Polycyclic aromatic hydrocarbons remobilization from contaminated porous media by (bio)surfactants washing.

Biosurfactants, surface-active agents produced by microorganisms, are increasingly studied for their potential use in soil remediation processes because they are more environmentally friendly than their chemically produced homologues. In this work, we report on the use of a crude biosurfactant produced by a bacterial consortium isolated from a PAHs-contaminated soil, compared with other (bio)surfactants (Tween80, Sodium dodecyl sulfate - SDS, rhamnolipids mix), to wash PAHs from a contaminated porous media. Assays were done using columns filled with sand or sand-clay mixtures (95:5) spiked with four model PAHs. The crude biosurfactant showed less adsorption to the [sand] and the [sand + clay] columns compared to Tween 80, SDS and the rhamnolipid mix. The biosurfactant showed the second best capacity to remove PAHs from the columns (as dissolved and particulate phases), both from [sand] and [sand + clay], after SDS when applied at lower concentrations than the other sufactants. The effluent concentrations of phenanthrene (PHE), pyrene (PYR) and benzo[a]pyrene (BAP) increased in the presence of the crude biosurfactant. Compared to the control experiment using only water, the global PAHs washed mass (amount of PAHs removed from the columns) increased between 9 and 1000 times for PHE and BAP in the [sand] column, and between 55 and 6000 times respectively for PHE and BAP in the [sand + clay] columns. Moreover, in the [sand + clay] columns, leaching of a part of the clays was observed in the SDS and the biosurfactant injections assays. This clay leaching resulted in higher PAHs removal, due not to desorption but rather to particulate transport. In the context of washing PAH-contaminated soils in biopiles or subsurface remediation, our results could help in sizing the remediation approach using an environmental friendly biosurfactant, before a pump-and-treat process.

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

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

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.

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.

Production of biosurfactant using the endemic bacterial community of a PAHs contaminated soil, and its potential use for PAHs remobilization.

Biosurfactants are surface-active agents produced by microorganisms whose use in soil remediation processes is increasingly discussed as a more environmentally friendly alternative than chemically produced surfactants. In this work, we report the production of a biosurfactant by a bacterial community extracted from a polluted soil, mainly impacted by PAHs, in order to use it in a soil-washing process coupled with bioremediation. Nutrient balance was a critical parameter to optimize the production. Best conditions for biosurfactant production were found to be 20 g/L of glucose, 2 g/L of NH4NO3, and 14.2 g/L of Na2HPO4, corresponding to a C/N/P molar ratio equal to 13/1/2. Purification of the produced biosurfactant by acidification and double extraction with dichloromethane as a solvent allowed measuring the Critical Micellar Concentration (CMC) as equal to 42 mg/L. The capacity of the purified biosurfactant to increase the apparent solubility of four reference PAHs (naphthalene, phenanthrene, pyrene and benzo[a]pyrene) was completed. The solubilisation ratios, in mg of PAH/g of biosurfactant for phenanthrene, pyrene and benzo[a]pyrene are 0.214, 0.1204 and 0.0068, respectively. Identification of the bacteria found in the colony producing the biosurfactant showed the presence of bacteria able to produce biosurfactant (Enterobacteriaceae, Pseudomonas), as well as, others able to degrade PAHs (Microbacterium, Pseudomonas, Rhodanobacteraceae).

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.

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.

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.

Occurrence of 1,1'-dimethyl-4,4'-bipyridinium (Paraquat) in irrigated soil of the Lake Chad Basin, Niger.

Increased use of agrochemical products to improve yields for irrigated crops in sub-Saharan Africa has been accompanied by a significant increase in the risk of environmental contamination. Detailed examples of the fate of pesticides after initial spreading on crop fields are scarce in tropical regions, where safe practices and related health risks are poorly understood by smallholder farmers. In the semi-arid environment of the Lake Chad Basin, SE Niger, both intrinsic properties of pesticides and extrinsic factors such as soil and climate helped to characterize processes leading to an accumulation of pesticides in soils. Analysis by HPLC-UV of a 6 m deep soil profile showed the presence of Paraquat at concentrations from 953 ± 102 µg kg(-1) to 3083 ± 175 µg kg(-1) at depths between 0.80 and 2.75 m below the land surface. Soil analysis revealed that up to approximately 15 % of the total soil matrix consists of smectites, a clay mineral capable of retaining cationic pesticides such as Paraquat, and a very low content of organic matter (<0.15 wt.% TOC). Paraquat could be stored and not bioavailable in a clayey barrier at approximately 2-m depth and therefore does not represent an immediate risk for populations or environment in this form. However, if the Paraquat application rate remains constant, the clayey barrier could reach a saturation limit within 150-200 years and 180-220 years if we consider a DT50 in soil of ~1,000 days (FAO). Consequently, it could lead to a deeper infiltration and so a pollution of groundwater. Such a scenario can represent a health risk for drinking water and for the Lake Chad, which is a major resource for this densely populated region of semi-arid Africa. Further analyses should focus on deeper layers and groundwater Paraquat contents to validate or invalidate the hypothesis of storage in this clay-rich layer.

GammaProteobacteria as a potential bioindicator of a multiple contamination by polycyclic aromatic hydrocarbons (PAHs) in agricultural soils.

The impact of a multiple contamination by polycyclic aromatic hydrocarbons (PAHs) was studied on permanent grassland soil, historically presenting low contamination (i.e. less than 1 mg kg(-1)). Soil microcosms were spiked at 300 mg kg(-1) with either single or a mixture of seven PAHs. While total dissipation of the phenanthrene was reached in under 90 days, only 60% of the PAH mixture were dissipated after 90 days. Interestingly, after 30 days, the abundance of the GammaProteobacteria class (assessed by qPCR) become significantly higher in microcosms spiked with the PAH mixture. In addition, the specific abundance of the cultivable Pseudomonas spp., which belong to the GammaProteobacteria class, increased earlier and transiently (after 8 days) in the microcosms spiked with the PAH mixture. Consequently, we propose to use the GammaProteobacteria as a bioindicator to detect the impact on the bacterial community of a multiple contamination by PAHs in agricultural soils.