In 2008, as part of a feasibility study for radioactive waste disposal in deep geological formations, the French National Radioactive Waste Management Agency (ANDRA) drilled several boreholes in the transposition zone in order to define the potential variations in the properties of the Callovo–Oxfordian claystone formation. This consisted of a rare opportunity to investigate the deep continental biosphere that is still poorly known. Four rock cores, from 1709, 1804, 1865, and 1935 m below land surface, were collected from Lower and Middle Triassic formations in the Paris Basin (France) to investigate their microbial and geochemical composition. Rock leachates showed high salinities ranging from 100 to 365 g·L−1 NaCl, current temperatures averaging 65 °C, no detectable organic matter, and very fine porosity. Microbial composition was studied using a dual cultural and molecular approach. While the broad-spectrum cultural media that was used to activate microbial communities was unsuccessful, the genetic investigation of the dominant 16S rRNA gene sequences revealed eight bacterial genera considered as truly indigenous to the Triassic cores. Retrieved taxa were affiliated to aerobic and facultative anaerobic taxon, mostly unknown to grow in very saline media, except for one taxon related to Halomonas. They included Firmicutes and α-, β-, and γ-Proteobacteria members that are known from many subsurface environments and deep terrestrial and marine ecosystems. As suggested by geochemical analyses of rocks and rock leachates, part of the indigenous bacterial community may originate from a cold paleo-recharge of the Trias aquifer with water originating from ice melting. Thus, retrieved DNA would be fossil DNA. As previously put forward to explain the lack of evidence of microbial life in deep sandstone, another hypothesis is a possible paleo-sterilisation that is based on the poly-extremophilic character of the confined Triassic sandstones, which present high salinity and temperature.

In situ biofilm sampling is a key step for the study of natural biofilms and using methodologies that reflect natural diversity is necessary to guarantee representative sampling. Here, we focalise on the impact of the type of substrata on which biofilms grow on bacterial and fungal communities' structure. The indirect molecular approach, Denaturing Gel Gradient Electrophoresis (DGGE) of a gene fragment coding for either 16S rRNA or 28S rRNA, for bacteria or fungi respectively, was used to evaluate the variability of microbial community structures among different biofilm substrata: natural (pebbles, live plants, wood and sediment), or artificial (glass, Plexiglas(®) and sterile wood), in a small river (the Loiret, France). Multivariate statistics, band richness and diversity indexes (Shannon and Simpson) were used to highlight variations in community structure between substrata. Results showed variations of bacterial and fungal diversity between different substrata according to substratum properties/origin (natural or artificial, organic or inorganic) but there was no optimal substratum for sampling, and artificial substrata were not significantly less applicable than natural substrata. Pooling 4 different substrata types allowed a higher bacterial and fungal biodiversity recovery. Point contact sampling may thus gain in robustness by increasing the number of substrata considered. Fungal species richness was similar to the bacterial one on most substrata which suggested they should be more frequently considered in riverine biofilm studies.

Mercury (Hg) mobility and speciation in subsurface aquifers is directly linked to its surrounding geochemical and microbial environment. The role of bacteria on Hg speciation (i.e., methylation, demethylation and reduction) is well documented, however little data is available on their impact on Hg mobility. The aim of this study was to test if (i) Hg mobility is due to either direct iron oxide reduction by iron reducing bacteria (IRB) or indirect iron reduction by sulfide produced by sulfate reducing bacteria (SRB), and (ii) to investigate its subsequent fate and speciation. Experiments were carried out in an original column setup combining geochemical and microbiological approaches that mimic an aquifer including an interface of iron-rich and iron depleted zones. Two identical glass columns containing iron oxides spiked with Hg(II) were submitted to (i) direct iron reduction by IRB and (ii) to indirect iron reduction by sulfides produced by SRB. Results show that in both columns Hg was leached and methylated during the height of bacterial activity. In the column where IRB are dominant, Hg methylation and leaching from the column was directly correlated to bacterial iron reduction (i.e., FeII release). In opposition, when SRB are dominant, produced sulfide induced indirect iron oxide reduction and rapid adsorption of leached Hg (or produced methylmercury) on neoformed iron sulfides (e.g., Mackinawite) or its precipitation as HgS. At the end of the SRB column experiment, when iron-oxide reduction was complete, filtered Hg and Fe concentrations increased at the outlet suggesting a leaching of Hg bound to FeS colloids that may be a dominant mechanism of Hg transport in aquifer environments. These experimental results highlight different biogeochemical mechanisms that can occur in stratified sub-surface aquifers where bacterial activities play a major role on Hg mobility and changes in speciation.

This study aimed at evaluating potential arsenic (As) mobility in an industrially contaminated soil (64mg/kg of As) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobic conditions using a laboratory batch experiment. In the presence of exogenous carbon sources, As-V initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides were solubilized and mainly reduced to As-III by indigenous soil microflora. After a 1-month incubation period in these biotic conditions, As-III accounted for 80-85% of the total dissolved As and more than 60% of the solid-phase As. Bacterial community structure (i.e., 16S rDNA-based capillary electrophoresis single-strand conformation polymorphism profiles) changed with incubation time and As amendment. The detection of distantly related arsenate respiratory reductase genes (arrA), as functional markers of As-V respirers, indicates that novel dissimilatory As-V-reducing bacteria may be involved in As biotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, a crucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible for heterotrophic As-V reduction to As-III, which may increase As toxicity and mobility in the contaminated soils.

In the context of artificial groundwater recharge, a reactive soil column at pilot-scale (4.5 m depth and 3 m in diameter) fed by treated wastewater was designed to evaluate soil filtration ability. Here, as a part of this project, the impact of treated wastewater filtration on soil bacterial communities and the soil's biological ability for wastewater treatment as well as the relevance of the use of multi-bioindicators were studied as a function of depth and time. Biomass; bacterial 16S rRNA gene diversity fingerprints; potential nitrifying, denitrifying, and sulfate-reducing activities; and functional gene (amo, nir, nar, and dsr) detection were analyzed to highlight the real and potential microbial activity and diversity within the soil column. These bioindicators show that topsoil (0 to 20 cm depth) was the more active and the more impacted by treated wastewater filtration. Nitrification was the main activity in the pilot. No sulfate-reducing activity or dsr genes were detected during the first 6 months of wastewater application. Denitrification was also absent, but genes of denitrifying bacteria were detected, suggesting that the denitrifying process may occur rapidly if adequate chemical conditions are favored within the soil column. Results also underline that a dry period (20 days without any wastewater supply) significantly impacted soil bacterial diversity, leading to a decrease of enzyme activities and biomass. Finally, our work shows that treated wastewater filtration leads to a modification of the bacterial genetic and functional structures in topsoil.

Artificially synthetized chloride-based-oxyanions such as perchlorate (ClO4-) and chlorate (ClO3-), are used in a vast number of applications such as military and aerospace industry; they are also used as herbicides and in pyrotechnic applications. Due to their very high solubility, perchlorate and chlorate are readily transported in water systems and can thus end up in drinking water. Ingestion of perchlorate may affect iodine uptake by the human thyroid and thus thyroidal hormone production. Pollution by these oxyanions is an emerging problem in France and recent events of ground water contamination have increased concerns on the fate of these substances, thus encouraging research on its fate in the environment and effect on human health. As the transport of (liquid and gaseous) chloride is highly dangerous, it is generally transformed directly on site and thus other chloride molecules such as 1, 2 dichloroethane (DCA) which is a precursor of vinyl chloride (VC), can be found in the same locations. The work we present here involves a site that once produced perchlorate, chlorate, DCA and VC. Due to historical losses, all of these substances are present in the groundwater. Following groundwater characterisation, the aim of the present study was to identify a naturel attenuation potential on site by researching specific genes involved in chlorate and perchlorate reduction and DCA or VC dehalogenation and then linking gene presence to activity using laboratory batch cultures. After collecting biomass from 36 ground-water-samples, DNA was extracted and PCRs (polymerase chain reactions) were carried out to amplify genes coding for several enzymes; the pcaA gene coding for a alkane dehydrogenase, the dhLA gene coding for a haloalkanedehalogenase, the pcrA gene coding for a perchlorate reductase and the cld gene coding for a chlorite reductase. Moreover, global bacterial diversity was studied by amplifying the gene coding for RNA 16S and analysing diversity with an electrophoresis approach (DGGE, Denaturing Gel Gradient Electrophoresis). Bands of interest were purified and sequenced. The gene-screening-results closely recovered zones where the chemicals had been detected in concentrations over 10µg/l and were especially precise for DCA and CV whereas for perchlorate and chlorate it suggested that the pollution span was once larger that presently. Degradation potentials, in batchs incubated at 15°C, demonstrated first chlorate reduction then perchlorate reduction after a week's incubation in presence of an available carbon source (acetate and lactate) but no degradation in autotrophic conditions. DCA amounts also decreased in batchs but over a longer time span. Due to the pollution layout, where the perchlorate and chlorate plume encounters the DCA and VC one, experiments are presently being carried out to assess whether these organochlorides and/or their dechlorination metabolites can be used as a carbon source to fuel perchlorate and chlorate reduction, thus actively contributing to a naturel attenuation of this pollution. This potential could then be stimulated and used for in situ bioremediation. Indeed, total degradation of these molecules produces Cl-, CO2 and H2O and which are harmless for the environment.

The purpose of this study was to identify and quantify the fate and speciation of carbon that can occur in mixtures of geological media (crushed rock) and autotrophic microbial communities. A sulfate reducing bacterium (Desulfotomaculum geothermicum) and a methanogenic archaeon (Methanothermococcus thermolithotrophicus) were both tested separately and together, with and without crushed sedimentary rock (carbonaceous sandstone) for different CO2 partial pressures (0.22, 0.88, 3.52, and 8bar) at 54°C in saline artificial groundwater. In order to quantify the respective metabolic activities, the inorganic gases of interest (H2, CH4, H2S and CO2) were measured and the speciation of carbon was assessed by measuring volatile, non-purgeable, total and dissolved organic carbon as well as total and dissolved inorganic carbon. Despite a protective effect of the mineral matrix, the results showed a high sensitivity of autotrophic microorganisms to the stress induced by pressures of CO2 superior to one bar and revealed that a part of this stress was due to direct toxic effects. M. thermolithotrophicus demonstrated a better tolerance to CO2 and was dominating the consortia. This ascendancy was interpreted as resulting from equilibrium displacement due to transport effects of methane between the liquid and gas phases. Abiotic dissolution was observed but some biomineralization processes of carbonates were also identified for D. geothermicum. Both strains displayed very different patterns in their conversion of inorganic carbon: while M. thermolithotrophicus was mainly producing methane, D. geothermicum induced the formation of biomass. The availability of crushed rock increased the proportion of sessile biofilms. All these results were analyzed in correlation with a successful PHREEQC simulation and demonstrate the strong influence of the microbial activities and diversity on the carbon fate in the immediate surroundings of geological CCS storage zones.

Arsenic (As) pollution in soils is a major health and environmental threat to water resources. Arsenic bioavailability in the environment is directly influenced by communities of As(III) oxidizing and As(V) reducing bacteria that catalyse the transformation of As(III) to As(V), a form easily precipitated, and the transformation of As(V) into As(III), the more mobile and toxic form, respectively. The objective of our work is to define molecular bio-indicators of arsenic mobility, in parallel with existing physic-chemical methods, and to evaluate their ability to predict As behaviour in the environment. Incubations of two soil samples from an industrial waste land containing different amounts of As (about 500 and 1600 ppm) showed that in aerobic conditions, favorable to As(III) oxidation, the microbial community stabilizes the arsenic by maintaining it as As(V). In anaerobic conditions, with the addition of a source of exogenic carbon to stimulate heterotrophic bacteria, the decrease in redox potential induces conditions in favor of As(V) reduction: 70% of the initial As(V) is solubilized. Without carbon addition, As solubilisation and As(V) reduction occur simultaneously in conditions where the redox potential is not favorable for As(V) respiration. The different As(III) oxidizing and As(V) reducing-bacterial communities issue from these incubations were studied, aiming in particular an As(III)-oxidase gene, aioB and an As(V)-reductase, coded by arrA. The pattern of these genes was also investigated in a set of 10 soil samples from the same waste land, covering a broader range of total As content (100 to 10000 ppm). Overall, a link between the evolution of these functional genes (quantity, diversity, ratio) and content, speciation and behavior of inorganic As in soil will be discussed.

Biochemical methods were selected to evaluate the role of exopolymeric substances in the stability of biofilms used in bioremediation processes. Biofilms of Thiomonas arsenivorans formed on pozzolana were thus treated with pronase (protein target), lectins (Con A or PNA), calcofluor or periodic acid (polysaccharides target), DNase (DNA target), and lipase (triglycerides target). Neither protease nor DNase treatments had any effect on bacterial adhesion. Lectins and calcofluor treatments mainly affected young biofilms. Lipase treatment had a noticeable effect on biofilm stability whatever the biofilm age. Results suggest that it would be an increased resistance of mature biofilms that protects them from external attacks.

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR (qPCR) were successfully developed to monitor functional aoxB genes as markers of aerobic arsenite oxidizers. DGGE profiles showed a shift in the structure of the aoxB -carrying bacterial population, composed of members of the Alpha -, Beta - and Gammaproteobacteria , depending on arsenic (As) and E h levels in Upper Isle River Basin waters. The highest aoxB gene densities were found in the most As-polluted oxic surface waters but without any significant correlation with environmental factors. Arsenite oxidizers seem to play a key role in As mobility in As-impacted waters.

The colonization of pozzolana by an As(III)-oxidizing bacterial consortium was monitored from the first hours of bacterial adhesion to 6 weeks of development under fed-batch conditions, using adapted ultrasonic dislodging and crystal-violet staining procedures to determine the biofilm adhering to the complex surfaces. The effect of temperature, arsenic concentration, and presence or absence of yeast extract (YE) on the amount of biofilm biomass and on the As(III)-oxidation were assessed to test the biofilm’s resilience and optimize the colonization. Fed-batch cultures allow twice as much pozzolana colonization as that obtained under batch conditions. In addition, As(III) oxidation and the quantities of biomass under fed-batch culture conditions were the same at 14°C and 25°C. Whereas YE improves (+150%) bacterial adhesion during the first 2 h, its impact in the longer term appears to be less significant—biofilm formation in presence of YE after 5 weeks was no greater than biofilm formation in the absence of YE. Finally, YE involves a drastic (−70%) decrease of As(III) oxidation. Preliminary tests for drinking-water bioremediation revealed the ability of Chéni Arsenic Oxidizing 1 biofilms to remain and retain As(III) oxidation activity at low As(III) concentrations (50 µg l−1).

A continuous bioleaching operation was carried out in a laboratory-scale unit using a cobaltiferous pyrite concentrate. The objective was to investigate mechanisms of microbial activity and mineral oxidation for a better understanding and optimisation of biomining operations, particularly the ones using stirred tank technology. A combination of scientific and technical approaches (molecular ecology and biochemistry) was used and various key operating parameters were tested (temperature, nitrogen source, CO2 availability).The bacterial strains that dominate the culture were present in the concentrate and originated from the deposit on the mining site. Whatever the operating conditions tested, the culture composition was very stable. The iron-oxidiser Leptospirillum ferriphilum BRGM1 was the dominant organism in standard (not limiting) conditions, and was always very well represented during the first 3-4 days of residence time. Sulfobacillus sp. BRGM2 also played a significant role in the process. The role and the presence of Acidithiobacillus caldus BRGM3 seemed of lesser importance.An increase in operating temperature from 35 degrees C to 45 degrees C had no major impact on bioleaching efficiency. When the nitrogen source was limiting, there was a negative impact on both bacterial growth and bioleaching efficiency. This was the result of a combination of factors such as less precipitate formation and decreased bacterial attachment to the pyrite surface. CO2 limitation had a very significant negative effect on bacterial productivity and consequently on bioleaching efficiency. However, when CO2 was a limiting factor, the population composition remained unchanged but a significant decrease in exopolysaccharide production was observed.This study gives new insights for the application of stirred tank technology to the processing of sulphide concentrates, and more specifically on the impact of key operating parameters on bioleaching performances, population dynamics and attachment of bacteria to the solid surfaces. (C) 2008 Elsevier B.V. All rights reserved.

ABSTRACT A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.

In the frame of a European project (BioMinE - FP6), a continuous bioleaching operation was carried out in a laboratory-scale unit using a cobaltiferous pyrite. The objective of the work was to use this system to investigate mechanisms of microbial activity and mineral oxidation in continuous stirred bioreactors (1x50L - 3x20L). A combination of scientific and technical approaches (molecular ecology, biochemistry and microscopy) was used and various key operating parameters were tested (temperature, nitrogen source, CO2 availability, designed consortia). An increase of temperature of 10°C (35°C to 45°C) had no major influence on the bioleaching efficiency. When the ammonium source was limiting, there was a negative influence on both bacterial growth and bioleaching efficiency. This result was related to a combination of factors such as less bacterial attachment to the pyrite surface and less precipitate formation. CO2 limitation had a very significant negative effect on the bacterial productivity and consequently on the bioleaching efficiency. Nevertheless, the population composition remained unchanged. An important decrease of EPS (sugar) production was also observed. The bacterial strains, that dominate the culture, originated from the deposit in Uganda. It seems that their bioleaching ability was improved over the time when cultured in continuous mode. The culture composition was very stable. The iron-oxidizer L. ferriphilum was the dominant organism in standard (not limiting) conditions, and was always very well represented during the first 3-4 days of residence time. Sulfobacillus sp. BRGM2 also played an important role in the process. This study gives new insights for the application of this technology, and more specifically on the influence of key operating parameters on bioleaching performances, population dynamics and attachment of bacteria to the solid surfaces.