Catherine Joulian

Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria

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.

Oxidation of arsenite by Thiomonas strains and characterization of Thiomonas arsenivorans sp. nov.

A novel bacterium, strain b6T (T=type strain), was isolated from a disused mine site by growth using arsenite [As(III)] as energy source in a simple mineral medium. Cells of strain b6T were rod-shaped, Gram-negative, non-sporulating and motile. Optimum growth occurred at temperatures between 20 and 30xa0°C, and at pH between 4.0 and 7.5. Strain b6T grew chemoautotrophically on As(III), sulphur and thiosulphate, and also heterotrophically on yeast extract and a variety of defined organic compounds. Several other Thiomonas strains, including the type species Thiomonas (Tm.) intermedia, were able to oxidize As(III), though only strain b6T and strain NO115 could grow using As(III) as sole energy source in the absence of any organic compound. The G+C content of the DNA of strain b6T was 65.1xa0molxa0%. Comparative small subunit (SSU) ribosomal RNA (rRNA) analysis indicated that strain b6T belongs to the genus Thiomonas in the β-subdivision of the Proteobacteria. It was closely related to an unnamed Thiomonas strain (NO115) isolated from a Norwegian mining site, though sequence identities between strain b6T and characterized Thiomonas species were less than 95%. DNA–DNA hybridization between strain b6T and the type species of the genus Tm. intermedia showed less than 50% homology. On the basis of phylogenetic and phenotypic characteristics, strain b6T (DSM 16361T, LMG 22795T) is proposed as the type strain of the new species Thiomonas arsenivorans, sp. nov.

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.

Decoupling of arsenic and iron release from ferrihydrite suspension under reducing conditions: a biogeochemical model

High levels of arsenic in groundwater and drinking water are a major health problem. Although the processes controlling the release of As are still not well known, the reductive dissolution of As-rich Fe oxyhydroxides has so far been a favorite hypothesis. Decoupling between arsenic and iron redox transformations has been experimentally demonstrated, but not quantitatively interpreted. Here, we report on incubation batch experiments run with As(V) sorbed on, or co-precipitated with, 2-line ferrihydrite. The biotic and abiotic processes of As release were investigated by using wet chemistry, X-ray diffraction, X-ray absorption and genomic techniques. The incubation experiments were carried out with a phosphate-rich growth medium and a community of Fe(III)-reducing bacteria under strict anoxic conditions for two months. During the first month, the release of Fe(II) in the aqueous phase amounted to only 3% to 10% of the total initial solid Fe concentration, whilst the total aqueous As remained almost constant after an initial exchange with phosphate ions. During the second month, the aqueous Fe(II) concentration remained constant, or even decreased, whereas the total quantity of As released to the solution accounted for 14% to 45% of the total initial solid As concentration. At the end of the incubation, the aqueous-phase arsenic was present predominately as As(III) whilst X-ray absorption spectroscopy indicated that more than 70% of the solid-phase arsenic was present as As(V). X-ray diffraction revealed vivianite Fe(II)3(PO4)2.8H2O in some of the experiments. A biogeochemical model was then developed to simulate these aqueous- and solid-phase results. The two main conclusions drawn from the model are that (1) As(V) is not reduced during the first incubation month with high Eh values, but rather re-adsorbed onto the ferrihydrite surface, and this state remains until arsenic reduction is energetically more favorable than iron reduction, and (2) the release of As during the second month is due to its reduction to the more weakly adsorbed As(III) which cannot compete against carbonate ions for sorption onto ferrihydrite. The model was also successfully applied to recent experimental results on the release of arsenic from Bengal delta sediments.

Sulfobacillus benefaciens sp. nov., an acidophilic facultative anaerobic Firmicute isolated from mineral bioleaching operations.

Gram-positive bacteria found as the sole Firmicutes present in two mineral bioleaching stirred tanks, and a third bacterium isolated from a heap leaching operation, were shown to be closely related to each other but distinct from characterized acidophilic iron- and sulfur-oxidizing bacteria of the genus Sulfobacillus, to which they were affiliated. One of the isolates (BRGM2) was shown to be a thermo-tolerant (temperature optimum 38.5°C, and maximum 47°C) obligate acidophile (pH optimum 1.5, and minimum 0.8), and also noted to be a facultative anaerobe, growing via ferric iron respiration in the absence of oxygen. Although isolates BRGM2 and TVK8 were able to metabolize many monomeric organic substrates, their propensity for autotrophic growth was found to be greater than that of Sulfobacillus thermosulfidooxidans and the related acidophile, Sb. acidophilus. Faster growth rates of the novel isolates in the absence of organic carbon was considered to be a major reason why they, rather than Sb. thermosulfidooxidans (which shared many physiological characteristics) more successfully exploited conditions in the stirred tanks. Based on their phylogenetic and phenotypic characteristics, the isolates are designated strains of the proposed novel species, Sulfobacillus benefaciens, with isolate BRGM2 nominated as the type strain.

Population structure and abundance of arsenite-oxidizing bacteria along an arsenic pollution gradient in waters of the Upper Isle River Basin, France.

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 Eh 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.

AsIII oxidation by Thiomonas arsenivorans in up-flow fixed-bed reactors coupled to As sequestration onto zero-valent iron-coated sand.

The combined processes of biological As(III) oxidation and removal of As(III) and As(V) by zero-valent iron were investigated with synthetic water containing high As(III) concentration (10 mg L(-1)). Two up-flow fixed-bed reactors (R1 and R2) were filled with 2 L of sieved sand (d = 3 ± 1 mm) while zero-valent iron powder (d = 76 μm; 1% (w/w) of sand) was mixed evenly with sand in R2. Thiomonas arsenivorans was inoculated in the two reactors. The pilot unit was studied for 33 days, with HRT of 4 and 1 h. The maximal As(III) oxidation rate was 8.36 mg h(-1) L(-1) in R1 and about 45% of total As was removed in R2 for an HRT of 1 h. A first order model fitted well with the As(III) concentration evolution at the different levels in R1. At the end of the pilot monitoring, batch tests were conducted with support collected at different levels in R1. They showed that bacterial As(III) oxidation rate was correlated with the axial length of reactor, which could be explained by biomass distribution in reactor or by bacterial activity. In opposition, As(III) oxidation rate was not stable in R2 due to the simultaneous bacterial As(III) oxidation and chemical removal by zero-valent iron and its oxidant products. However, a durable removal of total As was realized and zero-valent iron was not saturated by As over 33 days in R2. Furthermore, the influence of zero-valent iron and its oxidant corrosion products on the evolution of As(III)-oxidizing bacteria diversity was highlighted by the molecular fingerprinting method of PCR-DGGE using aoxB gene as a functional marker of aerobic As(III) oxidizers.

A Simple Biogeochemical Process Removing Arsenic from a Mine Drainage Water

Arsenic is a toxic element commonly found in mining environments. The present study aimed to determine the influence of the indigenous bacterial population on the biogeochemical evolution of arsenic concentration in a mine drainage water. Biological As(III)-oxidizing activity was detected in diverse micro-environments along the water stream, from the source to the discharge point. Laboratory experiments showed that the bacterial population promoted As(III) and Fe(II) oxidation in conditions close to those of the site, i.e., temperature, water composition and oxygen availability. Immobilization of bacteria on pozzolana in a column bioreactor increased oxidation rates compared to on-site natural conditions: As concentration in the bioreactor outlet was less than 50 μg L −1 , whereas on-site total As concentration at the discharge point is close to 100 μg L −1 . The highest As(III) removal rate in the inoculated column reached 1900 μg L −1 h −1 and As(III) removal rate in the non-inoculated blank column was 42 μg L −1 h −1 . Two strains of As(III)-oxidizing bacteria, related to Variovorax paradoxus and Leptothrix cholodnii, were respectively isolated from a laboratory reactor and directly from site sludge. A diverse population of bacteria affiliated to nine phylogenetic groups belonging to the Proteobacteria and to the Bacteroidetes was identified in a laboratory reactor by molecular analyses of 16S rRNA genes. Amongst these, microorganisms already known to be potentially responsible for As(III) or Fe(II) oxidation, as well as for As(V) reduction co-existed: they included members of the genera Gallionella, Pseudomonas, Ralstonia, Sphingomonas, Methylobacterium and Methylophilus. On-site experiments confirmed the laboratory results, i.e. the removal of As from the contaminated effluent, and a passive on site treatment is currently developed for improving the quality of the discharged water.

Arsenite-induced changes in abundance and expression of arsenite transporter and arsenite oxidase genes of a soil microbial community

We describe a real-time PCR assay for the quantitative detection of arsB and ACR3(1) arsenite transporter gene families, two ubiquitous and key determinants of arsenic resistance in prokaryotes. The assay was applied in batch growth experiments using a wasteland soil bacterial community as an inoculum to investigate the effect of increasing arsenite [As(III)] concentrations on genes and transcript abundances. The aioA gene encoding the large subunit of arsenite oxidase was monitored in parallel. Results showed that arsB and ACR3(1) gene abundances correlated positively with the As(III) concentration. Both genes showed similar transcription patterns and strong upregulation by arsenic. Microbial As(III) oxidation occurred in As(III) spiked cultures and was associated with expression of the aioA gene in most cases. However, aioA was also expressed in several non-amended culture replicates. Analysis of cDNA clone libraries revealed that Pseudomonas was the dominant metabolically active genus whatever the As(III) concentration. Expressed arsB and ACR3(1) gene sequences were also affiliated with those from Pseudomonas, while expressed aioA sequences were more taxonomically diverse. The study suggests that arsenite transporter genes are appropriate biomarkers of arsenic stress that may be suitable for further exploring the adaptive response of bacterial communities to arsenic in contaminated environments.

Complete removal of arsenic and zinc from a heavily contaminated acid mine drainage via an indigenous SRB consortium

Acid mine drainages (AMD) are major sources of pollution to the environment. Passive bio-remediation technologies involving sulfate-reducing bacteria (SRB) are promising for treating arsenic contaminated waters. However, mechanisms of biogenic As-sulfide formation need to be better understood to decontaminate AMDs in acidic conditions. Here, we show that a high-As AMD effluent can be decontaminated by an indigenous SRB consortium. AMD water from the Carnoulès mine (Gard, France) was incubated with the consortium under anoxic conditions and As, Zn and Fe concentrations, pH and microbial activity were monitored during 94days. Precipitated solids were analyzed using electron microscopy (SEM/TEM-EDXS), and Extended X-Ray Absorption Fine Structure (EXAFS) spectroscopy at the As K-edge. Total removal of arsenic and zinc from solution (1.06 and 0.23mmol/L, respectively) was observed in two of the triplicates. While Zn precipitated as ZnS nanoparticles, As precipitated as amorphous orpiment (am-AsIII2S3) (33-73%), and realgar (AsIIS) (0-34%), the latter phase exhibiting a particular nanowire morphology. A minor fraction of As is also found as thiol-bound AsIII (14-23%). We propose that the formation of the AsIIS nanowires results from AsIII2S3 reduction by biogenic H2S, enhancing the efficiency of As removal. The present description of As immobilization may help to set the basis for bioremediation strategies using SRB.