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International audience; A bacterial strain B2 that oxidizes arsenite into arsenate was isolated from the biofilm growing in a biological groundwater treatment process used for Fe removal. This strain is phylogenetically and morphologically different from the genus Leptothrix commonly encountered in biological iron oxidation processes. T-RFLP fingerprint of the biofilm revealed that this isolated strain B2 corresponds to the major population of the bacterial community in the biofilm. Therefore, it is probably one of the major contributors to arsenic removal in the treatment process.

International audience; The microbial communities have been investigated in the subsurface waters of the Carnoulès pyrite-rich tailings impoundment (France) for two hydrological situations characterized by the presence of oxygenated waters during winter and suboxic conditions in early autumn. In these acidic waters (2-5) characterized by elevated concentrations of Fe (1608-3354 mg·1-1), As (130-434 mg·1-1) and sulfates (5796-14318 mg·1-1) and variable dissolved oxygen content, the cultivable bacteria found in these system are Thiomonas and Acidithiobacillus ferrooxidans. Molecular methods, Terminal-Restriction Fragment Length Polymorphism (T-RFLP), and 16S rRNA encoding gene library analysis indicate low diversity. The environment is dominated by only a few types of microorganisms, with 70-80% of the whole bacterial population assigned to two or three Terminal-Restriction Fragments (T-RFs). Most of these organisms are uncultured, newly described, or recently associated with acid mine drainage. Modifications of the community structure are observed as a function of the sampling period and seem to be related to the aqueous chemistry of the tailings water. At low Dissolved Oxygen (DO = 1 mg·1-1) concentrations and moderately acidic conditions (pH = 5.7), the dominant organisms are related to the uncultured clone BA31 affiliated with Desulfosarcina variabilis, a sulfate-reducing bacteria (SRB), Acidithiobacillus ferrooxidans and the uncultured clone BVB20, closely related to Thiobacillus. At high (12 mg·1-1) DO concentrations and low (

International audience; The distribution of arsenic (As(III), As(V)) and iron (Fe(II), Fe(III)) species was monitored during 1 year in a borehole drilled in the Carnoulès tailings impoundment which contains As-rich pyrite. The concentrations of total As and Fe in subsurface waters exhibited strong variations over one year, which were controlled by dissolved oxygen concentrations. At high oxygen levels, extremely high As (up to 162 mM) and Fe (up to 364 mM) concentrations were reached in the borehole, with the oxidised species predominant. As and Fe concentrations decreased 10-fold under oxygen-deficient conditions, as a result of pH increase and subsequent precipitation of As(V) and Fe(III). From drill core sections, it appeared that at low dissolved oxygen levels, As(III) was primarily released into water by the oxidation of As-rich pyrite in the unsaturated zone. Subsequent As and Fe precipitation was promoted during transport to the saturated zone; this reaction resulted in As enrichments in the sediment below the water table compared to the original content in pyrite, together with the formation of As-rich (up to 35 wt% As) ferruginous material in the unsaturated zone. High amounts of As(V) were released from these secondary phases during leaching experiments with oxygenated acid sulfate-rich waters; this process is believed to contribute to As(V) enrichment in the subsurface waters of the Carnoulès tailings during periods of high dissolved oxygen level.

The oxidation of Fe(II) in acid mine drainage (AMD) leads to the precipitation of Fe(III) compounds which may incorporate toxic elements, such as arsenic (As), within their structure or adsorb them at their surface, thus limiting their mobility. The present work provides evidence for spatial and seasonal variations of microbial activity that influence arsenite oxidation and As immobilization in the heavily contaminated AMD from the Carnoulès mine, Gard, France ([As III] = 80 to 280 mg x L(-1) in the acidic spring draining the waste-pile). In the first tens of meters of the AMD, the rapid oxidation of Fe(II) leads to the coprecipitation of large amounts of As with Fe(III) in bacterial mats. XRD, XANES, and SEM analyses of sediments and stromatolite samples revealed the unusual formation of As(III)-rich compounds, especially nanocrystalline tooeleite, Fe6(AsO3)4(SO4)(OH)4 x 4H2O, a rare ferric arsenite sulfate oxy-hydroxide mineral, together with XRD-amorphous mixed As(III)/As(V)-Fe(III) oxy-hydroxide compounds. In the wet season, the suspended sediments of the upstream zone essentially consist of tooeleite associated with am-As(III)-Fe(III) oxy-hydroxides, while am-As(V)-Fe(III) oxy-hydroxides, having As:Fe molar ratios as high as 0.6-0.8, dominate in the dry season. Comparing natural and bioassay samples revealed that the formation of As(III)-rich compounds in the wet season may be related to the metabolic activity of bacterial strains able to oxidize Fe(II) but not As(III). One of these strains, having an Acidithiobacillus ferrooxidans genotype, has been isolated from the Carnoulès AMD. In contrast, the formation of As(V)-rich compounds in the dry season can be related to both biotic and abiotic oxidation of As(III) to As(V). Some Thiomonas strains isolated from the Carnoulès AMD were shown to be able to catalyze the oxidation of As(III) to As(V) in solution. Therefore, they can promote the formation of mixed As(V)-Fe(III) oxy-hydroxides, provided enough Fe(II) oxidizes. These results yield a better understanding of natural processes at this site and may help in designing efficient As-removal processes.

Karst aquifers, because of their unique hydrogeologic characteristics, are extremely susceptible to contamination by pathogens. Here we present the results of an investigation of contamination of a karst aquifer by fecal indicator bacteria. Two wells intercepting zones with contrasting effective hydraulic conductivities, as determined by pump test, were monitored both during the dry season and in response to a rain event. Samples were also collected from the adjacent ephemeral surface stream, which is known to be impacted by an upstream wastewater treatment plant after rainfall. Whole water and suspended sediment samples were analyzed for fecal coliforms and enterococci. During the dry season, pumping over a 2-day period resulted in increases in concentrations of fecal coliforms to greater than 10,000 CFU/100 ml in the high-conductivity well; enterococci and total suspended solids also increased, to a lesser degree. Toward the end of the pumping period, as much as 50% of the fecal coliforms were associated with suspended sediment. Irrigation of an up-gradient pine plantation with primary-treated wastewater is the probable source of the bacterial contamination. Sampling after a rain event revealed the strong influence of water quality of the adjacent Terrieu Creek on the ground water. Bacterial concentrations in the wells showed a rapid response to increased concentrations in the surface water, with fecal coliform concentrations in ground water ultimately reaching 60,000 CFU/100 ml. Up to 100% of the bacteria in the ground water was associated with suspended sediment at various times. The results of this investigation are evidence of the strong influence of surface water on ground water in karst terrain, including that of irrigation water. The large proportion of bacteria associated with particulates in the ground water has important implications for public health, as bacteria associated with particulates may be more persistent and more difficult to inactivate. The high bacterial concentrations found in both wells, despite the difference in hydraulic conductivity, demonstrates the difficulty of predicting vulnerability of individual wells to bacterial contamination in karst. The extreme temporal variability in bacterial concentrations underscores the importance of event-based monitoring of the bacterial quality of public water supplies in karst.

In fissured aquifers, hydrodynamic phenomena combined with a network of permeable fissures imparts varying degrees of vulnerability depending on the type of contaminant. The study presented here examines the impact on a karst aquifer of a river which receives effluents from a sewage treatment plant just upstream from the point of recharge. This recharge constitutes a source of bacteriological and chemical pollution. The aquifer is accessible by boreholes at discrete points, facilitating investigation of the evolution of three of the contaminants characteristic of treatment station effluents, NH4+ and two biological contaminants with different survival capacities. The study was undertaken both under normal climatic conditions and during a flood occurring after a long dry period. The rate of infiltration, of subsurface transport, and of dissemination of contaminants into a zone usually protected from contamination was recorded. The results confirm the vulnerability of fissured aquifers to contamination.

cited By 15; International audience; The Carnoulès mine is an extreme environment located in the South of France. It is an unusual ecosystem due to its acidic pH (2–3), high concentration of heavy metals, iron, and sulfate, but mainly due to its very high concentration of arsenic (up to 10 g L−1 in the tailing stock pore water, and 100–350 mg L−1 in Reigous Creek, which collects the acid mine drainage). Here, we present a survey of the archaeal community in the sediment and its temporal variation using a culture-independent approach by cloning of 16S rRNA encoding genes. The taxonomic affiliation of Archaea showed a low degree of biodiversity with two different phyla: Euryarchaeota and Thaumarchaeota. The archaeal community varied in composition and richness throughout the sampling campaigns. Many sequences were phylogenetically related to the order Thermoplasmatales represented by aerobic or facultatively anaerobic, thermoacidophilic autotrophic or heterotrophic organisms like the organotrophic genus Thermogymnomonas. Some members of Thermoplasmatales can also derive energy from sulfur/iron oxidation or reduction. We also found microorganisms affiliated with methanogenic Archaea (Methanomassiliicoccus luminyensis), which are involved in the carbon cycle. Some sequences affiliated with ammonia oxidizers, involved in the first and rate-limiting step in nitrification, a key process in the nitrogen cycle were also observed, including Candidatus Nitrososphaera viennensis and Candidatus nitrosopumilus sp. These results suggest that Archaea may be important players in the Reigous sediments through their participation in the biochemical cycles of elements, including those of carbon and nitrogen.

cited By 22; International audience; The acid waters (pH=2.73-3.4) that originate from the Carnoulès mine tailings (France) are known for their very high concentrations of As (up to 10,000 mg l(-1)) and Fe (up to 20,000 mg l(-1)). To analyze the composition of the archaeal community, (their temporal variation inside the tailing and spatial variations all along the Reigous Creek, which drains the site), seven 16S rRNA gene libraries were constructed. Clone analysis revealed that all the sequences were affiliated to the phylum Euryarchaeota, while Crenarchaeota were not represented. The study showed that the structure of the archaeal community of the aquifer of the tailing stock is different to that of the Reigous Creek. Irrespective of the time of sampling, the most abundant sequences found inside the tailing stock were related to Ferroplasma acidiphilum, an acidophilic and ferrous-iron oxidizing Archaea well known for its role in bioleaching. Inversely, in Reigous Creek, a sequence affiliated to the uncultured Thermoplasmatales archaeon, clone YAC1, was largely dominant. This study provides a better understanding of the microbial community associated with an acid mine drainage rich in arsenic.

Legionella bacterium is present naturally in ground water and has been described as a new pathogen growing in water distribution systems. Real-time PCR is a technology that permits us to quantify, in a rapid, sensitive, and reliable way, the genomic units of bacteria in water. The authors of this chapter have used real-time PCR to study the presence of Legionella bacteria in the environment and in water distribution networks. They have quantified Legionella spp. and L. pneumophila in natural water, water distributed in the urban network following disinfectant treatment, and waste water from domestic and industrial use. The water microbiology was studied by ATP measurements and enumeration of Legionella by culture and by real-time PCR. Thirty natural water samples were collected along the Vidourle river. Six waste water samples were collected in the three towns. PCR results were higher in natural water than in distributed water and lower than in waste water. In this study, real-time PCR shed light on the presence of Legionella species flora in natural water along Vidourle Valley watershed. Proliferating in biofilms and in amoebae, the Legionella species may be a useful indicator for the estimation of the total microbiological quality of water. ATP-metry can also be an interesting tool for the characterization of the microbiological quality of water.

The acid waters (pH 2.7 to 3.4) originating from the Carnoulès mine tailings contain high concentrations of dissolved arsenic (80 to 350 mg · liter −1 ), iron (750 to 2,700 mg · liter −1 ), and sulfate (2,000 to 7,500 mg · liter −1 ). During the first 30 m of downflow in Reigous creek issuing from the mine tailings, 20 to 60% of the dissolved arsenic is removed by coprecipitation with Fe(III). The microbial communities along the creek have been characterized using terminal-restriction fragment length polymorphism (T-RFLP) and 16S rRNA gene library analyses. The results indicate a low bacterial diversity in comparison with unpolluted water. Eighty percent of the sequences obtained are related to sequences from uncultured, newly described organisms or recently associated with acid mine drainage. As expected owing to the water chemistry, the sequences recovered are mainly related to bacteria involved in the geochemical Fe and S cycles. Among them, sequences related to uncultured TrefC4 affiliated with Gallionella ferruginea , a neutrophilic Fe-oxidizing bacterium, are dominant. The description of the bacterial community structure and its dynamics lead to a better understanding of the natural remediation processes occurring at this site.

Experiments were carried out to investigate the potential for bacterial colonization of different substrates in karst aquifers and the nature of the colonizing bacteria. Laboratory batch experiments were performed using limestone and PVC as substrates, a natural bacterial isolate and a known laboratory strain (Escherichia coli [E. coli]) as inocula, and karst ground water and a synthetic formula as growth media. In parallel, fragments of limestone and granite were submerged in boreholes penetrating two karst aquifers for more than one year; the boreholes are periodically contaminated by enteric bacteria from waste water. Once a month, rock samples were removed and the colonizing bacteria quantified and identified. The batch experiments demonstrated that the natural isolate and E. coli both readily colonized limestone surfaces using karst ground water as the growth medium. In contrast, bacterial colonization of both the limestone and granite substrates, when submerged in the karst, was less intense. More than 300 bacterial strains were isolated over the period sampled, but no temporal pattern in colonization was seen as far as strain, and colonization by E. coli was notably absent, although strains of Salmonella and Citrobacter were each observed once. Samples suspended in boreholes penetrating highly fractured zones were less densely colonized than those in the borehole penetrating a less fractured zone. The results suggest that contamination of karst aquifers by enteric bacteria is unlikely to be persistent. We hypothesize that this may be a result of the high flow velocities found in karst conduits, and of predation of colonizing bacteria by autochthonous zooplankton.

Reigous acid creek originating from the Carnoulès tailings impoundment supplies high concentrations of arsenic under soluble (up to approximately 4 mg/l) and particulate (up to 150 mgAs/g) phases to the Amous river, situated at the drainage basin of the Rhône river (Southern France). The metalloid is present as As(III) (95%) in Reigous creek water while As(V) predominates (50-80%) in the solid phase, i.e. schwertmannite. At the confluence between acid (pH5) creek and alkaline Amous river, As(III) concentrations decrease ten-fold through dilution and formation of As-rich ferrihydrite (As/Fe=0.02-0.1) containing 10-30% As(III). However, these attenuation processes are not efficient in the summer heatwave of 2003 since As concentrations in Amous river water (or=20 microg/l) and As/Fe ratios in particulate matter (or=0.07) are closed to those of Reigous creek (or=22 microg/l andor=0.02, respectively) or even higher. Downstream the confluence, processes involved in the transport of aqueous As along Amous river flowpath vary seasonally. Arsenic is transported conservatively in the aqueous phase away from the confluence in the cooler months; thus, dilution by unpolluted tributaries is the only process that decreases As concentrations. However, As(III) is rapidly oxidized and As(V) remains in solution. In contrast, during the warm season, desorption from As-rich sediment occurs which results in an increase of As(V) and As(III) concentrations along Amous river flow until they reach up to approximately 20 microg/l each. Therefore, Amous river seems not to be totally recovered from mine-related arsenic contamination after 3.5 km and may affect freshwater resources further downstream.

The unusual characteristics of subterranean flow in karst aquifers allow for the transport of sediment. Karst ground-water systems are created by dissolution of the bedrock matrix coupled with structural and stratigraphic controls. As a result, high flow velocities, large-diameter openings, and turbulent flow, all necessary for the entrainment and transport of particles, are present — at least episodically — in most karst systems. A better understanding of sediment transport in karst is needed to protect these vulnerable systems. Deposition of sediments within the aquifer may decrease aquifer permeability, fill in wells, and cause pumps to seize; their deposition internally or at spring mouths may destroy species habitat; and their presence impairs the esthetic appearance of spring water. Sediment is itself considered a contaminant — the U.S. Environmental Protection Agency (USEPA) (2000) has recently ruled that total maximum daily loads (TMDLs) are required even for stream segments impacted only by high suspended sediment concentrations. Furthermore, mobile sediment in karst may act as a vector for the transport of hydrophobic (“water fearing”) contaminants (Ford and Williams, 1989).

Weathering of the As-rich pyrite-rich tailings of the abandoned mining site of Carnoulès (southeastern France) results in the formation of acid waters heavily loaded with arsenic. Dissolved arsenic present in the seepage waters precipitates within a few meters from the bottom of the tailing dam in the presence of microorganisms. An Acidithiobacillus ferrooxidans strain, referred to as CC1, was isolated from the effluents. This strain was able to remove arsenic from a defined synthetic medium only when grown on ferrous iron. This A. ferrooxidans strain did not oxidize arsenite to arsenate directly or indirectly. Strain CC1 precipitated arsenic unexpectedly as arsenite but not arsenate, with ferric iron produced by its energy metabolism. Furthermore, arsenite was almost not found adsorbed on jarosite but associated with a poorly ordered schwertmannite. Arsenate is known to efficiently precipitate with ferric iron and sulfate in the form of more or less ordered schwertmannite, depending on the sulfur-to-arsenic ratio. Our data demonstrate that the coprecipitation of arsenite with schwertmannite also appears as a potential mechanism of arsenite removal in heavily contaminated acid waters. The removal of arsenite by coprecipitation with ferric iron appears to be a common property of the A. ferrooxidans species, as such a feature was observed with one private and three collection strains, one of which was the type strain.

International audience; Aims: To isolate, identify, and characterize heterotrophic bacteria in acid-mine drainage that mediate oxidation of As(III). Methods and Results: Samples of acid-mine drainage were collected over a period of 14 months. Heterotrophic and non-obligatory acidophilic bacteria in the samples were cultured on a solid medium (pH 7AE0-7AE2), and three strains were isolated. The three different strains belong to the genus Thiomonas, and have more than 99% homology with the group Ynys1. Culturing in mineral media demonstrated that the isolated strains used thiosulphate as an energy source, and oxidized iron in the presence of thiosulphate. However, none of the strains were able to oxidize arsenic in the presence of thiosulphate, nor could they use iron or arsenic alone as an energy source. In vitro experiments demonstrated that two of the Thiomonas strains were able to oxidize more than 90% of the As(III) present in the acid-mine drainage, whereas no abiotic oxidation of arsenic occurred. Conclusions: Two strains of newly identified Thiomonas sp. found in acid-mine drainage are capable of oxidizing arsenic. Significance and Impact of Study: These results represent the first reported oxidation of arsenic by Thiomonas sp. Biologically mediated oxidation and subsequent immobilization of arsenic is of great interest for the remediation of contaminated mine sites.

International audience; In the acid stream (pH 2.5–4.7) originating from the Carnoulès mine tailings, the acidophilic protozoan Euglena mutabilis grows with extremely high sulfate (1.9–4.9 g/l), iron (0.7–1.7 g/l) and arsenic concentrations (0.08–0.26 g/l). Strong variations in flow rate and high sulfate concentrations (up to 4.9 g/l) have been registered in early winter and might be the reason for the reduction in cell number of the protozoan from October to December 2001. No relation was established between arsenic concentration and/or speciation and abundance of the protozoan in the stream. Arsenite, which is the most toxic form, predominates in water. The oxidation of arsenite to arsenate occurred within a few days in laboratory experiments when E. mutabilis was present in Reigous Creek water and synthetic As(III)-rich culture medium. Methylated compounds (MMA, DMA) were not identified in the culture media. The protozoan bioaccumulated As in the cell (336±112 μg As/g dry wt.) as inorganic arsenite (105±52 μg As/g dry wt.) and arsenate (231±112 μg As/g dry wt.). Adsorption of As at the cell surface reached 57 mg/g dry wt. in the As(V) form for E. mutabilis grown in 250 mg/l As(III) synthetic medium. Both intracellular accumulation and adsorption at the cell surface increased for increasing As(III) concentration in the medium but the concentration factor in the cell relative to soluble As decreased.