Desulfocurvus vexinensis gen. nov., sp. nov., a sulfate-reducing bacterium isolated from a deep subsurface aquifer.

A novel anaerobic, chemo-organotrophic bacterium, designated VNs36(T), was isolated from a well that collected water from a deep saline aquifer used for underground gas storage at a depth of 830 m in the Paris Basin, France. Cells were curved motile rods or vibrios (3.0-5.0x0.5 microm). Strain VNs36(T) grew at temperatures between 20 and 50 degrees C (optimum 37 degrees C) and at pH values between 5.0 and 9.0 (optimum 6.9). It did not require salt for growth, but tolerated up to 20 g NaCl l(-1) (optimum 2 g l(-1)). In the presence of sulfate, strain VNs36(T) used lactate, formate and pyruvate as carbon and energy sources. The main fermentation products from lactate were acetate, H(2) and CO(2). Sulfate, thiosulfate and sulfite were used as electron acceptors, but not sulfur. The genomic DNA G+C content of strain VNs36(T) was 67.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain VNs36(T) was affiliated with the family Desulfovibrionaceae within the class Deltaproteobacteria. On the basis of 16S rRNA gene sequence comparisons, DNA G+C content and the absence of desulfoviridin in cell extracts, it is proposed that strain VNs36(T) be assigned to a new genus, Desulfocurvus gen. nov., as a representative of a novel species, Desulfocurvus vexinensis sp. nov. The type species of this genus is Desulfocurvus vexinensis with the type strain VNs36(T) (=DSM 17965(T)=JCM 14038(T)).

Characterization by culture and molecular analysis of the microbial diversity of a deep subsurface gas storage aquifer.

The bacterial diversity of a subsurface water sample collected from a gas storage aquifer in an Upper Jurassic calcareous formation was investigated by culture of microorganisms and construction of a 16S rRNA gene library. Both culture and molecular techniques showed that members of the phyla Firmicutes and class delta-proteobacteria dominated the bacterial community. The presence of hydrogen-utilizing autotrophic bacteria including sulfate reducers (e.g. Desulfovibrio aespoeensis) and homoacetogens (e.g. Acetobacterium carbinolicum) suggested that CO(2) and H(2) are the main carbon and energy sources sustaining a nutrient-limited subsurface lithoautotrophic microbial ecosystem (SLiME). Gram-positive SRB belonging to the genus Desulfotomaculum, frequently observed in subsurface environments, represented 25% of the clone library and 4 distinct phylotypes. No Archaea were detected by both experimental approaches. Water samples were collected in an area of the rauracian geological formation located outside the maximum seasonal extension of underground gas storage. Considering the observed microbial diversity, there is no evidence of any influence on the microbial ecology of the aquifer in the surroundings of maximum extension reached by the gas bubble of the underground storage, which should have resulted from the introduction of exogenous carbon and energy sources in a nutrient-limited ecosystem.

The effect of cleaning and disinfecting the sampling well on the microbial communities of deep subsurface water samples.

Our knowledge of the microbial characteristics of deep subsurface waters is currently very limited, mainly because of the methods used to collect representative microbial samples from such environments. In order to improve this procedure, a protocol designed to remove the unspecific, contaminant biofilm present on the walls of an approximately 800 m deep well is proposed. This procedure included extensive purges of the well, a mechanical cleaning of its wall, and three successive chlorine injections to disinfect the whole line before sampling. Total bacterial counts in water samples decreased from 2.5 x 10(5) to 1.0 x 10(4) per millilitre during the cleaning procedure. Culture experiments showed that the first samples were dominated by sulfate-reducers and heterotrophs, whereas the final sample was dominated by oligotrophic and hydrogenotrophic bacteria. Community structures established on the diversity of the 16S rRNA genes and data analysis revealed that the water sample collected, after a purge without removal of the biofilm, was characterized by numerous phyla which are not representative of the deep subsurface water. On the other hand, several bacterial phyla were only detected after the full cleaning of the well, and were considered as important components of the subsurface ecosystem which would have been missed in the absence of well cleaning.

Desulfovibrio putealis sp. nov., a novel sulfate-reducing bacterium isolated from a deep subsurface aquifer.

A novel sulfate-reducing bacterium was isolated from a well that collected water from a deep aquifer at a depth of 430 m in the Paris Basin, France. The strain, designated B7-43T, was made up of vibrioid cells that were motile by means of a single polar flagellum. Cells contained desulfoviridin. In the presence of sulfate, the following substrates were used as energy and carbon sources: lactate, pyruvate, malate, fumarate, ethanol, butanol, acetate/H2 and glycine. Sulfite and thiosulfate were also used as electron acceptors in the presence of lactate. In the absence of electron acceptors, pyruvate, malate and fumarate were fermented. Optimal growth was obtained in 1 g NaCl l(-1) and at pH 7. On the basis of 16S rRNA gene sequence analysis, the isolate was most closely related to members of the genus Desulfovibrio (90 % similarity). It is thus proposed that strain B7-43T (=DSM 16056T=ATCC BAA-905T) represents a novel species within this genus, Desulfovibrio putealis sp. nov.

Desulfovibrio bastinii sp. nov. and Desulfovibrio gracilis sp. nov., moderately halophilic, sulfate-reducing bacteria isolated from deep subsurface oilfield water.

Two moderately halophilic, mesophilic, sulfate-reducing bacteria were isolated from production-water samples from Emeraude Oilfield, Congo. Motile, vibrioid cells of SRL4225T grew optimally at a concentration of 4 % NaCl, at pH 5.8-6.2, with a minimal pH for growth of 5.2, showing that it is a moderately acidophilic bacterium. Cells of SRL6146T were motile, curved or vibrioid, long and thin rods. Optimal growth was obtained at a concentration of 5-6 % NaCl, at pH 6.8-7.2. The nutritional requirements showed that many of the characteristics of these strains overlap with those of known Desulfovibrio species. On the basis of 16S rRNA gene sequence analysis and DNA-DNA hybridization studies, both strains are members of the genus Desulfovibrio. However, they are not closely related to any species of the genus that have validly published names. It is therefore proposed that the two strains are members of two novel species of the genus Desulfovibrio with the names Desulfovibrio bastinii sp. nov. (type strain SRL4225T = DSM 16055T = ATCC BAA-903T) and Desulfovibrio gracilis sp. nov. (type strain SRL6146T = DSM 16080T = ATCC BAA-904T).