Towards an understanding of the factors controlling bacterial diversity and activity in semi-passive Fe- and As-oxidizing bioreactors treating arsenic-rich acid mine drainage.

Semi-passive bioreactors based on iron and arsenic oxidation and coprecipitation are promising for the treatment of As-rich acid mine drainages. However, their performance in the field remains variable and unpredictable. Two bioreactors filled with distinct biomass carriers (plastic or a mix of wood and pozzolana) were monitored during 1 year. We characterized the dynamic of the bacterial communities in these bioreactors, and explored the influence of environmental and operational drivers on their diversity and activity. Bacterial diversity was analyzed by 16S rRNA gene metabarcoding. The aioA genes and transcripts were quantified by qPCR and RT-qPCR. Bacterial communities were dominated by several iron-oxidizing genera. Shifts in the communities were attributed to operational and physiochemical parameters including the nature of the biomass carrier, the water pH, temperature, arsenic, and iron concentrations. The bioreactor filled with wood and pozzolana showed a better resilience to disturbances, related to a higher bacterial alpha diversity. We evidenced for the first time aioA expression in a treatment system, associated with the presence of active Thiomonas spp. This confirmed the contribution of biological arsenite oxidation to arsenic removal. The resilience and the functional redundancy of the communities developed in the bioreactors conferred robustness and stability to the treatment systems.

Contrasting arsenic biogeochemical cycling in two Moroccan alkaline pit lakes.

Pit lakes resulting from the flooding of abandoned mines represent a valuable freshwater reserve. However, water contamination by toxic elements, including arsenic, compromises their use for freshwater supply. For a better management of these reserves, our aim was to gain insight into arsenic cycling in two Moroccan alkaline pit lakes. We first showed that dimethylarsenic dominated in stratified lake ZA whereas in lake ZL1, As(V) was prevailing. Because microbially mediated processes largely contribute to arsenic cycling, the diversity of arsenic-methylating and -oxidizing bacteria was determined through the sequencing of arsM and aioA genes. Diverse arsM-carrying bacteria were thriving in ZA while a low diversity of aioA genes was detected in ZL1. We also determined the structure of the total bacterial communities by fingerprinting (ARISA). Contrasting arsenic speciation and bacterial communities in the two lakes were associated with differences of conductivity, Total Organic Carbon and temperature. In ZA, dissolved oxygen and redox potential were the main factors driving the total bacterial community structure and the ArsM diversity. In ZL1, stable bacterial communities were associated with limited water physico-chemistry variations. Our study provides new insights into the biogeochemical behavior of arsenic and the role of arsenic transforming bacteria in alkaline pit lakes.

Hydraulic retention time affects bacterial community structure in an As-rich acid mine drainage (AMD) biotreatment process.

Arsenic removal consecutive to biological iron oxidation and precipitation is an effective process for treating As-rich acid mine drainage (AMD). We studied the effect of hydraulic retention time (HRT)-from 74 to 456 min-in a bench-scale bioreactor exploiting such process. The treatment efficiency was monitored during 19 days, and the final mineralogy and bacterial communities of the biogenic precipitates were characterized by X-ray absorption spectroscopy and high-throughput 16S rRNA gene sequencing. The percentage of Fe(II) oxidation (10-47%) and As removal (19-37%) increased with increasing HRT. Arsenic was trapped in the biogenic precipitates as As(III)-bearing schwertmannite and amorphous ferric arsenate, with a decrease of As/Fe ratio with increasing HRT. The bacterial community in the biogenic precipitate was dominated by Fe-oxidizing bacteria whatever the HRT. The proportion of Gallionella and Ferrovum genera shifted from respectively 65 and 12% at low HRT to 23 and 51% at high HRT, in relation with physicochemical changes in the treated water. aioA genes and Thiomonas genus were detected at all HRT although As(III) oxidation was not evidenced. To our knowledge, this is the first evidence of the role of HRT as a driver of bacterial community structure in bioreactors exploiting microbial Fe(II) oxidation for AMD treatment.

Temperature and nutrients as drivers of microbially mediated arsenic oxidation and removal from acid mine drainage.

Microbial oxidation of iron (Fe) and arsenic (As) followed by their co-precipitation leads to the natural attenuation of these elements in As-rich acid mine drainage (AMD). The parameters driving the activity and diversity of bacterial communities responsible for this mitigation remain poorly understood. We conducted batch experiments to investigate the effect of temperature (20 vs 35 °C) and nutrient supply on the rate of Fe and As oxidation and precipitation, the bacterial diversity (high-throughput sequencing of 16S rRNA gene), and the As oxidation potential (quantification of aioA gene) in AMD from the Carnoulès mine (France). In batch incubated at 20 °C, the dominance of iron-oxidizing bacteria related to Gallionella spp. was associated with almost complete iron oxidation (98%). However, negligible As oxidation led to the formation of As(III)-rich precipitates. Incubation at 35 °C and nutrient supply both stimulated As oxidation (71-75%), linked to a higher abundance of aioA gene and the dominance of As-oxidizing bacteria related to Thiomonas spp. As a consequence, As(V)-rich precipitates (70-98% of total As) were produced. Our results highlight strong links between indigenous bacterial community composition and iron and arsenic removal efficiency within AMD and provide new insights for the future development of a biological treatment of As-rich AMD.

Dynamics of Bacterial Communities Mediating the Treatment of an As-Rich Acid Mine Drainage in a Field Pilot.

Passive treatment based on iron biological oxidation is a promising strategy for Arsenic (As)-rich acid mine drainage (AMD) remediation. In the present study, we characterized by 16S rRNA metabarcoding the bacterial diversity in a field-pilot bioreactor treating extremely As-rich AMD in situ, over a 6 months monitoring period. Inside the bioreactor, the bacterial communities responsible for iron and arsenic removal formed a biofilm ("biogenic precipitate") whose composition varied in time and space. These communities evolved from a structure at first similar to the one of the feed water used as an inoculum to a structure quite similar to the natural biofilm developing in situ in the AMD. Over the monitoring period, iron-oxidizing bacteria always largely dominated the biogenic precipitate, with distinct populations (Gallionella, Ferrovum, Leptospirillum, Acidithiobacillus, Ferritrophicum), whose relative proportions extensively varied among time and space. A spatial structuring was observed inside the trays (arranged in series) composing the bioreactor. This spatial dynamic could be linked to the variation of the physico-chemistry of the AMD water between the raw water entering and the treated water exiting the pilot. According to redundancy analysis (RDA), the following parameters exerted a control on the bacterial communities potentially involved in the water treatment process: dissolved oxygen, temperature, pH, dissolved sulfates, arsenic and Fe(II) concentrations and redox potential. Appreciable arsenite oxidation occurring in the bioreactor could be linked to the stable presence of two distinct monophylogenetic groups of Thiomonas related bacteria. The ubiquity and the physiological diversity of the bacteria identified, as well as the presence of bacteria of biotechnological relevance, suggested that this treatment system could be applied to the treatment of other AMD.

Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage.

The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ.

Diversity and Distribution of Arsenic-Related Genes Along a Pollution Gradient in a River Affected by Acid Mine Drainage.

Some microorganisms have the capacity to interact with arsenic through resistance or metabolic processes. Their activities contribute to the fate of arsenic in contaminated ecosystems. To investigate the genetic potential involved in these interactions in a zone of confluence between a pristine river and an arsenic-rich acid mine drainage, we explored the diversity of marker genes for arsenic resistance (arsB, acr3.1, acr3.2), methylation (arsM), and respiration (arrA) in waters characterized by contrasted concentrations of metallic elements (including arsenic) and pH. While arsB-carrying bacteria were representative of pristine waters, Acr3 proteins may confer to generalist bacteria the capacity to cope with an increase of contamination. arsM showed an unexpected wide distribution, suggesting biomethylation may impact arsenic fate in contaminated aquatic ecosystems. arrA gene survey suggested that only specialist microorganisms (adapted to moderately or extremely contaminated environments) have the capacity to respire arsenate. Their distribution, modulated by water chemistry, attested the specialist nature of the arsenate respirers. This is the first report of the impact of an acid mine drainage on the diversity and distribution of arsenic (As)-related genes in river waters. The fate of arsenic in this ecosystem is probably under the influence of the abundance and activity of specific microbial populations involved in different As biotransformations.

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.

Fate of Sb(V) and Sb(III) species along a gradient of pH and oxygen concentration in the Carnoulès mine waters (Southern France).

The speciation and behaviour of antimony were investigated in surface waters downstream from the abandoned Pb-Zn Carnoulès mine (Gard, France). These waters exhibit a permanent gradient of oxygen concentration and pH, ranging from acid suboxic in Reigous Creek at the outlet of sulfide tailings impoundment, to near neutral oxygenated at downstream sites along the rivers Amous and Gardon. The concentration of total dissolved (<0.22 µm) antimony, acquired through a seven-year monitoring, decreased from 7.7-409.9 µg L(-1) at the source of Reigous Creek to 0.22-0.45 µg L(-1) in the Gardon River, showing natural Sb attenuation. Speciation analysis carried out during three surveys indicated that Sb(III) represented up to 70% of the total dissolved Sb concentration at the source of Reigous Creek, while Sb(V) represented less than 50%. Field characterization showed that Sb(III) and Sb(V) species were attenuated through dilution and were also removed from the dissolved phase during downstream transport. Speciation analysis in suspended particulate matter extracts gave a distribution of particulate Sb into 70 to 100% of Sb(III) and less than 30% of Sb(V). The removal of Sb(III) and Sb(V) species from the dissolved phase was concordant with the oversaturation of Reigous Creek water relative to Sb(III)- and Sb(V)-oxides and Sb(III)- and Sb(V)-Fe oxides. Sb(III) was more efficiently removed than Sb(V) or As(III) and it was no more detectable in the dissolved phase at downstream sites in the rivers Amous and Gardon. Conversely, the concentration of Sb(V) in the rivers Amous and Gardon still denoted contamination arising from the Carnoulès mine. The range of log Kd values, from 2.4 L kg(-1) to 4.9 L kg(-1), indicated that Sb was mainly transported in the dissolved phase downstream the Reigous Creek input. Altogether, these results give a better understanding of the fate of Sb downstream from sulfide-rich mining wastes.

Efficient neuronal in vitro and in vivo differentiation after immunomagnetic purification of mESC derived neuronal precursors.

The cellular heterogeneity that is generated during the differentiation of pluripotent stem cells into specific neural subpopulations represents a major obstacle for experimental and clinical progress. To address this problem we developed an optimized strategy for magnetic isolation of PSA-NCAM positive neuronal precursors from embryonic stem cells (ESCs) derived neuronal cultures. PSA-NCAM enrichment at an early step of the in vitro differentiation process increased the number of ES cell derived neurons and reduced cellular diversity. Gene expression analysis revealed that mainly genes involved in neuronal activity were over-represented after purification. In vitro derived PSA-NCAM(+) enriched precursors were characterized in vivo through grafting into the forebrain of adult mice. While unsorted control cells 40 days post graft gave rise to a mixed population composed of immature precursors, early postmitotic neurons and glial cells, PSA-NCAM(+) enriched cells differentiated predominantly into NeuN positive cells. Furthermore, PSA-NCAM enriched population showed efficient migration towards the olfactory bulb after transplantation into the rostral migratory stream of the forebrain neurogenic system. Thus, enrichment of neuronal precursors based on PSA-NCAM expression represents a general and straightforward approach to narrow cellular heterogeneity during neuronal differentiation of pluripotent cells.

Agrin-signaling is necessary for the integration of newly generated neurons in the adult olfactory bulb.

In the adult forebrain, new interneurons are continuously generated and integrated into the existing circuitry of the olfactory bulb (OB). In an attempt to identify signals that regulate this synaptic integration process, we found strong expression of agrin in adult generated neuronal precursors that arrive in the olfactory bulb after their generation in the subventricular zone. While the agrin receptor components MuSK and Lrp4 were below detection level in neuron populations that represent synaptic targets for the new interneurons, the alternative receptor α3-Na(+)K(+)-ATPase was strongly expressed in mitral cells. Using a transplantation approach, we demonstrate that agrin-deficient interneuron precursors migrate correctly into the OB. However, in contrast to wild-type neurons, which form synapses and survive for prolonged periods, mutant neurons do not mature and are rapidly eliminated. Using in vivo brain electroporation of the olfactory system, we show that the transmembrane form of agrin alone is sufficient to mediate integration and demonstrate that excess transmembrane agrin increases the number of dendritic spines. Last, we provide in vivo evidence that an interaction between agrin and α3-Na(+)K(+)-ATPase is of functional importance in this system.

Coupling between hydrodynamic forces and planar cell polarity orients mammalian motile cilia.

In mammals, motile cilia cover many organs, such as fallopian tubes, respiratory tracts and brain ventricles. The development and function of these organs critically depend on efficient directional fluid flow ensured by the alignment of ciliary beating. To identify the mechanisms involved in this process, we analysed motile cilia of mouse brain ventricles, using biophysical and molecular approaches. Our results highlight an original orientation mechanism for ependymal cilia whereby basal bodies first dock apically with random orientations, and then reorient in a common direction through a coupling between hydrodynamic forces and the planar cell polarity (PCP) protein Vangl2, within a limited time-frame. This identifies a direct link between external hydrodynamic cues and intracellular PCP signalling. Our findings extend known PCP mechanisms by integrating hydrodynamic forces as long-range polarity signals, argue for a possible sensory role of ependymal cilia, and will be of interest for the study of fluid flow-mediated morphogenesis.

Efficient in vivo electroporation of the postnatal rodent forebrain.

Functional gene analysis in vivo represents still a major challenge in biomedical research. Here we present a new method for the efficient introduction of nucleic acids into the postnatal mouse forebrain. We show that intraventricular injection of DNA followed by electroporation induces strong expression of transgenes in radial glia, neuronal precursors and neurons of the olfactory system. We present two proof-of-principle experiments to validate our approach. First, we show that expression of a human isoform of the neural cell adhesion molecule (hNCAM-140) in radial glia cells induces their differentiation into cells showing a neural precursor phenotype. Second, we demonstrate that p21 acts as a cell cycle inhibitor for postnatal neural stem cells. This approach will represent an important tool for future studies of postnatal neurogenesis and of neural development in general.

Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain.

In the adult mouse forebrain, large numbers of neuronal precursors, destined to become GABA- and dopamine-producing interneurons of the olfactory bulb (OB), are generated in the subventricular zone (SVZ). Although this neurogenic system represents a potential reservoir of stem and progenitor cells for brain repair approaches, information about the survival and differentiation of SVZ-derived cells in ectopic brain regions is still fragmentary. We show here that ectopic grafting of SVZ tissue gave rise to two morphologically distinguishable cell types displaying oligodendrocytic or astrocytic characteristics. Since SVZ tissue contains neuronal and glial progenitors, we used magnetic cell sorting to deplete A2B5+ glial progenitors from the dissociated SVZ and to positively select cells that express PSA-NCAM. This procedure allowed the purification of neuronal precursors expressing TUJ1, DCX and GAD65/67. Transplantation of these cells led again to the generation of the same two glial cell types, showing that committed interneuron precursors undergo glial differentiation outside their normal environment.