Temporal evolution of surface and sub-surface geochemistry and microbial communities of Pb-rich mine tailings during phytostabilization: A one-year pilot-scale study.

Old mine waste repositories can present health and/or environmental issues linked to their erosion, inducing dissemination of metals and metalloids in air and water that can be attenuated through phytostabilization. Here, the effect of this widespread phytomanagement option on the biogeochemistry of a Pb-rich mine waste was evaluated with a laboratory pilot-scale experiment giving access to the non-saturated and saturated zones below the rhizosphere compartment. Amendment of the tailings surface with biochar, manure and iron-oxide-rich ochre promoted growth of the seeded Agrostis capillaris plants. These events were accompanied by an increase of pH and a decrease of Pb concentration in pore water of the surface layer, and by a transient increase of Pb, Zn, and Ba concentrations in the deeper saturated levels. Macroscopic and microscopic observations (SEM) suggest that Pb was immobilized in A. capillaris rhizosphere through mechanical entrapment of tailing particles. Microbial taxonomic and metabolic diversities increased in the amended phytostabilized surface levels, with a rise of the proportion of heterotrophic micro-organisms. Below the surface, a transient modification of microbial communities was observed in the non-saturated and saturated levels, however 11 months after seeding, the prokaryotic community of the deepest saturated zone was close to that of the initial tailings. pH and water saturation seemed to be the main parameters driving prokaryotic communities' structures. Results obtained at pilot-scale will help to precisely evaluate the impacts of phytostabilization on the temporal evolution of reactions driving the fate of pollutants inside the tailings dumps.

Co-culture of Salix viminalis and Trifolium repens for the phytostabilisation of Pb and As in mine tailings amended with hardwood biochar.

Metal(loid) soil pollution causes environmental and health issues, and thus those sites need to be remediated. This can be done through phytostabilization, in combination with biochar amendment. The objectives were to investigate the potential of Salix viminalis L. associated with Trifolium repens L. for the phytostabilization of biochar-amended contaminated soils by assessing (1) the tolerance of both plants to metal(loid)s, through the biomass production, (2) the concentrations of metal(loid)s in plant parts and (3) the concentrations of metal(loid)s in soil pore water and percolation waters. Results showed that plant growth affected soil pore water Physico-chemical properties and metal(loid) mobility. When comparing the mono- and poly-cultures, although pH was higher with the polyculture than the monoculture, the decrease in Pb mobility did not differ. Moreover, the leachate analysis showed that As concentration in the soil particles leached from the soil was higher in the polyculture condition, while Pb concentration was the highest in the willow vegetated condition. Finally, willow dry weight was not affected by the presence of clover, while clover dry weight was lower when it was grown with willow. In conclusion, the results showed that the willow and clover polyculture was not better than the monoculture of these two species for the phytomanagement of a former mine site amended with biochar.

Mobility of Pb, Zn, Ba, As and Cd toward soil pore water and plants (willow and ryegrass) from a mine soil amended with biochar.

Mine soils often contain metal(loid)s that may lead to serious environmental problems. Phytoremediation, consisting in covering the soil with specific plants with the possible addition of amendments, represents an interesting way of enhancing the quality of mine soils by retaining contaminants and reducing soil erosion. In order to study the effect of an assisted phytoremediation (with willow and ryegrass) on the properties of soil pore water (SPW), we investigated the impact of amendment with biochar (BC) combined with the planting of willow and ryegrass on the behavior of several metal(loid)s (Pb, Zn, Ba, As, and Cd) in a mine soil. Data on the physicochemical parameters and concentrations of the different metal(loid)s in both SPW and in plant tissues of willow and ryegrass highlight the importance of BC for SPW properties in terms of reductions in soluble concentrations of Pb and Zn, although there was no effect on the behavior of As and Cd. BC also increased soluble concentrations of Ba, probably related to ion release by the BC. By improving major ions available in mine soil, BC improved the lifetime of plants and enhanced their growth. Plant development did not appear to significantly affect the physicochemical parameters of SPW. Willow and ryegrass growing on soil with BC incorporated Cd and Ba into their tissues. The influence of plants on the behavior of metal(loid)s was noticeable only for ryegrass growing in soil with 2% BC, where it modified the behavior of Pb and Ba.

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.

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.

Microbial Sulfate Reduction Enhances Arsenic Mobility Downstream of Zerovalent-Iron-Based Permeable Reactive Barrier.

We assessed the potential of zerovalent-iron- (Fe(0)) based permeable reactive barrier (PRB) systems for arsenic (As) remediation in the presence or absence of microbial sulfate reduction. We conducted long-term (200 day) flow-through column experiments to investigate the mechanisms of As transformation and mobility in aquifer sediment (in particular, the PRB downstream linkage). Changes in As speciation in the aqueous phase were monitored continuously. Speciation in the solid phase was determined at the end of the experiment using X-ray absorption near-edge structure (XANES) spectroscopy analysis. We identified thio-As species in solution and AsS in solid phase, which suggests that the As(V) was reduced to As(III) and precipitated as AsS under sulfate-reducing conditions and remained as As(V) under abiotic conditions, even with low redox potential and high Fe(II) content (4.5 mM). Our results suggest that the microbial sulfate reduction plays a key role in the mobilization of As from Fe-rich aquifer sediment under anoxic conditions. Furthermore, they illustrate that the upstream-downstream linkage of PRB affects the speciation and mobility of As in downstream aquifer sediment, where up to 47% of total As initially present in the sediment was leached out in the form of mobile thio-As species.

Metagenomic insights into microbial metabolism affecting arsenic dispersion in Mediterranean marine sediments.

Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over-represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over-represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate-reducing bacteria and sulphur-oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico-chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage.

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.

Bio-precipitation of arsenic and antimony in a sulfate-reducing bioreactor treating real acid mine drainage water.

Arsenic (As) and antimony (Sb) from mining sites can seep into aquatic ecosystems by acid mine drainage (AMD). Here, the possibility of concomitantly removing As and Sb from acidic waters by precipitation of sulfides induced by sulfate-reducing bacteria (SRB) was investigated in a fixed-bed column bioreactor. The real AMD water used to feed the bioreactor contained nearly 1 mM As, while the Sb concentrations were increased (0.008 ± 0.006 to 1.01 ± 0.07 mM) to obtain an Sb/As molar ratio = 1. Results showed that the addition of Sb did not affect the efficiency of As bio-precipitation. Sb was removed efficiently (up to 97.9% removal) between the inlet and outlet of the bioreactor, together with As (up to 99.3% removal) in all conditions. Sb was generally removed as it entered the bioreactor. Appreciable sulfate reduction occurred in the bioreactor, which could have been linked to the stable presence of a major SRB operational taxonomic unit affiliated with the Desulfosporosinus genus. The bacterial community included polymer degraders, fermenters, and acetate degraders. Results suggested that sulfate reduction could be a suitable bioremediation process for the simultaneous removal of Sb and As from AMD.

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.

Evaluation of antimony availability in a mining context: Impact for the environment, and for mineral exploration and exploitation.

This work aims to establish Sb mobility, its transfer to biota and its effect on soil health in a semi-arid climate. The results show the presence of stibnite (Sb2S3) as the main primary Sb compound, bindhemite (Pb2Sb2O6(O,OH)), and minor proportions of stibiconite (Sb3+(Sb5+)2O6(OH)) as oxidised Sb species. This research also observes very high total Sb contents in mining materials (max: 20,000 mg kg-1) and soils (400-3000 mg kg-1), with physical dispersion around mining materials restricted to 450 m. The soil-to-plant transfer is very low, (bioaccumulation factor: 0.0002-0.1520). Most Sb remains in a residual fraction (99.9%), a very low fraction is bound to Fe and Mn oxy-hydroxides or organic matter, and a negligible proportion of Sb is leachable. The higher Sb mobility rates has been found under oxidising conditions with a long contact time between solids and water. The main factors that explain the poor Sb mobility and dispersion in the mining area are the low annual rainfall rates that slow down the Sb mobilisation process and the scarce formation of oxidised Sb compounds. All these data suggest poor Sb (III) formation and a low toxicological risk in the area associated with past mining activities. The low mobility of Sb suggests advantages for future sustainable mining of such ore deposits in a semi-arid climate and is also indicative of the limitations of geochemical exploration in the search for new Sb deposits.

Mobility of antimony in contrasting surface environments of a mine site: influence of redox conditions and microbial communities.

Microbial processes can influence the complex geochemical behaviour of the toxic metalloid antimony (Sb) in mining environments. The present study is aimed to evaluate the influence of microbial communities on the mobility of Sb from solid phases to water in different compartments and redox conditions of a mining site in southwest (SW) Spain. Samples of surface materials presenting high Sb concentrations, from two weathered mining waste dumps, and an aquatic sediment were incubated in slurries comparing oxic and anoxic conditions. The initial microbial communities of the three materials strongly differed. Incubations induced an increase of microbial biomass and an evolution of the microbial communities' structures and compositions, which diverged in different redox conditions. The presence of active bacteria always influenced the mobility of Sb, except in the neutral pH waste incubated in oxic conditions. The effect of active microbial activities in oxic conditions was dependent on the material: Sb oxic release was biologically amplified with the acidic waste, but attenuated with the sediment. Different bacterial genera involved in Sb, Fe and S oxidation or reduction were present and/or grew during incubation of each material. The results highlighted the wide diversity of microbial communities and metabolisms at the small geographic scale of a mining site and their strong implication in Sb mobility.

Water and soil contaminated by arsenic: the use of microorganisms and plants in bioremediation.

Owing to their roles in the arsenic (As) biogeochemical cycle, microorganisms and plants offer significant potential for developing innovative biotechnological applications able to remediate As pollutions. This possible use in bioremediation processes and phytomanagement is based on their ability to catalyse various biotransformation reactions leading to, e.g. the precipitation, dissolution, and sequestration of As, stabilisation in the root zone and shoot As removal. On the one hand, genomic studies of microorganisms and their communities are useful in understanding their metabolic activities and their interaction with As. On the other hand, our knowledge of molecular mechanisms and fate of As in plants has been improved by laboratory and field experiments. Such studies pave new avenues for developing environmentally friendly bioprocessing options targeting As, which worldwide represents a major risk to many ecosystems and human health.

Biogeochemical behaviour of geogenic As in a confined aquifer of the Sologne region, France.

Arsenic (As) is one of the main toxic elements of geogenic origin that impact groundwater quality and human health worldwide. In some groundwater wells of the Sologne region (Val de Loire, France), drilled in a confined aquifer, As concentrations exceed the European drinking water standard (10 µg L-1). The monitoring of one of these drinking water wells showed As concentrations in the range 20-25 µg L-1. The presence of dissolved iron (Fe), low oxygen concentration and traces of ammonium indicated reducing conditions. The δ34SSO4 was anticorrelated with sulphate concentration. Drilling allowed to collect detrital material corresponding to a Miocene floodplain and crevasse splay with preserved plant debris. The level that contained the highest total As concentration was a silty-sandy clay containing 26.9 mg kg-1 As. The influence of alternating redox conditions on the behaviour of As was studied by incubating this material with site groundwater, in biotic or inhibited bacterial activities conditions, without synthetic organic nutrient supply, in presence of H2 during the reducing periods. The development of both AsV-reducing and AsIII-oxidising microorganisms in biotic conditions was evidenced. At the end of the reducing periods, total As concentration strongly increased in biotic conditions. The microflora influenced As speciation, released Fe and consumed nitrate and sulphate in the water phase. Microbial communities observed in groundwater samples strongly differed from those obtained at the end of the incubation experiment, this result being potentially related to influence of the sediment compartment and to different physico-chemical conditions. However, both included major Operating Taxonomic Units (OTU) potentially involved in Fe and S biogeocycles. Methanogens emerged in the incubated sediment presenting the highest solubilised As and Fe. Results support the hypothesis of in-situ As mobilisation and speciation mediated by active biogeochemical processes.

Risk management for arsenic in agricultural soil-water systems: lessons learned from case studies in Europe.

Chronic exposure to arsenic may be detrimental to health. We investigated the behaviour, remediation and risk management of arsenic in Freiberg, Germany, characterized by past mining activities, and near Verdun in France, where World War I ammunition was destroyed. The main results included: (1) pot experiments using a biologically synthesized adsorbent (sorpP) with spring barley reduced the mobility of arsenic, (2) the Omega-3 Index ecotoxicological tests verified that sorpP reduced the uptake and toxicity of arsenic in plants, (3) reverse osmosis membrane systems provided 99.5% removal efficiency of arsenic from surface water, (4) the sustainability assessment revealed that adsorption and coagulation-filtration processes were the most feasible options for the treatment of surface waters with significant arsenic concentrations, and (5) a model was developed for assessing health risk due to arsenic exposure. Risk management is the main option for extensive areas, while remediation options that directly treat the soil can only be considered in small areas subject to sensitive use. We recommend the risk management procedure developed in Germany for other parts of the world where both geogenic and anthropogenic arsenic is present in agricultural soil and water. Risk management measures have been successful both in Freiberg and in Verdun.

Proposal that the arsenite-oxidizing organisms Thiomonas cuprina and 'Thiomonas arsenivorans' be reclassified as strains of Thiomonas delicata, and emended description of Thiomonas delicata.

The three As(III)-oxidizing members of the class Betaproteobacteria Thiomonas delicata, Thiomonas cuprina and 'Thiomonas arsenivorans' were isolated from mining sites in geographically distinct areas, namely Japan, Germany and France, respectively. They are all able to oxidize As(III) but only 'T. arsenivorans' and T. cuprina show efficient autotrophic growth with As(III) and are able to grow on a sole carbon source. These two organisms are also motile, whereas T. delicata is not. Only T. cuprina can grow autotrophically on chalcopyrite. The three strains share >99% gene sequence similarity with each other based on their 16S rRNA genes and 16S-23S ITS regions. DNA-DNA hybridization results are above, or close to, the threshold value of 70% recommended for the definition of bacterial species. The three taxa show very similar fatty acid profiles with differences only in five minor fatty acid components. They possess phylogenetic and chemotaxonomic similarities supporting the reclassification of these taxa as a single species. We propose that 'T. arsenivorans' and T. cuprina be reassigned as strains of T. delicata (type strain DSM 17897(T)).

Influence of agricultural amendments on arsenic biogeochemistry and phytotoxicity in a soil polluted by the destruction of arsenic-containing shells.

Agricultural soils can contain high arsenic (As) concentrations due to specific geological contexts or pollution. Fertilizer amendments could influence As speciation and mobility thus increasing its transfer to crops and its toxicity. In the present study, field-relevant amounts of fertilizers were applied to soils from a cultivated field that was a former ammunition-burning site. Potassium phosphate (KP), ammonium sulfate and organic matter (OM) were applied to these soils in laboratory experiments to assess their impact on As leaching, bioavailability to Lactuca sativa and microbial parameters. None of the fertilizers markedly influenced As speciation and mobility, although trends showed an increase of mobility with KP and a decrease of mobility with ammonium sulfate. Moreover, KP induced a small increase of As in Lactuca sativa, and the polluted soil amended with ammonium sulfate was significantly less phytotoxic than the un-amended soil. Most probable numbers of AsIII-oxidizing microbes and AsIII-oxidizing activity were strongly linked to As levels in water and soils. Ammonium sulfate negatively affected AsIII-oxidizing activity in the un-polluted soil. Whereas no significant effect on As speciation in water could be detected, amendments may have an impact in the long term.

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

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 E(h) 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.

Desulfosporosinus acidiphilus sp. nov.: a moderately acidophilic sulfate-reducing bacterium isolated from acid mining drainage sediments : New taxa: Firmicutes (Class Clostridia, Order Clostridiales, Family Peptococcaceae).

An obligately anaerobic, spore-forming, acidophilic sulfate-reducing bacterium, strain SJ4(T), was isolated from an acid mining effluent decantation pond sediment sample (pH around 3.0). Cells were Gram negative, non-motile, curved rods occurring singly. Strain SJ4(T) grew at pH 3.6-5.5 with an optimum at pH 5.2. Strain SJ4(T) utilized H(2), lactate, pyruvate, glycerol, glucose, and fructose as electron donors. Lactate and glucose were weakly used. Sulfate was used as electron acceptors, but not sulfite, elemental sulfur, arsenate (V), and fumarate. The G + C content of genomic DNA was 42.3 mol% (HPLC). 16S rRNA gene sequence analysis indicated that strain SJ4(T) belonged to the genus Desulfosporosinus within the family Peptococcaceae in the phylum Firmicutes. The level of 16S rRNA gene sequence similarity with other Desulfosporosinus species was 94.7-96.2%, D. orientis DSM 765(T) (similarity of 96.2%) and D. auripigmenti DSM 13351(T) (similarity of 95%) being its closest relatives. DNA-DNA relatedness values with D. orientis and D. auripigmenti were 16.5 and 31.8%, respectively. On the basis of phenotypic, phylogenetic, and genetic characteristics, strain SJ4(T) represents a novel species within the genus Desulfosporosinus, for which the name Desulfosporosinus acidiphilus sp. nov. is proposed. The type strain is SJ4(T) (=DSM 22704(T) = JCM 16185(T)).

Rapid and simple As(III) quantification using a turbidimetric test for the monitoring of microbial arsenic bio-transformation.

A turbidimetric test for rapid quantification of As(III) (detection limit of 3 mg/L, quantification range of 10-100 mg/L) in liquid growth medium was developed for assessing and monitoring microbial As(III)-oxidizing and As(V)-reducing activities. This test is based on As(III) chelation with pyrrolidine dithiocarbamate followed by spectrometric measurement of absorbance, and was validated by comparison with AAS quantification of As after As(III)/As(V) separation.