Ali Mouhamad

Benedicte Ahmad

Jerzy Prelot

Zajac

J Zajac

Calorimetric screening of co-operative effects in adsorption of Co(II) on γ-alumina surface in the presence of Co-complexing anions in aqueous solution

Keywords: -alumina, cobalt adsorption, organic ligands, isothermal titration calorimetry, co-operative effects

Calorimetric screening of co-operative effects in adsorption of Co(II) on γ-alumina surface in the presence of Co-complexing anions in aqueous solution

Introduction

Alumina-supported cobalt catalysts, such as Co(II)/-Al2O3, offer promising perspectives for mitigation of nitrogen oxides, oxidation of volatile organic compounds, hydrodesulfurization reactions, or Fischer-Tropsch production of clean fuels while reducing energy inputs and CO2 emissions [START_REF] Maunula | Reaction mechanism and kinetics of NOx reduction by propene on CoOx/alumina catalysts in lean conditions[END_REF][START_REF] Luo | Catalyst characterization and activity of Ag-Mn, Ag-Co and Ag-Ce composite oxides for oxidation of volatile organic compounds[END_REF][START_REF] Topsøe | Progress in the Design of Hydrotreating Catalysts Based on Fundamental Molecular Insight[END_REF][START_REF] Bechara | Catalytic properties of Co/Al2O3 system for hydrocarbon synthesis[END_REF][START_REF] Dry | Practical and theoretical aspects of the catalytic Fischer-Tropsch process[END_REF][START_REF] Adesina | Hydrocarbon synthesis via Fischer-Tropsch reaction: travails and triumphs[END_REF][START_REF] Hu | Application of Fischer-Tropsch Synthesis in Biomass to Liquid Conversion[END_REF]. It is commonly admitted that the catalytic activity chiefly depends on metal-support interactions as a function of such factors as the concentration and dispersion of cobalt species, surface chemistry of the support, and the type of functionalization method or thermal treatment applied. One notable example is the impregnation of alumina with small amounts of Co(II) nitrate solution, followed by a thermal treatment in air and subsequent reduction in hydrogen. The addition of oxygenated organic additives is probably the most effective way to control and modulate the deposition of the cobalt species, thereby enhancing the catalytic performance of Co(II)/-Al2O3 catalysts [START_REF] Jean-Marie | Influence of substoichiometric sorbitol addition modes on the structure and catalytic performance of aluminasupported cobalt Fischer-Tropsch catalysts[END_REF][START_REF] Girardon | Cobalt dispersion, reducibility, and surface sites in promoted silica-supported Fischer-Tropsch catalysts[END_REF][START_REF] Boily | Experimental study of cadmium-citrate co-adsorption onto α-Al2O3[END_REF][START_REF] Davis | Complexation of trace metals by adsorbed natural organic matter[END_REF][START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF].

In order to consider the potential impact that Co-complexing additives may have on the metal binding to the catalytic support, it is necessary to study the retention of cobalt under the conditions similar to those used during the supported catalyst preparation. In the previous paper [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF], Co(II) retention on -Al2O3 in the presence of acetate, citrate, tricarballylate, and nitrilotriacetate anions was quantified through measurements of the isotherms of individual adsorption of the components in bi-solute systems under the unadjusted pH condition. To mimic the usual preparation procedures, cobalt and additive species were adsorbed simultaneously from equimolar aqueous solutions (N-ligand-Co systems) or the adsorption of cobalt from a single-component solution was preceded by pre-loading -alumina with an aqueous solution containing only the additive (S-ligand-Co systems); sample drying was also carried out as an intermediate step (D-ligand-Co systems). In all cases, both the co-operative and competitive adsorption of the ligand and cobalt were evidenced on the basis of a comparison among the experimental adsorption isotherms, in accordance with the observed small changes in the pH of the equilibrium solid-liquid suspension and the results of potentiometric titrations. In accordance with published literature on the subject [START_REF] Schindler | Co-adsorption of metal ions and organic ligands; formation of ternary surface complexes[END_REF][START_REF] Fein | The effects of ternary surface complexes on the adsorption of metal cations and organic acids on mineral surfaces[END_REF], the formation of ternary surface complexes of type A (i.e., solid-metal-ligand) or type B (i.e., solid-ligand-metal) was considered to describe the co-operative adsorption at least in a qualitative manner.

The objective of the present study was to employ the isothermal titration calorimetry (ITC) to quantify the enthalpy effects accompanying the co-operative adsorption from aqueous solutions containing equimolar quantities of Co(II) cations and acetate or citrate anions. The resulting calorimetry data were intended to be used as a basis for shedding more light on the nature of interactions involved in the formation of surface complexes. In order to attain this objective, the calorimetry operating procedures were adjusted accordingly to reproduce, at best, the experimental conditions under which the previous adsorption study was made [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. Among others, it was necessary to develop a new data processing procedure allowing the formation of a cobalt-ligand complex in the bulk solution to be taken into account. The analysis of the calorimetric data was based on the adsorption isotherms measured previously for the N-ligand-Co systems. The research strategy applied here relied on a comparison between the cumulative enthalpy of displacement accompanying adsorption from aqueous solutions of cobalt cations and acetate or citrate anions and the analogous quantities obtained when each solute was retained on the solid surface from a single-solute solution.

Interpreting the heat quantities measured upon adsorption from ionic solutions at an electrified solid-liquid interface is never an easy task since the total thermal effect is composed of several hardly separable contributions which may be exothermic or endothermic, often depending on the nature of the adsorbate-adsorbent couple, surface coverage ratio, or pH of the solid suspension [START_REF] Prelot | What are the main contributions to the total enthalpy of displacement accompanying the adsorption of some multivalent metals at the silica-electrolyte interface?[END_REF][START_REF] Prelot | Effect of nanoscale pore space confinement on cadmium adsorption from aqueous solution onto ordered mesoporous silica: a combined adsorption and flow calorimetry study[END_REF][START_REF] Prelot | Contribution of calorimetry to the understanding of competitive adsorption of calcium, strontium, barium, and cadmium onto 4A type zeolite from two-metal aqueous solutions[END_REF][START_REF] Lanas | Efficient fluoride adsorption by mesoporous hierarchical alumina microspheres[END_REF]. This usually includes the heat effects related to: (i) adsorption of solute molecules or ions, as well as co-adsorption of counter-ions, (ii) desorption of solvent molecules, other molecules or ions pre-adsorbed at the solid-liquid interface, (iii) changes in the structure of the vicinal water (e.g., surface dewetting), (iv) changes in the hydration layers of the adsorbing and desorbing species, (v) changes in the electrical charge and potential of the solid surface. It is possible to conclude correctly on the affinity between a given solute and the solid surface if the related contribution is dominant over the others or/and the other contributions are constant over the adsorption range studied. This is argued to be true for the systems studied here and used as a key prerequisite for further analysis of the experimental enthalpy of displacement.

Materials and Methods

Materials

Gamma-alumina (-Al2O3) was prepared by calcination, in air at 500 °C for 4 h, of Pural SB3 boehmite supplied by Sasol Germany Ltd. The powdered sample had a BET specific surface area of 212 ± 11 m 2 g -1 [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. Cobalt(II) nitrate hexahydrate (purity  98%), ammonium acetate (purity  98%), and ammonium citrate tribasic (purity  97%) were purchased from Sigma-Aldrich. All chemicals were used without further purification. MilliQ water (18.2 M cm) was used to prepare aqueous solutions.

Calorimetric measurements of the enthalpy of displacement

The individual adsorption isotherms for cobalt and additive species from single-solute and bi-solute aqueous solutions at 298 K were determined previously under the unadjusted pH condition [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. In the present paper, the corresponding enthalpy changes were monitored by means of a TAM III multi-channel calorimetric device described elsewhere [START_REF] Prelot | On the real performance of cation exchange resins in wastewater treatment under conditions of cation competition: the case of heavy metal pollution[END_REF].

Prior to each adsorption run, a single-solute (cobalt or anionic ligand) or bi-solute (cobalt with a given anionic ligand) stock solution was placed in the micro-syringe. This stock solution was prepared by dissolving appropriate salts in ultrapure water. Stock solutions corresponding to the following concentrations were used in adequate calorimetry runs: 30 mmol L -1 for cobalt, 80 mmol L -1 for acetate, and 50 mmol L -1 for citrate salts. In the case of bi-solute solutions, cobalt and anionic ligand were mixed together in equimolar proportions. The concentrations of the two solutes in the bi-solute stock solutions were as follows: 50 mmol L -1 for cobalt-citrate system and 30 mmol L -1 for the other. All these concentrations were adjusted accordingly in order to cover the range of low and moderate adsorption quantities where the individual adsorption isotherms represented monotonously increasing functions of the concentration (i.e., the competitive effects did not manifest themselves on the adsorption isotherms).

In a typical adsorption experiment, about 800 µL of deionized water and 10 mg of γ-Al2O3 were poured into a 1 ml glass ampoule serving as a calorimetric cell. After the attainment of the thermal equilibrium inside the calorimetric device at 298 K, successive injections of the 10-μL aliquots of a given stock solution during 10 sec were performed, thus resulting in the heat flows recorded with an equilibration time of 45 min between 2 injections. The data processing procedures resulting in plots of the cumulative enthalpy of displacement, ∆ 𝐻 , as a function of the total amount adsorbed in single-solute and bi-solute systems are detailed in Supplementary material (see sections S1 and S2).

Generally, special attention should be paid when determining the heat contribution due to the dilution phenomenon. In the present case of alumina-cobalt or alumina-ligand systems, the adsorption of metal or ligands ions was accompanied by the transfer of potential-determining H3O + and OH -ions between the solid-liquid interface and the supernatant solution, thereby causing progressive changes in the solution pH. Nevertheless, the observed pH changes were rather small in the adsorption range covered by the calorimetry experiments. In consequence, the thermal effects recorded upon dilution of cobalt or ligand solution in ultrapure water may be thus considered as a good estimate of the enthalpy of dilution.

The same isothermal titration calorimeter by TAM Instruments was used to determine the equilibrium dissociation constants for Co-ligand aqueous complexes. For this purpose, a Co(NO3)2ꞏ6H2O stock solution was placed in the syringe and about 800 µL of an aqueous NH4CH3CO2 or HOC(CO2NH4)(CH2CO2NH4)2 solution were introduced into the calorimetric cell. Successive injections of the 10-μL aliquots of the Co-containing stock solution resulted in the absorbed or released heat recorded in the form of peaks. These thermal peaks were further integrated to obtain the corresponding values of heat of injection.

Given the complexity of the data processing procedures applied in the present study, the measurement uncertainty was not determined on the basis of error propagation law. Instead, the uncertainty in measured adsorption and enthalpy quantities was assessed directly by duplicating the adsorption and calorimetry experiments [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF][START_REF] Prelot | On the real performance of cation exchange resins in wastewater treatment under conditions of cation competition: the case of heavy metal pollution[END_REF]. The repeatability-reproducibility was found to be within:

-5%, for the measurement of the amount adsorbed, -7%, for the determination of the dissociation constants, -10%, for the determination of the enthalpy of displacement.

Results and Discussion

The detailed analysis of the calorimetric results obtained with the aid of an Isothermal Titration Calorimetry (ITC) device will be separated in two subsections devoted, respectively, to single-solute and bi-solute systems. The principal motivation behind this separation is the difference in the data processing procedures applied in each case.

Enthalpy effects of displacement in single-solute systems

In principle, the calorimetry measurements do not involve too much difficulty and the cumulative enthalpy curves may be quite easily obtained based on the routine processing procedures detailed in Supplementary material (see section S1). The enthalpy change accompanying the adsorption from single-solute aqueous solution represents the displacement effect according to the competitive character of the phenomenon. In consequence, such a displacement may be exothermic or endothermic and the results of ITC measurements usually differ greatly from the isosteric heats of adsorption which are calculated from the temperature dependency of the individual adsorption isotherms [START_REF] Builes | Isosteric Heats of Gas and Liquid Adsorption[END_REF][START_REF] Anastopoulos | Are the thermodynamic parameters correctly estimated in liquid-phase adsorption phenomena?[END_REF][START_REF] Zafar | Kinetics and Thermodynamics Studies of Cobalt (II) Adsorption onto Alumina[END_REF]. A recent study of fluoride adsorption onto mesoporous hierarchical alumina microspheres provides new experimental arguments for the direct determination of the heat of adsorption by ITC against the van't Hoff procedure [START_REF] Lanas | Efficient fluoride adsorption by mesoporous hierarchical alumina microspheres[END_REF].

Figure 1 shows the plots of the cumulative enthalpy of displacement, ∆ 𝐻 , as a function of the relative solute adsorption onto -alumina from single-solute solutions. Note that the relative solute adsorption was calculated by dividing the adequate amount adsorbed by its maximum value measured in the concentration range studied, according to the data reported in the previous article [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. The relative adsorption scale has been chosen to draw all enthalpy curves on one graph with the same x-axis for comparison. Otherwise, the absolute adsorption ranges are quite different for various systems, namely up to: 0.092 mmol g -1 , cobalt; 0.017 mmol g -1 , acetates; 0.14 mmol g -1 , citrates. Moreover, the differential enthalpy values indicated in Fig. 1 have been inferred from the plots of ∆ 𝐻 against the absolute quantity of adsorption (expressed in mmol g -1 ).

In the case of cobalt cations and citrate anions, the displacement phenomenon appears exothermic (i.e., negative ∆ 𝐻 values) in the whole adsorption range. Even though the enthalpy of displacement cannot be practically decomposed into individual enthalpy terms, the most plausible explanation here is that the strong solute-surface interaction is dominant over all other contributions. Furthermore, the enthalpy curves take the form of straight lines and a change in the slope is observed for higher adsorption values, particularly visible for citrates.

The linearity of ∆ 𝐻 vs. adsorption plots gives a strong indication that the partial mechanisms provide rather constant contributions to the total enthalpy. Each slope value is related to the differential molar enthalpy of displacement, thus giving an idea about the intensity of solute-surface interactions [START_REF] Penn | Investigation of Atrazine Sorption to Biochar With Titration Calorimetry and Flow-Through Analysis: Implications for Design of Pollution-Control Structures[END_REF]. The method of least squares for fitting straight lines to experimental data has been employed and the resulting differential enthalpy values are as follows (together with the maximum measurement uncertainty): 1.5 0.2 kJ mol -1 and 12.4 1.9 kJ mol -1 for Co(II); 12.5 1.9 kJ mol -1 and 25.4 3.8 kJ mol -1 for citrates.

The exothermic character of the competitive adsorption of cobalt or citrate species is consistent with the hypothesis of the formation of inner-sphere complexes induced by strong specific interactions between Co(H2O)6 2+ or C6H5O7 3-ions and the surface of γ-Al2O3 [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. It is worth noting that the cumulative enthalpy of displacement is much more negative for citrate anions than that of cobalt cations. Since the pH conditions are not far from the point of zero charge of the solid sample, the sign and the density of the surface charge may vary according to the charge regulation mechanism. In consequence, the retention of both cations and anions on the surface is possible. Besides the difference in affinity for solid surface between the two types of ions, the proton release to the equilibrium aqueous phase accompanying the retention of cobalt cations or the proton uptake paralleling the adsorption of citrate anions obviously contribute, in an opposite manner, to the overall enthalpy effect. Indeed, for mineral oxides, the formation of negative surface charge by desorption of protons from surface hydroxyl groups is endothermic, whereas the surface protonation is exothermic [START_REF] Casey | Enthalpy Changes for Brønsted Acid-Base Reactions on Silica[END_REF]. Furthermore, partial dehydration of a cobalt cation upon adsorption is certainly more endothermic than that of a citrate anion, firstly, due to the presence of a hydrophobic moiety in the latter and, secondly, because cations are generally characterized by much higher enthalpies of hydration compared to anions [START_REF] Marcus | A simple empirical model describing the thermodynamics of hydration of ions of widely varying charges, sizes, and shapes[END_REF].

The change in the slope of the linear plot of ∆ 𝐻 against the relative solute adsorption occurs at a relative value of 0.48 (i.e., 0.086 mmol g -1 ) for Co(II) and 0.54 (i.e., 0.093 mmol g -1 ) for citrates. Beyond these limit values, the intensity of the exothermic character of the competitive adsorption greatly increases for both types of species. This may suggest the formation of multi-dentate surface complexes within the ionic double layer. According to the literature of the subject [START_REF] Bourikas | Adsorption of cobalt species on the interface, which is developed between aqueous solution and metal oxides used for the preparation of supported catalysts: a critical review[END_REF], divalent cobalt cations are capable of forming mono-and bidentate inner-sphere complexes with negatively charged surface hydroxyl groups, whereas trivalent citrate anions may be attached to the alumina surface by means of one or two carboxyl groups and a hydroxyl one.

The precise mode of ion binding to the surface has a very important impact on the eventual formation of ternary surface complexes in bi-solute systems. In view of further discussion, it should be mentioned here that the existence of bidentate cobalt and tridentate citrate complexes on the solid surface would diminish the bridging capacity of the adsorbate species and thus greatly reduce the occurrence of ternary surface complexes.

The case of monovalent acetate anion, C2H3O2 -, appears somewhat more complicated to rationalize. Firstly, the displacement phenomenon is endothermic in a relative adsorption range up to 0.31 (i.e., 0.013 mmol g -1 ). Here, the differential molar enthalpy of displacement is 8.7 1.3 kJ mol -1 . The observed endothermic character of the overall displacement phenomenon points out rather weak acetate binding to the surface γ-Al2O3, which is in line with the formation of outer-sphere surface complexes, as has been envisaged previously [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. It seems likely that the exothermic effect of hydrogen bonding of acetate anions at a greater distance of approach to the alumina surface is over-compensated by some endothermic phenomena, which also result in an increase in the entropy of the system. These endothermic phenomena may be related to: (i) the displacement of some other anionic species (e.g., hydroxide anions) from the interface so as to maintain the electroneutrality of the ionic double layer and (ii) the displacement of vicinal water necessary for the formation of a cavity to make space for an oncoming acetate anion. Additionally, each acetate moiety in outer-sphere complexes may potentially induce an endothermic deprotonation of a surface hydroxyl, since it has been proven to be capable of capturing a proton from the surface group to form an acid molecule [START_REF] Liu | Surface complexes of acetate on edge surfaces of 2:1 type phyllosilicate: Insights from density functional theory calculation[END_REF].

Then, the slope of the linear plot surprisingly changes from positive to negative with the concomitant change in the differential molar enthalpy of displacement from 8.7 1.3 to 23.8 3.6 kJ mol -1 . The hypothesis of adsorption via inner-sphere complexation becomes plausible here, especially in the light of the conclusions drawn from density functional theory calculation of surface complexation of acetate on aluminosilicate surfaces: in addition to the covalent bonding of one carboxylate oxygen to a surface aluminium atom, the other oxygen atom may either form another covalent bond with the same Al atom or interact via hydrogenbonding with a surface OH group attached to this surface atom [START_REF] Liu | Surface complexes of acetate on edge surfaces of 2:1 type phyllosilicate: Insights from density functional theory calculation[END_REF].

Enthalpy effects of displacement in bi-solute systems

In the case of adsorption from bi-solute solution onto -alumina, the data processing becomes more difficult on account of the occurrence of Co-ligand complexes already in the aqueous phase before it enters into contact with the solid surface. Therefore, the enthalpy effects of dilution for any aqueous solution containing equimolar quantities of Co(II) and ligand species may also include a contribution coming from the dilution of these complexes. The adequate Coligand speciation diagrams simulated with a Medusa modelling software (e.g., see Supplementary Material section in the previous article [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]) have revealed the presence of the following predominant species in the pH range applied for each Co-ligand pair: Co(H2O)6 2+ but also Co(CH3COO) + to a much smaller extent (up to 30%) for the cobalt-acetate systems, Co(C6H5O7) -for the cobalt-citrate systems. As a consequence of this complexation in the bi-solute systems, the stock solution placed in the micro-syringe, the solution collected in the calorimetric cell after each injection during the dilution experiment, or the supernatant solution remaining in equilibrium with the currently adsorbed species onto solid particles during the adsorption experiment may contain some quantity of Co-ligand complex in addition to free cobalt or ligand species.

The knowledge of dissociation constants (Kd) in the systems studied is thus necessary to extract the concentration of free and complex species from the overall concentrations of cobalt and ligand in various solutions. On the basis of the partial concentrations of free and complex species, it was possible to determine the corresponding dilution effects contributing to the total enthalpy change recorded in each displacement experiment with the use of a bi-solute solution (see section S2

in Supplementary material). The cumulative enthalpies of displacement corrected for dilution effects have been collected in Fig. 3. They are plotted on the relative adsorption scale constructed by dividing the sum of the quantities of cobalt and ligand adsorption by its maximum value obtained in the concentration range studied [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. Since cobalt is a common component of the two bi-solute systems, the variations of ∆ 𝐻 recorded upon its adsorption from single-solute solution have been added to Fig. 3 for comparison purposes.

In order to rationalise these experimental results on more theoretical grounds, two alternative hypotheses are compared here: (1) independent adsorption of both components on the solid surface and (2) co-operative adsorption of the two components. To a first approximation, the assumption of the additivity of enthalpy contributions given individually by each solute component to the total displacement effect has been taken as a starting point for comparison. In consequence, the theoretical enthalpy of displacement in a bi-solute system may be calculated from the following expression:

∆ 𝐻 ∆ 𝑛 • ∆ ℎ ∆ 𝑛 • ∆ ℎ (1)
where ∆ 𝑛 and ∆ 𝑛 correspond to the quantities of individual adsorption for cobalt and ligand, respectively, onto -alumina in the bi-solute system (taken from the appropriate experimental curves), ∆ ℎ and ∆ ℎ denote the molar enthalpies of displacement accompanying the adsorption of each solute from bi-solute solutions. The latter may be approximated by the molar enthalpies of displacement measured in single-solute systems (i.e., the slopes of the various linear segments in Fig. 1) or they may be calculated from the comparison between the experimental enthalpy values and those obtained by imposing a particular mode of ligand and metal attachment to the surface of -alumina.

In the two bi-solute systems, the displacement is clearly an exothermic process in line with the exothermic effects accompanying the attachment of cobalt and citrate species from single solute solutions and contrary to the endothermic effects when acetates alone form outersphere complexes on the oxide surface. Within the framework of the hypothesis of independent adsorption, the enthalpy contribution due to the ligand, ∆ ℎ , should be thus negative for Eq. 1 to reproduce correctly the negative values of ∆ 𝐻 . Nevertheless, further data analysis has revealed that none of the combinations based on the slopes of the linear segments in Fig. 1 can reproduce well the experimental results in the whole adsorption range. Figure 4 illustrates the lack of concordance between ∆ 𝐻 and ∆ 𝐻 for the cobalt-citrate system in which the metal and ligand ions seem to adsorb in an independent manner only in the very beginning (at the most, the two first points on the enthalpy curve). The theoretical enthalpy values become far too negative for further points, (i.e., the exothermic character of the phenomenon is greatly overestimated).

The enthalpy modelling on the basis of the hypothesis of the co-operative adsorption has been more successful, as illustrated in Fig. 4. Indeed, the differences between the two types of enthalpy values are within experimental error for most experimental points. Here the theoretical enthalpy of displacement is obtained by imposing firstly a particular mode of attachment to the surface of -alumina via the metal (or ligand) and calculating its contribution to the experimental enthalpy of displacement as ∆ 𝑛 • ∆ ℎ (or ∆ 𝑛 • ∆ ℎ ), where ∆ ℎ (or ∆ ℎ ) is taken as corresponding to the first or the second linear segment of the enthalpy curve in Fig. 1. The procedure applied to determine the quantities of adsorption ∆ 𝑛 and ∆ 𝑛 from the individual adsorption isotherms in the bi-solute systems is detailed in Supplementary material (see section S2). Then an estimation of the molar enthalpy contribution for the other solute, i.e., ∆ ℎ or ∆ ℎ , is deduced directly from Eq.1 developed for ∆ 𝐻 determined experimentally. For example, when considering the direct metal attachment to the oxide surface as the starting hypothesis, one can obtain for the ligand enthalpy contribution in the bi-solute system:

∆ ℎ ∆ ∆ •∆ ∆ (2)
Nevertheless, the question arises as to whether these simplified models can explain the observed behaviour of each metal-ligand couple in an adequate way.

The case of cobalt-acetate couple is considered first. The best fitting model was related to the hypothesis of co-operative adsorption and it took into account the bridging role of the cobalt species directly attached to the solid surface. The theoretical metal contribution to the total enthalpy of displacement recorded in the cobalt-acetate system was fixed at a value equal to the differential enthalpy corresponding to the first linear segment of the enthalpy curve in Fig. 1, i.e., ∆ ℎ 1.5 kJ mol -1 . The fitting of Eq. 1 to the ∆ 𝐻 data resulted in a theoretical ligand contribution, ∆ ℎ , being equal to about 7.5 1.2 kJ mol -1 over the whole ∆ 𝑛 interval. Figure 4 shows that there is quite good concordance between the experimental ∆ 𝐻 and theoretical ∆ 𝐻 values.

It is worth noting that the stock solution used in the present calorimetry experiments has allowed the following maximum amounts adsorbed to be obtained: 0.086 mmol g -1 for Co (i.e., the end of the first linear segment of the enthalpy curve in Fig. 1) and 0.071 mmol g -1 for acetates. Within these adsorption ranges, the individual adsorption of the metal does not show much change compared to the single-solute system (see the inset graph in Fig. 3). Furthermore, the pH of the solid suspension is practically the same as that observed when only cobalt is present in the aqueous phase. Hence there should not be much difference in the cobalt interaction with the surface of -alumina between the two types of system and it is realistic to postulate the mono-dentate scheme of the metal attachment to the surface. On the other side, the individual adsorption of acetate anions is greatly enhanced compared to the single solute system. The ratio between the amounts in moles of cobalt and the ligand adsorbed on the oxide surface is on average close to 1.3 and it hardly changes over the adsorption range studied. All of this suggests that one ligand unit is bound preferentially to more than one adsorbed cobalt cations. Nevertheless, the negative value of ∆ ℎ is not fully consistent with the previously established image of a ternary surface complex of type A having a stoichiometry of 1:1 in which the ligand does not interact directly with the solid surface. The comparison with the two characteristic enthalpy values reported in Fig. 1 for acetate species alone in the aqueous phase gives an indication that acetate units interact additionally with some neighbouring surface sites.

Such an interaction should not be of electrostatic type and thus imply changes in the pH of the suspension. Already considered in the previous section, hydrogen bonding between acetate units and surface OH groups may be advanced in order to account for this effect.

The case of cobalt-citrate couple is different. First of all, the maximum quantities of adsorption attained in this system are equal to 0.333 and 0.235 mmol g -1 for cobalt and citrates, respectively. These adsorption ranges extend far beyond the maximum adsorption values recorded in the corresponding single-solute systems and thus the individual adsorption of both the metal and the ligand is greatly enhanced in the bi-solute system (e.g., see the adsorption curve for cobalt in the inset of Fig. 3). The best fitting models predict the direct attachment to the -Al2O3 surface for all citrate anions and, at least, 30% of the cobalt cations. The two solutes should thus interact independently with the appropriate surface sites in accordance with the local electric charge of the -alumina surface and this explains why the pH of the solid suspension lies somewhere between the values observed when only cobalt or citrate species are present in the aqueous phase [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF].

Up to the second point on the enthalpy curve (∆ 𝑛 0.99 mmol g -1 and ∆ 𝑛 0.077 mmol g -1 ), the metal and ligand ions are regarded as being adsorbed fully independently, with the differential molar enthalpies of displacement corresponding to the first linear segments in Fig. 1. Then, the best fitting model based on the hypothesis of cooperative adsorption foresees the formation of a ternary complex having a stoichiometry of 1:1 (i.e., one cobalt cation for each citrate anion). To construct this model, the theoretical molar enthalpy contribution due to citrate has been taken as being equal to 12.5 kJ mol -1 (i.e., the first linear segment in Fig. 1).

When applying Eq. 1, the individual enthalpy contribution ascribed to cobalt involved in these complexes is about 10.5 1.6 kJ mol -1 . In line with the endothermic displacement effects observed in this work for the formation of outer-sphere complexes in the single-solute systems, this molar enthalpy value at least suggests that the corresponding cobalt species are retained at a farer distance from the solid surface. Such particular modes of cobalt and ligand adsorption onto -alumina may be interpreted to be consistent with the formation of a ternary complex of type B in which citrate anions form the bidentate inner-sphere complexes, thereby providing negative sites for binding cobalt cations [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]. Given the stoichiometry of such a ternary complex, its formation represents an exothermic process with a total enthalpy balance of about -2 kJ mol -1 . Additional cobalt species (almost 0.1 mmol g -1 at the end of the adsorption range studied here) appear to bind to the surface by forming bidentate inner-sphere complexes. It is important to note that the enthalpy value imposed for this attachment (i.e., ∆ ℎ 12.4 kJ mol -1 ) is of the same order of magnitude as that of citrate anion alone and much more negative than that of the ternary complex. For this reason, it is understandable that some cobalt cations continue to adsorb on adequate binding sites even in the presence of citrate species.

Note that the formation of the bidentate inner-sphere complexes prevents the construction of solid-metal-ligand structures on the -alumina surface.

Conclusion

By applying the isothermal titration calorimetry (ITC) for a determination of the cumulative enthalpy of displacement, ∆ 𝐻 , as a function of the quantity of adsorption of cobalt cations and acetate or citrate anions onto -alumina from single-solute and equimolar bisolute solutions in water, a net progress in the description of the co-operative adsorption effects could be achieved. In the case of single-solute systems, the apparent linearity of the enthalpy plots in two successive intervals was firstly interpreted as an indication of the formation of inner-sphere (exothermic) and outer-sphere (endothermic) surface complexes. Based on the resulting differential enthalpy values, a simple modelling was proposed to rationalise the experimental results in the two bi-solute systems where the displacement appeared to be an exothermic process in the whole adsorption range studied. The mono-dentate scheme of the metal attachment to the surface in the cobalt-acetate system was considered to be hardly

Captions to Figures

Fig. 1 Variations of the cumulative enthalpy of displacement accompanying the individual adsorption of cobalt (circles) and anionic ligands onto -alumina from single-solute aqueous solutions at 298 K under the unadjusted pH condition: acetates (stars) and citrates (triangles). The numbers represent the differential molar enthalpy of displacement (in kJ mol -1 ) corresponding to each of the two linear parts (dashed lines) of the enthalpy curve; the non-zero values of y-intercepts are given in brackets. The inset shows the related individual adsorption isotherms published previously [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]; the dashed horizontal lines mark the upper limits of the adsorption range covered here by the enthalpy data for each system.

Fig. 2 ITC heat data corresponding to successive injections of a Co-containing stock solution into a ligand-containing aqueous solution placed in the calorimetric cell at 298 K; the solute concentrations, respectively, in the syringe and in the cell are as follows: 2 mol L -1 Co and 300 mmol L -1 actetate solutions (upper panel), 60 mmol L -1 Co and 5 mmol L -1 citrate solutions (lower panel). The two dissociation constants, 𝐾 , obtained by fitting the heat experimental values (filled circles) to the independent site binding model (solid lines) are indicated.

Fig. 3 Variations of the cumulative enthalpy of displacement accompanying the solute adsorption onto -alumina from single-solute and bi-solute aqueous solutions at 298 K under the unadjusted pH condition: cobalt alone (circles), cobalt-acetate (stars), and cobalt-citrate (triangles). The inset shows the individual isotherms of cobalt adsorption from single solute (circles) and bi-solute (stars and triangles) solutions [START_REF] Ahmad | The effect of chelating anions on the retention of Co(II) by γ-alumina from aqueous solutions under the unadjusted pH condition of supported catalyst preparation[END_REF]; the dashed horizontal lines mark the upper limits of the metal adsorption range covered here by the enthalpy data for each system.

Figure 2

 2 presents the results of additional ITC experiments carried out to determine the heat of successive injections of a Co-containing stock solution into a ligand-containing aqueous solution placed in the calorimetric cell. Here the soobtained heats of injection are plotted as a function of the Co-to-ligand molar ratio. The experimental heat values were fitted to the independent site binding model by using NanoAnalyzeTM Software offered by TA Instruments, thus leading to the determination of the equilibrium dissociation constants for both types of Co-ligand complex. The best fitting curves are shown in Fig. 2 together with the resulting dissociation constants: 𝐾 4.2 0.3 10 mol L -1 for the Co-acetate system; 𝐾 2.7 0.2 10 mol L -1 for the Co-citrate system.

Fig. 4

 4 Fig.[START_REF] Bechara | Catalytic properties of Co/Al2O3 system for hydrocarbon synthesis[END_REF] Comparison between the experimental enthalpy values, ∆ 𝐻 , measured for cobaltacetate (stars) or cobalt-citrate (triangles) systems and the theoretical ones, ∆ 𝐻 , corresponding to the best results of enthalpy modelling on the basis of Eq. 1: independent adsorption model (circles connected by dash-dot lines drawn to guide the eye), co-operative adsorption model (circles connected by dash lines drawn to guide the eye). The uncertainty in the enthalpy measurement is represented by error bars. The enthalpy values shown inside the panel indicate the differential molar enthalpy of displacement (in kJ mol -1 ) for ligand or cobalt imposed or estimated in the numerical calculations.

Acknowledgment

The authors greatly acknowledge the financial support of this work by the ANR SLIMCAT project 'Solid-Liquid Interface at Molecular-scale for Catalysis' (ANR-14-CE08-0019).

perturbed in comparison with that observed when the metal was retained from the single-solute solution. This provided the positively charged binding sites for the ligand units which additionally interacted with some electrically neutral binding sites on the -alumina surface. In the case of cobalt-citrate system, the metal and the ligand units were postulated to adsorb independently in the range of low adsorption values. Then the two solutes likely formed the ternary surface complexes having a stoichiometry of 1:1, with only the ligand units being attached directly to the oxide surface. Other cobalt cations were envisaged to form bidentate inner-sphere complexes.

The picture above constitutes a much more detailed description of the phenomenon in the range of low and moderate quantities of adsorption compared to that gained previously from the data analysis, relying mostly on the adsorption isotherms, pH changes, and potentiometric titration curves.

CRediT Author Statement

Mouhamad Ali Ahmad: investigation, data curation. Benedicte Prelot: conceptualization, project administration, methodology, supervision. Jerzy Zajac: methodology, visualization, writing-reviewing, editing.

Supplementary Material:

The online version of this article contains supplementary data, which are available to authorized users.


Haruna Abdullahi

Ka-Lun Wong

Keat Gin

Lim

Hussein Awala

Aurélie Vicente

Tau Chuan Ling

Svetlana Mintova

Eng-Poh Ng
email: epng@usm.my

Effects of Various Alkali Metal Cations on the Synthesis, Crystallization and Catalytic Properties of NKX-2 Aluminophosphites

Keywords: Alkali metal cation containing NKX-2, Organotemplate-free synthesis, Aluminophosphite, Crystal growth, Cyanoethylation reaction 1

Alkali metal-containing NKX-2 (M-NKX-2) aluminophosphite crystals were hydrothermally synthesized in the Al2O3-P2O5-Me2O-H2O system for the first time using various alkali metal hydroxides (MOH: M = Li, Na, K, Cs). The results showed that NKX-2 crystalline phase could be obtained within several hours without using harmful organic amine structure-directing agent.

In addition, the alkali metal hydroxides played an important role in controlling the crystallization rate and the morphology of M-NKX-2 particles in aqueous medium. The resulting M-NKX-2 solids possessed basicity which was catalytically active in base-catalyzed cyanoethylation reaction of methanol.

Introduction

Zeotype materials with aluminophosphate (AlPO-n) and silicoaluminophosphate (SAPO-n) are a class of microporous zeolite analogues widely used in adsorption, separation and catalysis [START_REF] Ahmad | Modification of gas selective SAPO zeolites using imidazolium ionic liquid to develop polysulfone mixed matrix membrane for CO2 gas separation[END_REF][START_REF] Wang | Synthesis, characterization, and catalytic application of hierarchical SAPO-34 zeolite with threedimensionally ordered mesoporous-imprinted structure[END_REF][START_REF] Tosheva | AlPO4-18 Seed Layers and Films by Secondary Growth[END_REF]. Typically, they are crystallized under hydrothermal conditions with water in a sealed autoclave under autogeneous pressure [START_REF] Khoo | Ionothermal approach for synthesizing AlPO-5 with hexagonal thin-plate morphology influenced by various parameters at ambient pressure[END_REF]. The synthesis of AlPO-n materials is also realized by using other methods such as solvothermal synthesis [START_REF] Jiao | Solvothermal synthesis and characterization of TiAPO-41[END_REF], ionothermal approach [START_REF] Azim | Ionothermal synthesis of crystalline microporous aluminophosphates: Systematic study on the conditions affecting the framework type[END_REF], dry-gel conversion [START_REF] Tao | Confined-space synthesis of hierarchical MgAPO-11 molecular sieves with good hydroisomerization performance[END_REF], microwave-assisted synthesis [START_REF] Ng | Microwave synthesis of colloidal stable AlPO-5 nanocrystals with high water adsorption capacity and unique morphology[END_REF], etc. [START_REF] Zhao | Solventless synthesis of AEL-type aluminophosphate molecular sieve from mechanochemically pretreated low-templated reactants[END_REF]. Usually, amines or quaternary ammonium salts are added to direct the formation of a particular porous framework structure [START_REF] Wang | Fabrication of BEA/MFI zeolite nanocomposites by confined space synthesis[END_REF][START_REF] Chen | Synthesis of ZSM-23 zeolite with dual structure directing agents for hydroisomerization of n-hexadecane[END_REF][START_REF] Wang | A facile route to encapsulate ultrasmall Ni clusters within the pore channels of AlPO-5[END_REF]. So far, there are more than 40 AlPO-n based porous frameworks that have been identified [START_REF]IZA-SC Database of zeolite structures[END_REF].

In general, phosphoric acid (H3PO4) is employed as the phosphorus source. The use of phosphorous acid (H3PO3) as an alternative phosphorus source has recently been reported, offering a new synthesis route of aluminophosphate materials. In this new synthesis process, NKX-2 which is an aluminophosphite, is formed as an important intermediate for synthesizing many useful microporous aluminophosphates (SAPO-46, AlPO-47, AlPO-41, SAPO-31, AlPO-11, AlPO-5 and AlPO-CJ2) [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF][START_REF] Li | Synthesizing large single crystals of aluminophosphate molecular sieves from a novel route[END_REF]. NKX-2 is an aluminophosphite with one-dimensional microporous channels. It was first prepared and reported in 2006 by Li et al. [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF]. As like AlPO-n materials, NKX-2 aluminophosphite is formed with the aid of amines or ionic liquids as structural directing agents (SDAs) and H3PO3 as the phosphorus source [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF][START_REF] Shi | Ionothermal synthesis of aluminophosphite NKX-2[END_REF]. Recently, we have reported the use of NaOH as an inorganic base to crystallize the NKX-2 particles without the addition of any harmful and expensive organic SDA [START_REF] Gopal | Organotemplate-free hydrothermal synthesis of NaNKX-2 aluminophosphite base catalyst[END_REF]. The resulting sodium containing NKX-2, surprisingly, possesses basicity and can be used as a solid base catalyst. In addition, a change in the crystallization profiles of NKX-2 is also observed in the SDA-free synthesis system due to the use of alkaline synthesis medium as compared to the weakly alkaline medium (with the presence of amines or ionic liquids) used in previous synthesis systems. It is shown that Na + ions have significant impact in the formation of NKX-2 crystalline phase, but the chemistry of alkali cations in the formation of NKX-2 is still not clear and hence, it is worth to be further explored.

In this work, NKX-2 aluminophosphite is synthesized in organotemplate-free Me2O-Al2O3-P2O5-H2O precursor system using various alkali metal hydroxides (MOH = LiOH, NaOH, KOH, CsOH) and H3PO3 as the mineralizing agents and the phosphorus source, respectively. The crystallization profiles and the morphological properties of the resulting M-NKX-2 such as crystallization rate, degree of crystallinity, morphology, crystallite size, elemental composition and crystallography structure are studied by microscopic and spectroscopic methods. Furthermore, the catalytic properties of the materials are also studied in the cyanoethylation reaction of methanol.

Experimental

Chemicals

NaOH (99%), KOH (85%), ethanol (99.9%) and acrylonitrile (99%) were purchased from Merck Chemicals GmbH. LiOH (98%) was purchased from Acros Organics while CsOH•H2O (≥99.5%), H3PO3 (98%) and aluminum isopropoxide (98%) were purchased from Sigma-Aldrich. All chemicals were used without additional purification.

Hydrothermal synthesis of M-NKX-2 crystals (M = Li, Na, K, Cs)

For the synthesis of Na-NKX-2, H3PO3 (0.744 g), aluminum isopropoxide (0.416 g) and distilled water (11.713 g) were mixed and stirred in order to obtain a clear solution. NaOH solution (1.520 g, 5 M) was then slowly added into the mixture. A cloudy solution with a molar composition of 1Al2O3:4.5P2O5:3.8Na2O:720H2O was obtained after the mixing process. The mixture was then transferred into a 50 mL Teflon-lined stainless steel autoclave before heating at 180 C at various times for crystallization. The autoclave was cooled down to room temperature. The solid product was filtered and washed until pH 7 before drying in an oven at 80 C overnight. Similar procedure was used to prepare Li-, K-and Cs-NKX-2, except the respective metal hydroxides were used instead of NaOH.

Characterization of solids

The crystallinity and purity of the M-NKX-2 powder samples were studied using a PANalytical X'Pert PRO diffractometer with a Cu Kα (λ = 1.5406 Å) radiation source. The XRD patterns were obtained in the 2θ range 5-40 with a step size of 0.02° and a scan speed of 0.2°/min. For identification, the powder XRD patterns were compared with the powder diffraction data (including the 2θ-values for CuKα radiation, d-spacings and relative intensities) reported in the reference [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF]. The degree of crystallinity was determined by evaluating the intensity of the two most intense XRD peaks at 2θ of 13.05 [010], 25.95 [111] and 29.12

[021], in comparison to a reference sample (Cs-NKX-2 synthesized for 2 h). The crystal structure determination was further confirmed by using Rietveld refinement via GSAS software.

The morphological characteristics and the average particle size distribution were evaluated by Scanning Electron Microscopy using a FESEM (Leo Supra 50VP) microscope with an accelerating voltage of 20 kV. The chemical composition of samples was measured by a Philips PW2404 XRF instrument. The elemental composition mapping of the solid samples was obtained using a FESEM (Leo Supra 50VP) equipped with an Oxford Instruments X-Max 80 mm 2 Solid State EDX detector. The infrared (IR) spectra of the solids were recorded with a Perkin Elmer spectrometer (System 2000) within the range of 4000-400 cm -1 using KBr pellet technique (KBr: sample weight ratio = 200:1). 27 Al and 31 P solid state magic angle spinning (MAS) NMR spectroscopy analyses were performed using a Bruker Ultrashield 500 spectrometer at MAS frequencies between 5 and 15 kHz. The 27 Al and 31 P MAS NMR spectra were recorded with 10000 scans by single-pulse excitation using π/12 (0.6 μs) and π/2 (3.0 μs) pulses, respectively. The basicity property of solid sample was determined by using back titration method [START_REF] Othman | Synthesis of mesoporous sodalite by mixed quaternary ammonium cation templates for knoevenagel condensation reaction[END_REF]. Typically, 0.200 g of dried solid sample was suspended in 10 mL of distilled water. The solution was magnetically stirred for 24 h before it was centrifuged (10000 rpm, 10 min). The separated solution was then mixed with 5 mL of 0.05 M HCl. The resulting solution was titrated with 0.02 M NaOH solution while phenolphthalein was used as the indicator. End point was noted when the colorless solution turned to pink. The readings were performed in triplicate for each sample.

Catalytic study

Cyanoethylation reaction of methanol was performed as follows: M-NKX-2 solid (0.20 g) was first dehydrated at 100 C for 3 h. The catalyst was then loaded into a 15 mL Teflon lined autoclave containing ethanol (76 mmol, 99%, Merck) and acrylonitrile (19 mmol, 99%, Merck). The autoclave was capped, sealed and heated at 150 C under magnetically stirring (400 rpm). The products were withdrawn after the reaction and analyzed using a GC (Hewlett-Packard 5880) and GC-MS (Perkin-Elmer GC-IR 2000 system) equipped with a HP-5 capillary column where n-nonane was used as an internal standard.

Results and discussion

Synthesis of M-NKX-2 (M = Li, Na, K, Cs)

The [210]/[120], which are consistent to those that have been reported in the reference, indicating the formation of pure NKX-2 crystalline phase [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF]. In addition, all samples experience different degrees of peak broadening due to the effect of different crystallite size [START_REF] Burton | Powder diffraction in zeolite science -an introductory guide[END_REF]. Thus, the results suggest that Li-NKX-2 aluminophosphite has the smallest crystals as it exhibits the broadest XRD peaks. This effect, nevertheless, is becoming less significant for Na-, K-and Cs-NKX-2. From the XRD analysis, the crystallite size of M-NKX-2 increases following the order Li > Na > K > Cs.

The M-NKX-2 is isostructure to the reported NKX-2 aluminophosphite according to the crystallography study [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF] (Fig. 3, Table 1). The structures do not expand significantly on the additional of alkali cations into the NKX-2 structure. Instead, the M-NKX-2 structures are more compact than that of NKX-2 structure, leading to a slight decrease in the structure volume.

Previous study has reported that the pure NKX-2 contains a three-linkage O atom linked by two adjacent AlO6 octahedrals and a distorted HPO3 2-tetrahedral where Al-O-Al linkages, which are forbidden based on the Lowensteins' rule, are formed [START_REF] Busca | Acidity and basicity of zeolites: A fundamental approach[END_REF]. In contrast, all four M-NKX-2 solids reported in this study follow the Lowenstein distribution. Six AlO6 octahedrals are linked to six HPO3 2-tetrahedrals to form 1D 12-MR channel system of NKX-2. In addition, the alkali cations are counter-balanced, (Al-O-P-O)2 -M + , at the electron-rich 4-numbered ring pockets located near the spacious 12-MR channel along the c-axis (inset of Fig. 3).

Alkali metal cations are also found to have profound effect on the particle morphology.

As seen from the SEM micrographs, the use of weak base LiOH results in the formation of Li-NKX-2 crystals with novel acicular (needle-shape) morphology (Fig. 4a). The length of the crystals range from 7.9 to 13.1 μm and some of them are intergrown. The core of Li-NKX-2 crystal is revealed in Fig. 4b, showing that the micron-sized Li-NKX-2 crystals are made up of smaller primary crystals with a size of ca. 175 nm. This result is in agreement with the XRD peak broadening observation for this sample. The morphology changes dramatically to bird nest-like shape for Na-NKX-2 (Fig. 4c &d). As can be seen, the secondary crystal size reduces to ca. 8  3.5 μm 2 . Long needle-shaped primary Na-NKX-2 nanocrystals can be observed growing on the surface of the bulk crystals. For K-NKX-2, these primary crystals (1.3  0.4 μm 2 ) intergrow and stack in a layer by layer fashion, producing the secondary crystals with grain shape and a size of 9  5.2 μm 2 (Fig. 4e &f). When the alkali hydroxide is replaced with CsOH, Cs-NKX-2 crystals of grain shape (10.5  6.7 μm 2 ) are produced (Fig. 4g &h). The surface of the crystals is smooth and no primary small crystals can be observed, thus agreeing well with the XRD results.

FTIR spectroscopy study has been performed to study the M-NKX-2 porous framework and the effect of the presence of different alkaline cations in the framework (Fig. 5). In general, all four fully crystalline M-NKX-2 samples show almost similar IR pattern as that of pure NKX-2 aluminophosphite [START_REF] Gopal | Organotemplate-free hydrothermal synthesis of NaNKX-2 aluminophosphite base catalyst[END_REF]. The broad band at 3458 cm -1 is due to the O-H stretching from water. The bands at 2481 and 1039 cm -1 are assigned to the P-H stretching and deformation modes of HPO3 2-, respectively [START_REF] Azim | Ionothermal synthesis of crystalline microporous aluminophosphates: Systematic study on the conditions affecting the framework type[END_REF]. The IR bands resonate at 1229 and 1139 cm -1 are attributed to the T-O (T = Al, P) asymmetric stretching vibration [START_REF] Ng | Synthesis of colloidal stable Linde Type J (LTJ) zeolite nanocrystals from rice husk silica and their catalytic performance in Knoevenagel reaction[END_REF]. Meanwhile, four characteristic bands at 594, 560, 517 and 445 cm -1 , which are due to the T-O-T vibrations, are also detected confirming the formation of the framework structure of NKX-2 [START_REF] Azim | Ionothermal synthesis of crystalline microporous aluminophosphates: Systematic study on the conditions affecting the framework type[END_REF].

The chemical environment of M-NKX-2 has also been studied using 27 Al and 31 P MAS NMR spectroscopy. For 27 Al MAS NMR spectra, two broad bands with a shoulder band are observed for all M-NKX-2 samples (Fig. 6A). These signals can be deconvoluted into three symmetry peaks, located at ca. 8.0 ppm,-7.0 ppm and -14.8 ppm. The first weak peak at ca. [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF]. On the other hand, only one nonsymmetry 31 P MAS NMR peak is shown by M-NKX-2 (Fig. 6B). This peak can be deconvoluted into two peaks. The strong band at ca. -14.0 ppm is assigned to the P(H)(M)(OAlOct)2 tetrahedral species while the peak at ca. -6.4 ppm corresponds to the lower condensation degree of phosphorus species, which is in agreement with the IR spectroscopy analysis. These results confirm the existence of the P-H bond in M-NKX-2 solids [START_REF] Sayari | Synthesis of Mesostructured Lamellar Aluminophosphates Using Supramolecular Templates[END_REF][START_REF] Luan | Characterization of Aluminophosphate-Based Tubular Mesoporous Molecular Sieves[END_REF].

The chemical compositions of the M-NKX-2 samples have been investigated by XRF spectroscopy. No significant difference on the P/Al/M molar ratios are observed when different alkali metal hydroxides are used to prepare M-NKX-2 aluminophosphites (Table 2). The M-NKX-2 solids have a P/Al ratio of ~1.55, which is slightly higher than that of the pure NKX-2 [START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF]. This suggests that the M-NKX-2 aluminophosphites contain excessive P sites that contribute to surface negative charge. The created surface charge is then counter-balanced by the extraframework alkali metal cations.

The elemental distribution of M-NKX-2 has been studied by EDX elemental mapping (Fig. 7). As can be seen, P, O and Al elements, which are the basic building elements of NKX-2 framework structure, are found in the entire region. In addition, Na, K and Cs elements are also abundant and evenly distributed throughout the samples. No Li element is shown because it has very low energy of characteristic radiation that is not easy to be detected by EDX detector [START_REF] Muto | Detection of local chemical states of lithium and their spatial mapping by scanning transmission electron microscopy, electron energy-loss spectroscopy and hyperspectral image analysis[END_REF].

The XRF and EDX elemental analyses reveal that the M-NKX-2 might have basicity due to the presence of alkali metal extraframework cations. Hence, back titration experiment is used to evaluate the amount of basicity of the samples. The results show that Cs-NKX-2 possesses the highest amount of basicity (310 mol g -1 ), which is nearly 11 times more than Li-NKX-2 (28 mol g -1 ). On the other hand, K-NKX-2 has 226 mol g -1 of basicity which is higher than Na-NKX-2 (193 mol g -1 ). These titration results hence reveal that M-NKX-2, especially Cs-NKX-2 can be a potential solid base catalyst.

Tables

  crystallization of M-NKX-2 involves a two-step process, namely nucleation and crystal growth. The sigmoidal crystallization curves extracted from XRD data for Li-, Na-, Kand Cs-NKX-2 are shown in Fig.1. It can be seen that the induction period of Cs-NKX-2 is much shorter than that of the other three counterparts. For the P2O5-Al2O3-Li2O-H2O system, diffraction peaks that indicating crystal formation of Li-NKX-2 is only observed after 6 h of hydrothermal treatment; fully crystalline sample is obtained when the hydrothermal treatment time reached 8 h. In contrast, when the aluminophosphite is prepared using more alkaline CsOH, highly crystalline Cs-NKX-2 can be produced within 2 h. Hence, the results suggest that the basicity of the alkali hydroxide used does play an important role in accelerating the nucleation stage during the crystallization process (LiOH < NaOH < KOH < CsOH). The XRD patterns of fully crystalline M-NKX-2 solids are shown in Fig. 2. All samples exhibit XRD peaks at 2θ = 13.01 [010], 25.90 [111], 29.10 [021], 34.01 [112], 34.81

8. 0

 0 ppm is assigned to the pentahedral aluminium species due to Al(PO)4(OH2). The second peak resonated at ca. -7.0 ppm is attributed to the [Al2(HPO3)3] species, viz. the basic building units of M-NKX-2 crystalline materials. The third peak at ca. -14.8 ppm is due to the octahedral AlO6 species, which share three Al-O corners with three HPO3 2-tetrahedra and three Al-O corners with one adjacent AlO6 octahedron

Table 1 :

 1 Crystal data of pure NKX-2 and M-NKX-2 from Rietveld refinement.

		NKX-2 a	Li-NKX-2 Na-NKX-2 K-NKX-2 Cs-NKX-2
	Space group	P63/m	P63/m	P63/m	P63/m	P63/m
	a (Å)	7.8762(11) 7.8549(7)	7.8755(5)	7.8766(5) 7.86290(7)
	b (Å)	7.8762	7.8549	7.8755	7.8766	7.8629
	c (Å)	7.1237(14) 7.0936(7)	7.0994(5)	7.1005(4) 7.09166(7)
	α ( o )	90	90	90	90	90
	β ( o )	90	90	90	90	90
	γ ( o )	120	120	120	120	120
	Volume (Å 3 )	382.71	379.034	381.337	381.502	379.703

a Data from Ref.

[START_REF] Li | Capturing an Aluminophosphite Intermediate in the New Route of Synthesizing Zeolite-like Aluminophosphates[END_REF]

.

Table 2 .

 2 Properties of M-NKX-2 solid products.

			XRF spectroscopy analysis a			P/Al molar
	Samples	P2O5 (%) Al2O3 (%) M2O (%)	Others (%)	P/Al/M molar ratio	ratio
	Li-NKX-2	66.11	30.48	3.33	0.08	0.93/0.60/0.22	1.56
	Na-NKX-2	63.30	29.49	7.06	0.15	0.89/0.58/0.23	1.54
	K-NKX-2	60.52	28.46	10.88	0.14	0.85/0.56/0.23	1.53
	Cs-NKX-2	50.90	23.60	25.38	0.12	0.72/0.46/0.18	1.55

a in mass%

Acknowledgement

The financial support from FRGS (203/PKIMIA/6711495, 203/PKIMIA/6711461) and RUI (1001/PKIMIA/8011012) grants is gratefully acknowledged. H. Abdullahi also thanks Tertiary Education Trust Fund (TETFund) for a doctoral scholarship.

Catalytic behaviour of M-NKX-2

The catalytic behaviour of M-NKX-2 (M = Li, Na, K, Cs) has been tested on cyanoethylation reaction of methanol, under autogeneous condition and free of organic solvent.

No conversion is observed when the reaction is conducted in the absence of M-NKX-2 catalyst.

Hence, this indicates that cyanoethylation of methanol is an activated catalytic reaction. The catalytic study shows that the cyanoethylation reaction is more favorable when Cs-NKX-2 is used as a catalyst (Fig. 8). The conversion reaches 89.94% after 14 h of reaction with 100% selectivity to 3-ethoxy-propionitrile. The catalytic reactivity, however, decreases orderly when the catalyst is replaced with K-, Na-and Li-NKX-2. Only 69.22%, 58.86% and 13.97% of conversion are measured when K-, Na-and Li-NKX-2 are used, respectively. The results reveal that the catalytic performance follow the basicity of the cations present in the M-NKX-2:

Catalyst reusability is a major problem for solid catalysts in liquid phase reaction [25].

Therefore, Cs-NKX-2 has been chosen for the catalytic reusability test. The results show that Cs-NKX-2 is recyclable since the reactivity of the recovered solid can be preserved at least up to six consecutive runs (conversion ca. 86.57% and selectivity to 3-ethoxy-propionitrile remains 100%) (Fig. 9). A slight decrease in the catalytic conversion is observed after each cycle of reaction which can be due to the physical loss of Cs-NKX-2 during catalyst recovery by centrifugation. The results also suggest that the extraframework Cs + alkali cations are strongly bounded to the NKX-2 surface via electrostatic attraction force, and hence it remains catalytically active to the cyanoethylation reaction even after several reaction runs.

Conclusions

In conclusion, M-NKX-2 containing various inorganic cations (M = Li, Na, K and Cs)

have been successfully synthesized from organotemplate-free hydrogel systems. Compared to pure NKX-2, the M-NKX-2 can be crystallized within several hours without using aminic organic template. Furthermore, the crystallization kinetics of M-NKX-2 is found to be significantly affected by the type of metal hydroxide used; CsOH crystallizes Cs-NKX-2 within The Cs-NKX-2 catalyst can be a promising base catalyst in cyanoethylation reaction under autogenic pressure since the solid can be easily separated from the reaction media. The solid catalyst is recyclable with small loss of activity up to six cycles.

[

Graphical abstract

Effects of Various Alkali Metal Cations on the Synthesis, Crystallization and Catalytic Properties of NKX-2 Aluminophosphites

Research Highlights

• Alkali metal containing NKX-2 is synthesized in Al2O3-P2O5-Na2O-H2O system.

• The use of different metal hydroxides significantly alters the crystallization time.

• The morphological and crystallographic properties are also changed.

• The solids exhibit basic character and are active in cyanoethylation of methanol.


S Proa-Coronado

C Séverac

A Martinez-Rivas

E Dague
email: edague@laas.fr

Beyond the paradigm of nanomechanical measurements on cells using AFM: An automated methodology to rapidly analyse thousands of cells

Nanomechanical properties of cells could be considered as cellular biomarkers. The main method used to access the mechanical properties is based on nanoindentations measurements, performed with an operator manipulated Atomic Force Microscope (AFM) which is time-consuming, and expensive. This is one of the reasons preventing the transfer of AFM technology into clinical laboratories. In this paper we report a methodology which includes an algorithm (transferred to a script, executed on a commercial AFM) able to automatically move the tip onto a single cell and through several cells to record force curves combined with a smart strategy of cell immobilization. Cells are placed into microwells of a microstructured polydimethylsiloxane (PDMS) stamp. Inside a classical 100x100 µm 2 AFM field, 100 cells can be immobilized. In an optimal configuration we were able to measure, within 4 h, a population of 900 Candida albicans cells both native and caspofungin treated, which represents an unprecedented performance. We discovered that the population is heterogeneous and can be divided, on the basis of nanomechanical properties, into 2 subgroups.

Introduction

Medical doctors constantly have to face the issues of diagnostic, prognostic or evaluation of treatment efficiency. To tackle this question there is a constant need to develop and adapt new, more accurate and sensitive biomarkers, able to help in differential diagnostic or be predictive as early as possible of the disease evolution. In this aspect cell mechanical properties have the potential of being used as label free biomarkers for some pathologies 1 . Indeed, cell mechanical properties have the potential to address the diagnostic of cancer [1][2][3][4] as it has been reported that cancerous cells change their mechanical phenotype, presenting a lower Young modulus [5][6][7] and adhesion [7][8][9] than normal cells. Other authors have reported that cell mechanical properties are modified during proliferation 10 , by comparing their elastic modulus to differentiate normal cells from cancerous cells 11 or normal cells from cells treated for example with H 2 O 2 , N-ethylmaleimide and chymotrypsin 12 . In the field of cardiology, it is also known that erythrocytes interactions with fibrogen, as probed by AFM, are modified in ischemia and that red blood cells stiffness is altered 13 . As for the cardiology area for example cardiomyocytes are difficult to handle, and then works have reported the characterization of 1 to 30 cardiomyocytes [14][15][16][17][18][19][20] in about 6-8 h. which is a too small amount of cells to make a statistically relevant information in the context of human diseases and therefore will never be reliable enough for clinicians. Mechanical properties have also help understanding the effects of antimicrobial molecules on bacteria or yeast cell's walls 21 . Another example in the bacteria field is the work of Francius et.al 22 . They reported that S. aureus exposed to lysostaphin, presented a decrease in elasticity and stiffness of its cell membrane. Also, Feuillie et.al [START_REF] Feuillie | Proc. Natl. Acad. Sci[END_REF] reported in their paper that when treating bacteria with a peptide derived from β-neurexin, it blocks the surface protein SdrC which impacts on the cell adhesion. Formosa et.al [START_REF] Formosa | [END_REF] reported the increase of the cell wall elastic modulus and an overexpression of the adhesive protein Als1p when exposing Candida albicans to some incremental doses of caspofungin. Mechanical properties help understanding the microorganisms cell wall structure, their resistance mechanisms, and adhesion processes 25,26 .

Atomic Force Microscopy (AFM) 27 is the technique of reference to acquire mechanical properties of cells 28,29 . The classical procedure to obtain force curves from an AFM can be described by the following steps: first the tip is calibrated, then the sample is placed on the microscope stage, next a topographic image is acquired to determine the position of the cells, the tip is moved to the central region of each cell. AFM indentations at different locations of the cell are performed, and force curves are obtained and recorded. Finally, when all the cells are measured, the stage is moved so that new cells are brought into the AFM field of view. This cycle is performed manually, and its throughput is low (<1 cell/10 min.) 30 . Up to this time the technique is neither used in the pharmaceutical industry nor in the antimicrobial drug discovery process. AFM is perceived as a research tool and indeed, the works reported so far have one common factor, which is the limited number of cells analyzed by an AFM. This limited number prevents the analysis of a cell population and therefore prohibits statistically relevant general conclusions or decisions. To be able to transfer the AFM technology to hospitals or pharmaceutical industry a mandatory step is to achieve high throughput results in order to analyze cell populations rather than single cells 21,31 .

Wang et.al 32 developed an automated system which uses image processing to identify Raji cell locations so the AFM tip can move exactly above the cells and take measurements. The location and measurement of the cells are done within 3 s per cell, but their system had some requirements: the cells needed to be round shape (which is usually the sign of dying cells), as the algorithm could only recognize round shape cells confining the system to a specific cell geometry. Moreover, the cells substrate had to be completely flat and the agglomeration of cells were to be avoided, because the system did not withdraw the tip from the sample. Finally, the authors tested their system with 4 cells per scanning area, but they did not report the number of cells analyzed per hour. In another effort to develop AFM measurements on tissues Roy et.al 33 developed a system that used image processing to align the AFM probe with a tissue of interest, they were able to obtain in an area of 80 μm x 150 μm up to 480 force curves in ~80 min. Nevertheless, the aim of their semi-automated system consisted in analyzing changes in tissue architecture not being adapted for single cell analysis. Another approach reported by Favre et.al 34 , focused on maximizing the number of cells analyzed by AFM. They developed an array of cantilevers that are controlled by one AFM acquiring images from different regions of a sample at the same time. However, to apply this technology to a cell population the cantilever arrays should be fabricated with the same dimensions as the cell arrays. Another example was the parallelism of AFM which was reported by Sadeghian et.al 35 . Their tool involved the miniaturization of AFMs, they reported a maximum of 44 miniaturized AFM in an area of 450 mm (wafer like area). Each AFM was capable of working independently from the others having different kinds of analysis. The authors tested the system by obtaining topographical images of colloidal gold nanoparticles (10 nm in diameter) deposited on mica. However, the AFM heads distribution is very particular, and the cell array had to be adapted. Moreover, the regions of interest locations are determined manually, meaning that the human intervention is still predominant. Both parallel solutions did not consider a way to automatically bring new cells (or another cell array) inside the field of view of the AFM, consuming a vast amount of time.

Very recently Antoine Dujardin et.al 36 reported a solution where an automated procedure allows an AFM to obtain biomechanical analysis on prokaryotes. A python script was implemented in a Dimension Fast Scan-Bio AFM (Bruker, Santa Barbara, CA, USA), however this process takes considerable time to realign the photodetector and perform the engagement each time to finally analyze 501 areas in 8 h 35 min. They tested the system with fixed Yersinia pseudotuberculosis and living Mycobacterium bovis BCG bacteria. To identify the bioelements, a force volume image was performed in each well. This image was used to determine the bacteria positions (on the basis of their height) and the identified positions were imaged (2x2 µm 2 area). The reported images are height and Peak Force-error signals discarding the option to perform a mechanical analysis on the analyzed bacteria.

In the present work an original automated methodology, previously submitted as patent 31 to measure cellular mechanical properties is reported. Our methodology combines a purposely developed copyright algorithm [START_REF] Etienne | AUTOMATIP: Automation of Biophysical measurements on cells by Atomic Force Microscope (AFM)[END_REF] executed as a script on commercial AFMs with a smart strategy of cell immobilization (supp. Figure S2). The script automatically moves the tip from cell to cell to record force curves of each cell of a cell population. Cells are immobilized at known locations into microwells of a microfabricated PDMS stamp [START_REF] Formosa | [END_REF] . Once the tip has scanned all the cells of the scanning area, a motor stage moves automatically and brings a new cell array into the scanning area to re-initiate the methodology.

We tested this AFM based automated methodology on eukaryotes C. albicans because they are known as an opportunistic pathogenic yeast which represents one of the main hospitalacquired infections. We decided to compare native C. albicans cells with caspofungin treated C. albicans cells. Caspofungin is a last change antifungal drug form the echinocandin class, know to modify the yeast cell wall mechanical properties [START_REF] Formosa | [END_REF] . In our test, the automated methodology takes an average time of 12 seconds to perform 9 nanoindentations per cell and per microwell, giving a large number of force curves that is between 8,000 to 9,000 in 4 h, providing a method for high throughput measurements of a cell population. This automated process can be considered as the first step for a viable future diagnostic tool 31 . Thanks to this development we present for the first time the mechanical properties of a cell population (800-900 cells) measured by AFM pointing out that the mechanical properties within the cell population is not homogeneous and may explain conflicting results from literature.

Results and discussion

Figure 1 shows the execution of the program developed for this work (recommendations to initialize the algorithm: supp. figure S1 and description of the algorithm in supp. Figure S2 and supp. Figure S3), 1A and 1B shows how the cantilever moves from one scanning area to another. The centering algorithm takes ~40 s to be executed on each microwell. Figure 1C shows the displacement among the microwells in one scanning area, the movement is from the center of one microwell to the center of other microwell. The program takes ~12 s to finish the 9 indentations per microwell and ~13 min. per area, that is 64 wells per scanning area. Then Figure 1D shows the nanoindentation on different regions inside a microwell. This result can be seen in the video on the supp. data information. The force curves were obtained from C. albicans cells immobilized inside the microfabricated wells. Four experiments were conducted with the objective to establish the repeatability and reliability of the results. Native and caspofungin treated cells were independently prepared as mentioned in the materials & method section and immobilized the day they were used. The decision to use caspofungin is because its action on the yeast cell wall is still under debate [START_REF] Formosa | [END_REF]42 . The script was executed and 1021 cells were analyzed for the first experiment (native cells), 957 cells for the second experiment (native cells), 1000 cells for the third (caspofungin), and 574 cells for the fourth experiment (caspofungin). For the experiments 1 and 3, 16 indentations per cells were taken, meanwhile for experiments 2 and 4, 9 nanoindentations were taken.

Experiments 1, 2, 3, and 4 are independent duplicates. The cell cultures were independent and were not performed the same day. Four experiments were performed, two of them (experiments 1 and 2) with native cells and two (experiments 3 and 4) with caspofungin treated cells. The objective of this setup was to obtain a comparable number of analyzed cells (for native and treated) and to determine the maximum number of cells analyzed in a fixed time (4 h).

Following the previous criteria Table 1 presents the number of cells analyzed, the number of force curves discarded, and the time taken to analyze each well. The force curves obtained were analyzed using the JPK data processing software, based on the work published by El-Kirat-Chatel 42 we extracted the cell spring constant from all the force curves. However, the filling rate of the PDMS stamp is not 100 % (actually ~86 %). In the sup. data section dealing with force curves acquisition and analysis, the parameters used to exclude curves recorded out of the cells are described (supp. Figure S4). To filter the force curves the following criteria was implemented:

 The contact point is used to determine if the force curves are from the bottom of the well, so all the curves with a contact point value below 4.15 µm are discarded.  Curves with a negative slope are discarded.  We assumed that the cell spring constant should be lower than that of the PDMS measured at 150 pN/nm, hence we discarded all force curve giving a spring constant higher than 150 pN/nm.

albicans, native and treated with caspofungin. A and B (left) show the spring constant histograms for experiments 1 and 2 of native C. albicans cells (1021 and 959 cells analyzed respectively). While C and D (left) show the spring constant histograms for the experiments 3 and 4 of treated C. albicans cells (1018 and 579 cells analyzed respectively). A and B (right) show the results obtained from the adhesion analysis on experiments 1 and 2. C and D show the adhesion results for experiments 3 and 4. E and F

show the one-way ANOVA test performed using the spring constant and adhesion data from 1-3 and 2-4 respectively. *** = p value < 0.001, NS = no significant difference. Bin width was determined by Freedman-Diaconis rule.

Figure 2A-right shows that the adhesion force between the bare tip and native cells was 0.64 ±0.6 nN in the first experiment, while in the second experiment still on native cells, 2 subpopulations were found: the first has a mean adhesion force of 0.7±1.4 nN while the second is 4.5±1.5 nN. The treatment with caspofungin has no significant effect on the adhesion if experiment 1 and 3 are considered (one way ANOVA test, figure 2E-right shows no significant difference) but it seems that caspofungin induces a decrease in the adhesion to the tip and a reduction of the population adhesion heterogeneity.

Discussion

The protocol used to immobilize the cells is the one described by Formosa et. al [START_REF] Formosa | [END_REF] , nevertheless in this work a modification was made to go from ~50 % to ~85 % of microwells filled with cells (see materials & methods section). The number of nanoindentations were 16 for experiments one and three and 9 for experiments two and four. The objective of varying the number of nanoindentations was to observe if a significant change will be present in the histograms, and as can be seen the two subpopulations start to merge when we decrease the number of measurements, however, with nine indentations it is still possible to see the two subpopulations (Figure 2).

We extracted the spring constant from every force curve we obtain with the automated procedure. Based on the results published by El-Kirat-Chatel 42 a shift on the results for the treated cells with respect to the native cells was expected, these can be confirmed by looking at Figure 2 (A andC, B andD). On the contrary, the presence of the two peaks in the histograms that we observed in all four experiments were unexpected. Indeed, experiments performed on single cell [START_REF] Formosa | [END_REF]42) only demonstrated homogeneous distribution of the nanomechanical properties. The difference in the spring constant absolute value for the two independent experiments on native cells may come from uncontrollable differences in the cell cultures. Indeed, the maximum applied force, the tip velocity, the cantilever spring constant, the buffer, the temperature, etc were the same. It is important to note that C. albicans is actually an extremely versatile microbe 43 able to sense and adapt to its environment. As a consequence, the growth phases of C. albicans are difficult to control and an unmeasurable difference in the initial culture conditions may result through the butterfly effect to the difference that we observe in between experiment 1 and 3. A further interesting experiment would be to follow a cell culture in order to monitor it's evolution throughout the time. But, most importantly we reproduced, two times, the distribution of the cells into two distinct populations. Dague E. et.al 44 reported an heterogeneity on the young modulus of Saccharomyces cerevisiae cells analyzed in the same conditions. Nevertheless, the numbers were really low (5 cells) and it was therefore impossible to draw a general conclusion at the population scale. Sub-populations in C. albicans have been described since more than ten years in the context of biofilms. They are reported to be responsible of biofilm resistance to chelating agents 45 and to antifungal drugs like amphotericin B 46 . In this last publication, the authors demonstrated that the sub-population was associated to ergosterol and beta 1-6 glucan pathway genes. They both are important component of the fungal cell wall and we know from previous investigation of the team 47 that their expression level is correlated with the nanomechanical properties of the cells. More recently, Rosenberg et al. showed that antifungal tolerance was a sub-population effect 48 . It therefore seems that sub-populations in C. albicans are common. In our work there is no a particular reason responsible for the 2 sub-populations, and we must admit that we have no clue of its origin. Globally, we can hypothesize that sub-populations, in C. albicans are an adaptation mechanism probably responsible for the remarkable expansion of this microbe, usually being commensal but also opportunistic pathogen.

To our knowledge this work is the first that reports the use of AFM to analyze hundreds of cells which demonstrate that a microbe population is mechanically heterogeneous. Supp Figure S6. (A and B) show the presence of the two populations at all times during the experiment. This means that the sub-populations are not due to the alteration or modification of the cells during the experiments. The two populations exist from the beginning to the end of the experiments. We also wondered if the sub populations could be due to an artifact linked with the tip position above the cell/well. To eliminate this hypothesis, we examined the distribution of stiffness constants for each position of the tip. This analysis, presented in Figures S6C and S6D (supp. Figure S6.), shows that the spring constants are distributed according to the same sub-population distribution for each analysis point. There is, therefore, no one position category that contributes to one sub-population and another position category that contributes to the other sub-population. These subpopulations are really linked to differences in spring constant between cells and are really reflecting the biological reality, complexity, variability of a Candida albicans cell population.

To be sure we also calculated the average value of each cell and represented these values on a histogram (supp. Figure S5). These representations show 2 sub-populations, centered on the same values as those of the global distribution histograms.

Our strategy to achieve a few force curves (9 to 16) over a large number of cells marks a break with the traditional approach to mechanical measurements, by AFM, on living cells. Nevertheless, we compared our results with those in the literature to validate the approach. Indeed, if the measurement on hundreds of cells were theoretically to open the door to the observation of sub-population, the decrease in the number of force curves, per cell, may also have negative effects. El Kirat-Chatel et.al 42 performed 256 nanoindentations on single cell, they found a stiffness value of 51+/-9 pN/nm for cells not treated with caspofungin and 27+/-10 pN/nm for cells treated with caspofungin. Our results are of the same order of magnitude (spring constant ranging from 21+/-6 to 81+/-19 pN/nm for native cells) and we observe the same tendency to decrease the spring constant with caspofungin treatment (spring constant ranging from 13 to 52 pN/nm, in our experiments, for caspofungin treated cells).

In another paper by Formosa et.al [START_REF] Formosa | [END_REF] showed that Candida albicans cells treated with caspofungin became harder. Formosa results are based on the analysis of 1024 nanoindentations performed on a cell. This inconsistency could be explained if authors selected an untreated cell from the softest subpopulation and a treated cell from the hardest subpopulation. Having no means at that time to access the sub-populations, the 2 results were accurate but incomplete. These inconsistencies are numerous in the literature and have partly motivated our work.

The results of the adhesion measures are particularly interesting and highlight the limit with which our method could be confronted. Indeed, it is known that Candida albicans is able to express on its surface a large number of different adhesins, in variable quantities 43,49 . The conditions of expression of some of them are known but for example the conditions of amyloid aggregation described in [START_REF] Formosa | [END_REF] have not been demonstrated. It seems that it could be triggered by mechanical stimulation (force induced nanodomain Alsteens PNAS ALS5 [START_REF] Alsteens | Proc. Natl. Acad. Sci[END_REF] ) but this has not been demonstrated in Candida. Our adhesion results are different from one experiment to another, although the culture conditions were the same or at least we had the impression that they were the same, the usual microbiological techniques being implemented. This probably means that the expression of adhesins and or their organization on the cell surface in experiments 1 and 2 were different. Our method is not designed to analysis in high details single cells and therefore the detection of nanodomains is impossible. It means that the traditional approach is not antagonist with our new method and that they provide additional information. Moreover, the differences in spring constant between the nanodomains and the "normal" cell wall are not in the order of magnitude of the 2 sub-populations. Stiffer nanodomains are 13.4 +/-0.2 nN/µm when the rest of the cell wall is 12.4 +/-0.3 nN/µm [START_REF] Formosa | [END_REF] . In the present work we report a sub-population at 21+/-6 nN/µm and the second at 48 +/-9 nN/µm. It means that the difference due to the nanodomains are included in the error bar of our measurement.

Thus, and that is a potential new limit, our results would no longer be incomplete because they would not consider the heterogeneity of the cell population but incomplete because we would lack control over the biological sample produced. Candida albicans is known for its versatility 43 and in this context serves this demonstration better than any other cell model would have done.

Conclusions

An automated methodology for AFM force curves acquisition on cell population was successfully developed and implemented on a JPK Nanowizard II. The portability of the algorithm was tested on a JPK Nanowizard III. The results demonstrate that increasing drastically the number of cells analyzed (from tens to hundreds) makes it possible to describe a cell population from the nanomechanical point of view. Moreover, we showed that the number of measurements per cell has no impact on the significance of the result.

Our results, in addition to being consistent with those in the literature, show for the first time the presence of at least 2 subcellular populations. These are distinguished by differences in mechanical properties and cell wall adhesion. This discovery could have important implications for understanding the pathogenicity of Candida albicans. Indeed, the adhesion of cells to the host represents the first stage of infection and the mechanical environment of Candida is known to induce transformation from the yeast form to the invasive hypha form. Different subpopulations in terms of adhesion and mechanical properties are thus potentially responsible for one or the other of these key stages of Candida albicans infection.

Methods

Cell culture

The cell cultures were prepared as previously reported [START_REF] Formosa | [END_REF][START_REF] Formosa | [END_REF] . C. albicans was stored at -80°C, four independent cultures were prepared with the C. albicans revivified on Yeast. Peptone Dextrose (YPD) agar, and each were grown in 5 ml YPD broth for 20 h at 30°C under static conditions. In two of these four independent cultures 9.4 µl of caspofungin at 0.1 mg/ml (4xMIC) concentration were added and let under static conditions for 24 h at 30°C. Yeast cells (native and treated) were concentrated by centrifugation, washed two times in acetate buffer, and resuspended in acetate buffer just before performing AFM experiments.

Sample preparation

600 µL were taken from the resuspended cell solution and centrifuged, to separate the buffer from the cells. The supernatant is deposited onto a PDMS stamp, prepared as described in [START_REF] Formosa | [END_REF] and degassed for about 40 min. After 40 min. the buffer is removed from the PDMS surface, 200 µl of the cells solution are deposited and allowed to stand for 15 min. at room temperature. The cells were then placed into the microstructures of the stamp by convective/capillary assembly as described in [START_REF] Formosa | [END_REF] .

The PDMS stamp with cells was finally fixed on a Petri dish (FluoroDish FD35-100) and it was filled with 5 ml of acetate buffer solution to maintain the cells in liquid media.

AFM measurements

For all experiments, commercially available silicon nitride triangular cantilevers (Bruker MLCT) with spring constants and sensitivity ranging respectively from 0.0110 N/m to 0.0405 N/m and from 31.8 nm/V to 54.2 nm/V were used. Cantilevers were calibrated using the thermal tune method. The parameters used to engage the tip on the stamp surface were as follows: IGain= 70 Hz, PGain= 0.002, Setpoint=0.559 nN. The topographic image used to determine W1 and W2 coordinates was recorded in the force map mode, (64 x64 pixels, maximum applied force 1nN, tip velocity 10 µm.s -1 ). The maximum applied force used to record force curves was set to 1 nN and the piezo and motor stage speed were 10 µm/s and 200 µm/s respectively. The AFM field was 10,000 µm 2 (100 µm x 100 µm).

The AFM automation has been implemented on a JPK Nanowizard II, with a motorized precision stage MotStage Zeiss AxioObserver (S/N SM-01-0017) on an inverted optical microscope Zeiss Axiovert 200M. The AFM control software (SPM version 4) runs under Ubuntu 10.04 LTS (Lucid Lynx), the script was executed by using the experiment planner module, included in the JPK SPM software control. Indeed, the experiment planner mode offers a Jython scripting interface to control the AFM, hence the software programs for automation has been developed in Jython programming language.

Statistical analysis: k-means method

The k-means method has been used to group the subpopulations observed in the results. The method is based in the Hartigan and Wong algorithm 39 , it divides M points in N dimensions into K clusters, the clusters centers are at the mean of their Voronoi set [START_REF] Core | R: A Language and Enviroment for Statistical Computing[END_REF] (the set of data points which are nearest to the group center). The procedure is to minimize the within-cluster sum of squares so the dimension of the clusters will be changed until the items in the same cluster are similar as possible and items in different clusters are different as possible.

The k-means method was used to divide the stiffness and adhesion results into groups for analysis.

Figure 1 .

 1 Figure 1. Algorithm execution. Screenshots taken from the Supp data video. A: Location of the center of the first microwell in the current scanning area. B: Position of the tip after moving the motor stage, area 2 is the active scanning area at this moment. C: Shows the data acquisition through different microwells (i-iii). D: Shows the data acquisition inside one microwell, the indentation is performed in different regions of the same microwell (magenta/green/blue).

Figure 2

 2 Figure 2 presents the spring constant and adhesion histograms for C. albicans cells in native conditions (A and B) and treated with caspofungin (C and D). 2A and B, left, show the spring constant histograms, the number of cells analyzed in the first two experiments were 1021 and 959 respectively; both are obtained by analyzing independently the cultured native cells. Analyzing the two histograms with the k-means method they can be deconvoluted into 2 populations that are slightly different in the 2 experiments. The first population has a mean

Figure 2 .

 2 Figure 2. Spring constant histograms for C. albicans, native and treated with caspofungin. A and B (left) show the spring constant histograms for experiments 1 and 2 of native C. albicans cells (1021 and 959 cells analyzed respectively). While C and D (left) show the spring constant histograms for the experiments 3 and 4 of treated C. albicans cells (1018 and 579 cells analyzed respectively). A and B (right) show the results obtained from the adhesion analysis on experiments 1 and 2. C and D show the adhesion results for experiments 3 and 4. E and Fshow the one-way ANOVA test performed using the spring constant and adhesion data from 1-3 and 2-4 respectively. *** = p value < 0.001, NS = no significant difference. Bin width was determined by Freedman-Diaconis rule.

Table 1 .

 1 Summary of the information derived from the experiments.

		Experiment Force Curves	Wells analyzed	Cells analyzed	Time per well(s)	Discarded force curves (%)
	Native cells	1 2	15927 8620	1021 959	1021 957	9 12	4.31 12.87
	Treated cells	3 4	15457 5180	1018 579	1000 574	9 12	8.19 20.88

Acknowledgements

We want to acknowledge FONCYCYT of CONACYT (Mexico), the ministry of Foreign affairs of France and the Université Paris 13, though the financial support of the international collaborative ECOS-NORD project named Nano-palpation for diagnosis, No. 263337 (Mexico) and MI5P02 (France). AMR would like to thank the financial support of SIP-IPN through the project No. 20195489. SPC is supported by a PhD fellowship from CONACYT (No. 288029) and IPN through the cotutelle agreement to obtain double PhD certificate (IPN-UPS). ED is researcher at Centre National de la Recherche Scientifique (CNRS).

Authors contribution

The authors declare no conflict of interest. This paper is not submited elsewhere and has not been considered for publication in any other journal.

Conflicts of interest

There are no conflicts to declare.