Pleomorphochaeta naphthae sp. nov., a new anaerobic fermentative bacterium isolated from an oil field.

A novel anaerobic fermentative bacterium, strain SEBR 4209T, was isolated from a water sample of a Congolese oil field. Strain SEBR 4209T is phylogenetically related to the genus Pleomorphochaeta, in the family Spirochaetaceae. Its closest relatives are Pleomorphochaeta caudata SEBR 4223T (94.5 % 16S rRNA gene sequence similarity) and Pleomorphochaeta multiformis MO-SPC2T (94.3 % similarity). Like the other members of this genus, cells have a pleomorphic morphology, in particular an annular shape and long stalks. Optimal growth was observed at 37 °C, at pH between 6.8 and 7.0, and with 40 g l-1 NaCl. This strain was only able to grow by fermentation of carbohydrates. The fermentation products from glucose utilization were acetate, ethanol, CO2 and H2. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c. The major polar lipids were phosphoglycolipids, phospholipids and glycolipids. The G+C content of the DNA was 29.6 mol%. Based on phenotypic characteristics and phylogenetic traits, strain SEBR 4209T is considered to represent a novel species of the genus Pleomorphochaeta, for which the name Pleomorphochaetanaphthae sp. nov. is proposed. The type strain is SEBR 4209T (=DSM 104684T=JCM 31871T).

Pleomorphochaeta caudata gen. nov., sp. nov., an anaerobic bacterium isolated from an offshore oil well, reclassification of Sphaerochaeta multiformis MO-SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov. as the type strain of this novel genus and emended description of the genus Sphaerochaeta.

A strictly anaerobic Gram-stain-negative bacterium, designated strain SEBR 4223T, was isolated from the production water of an offshore Congolese oil field. Cells were non-motile, pleomorphic and had spherical, annular or budding shapes, often exhibiting long stalks. Strain SEBR 4223T grew on a range of carbohydrates, optimally at 37 °C and pH 7, in a medium containing 40 g l-1 NaCl. Predominant fatty acids were C14 : 0, C14 : 0 DMA, C16 : 0 and C16 : 1ω7c and the major polar lipids were phosphoglycolipids, phospholipids, glycolipids and diphosphatidylglycerol. The G+C content of the DNA was 28.7 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that strain SEBR 4223T and Sphaerochaeta multiformis MO-SPC2T formed a cluster with similarity to other species of the genus Sphaerochaeta of of less than 86 %. On the basis of the phenotypic characteristics and taxonomic analyses, we propose a novel genus, Pleomorphochaeta gen. nov., to accommodate the novel species Pleomorphochaeta caudata sp. nov., with SEBR 4223T (=DSM 103077T=JCM 31 475T) as the type strain. We also propose the reclassification of Sphaerochaeta multiformis MO SPC2T as Pleomorphochaeta multiformis MO-SPC2T comb. nov., the type strain of this novel genus and emend description of the genus Sphaerochaeta.

Desulfotomaculum aquiferis sp. nov. and Desulfotomaculum profundi sp. nov., isolated from a deep natural gas storage aquifer.

Two novel strictly anaerobic bacteria, strains Bs105T and Bs107T, were isolated from a deep aquifer-derived hydrocarbonoclastic community. The cells were rod-shaped, not motile and had terminal spores. Phylogenetic affiliation and physiological properties revealed that these isolates belong to two novel species of the genus Desulfotomaculum. Optimal growth temperatures for strains Bs105T and Bs107T were 42 and 45 °C, respectively. The estimated G+C content of the genomic DNA was 42.9 and 48.7 mol%. For both strains, the major cellular fatty acid was palmitate (C16 : 0). Specific carbon fatty acid signatures of Gram-positive bacteria (iso-C17 : 0) and sulfate-reducing bacteria (C17 : 0cyc) were also detected. An insertion was revealed in one of the two 16S rRNA gene copies harboured by strain Bs107T. Similar insertions have previously been highlighted among moderately thermophilic species of the genus Desulfotomaculum. Both strains shared the ability to oxidize aromatic acids (Bs105T: hydroquinone, acetophenone, para-toluic acid, 2-phenylethanol, trans-cinnamic acid, 4-hydroxybenzaldehyde, benzyl alcohol, benzoic acid 4-hydroxybutyl ester; Bs107T: ortho-toluic acid, benzoic acid 4-hydroxybutyl ester). The names Desulfotomaculum aquiferis sp. nov. and Desulfotomaculum profundi sp. nov. are proposed for the type strains Bs105T (=DSM 24088T=JCM 31386T) and Bs107T (=DSM 24093T=JCM 31387T).

New Bio-Indicators for Long Term Natural Attenuation of Monoaromatic Compounds in Deep Terrestrial Aquifers.

Deep subsurface aquifers despite difficult access, represent important water resources and, at the same time, are key locations for subsurface engineering activities for the oil and gas industries, geothermal energy, and CO2 or energy storage. Formation water originating from a 760 m-deep geological gas storage aquifer was sampled and microcosms were set up to test the biodegradation potential of BTEX by indigenous microorganisms. The microbial community diversity was studied using molecular approaches based on 16S rRNA genes. After a long incubation period, with several subcultures, a sulfate-reducing consortium composed of only two Desulfotomaculum populations was observed able to degrade benzene, toluene, and ethylbenzene, extending the number of hydrocarbonoclastic-related species among the Desulfotomaculum genus. Furthermore, we were able to couple specific carbon and hydrogen isotopic fractionation during benzene removal and the results obtained by dual compound specific isotope analysis (𝜀C = -2.4‰ ± 0.3‰; 𝜀H = -57‰ ± 0.98‰; AKIEC: 1.0146 ± 0.0009, and AKIEH: 1.5184 ± 0.0283) were close to those obtained previously in sulfate-reducing conditions: this finding could confirm the existence of a common enzymatic reaction involving sulfate-reducers to activate benzene anaerobically. Although we cannot assign the role of each population of Desulfotomaculum in the mono-aromatic hydrocarbon degradation, this study suggests an important role of the genus Desulfotomaculum as potential biodegrader among indigenous populations in subsurface habitats. This community represents the simplest model of benzene-degrading anaerobes originating from the deepest subterranean settings ever described. As Desulfotomaculum species are often encountered in subsurface environments, this study provides some interesting results for assessing the natural response of these specific hydrologic systems in response to BTEX contamination during remediation projects.

Desulfotomaculum spp. and related gram-positive sulfate-reducing bacteria in deep subsurface environments.

Gram-positive spore-forming sulfate reducers and particularly members of the genus Desulfotomaculum are commonly found in the subsurface biosphere by culture based and molecular approaches. Due to their metabolic versatility and their ability to persist as endospores. Desulfotomaculum spp. are well-adapted for colonizing environments through a slow sedimentation process. Because of their ability to grow autotrophically (H2/CO2) and produce sulfide or acetate, these microorganisms may play key roles in deep lithoautotrophic microbial communities. Available data about Desulfotomaculum spp. and related species from studies carried out from deep freshwater lakes, marine sediments, oligotrophic and organic rich deep geological settings are discussed in this review.

Mesotoga infera sp. nov., a mesophilic member of the order Thermotogales, isolated from an underground gas storage aquifer.

Strain VNs100(T), a novel mesophilic, anaerobic, rod-coccoid-shaped bacterium, having a sheath-like outer structure (toga), was isolated from a water sample collected in the area of an underground gas storage aquifer. It was non-motile with cells appearing singly (2-4 µm long × 1-2 µm wide), in pairs or as long chains and stained Gram-negative. Strain VNs100(T) was heterotrophic, able to use arabinose, cellobiose, fructose, galactose, glucose, lactose, lactate, mannose, maltose, raffinose, ribose, sucrose and xylose as energy sources only in the presence of elemental sulfur as terminal electron acceptor. Acetate, CO2 and sulfide were the end products of sugar metabolism. Hydrogen was not detected. Elemental sulfur, but not thiosulfate, sulfate or sulfite, were reduced to sulfide. Strain VNs100(T) grew at temperatures between 30 and 50 °C (optimum 45 °C), at pH values between 6.2 and 7.9 (optimum 7.3-7.5) and at NaCl concentrations between 0 and 15 g l(-1) (optimum 2 g l(-1)). The DNA G+C content was 47.5 mol%. The main cellular fatty acid was C16 : 0. Phylogenetic analysis of the small subunit rRNA gene sequence indicated that strain VNs100(T) had as its closest relatives 'Mesotoga sulfurireducens' (97.1 % similarity) and Mesotoga prima (similarity of 97.1 % and 97.7 % with each of its two genes, respectively) within the order Thermotogales. Hybridization between strain VNS100(T) and 'M. sulfurireducens' and between strain VNS100(T) and M. prima showed 12.9 % and 20.6 % relatedness, respectively. Based on phenotypic, phylogenetic and taxonomic characteristics, strain VNs100(T) is proposed as a representative of a novel species of the genus Mesotoga in the family Thermotogaceae, order Thermotogales. The name Mesotoga infera sp. nov. is proposed. The type strain is VNs100(T) (= DSM 25546(T) = JCM 18154(T)).

Pedobacter tournemirensis sp. nov., isolated from a fault water sample of a deep Toarcian argillite layer.

A Gram-negative bacterium, designated TF5-37.2-LB10(T), was isolated from subsurface water of the Toarcian geological layer of Tournemire, France. Cells were non-motile straight rods that formed cream to light pink colonies on 10-fold diluted LB agar. Strain TF5-37.2-LB10(T) contained menaquinone 7 and its major fatty acids were iso-C(15 : 0), summed feature 3 (iso-C(15 : 0) 2-OH and/or C(16 : 1)ω7c), iso-C(17 : 0) 3-OH and iso-C(17 : 1)ω9c. The G+C content of the genomic DNA was 46 mol%. Phylogenetic analysis of the 16S rRNA gene sequence placed strain TF5-37.2-LB10(T) within the genus Pedobacter, family Sphingobacteriaceae. Pedobacter composti TR6-06(T) and Pedobacter oryzae DSM 19973(T) were the closest phylogenetic relatives (93.5 and 93.3 % 16S rRNA gene sequence similarity, respectively). On the basis of 16S rRNA gene sequence comparison and physiological and biochemical characteristics, strain TF5-37.2-LB10(T) represents a novel species of the genus Pedobacter, for which the name Pedobacter tournemirensis sp. nov. is proposed. The type strain is TF5-37.2-LB10(T) (= DSM 23085(T) = CIP 110085(T) = MOLA 820(T)).

Anaerobic biodegradation of BTEX by original bacterial communities from an underground gas storage aquifer.

BTEX biodegradation by an indigenous deep subsurface microbial community was evaluated in a water sample collected in the area of an underground gas storage. Five different sulfate-reducing microbial communities able to use at least either benzene, toluene, ethylbenzene, or xylene (BTEX) compounds were studied. A total of 21 different bacterial phylotypes were identified, each community containing three to nine bacterial phylotypes. Archaeal phylotypes were retrieved from only three communities. The analysis of 16S rRNA gene sequences showed that i) these consortia were mainly composed of novel species, some of which belonging to bacterial groups not previously suspected to be involved in BTEX anaerobic degradation, ii) three consortia were dominated by an uncultured Pelobacter sp. previously detected in biodegraded oil reservoirs, iii) a deeply branching species distantly affiliated to Thermotogales was abundant in two consortia, and that iv) Firmicutes related to the Desulfotomaculum and Carboxydocella genera represented the only three detectable phylotypes in a toluene-degrading consortium. This work shows that subdominant microbial populations present in a deep subsurface aquifer used for seasonal underground gas storage could be involved in the natural attenuation of the traces of BTEX coinjected with methane in the deep subsurface.

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.

Geosporobacter subterraneus gen. nov., sp. nov., a spore-forming bacterium isolated from a deep subsurface aquifer.

A novel, strictly anaerobic, chemo-organotrophic bacterium, designated strain VNs68(T), was isolated from a well that collected water from a deep aquifer at a depth of 800 m in the Paris Basin, France. Cells were thin, non-motile, Gram-positive rods forming terminal endospores (3.0-5.0 x 0.5 microm). Strain VNs68(T) grew at temperatures between 30 and 55 degrees C (optimum 42 degrees C) and at pH 5.6-8.4 (optimum pH 7.3). It did not require salt for growth but tolerated up to 40 g NaCl l(-1). Strain VNs68(T) was an obligate heterotroph fermenting carbohydrates such as glucose, xylose, fructose, ribose and cellobiose. Casamino acids and amino acids (arginine, serine, lysine, alanine, aspartate, asparagine, isoleucine, histidine) were also fermented. The main fermentation products from glucose were acetate with H(2) and CO(2). Sulfate, sulfite, thiosulfate, elemental sulfur, nitrate and nitrite were not used as electron acceptors. The G+C content of the genomic DNA was 42.2 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain VNs68(T) was affiliated to cluster XI, order Clostridiales, domain Bacteria. On the basis of 16S rRNA gene sequence comparisons and physiological characteristics, strain VNs68(T) is considered to represent a novel species of a new genus, for which the name Geosporobacter subterraneus gen. nov., sp. nov. is proposed. The type strain of Geosporobacter subterraneus is VNs68(T) (=DSM 17957(T) =JCM 14037(T)).

Petrotoga halophila sp. nov., a thermophilic, moderately halophilic, fermentative bacterium isolated from an offshore oil well in Congo.

A novel thermophilic, moderately halophilic, rod-shaped bacterium, strain MET-B(T), with a sheath-like outer structure (toga) was isolated from an offshore oil-producing well in Congo, West Africa. Strain MET-B(T) was a Gram-negative bacterium with the ability to reduce elemental sulfur, but not sulfate, thiosulfate or sulfite into sulfide. The optimum growth conditions were 60 degrees C, pH 6.7-7.2 and 4-6 % NaCl. The DNA G+C content was 34.6 mol%. Strain MET-B(T) was phylogenetically related to members of the genus Petrotoga; Petrotoga miotherma, Petrotoga olearia and Petrotoga mexicana were the closest relatives, with type strains exhibiting more than 99 % identity in an analysis of small-subunit rRNA gene sequences. The values for DNA-DNA relatedness between the type strains of these three species and strain MET-B(T) were less than 42 %. As MET-B(T) was found to be genetically and physiologically different from other species of the genus Petrotoga, this strain is proposed as representing a novel species, for which the name Petrotoga halophila sp. nov. is proposed. The type strain is MET-B(T) (=DSM 16923(T)=CCUG 50214(T)).

rdlA, a new gene encoding a rhodanese-like protein in Halanaerobium congolense and other thiosulfate-reducing anaerobes.

The recently described anaerobic moderately halophilic bacterium Halanaerobium congolense has been shown to reduce thiosulfate and sulfur-but not sulfate-into sulfide. When cultivated in the presence of thiosulfate as terminal electron acceptor, H. congolense possesses a highly active thiosulfate:cyanide sulfur-transferase activity (rhodanese-like enzyme). A gene library of H. congolense (DSM 11287T) was constructed, and a 3.1-kb Sau3A DNA that encompassed a thiosulfate:cyanide sulfur-transferase-encoding gene was isolated in Escherichia coli. This fragment contains 2 orfs, which were separately subcloned in E. coli. The 900-bp gene encoding the rhodanese-like protein was named rdlA. RdlA differs from other known rhodanese-like proteins by having two potential catalytic sites, one N-terminal and one C-terminal, both harboring a cysteine. The two putative active sites are preceded by a highly-conserved region of unknown function. Closely related genes were also characterized in other thiosulfate-reducing non-sulfate-reducing anaerobes belonging to phylogenetically distant microorganisms, thus suggesting that RdlA is of importance in the mechanism of thiosulfate reduction by numerous members of the domain Bacteria.

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).