Ashley P Black

Carlos Frontera

Arturo Torres

Miguel Recio-Poo

Patrick Rozier

Juan D Forero-Saboya

François Fauth

Esteban Urones-Garrote

M Elena

Arroyo-De Dompablo

M Rosa Palacín

M Elena Arroyo-De Dompablo

Elucidation of the redox activity of Ca2MnO3.5 and CaV2O4 in calcium batteries using operando XRD: charge compensation mechanism and reversibility

Keywords:

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Introduction

Li-ion batteries (LIB) have been continuously improving performance and reducing cost since its commercialization [START_REF] Placke | Lithium Ion, Lithium Metal, and Alternative Rechargeable Battery Technologies: The Odyssey for High Energy Density[END_REF] and are currently expanding their field of application from electronics to transportation and even grid storage. Nonetheless, the technology is now reaching its fundamental limits in terms of energy density. The "holy grail " to leap-frog present values would be to develop rechargeable batteries using metals as negative electrodes. Besides the intensive research on concepts based on lithium metal such as Li-S and Li-air, recent efforts are targeting the exploration of divalent metals [START_REF] Ponrouch | Multivalent Rechargeable Batteries[END_REF] . In contrast to lithium, Ca and Mg are both very abundant in the Earth's crust, ranked 5 th and 8 th , respectively, with the raw materials being lower in price than the lithium counterparts. Their capacities, despite lower than lithium metal, are much higher than those of the graphite based negative electrodes typically employed in LIBs. Proof-of-concept of a Mg metal based battery was provided already in 2000 [START_REF] Aurbach | Prototype Systems for Rechargeable Magnesium Batteries[END_REF] , but its commercialization has remained elusive due to the corrosive nature and low stability of the electrolytes employed and the fundamental limited diffusion of highly polarizing Mg 2 + ions. Ca metal based batteries have been much less explored but recent proof-of-concept of electrolytes enabling successful plating and stripping of the metal catalyzed activity in the field [START_REF] Arroyo-De Dompablo | Achievements, Challenges, and Prospects of Calcium Batteries[END_REF] . Indeed, estimates of prospective figures of merit at the cell level using open energy-cost models [START_REF] Berg | Rechargeable Batteries: Grasping for the Limits of Chemistry[END_REF] indicate that performances comparable to state-of-the art LIB could be achieved with positive electrodes exhibiting moderate potential (2.5V) and a capacity of 200 mAh/g, and that volu-metric energy densities above 1000 Wh/l would be possible considering the same capacity but operation at 3V [START_REF] Monti | Multivalent Batteries -Prospects for High Energy Density: Ca Batteries[END_REF] .

In spite of such nice expectations, the investigation of suitable positive electrode materials has been plagued with a number of hurdles. On one hand, the lack of standard electrolytes and protocols may result in side reactions inducing misinterpretation of the electrochemical response achieved [START_REF] Lipson | Current Collector Corrosion in Ca-Ion Batteries[END_REF][START_REF] Verrelli | On the Study of Ca and Mg Deintercalation from Ternary Tantalum Nitrides[END_REF][START_REF] Dugas | Methods and Protocols for Reliable Electrochemical Testing in Post-Li Batteries (Na, K, Mg, and Ca)[END_REF] , which makes real bulk activity difficult to elucidate unless complementary characterization techniques are used [START_REF] Tchitchekova | Electrochemical Intercalation of Calcium and Magnesium in TiS 2 : Fundamental Studies Related to Multivalent Battery Applications[END_REF] . On the other, testing of materials similar to those used in the LIB field which would enable reversible intercalation/de-intercalation of calcium has to date led to the identification of few active compounds of limited performance (V 2 O 5 , TiS 2 , MoO 3 , Prussian blue analogs…). Besides those, some structural frameworks were identified which enable electrochemical de-intercalation of calcium, such as 1D Ca 3 CoMO 6 (M = Co, Mn) [START_REF] Black | Operando Synchrotron X-Ray Diffraction in Calcium Batteries: Insights into the Redox Activity of 1D Ca 3 CoMO 6 (M = Co and Mn)[END_REF] , for which the process was found to be irreversible likely due to the structural changes taking place upon reduction resulting in too high migration barriers for calcium ions [START_REF] Torres | Evaluation of Cobalt Oxides for Calcium Battery Cathode Applications[END_REF] . These findings highlight the complexity of achieving materials with reversible Ca 2 + intercalation and acceptable performance.

In contrast, successful calcium intercalation into chemically or electrochemically oxidized polyanionic frameworks (e.g. Na 1.5-x VPO 4.8 F 0.7 , Na 3-y V 2 (PO 4 ) 3 or Li 1-z FePO 4 ) has been recently reported [START_REF] Jeon | Reversible Calcium-Ion Insertion in NASICON-Type NaV 2 (PO 4 ) 3[END_REF][START_REF] Kim | High-Voltage Phosphate Cathodes for Rechargeable Ca-Ion Batteries[END_REF][START_REF] Xu | A New High-Voltage Calcium Intercalation Host for Ultra-Stable and High-Power Calcium Rechargeable Batteries[END_REF] , with the positions of calcium in the crystal structure being elucidated in the case of the vanadium containing materials. Yet, since these compounds do contain lithium or sodium, an additional treatment, either chemical or electrochemical, is required to remove these alternative ions, which is an extra hurdle for practical applications. Otherwise, the exploration of organic electrode materials, not involving an intercalation redox mechanism, seems also promising [START_REF] Bitenc | Electrochemical Performance and Mechanism of Calcium Metal-Organic Battery[END_REF] .

Within this scenario, a DFT driven research approach is clearly a powerful strategy to shortcut the meandrous that a classical trial and error method will encounter along its way. Several works [START_REF] Zhang | Computational Screening of Layered Materials for Multivalent Ion Batteries[END_REF][START_REF] Torres | Analysis of Minerals as Electrode Materials for Ca-Based Rechargeable Batteries[END_REF][START_REF] Torres | Enlisting Potential Cathode Materials for Rechargeable Ca Batteries[END_REF][START_REF] Lu | Searching Ternary Oxides and Chalcogenides as Positive Electrodes for Calcium Batteries[END_REF] deal with the systematic search of potential positive electrodes for calcium batteries among large databases of compounds, such as the Inorganic crystal data base (ICSD), the Materials Project (MP) or the American Mineralogist Crystal Structure Database (AMCSD). From this compendium of computational results, it is concluded that the major obstacle for viable Ca-cathodes is the sluggish diffusion of Ca 2 + ions in inorganic structures. Computationally, the ionic diffusivity (D) can be inferred from the calculated migration barrier energy (Em) [START_REF] Morgan | Li Conductivity in Li x MPO 4 (M = Mn, Fe, Co, Ni) Olivine Materials[END_REF] . . Canepa et al . explored the tolerable energy barrier for an optimum electrode performance as a function of the C-rate, temperature and particle size [20 , 22] . For a nanosized compound, an energy barrier below 0.65 eV is required to achieve a C/2 battery operation rate (here D ∼ 10 -12 cm 2 s -1 ) at room temperature. The tolerable energy barrier can be increased by lowering the rate / particle size or increasing the temperature. For instance, the tolerable energy for 10 nm particles operating at 66 °C at a C/6 rate raises to 0.895 eV . Full details of the relationship between the migration barrier and the maximum particle size permitting reasonable diffusivity, at various variation C-rates and temperatures, are given by Lu et al . [START_REF] Lu | Searching Ternary Oxides and Chalcogenides as Positive Electrodes for Calcium Batteries[END_REF] and Canepa et al. [START_REF] Canepa | Odyssey of Multivalent Cathode Materials: Open Questions and Future Challenges[END_REF] . These values of tolerable migration energies are useful indicators in the quest for positive electrode materials. Indeed, experiments evidence that compounds with predicted energy barriers for calcium diffusion above ∼1 eV are inactive, even at very low C rates (C/100) and temperatures of ∼ 50 °C. Examples of those materials are perovskite-CaMoO 3 (Em = 1.8 eV) [START_REF] Arroyo-De Dompablo | In Quest of Cathode Materials for Ca Ion Batteries: The CaMO 3 Perovskites (M = Mo[END_REF] , CaMn 2 O 4 (Em = 1.3 eV) [START_REF] Dompablo | A Joint Computational and Experimental Evaluation of CaMn 2 O 4 Polymorphs as Cathode Materials for Ca Ion Batteries[END_REF] , CaV 2 O 5 (Em = 1.6-1.9) [START_REF] Verrelli | On the Strange Case of Divalent Ions Intercalation in V 2 O 5[END_REF][START_REF] Gautam | First-Principles Evaluation of Multi-Valent Cation Insertion into Orthorhombic V 2 O 5[END_REF][START_REF] Torres | DFT Investigation of Ca Mobility in Reduced-Perovskite and Oxidized-Marokite Oxides[END_REF] , brownmillerite-Ca 2 Fe 2 O 5 (Em = 1 eV) [27 , 28] , and CaTaN 2 (Em = 1.7 eV) [START_REF] Verrelli | On the Study of Ca and Mg Deintercalation from Ternary Tantalum Nitrides[END_REF] . On the other hand, when energy barriers are lower than 1 eV, some electrochemical activity is often observed, examples being TiS 2 (Em = 0.75 eV) [START_REF] Tchitchekova | Electrochemical Intercalation of Calcium and Magnesium in TiS 2 : Fundamental Studies Related to Multivalent Battery Applications[END_REF] , VOPO 4 (Em = 0.5 eV) [19 , 29] , Ca 4 Fe 9 O 17 (Em = 0.72 eV) [START_REF] Black | Appraisal of Calcium Ferrites as Cathodes for Calcium Rechargeable Batteries: DFT, Synthesis, Characterization and Electrochemistry of Ca 4 Fe 9 O 17[END_REF] or Ca 3 Co 2 O 6 (Em = 0.9 eV) [START_REF] Torres | Evaluation of Cobalt Oxides for Calcium Battery Cathode Applications[END_REF] .

It should be noted that the previous DFT-quests for Ca-cathodes mostly focus in materials that, as discussed above, can be synthetized in the discharged (or reduced) state, this is to say, containing Ca ions in the structure. Following with this research line, in this work we will show that DFT calculations enabled the identification of CaV 2 O 4 and Ca 2 MnO 3.5 as potential positive electrodes for calcium batteries [30 , 31] . The experimental investigation confirmed the viability and reversibility of deintercalation for CaV 2 O 4 , while in the case of Ca 2 MnO 3.5 , an alternative charge compensation mechanism for manganese oxidation was encountered.

Materials and methods

a) Computational

Calculations have been performed using the ab-initio total-energy and molecular dynamics program VASP (Vienna Ab-initio Simulation Program) developed at the Universität Wien [START_REF] Kresse | Efficient Iterative Schemes for Ab Initio Total-Energy Calculations Using a Plane-Wave Basis Set[END_REF] . Total energy calculations based on Density Functional Theory (DFT) were performed within the Generalized Gradient Approximation (GGA), with the exchange and correlation functional form developed by Perdew, Burke, and Ernzerhof (PBE) [START_REF] Perdew | Generalized Gradient Approximation Made Simple[END_REF] . The interaction of core electrons with the nuclei is described by the Projector Augmented Wave (PAW) method. The energy cut off for the plane wave basis set was kept fix at a constant value of 600 eV throughout the calculations. The integration in the Brillouin zone is done on appropriate k-point sets determined by the Monkhorst-Pack scheme. A convergence of the total energy close to 10 meV per formula unit is achieved with such parameters. Spin polarized calculations considering a ferromagnetic configuration were performed in all cases. All crystal structures were fully relaxed (atomic positions, cell parameters and volume). The final energies of the optimized geometries were recalculated so as to correct the changes in the basis set of wave functions during relaxation. To take electron correlation into account, the total energies of the GGA-optimized compounds have been calculated using the GGA + U method, with U eff of 3 and 4 eV for the d -states of V and Mn, respectively [START_REF] Jain | Commentary: The Materials Project: A Materials Genome Approach to Accelerating Materials Innovation[END_REF] .

The crystal models for the initial compounds were taken from the ICSD files 164185-CaV 2 O 4 35 and 62168-Ca 2 MnO 3.5 36 . The crystal models for the fully deintercalated phases were constructed removing all the Ca ions from the relaxed structures of the initial Ca-bearing compounds. To simulate the crystal structures of the possible partially deinserted compounds it is necessary to establish an ordered pattern of Ca ions and vacancies. Hence Ca-vacancy arrangements were created using the CASM (Cluster Approach to Statistical Mechanics) software package [START_REF] Van Der Ven | Linking the Electronic Structure of Solids to Their Thermodynamic and Kinetic Properties[END_REF] . Search for pseudo symmetry in the final optimized models was done using the PLATON software [START_REF] Spek | Structure Validation in Chemical Crystallography[END_REF] .

The average Ca intercalation voltages have been obtained from the calculated total energies of the calciated and decalciated forms, following the methodology described by Aydinol et al. [START_REF] Aydinol | Ab Initio Study of Lithium Intercalation in Metal Oxides and Metal Dichalcogenides[END_REF] . The Ca 2 + mobility is investigated using the Nudged Elastic Band method (NEB) as implemented in VASP [START_REF] Mills | Reversible Work Transition State Theory: Application to Dissociative Adsorption of Hydrogen[END_REF] . NEB calculations are usually carried out at the dilute limits, that is, either the migration of a single vacancy in an otherwise fully inserted structure or a single ion migration in an otherwise calcium-free host structure. To attain the dilute limit, we used a superstructure of the unit cell that guarantees a minimum interaction between defects. Constant volume calculations were performed within the GGA (and eventually GGA + U) approximation, considering a sufficient number of intermediate images initialized by linear interpolation between the two fully relaxed end points. To calculate the energy at the saddle point, cubic splines were fit through the images along each hop.

b) Experimental

Ca 2 MnO 4 was synthesized following the Pechini method [START_REF] Fawcett | Structure, Magnetism, and Properties of Ruddlesden -Popper Calcium Manganates Prepared from Citrate Gels[END_REF] X-Ray powder diffraction patterns were acquired on a Bruker D8 Advance A25 diffractometer in a Debye-Scherrer configuration equipped with a Mo K 𝛼1 radiation source ( 𝜆 = 0.7093 Å) and Johansson monochromator. Synchrotron X-ray diffraction (SXRD) patterns where collected on the MSPD beamline [START_REF] Fauth | The New Material Science Powder Diffraction Beamline at ALBA Synchrotron[END_REF] (ALBA synchrotron, Cerdanyola del Vallès, Spain) using the newly deployed position sensitive detector MYTHEN2 and 𝜆= 0.6200 Å. In both cases, the samples, either as prepared or recovered after running electrochemical experiments and dismantling cells inside an Ar filled glove box, were embedded in a 0.5 mm diameter borosilicate glass capillary and spun during data collection. Operando synchrotron diffraction experiments were carried out at the MSPD beamline using 5 mm diameter hole drilled 2032 coin cells equipped with a glass window and a setup enabling the alternative sequential measurement of eight cells in the same experiment [START_REF] Herklotz | A Novel High-Throughput Setup for in Situ Powder Diffraction on Coin Cell Batteries[END_REF] . Rietveld refinements and Fourier analysis were carried out with the Fullprof Suite program [45 , 46] .

High-Angle Annular Dark-Field (HAADF) Scanning-Transmission Electron Microscopy (STEM) images were acquired with an ARM200 cF microscope, fitted with a corrector of the spherical aberration (resolution of 0.08 nm). Electron Energy-Loss spectroscopy (EELS) was performed in the same microscope, using a GIF Quantum ER spectrometer. The Mn-L 2,3 edges were acquired ( 𝛽≈30 mrad, acquisition time of 1 second, dispersion of 0.1 eV/pixel) in order to quantify the average oxidation state according to the white-lines ratio method [START_REF] Schmid | Oxidation States of Mn and Fe in Various Compound Oxide Systems[END_REF] . The displacement of the absolute energy-loss value of the edge was compared between the different samples in order to check for the possible oxidation of Mn [START_REF] Egerton | Electron Energy-Loss Spectroscopy in the Electron Microscope[END_REF] . The average composition of the crystals was analyzed with X-Ray Energy Dispersive Spectroscopy (XEDS).

Tape casted electrodes were prepared from a mixture of as synthesized powder samples with carbon black (Super P, Timcal, Switzerland) and polyvinylidene difluoride (Arkema) in weight ratios of 80:10:10. A slurry was made by dispersing the active material, carbon and binder powders in N-methyl-2-pyrrolidone (Aldrich, ⩾99.9%) with a solid/solvent ratio of 100 mg/ml, which was then ball milled at 400 rpm for 1 h using a Retsch PM10 planetary mill, doctor blade casted (thickness 400 𝜇m) on a 18 𝜇m thick aluminum foil (Goodfellow, 99%) and vacuum dried at 120°C for 3 h. Electrode discs of 11 mm diameter with an average active material load of about 2 mg, were cut from the casted tape, pressed at 5 Tons and dried overnight in a vacuum chamber before use. Electrochemical tests were performed in three electrode Swagelok cells using activated carbon (AC, Kynol, ACC-509220) or Li metal as counter electrodes (CE) and an Ag wire was as reference electrode (RE), which was calibrated using a 10 mM ferrocene/ferrocenium solution in 0.45 Ca(BF 4 ) 2 in EC:PC following previously optimized protocols, its potential being 0.32 V vs NHE [START_REF] Verrelli | Steps Towards the Use of TiS 2 Electrodes in Ca Batteries[END_REF] . The electrolytes used were 0.45M Ca(BF 4 ) 2 dissolved in a 1:1 (v/v) mixture of ethylene carbonate (EC, Aldrich anhydrous 99.0%) and propylene carbonate (PC, Aldrich anhydrous 99.0%), prepared from Ca(BF 4 ) 2 •xH 2 O salt (Alfa Aesar), 1M LiPF 6 in 1:1 (v/v) mixture of ethylene carbonate (EC) and dimethyl carbonate (DMC) (Solvionic, denoted LP30), 1M Lithium bis oxalato borate (LiBoB, Chemetal, Battery grade) or 0.45M cesium fluoride (CsF, Aldrich, 99%) in 1:1 (v/v) mixture of EC and PC. The water content in all electrolytes was measured by Karl Fisher titration and found to be lower than 50 ppm. Electrolyte preparation and cell assembling were done in an Ar filled glove box with less than 1 ppm of O 2 and H 2 O. Prior to applying any current to the cells assembled, equilibration of the working electrode (WE) potential under the operating conditions at open circuit potential (OCP) was ensured. Tests were done using a Bio-Logic VMP3 potentiostat in galvanostatic mode with potential limitation (GCPL) at C/50 (equivalent to 1 mol of Ca + 2 or 2 moles of e -reacted in 50 h) at room temperature unless stated otherwise. Operando diffraction experiments were carried out at C/50 rate using the cells described in the previous sub-section containing 3 mg of powder electrode material prepared by mixing carbon black and the compound under study in weight ratios 25:75 and either activated carbon (AC) or Li metal as counter electrode and 0.45M Ca(BF 4 ) 2 in EC:PC or LP30 as electrolytes respectively. The calculated barriers of 1.1 and 1.2 eV in two different directions suggest that two-dimensional conductivity may take place. As discussed in the introduction, with in-plane energy barriers around 1 eV, Ca 2 MnO 3.5 falls in the border-line between electrochemically active and inactive materials. As expected, Ca-diffusion along the c -axis of Ca 2 MnO 3.5 is impeded as the calculated energy barriers are too high (above 1.8 eV, Fig. 1 C). Regarding the electronic properties, the calculated DOS suggest a semiconducting behavior (see Figure S1).

Results and discussion

Ca deinsertion from Ca 2 MnO 3.5 is likely limited to the Mn 3 + /Mn 4 + redox couple (maximum specific capacity 140 mAh/g), according to the reaction:

C a 2 Mn O 3 . 5 → 0 . 5 C a 2+ + e -+ C a 1 . 5 Mn O 3 . 5 (1)
For the Ca 1.5 MnO 3.5 composition, vacancies and Ca 2 + ions can adopt seven distinct arrangements within the primitive cell. The calculated average voltage taking the most stable configuration are 2.4 V (GGA) and 3.3 V (GGA + U). This would yield a maximum theoretical energy density of 462 mWh/g for this compound. Fig. 1 E compares the crystal structure of the most stable configuration for Ca 1.5 MnO 3.5 with that of the pristine material. A phase transformation is foreseen upon Ca-deintercalation, driven by the crystal field energies of Mn 3 + and Mn 4 + ions. Since Mn 4 + has a d 3 electronic configuration, it has a strong tendency to adopt an octahedral coordination. This change in the Mn-polyhedra is achieved via the rearrangement of the square pyramids present in the ab plane of Ca 2 MnO 3.5 ( Fig. 1 E left) so as to form double chains of square pyramids linked by corners to double chains of distorted octahedra in Ca 1.5 MnO 3.5 ( Fig. 1 E right) . Note that the oxygen content is not compatible with a Ca 1.5 MnO 3.5 structure consisting only of Mn-octahedra (K 2 NiF 4 type). The phase transformation supposes a minimum volume contraction of 2% within both the GGA and GGA + U approximations (see Table S1 for lattice parameters).

CaV 2 O 4 exhibits a crystal structure built up from edge-sharing chains of VO 6 octahedra, forming double rutile chains ( Fig. 2 A). Each double-rutile chain is connected to the adjacent chains through cornersharing oxygen. As a result, tunnels appear in the framework along the c axis (space group Pbnm ), where Ca ions occupy triangular prismatic sites with capped rectangular faces and Ca-O distances in the range 2.28-2.65 Å. Topotactic calcium deintercalation (V 3 + /V 4 + redox couple) would result in a specific capacity of 260 mAh/g, following the reaction:

Ca V 2 O 4 → C a 2+ + 2 e -+ V 2 O 4 (2)
The Expanded regions of the patterns are depicted in (C) and (D) so that the differences can be more clearly seen. lated average deintercalation voltage (GGA + U) for reaction (2) is 2.48 V, in good agreement with previous DFT calculations [START_REF] Lu | Searching Ternary Oxides and Chalcogenides as Positive Electrodes for Calcium Batteries[END_REF] . This value results in an appealing theoretical energy density of 650 mWh/g for this compound.

The pathway for Ca 2 + diffusion in CaV 2 O 4 is along the tunnel built up by interconnected Ca sites ( c axis in Fig. 2 A). In this tunnel adjacent Ca sites share a triangular face and therefore on going from one site to the adjacent site the diffusing Ca ions must pass across this triangular window. The calculated energy barriers for Ca diffusion ( Fig. 2 B) are 0.60 eV (GGA) and 0.71 eV (GGA + U), similar to the value reported by Canepa´s group (GGA-Em of 0.654 eV) [START_REF] Lu | Searching Ternary Oxides and Chalcogenides as Positive Electrodes for Calcium Batteries[END_REF] . NEB calculations for the fully deintercalated phase (V 2 O 4 ) yield a migration barrier of 1.05 eV ( Fig. 2

b) Experimental

Synthesis

The synthesis of Ca 2 MnO 3.5 by low temperature reduction of the Ruddlesden-Popper (n = 1) Ca 2 MnO 4 compound was reported by Poeppelmeier et al . [36 , 50] and found to require careful control of both the temperature and an appropriate reducing agent, with Ca 2 MnO 3.5 being metastable and achievable only in a very narrow range of temperatures. The reduction of Ca 2 MnO 4 , synthesized as described above, was attempted at temperatures ranging between 300°C and 430°C with different reaction times under a variety of reducing atmospheres and gas flows. In 5% H 2 /Ar atmosphere no evidence of the reduced Ca 2 MnO 3.5 phase could be observed in the XRD pattern of the samples achieved after treatments at temperatures between 300°C and 350°C, and even after several 48h steps with intermediate regrinding Ca 2 MnO 4 remained unreacted. At 380°C the characteristic reflections of the reduced phase started to appear with its amount being rather low even after consecutive regrinding and thermal treatment steps. Reaction of Ca 2 MnO 4 at 400°C for 18h in 5% H 2 /Ar led to the formation of a larger quantity of Ca 2 MnO 3.5 together with a significant amount of the over reduced Mn 2 + containing the rock salt-type Ca 2 MnO 3 phase. Analogous treatments were performed under 10% H 2 /Ar leading to similar results. In light of such findings, the use of a less reducing atmosphere was deemed necessary and NH 3 was selected to that purpose. The strategy was successful, despite optimization of the protocol was required to minimize the amount of impurities (either Ca 2 MnO 4 precursor and/or over reduced Ca 2 MnO 3 , both confirmed to be electrochemically inactive). Within the range of conditions systematically explored (see Figures S3-6 in S.I.) (temperature between 380 and 550°C, NH 3 flow between 50 and 1200 ml/min and time in the range from 4h to 50h), the best Ca 2 MnO 3.5 batches obtained, and used for electrochemical characterization, contain less than 5% impurities (as deduced from Rietveld refinement) and were prepared by a short treatment (4h) under high ammonia flux (1200 ml/min) at 420°C.

Electrochemical characterization

In order to assess their electroactivity, attempts to extract Ca 2 + from Ca 2 MnO 3. 5 and CaV 2 O 4 were carried out in either Li or Ca cells at C rates ranging between 1C and C/100). Fig. 3 A shows the characteristic voltage vs capacity profiles of Ca 2 MnO 3.5 and CaV 2 O 4 electrodes subjected to GCPL tests in three-electrode cells. Both exhibit an electrochemical response, with the potential being significantly higher for Ca 2 MnO 3.5 , in agreement with DFT results. The potential vs . capacity profile for the first oxidation of CaV 2 O 4 follows a two-region trend, with a first steep sloping region from 0.25 V vs Ag QRE (equivalent to 3.5 V vs Ca + 2 /Ca) up to 1.5 V and 130 mAh/g capacity and a second region extending to 260 mAh/g and 1.8 V vs Ag QRE. In contrast, for Ca 2 MnO 3.5 , the potential first increases rapidly to 1.4 V vs Ag QRE and then evolves gradually to reach 281 mAh/g at 1.9 V vs Ag QRE. The potential at the end of oxidation would be close to 5 V vs. Ca 2 + /Ca in both cases and hence significant electrolyte oxidation can be expected to take place.

The ex-situ XRD data corresponding to the samples recovered after first oxidation to their nominal theoretical capacity are displayed in Fig. 3 B. Patterns corresponding to oxidized CaV 2 O 4 and Ca 2 MnO 3.5 present significant changes when compared to those corresponding to the pristine compounds, which indicates that structural changes take place and would confirm the redox activity of these materials. In both cases, the formation of a new phase is observed upon oxidation with main reflections at 2 𝜃 (d) = 8.7°(4.62 Å), [START_REF] Xu | A New High-Voltage Calcium Intercalation Host for Ultra-Stable and High-Power Calcium Rechargeable Batteries[END_REF] and Ca 2 MnO 3.5 respectively. A fraction of the pristine compound is still present in the XRD pattern collected after oxidation to the theoretical capacity value (considering Mn + 4 and V + 4 as the highest achievable oxidation states), in agreement with a certain contribution to the observed values of capacity resulting from electrolyte decomposition side reactions. These same new phases were also observed when electrochemical oxidation was performed in Li cells (LP30 electrolyte) for comparative purposes (see Figures S7 andS8 in the S.I.).

Operando diffraction studies and charge compensation mechanism in Ca 2 MnO 3.5

The structural evolution of Ca 2 MnO 3.5 electrodes was assessed by operando XRD using synchrotron radiation. Fig. 4 A depicts the potential versus capacity profile and the corresponding XRD patterns. Oxidation of Ca 2 MnO 3.5 was conducted for 40 h (equivalent to a capacity 220 mAh/g) before reversing the current upon reduction for the following 24h (equivalent to 140 mAh/g). The changes observed on the XRD patterns collected throughout the experiment highlight the progressive growth of peaks at 2 𝜃 = 17.37 0 and 18.15 0 upon oxidation to the expense of reflections at 2 𝜃 = 17.50 0 , 17.96 0 and 18.96 0 (see Region I, Fig. 4 A). Upon reduction, no changes in the patterns are evidenced, which point at the irreversibility of the redox process and therefore the capacity achieved being most likely related to side reactions involving the electrolyte. Further structural characterization of the new phase formed (denoted Ox-Ca 2 MnO 3.5 from here on) was conducted by Rietveld refinement of the patterns obtained at the end of oxidation.

Fig. 4 B represents the observed and calculated X-ray diffraction patterns of Ox-Ca 2 MnO 3.5 . Its crystal structure seems to be closely related to that of Ruddlesden-Popper Ca 2 MnO 4 , (space group I4 1 /acd). (See Tables S2 to S5 for detailed structural and refinement parameters for Ca 2 MnO 4 and Ca 2 MnO 3.5 ). A first refinement of the Ox-Ca 2 MnO 3.5 diffraction pattern performed in the tetragonal I4 1 /acd space group resulted in cell parameter values of a = 5.2681(2) Å and c = 24.3730( 16) Å and a cell volume of 676.41(6) Å 3 (V/Z = 84.553 Å 3 ), representing a volume expansion of nearly 4% with respect to Ca 2 MnO 4 and Ca 2 MnO 3.5 which have cell volumes of 650.28(3) Å 3 (V/Z = 81.283 Å 3 ) and 651.77(4) Å 3 (V/Z = 81.471 Å 3 ) respectively. Note that the volume difference between Ca 2 MnO 4 and Ca 2 MnO 3.5 can be ascribed to the larger volume of Mn + 3 polyhedron (compared to that of Mn 4 + ), although it is partially compensated by lower oxygen content. In contrast, the volume expansion upon oxidation of Ca 2 MnO 3.5 is contrary to what would be expected for Ca deinsertion concomitant to Mn 3 + to Mn 4 + oxidation, and also to DFT predictions. The refinement of the calcium content in Ox-Ca 2 MnO 3.5 renders, within the error, no difference with respect to Ca 2 MnO 3.5 , which brings us to consider charge compensation mechanisms for manganese oxidation different from Ca extraction.

Due to the anion deficient nature of Ca 2 MnO 3.5 and considering the evolution towards a crystal structure closely related to the Ca 2 MnO 4 upon oxidation (consistent with the rearrangement predicted by DFT), anion intercalation appears as a reasonable alternative. This process might involve either large anions present in the electrolyte (BF 4

-or PF 6

-), which would likely induce larger structural changes, or other anions produced by their decomposition, such as F -which would seem a priori more plausible. In order to further elucidate these aspects, Ca 2 MnO 3.5 and Ox-Ca 2 MnO 3.5 were characterized by HAADF-STEM, EDS and EELS. Ca 2 MnO 3.5 consists of thick particles of 150-200 nm (see S.I.) and the HAADF-STEM images taken along the [-110] and [010] zone axis evidence a well crystallized sample ( Fig. 5 A). The characteristic lamellar structure of a Ruddlesden-Popper phase is identified with the intense bright spots corresponding to Mn atoms and the slightly less intense spots corresponding to the Ca atoms. The EDS measurements indicate a Ca:Mn atomic ratio consistent with the Ca 2 MnO 3.5 formula. Upon oxidation, less order is observed (see Figure S10 in the S.I.). The Ca:Mn ratio of the pristine sample is preserved in most of the Ox-Ca 2 MnO 3.5 crystallites; noteworthy, fluorine is clearly detected with approximate contents ranking from 6 to 12 % (see S.I. for EDS spectra). The EELS spectra depicted in Fig. 5 B indicate a shift in the Mn-L 2,3 edge when comparing Ox-Ca 2 MnO 3.5 and Ca 2 MnO 3.5 , which is consistent with Mn 3 + oxidation during the electrochemical treatment. Nevertheless, the degree of Mn oxidation is inhomogeneous, in agreement with the variable F content detected in the Ox-Ca 2 MnO 3.5 crystallites.

Attempting to confirm fluoride intercalation as the charge compensation mechanism upon oxidation, additional experiments were carried out involving the electrochemical oxidation of Ca 2 MnO 3.5 in a fluorine free electrolyte (1M LiBoB in EC:PC). No changes in the diffraction pattern of the pristine material were detected even after prolonged oxidation. This suggests that Ca ions are not deintercalated from the crystal structure of Ca 2 MnO 3.5 , which is in agreement with the high DFTmigration barriers ( > 1 eV). In contrast, electrochemical oxidation in an alternative F -containing electrolyte (0.45M CsF in EC:PC) resulted in the observation of the peaks characteristic of Ox-Ca 2 MnO 3.5 , which confirms that the redox activity is related to fluoride intercalation in the crystal structure (see Figure S11). These findings are consistent with the known behavior of Ruddlesden-Popper type structures when tested as positive electrode materials in fluoride ion batteries (FIB) [START_REF] Nowroozi | Fluoride Ion Batteries -Past, Present, and Future[END_REF] .

Taking into account the proposed insertion of fluoride ions in Ca 2 MnO 3.5 , the synchrotron XRD powder diffraction pattern of Ox-Ca 2 MnO 3.5 was refined in the orthorhombic Bbcm space group and the difference Fourier map calculated in order to identify possible sites for fluorine (Figure S12). Fluorine occupancies were then refined in the crystallographic sites corresponding to the anion "vacancy " position (Wyckoff position 4b 1 2 ,0,0). Fourier maps also show some significant missing electron density between CaO 2 planes, where some F -insertion has been reported for Ruddlesden-Popper phases as well [START_REF] Nowroozi | Fluoride Ion Batteries -Past, Present, and Future[END_REF] , as this position is suitable for the accommodation of additional anions [START_REF] Aikens | The Synthesis and Structure of a New Oxide Fluoride, LaSrMnO 4 F, with Staged Fluorine Insertion[END_REF] The structural evolution of CaV 2 O 4 upon electrochemical oxidation using 0.45 M Ca(BF 4 ) 2 in EC:PC as electrolyte was followed by operando synchrotron XRD. Fig. 6 A shows the full patterns and expanded views of main diffraction peaks in regions I and II. Dark blue stripes in region II correspond to time in which diffraction data collection was unfortunately lost. The XRD patterns indicate gradual structural evolution of CaV 2 O 4 upon oxidation and further reduction. In agreement with results achieved ex situ , new peaks appear at 2 𝜃 = 13.26 0 and 14.34 0 growing progressively upon oxidation to the expense of those at 2 𝜃 = 13.34 0 and 14.11 0 , which gradually decrease in intensity indicating that a twophase redox process takes place. Due to limited beamtime, the cell was switched to reduction without the oxidation being complete. The new peaks were seen to gradually shift to lower angles upon reduction, which would be consistent with the formation of a solid solution.

The refinement of a diffraction pattern of fully oxidized CaV 2 O 4 achieved after a prolonged ex-situ oxidation is depicted in Fig. 6 B and is consistent with no major structural changes taking place during oxidation and the space group being Pbnm as for the pristine compound . In contrast to what is observed for Ca 2 MnO 3.5 , the same phase is observed to form upon oxidation with all electrolyte compositions tested (with Ca(BF 4 ) 2 , LiPF 6 or LiBoB as salts), which points at the charge compensation mechanism not involving fluoride ions for the case of CaV 2 O 4 . The refined values of the cell parameters for the oxidized phase are a = 10.72008(9) Å, b = 9.20213(2) Å and c = 2.89418(3) Å and the cell volume is 285.504(5) Å 3 . This represents an expansion of 0.05 Å of the a lattice parameter, a c lattice parameter contraction = 0.09 Å and a volume contraction of 2.73 % respect to the pristine CaV 2 O 4 . This value is lower than the 11% contraction estimated by DFT for full oxidation of vanadium and removal of all calcium ions, but the lattice parameter variations are consistent with the extraction of calcium from the crystal structure (see Fig. 6 C). The contraction of c likely results from the shrinking of VO 6 octahedra due to oxidation of vanadium. The expansion of the a lattice parameter is attributable to an increase of electrostatic repulsion between the anions as the shielding effect of Ca + 2 ions decreases upon calcium extraction. The refinement of calcium occupancies in the oxidized phase renders a calcium content of 0.69 mol, indicating that ca. 0.3 mols of Ca + 2 have been extracted from the crystal structure of CaV 2 O 4 . This value is consistent with the smaller contraction of the unit cell observed experimentally with respect to the DFT calculated value for V 2 O 4 . Fig. 6 D depicts the fraction of pristine CaV 2 O 4 and oxidized Ca x V 2 O 4 obtained by sequential refinement of operando XRD data (see some examples in the SI, Figure S13). The amount of Ca x V 2 O 4 is found to gradually increase upon oxidation to the expense of CaV 2 O 4 . Fig. 6 E depicts the evolution of the total Ca:V 2 O 4 ratio and the value of x in the Ca x V 2 O 4 oxidized phase, except for the initial patterns where its fraction is too low to obtain a reliable occupancy of Ca. Upon oxidation, a gradual decrease in the total amount of calcium is observed, while no changes are detected in x, in agreement with a two phase process taking place. Despite the current being switched upon reduction at t = 32h, changes in the trend of the patterns are not clearly visible until a few hours later, which is likely due to sluggish reaction kinetics, as denoted by the significant cell polarization. Upon reduction, no significant changes in the relative amounts of Ca x V 2 O 4 and CaV 2 O 4 can be detected. Within the sloping region above -1V a slow and gradual increase in the value of x is observed, which further continues when the low voltage plateau is reached (t = 47h), at a faster pace, with the shift of the (021)-(211) doublet at 14.42 o to lower angles being clearly visible. The cell expansion associated to this shift occurs without any apparent diminution of peak intensity, in full agreement with a solid solution mechanism taking place in this region instead of a two-phase mechanism to fully revert to the pristine phase. Overall these findings would suggest a different mechanism upon oxidation (two-phase) and reduction (single phase). While it cannot be excluded that the energy barrier to reform the pristine structure is too high, the behavior may be different depending on the current applied if kinetic factors play a role.

Electrochemical testing of CaV 2 O 4

The reversibility of the redox processes prompted us to carry out further electrochemical cycling tests on CaV 2 O 4 ( Fig. 7 ) in three electrode cells. To avoid excessive electrolyte degradation, capacity was limited between x = 0 and x = 0.25 in Ca 1-x V 2 O 4 (65 mAh/g). Interestingly, the potential vs capacity profiles depict a significant decrease in the polarization after the first cycle, as the second oxidation starts at a potential ca. 500 mV lower than that of the first oxidation. For subsequent cycles the oxidation profile exhibits three distinct regions, with the length of the second plateau increasing upon cycling. It can be speculated that this could be related to enhanced active material utilization or to different reaction pathways ( i.e. solid solution vs . two phase), but further experiments are needed to confirm these hypotheses. The reduction profile involves mainly a gradual decrease of the potential followed by a plateau at ca. -0.5 V vs Ag QRE. Experiments involving a longer oxidation step (between x = 0 and x = 0.60 (155mAh/g)), carried out at 50°C to enhance kinetics, yielded significantly larger capacities while keeping rather similar electrochemical responses. The most relevant difference between the profiles depicted in Fig. 7 A and B is the clearer observation of diversesteps upon reduction for the latter, likely as a result of the oxidation proceeding to a larger extent. Overall, these preliminary experiments indicate that the redox mechanism deserves further investigation, including tests at different rates and temperatures, possibly involving also further operando studies.

Conclusion

DFT calculations have enabled to identify Ca 2 MnO 3.5 and CaV 2 O 4 as potential positive electrode materials for calcium cells, and also provided some clues to sketch possible redox mechanisms. After synthesis of such compounds and complementary to electrochemical measurements, operando XRD has been critical to probe the charge compensation mechanism and reversibility of the redox reactions taking place.

Ca 2 MnO 3.5 is an oxygen deficient compound (Mn is five-fold coordination) obtained by reduction of the Ruddlesden-Popper phase Ca 2 MnO 4 (Mn in octahedral sites), which has been shown to oxidize in calcium cells, concomitant to a change in the crystal structure and the oxidation of manganese, as detected by EELS. Operando experiments enable to follow the oxidation process, which involves the formation of a new phase with no changes in the calcium stoichiometry. Fourier difference maps enable to detect the presence of additional fluoride ions in the crystal structure, which are generated through electrolyte decomposition of the electrolyte salt. The charge compensation mechanism for manganese oxidation is thus found to involve fluoride intercalation and not calcium extraction. This is fully consistent with DFT results disclosing the trend of Mn 3 + polyhedra in Ca 2 MnO 3.5 to regain the octahedral coordination upon Ca deintercalation and also with the high calculated energy diffusion barriers for calcium (above 1 eV).

In contrast, CaV 2 O 4 exhibits an appealing energy barrier for Ca diffusion ( ∼ 0.6 eV) which notoriously increases upon calcium extraction to yield virtual-V 2 O 4 ( ∼ 1 eV). Experimentally, the extraction of 0.3 moles of calcium upon oxidation was demonstrated by operando diffraction studies, with the formation of a new isostructural phase which upon reduction is found to re-intercalate calcium via a solid solution process. While further studies are needed to fully elucidate the redox mechanism, the results presented herein confirm the viability of reversibly extracting and reintercalating calcium ions in the crystal structure and call for further investigation of CaV 2 O 4 as positive electrode material in calcium cells.

Ca 2

 2 MnO 3.5 is an oxygen-deficient compound derived from the first member of the Ruddlesden-Popper family Ca n + 1 Mn n O 3n + 1, (Ca 2 MnO 4 , K 2 NiF 4 -type). The introduction of oxygen vacancies in the structure of Ca 2 MnO 4 results in Mn 3 + ions in square pyramidal coordination. Hence, the structure of Ca 2 MnO 3.5 (space group Bbcm ) consists of square pyramidal MnO 5 polyhedra linked by corners in the ab plane, forming rings of six MnO 5 units themselves sharing corners to form [Mn 2 O 7 ] layers ( Figs. 1 A-1 C). Along the c-axis, in between two sheets of [Mn 2 O 7 ], there are two consecutive layers of calcium atoms which form a 8-fold coordination polyhedron, with Ca-O distances 2.3 Å x 2, 2.5 Å x 2, 2.6 Å x 2 and 3 Å x 2. Fig. 1 D shows the calculated energy barriers of Ca migration in a 2 × 1 × 1 supercell of Ca 2 MnO 3.5 (Ca 31 Mn 16 O 56 composition). Ca diffusion in the ab plane can involve different pathways, as shown in Fig. 1 B.

Fig. 1 .

 1 Fig. 1. Crystal structure of Ca 2 MnO 3.5 (A). Investigated pathways for Ca diffusion in a 2 × 1 × 1 supercell. The initial Ca site is denoted by a dark-blue ball, and the final Ca sites by stripped balls. The numbers indicate the calculated energy migration barriers (in eV) (B,C). Calculated energy barriers for Ca diffusion in a Ca 2 MnO 3.5 (D). View on the ab plane of the structures of the pristine material Ca 2 MnO 3.5 and the deintercalated Ca 1.5 MnO 3.5 (E). Color code: Ca blue, Mn brown, O red.

Fig. 2 .

 2 Fig. 2. Crystal structure of CaV 2 O 4 and pathway for Ca diffusion (A). Calculated energy barriers for Ca diffusion in a Ca x V 2 O 4 (B). Color code: Ca blue, V purple, O red.

Fig. 3 .

 3 Fig. 3. Voltage vs capacity profiles for CaV 2 O 4 //AC cells (magenta curve) and Ca 2 MnO 3.5 //AC cells (green curve), using 0.45 M Ca(BF 4 ) 2 in EC:PC as electrolyte (A) and corresponding ex situ XRD patterns at the end of oxidation (B). The patterns corresponding to pristine CaV 2 O 4 and Ca 2 MnO 3.5 are also displayed for comparison. Expanded regions of the patterns are depicted in (C) and (D) so that the differences can be more clearly seen.

  B), this is to say, too high to expect good Ca mobility. The energy barrier increase respective to CaV 2 O 4 is related to the more constrained pathway for Ca diffusion in the lower volume cell of V 2 O 4 . Indeed, in the CaFe 2 O 4 -type compounds the GGA + U calculated migration barriers increase with the lower cell volume: CaV 2 O 4 0. 71 eV, CaFe 2 O 4 0.75 eV, CaCr 2 O 4 0.87 eV and CaCo 2 O 4 1.03 eV (see Figure S2). In addition, enhanced electrostatic repulsions between the Ca ions and the more oxidized V + 4 ions in V 2 O 4 might play a role in the energy barrier increase.

Fig. 4 .

 4 Fig. 4. Characteristic voltage versus time profile for a Ca 2 MnO 3.5 // AC cell using 0.45M Ca(BF 4 ) 2 in EC:PC as electrolyte and corresponding operando synchrotron XRD patterns (A). Calculated and observed XRD patterns for Ca 2 MnO 3.5 electrodes after oxidation in LP30 electrolyte (B). Structural model for Ca 2 MnO 3.5 F 0.6 (C) used for the calculated pattern.

  . The final refinement performed in the orthorhombic Bbcm space group resulted in cell parameter values of a = 5.2891(1) Å, b = 10.5509(2) Å and c = 12.1422(1) Å and a cell volume of 677.59(2) Å 3 . It is worth mentioning that the electron density in 4 b position of Bbcm space group is split, which could indicate that the true symmetry of the phase is lower or that some degree of F -disorder exists, but no further reliable conclusions could be extracted from the available data. Refinement of F occupancies rendered a final stoichiometry Ca 2 MnO 3.5 F 0.6 which would indicate a Mn oxidation state of about + 3.6, consistent with the larger volume with respect to Ca 2 MnO 4 , and with fluoride ions being present not only in the initial vacant anionic sites but also between CaO 2 planes.

Fig. 5 .

 5 Fig. 5. Characteristic HAADF-STEM images of pristine Ca 2 MnO 3.5 (A). ( EELS spectra of pristine Ca 2 MnO 3.5 (in red), and oxidized crystallites with EDS deduced ratios of Ca:Mn:F of 58.0:31.4:10.6 (in blue) and 60.1:33.6:6.3 (in back). In the oxidized crystallites, the respective Mn oxidation states are + 3.5 (blue spectra) and + 3.3 (black spectra). Note that the formation of a Ruddlesden-Popper-like phase via fluoride insertion in Ca 2 MnO 3.5 would yield the composition Ca 2 MnO 3.5 F 0.5 , i.e. Ca:Mn:F ratio of 55.4: 38.0: 6.6 and Mn in a formal oxidation state of + 3.5 (B)

Fig. 6 .

 6 Fig. 6. Characteristic voltage versus time profile for a CaV 2 O 4 // AC cell using 0.45M Ca(BF 4 ) 2 in EC:PC as electrolyte, and corresponding operando synchrotron XRD patterns (A). Calculated and observed patterns for CaV 2 O 4 electrodes after full oxidation ex-situ in LP30 electrolyte (B). Structural model ofor Ca 0.7 V 2 O 4 (C). Phase fraction (D), calcium occupancies (note that x in Ca x V 2 O 4 equals 2Ca Occ ), and the total amount of Ca (considering the two coexisting phases) (E) versus time obtained by sequential Rietveld refinement of operando XRD patterns.

Fig. 7 .

 7 Fig. 7. Characteristic capacity vs. potential profiles for CaV 2 O 4 // AC cells using 0.45M Ca(BF 4 ) 2 in EC:PC as electrolyte tested with capacity limitation at room temperature ( A ) and at 50°C ( B ) , respectively. The capacity values are also expressed as calculated x in Ca x V 2 O 4 (top axis), considering that these are exclusively related to calcium extraction from the crystal structure. Oxidation and reduction steps are denoted in magenta and red respectively.

  MnO 4 was obtained by calcination of the precursor at 1000°C for 24 h and used to achieve Ca 2 MnO 3.5 under NH 3 flow (Carburos Metálicos 99.9%) after optimizing synthesis conditions (see Section 3 b).CaV 2 O 4 was synthesized according to the method reported by Hastings et al .[START_REF] Hastings | Magnetic Structure of Calcium Vanadite[END_REF] . The CaV 2 O 6 precursor was prepared by solid state reaction between CaCO 3 and V 2 O 5 (650°C, 12 hours, air atmosphere) and treated under H 2 gas at 700°C for 4 hours to yield pure CaV 2 O 4 .

	mixture. Finally, polycrystalline Ca 2
	.
	Mn(NO 3 ) 2 ٠ 9H 2 O (Aldrich 99%), Ca(NO 3 ) 2 ٠ 4H 2 O (Aldrich 99.95%),
	C 6 H 8 O 7 (citric acid, Aldrich 99.5%) and C 2 H 6 O 2 (ethylene glycol) were
	subsequently dissolved in water in a molar ratio of Ca: Mn: C 6 H 8 O 7 :
	C 2 H 6 O 2 = 2: 1: 3: 1. The solution was kept under continuous stirring at
	80°C until complete evaporation. The resulting citrate complexes were
	decomposed in air at 500°C for 6 h to yield an amorphous carbonate

Acknowledgements

This research was funded by the European Union's Horizon 2020 research and innovation programme H2020 FETOPEN-1-2016-2017 (CARBAT, grant agreement no. 766617). The authors are grateful for access to the computational facilities from Universidad de Oviedo (MALTA-Consolider cluster) and to ALBA synchrotron for beamtime (proposal 2021024935). ICMAB-CSIC members thank the Spanish Agencia Estatal de Investigación Severo Ochoa Programme for Centres of Excellence in R&D (CEX2019-000917-S).

Declaration of Competing Interest

None.

CRediT authorship contribution statement

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.ensm.2022.02.017 .


Sophie Greillier

Laurent Daniel

Catherine Caillaud

Bertrand Dussol

Guy Touchard

Jean-Michel Goujon

Noémie Jourde-Chiche

Mickaël Bobot
email: mickael.bobot@gmail.com

First phenotypic description of a female patient with c.610 T > C variant of GLA: a renal-predominant presentation of Fabry disease

Keywords: Fabry disease, Female, Phenotype, GLA variant, lysoGb3, Renal involvement

Background: Fabry disease (FD) is an X-linked lysosomal storage disorder due to deficient alpha-galactosidase activity leading to intracellular glycosphingolipid accumulation. Multiple variants have been reported in the GLA gene coding for alpha-galactosidase, and the question of the pathogenicity of rare variants needs to be addressed, especially in patients with mild phenotypes. Case presentation: The patient, a 37-year-old female, presented with a persistent proteinuria after an otherwise uncomplicated first pregnancy. Renal biopsy showed both mild mesangial IgA deposits, and a striking vacuolization of podocytes and tubular cells consistent with Fabry disease. On electron microscopy, discrete but characteristic pseudo-myelinic lamellar inclusions were observed in the podocytes' lysosomes. A more detailed physical examination revealed an angiokeratoma, and medical history ancient acroparesthesia. There was no cardiac or cerebral involvement of Fabry disease on magnetic resonance imaging. While blood enzymatic activity of alpha-ga lactosidase was normal in this patient, lysoGb3 was elevated (3 N), and a rare heterozygous variant called c.610 T > C was documented in GLA gene. The patient was treated with an ACE inhibitor, with a rapid decrease in proteinuria. After a 5-year follow-up, her renal function has remained normal, with mild proteinuria, and normal cardiac echography. Conclusions: We report and phenotypically describe the first case of a Fabry disease female patient carrying the GLA c.610 T > C variant associated with a renal-predominant clinical presentation.

Background

Fabry disease (FD) is a rare systemic storage disorder resulting from a deficiency of lysosomal alpha-galactosidase A, which leads to the accumulation of sphingolipids (mainly globotriaosylceramide, Gb3) in endothelial cells and various tissues. The enzymatic deficiency is linked to pathogenic variants in the GLA gene, located on the X chromosome. The most frequently affected organs are the heart, kidneys, skin, gut and central nervous system [START_REF] Eng | Fabry disease: guidelines for the evaluation and management of multi-organ system involvement[END_REF].

In the "classical" form of FD, typically affecting hemizygous male patients, the first symptoms appear in childhood and associate acroparesthesia, fever, abdominal pain and arthralgia. The cardiac, neurologic and renal manifestations generally appear between the fourth and sixth decade with potentially serious consequences: hypertrophic cardiomyopathy leading to heart failure, end-stage renal disease, and strokes with severe neurological sequelae [START_REF] Mehta | Fabry disease defined: baseline clinical manifestations of 366 patients in the Fabry outcome survey[END_REF].

The severity of clinical presentation is usually correlated with the depth of enzyme deficiency (alpha-galactosidase activity, AGAL-A) [START_REF] Germain | Fabry's disease (alpha-galactosidase-a deficiency): physiopathology, clinical signs, and genetic aspects[END_REF], and symptoms in females, who are heterozygous, are usually less pronounced than in males [START_REF] Echevarria | X-chromosome inactivation in female patients with Fabry disease[END_REF]. However, the random silencing of one X chromosome in each cell of female carriers, called "X-chromosome inactivation" or "lyonization", leads to the sole expression of the genes from the other X chromosome. The proportion of cells in which the normal X chromosome is silenced is not always 50% (a phenomenon called "X-inactivation skewing"), and can vary among the different organs, which results in the various levels of enzymatic deficiency and the broad range of phenotypic patterns observed in females with FD [START_REF] Lyon | Gene action in the X-chromosome of the mouse (Mus musculus L.)[END_REF][START_REF] Sagie | Mary LYON (1925-2014) and the random inactivation of chromosome x[END_REF].

Moreover, several GLA variants are associated with residual enzyme activity in vitro, without typical symptoms of FD ("non-classical" FD) [START_REF] Smid | Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance[END_REF], and many recently identified GLA variants have undetermined clinical significance [START_REF] Sagie | Mary LYON (1925-2014) and the random inactivation of chromosome x[END_REF].

Here we describe the first case of a female patient with a rare GLA variant associated with a mild FD phenotype comprising proteinuria, acroparesthesia and angiokeratoma. We detail the renal pathological lesions observed, and discuss the pathogenicity of this GLA variant.

Case presentation

A 37-year-old female, native of Romania, was referred to our centre for evaluation of persistent albuminuria (1 g per day) after an otherwise uncomplicated first pregnancy, with normal kidney function (serum creatinine 65 μmol/L, estimated glomerular filtration rate (eGFR) 94 mL/min/1.73 m 2 with the CKD-Epi formula).

Kidney biopsy showed mild mesangial IgA deposits (+) in immunofluorescence, without mesangial proliferation, as well as extensive vacuolisation of the podocytes and tubular epithelial cells, suggestive of FD [START_REF] Schiffmann | Screening, diagnosis, and management of patients with Fabry disease: conclusions from a 'kidney disease: improving global outcomes' (KDIGO) controversies conference[END_REF]. In electron microscopy, cytoplasmic vacuoles were observed in the podocytes and tubules, with discrete pseudomyelinic figures (Fig. 1).

The patient's medical history was as follows: chronic gastritis, chronic hand pain, a posteriori characterized as neuropathic (acroparesthesia). A detailed skin examination revealed an angiokeratoma confirmed by biopsy. There was no obvious family history of kidney disease or FD, but the family could not be thoroughly explored.

Further investigations did not reveal any other organ involvement of FD: normal sudation, normal ophthalmologic examination, normal cardiac explorations (ECG, echocardiography, cardiac magnetic resonance imaging (MRI)), and normal cerebral MRI.

There was no leukocyte enzyme deficiency (AGAL-A was 70% of female normal controls, reference range: 104-236 nmol/h/mg protein), but an elevated level of lyso-Globotriaosylsphingosine (lyso-Gb3) was detected by tandem mass spectrometry on dried blood (10 ng/mL, normal 0-3.5).

Sanger sequencing based on the analysis of the seven GLA exons including exon-intron junctions revealed a heterozygous variant within exon 4 of the GLA gene (reference sequence: NM_000169.2): c.610 T > C or p.(Trp204Arg) (Supplementary Figure 1). This variant can be considered as likely pathogenic (class 4) according to the ACMG variant classification: PS3 (functional studies performed by Lukas et al.) [START_REF] Lukas | Functional and clinical consequences of novel α-Galactosidase a mutations in Fabry disease[END_REF], PM2 (absent in population databases), PP3 (multiple lines of computational evidence supporting a deleterious effect). Xinactivation study was not performed (not routinely done in our country).

Since the clinical presentation was not severe, with normal enzyme level and proteinuria rapidly decreasing with an angiotensin converting enzyme inhibitor, the patient was offered two therapeutic options: early enzyme replacement therapy (ERT), or regular monitoring. She chose monitoring.

After a 5-year follow-up, the patient's kidney function remained normal (serum creatinine 70 μmol/L, eGFR 87 ml/min/1.73m 2 ) with normal blood pressure and mild proteinuria (urinary protein/creatinine ratio 0.3 g/g). Annual echocardiography shows no myocardial hypertrophy. The patient's 5-year-old son, who is asymptomatic, without proteinuria, has not yet been tested for enzyme activity or genetic explorations and is monitored by paediatricians.

Discussion and conclusions

We report here the case of a female patient with a rare GLA variant (c.610 T > C) with elevated lysoGb3 despite a normal AGAL-A activity, sphingolipid accumulation in podocytes and tubular epithelial cells, angiokeratoma and neuropathic pain. According to FD definition criteria [START_REF] Smid | Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance[END_REF][START_REF] Van Der Tol | A systematic review on screening for Fabry disease: prevalence of individuals with genetic variants of unknown significance[END_REF], this patient suffers from a classical form of FD, with typical symptoms (neuropathic pains, angiokeratoma) and a documented variant of the GLA gene. However, this heterozygous variant has never been described in a female patient before.

The same variant was reported only once, in a series of new GLA variants, in a male hemizygous patient [START_REF] Lukas | Functional and clinical consequences of novel α-Galactosidase a mutations in Fabry disease[END_REF]. This patient was a carrier of the same c.610 T > C variant, with elevated blood levels of lyso-Gb3, but unlike our patient, no AGAL-A activity was detected in vitro. No clinical data were available for this case. In addition to the 0% enzymatic activity, the deleterious effect of the c.610 T > C variant is supported by predictive tools such as Poly Phen-2 (probably damaging, score 1) or SIFT (not tolerated) [START_REF] Lukas | Functional and clinical consequences of novel α-Galactosidase a mutations in Fabry disease[END_REF][START_REF] Adzhubei | A method and server for predicting damaging missense mutations[END_REF]. This variant has not been reported in the GnomAD database, but 3 occurrences can be found in ClinVar: one without clinical information, and two related to the case discussed above (Lukas et al. 2014) [START_REF] Lukas | Functional and clinical consequences of novel α-Galactosidase a mutations in Fabry disease[END_REF]. This is therefore the first c.610 T > C variant with a detailed phenotypic description, and the first case in a female patient.

The discrepancy observed in our female patient between the normal leucocyte enzyme activity in vitro and the elevated lyso-Gb3 with localized manifestations of FD is possibly related to a skewed X-chromosome inactivation (lyonization) [START_REF] Lyon | Gene action in the X-chromosome of the mouse (Mus musculus L.)[END_REF], with a patchy AGAL-A defect and glycosphingolipid accumulation within tissues [START_REF] Echevarria | X-chromosome inactivation in female patients with Fabry disease[END_REF].

Since the broader screening for non-classical FD in patients with late symptomatology or single organ involvement (cardiac, renal, cerebrovascular variants of FD) [START_REF] Doheny | Fabry disease: prevalence of affected males and heterozygotes with pathogenic GLA mutations identified by screening renal, cardiac and stroke clinics, 1995-2017[END_REF], many GLA variants of unknown significance have been reported [START_REF] Lukas | Functional and clinical consequences of novel α-Galactosidase a mutations in Fabry disease[END_REF]. These variants can be associated with classical FD, attenuated or non-classical FD, or absence of disease [START_REF] Smid | Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance[END_REF][START_REF] Van Der Tol | A systematic review on screening for Fabry disease: prevalence of individuals with genetic variants of unknown significance[END_REF]. In this context, and in asymptomatic female patients with normal AGAL-A [START_REF] Desnick | Fabry disease, an under-recognized multisystemic disorder: expert recommendations for diagnosis, management, and enzyme replacement therapy[END_REF] the lyso-Gb3 assay is of particular interest [START_REF] Smid | Plasma globotriaosylsphingosine in relation to phenotypes of Fabry disease[END_REF]: elevated lyso-Gb3 is highly suggestive of classical FD if a new variant is discovered without typical FD symptoms [START_REF] Niemann | Gene mutations versus clinically relevant phenotypes: lyso-Gb3 defines Fabry disease[END_REF].

In this respect, the renal involvement of this patient is both highly suggestive of FD (vacuolisation with pseudomyelinic bodies) and atypical (additional IgA mesangial deposits). The patient had never been treated with medications associated with pseudomyelinic inclusions (such as antimalarials) [START_REF] Linthorst | Chloroquine-induced phospholipidosis of the kidney mimicking Fabry's disease[END_REF][START_REF] Costa | Curvilinear bodies in hydroxychloroquine-induced renal phospholipidosis resembling Fabry disease[END_REF][START_REF] Serre | Hydroxychloroquine-induced podocytopathy mimicking Fabry disease[END_REF][START_REF] Smid | Diagnostic dilemmas in Fabry disease: a case series study on GLA mutations of unknown clinical significance[END_REF] or with lyso-Gb3 elevation (such as amiodarone) [START_REF] Smid | Diagnostic dilemmas in Fabry disease: a case series study on GLA mutations of unknown clinical significance[END_REF].

In this form of FD, with mild symptoms and normal AGAL-A, the benefit of enzyme replacement therapy (ERT) remains uncertain. ERT significantly reduces plasma lyso-Gb3 [START_REF] Schiffmann | Screening, diagnosis, and management of patients with Fabry disease: conclusions from a 'kidney disease: improving global outcomes' (KDIGO) controversies conference[END_REF] and pain scores [START_REF] Hoffmann | Nature and prevalence of pain in Fabry disease and its response to enzyme replacement therapy--a retrospective analysis from the Fabry outcome survey[END_REF]. A task force of the American College of Medical Genetics suggests that ERT should be initiated when organ damage is evident and at the discretion of the clinician in asymptomatic hemizygotes, considering family history [START_REF] Wang | ACMG work group on diagnostic confirmation of Lysosomal storage diseases. Lysosomal storage diseases: diagnostic confirmation and management of presymptomatic individuals[END_REF]. Hopkin et al. recommended initiating ERT in symptomatic patients of any age or sex, including patients with evidence of tissue damage on kidney biopsy; the decision is left to the clinician for asymptomatic patients [START_REF] Hopkin | The management and treatment of children with Fabry disease: a United Statesbased perspective[END_REF]. The European Consensus distinguishes between male and female patients and between classical and non-classical forms of the disease: for males with classical FD, ERT should be initiated from the age of 16 years; males with non-classical FD and females with classical FD should be treated at the onset of clinical symptoms; ERT may be considered in females with non-classical FD and clinical signs [START_REF] Biegstraaten | Recommendations for initiation and cessation of enzyme replacement therapy in patients with Fabry disease: the European Fabry working group consensus document[END_REF]. However early treatment of patients with no or mild symptoms remains controversial [START_REF] Smid | Uncertain diagnosis of Fabry disease: consensus recommendation on diagnosis in adults with left ventricular hypertrophy and genetic variants of unknown significance[END_REF].

Another treatment option in FD is the administration of a pharmacological chaperone, such as oral migalastat, to reduce the degradation of misfolded alpha-galactosidase and promote its trafficking from the endoplasmic reticulum to the lysosomes, in order to increase its residual lysosomal activity [START_REF] Mendre | Pharmacological chaperones: a potential therapeutic treatment for conformational diseases[END_REF]. However, not all mutated enzymes are migalastat-amenable, since the mutated enzyme must have retained its catalytic activity despite abnormal folding [START_REF] Mccafferty | Migalastat: a review in Fabry disease[END_REF]. According to predictive models described elsewhere [START_REF] Lukas | Functional and clinical consequences of novel α-Galactosidase a mutations in Fabry disease[END_REF][START_REF] Andreotti | Therapy of Fabry disease with pharmacological chaperones: from in silico predictions to in vitro tests[END_REF], no benefits were expected from the pharmacological chaperone with the current variant.

A follow-up strategy was therefore proposed and chosen for this patient, with no worsening of the renal phenotype and no new organ damage after 5 years. This is still insufficient to conclude on the long-term risk of worsening chronic kidney disease or extra-renal damage, and longterm annual follow-up is planned for this patient.

In conclusion, we described the first c.610 T > C GLA variant with a detailed phenotypic description of a renalpredominant presentation of Fabry disease, and the first case in a female patient.

FIG. 1

 1 FIG. 1 a: Enlarged podocytes with bubbly cytoplasm associated with involvement of few distal tubular cells (arrowheads). Masson's trichrome, × 200. b: Massive vacuolization of podocytes without endocapillary alterations. Jones silver staining, × 400. Inset: electron microscopic. Vacuoles are empty or contain few simplified pseudo-myelin strips without zebra bodies

Acknowledgements

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Funding

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Supplementary information

Supplementary information accompanies this paper at https://doi.org/10. 1186/s12881-020-01071-5.

Additional file 1.

Abbreviations AGAL-A: Alpha-galactosidase activity; eGFR: Estimated glomerular filtration rate; ERT: Enzyme replacement therapy; FD: Fabry disease; Gb3: Globotriaosylceramide; MRI: Magnetic resonance imaging Authors' contributions SG: Draft of the manuscript. NJC, MB: Supervision. LD: Figs. MB: Formatting of the manuscript. LD, CC, BD, GT, JMG, NJC, MB: Critical revision. NJC was in charge of the patient. SG, BD, NJC, MB: Clinical data. CC: Genetic analysis. LD: Renal pathology (optical microscopy and immunofluorescence). GT, JMG: Renal pathology (electron microscopy). All authors read and approved this manuscript.

Availability of data and materials

The datasets generated and analysed during the current study and the raw sequence data obtained as a result of Sanger sequencing (Supplementary Figure 1 Ethics approval and consent to participate Not applicable.

Consent for publication

The patient gave her written consent for the publication of this case report.

Competing interests NJC received accommodation during a scientific congress funded by Genzyme®. BD has served as a consultant and received honoraria from Amicus Therapeutics®. The other authors declare that they have no competing interests.

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