Odile Bruneel

Bacterial immobilization and oxidation of arsenic in acid mine drainage (Carnoulès creek, France).

The acid waters (pH=2.73-3.37) originating from the Carnoulès mine tailings contain high dissolved concentrations of arsenic (1-3.5 mmol l(-1)) and iron (20-40 mmol l(-1)). At the outlet, arsenite predominates. During the first 30 m of downflow, 20-60% is removed by coprecipitation with Fe(III). This process results from bacterially mediated As- and Fe-oxidation. The precipitation rates in the creek depend on the oxygen concentration in spring water and are lower during the dry summer period when the anoxic character of the spring water inhibits the activity of oxidizing bacteria. Ex situ experiments show that the presence of bacteria-rich precipitates increases the As- and Fe-removal rates. Three strains of bacteria promoting the oxidation of As have been isolated, and two of them have the characteristics of Thiomonas ynys1. The third strain, which is not identified yet, also catalyzes the oxidation of Fe.

Diversity of Microorganisms in Fe-As-Rich Acid Mine Drainage Waters of Carnoulès, France

ABSTRACT 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.

Metabolic diversity among main microorganisms inside an arsenic-rich ecosystem revealed by meta- and proteo-genomics

By their metabolic activities, microorganisms have a crucial role in the biogeochemical cycles of elements. The complete understanding of these processes requires, however, the deciphering of both the structure and the function, including synecologic interactions, of microbial communities. Using a metagenomic approach, we demonstrated here that an acid mine drainage highly contaminated with arsenic is dominated by seven bacterial strains whose genomes were reconstructed. Five of them represent yet uncultivated bacteria and include two strains belonging to a novel bacterial phylum present in some similar ecosystems, and which was named ‘Candidatus Fodinabacter communificans.’ Metaproteomic data unravelled several microbial capabilities expressed in situ, such as iron, sulfur and arsenic oxidation that are key mechanisms in biomineralization, or organic nutrient, amino acid and vitamin metabolism involved in synthrophic associations. A statistical analysis of genomic and proteomic data and reverse transcriptase–PCR experiments allowed us to build an integrated model of the metabolic interactions that may be of prime importance in the natural attenuation of such anthropized ecosystems.

Immobilization of Arsenite and Ferric Iron by Acidithiobacillus ferrooxidans and Its Relevance to Acid Mine Drainage

ABSTRACT 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.

Structure, Function, and Evolution of the Thiomonas spp. Genome

Bacteria of the Thiomonas genus are ubiquitous in extreme environments, such as arsenic-rich acid mine drainage (AMD). The genome of one of these strains, Thiomonas sp. 3As, was sequenced, annotated, and examined, revealing specific adaptations allowing this bacterium to survive and grow in its highly toxic environment. In order to explore genomic diversity as well as genetic evolution in Thiomonas spp., a comparative genomic hybridization (CGH) approach was used on eight different strains of the Thiomonas genus, including five strains of the same species. Our results suggest that the Thiomonas genome has evolved through the gain or loss of genomic islands and that this evolution is influenced by the specific environmental conditions in which the strains live.

Predominance of Aqueous Tl(I) Species in the River System Downstream from the Abandoned Carnoulès Mine (Southern France)

Thallium concentration reached up to 534 μg L(-1) in the Reigous acid mine drainage downstream from the abandoned Pb-Zn Carnoulès mine (Southern France). It decreased to 5.44 μg L(-1) in the Amous River into which the Reigous creek flows. Tl(I) predominated (>98% of total dissolved Tl) over Tl(III), mainly in the form of Tl(+). Small amounts of Tl(III) evidenced in Reigous Creek might be in the form of aqueous TlCl(2)(+). The range of dissolved to particulate distribution coefficients log K(d) = 2.5 L kg(-1) to 4.6 L kg(-1) indicated low affinity of Tl for particles, mainly ferrihydrite, formed in the AMD-impacted watershed. The low retention of Tl(+) on ferrihydrite was demonstrated in sorption experiments, the best fit between experimental and modeled data being achieved for surface complexation constants log K(ads) = -2.67 for strong sites and log K(ads) = -3.76 for weak sites. This new set of constants allowed reasonable prediction of the concentrations of aqueous and particulate Tl resulting from the mixing of water from Reigous Creek and the Amous River water during laboratory experiments, together with those measured in the Amous River field study.

Microbial Diversity in a Pyrite-Rich Tailings Impoundment (Carnoulès, France)

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 · l−1), As (130–434 mg · l−1) and sulfates (5796–14318 mg · l−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 · l−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 · l−1) DO concentrations and low (< 2) pH values, the microbial diversity is less important and 65% of the population is assigned to the uncultured bacterium clone AS6 related to Desulfosarcina variabilis.

Diversity and spatiotemporal dynamics of bacterial communities: physicochemical and other drivers along an acid mine drainage

Deciphering the biotic and abiotic factors that control microbial community structure over time and along an environmental gradient is a pivotal question in microbial ecology. Carnoulès mine (France), which is characterized by acid waters and very high concentrations of arsenic, iron, and sulfate, provides an excellent opportunity to study these factors along the pollution gradient of Reigous Creek. To this end, biodiversity and spatiotemporal distribution of bacterial communities were characterized using T-RFLP fingerprinting and high-throughput sequencing. Patterns of spatial and temporal variations in bacterial community composition linked to changes in the physicochemical conditions suggested that species-sorting processes were at work in the acid mine drainage. Arsenic, temperature, and sulfate appeared to be the most important factors that drove the composition of bacterial communities along this continuum. Time series investigation along the pollution gradient also highlighted habitat specialization for some major members of the community (Acidithiobacillus and Thiomonas), dispersal for Acidithiobacillus, and evidence of extinction/re-thriving processes for Gallionella. Finally, pyrosequencing revealed a broader phylogenetic range of taxa than previous clone library-based diversity. Overall, our findings suggest that in addition to environmental filtering processes, additional forces (dispersal, birth/death events) could operate in AMD community.

Arsenic scavenging by aluminum-substituted ferrihydrites in a circumneutral pH river impacted by acid mine drainage.

Ferrihydrite (Fh) is a nanocrystalline ferric oxyhydroxide involved in the retention of pollutants in natural systems and in water-treatment processes. The status and properties of major chemical impurities in natural Fh is however still scarcely documented. Here we investigated the structure of aluminum-rich Fh, and their role in arsenic scavenging in river-bed sediments from a circumneutral river (pH 6-7) impacted by an arsenic-rich acid mine drainage (AMD). Extended X-ray absorption fine structure (EXAFS) spectroscopy at the Fe K-edge shows that Fh is the predominant mineral phase forming after neutralization of the AMD, in association with minor amount of schwertmannite transported from the AMD. TEM-EDXS elemental mapping and SEM-EDXS analyses combined with EXAFS analysis indicates that Al(3+) substitutes for Fe(3+) ions into the Fh structure in the natural sediment samples, with local aluminum concentration within the 25-30 ± 10 mol %Al range. Synthetic aluminous Fh prepared in the present study are found to be less Al-substituted (14-20 ± 5 mol %Al). Finally, EXAFS analysis at the arsenic K-edge indicates that As(V) form similar inner-sphere surface complexes on the natural and synthetic Al-substituted Fh studied. Our results provide direct evidence for the scavenging of arsenic by natural Al-Fh, which emphasize the possible implication of such material for scavenging pollutants in natural or engineered systems.

Geochemical Processes Controlling the Formation of As-Rich Waters Within a Tailings Impoundment (Carnoulès, France)

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.