Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Linking denitrification and pesticide transformation potentials with community ecology and groundwater discharge in hyporheic sediments in a lowland stream.

Contamination of rivers by nitrate and pesticides poses a risk for aquatic ecosystems in lowland catchments that are often intensively used for agriculture. Here, the hyporheic zone, the streambed underneath the stream, plays a vital role due to its efficient self-purification capacity. The present study aims to evaluate the denitrification and transformation potential of 14 pesticides and three transformation products in the hyporheic sediment from a lowland stream with a high N load and by comparing an agricultural straightened section to a natural meandering part of the stream influenced by different groundwater discharges. Batch experiments were set up to evaluate the denitrification and pesticide transformation potentials in hyporheic sediment from two depths (5-15 cm (a) and 15-25 cm (b)). Our results revealed that (i) differences between the agricultural and natural sections of the river did not influence pollutant attenuation, (ii) both the nitrate and pesticide attenuation processes were more rapid in the upper "a" layer compared to the "b" layer due to higher microbial abundance, (iii) high groundwater discharge reduced the denitrification potential while pesticide transformation was unaffected, (iv) denitrification correlated with denitrifier abundance (nirK) in the "b" layer, while this correlation was not seen in the "a" layer, and (v) a microbial community with low diversity can explain limited transformation for the majority of tested pesticides. Overall, our results suggest that high groundwater discharge zones with reduced residence time in the hyporheic zone can be an important source of pesticides and nitrate to surface water.

Unlocking secrets of microbial ecotoxicology: recent achievements and future challenges.

Environmental pollution is one of the main challenges faced by humanity. By their ubiquity and vast range of metabolic capabilities, microorganisms are affected by pollution with consequences on their host organisms and on the functioning of their environment. They also play key roles in the fate of pollutants through the degradation, transformation, and transfer of organic or inorganic compounds. Thus, they are crucial for the development of nature-based solutions to reduce pollution and of bio-based solutions for environmental risk assessment of chemicals. At the intersection between microbial ecology, toxicology, and biogeochemistry, microbial ecotoxicology is a fast-expanding research area aiming to decipher the interactions between pollutants and microorganisms. This perspective paper gives an overview of the main research challenges identified by the Ecotoxicomic network within the emerging One Health framework and in the light of ongoing interest in biological approaches to environmental remediation and of the current state of the art in microbial ecology. We highlight prevailing knowledge gaps and pitfalls in exploring complex interactions among microorganisms and their environment in the context of chemical pollution and pinpoint areas of research where future efforts are needed.

Degradation potential of MCPA, metolachlor and propiconazole in the hyporheic sediments of an agriculturally impacted river.

Hyporheic sediments are influenced by physical, biological, and chemical processes due to the interactions with river water and has been shown to play an important role in the environmental fate of pesticides. Therefore, this study evaluated the bacterial degradation potential of MCPA, metolachlor and propiconazole in hyporheic sediments sampled along a 20 km long stretch of an agriculturally impacted river dominated primarily by water losing conditions. Water physicochemical parameters in the river and nearby groundwater wells were assessed along with pesticide sorption to sediments and bacterial community composition. Degradation and mineralisation batch experiments were set up from six locations (five water losing, one water gaining) using environmentally relevant concentrations of pesticides (10 µg kg-1). Highly variable DT50 values from 11 to 44 days for MCPA, 11-27 days for metolachlor (MTC) and 60-147 days for propiconazole were calculated based on ~140 day studies. Degradation of MTC led to accumulation of the transformation products MOA and MESA in batch experiments. Noteworthy, MESA was detected in the groundwater wells adjacent to the part of the river impacted by losing conditions suggesting that degradation processes in hyporheic sediments may lead to the formation of transformation products (TP) leaching towards groundwater. Further, from propiconazole was identified a persistent transformation product being different from 1,2,4-triazole. Specific calculated DT50 values could not the linked to bacterial diversity. However, generally all sediment samples were characterised by high bacterial diversity, where approximately 80% of the relative sequence abundances were < 1%, which may increase the likelihood of finding contaminant-degrading genes, thereby explaining the general high contaminant-degrading activity. The studied sediments revealed a high potential to degrade pesticides despite only being exposed to low diffuse pollutant concentrations that is similar to calculated DT50 values in agricultural soils.

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.

Degradation of tetrachloroethylene by zero valent iron nanoparticles in the presence of a natural groundwater bacterial biofilm in a sandy porous media.

Biofilms are naturally present in aquifers and can interact with zero valent iron nanoparticles (nZVI) used as remediation agents in contaminated groundwater; thereby they may alter nZVI reactivity towards targeted contaminants in porous media. Laboratory scale experiments using columns filled with sand (50 cm long and 5.2 cm in diameter) were performed to investigate the impact of natural biofilms on nZVI reactivity towards tetrachloroethylene (PCE) in conditions simulating an unconsolidated sandy aquifer. Solutions containing PCE were injected through the sand columns in the presence or absence of biofilm and nZVI. Concentrations in PCE and its metabolites were monitored during 45 days in dissolved and gas phases. PCE concentrations decreased at the column outlets due both to its reductive dechlorination by nZVI (~30% of injected PCE) and its sorption or deposition (as PCE-DNAPL) on sand (~35% of injected PCE). No significant differences in PCE concentrations were found in presence or absence of biofilm. However, biofilm presence affected the nature of PCE metabolites. A higher release of ethene in the column containing biofilm was observed, whereas ethane was dominant in the absence of biofilm. Microbes consumed H2 released by the corrosion of nZVI limiting the hydrogenation of ethene to ethane. The consequences of biofilm development in porous media should be taken into account when considering treatment with nZVI, as it may affect the nature of produced metabolites.

Resistance and Resilience of Soil Nitrogen Cycling to Drought and Heat Stress in Rehabilitated Urban Soils.

In the context of climate change and biodiversity loss, rehabilitation of degraded urban soils is a means of limiting artificialization of terrestrial ecosystems and preventing further degradation of soils. Ecological rehabilitation approaches are available to reinitiate soil functions and enhance plant development. However, little is known about the long-term stability of rehabilitated soils in terms of soil functions when further natural or anthropogenic perturbations occur. Based on rehabilitated urban soils, the present study sought to evaluate the resistance and resilience of soil functions linked to carbon cycling and phosphate dynamics in addition to nitrogen cycling and related microbial communities after a heat and drought stress. A laboratory experiment was conducted in microcosms under controlled temperature conditions, with four contrasted soils collected from a rehabilitated urban brownfield; an initial, non-rehabilitated soil (IS), a technosol with a high organic matter level (HO), and two technosols with less organic matter (LO1 and LO2), together with their respective controls (no stress). Changes in potential denitrification (PDR), nitrification (PNR) rates, and their interactive relationships with soil microbial activities and soil physicochemical properties were determined following a combined heat (40°C) and drought stress period of 21 days. Measurements were carried out immediately after the stress (resistance), and then also 5, 30, and 92 days after soil rewetting at 60% water holding capacity (resilience). Microbial activities involved in soil functions such as carbon cycling and phosphate dynamics proved to be of low resistance in all soils except for IS; however, they were resilient and recovered rapidly after rewetting. On the other hand, the microbial activities and gene abundances that were measured in relation to nitrogen cycling processes showed that for denitrification, activities were more rapidly resilient than gene abundances whereas for nitrification the activities and gene abundances were resilient in the same way. Results suggest that, unless the soils contain high amounts of organic matter, microbial communities in imported soils can be more vulnerable to environmental pressures such as drought and heat than communities already present. This should be considered when rehabilitating degraded soils.

Dynamics of Soil Microbial Communities During Diazepam and Oxazepam Biodegradation in Soil Flooded by Water From a WWTP.

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.

Microbial Transformation of Chlordecone and Two Transformation Products Formed During in situ Chemical Reduction.

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.

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.

Chlorinated ethene biodegradation and associated bacterial taxa in multi-polluted groundwater: Insights from biomolecular markers and stable isotope analysis.

Chlorinated ethenes (CEs) are most problematic pollutants in groundwater. Dehalogenating bacteria, and in particular organohalide-respiring bacteria (OHRB), can transform PCE to ethene under anaerobic conditions, and thus contribute to bioremediation of contaminated sites. Current approaches to characterize in situ biodegradation of CEs include hydrochemical analyses, quantification of the abundance of key species (e.g. Dehalococcoides mccartyi) and dehalogenase genes (pceA, vcrA, bvcA and tceA) involved in different steps of organohalide respiration (OHR) by qPCR, and compound-specific isotope analysis (CSIA) of CEs. Here we combined these approaches with sequencing of 16S rRNA gene amplicons to consider both OHRB and bacterial taxa involved in CE transformation at a multi-contaminated site. Integrated analysis of hydrogeochemical characteristics, gene abundances and bacterial diversity shows that bacterial diversity and OHRB mainly correlated with hydrogeochemical conditions, suggesting that pollutant exposure acts as a central driver of bacterial diversity. CSIA, abundances of four reductive dehalogenase encoding genes and the prevalence of Dehalococcoides highlighted sustained PCE, DCE and VC degradation in several wells of the polluted plume. These results suggest that bacterial taxa associated with OHR play an essential role in natural attenuation of CEs, and that representatives of taxa including Dehalobacterium and Desulfosporosinus co-occur with Dehalococcoides. Overall, our study emphasizes the benefits of combining several approaches to evaluate the interplay between the dynamics of bacterial diversity in CE-polluted plumes and in situ degradation of CEs, and to contribute to a more robust assessment of natural attenuation at multi-polluted sites.

Impact of Fe(III) (Oxyhydr)oxides Mineralogy on Iron Solubilization and Associated Microbial Communities.

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.

Physico-chemical and agronomic results of soil remediation by In Situ Chemical Reduction applied to a chlordecone-contaminated nitisol at plot scale in a French Caribbean banana plantation.

The In Situ Chemical Reduction (ISCR) process was tested in a nitisol in a French Caribbean banana plantation using five different soil amendments. The addition of 2.8% or 4.0% of Zero Valent Iron (ZVI; dw/dw, 2 different trial plots) in the 0-40-cm soil layer lowered the initial chlordecone (CLD) concentration by up to 74% or 69% in 37 days or 94 days, with 75% of the decrease achieved after only 21 or 24 days of treatment depending on the trial plot. The addition of commercially available Daramend® was also tested by applying the 6% dose (dw/dw) recommended by the manufacturer and using either the regular alfalfa-based product or a bagasse-based product specifically formulated for the study. Both significantly lowered CLD concentrations, but to a lesser extent than with the ZVI-only amendment. A bagasse-ZVI mixture prepared on site produced results slightly better than the two Daramend®. The percentage decreases in CLD concentrations were correlated with the negative redox potentials achieved. In all the trial plots, dechlorinated transformation products appeared in the soil and soil water as the CLD concentrations decreased, with H atoms replacing up to 4 and 7 of the 10 Cl atoms, respectively. None of these degradation products appeared to accumulate in the soil or soil water during the treatment. Instead, the reverse occurred, with an overall downward trend in their concentrations over time. The effects of ISCR treatment on agronomic and human health-related parameters were measured in three different crops. The radishes produced with some treatments were visually of lower quality or smaller in size than those grown in the control plots. Lower yields were observed for the cucumbers and sweet potatoes grown after applying the bagasse-based amendments. Mortality among cucumber seedlings was observed after treatment with ZVI only. Simple operational solutions should suffice to remedy these negative agronomic effects. As regards human health-related effects, the CLD concentrations in radishes grown with three of the amendments were significantly lower than in the two control plots and well below the maximum residue level (MRL), which was substantially exceeded in the radishes grown on untreated soil. For cucumbers, the treatments with regular Daramend® and with a local bagasse-ZVI mixture produced fruits with CLD below the MRL and also below the concentrations in one of the two control plots. As for the sweet potatoes, adding a bagasse-ZVI mixture had a significant positive effect by decreasing contamination below the levels in the two control plots and below the MRL.

Shift in Natural Groundwater Bacterial Community Structure Due to Zero-Valent Iron Nanoparticles (nZVI).

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 ( NO 3 - 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.

Microbial community response to environmental changes in a technosol historically contaminated by the burning of chemical ammunitions.

The burning of chemical weapons in the 1926-1928 period produced polluted technosols with elevated levels of arsenic, zinc, lead and copper. During an eight-month mesocosm experiment, these soils were submitted to two controlled environmental changes, namely the alternation of dry and water-saturated conditions and the addition of fragmented organic forest litter to the surface soil. We investigated, by sequencing the gene coding 16S rRNA and 18S rRNA, (1) the structure of the prokaryotic and eukaryotic community in this polluted technosol and (2) their response to the simulated environmental changes, in the four distinct layers of the mesocosm. In spite of the high concentrations of toxic elements, microbial diversity was found to be similar to that of non-polluted soils. The bacterial community was dominated by Proteobacteria, Acidobacteria and Bacteroidetes, while the fungal community was dominated by Ascomicota. Amongst the most abundant bacterial Operational Taxonomic Units (OTUs), including Sphingomonas as a major genus, some were common to soil environments in general whereas a few, such as organisms related to Leptospirillum and Acidiferrobacter, seemed to be more specific to the geochemical context. Evolution of the microbial abundance and community structures shed light on modifications induced by water saturation and the addition of forest litter to the soil surface. Co-inertia analysis suggests a relationship between the physico-chemical parameters total organic carbon, Zn, NH4+ and As(III) concentrations and the bacterial community structure. Both these results imply that microbial community dynamics linked to environmental changes should be considered as factors influencing the behavior of toxic elements on former ammunition burning sites.

Functional Genes and Bacterial Communities During Organohalide Respiration of Chloroethenes in Microcosms of Multi-Contaminated Groundwater.

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, TCE, 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 µM 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.

Do natural biofilm impact nZVI mobility and interactions with porous media? A column study.

Nanoparticles (NP) used as remediation agents for groundwater treatment may interact with biofilms naturally present, altering NP mobility and/or reactivity and thereby NP effectiveness. The influence of the presence of a multi species biofilm on the mobility of two types of zero-valent iron NP (nZVI; NANOFER 25S and optimized NANOFER STAR, NanoIron s.r.o. (Czech Republic)) was tested in laboratory experiments with columns mimicking aquifer conditions. Biofilms were grown in columns filled with sand in nitrate reducing conditions using groundwater from an industrial site as inoculum. After two months growth, they were composed of several bacterial species, dominated by Pseudomonas stutzeri. Biofilm strongly affected the physical characteristics of the sand, decreasing total porosity from ~30% to ~15%, and creating preferential pathways with high flow velocities. nZVI suspensions were injected into the columns at a seepage velocity of 10mday-1. Presence of biofilm did not impact the concentrations of Fe at the column outlet nor the amount of total Fe retained in the sand, as attested by the measurement of magnetic susceptibility. However, it had a significant impact on NP size sorting as well as on total Fe distribution along the column. This suggests nZVI-biofilm interactions that were confirmed by microscopic observations using SEM/STEM coupled with energy-dispersive X-ray spectroscopy. Our study shows that biofilm modifies the water flow velocity in the porous media, favoring the transport of large aggregates and decreased NP mobility due to physical and chemical interactions.

Experimental Column Setup for Studying Anaerobic Biogeochemical Interactions Between Iron (Oxy)Hydroxides, Trace Elements, and Bacteria.

Fate and speciation of trace elements (TEs), such as arsenic (As) and mercury (Hg), in aquifers are closely related to physio-chemical conditions, such as redox potential (Eh) and pH, but also to microbial activities that can play a direct or indirect role on speciation and/or mobility. Indeed, some bacteria can directly oxidize As(III) to As(V) or reduce As(V) to As(III). Likewise, bacteria are strongly involved in Hg cycling, either through its methylation, forming the neurotoxin monomethyl mercury, or through its reduction to elemental Hg°. The fates of both As and Hg are also strongly linked to soil or aquifer composition; indeed, As and Hg can bind to organic compounds or (oxy)hydroxides, which will influence their mobility. In turn, bacterial activities such as iron (oxy)hydroxide reduction or organic matter mineralization can indirectly influence As and Hg sequestration. The presence of sulfate/sulfide can also strongly impact these particular elements through the formation of complexes such as thio-arsenates with As or metacinnabar with Hg. Consequently, many important questions have been raised on the fate and speciation of As and Hg in the environment and how to limit their toxicity. However, due to their reactivity towards aquifer components, it is difficult to clearly dissociate the biogeochemical processes that occur and their different impacts on the fate of these TE. To do so, we developed an original, experimental, column setup that mimics an aquifer with As- or Hg-iron-oxide rich areas versus iron depleted areas, enabling a better understanding of TE biogeochemistry in anoxic conditions. The following protocol gives step by step instructions for the column set-up either for As or Hg, as well as an example with As under iron and sulfate reducing conditions.

Representative sampling of natural biofilms: influence of substratum type on the bacterial and fungal communities structure.

In situ biofilm sampling is a key step for the study of natural biofilms and using methodologies that reflect natural diversity is necessary to guarantee representative sampling. Here, we focalise on the impact of the type of substrata on which biofilms grow on bacterial and fungal communities' structure. The indirect molecular approach, Denaturing Gel Gradient Electrophoresis (DGGE) of a gene fragment coding for either 16S rRNA or 28S rRNA, for bacteria or fungi respectively, 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 (the Loiret, 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.

Mercury mobilization and speciation linked to bacterial iron oxide and sulfate reduction: A column study to mimic reactive transfer in an anoxic aquifer.

Mercury (Hg) mobility and speciation in subsurface aquifers is directly linked to its surrounding geochemical and microbial environment. The role of bacteria on Hg speciation (i.e., methylation, demethylation and reduction) is well documented, however little data is available on their impact on Hg mobility. The aim of this study was to test if (i) Hg mobility is due to either direct iron oxide reduction by iron reducing bacteria (IRB) or indirect iron reduction by sulfide produced by sulfate reducing bacteria (SRB), and (ii) to investigate its subsequent fate and speciation. Experiments were carried out in an original column setup combining geochemical and microbiological approaches that mimic an aquifer including an interface of iron-rich and iron depleted zones. Two identical glass columns containing iron oxides spiked with Hg(II) were submitted to (i) direct iron reduction by IRB and (ii) to indirect iron reduction by sulfides produced by SRB. Results show that in both columns Hg was leached and methylated during the height of bacterial activity. In the column where IRB are dominant, Hg methylation and leaching from the column was directly correlated to bacterial iron reduction (i.e., Fe(II) release). In opposition, when SRB are dominant, produced sulfide induced indirect iron oxide reduction and rapid adsorption of leached Hg (or produced methylmercury) on neoformed iron sulfides (e.g., Mackinawite) or its precipitation as HgS. At the end of the SRB column experiment, when iron-oxide reduction was complete, filtered Hg and Fe concentrations increased at the outlet suggesting a leaching of Hg bound to FeS colloids that may be a dominant mechanism of Hg transport in aquifer environments. These experimental results highlight different biogeochemical mechanisms that can occur in stratified sub-surface aquifers where bacterial activities play a major role on Hg mobility and changes in speciation.