Clarisse Balland-Bolou-Bi

Emile B Bolou-Bi
email: clarisse.bolou-bi@u-pec.fr

Nathalie Vigier

Christian Mustin

Anne Poszwa

Increased Mg release rates and related Mg isotopic signatures during bacteria-phlogopite interactions

Keywords: Phlogopite, Bacteria, Organic acids, Magnesium stable isotopes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Silicate minerals, which are a major component of the continental crust, represent the main reservoir of magnesium and calcium on the Earth's surface. The reaction of atmospheric CO2 with silicate minerals releases divalent cations (Mg 2+ and Ca 2+ ) into rivers, which will ultimately form carbonates in the ocean. This process results in the net removal of CO2 from the atmosphere [START_REF] Berner | Weathering, plants, and the long-term carbon cycle[END_REF]. Thus, at the global scale, silicate mineral weathering is linked to the carbon cycle, and it therefore influences climate change on geological timescales. On shorter timescales, the silicate-weathering rate can vary significantly, which may potentially affect the carbon cycle [START_REF] Bastian | Abrupt response of chemical weathering to Late Quaternary hydroclimate changes in northeast Africa[END_REF]. At the soil scale, mineral weathering plays a key role in soil genesis and vegetation growth by releasing elements and nutrients from minerals.

Silicate mineral weathering results from the combination of several steps, including physical disintegration and chemical dissolution. Both processes are more or less linked to biological processes induced by bacteria, which are rarely considered in geochemical or isotopic studies.

Many studies that have been carried out under experimental conditions have demonstrated that bacteria can amplify silicate-weathering reactions (Barker and Banfield, 1998;[START_REF] Hutchens | The role of heterotrophic bacteria in feldspar dissolution -an experimental approach[END_REF][START_REF] Leyval | Weathering of Micas in the Rhizospheres of Maize, Pine and Beech Seedlings Influenced by Mycorrhizal and Bacterial Inoculation[END_REF][START_REF] Robert | Role of biological and Biochemical factors in soil mineral weathering[END_REF][START_REF] Welch | The effect of microbial glucose metabolism on bytownite feldspar dissolution rates between 5° and 35°C[END_REF]. Some bacteria from diverse genera are characterized by their ability to solubilize minerals, as reported in the review of [START_REF] Uroz | Mineral weathering by bacteria: ecology, actors and mechanisms[END_REF] However, previous studies have not indicated whether silicate dissolution represents an indirect consequence of microbial metabolism (i.e., the release of protons, metabolites or specific ligands), or if it depends on a specific strategy (e.g., if dissolution serves some physiological function) that gives a competitive ecological advantage to colonizing bacteria [START_REF] Bennett | Silicates, silicate weathering, and microbial ecology[END_REF]. Few studies have focused on the quantitative and qualitative impact of microbial diversity on silicate weathering in soils [START_REF] Bennett | Silicates, silicate weathering, and microbial ecology[END_REF][START_REF] Bennett | Microbial colonization and weathering of silicates in a petroleum-contaminated groundwater[END_REF][START_REF] Rogers | Mineral stimulation of subsurface microorganisms: Release of limiting nutrients from silicates[END_REF]. Most experimental studies have concluded that mineral chemistry strongly influences the colonization patterns of mineral and rocks by bacteria, as well as their metabolic activities. These experimental investigations have mainly been based on (1) analyses of cations released in solution by minerals and rocks, (2) analyses of the structural and chemical modification of minerals observed using microscopy or X-ray diffraction and (3) the bacterial colonization patterns of minerals. In these studies, the direct effects of bacteria can hardly be distinguished from the indirect effects of the metabolites produced by these bacteria.

Recently, the stable isotopic compositions of metals have been used as an innovative tool to trace biogeochemical processes, including bacteria-mineral interactions, silicate mineral weathering, and the sources and transportation of cations in rivers. For example, some studies have shown that bacteria-mineral interaction processes can induce the fractionation of the stable isotopes of transition metals (e.g., Zn, Fe, Cu) (Anbar, 2004;[START_REF] Beard | Iron Isotope Biosignatures[END_REF][START_REF] Beard | Application of Fe isotopes to tracing the geochemical and biological cycling of Fe[END_REF][START_REF] Co Brantley | Fe isotopic fractionation during mineral dissolution with and without bacteria[END_REF][START_REF] Brantley | Fractionation of Fe isotopes by soil microbes and organic acids[END_REF][START_REF] Mathur | Cu isotopic fractionation in the supergene environment with and without bacteria[END_REF]. In particular, these shifts are observed during the bacteria-and ligand-promoted dissolution of Fe-containing minerals (i.e., hornblende or goethite): the Fe released into the solution by these phases is enriched in light isotopes, with greater fractionation observed in the presence of strong ligands [START_REF] Co Brantley | Fe isotopic fractionation during mineral dissolution with and without bacteria[END_REF][START_REF] Brantley | Fractionation of Fe isotopes by soil microbes and organic acids[END_REF][START_REF] Wiederhold | Iron isotope fractionation during proton-promoted, ligand-controlled, and reductive dissolution of goethite[END_REF]. Redox reactions may also induce some isotope fractionation in these transition elements [START_REF] Wiederhold | Iron isotope fractionation during proton-promoted, ligand-controlled, and reductive dissolution of goethite[END_REF].

In contrast to these cations, magnesium is an element that is much less sensitive to physicochemical conditions because it has an invariable oxidation state (+II). On the other hand, it has been demonstrated that magnesium is one of the most important cations for bacterial growth [START_REF] Pelmont | Bactéries et environnement[END_REF] because magnesium is essential for the normal cell division of bacteria, and it stimulates the growth of these organisms [START_REF] Maloney | The mgtC gene of Burkholderia cenocepacia is required for growth under magnesium limitation conditions and intracellular survival in macrophages[END_REF]. Thus, Mg is the most abundant divalent metal in bacteria cells, and it is required for many structural and enzymatic functions. At least three families of proteins function as magnesium transporters for many strains of bacteria. In recent years, it has been shown that a subset of these transport proteins in bacteria are regulated by magnesium-responsive genetic control elements [START_REF] Maguire | Magnesium transporters: properties, regulation and structure[END_REF][START_REF] Wakeman | Assessment of the requirements for magnesium transporters in bacillus subtilis[END_REF]. Consequently, bacterial processes in soil can be traced using variations and applications of Mg stable isotope ratios. Indeed, recent environmental applications of stable magnesium isotopes have demonstrated that this tool represents a good tracer of weathering and some biological processes. In nature, Mg isotopic compositions (δ 26 Mg) vary from +1.8 ‰ (granite) to -6 ‰ (carbonate) [START_REF] Brenot | Magnesium isotope systematics of the lithologically varied Moselle river basin, France[END_REF][START_REF] Galy | Mg isotopic composition of carbonate: Insight from speleothem formation[END_REF][START_REF] Huang | Adsorption-and desorptioncontrolled magnesium isotope fractionation during extreme weathering of basalt in Hainan Island, China[END_REF][START_REF] Li | Heterogeneous magnesium isotopic composition of the upper continental crust[END_REF][START_REF] Liu | High-temperature inter-mineral magnesium isotope fractionation in mantle xenoliths from the North China craton[END_REF][START_REF] Pogge Von Strandmann | Lithium, magnesium and uranium isotope behaviour in the estuarine environment of basaltic islands[END_REF]. This indicates that the processes controlling the release of Mg from the continental crust to the formation of carbonate in the ocean are associated with Mg isotopic fractionation. Several studies have investigated the role of silicate weathering in both natural ecosystems [START_REF] Bolou-Bi | Effects of biogeochemical processes on magnesium isotope variations in a forested catchment in the Vosges Mountains (France)[END_REF][START_REF] Brenot | Magnesium isotope systematics of the lithologically varied Moselle river basin, France[END_REF][START_REF] Mavromatis | Magnesium isotope fractionation during hydrous magnesium carbonate precipitation with and without cyanobacteria[END_REF][START_REF] Tipper | Mg isotope constraints on soil pore-fluid chemistry: Evidence from Santa Cruz, California[END_REF][START_REF] Tipper | Calcium and magnesium isotope systematics in rivers draining the Himalaya-Tibetan-Plateau region: Lithological or fractionation control? Geochim[END_REF][START_REF] Tipper | Riverine evidence for a fractionated reservoir of Ca and Mg on the continents: Implications for the oceanic Ca cycle[END_REF]) and using experimental leaching and clay synthesis analyses [START_REF] Ryu | Experimental investigation of Mg isotope fractionation during mineral dissolution and clay formation[END_REF][START_REF] Wimpenny | Using Mg isotope ratios to trace Cenozoic weathering changes: A case study from the Chinese Loess Plateau[END_REF]. [START_REF] Ryu | Experimental investigation of Mg isotope fractionation during mineral dissolution and clay formation[END_REF] highlighted the dichotomy between the field studies and experimental investigations of low-temperature processes. The role of biological reactions in natural systems may partly explain this gap. A few studies have focused on the role of vegetation [START_REF] Black | Magnesium-isotope fractionation during plant growth[END_REF][START_REF] Bolou-Bi | Magnesium Isotope Variations to Trace Liming Input to Terrestrial Ecosystems: A Case Study in the Vosges Mountains[END_REF], 2012, 2010). These authors observed significant Mg isotopic fractionation between plants and their nutrient sources, in which the enrichment of heavy isotopes in plants is mainly controlled by biogeochemical processes at the root level. These results were obtained under both experimental conditions and in the field, but they did not estimate the potential roles of microorganisms. Indeed, a recent study [START_REF] Fahad | Fractionation and assimilation of Mg isotopes by fungi is species dependent[END_REF] has highlighted that fungi are able to fractionate Mg isotopes.

In addition, it could help clarify the relationships between microorganisms and soil development.

The aim of this study was to experimentally investigate the impact of bacterial activity on Mg isotope signatures during the weathering of phlogopite minerals in a closed system and under aerobic conditions. Two types of leaching experiments were performed: (1) "biotic" experiments, which occurred in the presence of bacterial strains and Mg-free nutritive media, and (2) "abiotic" experiments, which were performed using nitric or organic acids in sterile mineral media. The two main strategies developed by bacteria were investigated, namely, proton-promoted dissolution and ligand-promoted dissolution.

MATERIALS AND METHODS

Several phlogopite leaching experiments were performed using 4 different bacterial strains ("biotic" experiments), which were previously isolated from plant rhizospheres and acidic soils. To compare and distinguish the particular chemical processes linked to the weathering ability of each isolated bacterial strain, leaching experiments without bacteria were also performed using various acids:

-citric acid (10 -3 M, pH adjusted at 3.5 to 6) -gluconic acid (10 -3 M, pH adjusted at 3.5 and 5) -nitric acid (concentration adjusted to reach pH values of 2.5, 3.5, 4 and 5.5).

All the materials and chemicals were sterilized prior to use for the experiments. The abiotic experimental conditions were selected based on preliminary results yielded by biotic experiments in order to compare them as precisely as possible [START_REF] Balland | Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity[END_REF].

Minerals

The natural 2:1 phyllosilicate used in this study is phlogopite (from Madagascar-Vohitrosy). The chemical composition of this mineral is defined as follows (g/kg): SiO2 390; Al2O3 150; Fe2O3 52; MnO 0.9;Ca0 2.5;MgO 294;K20 110;TiO2 11.3;Na2O 1.3;P2O5 1.1 (Leyval et al., 1990). After undergoing wet grinding and sieving, phlogopite minerals were rinsed under sonication with distilled water at an approximately neutral pH to remove ultrafine particles. The 50-100 µm size fractions of phlogopite were used to conduct experiments. A specific surface area of 0.5 (±0.3) m 2 .g -1 was determined using the Brunauer-Emmet-Teller N2 adsorption method. Prior to starting experiments, mineral powders were sterilized at 170°C for 2 hours under dry atmosphere conditions.

Bacterial strains and cultivation

Soil bacteria were preselected based on their contrasting weathering abilities, which represent two main processes: (1) proton-promoted dissolution by "acidifying" bacteria by producing acids such as lactic or acetic acids that do not chelate metals and (2) ligandpromoted dissolution through the production of low molecular mass organic acids that also acidify the medium and chelate metals. Three heterotrophic strains of bacteria (PA1, AR1, RA1) were previously isolated from the rhizospheres of beech, pine and wheat. A complementary strain (PS2) was isolated from an acidic soil, i.e., a podzol (WRB) located in the Vosges Mountains in northeastern France [START_REF] Balland | Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity[END_REF]Djihad Oulkadi et al., 2014). AR1 and PS2 dissolve minerals by acidifying the medium, whereas RA1 and PA1 produce organic acids [START_REF] Balland | Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity[END_REF][START_REF] Leyval | Weathering of a Mica by Roots and Rhizospheric Microorganisms of Pine[END_REF]Djihad Oulkadi et al., 2014).

The genus of each bacterial strain was determined by amplifying and sequencing 16S rRNA genes. Polymerase chain reactions were performed using a universal set of primers in a total reaction volume of 50 µl containing 1x PCR mastermix (Eppendorf ®), 0.1 µM of primers (966 F and 1401 F) and 1 µl of cell extract (Fleske 1998). Prior to being sequenced by the MWG Biotech Company (Courtaboeuf, France), the PCR products (PCR product size: 433 base pairs) were purified and concentrated using mini-columns (High PureTM PCR Product Purification Kit, Roche diagnostic). A blast program was then used to compare the sequences with those of the GenBank databases (www.nbci.mlm.nih.gov.blast). The following identifications were made, with a maximum identification of approximately 99 %:

Rhanella aquatilis (RA1) and Pantoae agglomerans (PA1), which are both anaerobic facultative bacteria, and Agrobacterium radiobacter (AR1) and Pseudomonas sp. (PS2), which are both aerobic bacteria.

All bacterial strains were cryopreserved at -80°C in 30 % glycerol and then precultivated on Petri dishes containing Luna and Bertani agar (LB 20 g.l -1 from Difco and 15 g.l -1 agar). Strains were grown at 28°C. Biotic experiments were conducted using an actively growing culture of bacteria that was freshly rinsed and conditioned as described below. Prior to their deposition in the batch reactors, bacterial isolates were recovered and cultivated in 2 ml of LB liquid medium for 1 day. Cells in suspension were rinsed three times in deionized water and then suspended in 10 ml of BHm media in order to obtain 10 7 cells per ml of suspension.

Experimental setup

All experiments (i.e., biotic, abiotic and chemical leaching (without bacteria) experiments) were performed in batch reactor closed systems. Each batch reactor was presterilized with culture media by autoclaving at 110°C for 30 min.

Culture media

In all experiments, the classical mineral medium for bacterial cultures (i.e., a Bushnell-Hass-like medium, BHm) was used as a source of essential mineral nutrients for bacteria in order to simulate the chemical composition of a soil solution. This medium was modified to lack iron and magnesium, and phlogopite particles represented the only source of these two bacterial nutrients. Thus, the composition of the Mg-and Fe-free medium was: 20 mg.l -1 KCl; 20 mg.l -1 NaH2PO4, 2H2O; 22.5 mg.l -1 Na2HPO4, 2H2O; 65 mg.l -1 (NH4)2SO4; 100 mg.l -1 KNO3; 20 mg.l -1 CaCl2 (which was dissolved in distilled deionized water to obtain a relatively low ionic strength (4.5 mM).

During the microbial weathering of phyllosilicates, glucose is the sole source of carbon and energy. Glucose solutions (20 g.l -1 , 0.8 mM of carbon) were thus prepared using ultrapure deionized water (18 MΩ.cm-1, Elga Purelab), and sterilized by filtration at 0.2 µm. All reactants were chosen according to their purity in order to minimize the contamination of Fe and Mg. Neither Fe nor Mg was detected in the medium or in the glucose solution using inductively coupled plasma atomic emission spectroscopy analysis (ICP AES Jobin-Yvon 238 at LIMOS, France), which has a detection limit of 5 ppb.

Experimental procedure

In addition to the sterility precautions taken throughout all of the experiments, all laboratory materials were precleaned in diluted HNO3. Biotic and abiotic experiments were run in batch reactors composed of glass flasks. Four leaching experiments (R1 to R4) were performed using RA1, which represents chelating bacteria. One experiment was performed in the presence of other chelating bacteria for comparison (PA1) (experiment #P1). Two phlogopite leaching experiments were performed using the acidifying strain PS2; these were stopped after either 6 days (B1) or 12 days (B2). Another experiment was performed in the presence of other acidifying bacteria, AR1, for comparison, which was stopped after 12 days (experiment #A1). Reactors were continuously agitated using an automatic shaker (Certomatu, Braun 120 rpm), except in experiments R1, R4, P1, A1, H3 and C2, in which flasks were manually agitated every day for approximatively 30 seconds. All experiments were conducted under aerobic conditions at a room temperature of 24°C. Each batch was "closed" with carded cotton and greaseproof paper to allow the circulation of ambient air but to protect against bacterial contamination.

For biotic experiments, each reactor was filled with 3 g of phlogopite powder, 10 ml of cell suspension, 80 ml of BHm and 10 ml of glucose solution (with a batch concentration of 2 g.l -1 ). Cell suspensions were incubated in reactors for a duration of 6 to 12 days.

The number of bacterial cells in suspension (OD600nm), the consumption of glucose, the nature and quantities of the metabolites that were released and the concentrations of some major elements measured in the leachates were observed as a function of time (Table 1).

Abiotic chemical weathering experiments were performed in the same types of glass flasks that were filled with the same amount of phlogopite (3 g) and the same mineral medium (100 ml). Specific acids were added in order to obtain the chosen pH value and concentrations: four experiments were performed using nitric acid (HNO3) at various pH values (experiments #H1 to #H4) for 6 days for H1 and H2 and for 9 days for H3 and H4.

Four experiments were performed using gluconic acid (experiments #G1 to #G4), which were stopped after 6 days for G1, G2 and G4 and after 9 days for G3. Six experiments were performed using citric acid (experiments #C1 to #C6), which were stopped after 6 days for C2 to C6 and after 12 days for C1. The leachates collected from both experiments were then filtered at 0.45 µm and stored at 4°C prior to undergoing chemical and isotopic analyses.

Elemental and physiological analyses

Determination of pH, glucose content and number of bacterial cells

The pH values of these solutions were determined by adding 10 µl of bromocresol green to 200 µl of filtrate. Bromocresol green (BG) is yellow (λmax = 440 nm) in acidic solutions (pH < 3.8) and blue (λmax = 620 nm) in more basic solutions (pH > 5.8). The pH of each leaching solution was then determined at two wavelengths that are close to both BG maxima (BESHIR et al., 2007). A microplate reader (SAFAS Xenius FLX) was used to measure the absorbance at these various wavelengths.

The consumption of glucose by bacteria was determined by measuring the glucose remaining in the 3 µl of filtered solution after the addition of 300 µl of GOD-PAP (enzymatic kit, BioLabo). After 20 min, the glucose concentration was measured at an absorbance of 520 nm. The detection limit for glucose concentration was 0.1 ppm.

The concentration of planktonic bacteria was typically determined by measuring the absorbance of the cell suspension at a wavelength of 600 nm. A correlation between the number of cells and the absorbance was realized by directly enumerating the number of cells with a Thoma cell under microscopic observation as a function of the optical density at 600 nm. For each bacterial strain, an equation was obtained to approximate the number of cells in suspension as a function of optical density (data not shown).

Metabolites analyses

The ten organic acids that are most commonly released by bacteria were realized and quantified only in experiments R1, B1 and B2 (Table 1). These organic acids are formic, gluconic, butyric, pyruvic, citric, oxalic, propionic, acetic, succinic, malic, lactic and malonic acids. Measurements of these acids were performed using ion chromatography with conductivity detection (ICS 3000, Dionex Corp.) and an analytical column (AS 11 HC, Dionex corp.). Each sample was eluted using a KOH solution of varying concentration (0.9 mM to 60 mM) over time (using a step gradient) with a flow of 1.3 ml/min. The synthetic reference materials used were sodium formate, D-gluconic acid, sodium butyrate, pyruvic acid sodium salt, sodium citrate (tribasic), sodium oxalate, sodium propionate, sodium acetate, succinic acid disodium salt, DL-malic acid disodium salt (from Sigma-Aldricht), sodium-L-lactate and malonic acid disodium salt (from Fluka). The uncertainties were better than 0.5 % for all measured organic acids.

Magnesium and Si concentrations

The concentrations of the cations (Mg and Si) released in the solution were analyzed using atomic absorption spectrometry (SAA Perkin Elmer 5000 at SARM-CRPG Nancy, CNRS National Facilities, France) and inductively coupled plasma atomic emission spectroscopy (ICP-AES Jobin-Yvon 238 at LIEC Nancy, France). All solution samples were acidified with HNO3 prior to their analysis. The detection limit for Mg and Si was 0.05 ppm. The uncertainties obtained using our medium were better than 5 %. Accuracy and reproducibility were controlled by repeated analyses of Mg and Si synthetic reference materials (Spex-Certiprep).

Magnesium isotope analyses

Magnesium isotopic compositions were determined for solutions collected from all experiments and the initial phlogopite powder. Samples were purified prior to isotopic analysis to reduce interference and matrix effects [START_REF] Galy | High-precision measurement of magnesium isotopes by multiple-collector inductively coupled plasma mass spectrometry[END_REF]. To purify samples, an aliquot of each experimental solution, containing 50 µg of Mg, was evaporated to dryness in a Teflon cup. The residue was then dissolved in a mixture of concentrated nitric acid (HNO3) and hydrogen peroxide (H2O2, 30 %) (4-2 ml) at 80°C overnight to eliminate glucose. The phlogopite sample was dissolved in a mixture of concentrated acid (HNO3-HF, 3-1 ml) for 48 h at 80°C. The mixtures were then evaporated to dryness. All obtained residues were dissolved in 1 ml of 7M HCl, and a few grains of boric acid were added to the solution of phlogopite to avoid any Mg isotope fractionation during this step because the reaction between HF and silicate produces a milky suspension. This is composed of fluoride of calcium and magnesium, together with some mixed fluorides. Boric acid added to digested solution to allow obtaining clear solution and formed the water-soluble fluoroborates [START_REF] Potts | A Handbook of Silicate Rock Analysis[END_REF][START_REF] Zeev | Preconcentration technique for trace elements[END_REF]. The method used to separate Mg from all other elements in this study is described in detail by [START_REF] Bolou-Bi | Magnesium isotope compositions of natural reference materials[END_REF]. Purified Mg samples were obtained using three steps of ion-exchange chromatography using a combination of AGMP1-X8 and AG50W-X12 resins.

The final purified Mg solutions were evaporated to dryness. The dry samples were diluted with 0.05N HNO3 at 250 ppb before being introduced into the Nu Instruments MC ICP-MS, which is located at the "Ecole Normale Supérieure" (National Facilities, ENS-Lyon, France). Magnesium isotopic ratios were measured and normalized relative to the DSM3 standard solution [START_REF] Galy | High-precision measurement of magnesium isotopes by multiple-collector inductively coupled plasma mass spectrometry[END_REF], using a standard-sample bracketing technique to correct for instrumental mass bias. These data are expressed in δ notation to reflect their deviation from the DSM3 composition in parts per thousand (‰):

3 3 24 24 10 1 Mg Mg Mg Mg Mg(‰) ×               -                 = DSM x Sample x x δ
where x is either the mass of 26 or 25. The Cambridge-1 pure Mg standard was repeatedly measured during the analytical period and yielded a value of -2.62‰±0.13 (2SD, n = 3), which is similar to the overall value of the Cambridge Mg standard in the literature (-2.63‰±0.11, n=1425, compiled from the literature). Moreover, during Mg isotope purification, a seawater standard (BCR 403) was treated as a sample; it yielded a δ 26 Mg value of -0.81‰±0.03 (2SD, n = 4), which is similar to the δ 26 Mg values of seawater that have been reported in the literature (-0.83‰±0.11, n > 200; [START_REF] Ling | Homogeneous magnesium isotopic composition of seawater: An excellent geostandard for Mg isotope analysis[END_REF]).

RESULTS

Bacterial growth, glucose consumption and pH variations

The concentration of planktonic bacteria measured during these experiments is given by the optical density measured at 600 nm (OD600nm) (Figure 1a, Table 1, 2). Immediately after inoculation, all reactors with bacteria showed an initial OD600nm value of 0.05, which corresponds to a cell number of ≈ 10 6 bacteria.ml -1 . In experiments using the chelating RA1 bacterial strain, cell numbers increased regularly to maximum OD values ranging from 0.21 (~ 5.10 7 bacteria.ml -1 ) for experiment R1 to 0.1 (≈ 10 7 bacteria.ml -1 ) for experiment R4. In experiment R3, the maximum OD value was reached at day 3, which then slightly decreased to 0.12 (1.10 7 bacteria.ml -1 ). In the presence of the PS2 bacterial strain, cell numbers increased only slightly over time, as the OD values of experiments B1 and B2 reached maximum variations of 0.06 (≈ 10 6 bacteria.ml -1 ) and 0.08 (5.10 6 bacteria.ml -1 ), respectively, by the end of their experiments. The acidifying PS2 bacterial strain displayed a different behavior: its cell growth rate remained weak and relatively constant as a function of time (Fig 1a).

In all biotic reactors, the glucose concentrations strongly decreased after day 0. In the presence of the chelating RA1 bacterial strain, glucose concentrations decreased as a function of time, from 2 g.l -1 to 1.02 g.l -1 and 1.07 g.l -1 for experiments R3 and R4, respectively, on the 6 th day (Table 2, Figure 1b). In experiment R1, glucose disappeared after day 6.

In the presence of the acidifying PS2 strain, glucose concentrations also decreased as a function of time, reaching values of less than 1 g.l -1 within the two first days, thus indicating that PS2 consumed more glucose than RA1 at the beginning of these experiments, despite its lower growth rates. The concentrations of glucose then decreased more slightly down to 0.42 g.l -1 and 0.29 g.l -1 for experiments B1 and B2, respectively, after the 6 th day. For chelating bacteria, the consumption of glucose and the increase in the number of cells in the solution were interpreted to reflect bacterial growth followed by a prolonged stationary phase that was most likely sustained by the internal recycling of organic matter and nutrients.

In all biotic reactors, the pH rapidly decreased within the two first days from an initial value of 6.5 (Figure 1c). On the second day, the pH reached values of 5.2, 5.0, 4.6 and 4.2 for experiments R1, R2, R3 and R4, respectively, in the presence of the chelating bacteria RA1.

For experiments with the acidifying bacteria PS2, the pH decreased to lower values of 3.5 and 3.3 for experiments B1 and B2, respectively. In all biotic experiments, between days 2 and 12, the pH remained constant except in experiment R1, where the pH again increased on the third day and remained at a relatively constant value of 6.5-6.7. This is linked to the almost complete consumption of glucose by bacteria in this specific experiment. In experiments P1 and AR1, the pH values after 12 days were 3.9 and 4.5, respectively. These results are directly linked to glucose consumption and acidification, which are dependent on bacterial metabolism. In our experiments, bacteria used glucose as both an energy source (i.e., electron donor) and a carbon source for biomass synthesis.

The decrease in pH observed at the onset of these experiments is likely due to the release of CO2 by bacteria during respiration and/or the production of organic acids. Low pH values are then maintained by viable bacteria, which release protons, organic acids and CO2 into solution [START_REF] Vandevivere | Enhanced dissolution of silicate minerals by bacteria at near-neutral pH[END_REF][START_REF] Welch | The effect of microbial glucose metabolism on bytownite feldspar dissolution rates between 5° and 35°C[END_REF]. As shown in the R1 experiment, in the absence of glucose, the decreasing number of bacteria cannot maintain the production of protons and other organic acids. As a consequence, the pH increases due to the consumption of H + that is produced by the chemical alteration of phlogopite. Thus, these data show that both the bacterial growth rate and the mineral alteration rate can affect the chemistry of leachate solutions, as the excretion rate of protons and organic acids is balanced by the release rate of soluble elements from the weathering of phlogopite grains.

Organic acids released by bacteria

In all experiments, the abundances of propionic, malic and malonic acids were below detection limits. In the experiments performed with the chelating RA1 bacterial strain (#R1), the maximum concentrations were reached between the second and third days for lactic acid (62 ppm), acetic acid (152 ppm), formic acid (16.8 ppm), succinic acid (23.5 ppm), oxalic acid (7.2 ppm) and citric acid (3.1 ppm). Between days 2 and 6, the concentrations of organic acids decreased slightly down to 37.1, 0, 8.3, 4.2 and 5.4 ppm for lactic, acetic, formic, succinic and oxalic acids, respectively. A few ppm of citric acid (3.1 ppm) were detected only on the first day. The bacterial strain PS2 (#B2) produced only gluconic and butyric acids. The concentrations of gluconic acid increased from 1600 ppm on day 2 to 2360 ppm on day 6 and 2486 ppm on day 12. The concentrations of butyric acid increased from 21 ppm on day 2 to reach 31 ppm on day 6 day and 54 ppm on day 12.

The production of organic acids depends on many factors, such as the nutritional, physiological and growth conditions of cultures. Previous studies have shown that the partial oxidation of glucose can lower the pH due to the production of gluconic acid by bacteria [START_REF] Balland-Bolou-Bi | Effect of calco-magnesian amendment on the mineral weathering abilities of bacterial communities in acidic and silicate-rich soils[END_REF][START_REF] Vandevivere | Enhanced dissolution of silicate minerals by bacteria at near-neutral pH[END_REF][START_REF] Welch | The effect of microbial glucose metabolism on bytownite feldspar dissolution rates between 5° and 35°C[END_REF][START_REF] Wu | Characterization of elemental release during microbe-granite interactions at T = 28 °C[END_REF]. In the presence of RA1, the organic acids that were produced included citric and oxalic acids and especially lactic, formic and acetic acids. In contrast, the only major organic acid produced by the acidifying PS2 aerobic bacteria was gluconic acid, which is consistent with its known behavior in natural and laboratory settings [START_REF] Grybos | Biodissolution of colloidal-size clay minerals entrapped in microporous silica gels[END_REF][START_REF] Welch | The effect of microbial glucose metabolism on bytownite feldspar dissolution rates between 5° and 35°C[END_REF]. During experiment R1, which was performed with the RA1 strain, glucose was quickly and entirely consumed after 3 days. At the end of day 3, due to the lack of a carbon source, bacterial activity strongly decreased and then stopped. The pH of the solution quickly increased to 7 due to the consumption of protons by the buffering capacity of the phlogopite grains being weathered.

Mg and Si contents in the leachates

The fractions of Mg and Si (FMg and FSi) leached from phlogopite during the biotic and abiotic experiments were determined and are presented in Table 1 and Fig. 3, respectively. Overall, the Mg and Si fractions are very low for all experiments, both biotic and abiotic, which primarily reflects low weathering rates at these temperatures (24°C). In all cases, FMg progressively increased as a function of time (Fig. 3) and stabilized after approximately 6 days.

Under abiotic conditions, in the experiments performed with HNO3 (Figure 3B), the dissolved fraction of Mg from phlogopite (FMg) increased as a function of time and acidity to reach the following values by the end of these experiments: 1.20‰ (H1, day 6); 0.81‰ (H4, day 21) and 0.20‰ (H2, day 6), which occurred at pH values of 2.5, 3.5 and 4.0, respectively. After 6 days, the experiments with the lowest pH caused more Mg to be released from phlogopite. In the experiments performed with gluconic acid (Figure 3A), the fractions of dissolved Mg also increased over time and reached values of 0.56‰ (G3, day 9) and 0.25‰ (G4, day 6) at pH values of ≈ 3.5 and 4.5, respectively. In the experiments performed with citric acid, the dissolved fraction of Mg from phlogopite (FMg) increased as a function of time and acidity to reach the following values at the end of these experiments: 0.85‰ (C4, day 12); 0.23‰ (C3, day 6) and 0.50‰ (C4, day 6) at pH values of 5.7, 5.7 and 3.6, respectively. (Fig. 3A).

Increases in FMg with decreases in pH were observed in the experiments performed using HNO3 and citric and gluconic acids. At an acidic pH (3.5) on day 6, the f(Mg) values of solutions are very close for all experiments (HNO3, gluconic and citric acids). These results indicate that gluconic and citric acids act as strong acids at a pH of 3.5, similar to nitric acid. Indeed, the pKa value of gluconic acid is 3.86, whereas those of citric acids are 3.13, 4.76, and 6.4; this indicates that their carboxylic functions are completely dissociated.

At a neutral pH (5-6) on day 6, the solutions from the citric acid experiments displayed three times more FMg than those observed in the HNO3 experiments, thus reflecting the "complexing effect" of citric acid. Similar to the dissolved Mg fractions in the leachates, the dissolved Si fractions (Table 2) (1) increased over time, (2) increased with decreasing pH in all experiments and (3) exhibited no variations in FSi between treatments on day 6 in low-pH vs high-pH solutions.

Under biotic conditions (Fig. 3C), FMg increased slightly over time in all treatments. In the experiments performed using the RA1, PA1, AR1 strains, FMg reached 0.6‰ (R3, day 13), 0.75‰ (P1, day 12) and 0.56‰ (A1, day 12) with decreases in pH to values of 4.5, 3.9 and 4.6, respectively. In experiments performed using the PS2 strain (in which the pH decreased to 3.5), FMg reached 0.70‰ (B1, day 6) and 1.25‰ (B2, day 12). As observed in abiotic experiments, the dissolved Mg fractions increased when the pH of the leachates decreased.

The dissolved Si fractions (FSi; Table 1) followed the same trend as FMg in the leachates. In addition, comparing bioleaching and chemical leaching experiments at similar pH values revealed that FMg was greater in the presence of bacteria or citric acid than in the presence of nitric and gluconic acids.

Mg isotopic signatures

The magnesium measured in the leachates comes exclusively from the weathering of the phlogopite grains introduced in the reactors because all of the nutritive solutions used in the experiments were Mg-free. Magnesium isotopic compositions were measured for 10 aliquots of phlogopite grains and the solutions of all experiments (see Tables 1 and2).

The average δ 26 Mg value obtained for the phlogopite grains is -1.22±0.08‰ (n = 10). The δ 26 Mg values of the solutions range from -1.52±0.06‰ to +0.82±0.02‰. Overall, the Mg isotopic compositions of the leachates produced during the bio-and chemical experiments are very close to those of phlogopite. However, two different tendencies as a function of the dissolved Mg fraction can be highlighted. First, the acidifying bacterial strains release the highest Mg fractions to the solution, and their corresponding δ 26 Mg values are slightly heavier than those of the initial phlogopite particles (with ∆ 26 Mgsolution-phlogopite = +0.13 to +0.40‰). In contrast, organic acids (except gluconic acid) and the chelating bacteria strains R1, P1 and A1 release lower amounts of Mg to the solution, and their corresponding δ 26 Mg values are slightly lighter than those of the phlogopite grains (with ∆ 26 Mg = -0.01 to -0.30‰).

Interestingly, The measurement uncertainties (±0.10‰) are similar to those of lixiviate solutions (with an average ∆ 26 Mg value of -0.16‰ for ryegrass and clover) produced during phlogopite weathering by plants [START_REF] Bolou-Bi | Experimental determination of magnesium isotope fractionation during higher plant growth[END_REF]. Solutions derived from gluconic acid dissolution experiments contain very low Mg, but their δ 26 Mg values are very slightly higher than those of the initial phlogopite particles (with ∆ 26 Mg values of up to 0.20‰).

DISCUSSION

Assessing congruency and weathering rates

The ratios of the elements released (Mg and Si), together with the elemental ratios of the initial phlogopite as a function of time, are illustrated in Figure 4 and Table 1.

Abiotic experiments

At pH values of 2.5 and 3.5 for experiments H1 and H4 performed by nitric acid, respectively, Mg/Si decreased slightly with increasing pH (to 0.98, 0.92 and 0.92 at pH values of 2.5, 3.5 and 4, respectively, at 6 days). However, they are similar to the bulk initial ratio of the phlogopite (0.97). These values of Mg/Si are in good agreement with those obtained by [START_REF] Kalinowski | Kinetics of muscovite, phlogopite, and biotite dissolution and alteration at pH 1-4, room temperature[END_REF] during the dissolution of phlogopite by HNO3.

In our study, during abiotic leaching in the presence of citric acid (experiments C3 and C4), the Mg/Si ratios (0.95 and 0.7, respectively) were slightly lower than the Mg/Si ratio of the phlogopite (0.97), thus indicating that in contrast to the observation of Barman et al., 1992, Si was preferentially released. At a pH of 3.5, the preferential elemental release that occurred was Fe > Al > Mg = Si, with FFe = 2.02; FAl = 1.82; FMg = 0.53 and FSi = 0.56‰. The difference between our results and those of [START_REF] Barman | Weathering of silicate minerals by organic acids. I. Nature of cation solubilisation[END_REF] can be explained by the fact that at a pH of 3.5, only two carboxylic groups of citric acid were dissociated (pKa= 3.13;4.76;6.4); it is known that citrates have a strong affinity for iron to the detriment of magnesium, in comparison to their experiments, where citric acid caused all of these carboxylic groups to dissociate. Nevertheless, the high concentrations of citric acid (1 mM) used in our chemical experiments do not reflect their concentrations in bioleaching experiments in the presence of RA1 (approximately 3 ppm or 0.02 mM). High concentrations of organic acids can enhance surface complexation processes, which can decrease the number of surface reactive sites and consequently lead to a saturation of rates [START_REF] Golubev | Effect of pH and organic ligands on the kinetics of smectite dissolution at 25 °C[END_REF].

These observations were confirmed by experiments C5 and C6, which were performed with high concentrations of citric acid (10 to 100 mM) that led to Mg/Si ratios in solution that were significantly lower (approximately 0.56) than those in experiment C3 (0.95), but with a similar Mg value of 1.5 ppm after 6 days.

Biotic experiments

All other experiments (bio-and chemical leaching) yielded Mg/Si values that were less than the bulk initial ratio of the phlogopite, except for the experiments performed with the chelating bacterial strain RA1 (which yielded a Mg/Si value of approximately 1.5). In the presence of RA1 (experiments R2 and R3), Mg was preferentially released compared to Si. This reflects the greater dissolution of octahedra compared with tetrahedra. [START_REF] Acker | The influence of pH on biotite dissolution and alteration kinetics at low temperature[END_REF] demonstrated that at a pH of greater than 3 (in the case of our experiments R1 to R4, which had pH values of close to 4.5), biotite octahedra were preferentially destroyed. Moreover, [START_REF] Barman | Weathering of silicate minerals by organic acids. I. Nature of cation solubilisation[END_REF] highlighted that biotite weathering in the presence of a strong chelating agent, such as citric or oxalic acid, preferentially released Mg into solution over Si; this was the case in our experiment R1, where low amounts of oxalic and citric acids were quantified (approximately 7 and 3 ppm for oxalic and citric acids, respectively). The results of [START_REF] Barman | Weathering of silicate minerals by organic acids. I. Nature of cation solubilisation[END_REF] indicated that chemical leaching in the presence of citric acid preferentially releases Mg compared to Si due to the high solubility of Mg and that the localization of Mg in octahedra makes Mg easily accessible to the solution. At a pH of 1, the authors demonstrated that preferential elemental release occurred in the following order: Mg> Fe > Al > Si.

In the experiments performed with the bacterial strain PS2 (B2) and gluconic acid (G4), the Mg/Si ratios were very close to the bulk ratio of phlogopite (0.97) and those obtained in the experiment performed with HNO3 (H4). This observation is consistent with the results of the Mg and Si release rates, which indicate that pH exerts a control on the leaching of Mg and Si from phlogopite, as well as the results of several works [START_REF] Grybos | Biodissolution of colloidal-size clay minerals entrapped in microporous silica gels[END_REF]Oulkadi et al., 2014b), which showed that Pseudomonas sp. can weather clay from both octahedral and tetrahedral layers. As mentioned above, at this acidic pH, gluconic acid acts similar to a mineral acid, such as nitric acid.

These different types of bacteria may play different roles in the dissolution rate of phlogopite and can therefore have different influences on the chemical composition of the solutions. To remind, AR1 and PS2 dissolve minerals by acidifying the medium, whereas RA1 and PA1 produce organic acids [START_REF] Balland | Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity[END_REF][START_REF] Leyval | Weathering of a Mica by Roots and Rhizospheric Microorganisms of Pine[END_REF]Djihad Oulkadi et al., 2014). Thus, for the facultative anaerobic bacteria (RA1 and PA1), under conditions of stress, the glucose oxidation continued beyond the glucose intermediate. The lack of required enzymes caused some bacteria to accumulate or expel various intermediate acids as waste [START_REF] Gottschalk | Bacterial metabolism[END_REF], such as fumaric, citric, and oxalic acids and especially lactic, pyruvic and acetic acids resulting from fermentation. Thus, these strains have an anabolism that requires little energy. Consequently, their catabolism is not very efficient, as it uses a small fraction of the initial carbon in the solution (i.e., glucose). In contrast, the PS2 strain is an aerobic bacterial strain that has an anabolism that requires more energy and an efficient catabolism that requires large amounts of carbon. Moreover, D. Oulkadi et al., 2014a showed that RA1 produces siderophores, whereas PS2 does not. Siderophores and oxalic and citric acids clearly displayed ligand-promoted dissolution in addition to proton-promoted dissolution.

Weathering rates

To more precisely assess the controls of Mg and Si release and compare them with published estimates, their concentrations were normalized to the specific surface area of phlogopite and time (rMg and rSi are both expressed in mol.m -2 .s -1 ). The influence of pH, organic acids and bacteria on the release rates of Mg and Si could therefore be evaluated using the linear rate law described by [START_REF] Malmström | Biotite dissolution at 25°C: The pH dependence of dissolution rate and stoichiometry[END_REF] and [START_REF] Malmström | The dissolution of biotite and chlorite at 25°C in the near-neutral pH region[END_REF], which is given by the following equation: rM = kH (aH+) n + kO + kOH (aOH) m + kL,M (aL) l (eq. 1)

where M is the released element (Mg or Si); k is the rate constant; aH+, aOH and aL are the activities of protons, hydroxyls and ligands, respectively; and n, m, and l are the orders of reactions that are experimentally obtained with respect to protons, hydroxyls and ligands, respectively. This model assumes that (1) ligand-promoted processes operate in parallel with and independently of hydroxyl-or proton-promoted processes, and (2) ligand-promoted rates are additive. In this study, because phlogopite weathering occurs in an acidic environment, the terms kO and kOH (aOH) m are negligible. Thus, experimental data can be fitted to the rate law to yield the following equation: rM = kH (aH+) n + kL,M (aL) l (eq. 2)

The form of this empirical release rate equation is characteristic of dissolution rate laws in which proton adsorption or ligand complexation occurs at the mineral surface. For phlogopite, the apparent dissolution rate constant, which is normalized to the surface area, is KH = 4.7 10 - good agreement with the values provided in the literature [START_REF] Balland | Dissolution rates of phyllosilicates as a function of bacterial metabolic diversity[END_REF][START_REF] Kalinowski | Kinetics of muscovite, phlogopite, and biotite dissolution and alteration at pH 1-4, room temperature[END_REF][START_REF] Rufe | Quantitative Assessment of Reactive Surface Area of Phlogopite During Acid Dissolution[END_REF][START_REF] Taylor | Kinetics of dissolution and Sr release during biotite and phlogopite weathering[END_REF].

In both biotic and abiotic experiments, the apparent initial release rates of Mg and Si were calculated using equation 2. In all experiments, the release rates of Mg and Si decreased slightly with time. This has frequently been observed in dissolution experiments using fresh mineral samples, as in the works of [START_REF] Furrer | The coordination chemistry of weathering: I. Dissolution kinetics of [delta]-Al2O3 and BeO[END_REF] and [START_REF] Malmström | Biotite dissolution at 25°C: The pH dependence of dissolution rate and stoichiometry[END_REF]. This could be attributed to the initial rapid dissolution of ultrafine particles, which is then followed by a decrease in the dissolving surface area and the creation of more reactive surface sites.

Figure 4 represents the Mg (rMg) and Si (rSi) release rates as functions of average pH.

The observed correlations confirmed that this elemental release was largely pH-controlled.

The highest elemental release rates occurred at low pH values, whereas the lowest rates occurred at nearly neutral pH values. It can also be observed that biotic and abiotic experiments performed with citric acid or with produced citric acid have higher release rates relative to abiotic experiments performed with HNO3 and gluconic acid. At a given pH, Mg release rates were ~ 2 to 3 times higher in the presence of bacteria compared to those observed during chemical leaching performed with HNO3. Silicon release rates were also ~ 1.5 times higher in the presence of bacteria. The organic acids released by bacteria play an important role in the release of Si. Acidifying bacteria (PS2 and AR1) amplified Mg and Si release rates compared to HNO3. As shown in the organic acid analyses, the PS2 strain produced approximately 1 mM of gluconic acid into solution, leading to a pH of 3.5. At this pH, Mg and Si release rates were lower and similar to those observed during chemical experiments performed only with gluconic acid (G1, G2 and G3 ~ 1.43.10 -10 mol.m -2 .s -1 ) and HNO3 (H4) (1.38.10 -10 mol.m -2 .s -1 ). Moreover, at this pH, gluconic acid (pKa = 3.86) acted as a strong acid. However, the enhanced elemental release rates in the presence of PS2 and AR1 suggest that other processes must be taken into account to interpret such variations. [START_REF] Vandevivere | Enhanced dissolution of silicate minerals by bacteria at near-neutral pH[END_REF] highlighted microreaction zones at the bacteria-mineral contact where the chemical compositions of the solution in these sites were strongly different compared to that of the bulk solution, with high concentrations of protons and organic acids.

All of the bacterial strains used in this work, including both acidifying (PS2 and AR1) and chelating (PA1 and RA1) bacteria, principally accelerate the release of elements (Mg and Si) from phlogopite by decreasing the solution pH and/or producing chelating agents, such as organic acids. Similar conclusions have previously been reported (Barker et al., 1998;[START_REF] Vandevivere | Enhanced dissolution of silicate minerals by bacteria at near-neutral pH[END_REF][START_REF] Wu | Characterization of elemental release during microbe-granite interactions at T = 28 °C[END_REF]. Thus, it seems that the production of organic acids and siderophores plays a crucial role in the mechanism and efficiency of clay dissolution.

Several studies [START_REF] Cheah | Steady-state dissolution kinetics of goethite in the presence of desferrioxamine B and oxalate ligands: Implications for the microbial acquisition of iron[END_REF]D. Oulkadi et al., 2014b;[START_REF] Reichard | Dissolution mechanisms of goethite in the presence of siderophores and organic acids[END_REF] have demonstrated that the presence of siderophores could be strongly affected by the presence of oxalic acid inducing the high dissolution of minerals.

The measured growth of bacteria was not correlated to the Mg and Si released into solution during the bioleaching of phlogopite. This suggests that the growth of bacteria was not a crucial factor in highlighting the effectiveness of bacteria to weather phlogopite, as indicated by the relationship between the cell numbers and the elemental release rate. Indeed, the cell number of PS2 was less important than that of RA1, while elemental release was more important in the presence of the PS2 bacterial strain (2.22.10 -10 and 2.51.10 -10 mol.m -2 .s -1 for Mg and Si release rates, respectively) than in the presence of the RA1 bacterial strain (1.06.10 -10 and 7.23.10 -11 mol.m -2 .s -1 ). However, only planktonic bacteria were enumerated in our experiments, whereas adhering bacteria are known to play an important role in enhancing mineral weathering [START_REF] Vandevivere | Enhanced dissolution of silicate minerals by bacteria at near-neutral pH[END_REF]. At this stage of our study, it is not possible to determine the number of bacteria cells that were adsorbed onto phlogopite particles.

Magnesium isotope variations

The During phlogopite leaching by ligand-promoted dissolution, the decrease in the solution pH is associated with a light δ 26 Mg value compared to fresh phlogopite. In contrast, during phlogopite leaching by proton-promoted dissolution (i.e., nitric acid and Pseudomonas bacterial strain), the decrease in the solution pH is associated with a heavy δ 26 Mg value compared to fresh phlogopite. These results are in agreement with results found during clay mineral formation [START_REF] Wimpenny | Using Mg isotope ratios to trace Cenozoic weathering changes: A case study from the Chinese Loess Plateau[END_REF]. In soil clay, some Mg can be bound to functional groups on the clay surface as exchangeable Mg in addition to existing as a structural part of the octahedral layer [START_REF] Drever | The Geochemistry of Natural Waters, 2nd Editio[END_REF][START_REF] Opfergelt | Mechanisms of magnesium isotope fractionation in volcanic soil weathering sequences, Guadeloupe[END_REF][START_REF] Wimpenny | Using Mg isotope ratios to trace Cenozoic weathering changes: A case study from the Chinese Loess Plateau[END_REF]. According to the classification of trioctahedral mica, we could assume based on studies of cation exchange capacity that Mg in phlogopite derived from both structural site located in the octahedral layer and on exchangeable site at the mineral surface. Exchangeable Mg is isotopically lighter than clay in soil and bulk soil [START_REF] Bolou-Bi | Effects of biogeochemical processes on magnesium isotope variations in a forested catchment in the Vosges Mountains (France)[END_REF][START_REF] Opfergelt | Mechanisms of magnesium isotope fractionation in volcanic soil weathering sequences, Guadeloupe[END_REF][START_REF] Uhlig | Quantifying nutrient uptake as driver of rock weathering in forest ecosystems by magnesium stable isotopes[END_REF]. In addition, the leaching of the exchangeable Mg on clay with organic acids shown that this fraction is isotopically lighter than clay [START_REF] Wimpenny | Using Mg isotope ratios to trace Cenozoic weathering changes: A case study from the Chinese Loess Plateau[END_REF], similarly to results found during the ligand-promoted experiment in this study (Table 1). The use of strong acid would induce the leaching of both exchangeable Mg and structural Mg during the experiment. During proton-promoted dissolution, the δ 26 Mg value of Mg released into solution increased with the increasing mineral dissolution rate, in contrast to experiments with ligand-promoted dissolution (Fig. 6), except those performed with gluconic acid, which acts as a mineral acid under these conditions. Magnesium located in structural sites is contained in the brucitic layer. The leaching of this structural Mg is associated with the release of other ions located in this octahedral layer, such as Al. Moreover, in these minerals, Si is located in polymerized sheets of silica tetrahedral. The release of Si in solution reflects the dissolution of this structure in addition to the octahedral layer, as indicated by the correlation between Si and Mg (Fig. 4). Various Mg isotopic analyses during the synthesis of brucite demonstrate that it is enriched in heavy isotope in comparison with the solution [START_REF] Li | Magnesium isotope fractionation between brucite [Mg(OH)2] and Mg aqueous species: Implications for silicate weathering and biogeochemical processes[END_REF][START_REF] Wimpenny | Using Mg isotope ratios to trace Cenozoic weathering changes: A case study from the Chinese Loess Plateau[END_REF]. Therefore, we could conceive that Mg leaching in the structural sites using a strong acid (proton) would be the origin of heavy Mg isotopes enrichment in solutions.

The fractionation of Mg isotopes by bacteria is a complex process especially since it is dependent on the bacterium studied. Our work has highlighted the non-negligible role of bacterial metabolism (through the production of organic acids) during silicate weathering at ambient temperature in batch reactor. Additional experiments are needed to increase the Mg leaching from phlogopite by increasing time reaction in order to measure Mg uptake by bacteria (and if there is some Mg isotopes fractionation during this process) and analyze the Mg isotopic ratio in the reacted phlogopite in order to see if there is a formation of secondary phase. Future work should focus on linking Mg isotopic fractionation to these reactions. Mg isotopes appear to be well-suited tools for the discovery of biological markers on Earth.

However, a variety of abiotic chemical reactions could play a complicated role.

CONCLUSIONS

This study demonstrates that the decreases in pH and metabolites produced during heterotrophic bacterial activities could greatly accelerate the release of Mg and Si from phlogopite. Comparing bioleaching and chemical leaching experiments performed at similar pH values reveals that more elements were released into solution in the presence of bacteria or citric acid compared with those released in the presence of nitric and gluconic acids.

Bacterial metabolism differs from one strain to another and induces substantial changes in solution pH; these changes had important effects on the rate of elemental release from phlogopite compared those observed under abiotic conditions. In our experiments, aerobic bacterial strains were more efficient than facultative anaerobe bacteria in leaching Mg during phlogopite weathering.

In most experiments, little to no Mg isotopic fractionation linked to phlogopite leaching was highlighted. However, two tendencies could be distinguished, especially in the highest leached Mg fractions obtained: (1) for experiments performed with HNO3 and the acidifying bacteria PS2, the Mg leached into solution was enriched in its heavy isotope compared to phlogopite; (2) for experiments performed with citric acid, the Mg leached into solution was enriched in its light isotope compared to phlogopite. However, the Mg isotopic fractionation factor was very low, thus indicating that little to no fractionation occurred during our experiments, which correspond to the first event of the bioleaching of phlogopite (in a closed system). Moreover, when high phlogopite dissolution rates are correlated with low pH values, the Mg isotopic composition of the leached solution is enriched in heavy isotopes, which reflects the leaching of structural Mg contained in the octahedra of phlogopite. In experiments performed with chelating bacteria and citric acid, high phlogopite dissolution rates were enriched in light isotopes, especially at weak acid pH (4-6) values, due to the formation of secondary minerals or the leaching of the Mg adsorbed on the minerals. General characteristics of reactor solutions in biotic conditions and abiotic conditions. * "ppm"; "-" below detection $ MC-ICP-MS data were obtained using the Nu instrument (ENS, Lyon, with a 2σ external precision of 0.15‰). Data in italics were obtained using the Isoprobe (CRPG, Nancy, with a 2 σ external precision of 0.20‰). Reported values represent the means of two or more replicate analyses of each sample. "#": Organic acid concentration of 0.001 M; "##": organic acid concentration of 0.01 M; "###": organic acid concentration of 0.1 M Table 2.

FIGURE CAPTIONS

Composition of bioreactor medium with two bacterial strains (Rhanella aquatilis RA1 and Pseudomonas sp. PS2), including optical density at 600 nm, glucose remaining in solution and organic acids produced (i.e., gluconic, lactic, acetic, butyric, formic, succinic, oxalic and citric acids).

2

  low dissolution rate associated with the low Mg fraction released during the dissolution experiment could be the reason of similar δ 26 Mg values of the solution and initial phlogopite in the measurement uncertainties. This similarly δ 26 Mg values suggests that no fractionation occurred during the acid congruent dissolution of phlogopite under these conditions in the measurement uncertainties. Although the δ 26 Mg values of solutions are close to the δ 26 Mg value of phlogopite, two tendencies can be distinguished (Fig. 6). Results indicate that more Mg is released in solution; more δ 26 Mg value of the solution is heavier compared to initial phlogopite (Fig 6), and vice versa. These δ 26 Mg values are strongly correlated to solution pH (Fig 7), which suggests at first approximation that the mode of medium acidification (i.e., proton-promoted vs ligand-promoted agent used to leach Mg from a mineral) could explain the various δ 26 Mg values observed in solutions independently of the vital effect.

Figure 1 .

 1 Figure 1.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4 .

 4 Figure 4.

Figure 6 .

 6 Figure 6.

δ

  26 Mg/ DSM3 values versus pH for all experimental reactors.

  Figure 1

  Figure 3.

  Figure 4.

  Figure 7.

TABLE CAPTIONS Table 1 .

 CAPTIONS1

TABLE 1 .

 1

	Weathering agent	Experiments names	Times (day)	pH	Mg *	Si *	Mg/Si	 26 Mg / DSM3	$   25 Mg / DSM3	$ 	 26 Mg (solution-solide) 
		phlogopite						-1.22 +/-0.08 -0.62 +/-0.08
	HNO 3	H1	6	2.5	6.30	6.41	0.98	-0.82 +/-0.02 -0.42 +/-0.06	0.40
	HNO 3	H2	3	4.0	0.92	1.01	0.91
			6	4.0	1.08	1.18	0.91
	HNO 3	H3	9	5.6	1.44
	HNO 3	H4	6	3.6	2.70	3.01	0.89
			12	3.5	3.30	3.60	0.92
	gluconate ##	G1	3	3.2	2.00	1.93	1.04
			6	3.5	2.35	2.25	1.04
	gluconate ##	G3	3	3.5	1.70	1.89	0.90
			6	3.4	2.55	2.81	0.91
			9	3.4	2.95	3.23	0.91	-1.02+/-0.25 -0.51+/-0.09	0.20
	gluconate #	G4	3	3.6	1.60	1.75	0.91
			6	3.6	2	2.18	0.92	-1.22 +/-0.05 -0.63 +/-0.06	0.00
	citrate #	C1	10	5.7	4.74	5.06	0.94
			13	6.2	4.70	5.29	0.89	-1.52 +/-0.06 -0.78 +/-0.09	-0.30
	citrate #	C3	3	4.6	1.30	1.28	1.01
			6	5.7	1.60	1.68	0.95	-1.43 +/-0.02 -0.74 +/-0.02	-0.21
	citrate #	C4	3	3.6	2.20	2.31	0.95				-0.20
			6	3.6	2.80	3.01	0.93	-1.42 +/-0.07 -0.73 +/-0.07	-0.21
	citrate ##	C5	3	5.3	1.10	2.01	0.55
			6	5.2	1.50	2.69	0.55
	citrate ###	C6	3	5.3	1.20	2.11	0.57
			6	5.3	1.60	2.78	0.57
	RA1	R1	3	6.7	1.00	1.25	0.8
			6	6.7	1.50	1.61	0.93

mol.m -2 .s -1 . The reaction order (n) with respect to hydrogen ion activity is 0.42, which is in

ACKNOWLEDGEMENTS

Philippe Telouk for CNRS-INSU facilities and help with isotopic analyses at ENS-Lyon. We are grateful to Editor and Josh Wimpenny for comments to improve this manuscript.


Aydar Rakhmatullin

Rudy Michel

Catherine Bessada

Structural characterization and chemistry of LiF-AlF 3 melts with addition of MgO and MgF 2

Keywords: system MgO/MgF2-LiF-xAlF3, molten salts, high temperature and solid state NMR, nuclear reactors

The LiF-AlF3 system with additions of MgO and MgF2 has been studied by 17 O, 25 Mg, 27 Al and 19 F in-situ high temperature NMR, ex-situ solid state NMR and X-ray powder diffraction for a wide range of compositions. The description of the speciation in the melts and its evolution with different operational parameters such as temperature, time and composition were obtained for MgF2/MgO-(1-x)LiF-xAlF3 for x=14.5, 25 and 35.5 mol% systems. The evolution of the signals provides evidence of a chemical reaction between the MgO and the salt.

Introduction

Numerous study and research projects have been conducted on the targets in the core of an SFR (sodium cooled nuclear reactors) (for example, see [START_REF] Fanghänel | Transuranium Elements in the Nuclear Fuel Cycle[END_REF][START_REF] Ferrer | Summary Report on New Transmutation Analysis for the Evaluation of Homogeneous and Heterogeneous Options in Fast Reactors[END_REF]). One of the main questions is to know if the potential extraction step of americium in LiF-AlF3 can be used with a CERCER (ceramic-ceramic) targets containing MgO matrix retreatment [START_REF] Fanghänel | Transuranium Elements in the Nuclear Fuel Cycle[END_REF][START_REF] Blomgren | Partitioning and transmutation current developments-2010[END_REF][START_REF] Ouvrier | Recycling of MgO, Mo & ZrO2 based Actinide-Bearing Matrices: Assessment of Reprocessing Feasibility & Waste Production[END_REF]. This process, known as liquid-liquid reductive extraction, consists of a molten metallic aluminum pool in contact with Am ions in the molten salt. The metals will be extracted from one phase to the other.

The phase diagram of the LiF-AlF3 system has been studied by several authors [START_REF] Holm | Thermodynamic properties of molten cryolite and other fluoride mixtures[END_REF][START_REF] Jenssen | Phase and Structure Relations for Some Alkali Aluminum Fluorides[END_REF][START_REF] Holm | Phase relations and thermodynamic properties in the ternary reciprocal system LiF-NaF-Na3AlF6-Li3AlF6[END_REF]. A compound Li3AlF6 as well as two eutectics at 14.5 and 35.5 mol% AlF3 have been reported. The MgF2-Li3AlF6 pseudo-binary is a eutectic system. According to Kasicova et al. [START_REF] Kašíková | Fázový diagram soustavy Li3AlF6-MgF2. Sborník věd. prací Vys. školy báňské[END_REF], eutectic point is located at 37 mol% MgF2 and 63 mol% Li3AlF6, with teut =748 °C, but up to now, no more details were collected on this system. In the literature, many studies were reported on the MgF2-Na3AlF6 system, because of its big interest in aluminium electrolysis. Several investigations [START_REF] Grjotheim | Aluminium electrolysis[END_REF][START_REF] Holm | [END_REF][START_REF] Kostyukov | [END_REF] of the MgF2-Na3AlF6 phase diagram have been published. Kostyukov and Karpov [START_REF] Kostyukov | [END_REF] have reported that this system does not form a simple binary system. By cooling a molten mixture with a low content of magnesium fluoride, the solid compound NaMgF3 precipitates:

Na3AlF6 + 3MgF2  AlF3 + 3NaMgF3 (1)
At higher MgF2 contents, the incongruently melting compound Na2MgAlF7 is formed, and presumably also Na2Mg2Al3F15. MgF2 forms a solid solution with sodium cryolite, the composition of which may be taken from their phase diagram [START_REF] Kostyukov | [END_REF]. The same feature was also 20-Nov-20 supported by Holm [START_REF] Holm | [END_REF] on the basis of thermal, X-ray and optical analysis of mixtures containing more than 75 mol% cryolite. Sokolov et al. [START_REF] Kostyukov | [END_REF] and Kostyukov et al. [START_REF] Kostyukov | Physico-chemical study of the system NaF-AlF3-CaF2 (MgF2)[END_REF] assumed that magnesium fluoride is present in sodium cryolite-based melts as MgF3 -anions. They suppose that the formation of these complex anions influences on the activity of aluminium complexes in the melt. In the reference [START_REF] Fellner | Complex Formation in the Molten Systems NaF-MgF2 and Na3AlF6-MgF2[END_REF] authors built different structural models for the MgF2 dissolution in molten sodium cryolite. Their calculated results indicate that the presence of MgF4 2-ions is more probable than MgF3 -or Mg 2+ . The in-situ high temperature (HT) Raman spectroscopic study of the MgF2-Na3AlF6 system was carried out by Gilbert et al. [START_REF] Gilbert | Structure and Thermodynamics of NaF-AlF3 Melts with Addition of CaF2 and MgF2[END_REF]. From their analysis, MgF2 is an acidic additive and tends to form MgF3 -or MgF4 2-species. Mg2F6 2-is always a minor species compared to the tetrahedral complex MgF4 2-. They have also reported the formation of a bridged complex, such as Mg2F6 2-, for high amount of MgF2.

Compare with the MgF2-Na3AlF6 system only a few studies have been made in MgO-Na3AlF6. An eutectic was found at approximately 30 mol% and 892 °C [START_REF] Hayakawa | The solubility of substances in molten salts. The determination of the solubility of metallic oxides in molten cryolite Science Reports of the Saitama Universityrsity[END_REF]. According to Kostyukov et al. [START_REF] Kostyukov | Physico-chemical study of the system NaF-AlF3-CaF2 (MgF2)[END_REF] MgO reacts rapidly with aluminium fluoride and forms magnesium fluoride and alumina.

High temperature NMR spectroscopy can provide an experimental description of different species existing in melts. This method is sensitive to the first coordination sphere around a given cation.

Because of the aggressivity and reactivity of molten fluorides at high temperature, their experimental characterization is difficult and only a few spectroscopies (Raman [START_REF] Bessada | Structural and Dynamic Approaches of Molten Salts by High Temperature Spectroscopies[END_REF][START_REF] Gilbert | Structure and Thermodynamics of NaF-AlF3 Melts with Addition of CaF2 and MgF2[END_REF], NMR [START_REF] Bessada | Structural and Dynamic Approaches of Molten Salts by High Temperature Spectroscopies[END_REF][START_REF] Bessada | Lanthanide and actinide speciation in molten fluorides: A structural approach by NMR and EXAFS spectroscopies[END_REF][START_REF] Lacassagne | Structure of High-Temperature NaF-AlF3-Al2O3 Melts: A Multinuclear NMR Study[END_REF][START_REF] Robert | Study of NaF-AlF3 Melts by High-Temperature 27 Al NMR Spectroscopy: Comparison with Results from Raman Spectroscopy[END_REF], and extended X-ray absorption fine structure (EXAFS) [START_REF] Bessada | Lanthanide and actinide speciation in molten fluorides: A structural approach by NMR and EXAFS spectroscopies[END_REF]) were able to extend the description of such systems.

Therefore, the aim of present study is focused on the fundamental description of the system LiF-AlF3 melts with addition of MgF2 and MgO. We have first performed high 20-Nov-20 temperature NMR (HT NMR) experiments on LiF-AlF3 melts with different additions of MgF2

and MgO in order to follow the modification of aluminium first coordination shell upon fluoride and oxide addition. In a second step, we used solid-state NMR and X-ray diffraction to identify and further characterize the samples after HT NMR experiments. The combination of these techniques has proved to be a powerful tool in elucidating the structure in complex crystalline and/or disordered materials.

Results and Discussion

MgF2 additions in molten LiF-AlF3 mixtures 2.1.1. High temperature NMR results

In the melt, the HT NMR spectra consist in a single Lorentzian line, characteristic of a rapid exchange between the different species. The measured chemical shift is the weighted average of the individual chemical shifts of all species present in the melt [START_REF] Lacassagne | Structure of High-Temperature NaF-AlF3-Al2O3 Melts: A Multinuclear NMR Study[END_REF]. From the knowledge of the individual chemical shifts of the different species present in the melt, it is thus possible to extract the distribution the different ionic configurations depending on the composition.

2.1.1.1. 85.5-14.5 mol% LiF-AlF3 + MgF2 and 75-25 mol% LiF-AlF3 + MgF2

We have followed the effect of MgF2 additions in the two LiF-AlF3 mixtures: 14.5 and 25 mol% AlF3 at 3 different temperatures in the melt (see Table 1). In the system Li3AlF6-MgF2 we observe a clear variation of the 27 Al chemical shifts (the position of the line) with a maximum observed between 4 and 6 mol% MgF2. We have reported in the Fig. 1 the 27 Al chemical shifts measured in pure molten Li3AlF6 at 800, 880 and 900 °C. We notice the effect of temperature with a systematic shift of 2 ppm/80 °C for each composition.

20-Nov-20

While the effect of composition (mol% MgF2) is visible by the particular evolution of the chemical shifts: an increase up to a maximum value at around 5 mol% MgF2 and then a decrease.

In order to go further we have compared these data with the information given by the other nuclei 19 F and 25 Mg. Even if 7 Li is observable by NMR and present in the sample, the chemical shifts range of diamagnetic lithium is too small to be informative. We don't observe any variation of the 19 F signal position with MgF2 addition, which stays at -183 ppm at 880 °C as in the pure Li3AlF6 20-Nov-20 without MgF2. We just mention a shift of -2 ppm with temperature between 800 and 960 °C for all compositions.

The 25 Mg signal is not influenced by the amount of MgF2 and appears at the same position (±1 ppm) than in pure molten MgF2 i.e. at -4.1 ppm in this range of composition. The resolution of the spectra is poor, but good enough to determine the position. Nevertheless it is difficult to obtain the 25 Mg NMR signal with good signal-to-noise ratio for MgF2 additions less than 10 % due to low natural abundance (10%) and small gyromagnetic ratio of magnesium nucleus [START_REF] Harris | NMR nomenclature. Nuclear spin properties and conventions for chemical shifts (IUPAC Recommendations 2001)[END_REF]. If we consider the phase diagram of this system, over this range of MgF2 addition, no specific feature is observed for the concentration of MgF2 below 5 mol%. The evolution observed for 27 Al chemical shifts can be attributed to a slight change on speciation in the melt. This change does not seem to influence the fluorine and magnesium signals.

In the system 85.5-14.5 mol% LiF-AlF3 + MgF2 the changes in the 27 Al signal position with MgF2 addition were less noticeable. We have observed ±1ppm chemical shift variation with

MgF2 additions and we notice the effect of temperature with a systematic shift of 1.5 ppm/80°C for each composition.

64.5-35.5 mol% LiF-AlF3 + MgF2

We performed in-situ NMR measurements at high temperature for 2, 4, 6, 8, 10, 15 mol%

MgF2 additions but we obtained a good Lorentzian line shape signal of "pure liquid" only for small concentrations of magnesium fluoride: 2 and 4 mol%. For higher concentrations 27 Al and 19 F spectra were rather complex with different peaks corresponding to a mixture of solid and liquid 20-Nov-20 phases. The system is not stable, even just above the liquidus temperatures with strong sublimation phenomena and rapid evolution of the bath chemistry.

X-ray results

After the HT NMR experiments the solidified ternary mixtures were systematically characterized by X-Rays diffraction at room temperature. In case of MgF2 for the concentrations up to 25 mol% we observe the presence of MgF2, AlF3 and as well as expected Li3AlF6. At high contents of MgF2, in addition with Li3AlF6, MgF2 and AlF3, the formation of a new phase LiMgAlF6 [START_REF] Tao | Synthesis and Luminescence Characteristics of LiMgAlF6:Ln 3+ (Ln = Ce, Eu, Tb)[END_REF] was detected.

MgO additions in molten LiF-AlF3 mixtures 2.2.1. High temperature NMR results

Thanks to the information given by 27 Al, 25 Mg, 19 F and 17 O we describe the effect of MgO additions in the melts i.e. with three different AlF3 contents. The NMR data were collected at 800 °C, 880 °C and 960 °C for 14.5 mol% and 25 mol% AlF3.

For 35.5 mol% AlF3 we obtained a good sharp signal corresponding to the melt at 900°C.

For the lower temperature, we observed the signals corresponding to a mixture of solid and liquid phases. For the temperatures higher that 900 °C, after about 10 minutes we have found new broad "solid" signal with full width at half maximum of few kHz due to strong sublimation phenomena.

20-Nov-20 We have reported on Fig. 2 the 27 Al chemical shifts measured in the systems with MgO additions. We observed for all systems, that the only visible modification occurring with the addition of MgO up to 1 mol% with a systematic increase of the chemical shift value. The chemical shifts are remaining constant for higher than 1 mol% MgO content. The range of chemical shifts, where the variation is discussed, is quite small 2-3 ppm, and it indicates that if modification occurs in the melt, it doesn't affect strongly the local environment of the aluminum.

The 17 O signal is detected in the melt even at very low MgO content. In all systems its position remains constant and only the intensity of the peak is varying, corresponding to more oxygen in the liquid phase. In the system with 14.5 mol% AlF3 the position is (38±1) ppm, while for highest 20-Nov-20

AlF3 content 25 mol% and 35.5 mol% the position is at (30±1) ppm and (26.7±0.5) ppm respectively, and with no influence of the temperature. We notice also that the magnesium oxide is solubilized in the bath, the 17 O signal intensities increase with MgO additions (Fig. 3).

The 25 Mg signal is slightly influenced by the amount of MgO compare with pure molten MgF2 i.e.

at -4.1 ppm. It appears at (-7.0±1) ppm in this range of composition. The spectral signal to noise ratio is low, but enough is retained to provide a reasonably accurate measurement of the position.

Nevertheless, it is difficult to obtain the signal for MgO additions less than 10 mol%. It should be noted that in the system MgO-Li3AlF6 a small effect of temperature was observed, the chemical shifts increase slightly (+1 ppm) for the changing of the temperature from 800 °C to 960 °C.

20-Nov-20

X-ray results

The X-ray pattern of samples, with 14.5 and 25 mol% AlF3 contains the lines of precursor LiF, Li3AlF6 and two new phases, namely magnesium fluoride and alumina. Some traces of BN powder are due to the crucible were observed. For the 35.5 mol% AlF3, we also detected LiMgAlF6 [START_REF] Tao | Synthesis and Luminescence Characteristics of LiMgAlF6:Ln 3+ (Ln = Ce, Eu, Tb)[END_REF].

Solid state NMR

Solid state 27 species in the melt and the increasing of amount of AlF4 -and/or free fluorine ions with increasing the temperature.

Recently Moussaed [START_REF] Moussaed | Apport de la RMN Haute température et de la Dynamique Moléculaire à l'étude de la fluoroacidité dans les fluorures fondus[END_REF] studied the structure and dynamics of KF-MgF2 and LiF-MgF2 melts over a wide range of composition and temperature by HT NMR and HT PFG NMR. 25 Mg and 19 F NMR experimental data were also compared with MD calculations. In the case of LiF-MgF2, 25 Mg chemical shifts decrease from 0.5 ppm at 5 mol% MgF2 to -5 ppm in pure MgF2. For the KF-MgF2 system, the 25 Mg chemical shift values vary from 7 ppm for 15 mol% to -5 ppm in pure MgF2. MD calculations in agreement with HT NMR experiments provide evidence for the coexistence of the different Mg-based complexes: MgF4 2-, MgF5 3-, MgF6 4-, MgF7 5-, and MgF8 6-.

The average coordination for the magnesium ion in LiF-MgF2 system is between 5.8 and 6.0. It is worth noting the important contribution of MgF6 4-complexes, around 60 % of total Mg 2+ species.

The average coordination depends on the concentration and temperature of the melt. In case of KF-MgF2 at 10 mol% MgF2 the average coordination is 4.8 with important contribution of MgF5 3- (~60 %), and it increases up to 6 with increasing MgF2 amount.

Based on literature data [START_REF] Freitas | Chapter 2 -Recent Advances in Solid-State 25 Mg NMR Spectroscopy[END_REF][START_REF] Moussaed | Apport de la RMN Haute température et de la Dynamique Moléculaire à l'étude de la fluoroacidité dans les fluorures fondus[END_REF][START_REF] Moussaed | Structure and Dynamics of Alkali and Alkaline Earth Molten Fluorides by High-Temperature NMR and Molecular Dynamics[END_REF][START_REF] Sadoc | NMR parameters in alkali, alkaline earth and rare earth fluorides from first principle calculations[END_REF] we have constructed a 25 The X-ray diffraction patterns obtained after cooling at room temperature confirm the transformation of MgO and show evidence of the existence of Li3AlF6, Al2O3, MgF2, and LiF. The presence of these compounds suggests the complete reaction of magnesium oxide according to the chemical equation:

3MgO + 2AlF3→ 3MgF2+Al2O3 (2)
This interpretation is in very good agreement with data reported by Kostyukov et al. in the system NaF-AlF3-MgO [START_REF] Kostyukov | Physico-chemical study of the system NaF-AlF3-CaF2 (MgF2)[END_REF].

We report on the Fig. 5, the different 17 O chemical shifts measured in the melt depending on the amount AlF3. We show also for comparison, the 17 O chemical shift value measured in solid MgO at 47 ppm [START_REF] Turner | Solid-state oxygen-17 nuclear magnetic resonance spectroscopic study of the group 11 oxides[END_REF] (the melting temperature of MgO, tm =2852 °C, is too high for NMR measurements).

20-Nov-20 All our NMR measurements are consistent well with the supposition that the oxyfluoroaluminate anionic species (Al2O2F4 2-) form with the additions of MgO.

20-Nov-20

From the XRD patterns recorded on the solidified mixtures of MgO-LiF-AlF3, we have to note that for 35.5 mol% AlF3 composition, we obtained after cooling a mixture involving LiMgAlF6 that we had observed at high contents of MgF2 in MgF2-LiF-AlF3. This result is in good agreement with the findings of Tao et al. [START_REF] Tao | Synthesis and Luminescence Characteristics of LiMgAlF6:Ln 3+ (Ln = Ce, Eu, Tb)[END_REF], which synthesized LiMgAlF6 with the excesses of MgF2 and AlF3 in the reaction mixture.

LiMgF3 and NaMgF3 are predicted to have negative enthalpies of formation from the binary fluorides -53 kJ×mol -1 and -30 kJ×mol -1 respectively [START_REF] Claeyssens | Ab initio predictions of ferroelectric ternary fluorides with the LiNbO3 structure[END_REF]. But in case of MgF2-Na3AlF6 the formation of NaMgF3 as well as Na2MgAlF7 were observed [START_REF] Kostyukov | [END_REF]. In our work for the analogous MgF2 containing systems we didn't observe the precipitation any others phases.

Conclusions

High-temperature NMR study of melts in MgF2-LiF-AlF3 and MgO-LiF-AlF3 systems, by means of observation of the different nuclei involved in these systems: 27 Al, 25 Mg, 19 F, and 17 O, provides a powerful tool for the structural description of such complex liquids. In MgF2-LiF-AlF3 system magnesium atoms are surrounded by fluorines and the magnesium keep its sixfold coordination in the melt. The addition of MgO in the aluminium fluoride melts leads to a strong reaction with AlF3 and to a formation of alumina. We have found a formation of a bridged complex Al2O2F4 2-in the bath, when the amount of AlF3 increases.

Experimental section

Chemicals.

20-Nov-20

LiF, MgF2 and AlF3 were purchased from Sigma-Aldrich Co., 99.9 %. In order to detect the 17 O signal on MgO dissolution, because of its very low abundance (0.04 %), we have enriched the sample in 17 O. 17 O is the only isotope of the oxygen observable by NMR, because its spin is non-zero. We start from 17 O labeled H2O (enrichment 40 %, CortecNet). The labeled water was added to a stoichiometric amount of magnesium ethoxide in tetrahydrofuran (THF), under dry argon. The mixture was stirred and heated for 16 hours at 55 °C. Mg(OH)2 precipitated as light brown powder. Finally, solvent was distilled off and the reaction product calcined at 1400 °C for half hours. The quality of MgO was checked by X-Ray Diffraction (XRD) analysis (Bruker D8 Advance). All chemicals were handled in a glovebox with dry argon containing less than 1 ppm of water vapor.

The choice of the composition

Our approach is centered on the experimental characterization of MgO and MgF2 additions in LiF-AlF3 melts. Based on the LiF-AlF3 phase diagram we propose to study the effect of the AlF3 composition and temperature, and we focus on three compositions: eutectics 14.5 and 35.5 mol% AlF3 with low melting temperature (707 °C) compare with the other composition, and cryolitic composition Li3AlF6, at 25 mol% AlF3, with a congruent melting at 785 °C. This composition interests different other applications (aluminium electrolysis, pyrochemical treatments of nuclear wastes) and it has been extensively studied. We have then followed the effect of MgO and MgF2 additions in the three LiF-AlF3 mixtures: 14.5, 25, and 35.5 mol% AlF3.

The compositions of samples and the temperatures are given in Table 1.

20-Nov-20

NMR measurements

The HT NMR spectra were acquired using the laser heated NMR system developed in CEMHTI Orleans [START_REF] Bessada | Structural and Dynamic Approaches of Molten Salts by High Temperature Spectroscopies[END_REF]. The BN crucible containing the sample is placed inside the RF-coil, in the center of cryomagnet. The sample is heated by two CO2 laser (Coherent Diamond 250W), passing axially through the NMR probe. The temperature cannot be measured inside the crucible during the spectra acquisition, so thanks to the calibration of the laser power, we can determine the temperature within ±10 °C. The melting point of the sample is clearly indicated by the modification of the shape and width of the line. The transition from solid to liquid state can be followed on 27 Al NMR spectra during the heating of the sample. The time "zero" corresponds to the laser power setting, the desired temperature is considered to be reached in the sample after 4-5 minutes. For example in case MgF2-Li3AlF6, on heating, first, we observe one weak and broad signal related to the solid Li3AlF6. After 4-5 minutes, when the temperature is stabilized, we observe only a sharp signal corresponding to the melt. During the NMR experiments, the peaks do not change with time once the sample is liquid.

20-Nov-20

The HT spectra were recorded on Bruker AVANCE 400 (9.4 T) spectrometers using single pulse excitation consisting of 20 μs pulses; recycle delays of 500 ms, and 16 to 4096 scans to obtain a reliable signal-to-noise ratio. Several samples were checked after the HT NMR experiments using Solid-state NMR and X-ray diffraction. Solid-state NMR experiments were performed using Bruker Avance III spectrometer operating at 20.0 T, using a 4 mm resonance probe at a MAS frequency 14 kHz. 17 O and 27 Al MAS NMR data were collected from a single pulse experiment, with π/12 pulse widths between 1.0 μs and 1.2 μs and recycle delays of 0.5 s.

The reported chemical shifts are referenced to 11 M solutions of MgCl2, 1 M Al(NO3)3 for 25 Mg, 27 Al respectively, CFCl3 for 19 F and tap water for 17 O. The NMR parameters (chemical shifts and line widths) were fitted by means of a DMfit program [START_REF] Massiot | Modelling one-and two-dimensional solid-state NMR spectra[END_REF].

X-ray diffraction

X-ray diffraction analysis were done at room temperature under air on a Bruker D8 Advance diffractometer (CuKα1,2 radiation).

Fig. 1 .

 1 Fig. 1. On the left: evolution of 27 Al chemical shifts for different addition of MgF2 (0 to 25 mol%)

Fig. 2 .

 2 Fig. 2. 27 Al chemical shifts for different addition of MgO (0 to 10 mol%) measured in the

Fig. 3 .

 3 Fig. 3. 17 O NMR spectra in Li3AlF6 with different MgO additions (from 2 to 15 mol%). The 17 O

  Al NMR spectra of the same solidified mixtures show 3 signals, one very broad signal with maximum at around 65 ppm from aluminium in tetrahedral and two intense symmetric lines at -2 ppm and -16 ppm with spinning sidebands associated with a relatively low quadrupolar interaction from aluminium in octahedral coordination, which can be attributed to the lithium cryolite and aluminium fluoride. Transition alumina phases contain aluminium ions in two different types of tetrahedral, signals with maximum at 65 ppm, and octahedral coordination which covered by intense cryolite signal [22]. The 17 O NMR spectra show two overlapping resonances near 40 ppm and 60 ppm.Simulation of these spectra as the sum of two broad Gaussian line shapes with full width at half maximum (FWHM) ca. 3700 and 2400 Hz as illustrated in Fig.4, presumably due to a broad distribution of quadrupole coupling parameters. We indicate that the ratio of the integrated intensities of the two resonances is approximately 1:1. The same distribution of the aluminium sites was founded for different alumina phases by Walter and Oldfield[START_REF] Walter | Magic angle spinning oxygen-17 NMR of aluminum oxides and hydroxides[END_REF]. We assign the narrower resonance at 40 ppm to the OAl3 sites and the broader peak at 60 ppm to the trigonal 20-Nov-20 OAl4 sites. The greater breadth of this latter resonance is attributed to a larger quadrupole coupling constant for these tetrahedral sites, although we cannot discern any second-order quadrupolar splitting in the line shape. NMR results are in good agreement with the XRD data.

Fig. 4 .

 4 Fig. 4. 17 O MAS NMR experimental (black line) spectrum and it simulation (color lines) of

  Mg chemical shifts scalefor the different magnesium local environments: magnesium atom surrounded by 6 fluorines at around ≈ -4 ppm, MgF5 coordination at ≈ 7 ppm, MgO6 at ≈ 26 ppm and MgO4 between 48-55 ppm. We see that a magnesium atom surrounded by 6 fluorines will have a very different chemical shift than a magnesium surrounded by 6 oxygens[START_REF] Freitas | Chapter 2 -Recent Advances in Solid-State 25 Mg NMR Spectroscopy[END_REF]. In our systems, the measured chemical shift is the range of -3.5 to 8 ppm. Our data show that it will tend to form species with a magnesium atom surrounded by 6 fluorines. For the additions of MgO, the chemical shifts, that are relative to pure molten MgF2 (-4.1 ppm), could be the sign of the formation of MgF5 3-and MgF6 4-. From25 Mg NMR data we can propose the presence in the melt of species in which 20-Nov-20 magnesium atoms are surrounded by fluorine in both cases MgF and MgO and we state the magnesium don't keep its oxygen environment with adding magnesia in the bath.

Fig. 5 .

 5 Fig. 5. Evolution of the 17 O chemical shift with AlF3 composition at 900 °C.

17

 17

[ 20 Fig. 6 .

 206 Fig. 6. The rectangles figure the ranges of 27 Al chemical shifts values measured for the different

Table 1

 1 Compositions of the samples and temperatures of the NMR measurements.

	LiF-AlF3/mol%	Additives/mol%	Temperature/°C
	85.5-14.5	MgF2: 0, 4, 10 MgO: 0, 1, 2, 4, 6, 8	740, 820, 900 800, 880, 960
	75-25	MgF2: 0, 1, 2, 4, 6, 8, 10, 15, 25 MgO: 0, 1, 2, 4, 6, 8, 10, 15	800, 880, 960 800, 880, 960
	64.5-35.5	MgF2: 0, 2, 4, 6, 8, 10, 15 MgO: 0, 1, 2, 4, 6, 8, 10	not measured 900

20-Nov-20Graphical abstract:

Acknowledgment

This study was financially supported by the Sacsess program (Grant Agreement number 323282). We thank Dr. E. Veron for X-ray measurements and Dr. F. Šimko for useful discussions.