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.

Effect of Jute Fibres on the Process of MICP and Properties of Biocemented Sand.

There has been increasing interest, in the past decade, in bio-mediated approaches to soil improvement for geotechnical applications. Microbially induced calcium carbonate precipitation (MICP) has been investigated as a potentially sustainable method for the strengthening and stabilisation of soil structures. This paper presents the results of a study on the effect of jute fibres on both the MICP process and properties of biocemented sand. Ureolytic Sporosarcina pasteurii has been used to produce biocemented soil columns via MICP in the laboratory. Results showed that columns containing 0.75% (by weight of sand) untreated jute fibres had unconfined compressive strengths approximately six times greater on average compared to biocemented sand columns without jute fibres. Furthermore, efficiency of chemical conversion was found to be higher in columns containing jute fibres, as measured using ion chromatography. Columns containing jute had calcimeter measured CaCO3 contents at least three times those containing sand only. The results showed that incorporation of jute fibres into the biocemented sand material had a beneficial effect, resulting in stimulation of bacterial activity, thus sustaining the MICP process during the twelve-day treatment process. This study also explores the potential of jute fibres in self-healing MICP systems.

Physiological Capabilities of Cryoconite Hole Microorganisms.

Cryoconite holes are miniature freshwater aquatic ecosystems that harbor a relatively diverse microbial community. This microbial community can withstand the extreme conditions of the supraglacial environment, including fluctuating temperatures, extreme and varying geochemical conditions and limited nutrients. We analyzed the physiological capabilities of microbial isolates from cryoconite holes from Antarctica, Greenland, and Svalbard in selected environmental conditions: extreme pH, salinity, freeze-thaw and limited carbon sources, to identify their physiological limits. The results suggest that heterotrophic microorganisms in cryoconite holes are well adapted to fast-changing environmental conditions, by surviving multiple freeze-thaw cycles, a wide range of salinity and pH conditions and scavenging a variety of organic substrates. Under oxic and anoxic conditions, the communities grew well in temperatures up to 30°C, although in anoxic conditions the community was more successful at colder temperatures (0.2°C). The most abundant cultivable microorganisms were facultative anaerobic bacteria and yeasts. They grew in salinities up to 10% and in pH ranging from 4 to 10.5 (Antarctica), 2.5 to 10 (Svalbard), and 3 to 10 (Greenland). Their growth was sustained on at least 58 single carbon sources and there was no decrease in viability for some isolates after up to 100 consecutive freeze-thaw cycles. The elevated viability of the anaerobic community in the lowest temperatures indicates they might be key players in winter conditions or in early melt seasons, when the oxygen is potentially depleted due to limited flow of meltwater. Consequently, facultative anaerobic heterotrophs are likely important players in the reactivation of the community after the polar night. This detailed physiological investigation shows that despite inhabiting a freshwater environment, cryoconite microorganisms are able to withstand conditions not typically encountered in freshwater environments (namely high salinities or extreme pH), making them physiologically more similar to arid soil communities. The results also point to a possible resilience of the most abundant microorganisms of cryoconite holes in the face of rapid change regardless of the location.

Genome Sequences of Two Choline-Utilizing Methanogenic Archaea, Methanococcoides spp., Isolated from Marine Sediments.

The genomes of two Methanococcoides spp. that were isolated from marine sediments and are capable of carrying out methanogenesis from choline and other methylotrophic substrates were sequenced. The average nucleotide identity and in silico DNA-DNA hybridization analyses demonstrate that they represent species different from those previously described.

Rock-crushing derived hydrogen directly supports a methanogenic community: significance for the deep biosphere.

Microbial populations exist to great depths on Earth, but with apparently insufficient energy supply. Earthquake rock fracturing produces H2 from mechanochemical water splitting, however, microbial utilization of this widespread potential energy source has not been directly demonstrated. Here, we show experimentally that mechanochemically generated H2 from granite can be directly, long-term, utilized by a CH4 producing microbial community. This is consistent with CH4 formation in subsurface rock fracturing in the environment. Our results not only support water splitting H2 generation as a potential deep biosphere energy source, but as an oxidant must also be produced, they suggest that there is also a respiratory oxidant supply in the subsurface which is independent of photosynthesis. This may explain the widespread distribution of facultative aerobes in subsurface environments. A range of common rocks were shown to produce mechanochemical H2 , and hence, this process should be widespread in the subsurface, with the potential for considerable mineral fuelled CH4 production.

Phylogeny and physiology of candidate phylum 'Atribacteria' (OP9/JS1) inferred from cultivation-independent genomics.

The 'Atribacteria' is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the 'Atribacteria' inclusive of OP9 and JS1. Additional conserved features within the 'Atribacteria' were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the 'Atribacteria' are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.

Complex coupled metabolic and prokaryotic community responses to increasing temperatures in anaerobic marine sediments: critical temperatures and substrate changes.

The impact of temperature (0-80°C) on anaerobic biogeochemical processes and prokaryotic communities in marine sediments (tidal flat) was investigated in slurries for up to 100 days. Temperature had a non-linear effect on biogeochemistry and prokaryotes with rapid changes over small temperature intervals. Some activities (e.g. methanogenesis) had multiple 'windows' within a large temperature range (∼10 to 80°C). Others, including acetate oxidation, had maximum activities within a temperature zone, which varied with electron acceptor [metal oxide (up to ∼34°C) and sulphate (up to ∼50°C)]. Substrates for sulphate reduction changed from predominantly acetate below, and H2 above, a 43°C critical temperature, along with changes in activation energies and types of sulphate-reducing Bacteria. Above ∼43°C, methylamine metabolism ceased with changes in methanogen types and increased acetate concentrations (>1 mM). Abundances of uncultured Archaea, characteristic of deep marine sediments (e.g. MBGD Euryarchaeota, 'Bathyarchaeota') changed, indicating their possible metabolic activity and temperature range. Bacterial cell numbers were consistently higher than archaeal cells and both decreased above ∼15°C. Substrate addition stimulated activities, widened some activity temperature ranges (methanogenesis) and increased bacterial (×10) more than archaeal cell numbers. Hence, additional organic matter input from climate-related eutrophication may amplify the impact of temperature increases on sedimentary biogeochemistry.

Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure.

Bacterial spores are widespread in marine sediments, including those of thermophilic, sulphate-reducing bacteria, which have a high minimum growth temperature making it unlikely that they grow in situ. These Desulfotomaculum spp. are thought to be from hot environments and are distributed by ocean currents. Their cells and spores upper temperature limit for survival is unknown, as is whether they can survive repeated high-temperature exposure that might occur in hydrothermal systems. This was investigated by incubating estuarine sediments significantly above (40-80 °C) maximum in situ temperatures (∼ 23 °C), and with and without prior triple autoclaving. Sulphate reduction occurred at 40-60 °C and at 60 °C was unaffected by autoclaving. Desulfotomaculum sp. C1A60 was isolated and was most closely related to the thermophilic D. kuznetsovii(T) (∼ 96% 16S rRNA gene sequence identity). Cultures of Desulfotomaculum sp. C1A60, D. kuznetsovii(T)and D. geothermicum B2T survived triple autoclaving while other related Desulfotomaculum spp. did not, although they did survive pasteurisation. Desulfotomaculum sp. C1A60 and D. kuznetsovii cultures also survived more extreme autoclaving (C1A60, 130 °C for 15 min; D. kuznetsovii, 135 °C for 15 min, maximum of 154 °C reached) and high-temperature conditions in an oil bath (C1A60, 130° for 30 min, D. kuznetsovii 140 °C for 15 min). Desulfotomaculum sp. C1A60 with either spores or predominantly vegetative cells demonstrated that surviving triple autoclaving was due to spores. Spores also had very high culturability compared with vegetative cells (∼ 30 × higher). Combined extreme temperature survival and high culturability of some thermophilic Desulfotomaculum spp. make them very effective colonisers of hot environments, which is consistent with their presence in subsurface geothermal waters and petroleum reservoirs.

Glycine betaine as a direct substrate for methanogens (Methanococcoides spp.).

Nine marine methanogenic Methanococcoides strains, including the type strains of Methanococcoides methylutens, M. burtonii, and M. alaskense, were tested for the utilization of N-methylated glycines. Three strains (NM1, PM2, and MKM1) used glycine betaine (N,N,N-trimethylglycine) as a substrate for methanogenesis, partially demethylating it to N,N-dimethylglycine, whereas none of the strains used N,N-dimethylglycine or sarcosine (N-methylglycine). Growth rates and growth yields per mole of substrate with glycine betaine (3.96 g [dry weight] per mol) were similar to those with trimethylamine (4.11 g [dry weight] per mol). However, as glycine betaine is only partially demethylated, the yield per methyl group was significantly higher than with trimethylamine. If glycine betaine and trimethylamine are provided together, trimethylamine is demethylated to dimethyl- and methylamine with limited glycine betaine utilization. After trimethylamine is depleted, dimethylamine and glycine betaine are consumed rapidly, before methylamine. Glycine betaine extends the range of substrates that can be directly utilized by some methanogens, allowing them to gain energy from the substrate without the need for syntrophic partners.

Choline and N,N-dimethylethanolamine as direct substrates for methanogens.

Choline (N,N,N-trimethylethanolamine), which is widely distributed in membrane lipids and is a component of sediment biota, has been shown to be utilized anaerobically by mixed prokaryote cultures to produce methane but not by pure cultures of methanogens. Here, we show that five recently isolated Methanococcoides strains from a range of sediments (Aarhus Bay, Denmark; Severn Estuary mudflats at Portishead, United Kingdom; Darwin Mud Volcano, Gulf of Cadiz; Napoli mud volcano, eastern Mediterranean) can directly utilize choline for methanogenesis producing ethanolamine, which is not further metabolized. Di- and monomethylethanolamine are metabolic intermediates that temporarily accumulate. Consistent with this, dimethylethanolamine was shown to be another new growth substrate, but monomethylethanolamine was not. The specific methanogen inhibitor 2-bromoethanesulfonate (BES) inhibited methane production from choline. When choline and trimethylamine are provided together, diauxic growth occurs, with trimethylamine being utilized first, and then after a lag (∼7 days) choline is metabolized. Three type strains of Methanococcoides (M. methylutens, M. burtonii, and M. alaskense), in contrast, did not utilize choline. However, two of them (M. methylutens and M. burtonii) did metabolize dimethylethanolamine. These results extend the known substrates that can be directly utilized by some methanogens, giving them the advantage that they would not be reliant on bacterial syntrophs for their substrate supply.

Enrichment and cultivation of prokaryotes associated with the sulphate-methane transition zone of diffusion-controlled sediments of Aarhus Bay, Denmark, under heterotrophic conditions.

The prokaryotic activity, diversity and culturability of diffusion-controlled Aarhus Bay sediments, including the sulphate-methane transition zone (SMTZ), were determined using a combination of geochemical, molecular (16S rRNA and mcrA genes) and cultivation techniques. The SMTZ had elevated sulphate reduction and anaerobic oxidation of methane, and enhanced cell numbers, but no active methanogenesis. The prokaryotic population was similar to that in other SMTZs, with Deltaproteobacteria, Gammaproteobacteria, JS1, Planctomycetes, Chloroflexi, ANME-1, MBG-D and MCG. Many of these groups were maintained in a heterotrophic (10 mM glucose, acetate), sediment slurry with periodic low sulphate and acetate additions (~2 mM). Other prokaryotes were also enriched including methanogens, Firmicutes, Bacteroidetes, Synergistetes and TM6. This slurry was then inoculated into a matrix of substrate and sulphate concentrations for further selective enrichment. The results demonstrated that important SMTZ bacteria can be maintained in a long-term, anaerobic culture under specific conditions. For example, JS1 grew in a mixed culture with acetate or acetate/glucose plus sulphate. Chloroflexi occurred in a mixed culture, including in the presence of acetate, which had previously not been shown to be a Chloroflexi subphylum I substrate, and was more dominant in a medium with seawater salt concentrations. In contrast, archaeal diversity was reduced and limited to the orders Methanosarcinales and Methanomicrobiales. These results provide information about the physiology of a range of SMTZ prokaryotes and shows that many can be maintained and enriched under heterotrophic conditions, including those with few or no cultivated representatives.

Tateyamaria pelophila sp. nov., a facultatively anaerobic alphaproteobacterium isolated from tidal-flat sediment, and emended descriptions of the genus Tateyamaria and of Tateyamaria omphalii.

A Gram-negative motile rod, strain SAM4T, was isolated from the highest positive dilution of a most probable number series inoculated with tidal-flat sediments from the German North Sea coast. The isolate grew at 4-35 degrees C and showed constant growth yields throughout almost the whole temperature range. Growth was observed between pH 6 and 9 and at salinities of 0.3-10.2%. Strain SAM4T required Na+ for growth, contained bacteriochlorophyll a and was catalase- and oxidase-positive. It was nutritionally versatile growing on a variety of carbon compounds including carbohydrates, amino acids and organic acids like lactate or succinate. It grew anaerobically on complex media such as marine broth, indicating fermentation, and by reducing trimethylammonium oxide. The dominant phospholipids were phosphatidylethanolamine and phosphatidylglycerol, whereas only traces of phosphatidylcholine and an unidentified lipid were found. The major fatty acid was n-C18:1omega7c. The DNA G+C content was 56.4 mol%. The isolate was identified as a member of the Roseobacter clade within the class Alphaproteobacteria. However, based on phylogenetic, phenotypic and physiological data, it clearly differs from its closest relative Tateyamaria omphalii. Therefore, a novel species is proposed: Tateyamaria pelophila sp. nov., with strain SAM4T (=DSM 17270T=LMG 23018T) as the type strain. Emended descriptions of the genus Tateyamaria and of Tateyamaria omphalii are also presented.

Culturable prokaryotic diversity of deep, gas hydrate sediments: first use of a continuous high-pressure, anaerobic, enrichment and isolation system for subseafloor sediments (DeepIsoBUG).

Deep subseafloor sediments may contain depressurization-sensitive, anaerobic, piezophilic prokaryotes. To test this we developed the DeepIsoBUG system, which when coupled with the HYACINTH pressure-retaining drilling and core storage system and the PRESS core cutting and processing system, enables deep sediments to be handled without depressurization (up to 25 MPa) and anaerobic prokaryotic enrichments and isolation to be conducted up to 100 MPa. Here, we describe the system and its first use with subsurface gas hydrate sediments from the Indian Continental Shelf, Cascadia Margin and Gulf of Mexico. Generally, highest cell concentrations in enrichments occurred close to in situ pressures (14 MPa) in a variety of media, although growth continued up to at least 80 MPa. Predominant sequences in enrichments were Carnobacterium, Clostridium, Marinilactibacillus and Pseudomonas, plus Acetobacterium and Bacteroidetes in Indian samples, largely independent of media and pressures. Related 16S rRNA gene sequences for all of these Bacteria have been detected in deep, subsurface environments, although isolated strains were piezotolerant, being able to grow at atmospheric pressure. Only the Clostridium and Acetobacterium were obligate anaerobes. No Archaea were enriched. It may be that these sediment samples were not deep enough (total depth 1126-1527 m) to obtain obligate piezophiles.

Gammaproteobacteria as a possible source of eicosapentaenoic acid in anoxic intertidal sediments.

Eicosapentaenoic acid (EPA; n-20:5omega3) was found to be a constituent of phospholipids in three mesophilic strains of Gammaproteobacteria, which were isolated from anoxic most probable number series prepared with sediments from an intertidal flat of the German North Sea coast. Their partial 16S rRNA gene sequences identified the isolates as close relatives of Shewanella colwelliana, Vibrio splendidus, and Photobacterium lipolyticum. So far, eicosapentaenoic acid has mainly been reported to occur in eukaryotes and some piezophilic or psychrophilic bacteria. With decreasing temperature, relative contents of EPA (up to 14% of total fatty acids) increased in all strains. Additionally, Shewanella and Vibrio spp. showed a significant increase in monounsaturated fatty acids with lower growth temperature. Analysis of the phospholipid compositions revealed that EPA was present in all three major phospholipid types, namely, phosphatidyl glycerol (PG), cardiolipin and phosphatidyl ethanolamine (PE). However, EPA was enriched in PG and cardiolipin relative to PE. In the tidal flat sediments from which the isolates were obtained, substantial amounts of EPA-containing PG were detected, whereas other typical microeukaryotic phospholipids-being also a possible source of EPA-were abundant at the sediment surface but were present in clearly lower amounts in the anoxic layers beneath 5 cm depth. Therefore, the EPA-containing PG species in the deeper layers in these sediments may indicate the presence of Gammaproteobacteria closely related to the isolates. These bacteria appear to be an important source of EPA in buried, anoxic sediments beneath the layers harboring significant populations of benthic eukaryotes.

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.

Identity and abundance of active sulfate-reducing bacteria in deep tidal flat sediments determined by directed cultivation and CARD-FISH analysis.

The identity and abundance of potentially active sulfate-reducing bacteria (SRB) in several metre deep sediments of a tidal sand flat in the German Wadden Sea were assessed by directed cultivation and cultivation-independent CARD-FISH analysis (catalysed reporter deposition fluorescence in situ hybridization). Presumably abundant SRB from different sediment layers between 0.5 and 4 m depth were selectively enriched in up to million-fold diluted cultures supplemented with lactate, acetate or hydrogen. Partial 16S rRNA gene sequences obtained from highest dilution steps showing sulfide formation indicated growth of deltaproteobacterial SRB belonging to the Desulfobulbaceae and the Desulfobacteraceae as well as of members of the Firmicutes. Subsequent isolation resulted in 10 novel phylotypes of both litho- and organotrophic sulfate-reducing Deltaproteobacteria. Molecular pre-screening identified six isolates as members of the Desulfobulbaceae, sharing highest identities with either candidatus 'Desulfobacterium corrodens' (95-97%) or Desulfobacterium catecholicum (98%), and four isolates as members of Desulfobacteraceae, being related to either Desulfobacter psychrotolerans (98%) or Desulfobacula phenolica (95-97%). Relatives of D. phenolica were exlusively isolated from 50 and 100 cm deep sediments with 10 and 2 mM of pore water sulfate respectively. In contrast, relatives of D. corrodens, D. psychrotolerans and D. catecholicum were also obtained from layers deeper than 100 cm and with less than 2 mM sulfate. The high in situ abundance of members of both families in sediment layers beneath 50 cm could be confirmed via CARD-FISH analysis performed with a set of six SRB-specific oligonucleotide probes. Moreover, SRB represented a numerically significant fraction of the microbial community throughout the sediment (up to 7%) and reached even higher cell numbers in deep, sulfate-poor layers than in the sulfate-rich surface sediment. This relatively large community size of potentially active SRB in deep sandy sediments might on the one hand be a result of their syntrophic association with other anaerobes. Our results furthermore support the hypothesis that enhanced advective pore water transport might supply nutrients to microbial communities in deep sandy sediments and point to their so far unrecognized contribution to biogeochemical processes in Wadden Sea sediments.

Diversity of Bacillus-like organisms isolated from deep-sea hypersaline anoxic sediments.

BACKGROUND: The deep-sea, hypersaline anoxic brine lakes in the Mediterranean are among the most extreme environments on earth, and in one of them, the MgCl2-rich Discovery basin, the presence of active microbes is equivocal. However, thriving microbial communities have been detected especially in the chemocline between deep seawater and three NaCl-rich brine lakes, l'Atalante, Bannock and Urania. By contrast, the microbiota of these brine-lake sediments remains largely unexplored. RESULTS: Eighty nine isolates were obtained from the sediments of four deep-sea, hypersaline anoxic brine lakes in the Eastern Mediterranean Sea: l'Atalante, Bannock, Discovery and Urania basins. This culture collection was dominated by representatives of the genus Bacillus and close relatives (90% of all isolates) that were investigated further. Physiological characterization of representative strains revealed large versatility with respect to enzyme activities or substrate utilization. Two third of the isolates did not grow at in-situ salinities and were presumably present as endospores. This is supported by high numbers of endospores in Bannock, Discovery and Urania basins ranging from 3.8 x 10(5) to 1.2 x 10(6) g(-1) dw sediment. However, the remaining isolates were highly halotolerant growing at salinities of up to 30% NaCl. Some of the novel isolates affiliating with the genus Pontibacillus grew well under anoxic conditions in sulfidic medium by fermentation or anaerobic respiration using dimethylsulfoxide or trimethylamine N-oxide as electron acceptor. CONCLUSION: Some of the halophilic, facultatively anaerobic relatives of Bacillus appear well adapted to life in this hostile environment and suggest the presence of actively growing microbial communities in the NaCl-rich, deep-sea brine-lake sediments.

Two distinct Photobacterium populations thrive in ancient Mediterranean sapropels.

Eastern Mediterranean sediments are characterized by the periodic occurrence of conspicuous, organic matter-rich sapropel layers. Phylogenetic analysis of a large culture collection isolated from these sediments revealed that about one third of the isolates belonged to the genus Photobacterium. In the present study, 22 of these strains were examined with respect to their phylogenetic and metabolic diversity. The strains belonged to two distinct Photobacterium populations (Mediterranean cluster I and II). Strains of cluster I were isolated almost exclusively from organic-rich sapropel layers and were closely affiliated with P. aplysiae (based on their 16S rRNA gene sequences). They possessed almost identical Enterobacterial Repetitive Intergenic Consensus (ERIC) and substrate utilization patterns, even among strains from different sampling sites or from layers differing up to 100,000 years in age. Strains of cluster II originated from sapropels and from the surface and carbon-lean intermediate layers. They were related to Photobacterium frigidiphilum but differed significantly in their fingerprint patterns and substrate spectra, even when these strains were obtained from the same sampling site and layer. Temperature range for growth (4 to 33 degrees C), salinity tolerance (5 to 100 per thousand), pH requirements (5.5-9.3), and the composition of polar membrane lipids were similar for both clusters. All strains grew by fermentation (glucose, organic acids) and all but five by anaerobic respiration (nitrate, dimethyl sulfoxide, anthraquinone disulfonate, or humic acids). These results indicate that the genus Photobacterium forms subsurface populations well adapted to life in the deep biosphere.

Distribution of candidate division JS1 and other Bacteria in tidal sediments of the German Wadden Sea using targeted 16S rRNA gene PCR-DGGE.

The bacterial candidate division JS1 dominates a number of 16S rRNA gene libraries from deep subseafloor sediments, yet its distribution in shallow, subsurface sediments has still to be fully documented. Sediment cores (down to 5.5 m) from Wadden Sea tidal flats (Neuharlingersieler Nacken and Gröninger Plate) were screened for JS1 16S rRNA genes using targeted PCR-denaturing gradient gel electrophoresis (DGGE), which also detects some other important Bacteria. Bacterial subpopulations at both sites were dominated by Gammaproteobacteria in the upper sediment layers (down to 2 m) and in deeper layers by members of the Chloroflexi. The deeper layers of Neuharlingersieler Nacken consisted of grey mud with low sulphate (0.1-10 mM), elevated total organic carbon (TOC) ( approximately 1-2%) and JS1 sequences were abundant. In contrast, the deeper sandy layers of Gröninger Plate, despite also having reduced sulphate concentrations, had lower TOC (<0.6%) with few detectable JS1 sequences. Results indicated that JS1 prefers muddy, shallow, subsurface sediments with reduced sulphate, whereas Chloroflexi may out-compete JS1 in shallow, sandy, subsurface sediments. Bacterial population changes at both sites ( approximately 2 m) were confirmed by cluster analysis of DGGE profiles, which correlated with increased recalcitrance of the organic matter. This study extends the biogeographical range of JS1. The presence of JS1 and Chloroflexi in Wadden Sea sediments demonstrates that subsurface tidal flats contain similar prokaryotic populations to those found in the deeper subseafloor biosphere.

Spore dipicolinic acid contents used for estimating the number of endospores in sediments.

Endospores are heat-resistant bacterial resting stages that can remain viable for long periods of time and may thus accumulate in sediments as a function of sediment age. The number of spores in sediments has only rarely been quantified, because of methodological problems, and consequently little is known about the quantitative contribution of endospores to the total number of prokaryotic cells. We here report on a protocol to determine the number of endospores in sediments and cultures. The method is based on the fluorimetric determination of dipicolinic acid (DPA), a spore core-specific compound, after reaction with terbium chloride. The concentration of DPA in natural samples is converted into endospore numbers using endospore-forming pure cultures as standards. Quenching of the fluorescence by sediment constituents and background fluorescence due to humic substances hampered direct determination of DPA in sediments. To overcome those interferences, DPA was extracted using ethyl acetate prior to fluorimetric measurements of DPA concentrations. The first results indicated that endospore numbers obtained with this method are orders of magnitude higher than numbers obtained by cultivation after pasteurization. In one of the explored sediment cores, endospores accounted for 3% of all stainable prokaryotic cells.