A multi-site approach to investigate the role of toxicity and confounding factors on plant bioassay results.

Development of organisms that live on contaminated soils depends on toxicity as well as several physical and chemical soil properties. We aimed to identify plant bioassays most responsive to contaminants and not to confounding factors due to soil type differences. We implemented a multi-site approach in seven contaminated sites and used different ordinary plant bioassays (fourteen-day-shoot biomass and five-day-root and shoot elongation). Most of the sites were contaminated with polycyclic aromatic hydrocarbons (PAHs), and soils were sampled from areas of both high and low contamination. Bioassays were performed on ninety soil samples and were carried out with six model species. We performed analyses of regulatory PAHs and their derivatives content in the samples. Fourteen-day-shoot biomass responses depended on the site's origin, with an intricate response of plants that faced contrasted soil pH and organic matter content and various contaminant levels. Five-day-shoot and root lengths were informative when considering the most heavily PAH-contaminated site, since both measures exhibited a close dose-dependent response to PAHs but not to soil pH or organic matter content. For the other sites, elongation tests revealed tenuous effects somehow related to the presence of PAHs or their derivatives. We propose that tests based on plant development during their autotrophic phase (the fourteen-day-shoot biomass test in this study) are likely more sensitive to environmental stressors but less specific for contaminant-induced effects. Comparatively, tests based on early and heterotrophic plant development could be particularly more specific for soil contaminants, but the associated responses may be of low sensitivity.

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.

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)).

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.

Kinetics of benzene biotransformation under microaerophilic and oxygen-limited conditions.

A special microbial consortium adapted to degrade petroleum hydrocarbons at limited availability of oxygen, transformed benzene, a highly toxic and carcinogenic contaminant of groundwater and soil, at low initial dissolved oxygen (DO) concentrations of 0.05-2 mg/L. The employed initial concentrations of dissolved oxygen were considerably lower than the previously reported values. Under these conditions, the overall transformation of benzene ranged from 34% +/- 1.7% to 100%, considerably higher than the theoretical predictions for complete mineralization of benzene based on the requirement of 3.08 mg oxygen/mg benzene. Unlike biotransformation that proceeded at the lowest examined DO concentration of 0.05 mg/L, the mineralization of benzene, defined by its conversion to CO(2) and water, required a minimum DO concentration of 0.2 mg/L. The mineralization of benzene under microaerophilic conditions (DO < 2 mg/L), ranged from 0.83% +/- 0.06% to 89% +/- 1.3%, which was less than the theoretical predictions at any given initial DO concentration. The regulatory effects of dissolved oxygen concentration or its partial pressure on the activities of enzymes catalyzing the biotransformation of aromatic hydrocarbons was postulated to account for the reduced mineralization of benzene. The ratio between the transformed benzene and the consumed oxygen increased with the decrease of initial DO concentration, reaching a value of 2.8, considerably higher than the theoretical value of 0.33 obtained for a complete aerobic oxidation of benzene. Phenol was the major and the most stable intermediate metabolite during the biotransformation of benzene at low concentrations of DO. While benzene transformation stopped after the depletion of oxygen in the experimental system, phenol continued to accumulate under strictly anaerobic conditions, indicating its formation from an alternative carbon source, possibly biomass.