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Chlordecone (CLD) is a very persistent synthetic organochlorine pesticide found in the French West Indies. Recently published work has demonstrated the potential of zero-valent iron to dechlorinate CLD by in situ chemical reduction (ISCR) in soils under water-saturated conditions, forming mono- to penta-dechlorinated CLD transformation products. These transformation products are more mobile than CLD and less toxic; however, nothing is known about their further degradation, although increasing evidence of CLD biodegradation by bacteria is being found. The present study began with the enrichment from wastewater sludge of a CLD-transforming community which was then inoculated into fresh media in the presence of either CLD or two of the main ISCR transformation products, 10-monohydroCLD (-1Cl-CLD) and tri-hydroCLD (-3Cl-CLD). Carried out in triplicate batches and incubated at 38°C under anoxic conditions and in the dark, the cultures were sampled regularly during 3 months and analyzed for CLD, -1Cl-CLD, -3Cl-CLD, and possible transformation products by gas chromatography coupled to mass spectrometry. All batches showed a decrease in the amended substrates (CLD or hydroCLD). CLD degradation occurred with concomitant formation of a nine-carbon compound (pentachloroindene) and two sulfur-containing transformation products (chlordecthiol, CLD-SH; methyl chlordecsulfide, CLD-SCH3), demonstrating competing transformation pathways. In contrast, -1Cl-CLD and -3Cl-CLD only underwent a sequential reductive sulfidation/S-methylation process resulting in -1Cl-CLD-SH and -1Cl-CLD-SCH3 on the one hand, and -3Cl-CLD-SH, -3Cl-CLD-SCH3 on the other hand. Some sulfur-containing transformation products have been reported previously with single bacterial strains, but never in the presence of a complex microbial community. At the end of the experiment, bacterial and archaeal populations were investigated by 16S rRNA gene amplicon sequencing. The observed diversity was mostly similar in the CLD and -1Cl-CLD conditions to the inoculum with a dominant archaea genus, Methanobacterium, and four OTU affiliated to bacteria, identified at the family (Spirochaetaceae) or genus level (Desulfovibrio, Aminobacterium, and Soehngenia). On the other hand, in the -3Cl-CLD condition, although the same OTU were found, Clostridium sensu stricto 7, Candidatus Cloacimonas, and Proteiniphilum were also present at > 2% sequences. Presence of methanogens and sulfate-reducing bacteria could contribute to sulfidation and S-methylation biotransformations. Overall, these results contribute to increasing our knowledge on the biodegradability of CLD and its transformation products, helping to progress toward effective remediation solutions.

The demand for energy and chemicals is constantly growing, leading to an increase of the amounts of contaminants discharged to the environment. Among these, pharmaceutical molecules are frequently found in treated wastewater that is discharged into superficial waters. Indeed, wastewater treatment plants (WWTPs) are designed to remove organic pollution from urban effluents but are not specific, especially toward contaminants of emerging concern (CECs), which finally reach the natural environment. In this context, it is important to study the fate of micropollutants, especially in a soil aquifer treatment (SAT) context for water from WWTPs, and for the most persistent molecules such as benzodiazepines. In the present study, soils sampled in a reed bed frequently flooded by water from a WWTP were spiked with diazepam and oxazepam in microcosms, and their concentrations were monitored for 97 days. It appeared that the two molecules were completely degraded after 15 days of incubation. Samples were collected during the experiment in order to follow the dynamics of the microbial communities, based on 16S rRNA gene sequencing for Archaea and Bacteria, and ITS2 gene for Fungi. The evolution of diversity and of specific operating taxonomic units (OTUs) highlighted an impact of the addition of benzodiazepines, a rapid resilience of the fungal community and an evolution of the bacterial community. It appeared that OTUs from the Brevibacillus genus were more abundant at the beginning of the biodegradation process, for diazepam and oxazepam conditions. Additionally, Tax4Fun tool was applied to 16S rRNA gene sequencing data to infer on the evolution of specific metabolic functions during biodegradation. It finally appeared that the microbial community in soils frequently exposed to water from WWTP, potentially containing CECs such as diazepam and oxazepam, may be adapted to the degradation of persistent contaminants.

Bioleaching is a promising strategy to recover valuable metals from spent printed circuit boards (PCBs). The performance of the process is catalyzed by microorganisms, which the toxic effect of PCBs can inhibit. This study aimed to investigate the capacity of an acidophilic iron-oxidizing culture, mainly composed of Leptospirillum ferriphilum, to oxidize iron in PCB-enriched environments. The culture pre-adapted to 1% (w/v) PCB content successfully thrived in leachates with the equivalent of 6% of PCBs, containing 8.5 g L–1 Cu, 8 g L–1 Fe, 1 g L–1 Zn, 92 mg L–1 Ni, 12.6 mg L–1 Pb, and 4.4 mg L–1 Co, among other metals. However, the inhibiting effect of PCBs limited the microbial activity by delaying the onset of the exponential iron oxidation. Successive subcultures boosted the activity of the culture by reducing this delay by up to 2.6 times under batch conditions. Subcultures also favored the rapid establishment of high microbial activity in continuous mode.

International audience; Microbial mats are commonly observed in estuaries and in salt marshes but they only rarely represent a significant surface involved in salt production. In the Sečovlje salt works in Northern Adriatic, a microbial mat known as the “petola” covers the bottom of salt crystallising pans, highly influencing salt composition and salt production processes. Throughout the year the petola is subjected to numerous co-varying factors that drive changes in its structure and the microbial community. Seasonal modifications were investigated via various methods (cryo-HRSEM, XRD, elemental analysis, carbohydrate content, bacterial community structure). This study provides knowledge on microbial mat compositional characteristics and functional roles in response to seasonal variation in environmental conditions. The in situ characterisation (close-to its natural hydrated state) of the three-dimensional microstructure provides precise information about dominating filamentous cyanobacterium Coleofasciculus chthonoplastes and extracellular polymer secretion (EPS) organisation. This is the first study to address how microbial mat composition and structure, especially 3D EPS network (and microbial diversity), affects the salt production processes within a hypersaline environment.

The impact of two pesticides (S-metolachlor and propiconazole) and their respective main metabolites (ESA-metolachlor and 1,2,4-triazole) on bacterial denitrification in groundwater was studied. For this, the denitrification activity and the bacterial diversity of a microbial community sampled from a nitrate-contaminated groundwater were monitored during 20 days in lab experiments in the presence or absence of pesticides or metabolites at 2 or 10 μg/L. The kinetics of nitrate reduction along with nitrite and N2O production all suggested that S-metolachlor had no or only little impact, whereas its metabolite ESA-metolachlor inhibited denitrification by 65% at 10 μg/L. Propiconazole and 1,2,4-triazole also inhibited denitrification at both concentrations, but to a lesser extent (29–38%) than ESA-metolachlor. When inhibition occurred, pesticides affected the reduction of nitrate into nitrite step. However, no significant differences were detected on the abundance of nitrate reductase narG and napA genes, suggesting an impact of pesticides/metabolites at the protein level rather than on denitrifying bacteria abundance. 16S rRNA gene Illumina sequencing indicated no major modification of bacterial diversity in the presence or absence of pesticides/metabolites, except for ESA-metolachlor and propiconazole at 10 μg/L that tended to increase or decrease Shannon and InvSimpson indices, respectively. General growth parameters suggested no impact of pesticides, except for propiconazole at 10 μg/L that partially inhibited acetate uptake and induced a decrease in microbial biomass. In conclusion, pesticides and metabolites can have side effects at environmental concentrations on microbial denitrification in groundwater and may thus affect ecosystem services based on microbial activities.

International audience; Acid mine drainage (AMD) still represents a huge environmental problem. Technical and scientific breakthroughs are still needed to decrease environmental damages, treatment cost, and waste production associated with AMD. The feasibility of combining continuously fed anaerobic sulfate-reducing bioreactor with downstream iron oxidation step was tested, at a laboratory scale, with two types of real arsenic-rich AMD waters from the site of Carnoulès (France), that differed in acidity (pH 3.3 and 4.0), arsenic (As, 18 and 174 mg.L−1), and metal concentrations. Iron remained in solution, while up to 99% of As was precipitated as amorphous orpiment in the anaerobic sulfate-reducing bioreactor. Zinc (Zn) precipitation was also observed, up to 99%; however, the efficiency of Zn precipitation was less stable than that of As. The anaerobic bioreactor presented a stable bacterial community including a Desulfosporosinus-related sulfate reducer. When the effluents from the anaerobic process step were treated in a laboratory aerobic bioreactor, iron was oxidized efficiently. The feasibility of efficient orpiment bio-precipitation coupled with downstream iron oxidation was shown, thus opening the perspective of a low-cost combination of treatment steps for the removal of arsenic, zinc, and iron in As-rich AMDs.

Pyrite-bearing coal wastes are responsible of the formation of acid mine drainage (AMD), and their management to mitigate environmental impacts is a challenge to the coal mine industry in Europe and worldwide. The European CEReS project sought to develop a generic co-processing strategy to reuse and recycle coal wastes, based on removal of AMD generating potential through bioleaching. Chemolitoautotrophic iron- and sulfur-oxidizing microbial consortia were enriched from a Polish coal waste at 30 °C and 48 °C, but not 42 °C. Pyrite leaching yield, determined from bioleaching tests in 2-L stirred bioreactors, was best with the 48 °C endogenous consortium (80%), then the 42 °C exogenous BRGM-KCC consortium (71%), and finally the 30 °C endogenous consortium (50%). 16S rRNA gene-targeted metagenomics from five surface locations on the dump waste revealed a microbial community adapted to the site context, composed of iron- and/or sulfur-oxidizing genera thriving in low pH and metal rich environments and involved in AMD generation. All together, the results confirmed the predisposition of the pyritic coal waste to bioleaching and the potential of endogenous microorganisms for efficient bioleaching at 48 °C. The good leaching yields open the perspective to optimize further and scale-up the bioleaching process.

Iron-reducing bacteria (IRB) are strongly involved in Fe cycling in surface environments. Transformation of Fe and associated trace elements is strongly linked to the reactivity of various iron minerals. Mechanisms of Fe (oxyhydr)oxides bio-reduction have been mostly elucidated with pure bacterial strains belonging to Geobacter or Shewanella genera, whereas studies involving mixed IRB populations remain scarce. The present study aimed to evaluate the iron reducing rates of IRB enriched consortia originating from complex environmental samples, when grown in presence of Fe (oxyhydr)oxides of different mineralogy. The abundances of Geobacter and Shewanella were assessed in order to acquire knowledge about the abundance of these two genera in relation to the effects of mixed IRB populations on kinetic control of mineralogical Fe (oxyhydr)oxides reductive dissolution. Laboratory experiments were carried out with two freshly synthetized Fe (oxyhydr)oxides presenting contrasting specific surfaces, and two defined Fe-oxides, i.e., goethite and hematite. Three IRB consortia were enriched from environmental samples from a riverbank subjected to cyclic redox oscillations related to flooding periods (Decize, France): an unsaturated surface soil, a flooded surface soil and an aquatic sediment, with a mixture of organic compounds provided as electron donors. The consortia could all reduce iron-nitrilotriacetate acid (Fe(III)-NTA) in 1–2 days. When grown on Fe (oxyhydr)oxides, Fe solubilization rates decreased as follows: fresh Fe (oxyhydr)oxides > goethite > hematite. Based on a bacterial rrs gene fingerprinting approach (CE-SSCP), bacterial community structure in presence of Fe(III)-minerals was similar to those of the site sample communities from which they originated but differed from that of the Fe(III)-NTA enrichments. Shewanella was more abundant than Geobacter in all cultures. Its abundance was higher in presence of the most efficiently reduced Fe (oxyhydr)oxide than with other Fe(III)-minerals. Geobacter as a proportion of the total community was highest in the presence of the least easily solubilized Fe (oxyhydr)oxides. This study highlights the influence of Fe mineralogy on the abundance of Geobacter and Shewanella in relation to Fe bio-reduction kinetics in presence of a complex mixture of electron donors.

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.

International audience; Microbial transformation of arsenic species and their interaction with the carbon cycle play a major role in the mobility of this toxic metalloid in the environment. The influence of simple or complex organic substrates on arsenic bio-oxidation was studied using two bacterial strains: one – the arsenivorans strain of Thiomonas delicata – is able to use AsIII as sole energy source; the other, Herminiimonas arsenicoxydans, is not. Experiments were performed at two AsIII concentrations (75 and 2 mg/L). At 75 mg/L As, for both strains, expression of aioA gene decreased when yeast extract concentration was raised from 0.2 to 1 g/L. At 2 mg/L As, the presence of either yeast extract or simple (succinate or acetate) organic substrates in the medium during bacterial growth decreased the AsIII-oxidation rate by both strains. When added specifically during oxidation test, yeast extract but not simple organic substrates seems to have a negative effect on AsIII oxidation. Taken together, results confirm the negative influence of simple or complex organic substrates on the kinetics of microbial AsIII oxidation and suggest that this effect results from different mechanisms depending on the type of organic substrate. Further, for the first time, the influence of a complex organic substrate, yeast extract, on aioA gene expression has been evidenced.

International audience

International audience; Microcosm experiments with CE-contaminated groundwater from a former industrial site were set-up to evaluate the relationships between biological CE dissipation, dehalogenase genes abundance and bacterial genera diversity. Impact of high concentrations of PCE on organohalide respiration was also evaluated. Complete or partial dechlorination of PCE, TOE, cis-DCE and VC was observed independently of the addition of a reducing agent (Na2S) or an electron donor (acetate). The addition of either 10 or 100 mu M PCE had no effect on organohalide respiration. qPCR analysis of reductive dehalogenases genes (pceA, tceA, vcrA, and bvcA) indicated that the version of pceA gene found in the genus Dehalococcoides [hereafter named pceA(Dhc)] and vcrA gene increased in abundance by one order of magnitude during the first 10 days of incubation. The version of the pceA gene found, among others, in the genus Dehalobacter, Sulfurospirillum, Desulfuromonas, and Geobacter [hereafter named pceA(Dhb)] and bvcA gene showed very low abundance. The tceA gene was not detected throughout the experiment. The proportion of pceA(Dhc) or vcrA genes relative to the universal 16S ribosomal RNA (16S rRNA) gene increased by up to 6-fold upon completion of cis-DCE dissipation. Sequencing of 16S rRNA amplicons indicated that the abundance of Operational Taxonomic Units (OTUs) affiliated to dehalogenating genera Dehalococcoides, Sulfurospirillum, and Geobacter represented more than 20% sequence abundance in the microcosms. Among organohalide respiration associated genera, only abundance of Dehalococcoides spp. increased up to fourfold upon complete dissipation of PCE and cis-DCE, suggesting a major implication of Dehalococcoides in CEs organohalide respiration. The relative abundance of pceA and vcrA genes correlated with the occurrence of Dehalococcoides and with dissipation extent of PCE, cis-DCE and CV. A new type of dehalogenating Dehalococcoides sp. phylotype affiliated to the Pinellas group, and suggested to contain both pceA(Dhc) and vcrA genes, may be involved in organohalide respiration of CEs in groundwater of the study site. Overall, the results demonstrate in situ dechlorination potential of CE in the plume, and suggest that taxonomic and functional biomarkers in laboratory microcosms of contaminated groundwater following pollutant exposure can help predict bioremediation potential at contaminated industrial sites.

International audience; Toxic and persistent contaminants in groundwater are technologically difficult to remediate. Remediation techniques using nanoparticles (NPs) such as nZVI (Zero-Valent Iron) are applicable as in situ reduction or oxidation agents and give promising results for groundwater treatment. However, these NP may also represent an additional contamination in groundwater. The aims of this study are to assess the impact of nZVI on the nitrate-reducing potential, the abundance and the structure of a planktonic nitrate-reducing bacterial community sampled in groundwater from a multicontaminated site. An active nitrate-reducing bacterial community was obtained from groundwater samples, and inoculated into batch reactors containing a carbon substrate, nitrate and a range of nZVI concentrations (from 0 to 70.1 mg Fe.L-1). Physical (pH, redox potential), chemical (NO3- concentrations) and biological (DNA, RNA) parameters were monitored during 1 week, as well as nZVI size distribution and mortality of bacteria. Nitrate-reducing activity was temporally stopped in the presence of nZVI at concentrations higher than 30 mg L-1, and bacterial molecular parameters all decreased before resuming to initial values 48 h after nZVI addition. Bacterial community composition was also modified in all cultures exposed to nZVI as shown by CE-SSCP fingerprints. Surprisingly, it appeared overall that bacteria viability was lower for lower nZVI concentrations. This is possibly due to the presence of larger, less reactive NP aggregates for higher nZVI concentrations, which inhibit bacterial activity but could limit cell mortality. After 1 week, the bacterial cultures were transplanted into fresh media without nZVI, to assess their resilience in terms of activity. A lag-phase, corresponding to an adaptation phase of the community, was observed during this step before nitrate reduction reiterated, demonstrating the community's resilience. The induction by nZVI of modifications in the bacterial community composition and thus in its metabolic potentials, if also occurring on site, could affect groundwater functioning on the long term following nZVI application. Further work dedicated to the study of nZVI impact on bacterial community directly on site is needed to assess a potential impact on groundwater functioning following nZVI application.

In 2008, as part of a feasibility study for radioactive waste disposal in deep geological formations, the French National Radioactive Waste Management Agency (ANDRA) drilled several boreholes in the transposition zone in order to define the potential variations in the properties of the Callovo&ndash, Oxfordian claystone formation. This consisted of a rare opportunity to investigate the deep continental biosphere that is still poorly known. Four rock cores, from 1709, 1804, 1865, and 1935 m below land surface, were collected from Lower and Middle Triassic formations in the Paris Basin (France) to investigate their microbial and geochemical composition. Rock leachates showed high salinities ranging from 100 to 365 g&middot, L&minus, 1 NaCl, current temperatures averaging 65 &deg, C, no detectable organic matter, and very fine porosity. Microbial composition was studied using a dual cultural and molecular approach. While the broad-spectrum cultural media that was used to activate microbial communities was unsuccessful, the genetic investigation of the dominant 16S rRNA gene sequences revealed eight bacterial genera considered as truly indigenous to the Triassic cores. Retrieved taxa were affiliated to aerobic and facultative anaerobic taxon, mostly unknown to grow in very saline media, except for one taxon related to Halomonas. They included Firmicutes and &alpha, &beta, and &gamma, Proteobacteria members that are known from many subsurface environments and deep terrestrial and marine ecosystems. As suggested by geochemical analyses of rocks and rock leachates, part of the indigenous bacterial community may originate from a cold paleo-recharge of the Trias aquifer with water originating from ice melting. Thus, retrieved DNA would be fossil DNA. As previously put forward to explain the lack of evidence of microbial life in deep sandstone, another hypothesis is a possible paleo-sterilisation that is based on the poly-extremophilic character of the confined Triassic sandstones, which present high salinity and temperature.

International audience; 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.

International audience; In bioleaching processes using autotrophic bacteria, carbon dioxide (CO2) is the carbon source for the growth of the microorganisms and its availability is dependent on gas mass transfer. The objective of this study was to investigate the demand in CO2 in complex sulfidic copper (Cu) concentrate bioleaching operations and to optimise CO2 supply. Batch tests in 2 l-stirred reactors at 10%w/v solid concentration were performed to study the need for CO2-enrichment and to determine the adequate CO2 partial pressure in the gas inlet. The results show that ferrous iron (Fe(II)) oxidation, and thus microbial activity, is delayed when air is injected without CO2-enrichment; the carbonates present in the solid are not sufficient to provide the CO2 required for the activity of Fe oxidising bacteria. CO2-enrichment improves leaching kinetics since the copper dissolution rate increases from 84 mg L−1 h−1 with air solely to 120 mg L−1 h−1 when CO2 is added to air. A CO2 enrichment influences both the composition of the bacterial community and the abundance of the bioleaching species. This study proposes also a methodology to determine gas/liquid transfer components and to assess CO2 limitations in the system. It shows that the microorganisms are not only sensitive to the transfer rate of CO2 from the gas to the liquid phase, but also to the availability of CO2 in solution.

Dichloromethane (DCM) is a widespread and toxic industrial solvent which often co-occurs with chlorinated ethenes at polluted sites. Biodegradation of DCM occurs under both oxic and anoxic conditions in soils and aquifers. Here we investigated in situ and ex situ biodegradation of DCM in groundwater sampled from the industrial site of Themeroil (France), where DCM occurs as a major co-contaminant of chloroethenes. Carbon isotopic fractionation (eC) for DCM ranging from −46 to −22‰ were obtained under oxic or denitrifying conditions, in mineral medium or contaminated groundwater, and for laboratory cultures of Hyphomicrobium sp. strain GJ21 and two new DCM-degrading strains isolated from the contaminated groundwater. The extent of DCM biodegradation (B%) in the aquifer, as evaluated by compound-specific isotope analysis (δ13C), ranged from 1% to 85% applying DCM-specific eC derived from reference strains and those determined in this study. Laboratory groundwater microcosms under oxic conditions showed DCM biodegradation rates of up to 0.1 mM·day−1, with concomitant chloride release. Dehalogenase genes dcmA and dhlA involved in DCM biodegradation ranged from below 4 × 102 (boundary) to 1 × 107 (source zone) copies L−1 across the contamination plume. High-throughput sequencing on the 16S rrnA gene in groundwater samples showed that both contaminant level and terminal electron acceptor processes (TEAPs) influenced the distribution of genus-level taxa associated with DCM biodegradation. Taken together, our results demonstrate the potential of DCM biodegradation in multi-contaminated groundwater. This integrative approach may be applied to contaminated aquifers in the future, in order to identify microbial taxa and pathways associated with DCM biodegradation in relation to redox conditions and co-contamination levels.

International audience; 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.

Two bacterial consortia prepared from a polychlorinated biphenyl (PCB)-contaminated soil enrichment were used as inocula, for aerobic PCB-degradation experiments in soil microcosms. The consortia were prepared either as a planktonic culture, or as a biofilm attached to granular activated carbon (GAC). Both consortia were mainly composed of members of the α-, β- and γ-subclasses of the phylum Proteobacteria. The most abundant bacteria, belonged to the genera Pseudomonas, Achromobacter, Ochrobactrum and Halomonas which are commonly associated with soil contaminated with biphenyl or PCBs. The decrease of the level of extractable PCB congeners was assessed in microcosms containing the same PCB-polluted soil from which the consortia were prepared and it was spiked or not with Aroclor 1242. When Aroclor 1242 was added to soil, mainly low-chlorinated congeners were removed, whereas in non-spiked soil, decreases of extractable PCBs levels were observed for a broader range of congeners. The biofilm-coated GAC was less efficient than the planktonic cells to decrease the total amount of extractable PCBs. This limitation was possibly due to the differences in the bacterial composition of the two inocula and to the reduced bioavailability the GAC-adsorbed PCBs. Nevertheless, the biofilm-coated GAC accelerated the aerobic removal of the extractable PCBs during the first three months of incubation, albeit limited in terms of total PCB-removal.

International audience; Passive water treatments based on biological attenuation can be effective for arsenic-rich acid mine drainage (AMD). However, the key factors driving the biological processes involved in this attenuation are not well-known. Here, the efficiency of arsenic (As) removal was investigated in a bench-scale continuous flow channel bioreactor treating As-rich AMD (∼30-40 mg L-1). In this bioreactor, As removal proceeds via the formation of biogenic precipitates consisting of iron- and arsenic-rich mineral phases encrusting a microbial biofilm. Ferrous iron (Fe(II)) oxidation and iron (Fe) and arsenic removal rates were monitored at two different water heights (4 and 25 mm) and with/without forced aeration. A maximum of 80% As removal was achieved within 500 min at the lowest water height. This operating condition promoted intense Fe(II) microbial oxidation and subsequent precipitation of As-bearing schwertmannite and amorphous ferric arsenate. Higher water height slowed down Fe(II) oxidation, Fe precipitation and As removal, in relation with limited oxygen transfer through the water column. The lower oxygen transfer at higher water height could be partly counteracted by aeration. The presence of an iridescent floating film that developed at the water surface was found to limit oxygen transfer to the water column and delayed Fe(II) oxidation, but did not affect As removal. The bacterial community structure in the biogenic precipitates in the bottom of the bioreactor differed from that of the inlet water and was influenced to some extent by water height and aeration. Although potential for microbial mediated As oxidation was revealed by the detection of aioA genes, removal of Fe and As was mainly attributable to microbial Fe oxidation activity. Increasing the proportion of dissolved As(V) in the inlet water improved As removal and favoured the formation of amorphous ferric arsenate over As-sorbed schwertmannite. This study proved the ability of this bioreactor-system to treat extreme As concentrations and may serve in the design of future in-situ bioremediation system able to treat As-rich AMD.

International audience; 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.

International audience; We assessed the effect of pesticides, especially commonly detected herbicides, on bacterial communities in groundwater. To this end, we used a combined approach with i) triazine-spiked experiments at environmentally relevant concentrations (1 and 10 mu g/L) in waters with contrasting contamination histories, and ii) in situ monitoring in a rural aquifer, where many additional biotic and abiotic parameters also affect the community. Microbial community was characterized by fingerprinting techniques (CE-SSCP), gene presence (atzA/B/C/D/F/F and amoA genes) and abundance (16S RNA, napA and narG genes). During triazine-spiked experiments, the bacterial community structure in reference water was modified following an exposure to atrazine (ATZ) and/or its metabolite desethylatrazine (DEA) at 1 mu g/L; in historically contaminated water, the bacterial community structure was modified following an exposure to 10 mu g/L ATZ/DEA. Similarly, biodiversity indices and biomass in the reference water appeared affected at lower triazine concentrations than in the historically-contaminated water, though these end-points are less sensitive than the community structure. Our results thus suggest that the history of contamination induced a community tolerance to the tested triazines. ATZ and DEA were not degraded during the experiment and this was consistent with the absence of atz genes involved in their degradation in none of the tested conditions. In field monitoring, triazines that represent a historical and diffuse contamination of groundwater, participate in the microbial community structure, confirming the triazine effect observed under laboratory conditions: Other herbicides, such as chloroacetanilides that are applied today, did not appear to affect the whole community structure; they however induced a slight, but significant, increase in the abundance of nitrate-reducing bacteria. To our best knowledge, this is the first study on the microbial ecotoxicology of pesticides and their metabolites at environmentally relevant concentrations in groundwater.

Microbiology of a hypersaline oil reservoir located in Central Africa was investigated with molecular and culture methods applied to preserved core samples. Here we show that the community structure was partially acquired during sedimentation, as many prokaryotic 16S rRNA gene sequences retrieved from the extracted DNA are phylogenetically related to actual Archaea inhabiting surface evaporitic environments, similar to the Cretaceous sediment paleoenvironment. Results are discussed in term of microorganisms and/or DNA preservation in such hypersaline and Mg-rich solutions. High salt concentrations together with anaerobic conditions could have preserved microbial/molecular diversity originating from the ancient sediment basin wherein organic matter was deposited.

International audience; This study aimed at evaluating potential arsenic (As) mobility in an industrially contaminatedsoil (64 mg As kg-1) of the Meuse River basin, and at identifying key bacterial groups that drive soil As dynamics. Both speciation and release of As from this soil was followed under anaerobicconditions using a laboratory batch experiment. In the presence of exogenous carbon sources,AsV initially present in the soil matrix and/or adsorbed on synthetic hydrous ferric oxides wassolubilized and mainly reduced into AsIII by indigenous soil microfora. After a one-monthincubation period in these biotic conditions, AsIII accounted for 80-85% of the total dissolved Asand more than 60% of the solid-phase As. Bacterial community structure (i.e. 16S rDNA-basedCE-SSCP profiles) changed with incubation time and As amendment. The detection of distantlyrelated arsenate respiratory reductase genes (arrA), as functional markers of AsV-respirers,indicates that novel dissimilatory AsV-reducing bacteria may be involved in Asbiotransformation and mobility in anoxic soils. Since As and iron were concomitantly released, acrucial role of indirect As-mobilizing bacteria on As behavior was also revealed. Our results show that the majority of As within the soil matrix was bioavailable and bioaccessible forheterotrophic AsV reduction to AsIII, which may increase As toxicity and mobility in thecontaminated soils.

[Departement_IRSTEA]Ecotechnologies [TR1_IRSTEA]TED; International audience; To reduce the environmental footprint of human activities, the quality of environmental media such as water, soil and the atmosphere should be first assessed. Microorganisms are well suited for a such assessment because they respond fast to environmental changes, they have a huge taxonomic and genetic diversity, and they are actively involved in biogeochemical cycles. Here, we review microbiological methods that provide sensitive and robust indicators for environmental diagnosis. Methods include genomics, transcriptomics, proteomics and metabolomics to study the abundance, diversity, activity and functional potentials of indigenous microbial communities in various environmental matrices such as water, soil, air and waste. We describe the advancement, technical limits and sensitivity of each method. Examples of method application to farming, industrial and urban impact are presented. We rank the most advanced indicators according to their level of operability in the different environmental matrices based on a technology readiness level scale.

A continuous bioleaching operation was carried out in a laboratory-scale unit using a cobaltiferous pyrite concentrate. The objective was to investigate mechanisms of microbial activity and mineral oxidation for a better understanding and optimisation of biomining operations, particularly the ones using stirred tank technology. A combination of scientific and technical approaches (molecular ecology and biochemistry) was used and various key operating parameters were tested (temperature, nitrogen source, CO 2 availability). The bacterial strains that dominate the culture were present in the concentrate and originated from the deposit on the mining site. Whatever the operating conditions tested, the culture composition was very stable. The iron-oxidiser Leptospirillum ferriphilum BRGM1 was the dominant organism in standard (not limiting) conditions, and was always very well represented during the first 3–4 days of residence time. Sulfobacillus sp. BRGM2 also played a significant role in the process. The role and the presence of Acidithiobacillus caldus BRGM3 seemed of lesser importance. An increase in operating temperature from 35 °C to 45 °C had no major impact on bioleaching efficiency. When the nitrogen source was limiting, there was a negative impact on both bacterial growth and bioleaching efficiency. This was the result of a combination of factors such as less precipitate formation and decreased bacterial attachment to the pyrite surface. CO 2 limitation had a very significant negative effect on bacterial productivity and consequently on bioleaching efficiency. However, when CO 2 was a limiting factor, the population composition remained unchanged but a significant decrease in exopolysaccharide production was observed. This study gives new insights for the application of stirred tank technology to the processing of sulphide concentrates, and more specifically on the impact of key operating parameters on bioleaching performances, population dynamics and attachment of bacteria to the solid surfaces.

A novel anaerobic, long-chain alkene-degrading, sulfate-reducing bacterium, strain LM2801T, was isolated from brackish sediment of a wastewater decantation facility of an oil refinery (Berre lagoon, France). Cells of strain LM2801T were Gram-negative, motile, slightly curved or vibrioid rods. Its optimum growth conditions were 30–36 °C, 6–10 g NaCl l−1 and pH 7.5. Strain LM2801T incompletely oxidized long-chain alkenes (from C14 to C23) and fatty acids (C14 to C24). The DNA G+C content was 45.5 mol%. Sequence analyses of the 16S rRNA and dsrAB genes indicated that the strain was a member of the family Desulfobacteraceae within the Deltaproteobacteria. This novel isolate possesses phenotypic and phylogenetic traits that do not allow its classification as a member of any previously described genus. Therefore, strain LM2801T is described as a member of a new genus, Desulfatiferula gen. nov., of which Desulfatiferula olefinivorans sp. nov. is the type species. The type strain of Desulfatiferula olefinivorans is LM2801T (=DSM 18843T =JCM 14469T).

International audience; 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

International audience; A batch upflow fixed-bed sulphate-reducing bioreactor has been set up and monitored for the treatment of synthetic solutions containing divalent iron (100 mg/L and 200 mg/L), zinc (100 mg/L and 200 mg/L), copper (100 mg/L and 200 mg/L), nickel (100 mg/L and 200 mg/L) and sulphate (1700 mg/L and 2130 mg/L) at initial pH 3-3.5, using ethanol as the sole electron donor. The reactor has been operated at the theoretical stoichiometric ethanol/sulphate ratio. Complete oxidation of ethanol has been achieved through complete oxidation of the intermediately, microbially produced acetate. This is mainly attributed to the presence of Desulfobacter postgatei species which dominated the sulphate-reducing community in the reactor. The reduction of sulphate was limited to about 85%. Quantitative precipitation of the soluble metal ions has been achieved. XRD and SEM-EDS analyses performed on samples of the produced sludge showed poorly crystalline phases of marcasite, covellite and wurtzite as well as several mixed metal sulphides.

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 17897T).

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47A degrees C with an optimum at 40-45A degrees C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H-2, and CO2. The predominant cellular fatty acids were non-branched fatty acids C-16:1, C-16:0, and C-14:0. The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).

International audience; During the Bioshale European project, a technoeconomic study of the bioleaching of a copper concentrate originating from a black shale ore was carried out. This concentrate is a multi-mineral resource in which the copper sulphides are mainly chalcocite, covellite, bornite and chalcopyrite. The experiments undertaken to produce the techno-economic data were also an opportunity to carry out more fundamental research. The objective of this work was to combine the results of the bioleaching experiments, in terms of copper recovery, with the results of bacterial community monitoring and mineralogy residue analysis. Batch and continuous bioleaching tests were carried out with 10% solids, at 42 °C and with a pH between 1.2 and 1.6. Final copper recovery was higher in batch cultures than in continuous mode (>95% vs. 91%). Mineralogical analysis showed that the limiting factor for copper recovery was incomplete chalcopyrite dissolution in both cases. However, chalcopyrite was even less dissolved in continuous conditions. This was also related to a variation in bacterial community structure. The population in all tests was composed of Acidithiobacillus caldus, Leptospirillum ferriphilum and one or two species of Sulfobacillus (Sulfobacillus thermosulfidooxidans and sometimes Sulfobacillus benefaciens), but Sulfobacillus and more generally sulphur oxidizers were more represented in batch mode. It was proposed that due to their capacity to reduce inorganic compounds, sulphur oxidizers may be efficient in limiting chalcopyrite surface hindering. It may help to better dissolve this mineral and reach a better copper recovery.

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.

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.

International audience; The characteristics of the biofilm and the solids formed during the operation of a sulphate-reducing fixed-bed reactor, fed with a moderately acidic synthetic effluent containing zinc and iron, are presented. A diverse population of δ-Proteobacteria SRB, affiliated to four distinct genera, colonized the system. The morphology, mineralogy and surface chemistry of the precipitates were studied by X-ray diffraction (XRD), scanning electron microscopy (SEM) and energy dispersive X-ray (EDX). The XRD patterns observed are characteristic of amorphous solid phases. Peaks corresponding to crystalline iron sulphide, marcasite, sphalerite and wurtzite were also identified. SEM-EDX results confirm the predominance of amorphous phases appearing as a cloudy haze. EDX spectra of spots on the surface of these amorphous phases reveal the predominance of iron, zinc and sulphur indicating the formation of iron and zinc sulphides. The predominance of these amorphous phases and the formation of very fine particles, during the operation of the SRB column, are in agreement and can be explained by the formation pathways of metal sulphides at ambient temperature, alkaline pH and reducing conditions. Solids are precipitated either as (i) amorphous phases deposited on the bed material, as well as on surface of crystals, e.g. Mg3(PO4)2 and (ii) as rod-shaped solids characterized by a rough hazy surface, indicating the encapsulation of bacterial cells by amorphous metal sulphides.

International audience; 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 degrees C, and maximum 47 degrees 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.

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., Author Summary Recent advances in the field of arsenic microbial metabolism have revealed that bacteria colonize a large panel of highly contaminated environments. Belonging to the order of Burkholderiales, Thiomonas strains are ubiquitous in arsenic-contaminated environments. The genome of one of them, i.e. Thiomonas sp. 3As, was deciphered and compared to the genome of several other Thiomonas strains. We found that their flexible gene pool evolved to allow both the surviving and growth in their peculiar environment. In particular, the acquisition by strains of the same species of different genomic islands conferred heavy metal resistance and metabolic idiosyncrasies. Our comparative genomic analyses suggest that the natural environment influences the genomic evolution of these bacteria. Importantly, these results highlight the genomic variability that may exist inside a taxonomic group, enlarging the concept of bacterial species.

International audience; In situ biofilm sampling is a key step for the study of natural biofilms and using methodologies that reflect naturaldiversity is necessary to guarantee representative sampling. Here, we focalise on the impact of the type of substrataon which biofilms grow on bacterial and fungal communities’ structure. The indirect molecular approach, DenaturingGel Gradient Electrophoresis (DGGE) of a gene fragment coding for either 16S rRNA or 28S rRNA, for bacteria or fungirespectively, was used to evaluate the variability of microbial community structures among different biofilm substrata:natural (pebbles, live plants, wood and sediment), or artificial (glass, Plexiglas® and sterile wood), in a small river (theLoiret, France). Multivariate statistics, band richness and diversity indexes (Shannon and Simpson) were used to highlight variations in community structure between substrata. Results showed variations of bacterial and fungal diversity between different substrata according to substratum properties/origin (natural or artificial, organic or inorganic) but there was no optimal substratum for sampling, and artificial substrata were not significantly less applicable than natural substrata. Pooling 4 different substrata types allowed a higher bacterial and fungal biodiversity recovery. Point contact sampling may thus gain in robustness by increasing the number of substrata considered. Fungal species richness was similar to the bacterial one on most substrata which suggested they should be more frequently considered in riverine biofilm studies.