The relative dominance of Euryarchaeota over Crenarchaeota in all layers has been reported for other gas-hydrate- or methane-bearing sediments as well (e.g. Inagaki et al., 2006; Parkes et al., 2007). The Euryarchaeota found were related to methane metabolism. In particular, ANME groups were retrieved at a high relative abundance (>50%) in all depth layers. At 25 cm b.s.f., the dominant phylotype (KZNMV-25-A23) was closely related to the H2-using methanogen Methanogenium marinum. Therefore, the archaeal communities of the top 30 cm in the Kazan MV are highly specialized and selected for life within gas-hydrate sediments. The fact that high concentrations of gas hydrate of the size of rice grains were present even close to the sediment surface can be interpreted as a very strong methane flux (Perissoratis, 2005), providing suitable substrate conditions for ANME.
The Deltaproteobacteria found were highly diverse (Fig. 3), even at the order level (Desulfobacterales, Synthrophobacterales, Bdellovibrionales and Myxococcales). Many phylotypes found at 5 cm b.s.f. fell into the cold seep-associated clades of SRB, SEEP-SRB1 (Desulfosarcina/Desulfococcus-related), SEEP-SRB3 (Desulfobulbus-related) and SEEP-SRB4 (Desulforhopalus-related). Members of SEEP-SRB1 are considered to be the sulphate-reducing partners of ANME-1 and ANME-2 (Knittel et al., 2003), while the ecological role of SEEP-SRB3 and SEEP-SRB4 remains unknown. The most abundant phylotype (KZNMV-5-B4, 50.8% of the Deltaproteobacteria phylotypes) was clustered within an as yet uncharacterized clade of Desulfobulbaceae, which is physically associated with ANME-2c consortia (Pernthaler et al., 2008). In addition, a few phylotypes were related to Desulfobacterium anilii and related environmental sequences. This group is known to include several cultivated and uncultivated hydrocarbon-degrading Bacteria capable of complete oxidation of various aromatic hydrocarbons (Harms et al., 1999).
Non-SRB sequences of the Deltaproteobacteria were also present at all depths. Phylotype KZNMV-10-B49 was related to Bdellovibrio sp. and Bacteriovorax sp. Both genera are aerobic, obligatory predators of Bacteria that forage on a wide variety of susceptible gram-negative microorganisms (Baer et al., 2000). Four phylotypes fell into the Myxococcales, a group often found in marine sediments (Ravenschlag et al., 1999; Wilms et al., 2006; Zhang et al., 2008). Members of this group are known nonobligate predators as well (Burnham et al., 1984). Finally, a singleton phylotype (KZNMV-5-B10) was related to Syntrophus aciditrophus, known to ferment benzoate and living often syntrophically with H2-consuming methanogens (Jackson et al., 1999; Becker et al., 2005).
Based on the archaeal and Deltaproteobacteria phylotypes found, the prokaryotic communities of the Kazan MV sediments consist of microorganisms involved in methane- and, presumably, sulphate-based biogeochemical processes, as is the case for the eastern Mediterranean MV cold seep sediments (Heijs et al., 2006, 2007; Kormas et al., 2008; Omoregie et al., 2008, 2009). The geochemical profiles (Kormas et al., 2008) support the notion that AOM is a metabolic process than takes place at 15 and 20 cm b.s.f., whereas earlier studies placed the AOM zone at the 7–20 cm b.s.f. (Werne et al., 2002, 2004; Haese et al., 2003).
The sequences of Gammaproteobacteria represented diverse organisms (Fig. 2), as is the case in several marine sediments (Li et al., 1999; Inagaki et al., 2003; Bowman et al., 2005). The dominance of this group has been observed in another case of gas-hydrate sediment and was attributed to the opportunistic life strategies of the Gammaproteobacteria (Jiang et al., 2007). Their ecophysiological role is also diverse, but several of the phylotypes recovered by this study were related to characterized sulphur and sulphide oxidizers or to environmental sequences originating from habitats where sulphur and sulphide oxidation occurred or was predicted to occur.
Phylotypes most closely aligned with psychrophilic Legionellales primarily isolated from deep-sea environments were also recovered. Other phylotypes belonging to families that are more physiologically constrained include the methane-oxidizing Methylococcaceae (Methylococcales) (e.g. KZNMV-0-B63, KZNMV-0-B33), which were found mainly at the top layer, as well as at 10 and 25 cm b.s.f., while sulphide-oxidizing Ectothiorhodospiraceae (Chromatiales) (KZNMV-0-B47 and KZNMV-0-B59) were only retrieved from the top layer. The rest of the phylotypes from the top, 10 and 25 cm b.s.f. layers belonged to the orders of Pseudomonadales, Enterobacterales, Legionellales and Thiotrichales or formed clusters that could not be firmly affiliated to any known taxonomic groups within the Gammaproteobacteria and included only uncultured representatives. These uncharacterized groups, however, were clustered with phylotypes recovered from cold seep environments, gas-hydrate-associated sediments, ocean crusts, marine sediments, characterized symbionts–epibionts of marine invertebrates and putative sulphur oxidizers, implying their involvement in the sulphur cycle.
The Epsilonproteobacteria phylotypes found in this work were related to phylotypes from deep-sea sediments, hydrothermal vents, cave waters and cold seeps. The only cultured isolate within this clade is Sulfurovum lithotrophicum, a sulphur-oxidizing chemolithoautotroph from hydrothermal sediments in a black smoker environment (Inagaki et al., 2004). Thus, Epsilonproteobacteria in the Kazan MV sediment were probably involved, together with Gammaproteobacteria, in sulphur cycling. Indeed, the composition of major elements in the sediments of the Anaximander MVs showed a clear enrichment in sulphur compared with the general composition of pelagic sediments of the East Mediterranean Sea (Perissoratis, 2005).
The site studied was characterized by the absence of overlying pelagic sediments, indicative of relatively recent mud flows, suggesting possible oxygen diffusion down to the sediment. At the bottom of the core (35–40 cm b.s.f.), larger hydrates were found. The possible outcropping of these hydrates could cause sediment resuspension and mixing, thus enhancing oxygen diffusion from the sediment surface and creation of microaerophilic conditions. This would also explain the presence and possible metabolic activity of aerobic, methylotrophic Alphaproteobacteria at the bottom (30 cm b.s.f.) layer. Moreover, methylotrophs form different kinds of resting stages such as cysts and endospores, which enables them to survive even long periods of anoxia or lack of methane (Whittenbury et al., 1970).
Among the nonproteobacterial phylotypes, sequences belonging to phylogenetic groups related to deep biosphere and gas hydrate sediments were retrieved. The same JS1 phylotype was present in all layers. Originally identified in Japan Sea sediments, members of candidate division JS1 have also been commonly recovered from methane-hydrate-associated marine sediments, such as sediments from the Nankai Trough, Hydrate Ridge and the Peru Margin (Webster et al., 2004, 2006, 2007; Fry et al., 2006; Inagaki et al., 2006; Parkes et al., 2007), but also from older Kazan MV sediments (Heijs et al., 2008). Sequences of Chloroflexi were recovered from all examined depths, except the top one, and were present in greatest abundance in our clone library from the 25 cm b.s.f. layer. Chloroflexi occur frequently in hydrocarbon-rich sediments and the deep subsurface (Kormas et al., 2003; Inagaki et al., 2006).
Cluster analysis was performed to reveal community similarity patterns, at both the phylotype and the phylogenetic group levels, between the different sediment layers. The results from both phylotype and phylum approaches were in agreement for the Archaea (Fig. 5c and d). For the Bacteria (Fig. 5a and b), however, the different layers showed different community similarities under the two different approaches. When phylotypes, especially the most abundant and/or the most dominant ones, are affiliated to a large, functionally diverse group (e.g. Gammaproteobacteria), cluster analysis may lead to ‘false’ community similarities (e.g. between the surface and the 30 cm b.s.f. layers). This highlights the need for diversity comparisons at the phylotype rather than at the phylum level, and that higher taxon analysis is appropriate only in highly specific communities.
The use of the Shannon–Wiener diversity index H in prokaryotic communities seems to be applicable and realistically informative, when clone library coverage is sufficiently large (Hill et al., 2003; Kemp & Aller, 2004), and can be used to compare the diversity between different sites or even studies. In addition, the application of such a universal index, which is fairly easily applied in meta-analysis of published data, will allow reasonable comparisons between hot-spot and non-hot-spot sites. In our study, the minimal H-values for both the Bacteria and the Archaea at 15/20 cm b.s.f. was further evidence that a unique, specialized assemblage was established there, related to AOM according to the inferred ecophysiology of the phylotypes found. Another pattern revealed was that the H-values for Bacteria and Archaea were lower than for previous works from sediments of the same and other MVs (Amsterdam and Napoli) in the eastern Mediterranean (Heijs et al., 2008), even though in that study, the more coarse vertical selection of the extracted sediment may have affected the overall values, but were comparable with hydrate sediments from the Gulf of Mexico (Mills et al., 2005). Bacterial diversity was greater than archaeal diversity for all layers, which is generally the case for bacterial and archaeal libraries constructed from the same sampling location (Aller & Kemp, 2008). The higher bacterial vs. archaeal diversity seems to apply to hydrate-bearing sediments (Mills et al., 2005), cold seeps (Reed et al., 2006) and MVs (Heijs et al., 2008), and is possibly a general trend in methane-related environments (Lanoil et al., 2001; Aloisi et al., 2002; Teske et al., 2002; Nauhaus et al., 2005; Lloyd et al., 2006; Lösekann et al., 2007) based on the relative abundance of phylotypes retrieved from each domain. The different ecophysiological roles of Bacteria and Archaea might primarily involve the use of available energy sources and adaptation to energy stress (Valentine, 2007). For methane oxidizers, extensive environmental, laboratory and modelling studies indicate that their mode of growth yields only small amounts of energy and often occurs at exceedingly slow rates. Valentine (2007) contends that the distribution of catabolic pathways among the Archaea results directly from their adaptation to chronic energy stress and that distinctive mechanisms of energy conservation allow many Archaea to adapt readily to environments of differing energy availabilities. In the case of methanogens and methane oxidizers, dominance is achieved through metabolic exclusivity, whereby these organisms have evolved to exclude or outcompete bacteria by the use of unique catabolic pathways. Bacteria, by contrast, seem to focus on exploiting new or variable resources.
Deep-sea sediment geochemical and physical data are acquired at a fine spatial scale more often than biological samples. For marine MVs, the studies available are characterized by a general lack of data on the temporal changes of their hosted microbial communities. In the case of the Kazan MV, however, Heijs et al. (2008) analysed the prokaryotic diversity in samples taken in 1999 from a site c. 30 m away – but still in the active site of the MV – from the one we analysed. Heijs et al. (2008) used a coarser sampling depth resolution (0–6, 6–22 and 22–34 cm b.s.f.). In addition, the different sediment mass used for DNA extraction and different PCR conditions and primers limit the safety of such comparisons. Because of the scarcity of such data, however, we believe that a comparison of relative abundances is feasible, and after pooling our results according to the depth layers of 0–5, 10–20 and 25–30 cm b.s.f., the two studies revealed very different biodiversities, especially in the top two layers. For Archaea, the top layer was dominated by Halobacteriales-related phylotypes (Heijs et al., 2008), but our study showed that it was dominated by ANME-2. The middle layer showed an overlap in ANME-2 (the second most dominant group in Heijs et al., 2008), although with different phylotypes. The bottom layer was dominated by ANME-2 (Heijs et al., 2008) and by ANME-1 and ANME-3 (present study). The dominant Bacteria in the top two layers were different in the two studies (Actinobacteria, Chloroflexi vs. Gamma- and Deltaproteobacteria). The bottom layer, although containing different dominant phyla in the two studies, showed some overlap for the Deltaproteobacteria and Chloroflexi. Such variations, apart from methodological and microscale differences, could be attributed to differences in the prevailing conditions, for example due to eruptions. We suggest that our sampling strategy revealed a higher prokaryotic diversity and also showed that the distribution of prokaryotes explains the geochemical features found. Future comparisons will confirm the persistent or the ephemeral aspect of these communities.
In conclusion, the present study showed that Bacteria and Archaea communities differ even every 5 cm of the top 30 cm in an active site of the Kazan MV with recent mud flow. The archaeal communities showed a lower diversity than the bacterial communities, but were more closely related to ANMEs, while the Bacteria included AOM-related phylotypes. Only at 15 and 20 cm b.s.f. did the two communities show the typical AOM-related profile, known to occur in similar habitats elsewhere, consistent with the prevailing methane–sulphate conditions.