A field-pilot for passive bioremediation of As-rich acid mine drainage.

A field-pilot bioreactor exploiting microbial iron (Fe) oxidation and subsequent arsenic (As) and Fe co-precipitation was monitored during 6 months for the passive treatment of As-rich acid mine drainage (AMD). It was implemented at the Carnoulès mining site (southern France) where AMD contained 790-1315 mg L-1 Fe(II) and 84-152 mg L-1 As, mainly as As(III) (78-83%). The bioreactor consisted in five shallow trays of 1.5 m2 in series, continuously fed with AMD by natural flow. We monitored the flow rate and the water physico-chemistry including redox Fe and As speciation. Hydraulic retention time (HRT) was calculated and the precipitates formed inside the bioreactor were characterized (mineralogy, Fe and As content, As redox state). Since As(III) oxidation improves As retention onto Fe minerals, bacteria with the capacity to oxidize As(III) were quantified through their marker gene aioA. Arsenic removal yields in the pilot ranged between 3% and 97% (average rate (1.8 ±â€¯0.8) ✕ 10-8 mol L-1 s-1), and were positively correlated to HRT and inlet water dissolved oxygen concentration. Fe removal yields did not exceed 11% (average rate (7 ±â€¯5) ✕ 10-8 mol L-1 s-1). In the first 32 days the precipitate contained tooeleite, a rare arsenite ferric sulfate mineral. Then, it evolved toward an amorphous ferric arsenate phase. The As/Fe molar ratio and As(V) to total As proportion increased from 0.29 to 0.86 and from ∼20% to 99%, respectively. The number of bacterial aioA gene copies increased ten-fold during the first 48 days and stabilized thereafter. These results and the monitoring of arsenic speciation in the inlet and the outlet water, provide evidences that As(III) oxidized in the pilot. The biotreatment system we designed proved to be suitable for high As DMA. The formation of sludge highly enriched into As(V) rather than As(III) is advantageous in the perspective of long term storage.

Spatial Distribution of Eukaryotic Communities Using High-Throughput Sequencing Along a Pollution Gradient in the Arsenic-Rich Creek Sediments of Carnoulès Mine, France.

Microscopic eukaryotes play a key role in ecosystem functioning, but their diversity remains largely unexplored in most environments. To advance our knowledge of eukaryotic microorganisms and the factors that structure their communities, high-throughput sequencing was used to characterize their diversity and spatial distribution along the pollution gradient of the acid mine drainage at Carnoulès (France). A total of 16,510 reads were retrieved leading to the identification of 323 OTUs after normalization. Phylogenetic analysis revealed a quite diverse eukaryotic community characterized by a total of eight high-level lineages including 37 classes. The majority of sequences were clustered in four main groups: Fungi, Stramenopiles, Alveolata and Viridiplantae. The Reigous sediments formed a succession of distinct ecosystems hosting contrasted eukaryotic communities whose structure appeared to be at least partially correlated with sediment mineralogy. The concentration of arsenic in the sediment was shown to be a significant factor driving the eukaryotic community structure along this continuum.

Archaeal diversity: temporal variation in the arsenic-rich creek sediments of Carnoulès Mine, France.

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.

Archaeal diversity in a Fe-As rich acid mine drainage at Carnoulès (France).

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.

Diversity of microorganisms in Fe-As-rich acid mine drainage waters of Carnoulès, France.

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.

A new bacterial strain mediating As oxidation in the Fe-rich biofilm naturally growing in a groundwater Fe treatment pilot unit.

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.

Sorption and redox processes controlling arsenic fate and transport in a stream impacted by acid mine drainage.

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 (pH<5) 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 (

Immobilization of arsenite and ferric iron by Acidithiobacillus ferrooxidans and its relevance to acid mine drainage.

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.

Mediation of arsenic oxidation by Thiomonas sp. in acid-mine drainage (Carnoulès, France).

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 7.0-7.2), 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.