflected in the Bio-Env results, which indicate that sulfate, TDS and methane are also important correlates on community structure in PC17 due to their coupled relationships in sediment diagenesis.
The biological features common to the SMTZ were elevated concentration of Deltaproteobacteria and JS1 candidate sequences relative to other depths (Fig. 4). The most abundant JS1 phylotype in this study was ≥98% similar to an isolate from hydrate-bearing sediments from the Chilean margin (GenBank accession EF093942). The Bio-Env analysis indicated that methane was the main abiotic driver on the SMTZ cluster. Although the biogeochemical role of JS1 candidates is unknown, it has been suggested that their abundance is controlled by the presence of methane (Webster et al., 2006) and low sulfate concentration (Parkes et al., 2007). In this study, the influence of methane on JS1 candidates cannot be ruled out because they were abundant in methane-rich sections of PC4, PC7 and PC17 (Fig. 4). However, because they were observed in PC14 (Fig. 4c), where methane was below the LOD, other factors must influence their presence. Similarly, if methane principally governed JS1 candidate abundance, it is expected that they would be most abundant in PC17, which had the highest methane concentrations; however, this was not the case. JS1 candidates are often observed in gas hydrate-containing sediments (Inagaki et al., 2006), although none were observed in this study. The distribution of JS1 candidates in this study suggests metabolic diversity in this group, which involves more than methane metabolism.
At the SMTZ Deltaproteobacteria were largely affiliated with the Desulfosarcina/Desulfococcus subgroup. Other studies report enrichment of this subgroup at the SMTZ (Mills et al., 2003; Inagaki et al., 2006; Hamdan et al., 2008) and on their suspected role in AOM. The appearance of these sequences outside of the SMTZ was limited, indicating that conditions at the SMTZ select for these phylotypes. Below the SMTZ, Deltaproteobacteria generally accounted for <1% of the sequences. Above, phylotypes related 98% to a Desulfobacter isolate (GenBank accession U85476) obtained from salt marsh sediments were abundant. The high concentration of Desulfobacterales– the order that encompasses most marine sulfate reducers, above the SMTZ is indicative of the SR zone (Arakawa et al., 2006).
The data for Deltaproteobacteria indicate that sulfate-dependent AOM and organoclastic SR likely occurred in cores from this study. Without a conservative tracer to differentiate physical mixing from biogeochemical processes, it is difficult to delineate SR processes (i.e. organoclastic SR vs. AOM) from the nonlinear sulfate profiles (Pohlman et al., 2008). However, sulfate and methane profiles, particularly from PC7 and PC17, the shallow depth of the SMTZ in these cores and the concentration of Desulfosarcina/Desulfococcus-related sequences suggest that SR associated with AOM was focused at the SMTZ.
Methane, DIC, TOC and DOC δ13C (Table 1) and methane and sulfate concentration data support the idea that both AOM and organoclastic SR impact sediment biogeochemistry in PC4, PC17 and PC7. TOC δ13C above the SMTZ in these cores fall within the range of pelagic organic matter (Claypool & Kaplan, 1974; Peterson et al., 1994; Niggemann & Schubert, 2006). Thus, above the SMTZ 13C-enriched DIC may have resulted from the metabolism of pelagic organic matter. However, at the SMTZ of PC4 and PC17 13C-depleted DIC could only result from oxidation of 13C-depleted methane because δ13C for TOC and DOC in these cores cannot explain the substantially 13C-depleted DIC. At the SMTZ in PC7 and PC17, the TIC concentration was elevated relative to PC4 and PC14 (Fig. 2e) and carbonate deposits were observed in PC17. Reduced porosity at the SMTZ was observed in both PC17 and PC7. These observations suggest carbonate precipitation in conjunction with AOM. In PC17, 13C-depleted TIC at the SMTZ can only be explained by AOM (Table 1). DOC δ13C indicate that 13C-depleted methane was incorporated into the organic carbon pool at the SMTZ of PC17 (Table 1). Both TIC and DOC were 13C-depleted at the SMTZ in PC7 (Table 1). Neither TIC accumulation nor TIC 13C-depletion was observed in PC4; however, 13C-depleted DIC was (Table 1). Because the abundance of Desulfosarcina/Desulfococcus at the SMTZ in PC4 was reduced compared with PC17 and PC7, this may be indicative of lower rates of AOM, and hence, a less distinct geochemical signature. However, the reduced supply of methane at PC4 relative to PC17 and PC7 may have driven the reduction in Desulfosarcina/Desulfococcus in the first place. As a whole, these data suggest that the local influence of methane on bacterial communities is significant in PC17 and PC7, reduced in PC4 and negligible in PC14.