Methanogen activity and microbial diversity in Gulf of Cádiz mud volcano sediments.

The Gulf of Cádiz is a tectonically active continental margin with over sixty mud volcanoes (MV) documented, some associated with active methane (CH4) seepage. However, the role of prokaryotes in influencing this CH4 release is largely unknown. In two expeditions (MSM1-3 and JC10) seven Gulf of Cádiz MVs (Porto, Bonjardim, Carlos Ribeiro, Captain Arutyunov, Darwin, Meknes, and Mercator) were analyzed for microbial diversity, geochemistry, and methanogenic activity, plus substrate amended slurries also measured potential methanogenesis and anaerobic oxidation of methane (AOM). Prokaryotic populations and activities were variable in these MV sediments reflecting the geochemical heterogeneity within and between them. There were also marked differences between many MV and their reference sites. Overall direct cell numbers below the SMTZ (0.2-0.5 mbsf) were much lower than the general global depth distribution and equivalent to cell numbers from below 100 mbsf. Methanogenesis from methyl compounds, especially methylamine, were much higher than the usually dominant substrates H2/CO2 or acetate. Also, CH4 production occurred in 50% of methylated substrate slurries and only methylotrophic CH4 production occurred at all seven MV sites. These slurries were dominated by Methanococcoides methanogens (resulting in pure cultures), and prokaryotes found in other MV sediments. AOM occurred in some slurries, particularly, those from Captain Arutyunov, Mercator and Carlos Ribeiro MVs. Archaeal diversity at MV sites showed the presence of both methanogens and ANME (Methanosarcinales, Methanococcoides, and ANME-1) related sequences, and bacterial diversity was higher than archaeal diversity, dominated by members of the Atribacterota, Chloroflexota, Pseudomonadota, Planctomycetota, Bacillota, and Ca. "Aminicenantes." Further work is essential to determine the full contribution of Gulf of Cádiz mud volcanoes to the global methane and carbon cycles.

Subsurface microbiology and biogeochemistry of a deep, cold-water carbonate mound from the Porcupine Seabight (IODP Expedition 307).

The Porcupine Seabight Challenger Mound is the first carbonate mound to be drilled (approximately 270 m) and analyzed in detail microbiologically and biogeochemically. Two mound sites and a non-mound Reference site were analyzed with a range of molecular techniques [catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH), quantitative PCR (16S rRNA and functional genes, dsrA and mcrA), and 16S rRNA gene PCR-DGGE] to assess prokaryotic diversity, and this was compared with the distribution of total and culturable cell counts, radiotracer activity measurements and geochemistry. There was a significant and active prokaryotic community both within and beneath the carbonate mound. Although total cell numbers at certain depths were lower than the global average for other subseafloor sediments and prokaryotic activities were relatively low (iron and sulfate reduction, acetate oxidation, methanogenesis) they were significantly enhanced compared with the Reference site. In addition, there was some stimulation of prokaryotic activity in the deepest sediments (Miocene, > 10 Ma) including potential for anaerobic oxidation of methane activity below the mound base. Both Bacteria and Archaea were present, with neither dominant, and these were related to sequences commonly found in other subseafloor sediments. With an estimate of some 1600 mounds in the Porcupine Basin alone, carbonate mounds may represent a significant prokaryotic subseafloor habitat.

Prokaryotic functional diversity in different biogeochemical depth zones in tidal sediments of the Severn Estuary, UK, revealed by stable-isotope probing.

Stable isotope probing of prokaryotic DNA was used to determine active prokaryotes using (13)C-labelled substrates (glucose, acetate, CO(2)) in sediment slurries from different biogeochemical zones of the Severn Estuary, UK. Multiple, low concentrations (5 x 100 microM) of (13)C-substrate additions and short-term incubations (7 days) were used to minimize changes in the prokaryotic community, while achieving significant (13)C-incorporation. Analysis demonstrated clear metabolic activity within all slurries, although neither the net sulphate removal nor CH(4) production occurred in the anaerobic sulphate reduction and methanogenesis zone slurries. Some similarities occurred in the prokaryotic populations that developed in different sediment slurries, particularly in the aerobic and dysaerobic zone slurries with (13)C-glucose, which were dominated by Gammaproteobacteria and Marine Group 1 Archaea, whereas both anaerobic sediment slurries incubated with (13)C-acetate showed incorporation into Epsilonproteobacteria and other bacteria, with the sulphate reduction zone slurry also showing (13)C-acetate utilization by Miscellaneous Crenarchaeotic Group Archaea. The lower potential energy methanogenesis zone slurries were the only conditions where no (13)C-incorporation into Archaea occurred, despite Bacteria being labelled; this was surprising because Archaea have been suggested to be adapted to low-energy conditions. Overall, our results highlight that uncultured prokaryotes play important ecological roles in tidal sediments of the Severn Estuary, providing new metabolic information for novel groups of Archaea and suggesting broader metabolisms for largely uncultivated Bacteria.

Biogeochemistry and biodiversity of methane cycling in subsurface marine sediments (Skagerrak, Denmark).

This biogeochemical, molecular genetic and lipid biomarker study of sediments ( approximately 4 m cores) from the Skagerrak (Denmark) investigated methane cycling in a sediment with a clear sulfate-methane-transition zone (SMTZ) and where CH(4) supply was by diffusion, rather than by advection, as in more commonly studied seep sites. Sulfate reduction removed sulfate by 0.7 m and CH(4) accumulated below. (14)C-radiotracer measurements demonstrated active H(2)/CO(2) and acetate methanogenesis and anaerobic oxidation of CH(4) (AOM). Maximum AOM rates occurred near the SMTZ ( approximately 3 nmol cm(-3) day(-1) at 0.75 m) but also continued deeper, overall, at much lower rates. Maximum rates of H(2)/CO(2) and acetate methanogenesis occurred below the SMTZ but H(2)/CO(2) methanogenesis rates were x 10 those of acetate methanogenesis, and this was consistent with initial values of (13)C-depleted CH(4) (delta(13)C c.-80 per thousand). Areal AOM and methanogenic rates were similar ( approximately 1.7 mmol m(-2) day(-1)), hence, CH(4) flux is finely balanced. A 16S rRNA gene library from 1.39 m combined with methanogen (T-RFLP), bacterial (16S rRNA DGGE) and lipid biomarker depth profiles showed the presence of populations similar to some seep sites: ANME-2a (dominant), ANME-3, Methanomicrobiales, Methanosaeta Archaea, with abundance changes with depth corresponding to changes in activities and sulfate-reducing bacteria (SRB). Below the SMTZ to approximately 1.7 m CH(4) became progressively more (13)C depleted (delta(13)C -82 per thousand) indicating a zone of CH(4) recycling which was consistent with the presence of (13)C-depleted archaeol (delta(13)C -55 per thousand). Pore water acetate concentrations decreased in this zone (to approximately 5 microM), suggesting that H(2), not acetate, was an important CH(4) cycling intermediate. The potential biomarkers for AOM-associated SRB, non-isoprenoidal ether lipids, increased below the SMTZ but this distribution reflected 16S rRNA gene sequences for JS1 and OP8 bacteria rather than those of SRB. At this site peak rates of methane production and consumption are spatially separated and seem to be conducted by different archaeal groups. Also AOM is predominantly coupled to sulfate reduction, unlike recent reports from some seep and gassy sediment sites.

Culturable phylogenetic diversity of the phylum 'Bacteroidetes' from river epilithon and coastal water and description of novel members of the family Flavobacteriaceae: Epilithonimonas tenax gen. nov., sp. nov. and Persicivirga xylanidelens gen. nov., sp. nov.

Members of the phylum 'Bacteroidetes' are important heterotrophs involved in cycling organic carbon in aquatic habitats. Their diversity has been studied by molecular methods in both freshwater and marine habitats and many novel genera and species within this phylum have been characterized in recent years. In this study, we examined the diversity of members of the 'Bacteroidetes' that could be readily isolated on solid media from river epilithon and coastal sea water. Most (93 %) of the 55 isolates confirmed as members of the 'Bacteroidetes' and examined by phylogenetic analysis of 16S rRNA gene sequences belonged to the Flavobacteriaceae. Furthermore, most (62 %) of these were almost certainly members of the genus Flavobacterium and all but one were from river epilithon. Conversely, the sea-water isolates were more widely distributed in clades containing other genera. Some of the isolates were deep-branching within phylogenetic trees and so could not be assigned to putative genera. Two of these deep-branching isolates were characterized by polyphasic taxonomy and are proposed as novel species within two new genera of the family Flavobacteriaceae. These are Epilithonimonas tenax gen. nov., sp. nov. (type strain EP105T = NCIMB 14026T = DSM 16811T) and Persicivirga xylanidelens gen. nov., sp. nov. (type strain SW256T = NCIMB 14027T = DSM 16809T).

A comparison of stable-isotope probing of DNA and phospholipid fatty acids to study prokaryotic functional diversity in sulfate-reducing marine sediment enrichment slurries.

Marine sediment slurries enriched for anaerobic, sulfate-reducing prokaryotic communities utilizing glucose and acetate were used to provide the first comparison between stable-isotope probing (SIP) of phospholipid fatty acids (PLFA) and DNA (16S rRNA and dsrA genes) biomarkers. Different 13C-labelled substrates (glucose, acetate and pyruvate) at low concentrations (100 microM) were used over a 7-day incubation to follow and identify carbon flow into different members of the community. Limited changes in total PLFA and bacterial 16S rRNA gene DGGE profiles over 7 days suggested the presence of a stable bacterial community. A broad range of PLFA were rapidly labelled (within 12 h) in the 13C-glucose slurry but this changed with time, suggesting the presence of an active glucose-utilizing population and later development of another population able to utilize glucose metabolites. The identity of the major glucose-utilizers was unclear as 13C-enriched PLFA were common (16:0, 16:1, 18:1omega7, highest incorporation) and there was little difference between 12C- and 13C-DNA 16S rRNA gene denaturing gradient gel electrophoresis (DGGE) profiles. Seemingly glucose, a readily utilizable substrate, resulted in widespread incorporation consistent with the higher extent of 13C-incorporation (approximately 10 times) into PLFA compared with 13C-acetate or 13C-pyruvate. 13C-PLFA in the 13C-acetate and 13C-pyruvate slurries were similar to each other and to those that developed in the 13C-glucose slurry after 4 days. These were more diagnostic, with branched odd-chain fatty acids (i15:0, a15:0 and 15:1omega6) possibly indicating the presence of Desulfococcus or Desulfosarcina sulfate-reducing bacteria (SRB) and sequences related to these SRB were in the 13C-acetate-DNA dsrA gene library. The 13C-acetate-DNA 16S rRNA gene library also contained sequences closely related to SRB, but these were the acetate-utilizing Desulfobacter sp., as well as a broad range of uncultured Bacteria. In contrast, analysis of DGGE bands from 13C-DNA demonstrated that the candidate division JS1 and Firmicutes were actively assimilating 13C-acetate. Denaturing gradient gel electrophoresis also confirmed the presence of JS1 in the 13C-DNA from the 13C-glucose slurry. These results demonstrate that JS1, originally found in deep subsurface sediments, is more widely distributed in marine sediments and provides the first indication of its metabolism; incorporation of acetate and glucose (or glucose metabolites) under anaerobic, sulfate-reducing conditions. Here we demonstrate that PLFA- and DNA-SIP can be used together in a sedimentary system, with low concentrations of 13C-substrate and overlapping incubation times (up to 7 days) to provide complementary, although not identical, information on carbon flow and the identity of active members of an anaerobic prokaryotic community.

Fluviicola taffensis gen. nov., sp. nov., a novel freshwater bacterium of the family Cryomorphaceae in the phylum 'Bacteroidetes'.

A novel, strictly aerobic, Gram-negative, yellow-orange-pigmented, motile, catalase-positive, oxidase-negative bacterium, RW262(T), was isolated from water of the River Taff, Cardiff, UK, during January 2000. Phylogenetic analysis of the 16S rRNA gene indicated that strain RW262(T) was a member of the family Cryomorphaceae within the phylum 'Bacteroidetes'. The DNA G+C content of strain RW262(T) was 37.2 mol%. The predominant fatty acid was the branched-chain saturated fatty acid i15:0 (44.2%). On the basis of polyphasic analysis of phenotypic, chemotaxonomic, genotypic and phylogenetic characteristics, it is proposed that this freshwater bacterium represents a novel genus and species within the family Cryomorphaceae, Fluviicola taffensis gen. nov., sp. nov. The type strain is RW262(T) (=NCIMB 13979(T)=DSM 16823(T)).