Battaglia-Brunet Fabienne

Naveau Aude

Cary Lise

Bueno Maïté

Briais Justine

Charron Mickael

Joulian Catherine

Thouin Hugues

Justine Briais

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

Keywords: groundwater, arsenic, speciation, microbial communities, sediment incubation, changing redox

Arsenic (As) is one of the main toxic elements of geogenic origin that impact groundwater quality and human health worldwide. In some groundwater wells of the Sologne region (Val de Loire, France), drilled in a confined aquifer, As concentrations exceed the European drinking water standard (10 µg L -1 ). The monitoring of one of these drinking water wells showed As concentrations in the range 20 to 25 µg L -1 . The presence of dissolved iron (Fe), low oxygen concentration and traces of ammonium indicated reducing conditions. The δ 34 SSO4 was anticorrelated with sulphate concentration. Drilling allowed to collect detrital material corresponding to a Miocene floodplain and crevasse splay with preserved plant debris. The level that contained the highest total As concentration was a silty-sandy clay containing 26.9 mg kg -1 As. The influence of alternating redox conditions on the behaviour of As was studied by incubating this material with site groundwater, in biotic or inhibited bacterial activities J o u r n a l P r e -p r o o f conditions, without synthetic organic nutrient supply, in presence of H2 during the reducing periods. The development of both AsV-reducing and AsIII-oxidising microorganisms in biotic conditions was evidenced. At the end of the reducing periods, total As concentration strongly increased in biotic conditions. The microflora influenced As speciation, released Fe and consumed nitrate and sulphate in the water phase. Microbial communities observed in groundwater samples strongly differed from those obtained at the end of the incubation experiment, this result being potentially related to influence of the sediment compartment and to different physico-chemical conditions. However, both included major Operating Taxonomic Units (OTU) potentially involved in Fe and S biogeocycles. Methanogens emerged in the incubated sediment presenting the highest solubilised As and Fe. Results support the hypothesis of in-situ As mobilisation and speciation mediated by active biogeochemical processes.

Introduction

The presence of arsenic (As) in groundwater is a widespread environmental issue which consequences affect millions of people's health [START_REF] Ravenscroft | Arsenic Pollution: A Global Synthesis, RGS-IBG Book Series[END_REF][START_REF] Chakraborti | Arsenic groundwater contamination and its health effects in Patna district (capital of Bihar) in the middle Ganga plain, India[END_REF]. Chronic exposure to As was reported to increase the risk of a range of illnesses including cancer [START_REF] Schmidt | Low-dose arsenic: in search of a risk threshold[END_REF][START_REF] Tsuji | Dose-response for assessing the cancer risk of inorganic arsenic in drinking water: the scientific basis for use of a threshold approach[END_REF]. High As concentrations exceeding the European drinking water standards (10 µg L -1 , EU Council Directive 98/83/EC) are generally linked to geological context in many regions of the world [START_REF] Podgorski | Arseniteinduced changes in abundance and expression of arsenite transporter and arsenite oxidase genes of a soil microbial community[END_REF]. This toxic element presents several oxidation states and compounds potentially changed by microbial activities according to redox conditions. Arsenic release in several sedimentary and alluvial aquifers is J o u r n a l P r e -p r o o f linked with low redox conditions (Ravencroft et al. 2009;[START_REF] Erickson | Drinking water quality in the glacial aquifer system, northern USA[END_REF] and reductive reactions catalysed by microbial processes (Deng et al., 2018). These bio-reactions are linked to the availability of electron donors, generally organic matter. Several studies reported the mobilisation of As from underground sediments fuelled by organic pollutants plumes [START_REF] Erickson | Arsenic Cycling in Hydrocarbon Plumes: Secondary Effects of Natural Attenuation[END_REF][START_REF] Ziegler | A mass balance approach to investigate arsenic cycling in a petroleum plume[END_REF]. However, the natural organic matter present in sediments, found both in active and ancient floodplains, also plays the role of electron donor in reactions that solubilise As [START_REF] Sahu | Role of shallow alluvial stratigraphy and Holocene geomorphology on groundwater arsenic contamination in the Middle Ganga Plain, India[END_REF][START_REF] J O U R N A L P R E -P R O O F Johannesson | Biogeochemical and reactive transport modeling of arsenic in groundwaters from the Mississippi River delta plain: An analog for the As-affected aquifers of South and Southeast Asia[END_REF]. The reductive dissolution of As-bearing FeIII oxides minerals was evidenced as one of the main bio-processes able to release As from sediments to groundwater [START_REF] Welch | Arsenic in ground water of the United States: occurrence and geochemistry[END_REF][START_REF] Nath | Mobility of arsenic in the sub-surface environment: An integrated hydrogeochemical study and sorption model of the sandy aquifer materials[END_REF][START_REF] Duan | Experimental constraints on redox-induced arsenic release and retention from aquifer sediments in the central Yangtze River Basin[END_REF]. However, sulphur biogeocycle may also influence As behavior: dissolved sulphide can contribute to FeIII reduction [START_REF] Xwang | Coupled iron, sulfur and carbon isotope evidences for arsenic enrichment in groundwater[END_REF] and As may be adsorbed on FeS precipitates.

Moreover, the formation of soluble thio-As complexes may increase As mobility through decreasing the adsorption of As on solid oxides [START_REF] Pi | Vertical variability of arsenic concentrations under the control of iron-sulfur-arsenic interactions in reducing aquifer systems[END_REF]. Several studies described the strong link between As, Fe and S biogeochemical cycles and As mobility and proposed geochemical models [START_REF] Xwang | Coupled iron, sulfur and carbon isotope evidences for arsenic enrichment in groundwater[END_REF][START_REF] Pi | Vertical variability of arsenic concentrations under the control of iron-sulfur-arsenic interactions in reducing aquifer systems[END_REF]. Influence of the bio-reduction of iron oxides and sulphate on As concentrations, related with fluctuations of groundwater level and redox conditions, was observed [START_REF] Schaefer | Aquifer Arsenic Cycling Induced by Seasonal Hydrologic Changes within the Yangtze River Basin[END_REF][START_REF] Zheng | Microbial sulfate reduction facilitates seasonal variation of arsenic concentration in groundwater of Jianghan Plain, Central China[END_REF]. Functional diversity of As-rich groundwater supported these hypotheses by the presence of microorganisms able to reduce AsV, either through AsV respiration or resistance mechanisms, AsIII-oxidising bacteria, FeIII-reducing and SO4-reducing bacteria (Paul et al., 2015;[START_REF] Cavalca | Exploring Biodiversity and Arsenic Metabolism of Microbiota Inhabiting Arsenic-Rich Groundwaters in Northern Italy[END_REF][START_REF] Zheng | Seasonal microbial variation accounts for arsenic dynamics in shallow alluvial aquifer systems[END_REF][START_REF] Wang | A strictly anaerobic betaproteobacterium Georgfuchsia toluolica gen. nov., sp. nov. degrades aromatic compounds with Fe(III), Mn(IV) or nitrate as an electron acceptor[END_REF]. In addition to field studies, a range of laboratory experiments were focused on the elucidation of biogeochemical processes in sediments from floodplains and deltas. Mechanisms of As release were studied through long-term [START_REF] Radloff | Mobilization of Arsenic During One-Year Incubations of Grey Aquifer Sands from Araihazar, Bangladesh[END_REF]Gillipsie et al., 2016;[START_REF] Duan | Experimental constraints on redox-induced arsenic release and retention from aquifer sediments in the central Yangtze River Basin[END_REF][START_REF] Gao | Microbially mediated mobilization of arsenic from aquifer sediments under bacterial sulfate reduction[END_REF] or short-term [START_REF] Schaefer | Aquifer Arsenic Cycling Induced by Seasonal Hydrologic Changes within the Yangtze River Basin[END_REF] incubations of underground sediments. Electron donors supply generally enhanced As release in biotic and J o u r n a l P r e -p r o o f anoxic conditions (Gillipsie et al., 2016;[START_REF] Wang | Sulfate enhances the dissimilatory arsenate-respiring prokaryotes-mediated mobilization, reduction and release of insoluble arsenic and iron from the arsenic-rich sediments into groundwater[END_REF]Deng et al., 2018), however results strongly depended on the sediment geochemistry (Deng et al., 2018). Arsenic mobility was hampered in presence of oxygen [START_REF] Radloff | Mobilization of Arsenic During One-Year Incubations of Grey Aquifer Sands from Araihazar, Bangladesh[END_REF] and mobilised during anoxic periods when redox conditions were alternated [START_REF] Duan | Experimental constraints on redox-induced arsenic release and retention from aquifer sediments in the central Yangtze River Basin[END_REF]. Microbial communities of incubated sediments were rarely described, although studies performed with core materials from the alluvial Jianghan plain in the middle reaches of the Yangtze River (China) indicated the presence of both FeIII-and SO4-reducing bacteria in anaerobic microcosms showing As release [START_REF] Duan | Experimental constraints on redox-induced arsenic release and retention from aquifer sediments in the central Yangtze River Basin[END_REF][START_REF] Gao | Microbially mediated mobilization of arsenic from aquifer sediments under bacterial sulfate reduction[END_REF]. However, complementary information on the influence of microbial community structure and activity on the amplitude of the bio-reactions mobilising As would help to improve models that predict changes in groundwater quality. In this context, the present research aimed to clarify the links between microbial processes and As release in groundwater, through the example of the Sologne region (Val de Loire, France), where As concentrations exceeding 10 µg L -1 were detected in 43 wells out of 135 that have been monitored (Supplementary Figure 1, https://ades.eaufrance.fr/), distributed in an area of approximately 5 000 km 2 , in a geological context of Miocene sedimentary formation [START_REF] Cary | Contexte, origines et biogéochimie de l'arsenic et du sélénium dans la nappe des calcaires de Beauce -Base de données des informations régionales disponibles et SIG[END_REF]. A drinking water well was monitored for two years, in order to study the evolution of water parameters. In parallel, core sediment material was characterised for mineralogy and geochemistry, then incubated in alternating redox conditions. Determination of microbial indicators and comparison of the evolution of physico-chemical parameters in biotic and abiotic conditions were performed to enlighten the biogeochemical processes influencing the mobility and speciation of arsenic.

Materials and Methods

Study site and sampling

The sampling site is located on the territory of Marcilly-en-Gault village, in Sologne (Supplementary Figure 1), a wetland-rich region in the center of France, South of Orleans city, J o u r n a l P r e -p r o o f where sands and clays of continental detrital origin (Burdigalian, Miocene) cover the calcareous aquifer of the Orléanais (Aquitanian, Miocene, Deprez and Martin, 1970). Water samplings were performed in the drinking water well (reference BSS 04611X0001/FAEP, BSS001FRCU, http://ficheinfoterre.brgm.fr/InfoterreFiche/ficheBss.action?id=04611X0001/FAEP), drilled in 1965, at 90.2 m total depth, the water level being at 21 m depth (without pumping) and the pumping performed at 70 m depth (from the ground surface). Total historical regulatory monitoring since 1986 indicates median As concentration of 23.0 µg L -1 with a standard deviation of 4.7 µg L -1 . Well water was sampled after pumping sufficiently to reach stable values of pH, redox potential, dissolved oxygen concentration and conductivity. Five sampling campaigns were performed at the following dates: June 2017 23 th ; September 2017 4 th ; December 2017 18 th ; February 2018 27 th , May 2018 29 th . Water samples were filtrated at 0.45 µm (except for total organic carbon) and conditioned for further chemical analyses (Supplementary table 1).

For molecular microbiological analyses, 10 L of groundwater were sampled at each sampling date in 2 L sterile polyethylene bottles, kept on ice until filtration at the laboratory within 12 hours after sampling. Water samples were filtrated on sterile 0.22 µm nitrocellulose filters, that were immediately stored at -20°C until microbial DNA extraction.

Core sampling was performed about 100 m North from the location of the well, with an auger drilling machine (diameter 152 mm) (Supplementary Figure 1B). Core was performed down to 48.5 m depth, in March 2017 (23-24 th ) without adding drilling fluid. Samples of each different geological facies were subsampled on-site with an alcohol-washed spatula, and immediately stored in sterile glass jars, flushed with N2, and kept on ice until storage at 4°C in the laboratory.

Jars for microbial analysis and experiments were always opened in sterile conditions. Samples for chemical and mineralogical analyses were kept frozen (-20°C) until analyses.

Mineralogical and petrographic analysis

J o u r n a l P r e -p r o o f Core samples were lyophilised and milled below 200 µm. Powder samples (infra 50 µm fraction) were analysed by X-ray diffraction (XRD) using a Phillips Panalytical X'Pert Pro apparatus in the same experimental conditions as described by [START_REF] Bassil | Determination of the distribution and speciation of selenium in argillaceous sample using chemical extractions and post-extractions analyses: application to the hydrogeological experimental site of Poitiers[END_REF]. Phase identification was made with the X'pert HighScore software using JCPDS Pdf2 mineralogical database (Supplementary Figure 5). After deconvolution and integration of the main peak area of each phase using Fytyk software, the semi-quantifications (Supplementary Table 5) were realised by applying modified RIR method (Chung, 1975).Thin sections (26 mm × 46 mm) were consolidated after impregnation at room temperature and under atmospheric pressure with a two-component epoxy resin (Araldite 2020) and 20% acetone. The thin sections were observed using an optical polarised light microscope (Nikon Eclipse E600 POL).

Batch experiments

Incubations of core solids sampled at 31.5 m depth, named M8, were prepared in 1 L-Shott bottles equipped with rubber stoppers and sterilised by autoclaving. Each batch was composed of 60 g solids and 600 mL of groundwater from the drinking water well (sampled on May 2018 29 th ). One batch was prepared with M8 core material submitted to γ-irradiation (Dagneux, France) in order to observe the evolution of the system when biological activities are inhibited (IN condition). The water was used as it was pumped for biotic experiments (BIO condition), and filtrated at 0.22 µm to remove bacteria for the IN condition. Three BIO flasks were prepared by distributing a single slurry of core sediment mixed with site water (magnetic stirring) in 3 bottles with a 25 mL sterile pipette. Alternate redox conditions were applied as follows: 3 months in anaerobic conditions (T0m to T3m), followed by 3 months in aerobic conditions (T3m to T6m) then again 3 months in anaerobic conditions (T6m to T9m). At the beginning of each anaerobic periods, the gas phase composition was imposed at 75% N2, 25% H2, and 1.25% CO2, at a total pressure of 1 bar over the atmospheric pressure. For establishing the intermediary aerobic conditions, the flasks were opened in order to replace the N2/H2/CO2 mixture by sterile J o u r n a l P r e -p r o o f air. Flasks were incubated at 20°C under reciprocal agitation. Sampling at the end of each anaerobic period was performed in a glovebox (N2 atmosphere) under agitation, in order to avoid change of the solids/water ratio. Just after sampling, pH, redox potential and conductivity were immediately measured. Samples (10 mL) were filtrated at 0.22 µm in a sterile Hungate tube filled with N2 and kept at 4°C until As speciation. Samples (15 mL) filtrated at 0.45 µm were acidified with HNO3 for analyses of total As and major cations. Samples (15 mL) filtrated at 0.45 µm but not acidified were kept for quantification of major anions (nitrate, sulphate).

Slurry samples (1 mL) were stored in sterile tubes for Most Probable Number (MPN) of Astransforming microbes analyses. At the end of experiment, slurries were centrifuged in sterile falcon tubes, and the pellets containing both bacteria attached on sediments and suspended in water were stored at -20°C until microbial DNA extraction.

Chemical analyses

Methods for analysing major chemical species and trace elements in water from the site and incubations are detailed in Supplementary Table 1. Separation of As species in groundwater samples was performed on site [START_REF] Kim | Separation of inorganic arsenic species in groundwater using ion exchange method[END_REF]. This method gives precise AsIII and AsV concentrations when only these two species are present, thus results are given as AsIII-like and AsV-like species, because other As forms might be present in groundwater. Isotopy of sulphur (δ 34 S) was measured by continuous flow isotope ratio mass spectrometry (CF-IRMS) after precipitation as BaSO4 and reduction/pyrolysis [START_REF] Kloppmann | Massive arrival of desalinated seawater in a regional urban water cycle: A multi-isotope study (B, S, O, H)[END_REF]. The total elemental composition of the core samples (Table 2) was determined, after digestion (HNO3/HF), by ICP-OES and ICP-MS in an accredited laboratory (CRPG-CNRS, Nancy). Organic carbon concentrations were determined by CHNS analysis (Flash 2000 Thermo, IC2MP, France). Total As and Se were determined by ICP-MS equipped with an octopole collision/reaction cell (C/RC) (Agilent 7500ce) after HNO3/HCl digestion. The C/RC was pressurised with helium and hydrogen delivered at 1 and 4 mL min -1 . Analyses of major cations and anions in water J o u r n a l P r e -p r o o f samples from incubation experiments were performed by ion chromatography equipped with conductivity detectors (Professional IC Vario, Metrohm). Anions were separated with a Metrosep A Supp 16 ionic resin column (150 mm × 4 mm) and cations with a Metrosep C6 (150 mm × 4 mm). Speciation of As in these samples was performed by HPLC (Agilent 1100 series HPLC pump) coupled to ICP-MS (Agilent 7500ce) with the same C/RC operating conditions as described for total analysis. Chromatographic separation was carried on using an anion exchange stationary phase (Agilent G3154-65001 column and G3154-65002 guard column) with an ammonium nitrate mobile phase (20 mmol L -1 , 2.5% (v/v) methanol and pH 8.5 adjusted with ammonia), delivered at 1 mL min -1 flow rate. The sample injection volume was 100 µL. These analytical conditions allow the separation of AsIII, AsV and mono-and dimethylated AsV species (MMA V and DMA V ) (see example chromatogram in Supplementary Figure 2).

Microbiological analyses 2.5.1. MPN of AsIII-oxidising and AsV-reducing bacteria

Most Probable Numbers (MPN) of active AsIII-oxidising and AsV-reducing microbes were determined in slurries by the methods described in [START_REF] Thouin | Characterization and mobility of arsenic and heavy metals in soils polluted by the destruction of arseniccontaining shells from the Great War[END_REF] and [START_REF] Thouin | Influence of environmental changes on the biogeochemistry of arsenic in a soil polluted by the destruction of chemical weapons: a mesocosm study[END_REF], with the following modification: the pH of the culture media was adjusted to 7 in order to fit with the groundwater pH.

Microbial DNA extraction

Microbial DNA was extracted from (i) the filters obtained from groundwater filtration and stored at -20°C, cut into strips with a sterile scalpel just prior extraction, and (ii) the slurries pellets from end of batch experiments. The FastDNA™ Spin Kit for Soil procedure (MP Biomedicals) was followed, using a FastPrep-24™ instrument at a speed of 5 m s -1 for 30 s.

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DNA extract concentration was measured using the Quantifluor dsDNA sample kit and the Quantus fluorimeter, according to the manufacturer's recommendations (Promega).

16S rRNA, aioA and arsB genes copy number monitoring

Abundance of bacterial 16S rRNA genes and of aioA and arsB functional genes, encoding respectively for the catalytic subunit of arsenite oxidase and the AsIII-efflux pump of Ars resistance system, were performed by quantitative PCR (qPCR). The main characteristics of qPCR reactive mixtures and programs are described in Supplementary Information 1, Supplementary Table 2, andin

16S rRNA gene metabarcoding

For next generation sequencing, amplicon libraries and sequences were generated by INRAE Transfert (Narbonne, France). Briefly, the V4-V5 region of the gene coding 16S rRNA (bacteria and archaea) was amplified using the barcoded, universal primer set 515WF/918WR (Wang et al., 2009). PCR reactions were performed using AccuStart II PCR ToughMix kit and cleaned (HighPrep PCR beads, Mokascience). Pools were submitted for sequencing on Illumina MiSeq instrument at GeT-PlaGe (Auzeville, France). Sequences were processed using the FROGS (v.3.2) bioinformatics pipeline [START_REF] Escudié | FROGS: Find, Rapidly, OTUs with Galaxy Solution[END_REF], implemented into the Genotoul plateform of the Galaxy server of Toulouse (details in Supplementary information 1). The raw datasets are available on the European Nucleotide Archive system under project accession number PRJEB53046.

Statistical analyses

Statistical tests were carried out using R 4.0.3 (www.r-project.org). Redundance analysis (RDA) with the vegan R package, was performed to identify the effects of environmental variables on bacterial composition structure, based on the relative abundance of the detected

J o u r n a l P r e -p r o o f

OTUs in water sample from the temporal evolution. This analysis was calculated based on Hellinger -transformed phylum abundance basis of 16S rRNA gene data [START_REF] Borcard | Numerical ecology with R[END_REF].

Environmental explanatory tested variables were standardised and the function ordistep in vegan package was used to identify the significant explanatory forward environmental variable.

The significance of the RDA model was tested by ANOVA based on Monte Carlo test with 999 permutations. This analysis helps to determine the most influential factors and the extent that various environmental parameters affected bacterial phylum, most abundant OTU composition.

Principal component analysis (PCA) was performed on biogeochemical parameters from the batch experiment.

Results

Temporal monitoring of groundwater

Groundwater level and geochemistry

The groundwater level variations (Fig. 1A andB) present well-marked annual cycles with high level observed in late spring and the lowest level in late summer. The total amplitude of these J o u r n a l P r e -p r o o f variations is in the range of 2 to 3 m. The total As concentration (Fig. 1A) remained between 20 and 25 µg L -1 along the monitoring period, with a mean of 23.6 µg L -1 and a standard deviation of 1.6 µg L -1 , lower than the measurement uncertainty (2.0-2.5 µg L -1 ). The speciation of As seemed to be more variable than total As concentration. Dissolved Fe concentration varied between 0.15 and 0.3 mg L -1 (± 35%), and SO4 concentration between 5.2 and 5.8 mg L -1 (± 5%). Ammonium was always detected, with concentrations generally in the range 0.07-0.1 mg L -1 (± 40%, Supplementary Tables 1 and3). For these three parameters, the temporal variations remained in the range of the analytical measurement uncertainty. Results of isotopic analyses of SO4-S showed variations of δ 34 S higher than the range of analytical standard deviation (Fig. 1B).

Microbial parameters

Rarefaction curves (Supplementary Figure 3) together with richness and diversity indexes (Supplementary Table 4) indicated a relatively low bacterial diversity. Bacterial communities of the groundwater were composed of a majority of Proteobacteria (Fig. 2A). The most abundant OTU found in groundwater was affiliated to Hydrogenophilaceae family, with an unknown genus representing 69 to 86% of the total sequences (Fig. 2B). The second main OTU belonged to the Gallionellaceae family, gathering autotrophic FeII-oxidisers [START_REF] Hallbeck | The Family Gallionellaceae[END_REF]. Autotrophic bacteria using FeII as energy source, identified at the genus level, (Gallionella, Ferriphaselus and Sideroxydans genera), were always present in groundwater samples (from 0.5 to 2.2 % of the sequences). Sulphate-reducing bacteria (SRB), including Desulfovibrio and Desulfatirhabdium genera were always present but in small abundance (total from 0.1 to 0.5 % of the sequences), and this abundance did not vary with seasons. Bacteria J o u r n a l P r e -p r o o f involved in denitrification and able to use sulphur compounds as energy source, identified at the genus level (Sulfuritalea, Denitratisoma, Sulfuricella) were always present as minor OTUs (total from 0.01 to 0.1% of the sequences) in groundwater. The global abundance of the universal bacterial 16S rRNA gene did not follow a clear seasonal tendency (Fig. 2C). The two functional genes, i.e. aioA and asrB genes, involved in As biotransformations were always present, and their abundance ranged between 1.6 × 10 6 and 2.6 × 10 8 gene copies L -1 for aioA, and between 5.8 × 10 3 and 2.2 × 10 5 gene copies L -1 for arsB. . A RDA was performed to identify the influence on microbial community structure of physicochemical characteristics of groundwater (Fig. 3). The results indicated that total As, total Fe and dissolved oxygen concentration were explanatory variables that significantly influenced the community structure at the phylum level (Fig. 3A), whereas only Mn concentration significantly influenced the community structure at the genus level (Fig. 3B).

J o u r n a l P r e -p r o o f

Solids description

Sedimentological description

J o u r n a l P r e -p r o o f

The sedimentological description (Supplementary Figure 4) begins at 13.5 m depth; this section shows at the base the dominant clays with roots traces and plant debris (48.0 m to 35.5 m).

These clays are followed by poorly sorted medium and coarse sands (35.5 m to 31.5 m). Finally, the youngest levels (31.5 m to 13.6 m) correspond to an alternation of well-sorted medium sands and clays with plant debris. The profile represents a part of "Sables et Argiles de Sologne" formation [START_REF] Desprez | Hydrogéologie du calcaire de Beauce sous la Sologne (Loiret Loir-et-Cher)[END_REF]) that covers the aquifer limestone formations "Marne de l'Orléanais" and "Calcaire de Pithiviers ». The sedimentary filling is characterised by clays and heterogeneous sands (well to poorly sorted fine to coarse sands). These clays with root traces, organic matter and plant debris correspond to floodplain deposits. The thick sandy part (poorly-sorted) probably corresponds to meandering channel (river) and the isolated sandy levels (well-sorted) in the floodplain clays corresponds to crevasse splay deposits.

Mineralogy

The mineralogy of the sediments was determined by XRD on 13 different samples corresponding to 13 visually different geological layers, with one of the layer (M9, 37.5 m deep) divided in two sub-samples (XRD spectra given in supplementary Figure 5). Total As concentration was also determined on these samples (Supplementary Table 5). The upper 8 layers (M1 -M8) represent a floodplain clays with crevasse splay sands. Two samples, M7 and M8 (floodplain and crevasse splay facies), present the two highest As concentrations of the profile, (> 20 ppm), and relatively high clay content. These two layers are visually characterised by different colours; blue for M7, and red-brown for M8. The M9 layer presents heterogeneous facies, marly in the upper part (M9a). This level is close to the interface between two different geological formations, the sands and clays of Sologne above, and the underlying calcareous formation. The deeper samples (M10-M13) are mostly calcareous facies (M11 and M13) or marl-calcareous (M10 and M12).

Chemical composition

J o u r n a l P r e -p r o o f

Three samples, corresponding to the 2 clayey levels with high As concentrations (M7 and M8), and one of the underlying calcareous levels (M12) were characterised in terms of major and trace elements contents (Supplementary Table 6 and Fig. 4 for M8). These analysis confirmed the siliceous feature of M7 and M8 compared with the characteristics of M12 that defined this level as calcareous. Iron, sulphur and organic carbon contents were similar in the 3 samples, with significant Fe contents but very low S and C concentrations. Arsenic and metals (Pb and U) were enriched in M7 and M8 compared with M12.

Petrographic description

Petrographic observations of resin-impregnated M8 sample (Fig. 4) showed that the material was mainly composed of beige siliceous clay showing numerous desiccation traces (arrow D in Fig. 4A), and a laminar structure (arrow L in Fig. 4A). Desiccation cracks form sub-polygonal structures where the siliceous matrix can be partially or entirely transformed into clay (Fig. 4B, arrows MA), as indicated by the presence of remaining white matrix in clayey phases (Fig. 4C,D,E). The laminations are composed of successive layers of clear siliceous material and clayey beige material. Small black, dark brown and red pellets are observed inserted into the layers (Fig. 4D).

J o u r n a l P r e -p r o o f

Batch experiments with M8

Water geochemistry

Total As concentrations increased during the anaerobic periods and decreased during the aerobic step in the BIO slurries (Fig. 5). These variations in As concentrations were not observed in the IN condition. At the end of the first anaerobic period (T3m), total As concentrations were in the range 100-150 µg L -1 in BIO slurries, and the three parallel microcosms behaved in the same way. Conversely, at the end of the second anaerobic period (T9m), the BIO B slurry presented higher As concentration (275 µg L -1 ) than A and C (between 150 and 200 µg L -1 ). Total dissolved Fe followed the same evolution as total As, with concentrations close to 900 µg L -1 at the end of the first anaerobic period, and up to 7000 µg L - J o u r n a l P r e -p r o o f 1 in B slurry at the end of the second anaerobic period. The redox potential was lower at the end of the second anaerobic period in all flasks, independently from the presence of active bacteria, and pH was higher in BIO than in IN conditions all along the experiment. During the anaerobic periods, pH tended to decrease, probably because of CO2 present in the gas phase. Acidification was less important in B slurry than in A and C, at the end of experiment. 3 months; T6m: 6 months; T9m: 9 months. Major phyla (E) and 10 major OTUs (F) found in the BIO slurries at the end of the second anaerobic period (T9m).

The incubation conditions influenced the speciation of As in the water phase (Table 1). In the IN condition, AsIII was the major initial species, then AsV became the main form of As until the end of experiment. In the BIO condition, at the end of the anaerobic periods, As-containing species were detected, with retention times that did not match any available standard. They could not be precisely quantified because of the lack of standards, however as estimated based J o u r n a l P r e -p r o o f on AsV standard additions slope, they represented more than 50% of total As in A, B and C at the end of the first anaerobic period and in A and C at the end of the second anaerobic period.

The unidentified As species were not detected at the end of the aerobic incubation period, however at the end of this experimental step, dimethyl-arsenate (DMA V ) was present in BIO condition, representing nearly 50% of total As. From the end of the first incubation period, dissolved As concentration remained very low but mainly in the form of AsV in the IN microcosm. This result might be linked to the adsorption of AsIII followed by abiotic oxidation at the surface of clay material (Lin and Puls, 2000). Concentrations of Cl -, Na + , Ca 2+ and Mg 2+ remained stable and identical in BIO and IN conditions all along experiment (Supplementary Figure 6), whereas NO3 -and SO4 2-tended to be more impacted by redox conditions in BIO than in IN condition, particularly between T0m and T6m.

Microbial parameters

The quantification of As-transforming microbes by MPN showed the growth of active AsIIIoxidising and AsV-reducing bacteria throughout the incubation (Table 1), independently of aerobic or anaerobic periods, whereas in IN condition the quantity of As-transforming microbes remained lower than the quantification limit all along the experiment. AsV-reducing bacteria were always more abundant than AsIII-oxidising micro-organisms. At the end of the second anaerobic period, the order of magnitude of MPN in sediments from parallel microcosms A, B and C was 10 6 g -1 for AsIII-oxidising bacteria and 10 7 g -1 for AsV-reducing bacteria.

The PCA built with all physico-chemical and biological parameters measured during the incubation (Supplementary Figure 7) illustrates the contrast between the evolution of IN and BIO slurries. Arsenic concentration was correlated with Fe concentration, and negatively correlated with sulphate concentration. Moreover, this analysis suggests an early divergence, detectable at the end of the first incubation period, between the microcosm B and the two microcosms A and C.

The bacterial diversity in the slurries at the end of the second anaerobic period (supplementary

Discussion

Geological context and origin of As in the confined aquifer

Macroscopic and microscopic observations of the core material suggest that the sands and clays covering the calcareous confined aquifer were deposited in a meandering river system environment composed of channels, crevasse splay and floodplain facies. These last two facies are enriched in As and other trace elements. The As enrichment in sedimentary facies linked with high groundwater As concentrations was already reported [START_REF] Chatterjee | Mobilization of arsenic in sedimentary aquifer vis-à-vis subsurface iron reduction processes[END_REF][START_REF] Sahu | Role of shallow alluvial stratigraphy and Holocene geomorphology on groundwater arsenic contamination in the Middle Ganga Plain, India[END_REF]. In these formations, As-rich levels are generally described as containing elevated organic matter concentrations. Here, the clayey levels presenting the highest As contents displayed low organic carbon concentrations (0.01 to 0.02%). However, macroscopic observation of plant debris in the core suggested that organic matter is present but distributed in nuggets.

As release mechanisms implying Fe and S cycles

J o u r n a l P r e -p r o o f

The geochemical composition of the confined groundwater, with absence of nitrate (< 0.5 mg L -1 ) and presence of ammonium, dissolved Mn and Fe, suggests anaerobic conditions. The dissolved O2 concentrations (0.9 to 2.3 mg L -1 ) might be related to the diffusion of this gas into the well which creates local suboxic conditions. Low concentrations of SO4 2-associated with high δ 34 S of sulphate (mean of 16.3 ‰) would be consistent with in-situ sulphate reduction (Stueben et al., 2003;[START_REF] Xwang | Coupled iron, sulfur and carbon isotope evidences for arsenic enrichment in groundwater[END_REF]. The evolution of the δ 34 SSO4 of the water sampled in the drinking water well of Marcilly-en-Gault according to total sulphate concentration suggests variations of the in-situ activity of SRB, that might be influenced by evolution of redox conditions or organic matter supply. These δ 34 S values are also associated to notable concentrations of As (mean 22.6 µg L -1 ) as already described by various authors e. g., (Fig. 1C; [START_REF] Pi | Vertical variability of arsenic concentrations under the control of iron-sulfur-arsenic interactions in reducing aquifer systems[END_REF], Stueben et al., 2003[START_REF] Xwang | Coupled iron, sulfur and carbon isotope evidences for arsenic enrichment in groundwater[END_REF]. In a complementary way, the development of active As-transforming microbes during batch incubations confirmed the presence of living micro-organisms in the confined aquifer. These results support the hypothesis of active biogeochemical reactions influencing the release and speciation of As in groundwater.

A link between biogeochemical processes and As mobilisation from sediments to groundwater, implying diverse mechanisms and especially iron and sulphur cycles, was evidenced in several other sites. The mechanism most often at the origin of As release is the biological reduction of Fe oxides [START_REF] Stuben | Arsenic enrichment in groundwater of West Bengal, India: geochemical evidence for mobilization of As under reducing conditions[END_REF][START_REF] Zheng | Redox control of arsenic mobilization in Bangladesh groundwater[END_REF][START_REF] Nath | Mobility of arsenic in the sub-surface environment: An integrated hydrogeochemical study and sorption model of the sandy aquifer materials[END_REF][START_REF] Erickson | Arsenic Cycling in Hydrocarbon Plumes: Secondary Effects of Natural Attenuation[END_REF][START_REF] Ziegler | A mass balance approach to investigate arsenic cycling in a petroleum plume[END_REF][START_REF] J O U R N A L P R E -P R O O F Johannesson | Biogeochemical and reactive transport modeling of arsenic in groundwaters from the Mississippi River delta plain: An analog for the As-affected aquifers of South and Southeast Asia[END_REF]. The confined groundwater of Marcilly-en-Gault contains dissolved Fe, the analysis of core materials indicated the presence of FeIII in the Asrich levels, and our slurry incubation experiment showed As and Fe release during anaerobic periods (Figure 5), and correlation between these two parameters (Supplementary Figure 7).

These results support the hypothesis that Fe oxides reduction plays a major role in the release of As. Sulphate reduction was also invoked in previous studies as possibly contributing to As release through (1) indirect chemical reduction of Fe oxides by H2S, and (2) the formation of J o u r n a l P r e -p r o o f soluble thio-arsenate complexes [START_REF] Xwang | Coupled iron, sulfur and carbon isotope evidences for arsenic enrichment in groundwater[END_REF][START_REF] Gao | Microbially mediated mobilization of arsenic from aquifer sediments under bacterial sulfate reduction[END_REF]. These processes might have modestly contributed to As mobility in our microcosms. [START_REF] Pi | Vertical variability of arsenic concentrations under the control of iron-sulfur-arsenic interactions in reducing aquifer systems[END_REF] observed profiles of biogeochemical Fe reduction and sulphate reduction controlling both As speciation and concentration in groundwater of Datong basin, China. They hypothesised that vertical variations of As concentrations and forms were associated with availability of organic matter, providing energy to microbial reactions. Our incubations of core materials showed that bacterial activities influenced As speciation, producing As species different from AsIII and AsV. In anaerobic conditions, non-identified species might correspond to thio-arsenate complexes that were already associated with bacterial sulphate reduction in core sediments microcosms [START_REF] Gao | Microbially mediated mobilization of arsenic from aquifer sediments under bacterial sulfate reduction[END_REF]. In aerobic conditions, up to 50% of total As was present as DMA V , a biologically produced methylated species [START_REF] Dombrowski | Thermodynamic Analysis of Arsenic Methylation[END_REF]). Yet, these species exclusively produced when bacterial communities are active could be less efficiently adsorbed on Fe oxides surfaces than AsV or AsIII [START_REF] Lafferty | Methyl Arsenic Adsorption and Desorption Behavior on Iron Oxides[END_REF][START_REF] Couture | Effect of microbially mediated iron mineral transformation on temporal variation of arsenic in the Pleistocene aquifers of the central Yangtze River basin[END_REF]. Thus, in addition to the reduction of iron oxides, bio-reduction of sulphate and direct As transformation by microbes could contribute to the mobility of this toxic element.

Microbial assemblages

Groundwater

The main major OTU found in Marcilly-en-Gault groundwater was affiliated to Hydrogenophilaceae family, currently composed of 5 genera, Thiobacillus, Hydrogenophilus, Sulfuricella, Petrobacter, and Tepidiphilus [START_REF] Garrity | Order II. Hydrogenophilales ord. nov[END_REF][START_REF] Orlygsson | Arsenic biotransformation and release by bacteria indigenous to arsenic contaminated groundwater[END_REF]. This family includes chemolithotrophic or mixotrophic bacteria able to oxidise inorganic substrates, such as sulphur compounds or hydrogen, possibly using nitrate as electron acceptor (Sulfuricella, [START_REF] Kojima | Sulfuricella denitrificans gen. nov., sp. nov., a sulfur-oxidizing autotroph isolated from a freshwater lake[END_REF]. The second main metabolic group found in J o u r n a l P r e -p r o o f groundwater is composed of OTUs affiliated to Galliollenaceae, gathering autotrophic microorganisms oxidising FeII in micro-aerophilic environments. Their abundance in the sampled groundwater is consistent with the presence of dissolved Fe, and it might be assumed that they could form biofilms onto surfaces of the drinking water well [START_REF] Stuetz | Impact of localised dissolved iron concentrations on the biofouling of environmental wells[END_REF]. They may also grow in areas of the aquifer where small quantities of oxygen could diffuse or be transported. Georgfuchsia was described as able to degrade aromatic compounds (Weelink et al., 2009), that could be an asset to use organic molecules derived from buried plant debris. Sphingopyxis, another heterotroph found in the groundwater, is also known for its ability to degrade complex molecules [START_REF] Sharma | The genus Sphingopyxis: Systematics, ecology, and bioremediation potential -A review[END_REF].

Batch experiments

The microbial communities retrieved at the end of the incubation experiment clearly differed from those observed in groundwater samples. This difference can be explained by the strong reducing conditions and hydrogen supply during the last incubation period, while in the drinking well, diffusion of oxygen could create microaerophilic conditions, specific to the local environment of this water source. Moreover, the sediment is a source of nutrients and carries some bacteria. Indeed, bacterial communities present in the water phase of aquifers may differ from those attached to solid phases [START_REF] Smith | Impact of hydrologic boundaries on microbial planktonic and J o u r n a l P r e -p r o o f biofilm communities in shallow terrestrial subsurface environments[END_REF]. In these incubated slurries, the two most abundant OTUs were affiliated, at the family level, with Rhodocyclaceae and Geobacteraceae. Rhodocyclaceae family includes many genera presenting diverse J o u r n a l P r e -p r o o f metabolisms; thus it is hardly possible to infer a role of members of this OTU. However, a recent report of microcosm experiment performed with groundwater and sediments supplied with hydrogen also led to enrichment of Rhodocyclaceae that could be related to the genus Dechloromonas, a hydrogenotrophic denitrifier [START_REF] Duffner | Dechloromonas and close relatives prevail during hydrogenotrophic denitrification in stimulated microcosms with oxic aquifer material[END_REF]. This OTU was clearly enriched during incubation, but was also present in all groundwater samples, in small abundance. Geobacteraceae members share the ability to use FeIII as terminal electron acceptor and can use either small organic compounds or hydrogen as electron source [START_REF] J O U R N A L P R E -P R O O F Röling | The Family Geobacteraceae[END_REF].

Their presence as a major OTU in our system can be related with the Fe oxides dissolution process which could mobilise As. This OTU was very rare in the groundwater samples, only be linked to the initial heterogeneity of the solid phase inherent in the nature of the actual material that induced heterogeneity in initial microbial communities. Such phenomenon was previously reported in parallel slurries of underground sediment mixed with ferrihydrite [START_REF] Kwon | Impact of Organic Carbon Electron Donors on Microbial Community Development under Iron-and Sulfate-Reducing Conditions[END_REF] which evolved differently in terms of geochemistry and microbial communities, this result being related with subtle differences in the initial composition of the microbial communities. High As concentrations in zones of active methanogenic activities have already been reported [START_REF] Wang | Sulfate enhances the dissimilatory arsenate-respiring prokaryotes-mediated mobilization, reduction and release of insoluble arsenic and iron from the arsenic-rich sediments into groundwater[END_REF][START_REF] Glodowska | Arsenic behavior in groundwater in Hanoi (Vietnam) influenced by a complex biogeochemical network of iron, methane, and sulfur cycling[END_REF] whereas the biogeochemical link between methanogenesis and As release is not clearly elucidated. According to [START_REF] Glodowska | Arsenic behavior in groundwater in Hanoi (Vietnam) influenced by a complex biogeochemical network of iron, methane, and sulfur cycling[END_REF], organic carbon input due to hydrogeological conditions fuels fermentation producing J o u r n a l P r e -p r o o f hydrogen, thus, indirectly, methanogenic metabolisms and efficient reduction of As-bearing Fe minerals. Methane oxidation might also be coupled to FeIII reduction. In our systems, hydrogen was provided, thus could be consumed for both FeIII reduction and methanogenesis. One important question would be the possible availability of hydrogen or other electron donors in the confined aquifer. Previous studies showed that intrinsic organic matter of aquifer sediments could fuel anaerobic respiration processes [START_REF] Duan | Experimental constraints on redox-induced arsenic release and retention from aquifer sediments in the central Yangtze River Basin[END_REF][START_REF] Gao | Microbially mediated mobilization of arsenic from aquifer sediments under bacterial sulfate reduction[END_REF]. However, organic carbon concentration was very low in M8 sample, (0.02%) compared with that reported in previous studies (0.16 to 0.70% in [START_REF] Duan | Experimental constraints on redox-induced arsenic release and retention from aquifer sediments in the central Yangtze River Basin[END_REF]0.22% in Gao et al., 2021). The classical hypothesis of hydrogen production through fermentation of organic matter could hardly be considered in our system except if nuggets of organic matter, such as the plant debris macroscopically observed in the sedimentary profile, could be used as organic substrates. As a fact, the microbial community of incubated slurries contained the fermentative genus Propionivibrio, in low abundance (0.1 to 0.6%) but in all microcosms. Species of this genus, belonging to Rhodocyclaceae, are aerotolerant anaerobes performing fermentation of aromatic compounds [START_REF] Brune | Propionivibrio limicola sp. nov., a fermentative bacterium specialized in the degradation of hydroaromatic compounds, reclassification of Propionibacter pelophilus as Propionivibrio pelophilus comb. nov. and amended description of the genus Propionivibrio[END_REF]; their emergence in the incubated slurries might indicate the intrinsic organic matter could be metabolised.

Linking microbial assemblages with biogeochemical processes

SRB were always detected both in groundwater samples and microcosms. However, they were present in relatively low abundance in groundwater, and in B microcosm in which the highest release of As was observed. In-situ evolution of SO4 and δ 34 S and the development of SRB in A and C slurries support the hypothesis of active sulphate-reduction in the confined aquifer.

Both groundwater and microcosms hosted microbial assemblages containing OTUs whose known members are involved in iron and sulphur cycles, but only a few methanogens except in one of the microcosms, where this metabolic group was abundant. All these types of microbial metabolisms were already observed in other As-bearing groundwater systems (Table 2).

J o u r n a l P r e -p r o o f

Globally, the major abundance of bacterial OTUs involved in Fe cycling, i.e. FeII oxidation in groundwater samples, and FeIII-reduction in all incubated slurries, strongly suggests a major role of Fe biogeocyle in the confined aquifer. The Geobacteraceae-affiliated OTU massively present at the end of slurry incubations, and putatively involved in Fe oxide reduction, was only sparsely detected in groundwater samples, possibly because they were not mobile but attached to solid phases, or because the local suboxic condition of the well was not suitable for their growth. However another OTU, affiliated to Georgfuchsia, a known FeIII reducer (Weelink et al., 2009), was present in low abundance but regularly along all seasons in groundwater.

Conclusion

The occurrence of As in the confined aquifer of Sologne appears to find its origins in anoxic bioprocesses, as already described in other sedimentary paleo-environments. In particular, the

  Michel et al. (2021), Fernandez-Rojo et al. (2017) and Poirel et al. (2013).

238Fig. 1 .

 1 Fig. 1. Evolution of groundwater geochemical parameters: (A) Total As (red) and As speciation

Fig. 2 .

 2 Fig. 2. Drinking water well of Marcilly-en-Gault, evolution of (A) main bacterial phyla; (B)

Fig. 3 .

 3 Fig. 3. RDA of sequence abundance assigned at phylum level (A) and genus level (B) of

Fig. 4 .

 4 Fig. 4. Microscopic petrographic observations of thin sections (A to D) and chemical

Fig. 5 .

 5 Fig. 5. Evolution of pH (A), redox potential (Eh, B), total As (C) and Fe (D) concentrations in

  sparsely detected in September 2016 and May 2018. Incubation also led to enrichment of Comamonadaceae, a very metabolically diverse family. The next groups of less abundant but major OTUs marked differences between microcosms: they included either SRB in A and C, or methanogens in B. This strong contrast between B compared with A/C microbial communities might be considered in the light of the difference in final geochemical profiles of the slurries: B presented much more dissolved Fe and As than A and C. In terms of sulphate concentrations, the three microcosms behaved similarly. The divergence of results in B might

Fe

  oxides detected in As-rich floodplain and crevasse splay clays (fluvial environment) covering the calcareous aquifer (lake environment) could be biologically reduced. The major role of FeIII reduction in As release was supported by high Fe concentrations in water and solid phases, and proportions of microbial groups involved in Fe bio-geocycle, both in groundwater samples and microcosms. Other microbial reactions could play non negligible roles in As mobilisation, namely sulphate reduction, AsV reduction, AsIII oxidation and As methylation.Corresponding bacterial activities drive the production of diverse As species whose proportions may fluctuate and influence the distribution of As between solids and water. Even if phenomena globally followed the same patterns in all microcosms, our results underline the possibility of strong variations in the amplitude of the bio-reactions driving As release from solids to water, linked to subtle heterogeneities that induced important divergence of microbial communities compositions. This phenomenon, generally ignored or underestimated, deserves to be explored further to improve the accuracy of predictive models taking into account biogeochemical J o u r n a l P r e -p r o o f reactions. In the context of global change and increasing anthropic pressure on water resources, managers will need reliable tools to anticipate the evolution of groundwater quality. At larger scale, important spatial heterogeneities of organic matter distribution and As concentrations, linked to geological context, i.e. sedimentary deposition in the meandering river system (channels, crevasse splay and floodplain), may occur and induce local variations of the intensity of biogeochemical processes. The mechanisms of As release in the sub-surface and their potential evolution with groundwater table level would need to be better understood through long-term monitoring of dissolved As species, and development of experimental systems and models taking into account heterogeneities at different scales.

Table 1 .

 1 Total As, As speciation in the water phases of incubated slurries, and MPN of AsIIIoxidising and AsV-reducing microbes.ND: not detected, (*) unidentified As-containing species, estimated indicative values calculated with AsV standard addition slope; LQ = 4.00×10 1 g -1 .

	Time Beginning (T0m) End of first	Condition Total As (µg L -1 ) BIO A 17.0 BIO B 16.7 BIO C 15.3 IN 14.3 BIO A 155.0 J o u r n a l P r e -p r o o f AsIII (%) AsV (%) DMA V (%) Un. species 1* (%) Un. species 2* (%) Un. species 3* (%) 40.0 60.0 ND ND ND ND 37.2 62.8 ND ND ND ND 36.9 63.1 ND ND ND ND 95.5 4.5 ND ND ND ND 4.8 0.8 ND 6.8 8.6 79.0	MPN AsIII-ox g -1 solids < LQ < LQ 8.00×10 1 < LQ 1.84×10 3	MPN AsV-red g -1 solids 3.16×10 3 3.76×10 3 9.20×10 2 < LQ 2.52×10 3
	anaerobic	BIO B	118.0	4.5	0.7	ND	9.8	7.9	77.0	9.60×10 3	5.20×10 3
	period (T3m)	BIO C	141.0	6.7	1.3	ND	7.7	9.0	75.3	3.60×10 3	9.60×10 4
		IN	4.4	3.5	96.5	ND	ND	ND	ND	< LQ	< LQ
	End of	BIO A	1.2	1.8	44.6	53.6	ND	ND	ND	5.20×10 5	9.60×10 5
	aerobic	BIO B	1.4	1.5	46.2	52.3	ND	ND	ND	1.32×10 5	1.96×10 5
	period	BIO C	1.1	1.7	50	48.3	ND	ND	ND	1.32×10 5	5.20×10 6
	(T6m)	IN	0.5	4.0	95.0	1.0	ND	ND	ND	< LQ	< LQ
	End of	BIO A	173.0	14.1	0.9	ND	10.3	5.9	68.8	1.96×10 5	3.68×10 7
	second	BIO B	278.0	88.7	2.3	ND	8.5	0.5	ND	9.60×10 5	6.80×10 6
	anaerobic	BIO C	193.0	25.7	1.6	ND	5.8	6.3	60.6	8.80×10 5	5.20×10 6
	period (T9m)	IN	4.2	10.6	89.4	ND	ND	ND	ND	< LQ	< LQ

Table 4

 4 profiles in batches A and C, that differed from batch B that contained Euryarchaeota (Archaea domain) in higher abundance, with a significant proportion of sequences affiliated to methanogenic micro-organisms. All slurries contained, as major OTUs, sequences that were only affiliated at the family level, first to Rhodocyclaceae, then to Geobacteraceae. Sum of sulphate-reducers sequences (Supplementary Table7) represented nearly 10% in A and C, but only 0.3% in B. Conversely, sum of methanogens sequences represented 37% of the sequences in B, but less than 0.2% in A and were not detected in C.

) was higher than that of the groundwater bacterial communities, in spite of a lower richness. The corresponding 16S rRNA gene sequences (Fig.

5 E and F

) showed close diversity J o u r n a l P r e -p r o o f

  [START_REF] Glodowska | Arsenic behavior in groundwater in Hanoi (Vietnam) influenced by a complex biogeochemical network of iron, methane, and sulfur cycling[END_REF] already quantified non negligible proportions of Galliollenaceae and other putative FeII-oxidisers in As-bearing groundwater where they are assumed to favour As immobilisation with FeIII oxides formation in environments of lowoxygen availability. Bacteria that could contribute directly or indirectly to iron oxides reduction, such a sulphate reducers (Desulfurivibrionaceae) and FeIII reducers (Georgfuchsia) were always detected in groundwater samples, but in relatively low abundance. Interestingly,

Table 2 .

 2 Main characters of microbial communities in arsenic-rich groundwater, groundwater-related sediments, and related laboratory experiments

	Environments Groundwater, Inner Mongolia J o u r n a l P r e -p r o o f Main OTUs (16S genus level) Main detected functions Pseudomonas, Acinetobacter, Brevundimonas, Aquabacterium, Psychrobacter, Geobacter, Arthrobacter, Massilia, Dietzia, Sphingomonas, Not mentioned Li et al., References 2013
		Planococcus, Methanosaeta,
		Nitrosophaera, Thermoprotei
	Holocene and	Geobacter, Propionivibrio, Aeromonas,	Not mentioned Hery et al.,
	Pleistocene	Acidobacteria, Pelagibius, Enterobacter,	2015
	sediments, Cambodia	Pseudomonas, Sphingomonas,
		Pseudolabrys, Methylobacterium,
		Microvirga, Delftia, Tumebacillus,
		Euzebya, Dietzia

Acknowledgements

This research was supported by the Water Agency Loire-Bretagne (decision n°2015C009), and by BRGM funding PEX BIODIV. We thank Luigi Ardito for performing core sampling operation, and the municipal authority of Marcilly-en-Gault for allowing our sampling campaigns.

 Zuzolo, D., Cicchella, D., Demetriades, A., Birke, M., Albanese, S., Dinelli, E., Lima, A., Valera, P., De Vivo, B., 2020


Arcuate Fasciculus' middle and ventral temporal connections undercut by tract-tracing evidence

Yannick Becker [START_REF] Vavassori | Hodology of the superior longitudinal system of the human brain: a historical perspective, the current controversies, and a proposal[END_REF][START_REF] Becker | The Arcuate Fasciculus and language origins: Disentangling existing conceptions that influence evolutionary accounts[END_REF] , Kep Kee Loh 1,2 , Olivier Coulon 2 , Adrien Meguerditchian 1,3 1 Laboratoire de Psychologie Cognitive, CNRS UMR 7290, Marseille, France 2 Institut de Neurosciences de la Timone, Aix-Marseille Univ, CNRS UMR 7289, Marseille, France [START_REF] Giampiccolo | Controversy over the temporal cortical terminations of the left arcuate fasciculus: a reappraisal[END_REF] Station de Primatologie CNRS-CELPHEDIA UAR846, 13790 Rousset, France

We are pleased to respond to the recent review article of Giampiccolo and Duffau (2022) published in Brain about the temporal terminations of the Arcuate Fasciculus (AF). The AF is regarded a major white matter pathway for language in the human brain [START_REF] Vavassori | Hodology of the superior longitudinal system of the human brain: a historical perspective, the current controversies, and a proposal[END_REF] . As such, there has been massive cross-disciplinary interest about the AF since its first discovery in the early 19 th century. However, as highlighted in our recent review article 2 , the anatomical definition of the Arcuate Fasciculus remains highly controversial, as its exact course and terminations, and how it differs between human versus nonhuman primate brains remain intensely debated in the literature.

Giampiccolo and Duffau 3 had proposed a novel model of the AF's temporal connections based on recent evidence from clinical cases and direct electrical stimulation studies. Their model depicted more extensive AF temporal terminations that reach anterior-ventrally beyond the posterior superior temporal cortex, into the middle temporal gyrus, and the inferior/basal temporal cortex (Figure 1 left panel). While this model may have important clinical implications, it is based on methods that are limited in providing the "ground truth" delineation of the AF tract. Specifically, we contend that fundamental findings about the AF's temporal connections from monkey tract-tracing studies, the gold-standard methodology for detecting intercortical connections) are incongruent with Giampiccolo and Duffau's model [START_REF] Giampiccolo | Controversy over the temporal cortical terminations of the left arcuate fasciculus: a reappraisal[END_REF] .

Precious knowledge about brain connections is derived from tract-tracing experiments in nonhuman primates, which many, including the authors themselves, acknowledge to be the gold-standard in the field [START_REF] Petrides | Neuroanatomy of Language Regions of the Human Brain[END_REF] . Indeed, in contrast with other in-vivo techniques, the tract-tracing approach has the advantage to reveal mono-synaptic connections (i.e., direct, single-axonal connections from one neuron to another) between cortical areas, and is therefore, the only technique that can reveal the true course and terminations of a single continuous white matter tract, such as the AF. This method involves the injection of anatomical tracers into a living brain, which are then transported by interconnecting axons from neurons in the injected site to other neurons. Critically, this is followed by histological analyses of the brain tissue postmortem, in order to visualise the axons, and the cell bodies of the neurons that have been labelled by the injected tracers. Unfortunately, this technique, requiring both invasive in-vivo cortical injections, and the eventual sacrifice of the subject for post-mortem histological analyses, is virtually impossible to conduct in humans.

Based on tract-tracing experiments in macaque monkeys, the AF's temporal terminations rarely exceeds the posterior third of the superior temporal gyrus anteriorly (i.e., at the level of the Planum Temporale), and the ventral bank of the STS, ventrally (Figure 1 left panel [START_REF] Petrides | Neuroanatomy of Language Regions of the Human Brain[END_REF] ).

Using dMRI tractography, recent researchers have performed the careful dissection of the human AF based on the macaque AF obtained via tract-tracing. This work had demonstrated that, in the human brain, the AF temporal terminations stay mostly in the posterior superior temporal gyrus/sulcus region (e.g., [START_REF] Barbeau | Dissociating the white matter tracts connecting the temporo-parietal cortical region with frontal cortex using diffusion tractography[END_REF][START_REF] Frey | Dissociating the Human Language Pathways with High Angular Resolution Diffusion Fiber Tractography[END_REF][START_REF] Luo | Middle longitudinal fascicle is associated with semantic processing deficits in primary progressive aphasia[END_REF][START_REF] Makris | Delineation of the Middle Longitudinal Fascicle in Humans: A Quantitative, In Vivo, DT-MRI Study[END_REF] ; Figure 1 approaches. Nevertheless, we cannot ignore the fact that these methods do not allow the distinction of mono-synaptical from poly-synaptical connections (i.e., indirect connections that involve one or more interconnecting neurons). In other words, these methods are not able to disentangle the AF fibres from the other temporal fibre pathways that build synapses with the AF in the temporal lobe [START_REF] Petrides | Neuroanatomy of Language Regions of the Human Brain[END_REF][START_REF] Latini | New Insights Into the Anatomy, Connectivity and Clinical Implications of the Middle Longitudinal Fasciculus[END_REF][START_REF] Wang | Rethinking the Role of the Middle Longitudinal Fascicle in Language and Auditory Pathways[END_REF] . It is thus possible that the more anterior and ventral temporal gyrus terminations proposed by Giampiccolo and Duffau 3 , might not reflect monosynaptic connections from the AF, but interactions with between the AF and fibres from other temporal pathways. As such, we feel the authors should nuance their position that their revised AF model represents the true human AF and discuss the potential disagreements between their model and other recent models of the human AF that have been derived based on macaque tract tracing studies [START_REF] Barbeau | Dissociating the white matter tracts connecting the temporo-parietal cortical region with frontal cortex using diffusion tractography[END_REF][START_REF] Frey | Dissociating the Human Language Pathways with High Angular Resolution Diffusion Fiber Tractography[END_REF][START_REF] Luo | Middle longitudinal fascicle is associated with semantic processing deficits in primary progressive aphasia[END_REF][START_REF] Makris | Delineation of the Middle Longitudinal Fascicle in Humans: A Quantitative, In Vivo, DT-MRI Study[END_REF] .

In conclusion, the human AF model proposed by Giampiccolo and Duffau 3 is based on the best available methods (dMRI tractography, electrical stimulation, blunt dissections) to study white matter connections in the human brain. These methods, unfortunately, do not warrant the detection of true monosynaptic connections, which is only possible using invasive tracttracing techniques. Importantly, their model is incongruent with existing human AF models that are based on macaque tract-tracing knowledge [START_REF] Petrides | Neuroanatomy of Language Regions of the Human Brain[END_REF][START_REF] Barbeau | Dissociating the white matter tracts connecting the temporo-parietal cortical region with frontal cortex using diffusion tractography[END_REF][START_REF] Frey | Dissociating the Human Language Pathways with High Angular Resolution Diffusion Fiber Tractography[END_REF][START_REF] Luo | Middle longitudinal fascicle is associated with semantic processing deficits in primary progressive aphasia[END_REF][START_REF] Makris | Delineation of the Middle Longitudinal Fascicle in Humans: A Quantitative, In Vivo, DT-MRI Study[END_REF] . As it stands, there is currently no method to determine which model depicts the true human AF, since tract-tracing studies are not possible in humans. Until that technology surfaces, the field is confronted with two competing hypotheses: 1) The human AF has indeed expanded anteriorly and ventrally into the middle and inferior temporal cortex as Giampiccolo and Duffau 3 depicts; or 2) The human AF is homologous to that of the monkey AF, and Giampiccolo and Duffau's observations 3 could be due to the fact that the human AF is entangled with other temporal tracts (e.g., the Inferior Longitudinal Fasciculus (ILF) Inferior Fronto-Occipital fasciculus (IFOF), the Middle Longitudinal Fasciculus (MdLF), Uncinate Fasciculus (UF), Extreme

Capsule Fasciculus (ECF) etc.).

  left panel), which runs contrary to the revised model proposed by Giampiccolo and Duffau 4 (Figure 1 right panel).

Figure 1 Contemporary

 1 Figure 1 Contemporary Models of the AF fibre pathways in humans. Left: Macaque tract-tracing based human tractography model. Figure adapted from 2 , based on 4 . Right: Giampiccolo and Duffau's model with extended temporal lobe terminations added onto the tract tracing model. AF=Arcuate Fasciculus; SFS=Superior Frontal Sulcus; SPrS=Superior Precentral Sulcus; IPrS=Inferior Precentral Sulcus; IFS=Inferior Frontal Sulcus; AS=Ascending branch of Sylvian Fissure; HS=Horizontal branch of Sylvian Fissure; IPS=Intra-Parietal Sulcus; Tpt=Temporal parietal area (BA22); aSMG=anterior Supra-Marginal Gyrus (BA40); pSMG=posterior Supra Marginal Gyrus (BA40); ANG=Angular Gyrus (BA39); SPL=Superior Parietal Lobule (BA7) ; MTG=Middle Temporal Gyrus; aITG=anterior Inferior Temporal Gyrus; pITG=posterior Inferior Temporal Gyrus


Arnaud Belcour

Baptiste Ruiz

Clémence Frioux

Samuel Blanquart

Anne Siegel

EsMeCaTa: Estimating metabolic capabilities from taxonomic affiliations

Predicting the functional potential of microorganisms in environmental samples from cultivation-independent techniques is a major challenge. A persistent difficulty lies in associating taxonomic profiles obtained from metabarcoding experiment with accurate functional profiles, particularly for poorly-resolved taxonomic groups. In this paper, we present EsMeCaTa a python package predicting shared proteins from taxonomic affiliations. EsMeCaTa relies on the UniProt database to retrieve the public proteomes associated with a taxon and then uses MMseqs2 in order to compute the set of proteins shared in the taxon. Finally, Es-MeCaTa extracts the functional annotations of these proteins to provide an accurate estimate of the functional potential associated to taxonomic affiliations.

Introduction

Sequencing of gene markers, such as 16S rRNA gene, is commonly applied to characterize the diversity of organisms in environmental samples. From these data, taxonomic affiliations, such as Operational Taxonomic Units (OTUs), can then be used to estimate the potential functions in the environment.

Various tools have been developed to estimate functional profiles associated with an OTU, such as PICRUSt2 [START_REF] Douglas | PICRUSt2 for prediction of metagenome functions[END_REF], Paprica [START_REF] Bowman | Microbial communities can be described by metabolic structure: A general framework and application to a seasonally variable, depth-stratified microbial community from the coastal west antarctic peninsula[END_REF], Tax4fun2 [START_REF] Wemheuer | Tax4fun2: prediction of habitat-specific functional profiles and functional redundancy based on 16s rRNA gene sequences[END_REF]. These tools have been developed mainly to proceed 16S rRNAs, although other gene markers can be considered (e.g. [START_REF] Ogier | rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing[END_REF]). Apart from marker genes, taxonomic 1 affiliations can be obtained from shallow whole genome sequencing data, but the association of function with these data remains uneasy.

We developed EsMeCaTa (Estimating Metabolic Capabilities from Taxonomic affiliations) as a method permitting the estimation of functions independently of the taxonomic assignment method used. EsMeCaTa is a python package that relies on the UniProt database to estimate the shared proteins associated with prokaryotic or eukaryotic taxa considered as input, providing insights into their putative metabolic capabilities.

Approach

EsMeCaTa takes as input a tabulated file containing two columns. The first column is an identifier and the second contains a taxonomic affiliation (starting with the highest taxonomic rank, such as kingdom, to the lowest taxonomic such as species) as defined by the NCBI Taxonomy database [START_REF] Schoch | NCBI taxonomy: a comprehensive update on curation, resources and tools[END_REF]. The outputs of the workflow are, for each taxon (1) fasta files of all proteomes selected by EsMeCaTa, (2) a fasta file of the shared proteins clustered by MMseqs2 from these proteomes, and (3) a tabulated file containing the functional annotations associated with these proteins (Gene Ontology Terms (GO), Enzyme Commission (EC)).

The first part of the workflow selects the lowest taxonomic rank of each input taxonomic affiliation for which the UniProt Proteomes database (The UniProt Consortium, 2021) contains at least one proteome exhibiting a BUSCO score higher than 80% and considered "non-redundant" and "not-excluded" by UniProt (column 'Proteomes selection' in Table 1). More precisely, the taxonomic affiliation is processed using the ete3 python package [START_REF] Huerta-Cepas | ETE 3: Reconstruction, analysis, and visualization of phylogenomic data[END_REF] in order to associate a taxon ID (from the NCBI taxonomy database) to each taxon from the affiliation. Using this ID, queries against the UniProt Proteomes database find the lowest-ranking taxon for which there is at least one reference or non-reference proteome in the database and download those proteomes. If the number of proteomes is greater than a threshold (100 by default), only a subsample is downloaded.

The second part of the workflow aims at identifying proteins shared by proteomes associated with a taxon. With the downloaded proteomes, EsMeCaTa performs protein clustering using MMseqs2 [START_REF] Steinegger | MMseqs2 enables sensitive protein sequence searching for the analysis of massive data sets[END_REF] and selects clusters such that proteins are shared by at least X% of the proteomes (see the column 'Protein clusters (MMseqs2)' in Table 1). The threshold X = 0 corresponds to the case where all protein clusters are selected (called 'Panproteome', abbreviated Pan-P, in reference to pan-genome). A second threshold at X = 0.95 retains only the clusters containing a protein originating from at least 95% of the proteomes (called 'Soft core proteome', abbreviated Soft-P).

A third threshold at X = 0.5 retains cluster containing proteins occurring in at least 50% of the proteomes (called 'Shell core proteome', abbreviated Shell-P). For each protein cluster, EsMeCaTa selects the representative protein (first sequence in the alignment made by MMseqs2) to represent the cluster. The 2 selected sequences of the representative proteins are stored in a fasta file using biopython [START_REF] Peter | Biopython: freely available python tools for computational molecular biology and bioinformatics[END_REF].

The final step of the workflow annotates the protein clusters by querying the UniProt database (GO, EC, see the columns 'Functional annotation of clusters' in the Table 1). Table 1: Result of EsMeCaTa on 13 taxonomic affiliations (described by names and ranks). Soft-P: Soft core proteome. Shell-P: Shell core proteome. Pan-P: Pan-proteome.

Results

Input

Table 1 shows the application of EsMeCaTa (with UniProt 2021 03 and MMseqs2 13.45111) to 13 different taxonomic affiliations (the lowest taxon in these affiliations is indicated in the 'Lowest taxon name' column) selected to cover both prokaryotic (Gammaproteobacteria) and eukaryotic (Alveolata) taxa with different taxonomic ranks (from class to genus), in order to illustrate the uncertainty in the input taxonomic affiliation, the available knowledge and the biases toward most documented clades.

For 9 taxonomic affiliations, proteomes were selected using the lowest taxonomic rank, whereas for the 4 other affiliations, EsMeCaTa selected a higher taxonomic rank (bold in the column 'Taxon rank used'). For the 13 selected taxa, UniProt contained from 1 to 8,271 proteomes. In two cases (Cronobacter and Lelliottia), no reference proteome was found and EsMeCaTa returned nonreference proteomes of Uniprot. In 9 cases (such as Escherichia), EsMeCaTa returned the reference proteomes found in Uniprot (from 1 to 48). In the last two cases (Enterobacterales and Gammaproteobacteria), more than 99 reference proteomes were found, of which 96 proteomes were selected by the subsampling procedure.

We observe that in general (in the column 'Protein clusters (MMseqs2)' of the table 1), the size of the Pan-P increases with the number of selected proteomes, while the size of the Soft-P decreases. The size of the Shell-P appears to be much more stable. The numbers of GOs and ECs recovered follow the same trends and are systematically lower than the Pan-P, Shell-P and Soft-P sizes (column 'Functional annotation of clusters' of Table 1).

To test EsMeCaTa on environmental samples, we analysed the taxonomic affiliations contained in the 16S rRNA (413 OTUs) and rpoB (309 OTUs) datasets provided in [START_REF] Ogier | rpoB, a promising marker for analyzing the diversity of bacterial communities by amplicon sequencing[END_REF]. Run of EsMeCaTa on the 722 OTUs took 2 days and 10 hours on a 20 CPU cluster. For the 16S and rpoB taxonomic affiliations respectively, means of 31 and 22 proteomes were recovered. Soft-P contained respectively 1,335 and 1,669 proteins clusters in average, associated with respectively 815 and 1,014 GOs, and 288 and 367 ECs in average. This suggests that EsMeCaTa can be used for the analysis of environmental samples by predicting proteins and functions. This paves the way to study the metabolic capabilities of taxa present in the sample.

Financial Support: This work was supported by Deep Impact ANR-20-PCPA-0004.

Conflict of Interest: none declared.

Acknowledgements: We acknowledge the GenOuest bioinformatics core facility at https://www.genouest.org.

EsMeCaTa is available at: https://github.com/AuReMe/esmecata under