Evidence of active methanogen communities in shallow sediments of the sonora margin cold seeps.

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.

Bacterial communities and syntrophic associations involved in anaerobic oxidation of methane process of the Sonora Margin cold seeps, Guaymas Basin.

SUMMARY: The Sonora Margin cold seeps present on the seafloor a patchiness pattern of white microbial mats surrounded by polychaete and gastropod beds. These surface assemblages are fuelled by abundant organic inputs sedimenting from the water column and upward-flowing seep fluids. Elevated microbial density was observed in the underlying sediments. A previous study on the same samples identified anaerobic oxidation of methane (AOM) as the potential dominant archaeal process in these Sonora Margin sediments, probably catalysed by three clades of archaeal anaerobic methanotrophs (ANME-1, ANME-2 and ANME-3) associated with bacterial syntrophs. In this study, molecular surveys and microscopic observations investigating the diversity of Bacteria involved in AOM process, as well as the environmental parameters affecting the composition and the morphologies of AOM consortia in the Sonora Margin sediments were carried out. Two groups of Bacteria were identified within the AOM consortia, the Desulfosarcina/Desulfococcus SEEP SRB-1a group and a Desulfobulbus-related group. These bacteria showed different niche distributions, association specificities and consortia architectures, depending on sediment surface communities, geochemical parameters and ANME-associated phylogeny. Therefore, the syntrophic AOM process appears to depend on sulphate-reducing bacteria with different ecological niches and/or metabolisms, in a biofilm-like organic matrix.

Methanococcoides vulcani sp. nov., a marine methylotrophic methanogen that uses betaine, choline and N,N-dimethylethanolamine for methanogenesis, isolated from a mud volcano, and emended description of the genus Methanococcoides.

A novel, strictly anaerobic, methylotrophic marine methanogen, strain SLH33(T), was isolated from deep sediment samples covered by an orange microbial mat collected from the Napoli Mud Volcano. Cells of strain SLH33(T) were Gram-stain-negative, motile, irregular cocci that occurred singly. Cells utilized trimethylamine, dimethylamine, monomethylamine, methanol, betaine, N,N-dimethylethanolamine and choline (N,N,N-trimethylethanolamine) as substrates for growth and methanogenesis. The optimal growth temperature was 30 °C; maximum growth rate was obtained at pH 7.0 in the presence of 0.5 M Na(+). The DNA G+C content of strain SLH33(T) was 43.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain SLH33(T) within the genus Methanococcoides. The novel isolate was related most closely to Methanococcoides methylutens TMA-10(T) (98.8% 16S rRNA gene sequence similarity) but distantly related to Methanococcoides burtonii DSM 6242(T) (97.6%) and Methanococcoides alaskense AK-5(T) (97.6%). DNA-DNA hybridization studies indicated that strain SLH33(T) represents a novel species, given that it shared less than 16% DNA-DNA relatedness with Methanococcoides methylutens TMA-10(T). The name Methanococcoides vulcani sp. nov. is proposed for this novel species, with strain SLH33(T) ( = DSM 26966(T) = JCM 19278(T)) as the type strain. An emended description of the genus Methanococcoides is also proposed.

Methanogenic diversity and activity in hypersaline sediments of the centre of the Napoli mud volcano, Eastern Mediterranean Sea.

Submarine mud volcanoes are a significant source of methane to the atmosphere. The Napoli mud volcano, situated in the brine-impacted Olimpi Area of the Eastern Mediterranean Sea, emits mainly biogenic methane particularly at the centre of the mud volcano. Temperature gradients support the suggestion that Napoli is a cold mud volcano with moderate fluid flow rates. Biogeochemical and molecular genetic analyses were carried out to assess the methanogenic activity rates, pathways and diversity in the hypersaline sediments of the centre of the Napoli mud volcano. Methylotrophic methanogenesis was the only significant methanogenic pathway in the shallow sediments (0-40 cm) but was also measured throughout the sediment core, confirming that methylotrophic methanogens could be well adapted to hypersaline environments. Hydrogenotrophic methanogenesis was the dominant pathway below 50 cm; however, low rates of acetoclastic methanogenesis were also present, even in sediment layers with the highest salinity, showing that these methanogens can thrive in this extreme environment. PCR-DGGE and methyl coenzyme M reductase gene libraries detected sequences affiliated with anaerobic methanotrophs (mainly ANME-1) as well as Methanococcoides methanogens. Results show that the hypersaline conditions in the centre of the Napoli mud volcano influence active biogenic methane fluxes and methanogenic/methylotrophic diversity.

Unveiling microbial life in new deep-sea hypersaline Lake Thetis. Part I: Prokaryotes and environmental settings.

In September 2008, an expedition of the RV Urania was devoted to exploration of the genomic richness of deep hypersaline anoxic lakes (DHALs) located in the Western part of the Mediterranean Ridge. Approximately 40 nautical miles SE from Urania Lake, the presence of anoxic hypersaline lake, which we named Thetis, was confirmed by swath bathymetry profiling and through immediate sampling casts. The brine surface of the Thetis Lake is located at a depth of 3258 m with a thickness of ≈ 157 m. Brine composition was found to be thalassohaline, saturated by NaCl with a total salinity of 348‰, which is one of highest value reported for DHALs. Similarly to other Mediterranean DHALs, seawater-brine interface of Thetis represents a steep pycno- and chemocline with gradients of salinity, electron donors and acceptors and posseses a remarkable stratification of prokaryotic communities, observed to be more metabolically active in the upper interface where redox gradient was sharper. [(14) C]-bicarbonate fixation analysis revealed that microbial communities are sustained by sulfur-oxidizing chemolithoautotrophic primary producers that thrive within upper interface. Besides microaerophilic autotrophy, heterotrophic sulfate reduction, methanogenesis and anaerobic methane oxidation are likely the predominant processes driving the ecosystem of Thetis Lake.

Archaeal populations in hypersaline sediments underlying orange microbial mats in the Napoli mud volcano.

Microbial mats in marine cold seeps are known to be associated with ascending sulfide- and methane-rich fluids. Hence, they could be visible indicators of anaerobic oxidation of methane (AOM) and methane cycling processes in underlying sediments. The Napoli mud volcano is situated in the Olimpi Area that lies on saline deposits; from there, brine fluids migrate upward to the seafloor. Sediments associated with a brine pool and microbial orange mats of the Napoli mud volcano were recovered during the Medeco cruise. Based on analysis of RNA-derived sequences, the "active" archaeal community was composed of many uncultured lineages, such as rice cluster V or marine benthic group D. Function methyl coenzyme M reductase (mcrA) genes were affiliated with the anaerobic methanotrophic Archaea (ANME) of the ANME-1, ANME-2a, and ANME-2c groups, suggesting that AOM occurred in these sediment layers. Enrichment cultures showed the presence of viable marine methylotrophic Methanococcoides in shallow sediment layers. Thus, the archaeal community diversity seems to show that active methane cycling took place in the hypersaline microbial mat-associated sediments of the Napoli mud volcano.

Distribution of anaerobic methane-oxidizing and sulfate-reducing communities in the G11 Nyegga pockmark, Norwegian Sea.

Pockmarks are seabed geological structures sustaining methane seepage in cold seeps. Based on RNA-derived sequences the active fraction of the archaeal community was analysed in sediments associated with the G11 pockmark, in the Nyegga region of the Norwegian Sea. The anaerobic methanotrophic Archaea (ANME) and sulfate-reducing bacteria (SRB) communities were studied as well. The vertical distribution of the archaeal community assessed by PCR-DGGE highlighted the presence of ANME-2 in surface sediments, and ANME-1 in deeper sediments. Enrichments of methanogens showed the presence of hydrogenotrophic methanogens of the Methanogenium genus in surface sediment layers as well. The active fraction of the archaeal community was uniquely composed of ANME-2 in the shallow sulfate-rich sediments. Functional methyl coenzyme M reductase gene libraries showed that sequences affiliated with the ANME-1 and ANME-3 groups appeared in the deeper sediments but ANME-2 dominated both surface and deeper layers. Finally, dissimilatory sulfite reductase gene libraries revealed a high SRB diversity (i.e. Desulfobacteraceae, Desulfobulbaceae, Syntrophobacteraceae and Firmicutes) in the shallow sulfate-rich sediments. The SRB diversity was much lower in the deeper section. Overall, these results show that the microbial community in sediments associated with a pockmark harbour classical cold seep ANME and SRB communities.

Active archaeal communities at cold seep sediments populated by Siboglinidae tubeworms from the Storegga Slide.

Siboglinid tubeworms in cold seep sediments can locally modify the geochemical gradients of electron acceptors and donors, hence creating potential microhabitats for prokaryotic populations. The archaeal communities associated with sediments populated by Oligobrachia haakonmosbiensis and Sclerolinum contortum Siboglinid tubeworms in the Storegga Slide were examined in this study. Vertical distribution of archaeal communities was investigated using denaturing gradient gel electrophoresis based on 16S rRNA genes. The active fraction of the archaeal community was assessed by using reverse-transcribed rRNA. Archaeal communities associated with sediments colonized by tubeworms were affiliated with uncultivated archaeal lineages of the Crenarchaeota and Euryarchaeota. The composition of the active archaeal populations changed with depth indicating a reorganization of microbial communities. 16S rRNA gene libraries were dominated by sequences affiliated to the Rice Cluster V which are unusual in marine sediment samples. Moreover, this study provides the first evidence of living Crenarchaeota of the Rice Cluster V in cold seep sediments. Furthermore, the Storegga Slide sediments harbored a high diversity of other minor groups of uncultivated lineages including Terrestrial Miscellaneous Euryarchaeotal Group, Marine Benthic Group (MBG)-D, MBG-E, Deep-Sea Hydrothermal Vent Euryarchaeotal Group, Lake Dagow Sediment, Val Kotinen Lake clade III, and Sippenauer Moor 1. Thus, we hypothesize that the vertical geochemical imprint created by the tubeworms could support broad active archaeal populations in the Siboglinidae-populated Storegga Slide sediments.

New Insights into the Ecology and Physiology of Methanomassiliicoccales from Terrestrial and Aquatic Environments.

Members of the archaeal order Methanomassiliicoccales are methanogens mainly associated with animal digestive tracts. However, environmental members remain poorly characterized as no representatives not associated with a host have been cultivated so far. In this study, metabarcoding screening combined with quantitative PCR analyses on a collection of diverse non-host-associated environmental samples revealed that Methanomassiliicoccales were very scarce in most terrestrial and aquatic ecosystems. Relative abundance of Methanomassiliicoccales and substrates/products of methanogenesis were monitored during incubation of environmental slurries. A sediment slurry enriched in Methanomassiliicoccales was obtained from a freshwater sample. It allowed the reconstruction of a high-quality metagenome-assembled genome (MAG) corresponding to a new candidate species, for which we propose the name of Candidatus 'Methanomassiliicoccus armoricus MXMAG1'. Comparison of the annotated genome of MXMAG1 with the published genomes and MAGs from Methanomassiliicoccales belonging to the 2 known clades ('free-living'/non-host-associated environmental clade and 'host-associated'/digestive clade) allowed us to explore the putative physiological traits of Candidatus 'M. armoricus MXMAG1'. As expected, Ca. 'Methanomassiliicoccus armoricus MXMAG1' had the genetic potential to produce methane by reduction of methyl compounds and dihydrogen oxidation. This MAG encodes for several putative physiological and stress response adaptations, including biosynthesis of trehalose (osmotic and temperature regulations), agmatine production (pH regulation), and arsenic detoxication, by reduction and excretion of arsenite, a mechanism that was only present in the 'free-living' clade. An analysis of co-occurrence networks carried out on environmental samples and slurries also showed that Methanomassiliicoccales detected in terrestrial and aquatic ecosystems were strongly associated with acetate and dihydrogen producing bacteria commonly found in digestive habitats and which have been reported to form syntrophic relationships with methanogens.

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin.

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYROT was isolated from a deep hypersaline anoxic basin called "Tyro" located in the Eastern Mediterranean Sea. Cells of SLHTYROT were motile cocci. The strain SLHTYROT grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L-1 NaCl (optimum 135 g L-1). Strain SLHTYROT was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYROT was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYROT was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYROT should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYROT (=DSM 108854 = JCM 32768 = UBOCC-M-3308).

Simultaneous analysis of microbial identity and function using NanoSIMS.

Identifying the function of uncultured microbes in their environments today remains one of the main challenges for microbial ecologists. In this article, we describe a new method allowing simultaneous analysis of microbial identity and function. This method is based on the visualization of oligonucleotide probe-conferred hybridization signal in single microbial cells and isotopic measurement using high-resolution ion microprobe (NanoSIMS). In order to characterize the potential of the method, an oligonucleotide containing iodized cytidine was hybridized on fixed cells of Escherichia coli cultured on media containing different levels of 13C or 15N. Iodine signals could clearly be localized on targeted cells and the isotopic enrichment could be monitored at the single-cell level. The applicability of this new technique to the study of in situ ecophysiology of uncultured microorganisms within complex microbial communities is illustrated.

Complete Genome Sequence of the Halophilic Methylotrophic Methanogen Archaeon Methanohalophilus portucalensis Strain FDF-1T.

We report here the complete genome sequence (2.08 Mb) of Methanohalophilus portucalensis strain FDF-1T, a halophilic methylotrophic methanogen isolated from the sediment of a saltern in Figeria da Foz, Portugal. The average nucleotide identity and DNA-DNA hybridization analyses show that Methanohalophilus mahii, M. halophilus, and M. portucalensis are three different species within the Methanosarcinaceae family.

Relative Abundance and Diversity of Bacterial Methanotrophs at the Oxic-Anoxic Interface of the Congo Deep-Sea Fan.

Sitting at ∼5,000 m water depth on the Congo-Angola margin and ∼760 km offshore of the West African coast, the recent lobe complex of the Congo deep-sea fan receives large amounts of fluvial sediments (3-5% organic carbon). This organic-rich sedimentation area harbors habitats with chemosynthetic communities similar to those of cold seeps. In this study, we investigated relative abundance, diversity and distribution of aerobic methane-oxidizing bacteria (MOB) communities at the oxic-anoxic interface of sedimentary habitats by using fluorescence in situ hybridization and comparative sequence analysis of particulate mono-oxygenase (pmoA) genes. Our findings revealed that sedimentary habitats of the recent lobe complex hosted type I and type II MOB cells and comparisons of pmoA community compositions showed variations among the different organic-rich habitats. Furthermore, the pmoA lineages were taxonomically more diverse compared to methane seep environments and were related to those found at cold seeps. Surprisingly, MOB phylogenetic lineages typical of terrestrial environments were observed at such water depth. In contrast, MOB cells or pmoA sequences were not detected at the previous lobe complex that is disconnected from the Congo River inputs.

Complete Genome Sequence of Methanohalophilus halophilus DSM 3094T, Isolated from a Cyanobacterial Mat and Bottom Deposits at Hamelin Pool, Shark Bay, Northwestern Australia.

The complete genome sequence of Methanohalophilus halophilus DSM 3094T, a member of the Methanosarcinaceae family and the Methanosarcianales order, consists of 2,022,959 bp in one contig and contains 2,137 predicted genes. The genome is consistent with a halophilic methylotrophic anaerobic lifestyle, including the methylotrophic and CO2-H2 methanogensis pathways.

Phylogenetic and functional diversity of microbial communities associated with subsurface sediments of the Sonora Margin, Guaymas Basin.

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.

Archaeal and anaerobic methane oxidizer communities in the Sonora Margin cold seeps, Guaymas Basin (Gulf of California).

Cold seeps, located along the Sonora Margin transform fault in the Guaymas Basin, were extensively explored during the 'BIG' cruise in June 2010. They present a seafloor mosaic pattern consisting of different faunal assemblages and microbial mats. To investigate this mostly unknown cold and hydrocarbon-rich environment, geochemical and microbiological surveys of the sediments underlying two microbial mats and a surrounding macrofaunal habitat were analyzed in detail. The geochemical measurements suggest biogenic methane production and local advective sulfate-rich fluxes in the sediments. The distributions of archaeal communities, particularly those involved in the methane cycle, were investigated at different depths (surface to 18 cm below the sea floor (cmbsf)) using complementary molecular approaches, such as Automated method of Ribosomal Intergenic Spacer Analysis (ARISA), 16S rRNA libraries, fluorescence in situ hybridization and quantitative polymerase chain reaction with new specific primer sets targeting methanogenic and anaerobic methanotrophic lineages. Molecular results indicate that metabolically active archaeal communities were dominated by known clades of anaerobic methane oxidizers (archaeal anaerobic methanotroph (ANME)-1, -2 and -3), including a novel 'ANME-2c Sonora' lineage. ANME-2c were found to be dominant, metabolically active and physically associated with syntrophic Bacteria in sulfate-rich shallow sediment layers. In contrast, ANME-1 were more prevalent in the deepest sediment samples and presented a versatile behavior in terms of syntrophic association, depending on the sulfate concentration. ANME-3 were concentrated in small aggregates without bacterial partners in a restricted sediment horizon below the first centimetres. These niche specificities and syntrophic behaviors, depending on biological surface assemblages and environmental availability of electron donors, acceptors and carbon substrates, suggest that ANME could support alternative metabolic pathways than syntrophic anaerobic oxidation of methane.

Methanogenic activity and diversity in the centre of the Amsterdam Mud Volcano, Eastern Mediterranean Sea.

Marine mud volcanoes are geological structures emitting large amounts of methane from their active centres. The Amsterdam mud volcano (AMV), located in the Anaximander Mountains south of Turkey, is characterized by intense active methane seepage produced in part by methanogens. To date, information about the diversity or the metabolic pathways used by the methanogens in active centres of marine mud volcanoes is limited. (14)C-radiotracer measurements showed that methylamines/methanol, H(2)/CO(2) and acetate were used for methanogenesis in the AMV. Methylotrophic methanogenesis was measured all along the sediment core, Methanosarcinales affiliated sequences were detected using archaeal 16S PCR-DGGE and mcrA gene libraries, and enrichments of methanogens showed the presence of Methanococcoides in the shallow sediment layers. Overall acetoclastic methanogenesis was higher than hydrogenotrophic methanogenesis, which is unusual for cold seep sediments. Interestingly, acetate porewater concentrations were extremely high in the AMV sediments. This might be the result of organic matter cracking in deeper hotter sediment layers. Methane was also produced from hexadecanes. For the most part, the methanogenic community diversity was in accordance with the depth distribution of the H(2)/CO(2) and acetate methanogenesis. These results demonstrate the importance of methanogenic communities in the centres of marine mud volcanoes.

Marinilactibacillus piezotolerans sp. nov., a novel marine lactic acid bacterium isolated from deep sub-seafloor sediment of the Nankai Trough.

A piezotolerant, mesophilic, marine lactic acid bacterium (strain LT20T) was isolated from a deep sub-seafloor sediment core collected at Nankai Trough, off the coast of Japan. Cells were Gram-positive, rod-shaped, non-sporulating and non-motile. The NaCl concentration range for growth was 0-120 g l(-1), with the optimum at 10-20 g l(-1). The temperature range for growth at pH 7.0 was 4-50 degrees C, with the optimum at 37-40 degrees C. The optimum pH for growth was 7.0-8.0. The optimum pressure for growth was 0.1 MPa with tolerance up to 30 MPa. The main cellular phospholipids were phosphatidylglycerols (25 %), diphosphatidylglycerols (34 %) and a group of compounds tentatively identified as ammonium-containing phosphatidylserines (32 %); phosphatidylethanolamines (9 %) were minor components. The fatty acid composition was dominated by side chains of 16 : 0, 14 : 0 and 16 : 1. The G+C content of the genomic DNA was 42 mol%. On the basis of 16S rRNA gene sequence analysis and the secondary structure of the V6 region, this organism was found to belong to the genus Marinilactibacillus and was closely related to Marinilactibacillus psychrotolerans M13-2(T) (99 %), Marinilactibacillus sp. strain MJYP.25.24 (99 %) and Alkalibacterium olivapovliticus strain ww2-SN4C (97 %). Despite the high similarity between their 16S rRNA gene sequences (99 %), the DNA-DNA hybridization levels were less than 20 %. On the basis of physiological and genetic characteristics, it is proposed that this organism be classified as a novel species, Marinilactibacillus piezotolerans sp. nov. The type strain is LT20T (=DSM 16108T=JCM 12337T).

Molecular monitoring of culturable bacteria from deep-sea sediment of the Nankai Trough, Leg 190 Ocean Drilling Program.

Culturable bacteria were detected in deep-sea sediment samples collected from the Nankai Trough site 1173 (Ocean Drilling Program, ODP, Leg 190) at 4.15 m below the seafloor with 4791 m of overlying water. In this deep ocean near surface sediment, mainly fermentative heterotrophs, autotrophic acetogens and sulfate-reducing bacteria were enriched by using two different non-selective enrichment culture media. Culturable bacterial population shifts within the deep marine sediment enrichments were monitored by using denaturating gradient gel electrophoresis (DGGE). DGGE analysis revealed a decrease in the number of 16S rRNA gene fragments from high to low carbon concentrations, and from low to high dilution of inoculum, suggesting that fast-growing bacteria were numerically dominant in enrichment culture samples. The dominant 16S rRNA fragments observed in DGGE gels were assigned to the Firmicutes, Proteobacteria (gamma and delta subgroups) and Spirochaeta phyla. Continual sub-culture and purification resulted in two isolates which were phylogenetically identified as members of the genera Acetobacterium and Marinilactibacillus. Our results, which combine enrichment culturing with DGGE analysis, indicated that enrichment cultures derived from inoculum dilution and media with various concentrations of carbon could facilitate the detection and isolation of a greater number of environmentally relevant bacterial species than when using traditional enrichment techniques alone.

Shewanella profunda sp. nov., isolated from deep marine sediment of the Nankai Trough.

A novel piezotolerant, mesophilic, facultatively anaerobic, organotrophic, polarly flagellated bacterium (strain LT13a(T)) was isolated from a deep sediment layer in the Nankai Trough (Leg 190, Ocean Drilling Program) off the coast of Japan. This organism used a wide range of organic substrates as sole carbon and energy sources: pyruvate, glutamate, succinate, fumarate, lactate, citrate, peptone and tryptone. Oxygen, nitrate, fumarate, ferric iron and cystine were used as electron acceptors. Maximal growth rates were observed at a hydrostatic pressure of 10 MPa. Hydrostatic pressure for growth was in the range 0.1-50 MPa. Predominant cellular fatty acids were 16 : 1omega7c, 15 : 0 iso, 16 : 0 and 13 : 0 iso. The G+C content of the DNA was 44.9 mol%. On the basis of 16S rRNA gene sequences, strain LT13a(T) was shown to belong to the gamma-Proteobacteria, being closely related to Shewanella putrefaciens (98 %), Shewanella oneidensis (97 %) and Shewanella baltica (96 %). Levels of DNA homology between strain LT13a(T) and S. putrefaciens, S. oneidensis and S. baltica were <20 %, indicating that strain LT13a(T) represents a novel species. Genetic evidence and phenotypic characteristics showed that isolate LT13a(T) constitutes a novel species of the genus Shewanella. Because of the deep origin of the strain, the name Shewanella profunda sp. nov. is proposed, with LT13a(T) (=DSM 15900(T)=JCM 12080(T)) as the type strain.