The sequence capture by hybridization: a new approach for revealing the potential of mono-aromatic hydrocarbons bioattenuation in a deep oligotrophic aquifer.

The formation water of a deep aquifer (853 m of depth) used for geological storage of natural gas was sampled to assess the mono-aromatic hydrocarbons attenuation potential of the indigenous microbiota. The study of bacterial diversity suggests that Firmicutes and, in particular, sulphate-reducing bacteria (Peptococcaceae) predominate in this microbial community. The capacity of the microbial community to biodegrade toluene and m- and p-xylenes was demonstrated using a culture-based approach after several hundred days of incubation. In order to reveal the potential for biodegradation of these compounds within a shorter time frame, an innovative approach named the solution hybrid selection method, which combines sequence capture by hybridization and next-generation sequencing, was applied to the same original water sample. The bssA and bssA-like genes were investigated as they are considered good biomarkers for the potential of toluene and xylene biodegradation. Unlike a PCR approach which failed to detect these genes directly from formation water, this innovative strategy demonstrated the presence of the bssA and bssA-like genes in this oligotrophic ecosystem, probably harboured by Peptococcaceae. The sequence capture by hybridization shows significant potential to reveal the presence of genes of functional interest which have low-level representation in the biosphere.

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)).