Mineral phase in shell repair of Manila clam Venerupis philippinarum affected by brown ring disease.

The mineral phase of shell repair in the Manila clam Venerupis philippinarum affected by brown ring disease (BRD) was characterised at various scales and at various stages of shell repair by confocal Raman microspectrometry and scanning electron microscopy. Spherulitic and quadrangular aragonite microstructures associated with polyene pigments were clearly observed. Von Kossa staining showed that at the beginning of shell repair, hemocytes are filled with insoluble calcium carbonate salts in all fluids and then are transported toward the extrapallial fluids and the repair sites. Our analyses suggest that after a Vibrio tapetis attack and BRD deposit some clams rapidly cover the deposit, resulting in a modification in the microstructure, which could be produced by the participation of both the mantle and hemocytes.

Investigations into the uptake of copper, iron and selenium by a highly sulphated bacterial exopolysaccharide isolated from microbial mats.

A bacterium isolated from microbial mats located on a polynesian atoll produced a high molecular weight (3,000 kDa) and highly sulphated exopolysaccharide. Previous studies showed that the chemical structure of this EPS consisted of neutral sugars, uronic acids, and high proportions of acetate and sulphate groups. The copper- and iron-binding ability of the purified pre-treated native EPS was investigated. Results showed that this EPS had a very high affinity for both copper (9.84 mmol g(-1) EPS) and ferrous iron (6.9 mmol g(-1) EPS). Amazingly, this EPS did not show any affinity for either ferric ions or selenium salts. This finding is one of the first steps in assessing the biotechnological potential of this polysaccharide.

Dissolved and particulate metals (Fe, Zn, Cu, Cd, Pb) in two habitats from an active hydrothermal field on the EPR at 13 degrees N.

The distribution of Fe, Cu, Zn, Pb, Cd between the dissolved (<2 microm) and the particulate (>2 microm) fractions was measured after in-situ filtration in two hydrothermal habitats. The total metal concentration ranges exhibit a clear enrichment compared with the seawater concentration, accounting for the hydrothermal input for all the metals considered. Iron is the predominant metal (5-50 microM) followed by Zn and Cu. Cd and Pb are present at the nM level. At the scale studied, the behavior of temperature, pH and dissolved iron is semi-conservative whereas the other dissolved and particulate metals are characterized by non-conservative patterns. The metal enrichment of the >2 microm fraction results from the settlement and accumulation of particulate matter close to the organisms, acting as a secondary metal source. The enrichment observed in the dissolved fraction can be related to the dissolution or oxidation of particles (mainly polymetallic sulfide) or to the presence of small particles and large colloids not retained on the 2 microm frit. SEM observations indicate that the bulk particulate observed is characteristic of crystalline particles settling rapidly from the high temperature smoker (sphalerite, wurtzite and pyrite), amorphous structures and eroded particles formed in the external zone of the chimney. Precipitation of Zn, Cu, Cd and Pb with Fe as wurtzite, sphalerite and pyrite is the main process taking place within the area studied and is semi-quantitative. The distribution of the dominant observed fauna has been related to the gradient resulting from the dilution process, with the alvinellids worms colonizing the hotter and more variable part of the mixing zone, but also to the metallic load of the mixing zone. Dissolved and particulate metal concentrations are therefore necessary abiotic factors to be studied in a multiparametric approach to understand the faunal distribution in hydrothermal ecosystems.

A novel mcl-PHA produced on coprah oil by Pseudomonas guezennei biovar. tikehau, isolated from a "kopara" mat of French Polynesia.

Pseudomonas guezennei biovar. tikehau was isolated from a microbial mat on the atoll of Tikehau in French Polynesia, and is able to synthesize medium chain length poly-beta-hydroxyalkanaote copolymers when grown on coprah oil. A two-step cultivation process was used and the biosynthesis of PHAs was followed along 52h by regular culture sampling. The polyester was purified from freeze-dried cells and analysed by nuclear magnetic resonance (NMR), Fourier transform infra red (FTIR), and gas chromatography mass spectrometries. The copolyester produced by P. guezennei biovar. tikehau from coprah oil mainly consisted of saturated monomers, i.e. 3-hydroxyoctanoate (3HO) and 3-hydroxydecanoate (3HD), and the monomeric composition of the polyester did not change during the fermentation process. However, yield of PHAs production varied from 4% of the cellular dry weight (CDW) to 63% obtained after 36h. Scan electron microscopy was used to study the morphology and organization of PHAs granules within the cells and revealed the presence of several granules occupying almost the entire cell volume.

Biogeographical distribution of Rimicaris exoculata resident gut epibiont communities along the Mid-Atlantic Ridge hydrothermal vent sites.

Rimicaris exoculata is a deep-sea hydrothermal vent shrimp whose enlarged gill chamber houses a complex trophic epibiotic community. Its gut harbours an autochthonous and distinct microbial community. This species dominates hydrothermal ecosystem megafauna along the Mid-Atlantic Ridge, regardless of contrasting geochemical conditions prevailing in them. Here, the resident gut epibiont community at four contrasted hydrothermal vent sites (Rainbow, TAG, Logatchev and Ashadze) was analysed and compiled with previous data to evaluate the possible influence of site location, using 16S rRNA surveys and microscopic observations (transmission electron microscopy, scanning electron microscopy and fluorescence in situ hybridization analyses). Filamentous epibionts inserted between the epithelial cell microvilli were observed on all examined samples. Results confirmed resident gut community affiliation to Deferribacteres, Mollicutes, Epsilonproteobacteria and to a lesser extent Gammaproteobacteria lineages. Still a single Deferribacteres phylotype was retrieved at all sites. Four Mollicutes-related operational taxonomic units were distinguished, one being only identified on Rainbow specimens. The topology of ribotype median-joining networks illustrated a community diversification possibly following demographic expansions, suggesting a more ancient evolutionary history and/or a larger effective population size at Rainbow. Finally, the gill chamber community distribution was also analysed through ribotype networks based on sequences from R. exoculata collected at the Rainbow, Snake Pit, TAG, Logatchev and Ashadze sites. Results allow the refining of hypotheses on the epibiont role and transmission pathways.

Acquisition of epibiotic bacteria along the life cycle of the hydrothermal shrimp Rimicaris exoculata.

The caridean shrimp Rimicaris exoculata dominates the fauna at several Mid-Atlantic Ridge hydrothermal vent sites. This shrimp has an enlarged gill chamber, harboring a dense ectosymbiotic community of chemoautotrophic bacteria associated with mineral oxide deposits. Until now, their acquisition is not fully understood. At three hydrothermal vent sites, we analyzed the epibionts diversity at different moult stages and also in the first stages of the shrimp life (eggs, hatched eggs (with larvae) and juveniles). Hatched eggs associated with young larvae were collected for the first time directly from gravid females at the Logachev vent site during the Serpentine cruise. An approach using 16S rRNA clone libraries, scanning and transmission electron microscopy, and fluorescent in situ hybridization was used. Molecular results and microscope observations indicated a switch in the composition of the bacterial community between early R. exoculata life cycle stage (egg libraries dominated by the Gammaproteobacteria) and later stages (juvenile/adult libraries dominated by the Epsilonproteobacteria). We hypothesized that the epibiotic phylotype composition could vary according to the life stage of the shrimp. Our results confirmed the occurrence of a symbiosis with Gammaproteobacteria and Epsilonproteobacteria, but more complex than previously assumed. We revealed the presence of active type-I methanotrophic bacteria colonizing the cephalothorax of shrimps from the Rainbow site. They were also present on the eggs from the Logachev site. This could be the first 'epibiotic' association between methanotrophic bacteria and hydrothermal vent crustacean. We discuss possible transmission pathways for epibionts linked to the shrimp life cycle.

Detection of polycyclic aromatic hydrocarbon (PAH) compounds in artificial sea-water using surface-enhanced Raman scattering (SERS).

This paper reports an accurate synthesis of surface-enhanced Raman scattering (SERS) active substrates, based on gold colloidal monolayer, suitable for in situ environmental analysis. Quartz substrates were functionalized by silanization with (3-mercaptopropyl)trimethoxysilane (MPMS) or (3-aminopropyl)trimethoxysilane (APTMS) and they subsequently reacted with colloidal suspension of gold metal nanoparticles: respectively, the functional groups SH and NH(2) bound gold nanoparticles. Gold nanoparticles were prepared by the chemical reduction of HAuCl(4) using sodium tricitrate and immobilized onto silanized quartz substrates. Active substrate surface morphology was characterized with scanning electron microscopy (SEM) measurements and gold nanoparticles presented a diameter in the range 40-100 nm. Colloidal hydrophobic films, allowing nonpolar molecule pre-concentration, were obtained. The surfaces exhibit strong enhancement of Raman scattering from molecules adsorbed on the films. Spectra were recorded for two PAHs, naphthalene and pyrene, in artificial sea-water (ASW) with limits of detection (LODs) of 10 ppb for both on MPMS silanized substrates.

Nautilia abyssi sp. nov., a thermophilic, chemolithoautotrophic, sulfur-reducing bacterium isolated from an East Pacific Rise hydrothermal vent.

A novel strictly anaerobic, thermophilic, sulfur-reducing bacterium, designated PH1209(T), was isolated from an East Pacific Rise hydrothermal vent (1 degrees N) sample and studied using a polyphasic taxonomic approach. Cells were Gram-negative, motile rods (approx. 1.60 x 0.40 microm) with a single polar flagellum. Strain PH1209(T) grew at temperatures between 33 and 65 degrees C (optimum 60 degrees C), from pH 5.0 to 8.0 (optimum 6.0-6.5), and between 2 and 4 % (w/v) NaCl (optimum 3 %). Cells grew chemolithoautotrophically with H(2) as an energy source, S(0) as an electron acceptor and CO(2) as a carbon source. Strain PH1209(T) was also able to use peptone and yeast extract as carbon sources. The G+C content of the genomic DNA was 35 mol%. Phylogenetic analyses based on 16S rRNA gene sequencing showed that strain PH1209(T) fell within the order Nautiliales, in the class Epsilonproteobacteria. Comparative 16S rRNA gene sequence analysis indicated that strain PH1209(T) belonged to the genus Nautilia and shared 97.2 and 98.7 % 16S rRNA gene sequence identity, respectively, with the type strains of Nautilia lithotrophica and Nautilia profundicola. It is proposed, from the polyphasic evidence, that the strain represents a novel species, Nautilia abyssi sp. nov.; the type strain is PH1209(T) (=DSM 21157(T)=JCM 15390(T)).

Recovery in culture of viable but nonculturable Vibrio parahaemolyticus: regrowth or resuscitation?

The objective of this study was to explore the recovery of culturability of viable but nonculturable (VBNC) Vibrio parahaemolyticus after temperature upshift and to determine whether regrowth or resuscitation occurred. A clinical strain of V. parahaemolyticus Vp5 was rendered VBNC by exposure to artificial seawater (ASW) at 4 degrees C. Aliquots of the ASW suspension of cells (0.1, 1 and 10 ml) were subjected to increased temperatures of 20 degrees C and 37 degrees C. Culturability of the cells in the aliquots was monitored for colony formation on a rich medium and changes in morphology were measured by scanning (SEM) and transmission (TEM) electron microscopy. Samples of VBNC cells were fixed and examined by SEM, revealing a heterogeneous population comprising small cells and larger, flattened cells. Forty-eight hours after temperature upshift to 20 degrees C or 37 degrees C, both elongation and division by binary fission of the cells were observed, employing SEM and TEM, but only in the 10-ml aliquots. The results suggest that a portion of VBNC cells is able to undergo cell division. It is concluded that a portion of VBNC cells of V. parahaemolyticus subjected to cold temperatures remain viable. After temperature upshift, regrowth of those cells, rather than resuscitation of all bacteria of the initial inoculum, appears to be responsible for recovery of culturability of VBNC cells of V. parahaemolyticus. Nutrient in filtrates of VBNC cells is hypothesized to allow growth of the temperature-responsive cells, with cell division occurring via binary fission, but also including an atypical, asymmetric cell division.

Caminibacter hydrogeniphilus gen. nov., sp. nov., a novel thermophilic, hydrogen-oxidizing bacterium isolated from an East Pacific Rise hydrothermal vent.

A novel thermophilic, anaerobic, hydrogen-oxidizing bacterium, designated strain AM1116T, was isolated from an East Pacific Rise hydrothermal vent sample. The cells were rod-shaped (1.01-5 x 0.5 microm), motile with polar flagella. They grew at temperatures between 50 and 70 degrees C (optimum 60 degrees C; doubling time approximately 1.5 h), at between pH 5.0 and 7.5 (optimum around pH 5.5-6.0) and in between 10 and 40 g NaCl l(-1) (optimum 20-25 g l(-1)). Cells grew chemolithoautotrophically in a H2/CO2 atmosphere (80:20; 200 kPa). Poor heterotrophic growth was observed on complex organic substrates. Elemental sulphur and nitrate served as electron acceptors, respectively yielding hydrogen sulphide and ammonia (doubling times were equal with the two electron acceptors). In contrast, when cystine was used as electron acceptor, growth was poor. The G+C content of the genomic DNA was 29 +/- 1 mol %. Phylogenetic analyses of the 16S rRNA gene located the strain within the epsilon-Proteobacteria, in the bacterial domain. On the basis of 16S rDNA sequence comparisons, physiological and biochemical characteristics, it is proposed that the isolate should be described as the type species of a new genus, Caminibacter gen. nov., as Caminibacter hydrogeniphilus sp. nov. The type strain is strain AM1116T (= DSM 14510T = CIP 107140T).

Caminicella sporogenes gen. nov., sp. nov., a novel thermophilic spore-forming bacterium isolated from an East-Pacific Rise hydrothermal vent.

A novel thermophilic, anaerobic, strictly chemoorganoheterotrophic bacterium, designated as AM1114T, was isolated from a deep-sea hydrothermal vent sample from the East-Pacific Rise (EPR 13 degrees N). The cells were long (3-10 microm) rods, motile with peritrichous flagella, and exhibited a gram-negative cell wall ultrastructure. In the late stationary phase of growth, cells formed an ovoid, refractile, terminal endospore. They grew at 45-65 degrees C inclusive (optimum 55-60 degrees C; doubling time approx. 45 min), at pH 4.5-8.0 inclusive (optimum pH 7.5-8.0) and at sea salt concentrations of 20-60 g l(-1) inclusive (optimum 25-30 g l(-1)). Strain AM1114T was an obligately heterotrophic bacterium able to ferment a mixture of 20 amino acids, complex proteinaceous substrates (such as yeast extract, brain-heart infusion or peptone), and carbohydrates such as glucose, galactose or maltose. The main fermentation products on glucose/yeast extract/peptone/sulfur medium were hydrogen, carbon dioxide, butyrate, ethanol, acetate, formate and L-alanine. The G+C content of the genomic DNA (determined by thermal denaturation) was 24.2+/-1 mol%. Phylogenetic analyses of the 16S rRNA gene located the strain within cluster XI of the lineage encompassing the genus Clostridium and related genera (sensu Collins et al., 1994), in the bacterial domain. On the basis of 16S rDNA sequence comparisons and physiological and biochemical characteristics, it is proposed that the isolate should be described as a novel genus, namely Caminicella gen. nov., of which Caminicella sporogenes sp. nov. is the type species. The type strain is AM1114T (= DSM 14501T = CIP 107141T).

Desulfurobacterium crinifex sp. nov., a novel thermophilic, pinkish-streamer forming, chemolithoautotrophic bacterium isolated from a Juan de Fuca Ridge hydrothermal vent and amendment of the genus Desulfurobacterium.

A novel thermophilic, chemolithoautotrophic bacterium, designated as NE1206(T), was isolated from a Juan de Fuca Ridge hydrothermal vent sample (tubes of the annelid polychaete Paralvinella sulfincola attached to small pieces of hydrothermal chimney). The cells were rod-shaped (1.2-3.5 x 0.4-0.7 microm), occurring as single motile rods or forming macroscopic aggregates visible as pinkish to brownish streamers. The new isolate was anaerobic. It grew between 50 and 70 degrees C (optimum 60-65 degrees C; doubling time approximately 1 h 15 min at 60 degrees C), between pH 5.0 and 7.5 (optimum pH around 6.0-6.5) and at sea salts concentrations between 20 and 40 g l(-1 )(optimum 30 g l(-1)). Cells grew chemolithoautotrophically in an H(2)/CO(2) atmosphere (80/20, v/v; 200 kPa). Molecular hydrogen was the sole electron donor used by the strain. Nitrate and elemental sulfur served as electron acceptors, yielding ammonia and hydrogen sulfide, respectively (nitrate reduction supported higher growth rates than sulfur reduction). The G+C content of the genomic DNA was 36.7+/-0.8 mol%. Phylogenetic analyses of the 16S rRNA gene located the strain within the genus Desulfurobacterium. However, the novel isolate possesses physiological and biochemical characteristics that differ from the previously described species of this genus. We propose that the isolate represents a novel species, Desulfurobacterium crinifex sp. nov. The type strain is NE1206(T) (DSM 15218(T), CIP 107649(T)). An amendment of the genus Desulfurobacterium description is proposed, based on the phenotypic characteristics of the novel species.