Jie Yu

Frederic Dappozze

Juan Martín-Gomez

Jesús Hidalgo-Carrillo

Alberto Marinas

Philippe Vernoux

Angel Caravaca
email: angel.caravaca@ircelyon.univ-lyon1.fr

Chantal Guillard
email: chantal.guillard@ircelyon.univ-lyon1.fr

photocatalytic oxidation of glycerol on WO3-based materials

Keywords: photocatalysis, valorization, glycerol, glyceraldehyde, WO3

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

Introduction

The current development of the economy has nowadays a significant ecological impact.

Therefore, the generation of clean energy, together with the production of high-value chemicals to replace the utilization of petroleum-derivate products via efficient/nonexpensive technologies, has become a major challenge for human beings. In particular, the current energy sector in this world mainly depends on limited sources of fossil fuels, which limits the long-term development of the economy. Thus, the production of biodiesel fuel derived from a renewable biomass source has become one of the main strategic alternatives to overcome these issues. As a matter of fact, according to the report provided by the Food and Agriculture Organization of the United Nations (OECD/FAO), the production of biodiesel is increasing year by year and it is forecasted to remain at a high level [START_REF]OECD/FAO, OECD-FAO Agricultural Outlook 2016-2025[END_REF], [START_REF] Paris | OECD-FAO Agricultural Outlook[END_REF].

However, the production of biodiesel is accompanied by a significant co-production of glycerol. Around 100 kg of glycerol (~110 kg of crude glycerol) is generated from 1 ton of biodiesel production [START_REF] Santibáñez | Residual glycerol from biodiesel manufacturing, waste or potential source of bioenergy: a review[END_REF], leading to a significant devaluation of this product on the market.

As a consequence, an important amount of glycerol is finally incinerated for heat energy production. Nevertheless, considering that biomass is the only alternative to fossil fuels for the production of chemicals, it would be more interesting to convert glycerol into valueadded products. Therefore, a better valorization of glycerol will have great economical and environmental benefits.

Glycerol, with three hydroxyl groups, can be transformed into a wide variety of value-added products, including dihydroxyacetone, glyceraldehyde, glyceric acid, tartronic acid, glycolic acid, formic acid and oxalic acid [START_REF] Pagliaro | From glycerol to value-added products[END_REF], [START_REF] Kwon | Combining voltammetry with HPLC: Application to electrooxidation of glycerol[END_REF]. All these products, except for oxalic acid and formic acid, with high production volumes, are economically more interesting than glycerol. For example, glyceraldehyde could be applied in the domains of cosmetic and pharmaceutical industries as well as organic chemistry [START_REF] Diguilio | Tuning product selectivity in the catalytic oxidation of glycerol by employing metal-ZSM-11 materials[END_REF]. Dihydroxyacetone could be used in cosmetics as an ingredient in tanning lotions [START_REF] Walgode | A review of aerobic glycerol oxidation processes using heterogeneous catalysts: a sustainable pathway for the production of dihydroxyacetone[END_REF]. Glyceric acid has found applications in the field of medicine to cure skin disorders [START_REF] Detoni | Effect of Pt/HZSM-5 dealumination by high temperature reduction on glycerol oxidation[END_REF]. Glycolic acid could be utilized in daily necessities such as degreasing agents, tanning agents, and skincare products [START_REF] Castillo | Chemical peels in the treatment of acne: patient selection and perspectives[END_REF]. Tartronic acid and oxalic acid are used as an agent of chelating in fine chemistry [START_REF] Coelho | Separation of tartronic and glyceric acids by simulated moving bed chromatography[END_REF]. Nevertheless, among them, glyceraldehyde is one of the most value-added products [START_REF] Mendoza | Selective production of dihydroxyacetone and glyceraldehyde by photo-assisted oxidation of glycerol[END_REF], [START_REF] Behr | Improved utilisation of renewable resources: New important derivatives of glycerol[END_REF]. Thus, selective catalytic oxidation of glycerol to glyceraldehyde possesses great commercial value.

To achieve the production of high value chemicals, different technologies have been applied in glycerol oxidation, such as thermo- [START_REF] Liang | Selective oxidation of glycerol with oxygen in a base-free aqueous solution over MWNTs supported Pt catalysts[END_REF], [START_REF] Kimura | Selective oxidation of glycerol on a platinum-bismuth catalyst[END_REF], [START_REF] Tan | Selective oxidation of glycerol to dihydroxyacetone over N-doped porous carbon stabilized CuxO supported Au catalysts[END_REF], photo- [START_REF] Mendoza | Selective production of dihydroxyacetone and glyceraldehyde by photo-assisted oxidation of glycerol[END_REF], [START_REF] Payormhorm | Synthesis of C-doped TiO2 by sol-microwave method for photocatalytic conversion of glycerol to value-added chemicals under visible light[END_REF], [START_REF] Imbault | Selective oxidation of crude glycerol to dihydroxyacetone in a biphasic photoreactor[END_REF], electro- [START_REF] Zhang | Electrocatalytic oxidation of glycerol on Pt/C in anionexchange membrane fuel cell: Cogeneration of electricity and valuable chemicals[END_REF], [START_REF] Liu | Selective electro-oxidation of glycerol to dihydroxyacetone by a non-precious electrocatalyst -CuO[END_REF], [START_REF] Lee | Atomic-layer-deposited SnO2on Pt/C prevents sintering of Pt nanoparticles and affects the reaction chemistry for the electrocatalytic glycerol oxidation reaction[END_REF], and photoelectrocatalytic technologies [START_REF] Raptis | Renewable energy production by photoelectrochemical oxidation of organic wastes using WO3 photoanodes[END_REF]. Among them, photocatalysis is considered a green and energy-saving technology that attracts scientists' attention all over the world, since it utilizes light (potentially sunlight) to generate charge carriers (holes and electrons) and active species, which can activate the target molecules [START_REF] Zhou | The preparation, and applications of g-C3 N4/TiO2 heterojunction catalysts-a review[END_REF], [START_REF] Qiu | Recent advances in three-dimensional graphene based materials for catalysis applications[END_REF], [START_REF]Heterogeneous Photocatalysis: From Fundamentals to Green Applications[END_REF]. In this sense, most photo-oxidation reactions usually take place at room temperature and atmospheric pressure, by using a semiconductor with the proper band gap to ensure a good potential application prospect [START_REF] Colmenares | Nanophotocatalysis in selective transformations of lignocellulosederived molecules: a green approach for the synthesis of fuels, fine chemicals, and pharmaceuticals[END_REF]. Since V. Maurino et al. [START_REF] Maurino | Glycerol transformation through photocatalysis: A possible route to value added chemicals[END_REF] firstly reported the photocatalytic oxidation of glycerol as a possible route to produced added-value chemicals in 2008, many studies have been carried out to investigate this reaction over different catalytic materials. Most of them were performed on the most popular photocatalytic material, i.e., TiO2. As an example, some of those works focused on the performance of glycerol oxidation over TiO2 or TiO2-doped with noble metals such as Au or Pt. [START_REF] Panagiotopoulou | Kinetics and mechanism of glycerol photo-oxidation and photo-reforming reactions in aqueous TiO2 and Pt/TiO2 suspensions[END_REF], [START_REF] De Oliveira Melo | Visible light-induced hydrogen production from glycerol aqueous solution on hybrid Pt--CdS--TiO2 photocatalysts[END_REF] V. Augugliaro and coworkers [START_REF] Augugliaro | Partial photocatalytic oxidation of glycerol in TiO2 water suspensions[END_REF] used commercial and home-made TiO2 samples in the anatase, rutile or anataserutile phases to investigate the glycerol oxidation products in an aqueous solution and showed that1,3-dihydroxyacetone (DHA), glyceraldehyde, formic acid and carbon dioxide are the main products. In their study, the highest selectivity of the value-added glyceraldehyde was 13% and that of DHA was 8% at a 35% glycerol conversion. C. Minero et al. [START_REF] Minero | Glycerol as a probe molecule to uncover oxidation mechanism in photocatalysis[END_REF] used two different commercial TiO2 powders (Degussa P25 and anatase Merck TiO2) to investigate the photocatalytic oxidation mechanism of glycerol and found that various TiO2 specimens could lead to different selectivities of products.

WO3 is an excellent metal oxide semiconductor photocatalyst [START_REF] Szilágyi | Photocatalytic properties of WO3/TiO2 core/shell nanofibers prepared by electrospinning and atomic layer deposition[END_REF], [START_REF] Szilágyi | WO3 photocatalysts: Influence of structure and composition[END_REF], with better visible light response than TiO2. [START_REF] Lin | Preparation and photocatalytic properties of biomorphic hierarchical WO3 based on bionic rice hull[END_REF] However, to the best of the authors´ knowledge, although it has been widely used in many fields such as H2 evolution by glycerol electroreforming [START_REF] Tahir | Au-NPs embedded Z-scheme WO3/TiO2 nanocomposite for plasmon-assisted photocatalytic glycerol-water reforming towards enhanced H2 evolution[END_REF], [START_REF] Stelmachowski | The photocatalytic conversion of (biodiesel derived) glycerol to hydrogen-A short review and preliminary experimental results part 1: A review[END_REF], the degradation of pollutants [START_REF] Liu | Synthesis of a WO3 photocatalyst with high photocatalytic activity and stability using synergetic internal Fe 3+ doping and superficial Pt loading for ethylene degradation under visible-light irradiation[END_REF], [START_REF] Wu | Preparation of WO3-modified TiO2 thin film by peroxo sol--gel method and its photocatalytic activity in degradation of methylene blue[END_REF], gas sensor [START_REF] Hung | Facile synthesis of ultrafine rGO/WO3 nanowire nanocomposites for highly sensitive toxic NH3 gas sensors[END_REF] and electrochemical energy storage [START_REF] Chen | Hierarchical nanostructured WO3 with biomimetic proton channels and mixed ionic-electronic conductivity for electrochemical energy storage[END_REF], the photocatalytic oxidation of glycerol towards high-value chemicals has never been investigated on WO3. Therefore, this work aims to study and thoroughly characterize a series of WO3-based materials for the photocatalytic oxidation of glycerol towards added-value chemicals. An outstanding performance was found towards the production of glyceraldehyde, one of the most valuable derivatives of glycerol. The activity and selectivity provided by such materials were compared with those of commercial TiO2-based photocatalysts.

Experimental 2.1 Photocatalytic materials and photocatalytic activity measurements

Several WO3-and TiO2-based materials were used in this study. First of all, a pure WO3 photocatalyst was prepared by a simple hydrothermal method. Firstly, 0.6 g of WCl6 was dissolved in 120 mL of Ethanol solution under constant stirring at room temperature for 10 min. Then, the obtained solution was transferred to a Teflon-lined stainless-steel autoclave (200 mL) and kept in an oven for 12 h (180 o C). Finally, after cooling down to room temperature, WO3 powder was withdrawn, washed with Ethanol (3 × 90 mL) and deionized water (3 × 90 mL), and dried at 80 o C in air overnight. This material was tested as prepared.

For comparison purposes, the as-prepared WO3 catalyst was also calcined at 450 o C.

In addition, a series of TiO2-based materials were acquired commercially, including Anatase (HPX-200), Rutile (HPX-400C) and DTW5 (5 wt% WO3/TiO2), all of them provided by Tronox®, while P25 (a very well-known heterojunction of anatase/rutile) was supplied by Evonik®.

A typical photocatalytic experiment was carried out according to the following protocol: 7.5 mg of the catalyst were introduced in a quartz reactor containing 30 mL of glycerol (in aqueous solution concentration of 1.10 mmol L -1 ). The photocatalytic reactions were conducted under ultraviolet light with a UV lamp (UV-A PL-L, 18 W) which delivers an irradiation intensity of 5.60 mW cm -2 . The reactor is open to the air without bubbling. Prior to the photocatalytic reaction, the suspension was stirred for 30 min in the dark to achieve the adsorption-desorption equilibrium of glycerol on the surface of catalysts. Every 30 min, a sample (0.5 mL) was taken from the mixture solution and immediately filtered by a Millipore 0.45 µm (hydrophilic PVDF) membrane using a plastic syringe. In the case of WO3, the sampling interval is 2 h and its photooxidation performance is tested by two parallel experiments owing to its low kinetic. The concentrations of glycerol and derivate products were analysed by a Shimadzu SPD-M20A prominence ultrafast high-performance liquid chromatography (HPLC) assembled with a Transgenomic ICSep ICE-COREGEL-87H3 organic acid column. The mobile phase is 5 mmol/L of H2SO4 and its flow rate is 0.7 mL/min. The temperature of the oven is 30 o C. The products of glycerol oxidation could be determined by the ultraviolet detector at the wavelength of 210 nm except from glyceric acid which is identified at 240 nm. Glycerol could be analyzed by the refractive index detector (RID).

The Glycerol conversion (in %) was calculated according to equation (1):

% = × 100 (1)
where C0 glycerol and Ct glycerol stand for the initial and real-time concentrations of glycerol, respectively.

The Yield (in %) of every product was calculated according to equation (2):

% = ! "# × 100 × (2)
where Ct product stands for the real-time concentration of the product and n is the ratio of the carbon number present in the product divided by the number of carbon present in the glycerol.

The Selectivity (in %) of every product was calculated according to equation (3):

$ % % % = ! "# × 100 × (3)
where all the parameters were already defined in equations ( 1) and (2).

Characterization

X-ray diffraction (XRD) was conducted with a Bruker D8 diffractometer using Cu Kα (λ= UV-vis diffuse reflectance spectroscopy (UV-vis DRS) measurements were conducted using an AvaSpec-2048 Fiber Optic Spectrometer. Spectra were recorded from 200 to 600 nm using a 2048 pixel CCD detector array. Barium sulfate (BaSO4) was used as a blank reference.

0
The electrochemical photocurrent experiments were carried out in a photoelectrochemical cell (supplied by redox.me ®) with a standard three-electrode system consisting of a Pt wire as the counter electrode, a saturated Ag/AgCl as the reference electrode and a working electrode prepared by the following process: 5 mg of the catalyst (as-prepared WO3 or commercial samples) were dispersed in 0.5 mL ethanol. Then, 20 μL of that dispersion were impregnated over an indium-tin oxide (ITO) substrate, with an active area of ca. 1 cm 2 , and dried at room temperature. Different electrical potentials were applied with a potentiostatgalvanostat (VoltaLab PGZ402).

The concentrations of total organic carbon (TOC) of the solutions before and after the photocatalytic tests on different materials were measured using a Shimadzu TOC-VCPN analyzer equipped with an auto-sampler.

Acid properties of the different samples were determined by using pyridine as a probe molecule. Pyridine adsorption was followed by Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), which were carried out on a Fourier-transform infrared spectroscopy (FTIR) instrument (Perkin Elmer Frontier) equipped with an environmental chamber (Harrick HVC-DRM). A resolution of 4 cm -1 was used with 125 scans averaged to obtain a spectrum from 4000 to 400 cm -1 . Prior to each experiment, pyridine adsorption was carried out at 100 °C for 45 min allowing the saturation of the catalyst surface. The physically adsorbed pyridine was then removed from the surface with an air flow (50 mL•min -1 ). Finally, the IR spectra were recorded at 100 °C. Bands at 1448 and 1537 cm -1 (corresponding to Lewis and Brönsted acid sites, respectively) were integrated and acidity was determined using the corresponding molar extinction coefficients.

Results and discussion

Catalysts characterization

First of all, the physicochemical properties of the materials proposed in this study were characterized by XRD and microscopy techniques (SEM and HRTEM). Fig. 1a shows the XRD patterns for Anatase, Rutile and P25, while Fig. 1b shows the patterns for the asprepared WO3 catalyst, together with the commercial DTW5 (5 % WO3 on TiO2). As expected, pure Anatase and Rutile samples only exhibit corresponding pure crystalline phases (PDF #89-4921 and #77-0440, respectively). In addition, P25, well known as a very efficient heterojunction of anatase and rutile for a wide variety of photocatalytic processes, exhibits diffraction peaks corresponding to both crystalline phases.

Figure 1

Fig. 1 b indicates that the as-prepared WO3 catalyst exhibits a rather amorphous behavior, although a well-defined peak at 2θ = 23.1 o could be identified, which belongs to the (001) phase of a monoclinic structure of WO3 (PDF #72-0677) [START_REF] Koo | In Situ Photoelectrochemical Chloride Activation Using a WO3 Electrode for Oxidative Treatment with Simultaneous H2 Evolution under Visible Light[END_REF]. Its low crystallinity (48.6 %, Table 1) compared to the commercial catalytic materials (80 -85 %, Table 1) could be attributed to the hydrothermal synthesis method, which was performed at low temperature (180 o C) without any additional calcination step. For comparison purposes, a similar WO3 material was calcined at 450 o C in air (1 h, 5 o C min -1 heating rate), and the XRD patterns are shown in Fig. S1. After the calcination step, the material evolved to a highly crystalline monoclinic catalyst. Finally, regarding the DTW5 sample, the titanium oxide phase belongs to anatase, and no peaks related to any WO3 crystalline phase could be identified by XRD. In principle, this could be attributed to a very high dispersion of small WO3 nanoparticles on the TiO2 (anatase) support, and/or to a very low crystallinity of WO3 compared to TiO2. All materials and phases present similar crystallite sizes in the range of 16-33 nm (Table 1).

To get more insights into the structural parameters of these catalysts, they were further characterized by HRTEM.

Figure 2

Fig. 2 reveals sharp and clear lattice fringes for all the materials, regardless of the crystallinity level. The lattice spacing of Anatase and Rutile is 0.367 nm and 0.249 nm, respectively, in good agreement with theoretical values of (101) plane of the anatase phase [START_REF] Ren | Lithium Intercalation into Mesoporous Anatase with an Ordered 3D Pore Structure[END_REF] and (101) plane of the rutile phase [START_REF] Miao | Preparation and characterization of polycrystalline anatase and rutile TiO2 thin films by rf magnetron sputtering[END_REF]. It is worth noting that for the commercial DTW5 sample, although there are no significant peaks of WO3 in the XRD pattern, the lattice spacing of 0.388 nm was found, corresponding to the (001) plane of WO3 [START_REF] Pokhrel | Growth of ultrafine single crystalline WO3 nanoparticles using flame spray pyrolysis[END_REF]. This clearly revealed the existence of well-dispersed WO3 with the size of ~14 nm on the TiO2 (anatase) matrix. Finally, the as-prepared WO3 synthesized using WCl6 as the precursor possesses a similar lattice spacing of 0.381 nm, which matches well with the (001) plane of the monoclinic WO3 previously reported with a similar precursor. [START_REF] Pokhrel | Growth of ultrafine single crystalline WO3 nanoparticles using flame spray pyrolysis[END_REF] In addition, the overall morphology of the catalyst grains was observed by SEM. As shown in Fig. S2, it was found that anatase, P25, DTW5 and WO3 presented spherical shapes while rutile shows rods-like shape agglomerates.

N2 adsorption-desorption was carried out to determine the specific surface area (SBET, Table 1) together with the pore size and the porous volume of each material. These parameters could eventually affect their performance for glycerol conversion, owing to the fact that the catalyst active sites along with the adsorption of glycerol as well as its oxidation products usually positively relate to the specific surface area. As shown in Fig. S3, type IV isotherms were obtained with a small hysteresis loop at a relative pressure of 0.7-1.00. Therefore, all materials exhibit, as expected, a mesoporous structure with pore sizes between 10-15 nm (Table 1). In addition, while Anatase, Rutile and DTW5 showed the highest SBET (78 -92 m 2 g -1 ), this value was slightly lower for P25 (63 m 2 g -1 ) and drastically decreased for the asprepared WO3 (33 m 2 g -1 ).

All in all, it seems that in general, the commercial materials exhibit a more crystallized structure with higher specific surface areas compared to the as-prepared WO3 catalyst.

Therefore, in principle, a lower overall catalytic performance could be expected for the latter to a certain extent. Nevertheless, other important features, such as their electronic properties (band gap, charges separation), and concentration of acid sites are known to play a key role in the performance of such photocatalytic materials.

The electronic properties of these materials were first investigated by diffuse reflectance spectroscopy (DRS) in the UV-Vis range. The obtained spectra of samples are shown in Fig. 3a. Comparing Anatase and Rutile samples, a slightly higher adsorption towards the visible range could be observed for the latter, as previously reported [START_REF] Elsellami | Highly photocatalytic activity of nanocrystalline TiO2 (anatase, rutile) powders prepared from TiCl4 by solgel method in aqueous solutions[END_REF]. In addition, as expected, it was found that all the samples could absorb UV light while the as-prepared WO3 could respond to not only UV light but also partially to visible light.

Figure 3

Fig. 3b shows the Tauc plots for band gap calculations using the Kubelka-Munk equation.

Briefly, an extrapolation of the linear part of the curve (α•hν) 1/2 (where α is the absorption coefficient) as a function of hν defines the band gap energy of the samples considered as indirect band gap semiconductors [START_REF] Fessi | Surface and Electronic Features of Fluorinated TiO2 and Their Influence on the Photocatalytic Degradation of 1-Methylnaphthalene[END_REF]. As previously mentioned, all the commercial TiO2based materials exhibit a band gap compatible with the use of UVA light (> 3.0 eV). Anatase and DTW5 showed an almost identical band gap value, which seems to indicate that the addition of 5 % WO3 on TiO2 (anatase) does not significantly affect its electronic properties.

In addition, P25, with an anatase/rutile heterojunction, exhibited a slightly smaller band gap, closer to that of rutile. Finally, for the as-prepared WO3 catalyst, its band gap was 2.61 eV, in good agreement with previous studies, which indicates that its response range to photoirradiation could be expanded to visible light [START_REF] Raptis | Renewable energy production by photoelectrochemical oxidation of organic wastes using WO3 photoanodes[END_REF]. According to these results, we chose UV light to compare the performance of glycerol photocatalytic oxidation as all catalysts respond to UV light.

In a new effort to characterize the electronic properties of the materials, photocurrent experiments were performed. These tests were carried out in the photoelectrochemical cell described in the experimental section, in neutral media (0.5 M Na2SO4 liquid electrolyte, in order to avoid any potential degradation of the materials in acid or alkaline media), upon the application of a constant potential of 0.9 V vs Ag/AgCl reference electrode. All materials were subjected to UV irradiation in step changes of 30 seconds. The higher the photocurrent intensity usually indicates the better separation situation of photo-generated electrons and holes, which generally contributes to the progress of photocatalytic oxidation. [START_REF] Wu | Ga-Doped and Pt-Loaded Porous TiO2-SiO2 for Photocatalytic Nonoxidative Coupling of Methane[END_REF], [START_REF] Hu | Construction of solid-state Z-scheme carbon-modified TiO2/WO3 nanofibers with enhanced photocatalytic hydrogen production[END_REF]. As shown in Fig. 4, the photocurrent intensity obtained upon irradiation follows this rank: P25>

Anatase≈Rutile≈DTW5>WO3. These results indicate that a better separation rate of photogenerated charges (electrons and holes) upon irradiation could be expected on TiO2-based materials.

Figure 4

Therefore, the addition of WO3 seemed to have a detrimental effect on such a phenomenon. If the separation of these photo-generated charges is related to the overall activity of oxidation on these materials, we could expect a higher glycerol photo-oxidation rate on TiO2 than on WO3-based catalysts.

Finally, the nature and relative amount of acid sites of P25, DTW5 and WO3 were characterized by adsorption of pyridine followed by diffuse reflection infrared spectroscopy (DRIFT). and 1542 cm -1 could be attributed to Lewis acid sites and to protonated pyridine molecules bound to surface Brönsted acid sites, respectively [START_REF] Pokhrel | Growth of ultrafine single crystalline WO3 nanoparticles using flame spray pyrolysis[END_REF], [START_REF] Elsellami | Highly photocatalytic activity of nanocrystalline TiO2 (anatase, rutile) powders prepared from TiCl4 by solgel method in aqueous solutions[END_REF]. In addition, Table 2 presents the calculated quantitative values of both acid sites, the overall amount and the ratio between them. If we compare the results for P25 (TiO2) and WO3, it can be clearly observed that WO3 possesses the highest percentage of Brönsted and total acid sites. This is expected considering that tungsten-based catalysts are well-known for their acidic properties. For instance, Ganji et al. [START_REF] Ganji | Trade-off between acidic sites and crystallinity of the WO3-TiO2 catalyst toward dehydration of glucose to 5-hydroxymethylfurfural[END_REF] reported an overall higher acidity of WO3 and WO3/TiO2 materials compared to TiO2, obtained by both pyridine-FTIR and NH3-TPD (temperature programmed desorption). That could explain that the DTW5, compared with P25, also exhibited a higher amount of Brönsted and total acid sites. These acidic properties are expected to play a very important role in the photocatalytic activity of these materials towards glycerol activation. For instance, Pt/WO3/Al2O3 catalysts have been reported as one of the most successful catalytic systems for the effective conversion of glycerol to 1,3-propanediol by thermal-hydrogenolysis [START_REF] Kurosaka | Production of 1,3-propanediol by hydrogenolysis of glycerol catalyzed by Pt/WO3/ZrO2[END_REF],

where Brønsted acid sites are considered to play a key role in the adsorption/activation of the secondary C-O bond in glycerol [START_REF] Aihara | Investigation of the mechanism of the selective hydrogenolysis of CO bonds over a Pt/WO3/Al2O3 catalyst[END_REF]. Also, in a previous study by Checa et al. dealing with the same reaction [START_REF] Pokhrel | Growth of ultrafine single crystalline WO3 nanoparticles using flame spray pyrolysis[END_REF], it was found that the addition of tungsten to a Pt/ZrO2 catalyst enhanced its selective performance of the target product 1,3-propanediol, most likely due to the creation of a higher concentration of Brönsted acid sites.

To summarize this section, the characterization results exhibit overall enhanced physicochemical properties of TiO2-based materials compared to WO3 catalysts in terms of specific surface area, crystallinity, and photo-generated charge separation. However, the WO3 -based materials are much more acidic, containing a higher amount of Brönsted acid sites.

Photocatalytic activity

All the catalysts previously characterized were tested for photocatalytic oxidation of glycerol.

Figure 6 shows the evolution of the glycerol conversion with time upon irradiation.

Regarding all the TiO2-based materials, significant oxidation of glycerol was achieved after 5 hours (Fig. 6a). However, for the as-prepared WO3 catalyst, the glycerol consumption rate is lower, and therefore the reaction time was extended up to 22 h (Fig. 6b). In order to prove that the activity was due to photocatalysis, blank experiments over P25, DTW5 and WO3 (in the dark, Fig. S4a-c) and direct photolysis of glycerol (without any catalyst in the light, Fig. S4d) were performed, where the glycerol conversion was negligible.

Figure 6

As observed in Fig. 6a, P25 seemed to be the most active material, achieving a complete glycerol conversion after 4 h. Therefore, the photocatalytic activity obtained upon irradiation for glycerol oxidation obeys the order P25>Anatase>Rutile>DTW5>WO3. With regards to P25, its outstanding activity has been already proven for a wide variety of photocatalytic processes, including glycerol photooxidation [START_REF] Qiu | Recent advances in three-dimensional graphene based materials for catalysis applications[END_REF], [START_REF] Maurino | Glycerol transformation through photocatalysis: A possible route to value added chemicals[END_REF], [START_REF] Panagiotopoulou | Kinetics and mechanism of glycerol photo-oxidation and photo-reforming reactions in aqueous TiO2 and Pt/TiO2 suspensions[END_REF]. P25 consists of a mixed phase of anatase and rutile in a ratio of about 4:1. In the so-called anatase/rutile heterojunctions, upon UV irradiation, the photogenerated electrons from the anatase phase are transferred to the rutile phase on account of its lower conduction band energy [START_REF] Subramanian | Sustainable hydrogen and/or syngas production: New approaches to reforming[END_REF]. This results in more efficient separation of the photogenerated charge carriers, as observed in the photocurrent experiments (Fig. 4). Another evidence of the creation of this heterojunction was observed in the DRS experiments, where the band gap for P25 was in-between that of anatase and rutile (Fig. 3b). One may expect a similar behavior for the DTW5 material. As a matter of fact, WO3/TiO2 composites have been previously developed and reported to enhance the separation of photogenerated charge carriers on the heterojunctions between both phases together with a modification of the band gap of TiO2 towards a more visible-light range [START_REF] Palmas | TiO2-WO3 nanostructured systems for photoelectrochemical applications[END_REF].

However, in this case, we could not observe a significant heterojunction, since the band gap for DTW5 was found to be almost identical to that of anatase (Fig. 3b). Furthermore, the photocurrent obtained for DTW5 was even slightly lower than that for Anatase (Fig. 4).

Therefore, we conclude that the low loading of WO3 (5 %) on anatase can neither enhance the separation of charge carriers nor the conversion of glycerol. It is worth noting therefore that the photocurrent activity observed in Fig. 4 obeys the same order as the overall glycerol conversion activity after 5 h of irradiation observed in Fig. 6. This suggests that the photocatalytic degradation of glycerol is strongly related to the separation of the electrons and holes produced upon irradiation.

This study aims to enhance the production of added-value products by photocatalytic oxidation of glycerol. Fig. 7 shows the distribution of products with time for all the materials.

Figure 7

The main oxidation products observed were glyceraldehyde, DHA, glycolic acid, tartronic acid, oxalic acid, formic acid and acetic acid. Nevertheless, the amount and distribution of each of these products are highly influenced by the nature of the photocatalytic material.

Starting with the most active material, P25, we can clearly observe that some of the main oxidation products, including glyceraldehyde, DHA and formic acid are produced during a short period of around 1 h. A similar effect was observed in the Anatase catalyst, although the products distribution and amount were different, as expected in view of the observed lower overall conversion (Fig. 6). On the other hand, the Rutile catalyst seemed to produce a significant amount of formic acid.

Our results for both, P25 and Anatase photocatalysts are in good agreement with the model proposed by Jedsukontorn et al. [START_REF] Ganji | Trade-off between acidic sites and crystallinity of the WO3-TiO2 catalyst toward dehydration of glucose to 5-hydroxymethylfurfural[END_REF], [START_REF] Kurosaka | Production of 1,3-propanediol by hydrogenolysis of glycerol catalyzed by Pt/WO3/ZrO2[END_REF] for glycerol conversion on TiO2 (anatase).

According to those studies, upon UV irradiation, glycerol acts as a hole scavenger of the photogenerated holes [START_REF] Jedsukontorn | Route of glycerol conversion and product generation via TiO2-induced photocatalytic oxidation in the presence of H2O2[END_REF], or reacts with the reactive oxidizing species [START_REF] Jedsukontorn | Mechanistic aspect based on the role of reactive oxidizing species (ROS) in macroscopic level on the glycerol photooxidation over defected and defected-free TiO2[END_REF], oxidizing it to both glyceraldehyde and DHA. Minero et al. [START_REF] Minero | Glycerol as a probe molecule to uncover oxidation mechanism in photocatalysis[END_REF] also proposed that the formation of these species could be mediated by ▪ OH radicals. Glyceraldehyde is produced when the primary -OH group is adsorbed and oxidized, while DHA is produced upon the oxidation of the secondary -OH group of glycerol. The glyceraldehyde could be then oxidized to produce glyceric acid, while the DHA could be rearranged into glyceraldehyde [START_REF] Jedsukontorn | Route of glycerol conversion and product generation via TiO2-induced photocatalytic oxidation in the presence of H2O2[END_REF]. It could be expected afterward that the products keep evolving into smaller organic molecules, such as tartronic acid, oxalic acid, and as observed in this study, formic acid. These reaction routes are in good agreement with the results obtained for Anatase and P25, since the observed reaction products evolved with time and then disappeared.

However, for Rutile, the main product seemed to be formic acid, and we could not observe a similar evolution for the other TiO2 catalysts. In this sense, Augugliaro et al. [START_REF] Augugliaro | Partial photocatalytic oxidation of glycerol in TiO2 water suspensions[END_REF] proposed a mechanism where all the species are produced in parallel on P25, anatase and rutile catalysts, including glyceraldehyde, DHA, formic acid and CO2. Minero et al. [START_REF] Minero | Glycerol as a probe molecule to uncover oxidation mechanism in photocatalysis[END_REF] also suggested a model where C2 + C1 products could also be produced in parallel by direct electron transfer (in this case over P25). We believe that these mechanisms are in good agreement with the results obtained for Rutile, where the formation of formic acid seemed to be the product of a parallel process.

The most interesting results were obtained with the WO3-based materials. Starting with the as-prepared WO3, a completely different behavior was observed. In this case, as opposed to Anatase and P25, the concentration of reaction products gradually increased with time. The main product was glyceraldehyde, followed by DHA and then by smaller quantities of further oxidized products. These results suggest a similar first oxidation step as previously explained for TiO2, where glycerol undergoes the first oxidation towards glyceraldehyde and DHA.

Considering that the former is the result of the oxidation of primary (terminal) -OH groups of glycerol and that we have two primary -OH groups its likelihood to be produced is much higher compared to that for the production of DHA, which is the result of the oxidation of the secondary -OH group in glycerol. Moreover, on the one hand, in presence of WO3, the ratio glyceraldehyde/DHA is higher than 2. It suggests that in the presence of WO3, a fraction of the DHA formed is probably converted into glyceraldehyde and on the other hand, these high added-value products seemed to desorb in the liquid phase, rather than to undergo further oxidation towards smaller molecules. These photocatalytic properties are of paramount importance for the development of novel catalysts with high selectivity towards glyceraldehyde. However, as discussed in Fig. 6, the photocatalytic reaction on this material is rather slow compared to that of Anatase, Rutile and P25, and longer reaction times were required with this system for practical purposes.

Before discussing the photocatalytic behavior of the DTW5 material, two parameters will be introduced to compare the performance of pure TiO2-based materials (Anatase, Rutile and P25) and the as-prepared WO3. Fig. 8 shows the variation of the selectivity of products while Fig. 9 shows the variation of the yield of products, both of them as a function of the glycerol conversion.

Figure 8

Figure 9 These figures allow us to compare the production of the added-value products of interest (in this case glyceraldehyde) at glycerol isoconversion conditions. As expected for P25 and Anatase according to the results discussed in Fig. 7, high selectivities and yields to oxidation products are only achieved at low conversion levels. Unfortunately, there was only one products analysis below 40% of conversion due to the fast kinetics on P25. As the system evolved in time, the conversion increased but the production of valuable products decreased.

In addition, since the sum of all the liquid products selectivity is far from 100 % at all the conversion levels, it could be assumed that CO2 could be also produced. This hypothesis is confirmed considering the TOC measured after about 45% of conversion (Fig. 10).

Figure 10

This was also the case for the Rutile catalyst which predominantly produces formic acid.

Therefore, none of the pure TiO2-based catalysts exhibited a significant performance towards the production of glyceraldehyde.

Nevertheless, in these figures, we could clearly observe that the as-prepared WO3 catalyst exhibited an outstanding activity towards the production of the desired product, glyceraldehyde, even under high conversion levels. As a matter of fact, with a relatively constant selectivity towards this product, its yield gradually increased with the overall glycerol conversion. In addition, the overall selectivity of liquid organic products over this catalyst is much higher than that of the TiO2 materials, which indicates that the total glycerol oxidation towards CO2 is much more unlikely as confirmed by TOC analysis (Fig. 10).

Besides, WO3 post-treated via calcination at 450 o C exhibited a slightly superior glycerol conversion owing to its higher crystallinity (Fig. S1), with similar values of glyceraldehyde selectivity and yield (Fig. S5). Therefore, TiO2-based materials (mainly Anatase and P25) exhibited, as expected, a great overall catalytic performance for the oxidation of glycerol, but a rather poor selectivity to glyceraldehyde, while the as-prepared (and calcined) WO3

catalysts exhibited an interesting selectivity/yield towards this product, even under high conversion levels. The main issue of this material is related to the rather slow kinetics, which could lower the added value of this material for practical purposes.

To overcome this issue, the DTW5 catalyst was tested for the photocatalytic oxidation of glycerol. In Figs. 789we can clearly observe that the presence of a small amount of WO3

(5 %) in the anatase support led to completely different behavior in the desired direction.

Hence, as opposed to the Anatase photocatalyst, the reaction products formed on DTW5 did not seem to disappear with time, but rather to increase in a similar way to that obtained with the as-prepared WO3 material. In addition, the selectivity/yield towards glyceraldehyde was much higher for DTW5 compared to the TiO2 catalysts. Table 3 shows the values for glyceraldehyde selectivity and yield at similar glycerol conversion values (43-47 %) over all the photocatalysts. While both parameters are rather low for TiO2 materials in our study (< 5 %) and for other noble metal-free TiO2-based photocatalysts in publications [START_REF] Augugliaro | Partial photocatalytic oxidation of glycerol in TiO2 water suspensions[END_REF], [START_REF] Minero | Glycerol as a probe molecule to uncover oxidation mechanism in photocatalysis[END_REF], [START_REF] Imbault | Photocatalytic production of dihydroxyacetone from glycerol on TiO2 in acetonitrile[END_REF],

[59], glyceraldehyde yields/selectivities of 13 %/29 % and 8 %/19 % were obtained for the as-prepared WO3 and for DTW5, respectively.

In addition, total organic carbon analysis (TOC) of the liquid solutions was performed for the different photocatalytic systems when the glycerol conversion achieved a value ~ 45 %. The obtained values were compared with the initial concentration of glycerol (Fig. 10). The results obtained allowed us to confirm the level of mineralization of glycerol over the different catalysts upon irradiation. In addition, as we discussed in the previous figures, the modification of anatase with WO3 (DTW5) allowed avoiding the mineralization compared to the Anatase catalyst, in good agreement with the higher selectivity exhibited by this material towards liquid added-value products, mainly glyceraldehyde.

Therefore, the photocatalytic activity results obtained in this study clearly demonstrated, for the first time in literature, an outstanding performance of WO3-based materials for the selective photocatalytic oxidation of glycerol to glyceraldehyde. In particular, the commercial 5 % WO3/TiO2 (anatase) catalyst allows combining the main features of anatase (fast kinetics for glycerol conversion) and WO3 (high selectivity towards glyceraldehyde). These results are in good agreement with the characterization study described in section 3.1. The textural parameters (crystallinity, crystal size and specific surface area, Table 1) for Anatase, Rutile, P25 and DTW5 were relatively similar and cannot explain the differences observed in photocatalytic properties. Nevertheless, the overall activity for glycerol conversion was strongly correlated with the electronic properties of these materials, namely the separation of photogenerated charge carriers (as observed by the photocurrent characterization tests, Fig. 4).

This phenomenon was previously observed in many photocatalytic processes, since an enhanced separation of e -and holes is indeed one of the most common strategies to improve the overall performance of the photocatalytic system. This is usually managed either by doping the semiconductor catalyst with a metal (e -trap), or by the formation of heterojunctions between different materials or different phases of the same material [START_REF] Subramanian | Sustainable hydrogen and/or syngas production: New approaches to reforming[END_REF].

However, the enhanced glyceraldehyde selectivity observed for DTW5 catalyst compared to Anatase (or any other TiO2-based catalyst) could not be explained by its electronic properties.

Considering that both materials exhibit an almost identical band gap (Fig. 3b), and the fact that DTW5 shows lower photocurrent activity (i.e., worse separation of photogenerated carriers, Fig. 4), the formation of a successful heterojunction between TiO2 and WO3 could be disregarded. We believe therefore that the outstanding performance of DTW5 towards glyceraldehyde production could be attributed to its enhanced surface acidity.

In this sense, previous studies dealing with the thermal catalytic hydrogenolysis of glycerol already proved that WO3 plays a role of paramount importance due to the presence of Bronsted sites, which allow for the selective activation of C-O bonds in glycerol [START_REF] Kurosaka | Production of 1,3-propanediol by hydrogenolysis of glycerol catalyzed by Pt/WO3/ZrO2[END_REF].

Furthermore, the obtained results could be correlated with a previous study by Lopez-Tenllado et al. [START_REF] López-Tenllado | A comparative study of Bi2WO6, CeO2, and TiO2 as catalysts for selective photo-oxidation of alcohols to carbonyl compounds[END_REF] dealing with the selective photooxidation of organic molecules (crotyl alcohol to crotonaldehyde). This reaction was performed on titania, ceria and bismuth tungstate catalysts, where the latter was the most selective. This phenomenon was attributed to the lowest adsorption of the aldehyde reaction product on the tungsten-based catalyst. In other words, this material seemed to favor the desorption of the desired product, avoiding its further photooxidation. Therefore, according to the obtained results and the above-mentioned studies, we suggest that the higher selectivity/yield achieved by the WO3-based catalysts can be attributed to the incorporation of additional acid sites to the anatase semiconductor. These new sites would allow enhancing the selective activation of primary C-O bonds in glycerol towards the production of glyceraldehyde and, on the other hand, to quickly desorb aldehyde products in the liquid phase. A possible path of reactions over TiO2-based and WO3-based materials has been put forward according to the above discussion (Scheme 1).

Scheme 1

All in all, this study demonstrates that WO3-based materials are active for the valorization of glycerol towards a high added-value product: glyceraldehyde. All these materials exhibit outstanding advantages. On the one hand, the as-prepared WO3 catalyst leads to a high yield of glyceraldehyde even at very high conversion levels. However, its applicability could be compromised by its slow overall kinetics. In this sense, the calcined WO3 catalyst, with improved crystallinity, seemed to enhance the kinetics without compromising the glyceraldehyde yield, although the calcination step could eventually increase the overall production cost of this material. Finally, commercial DTW5 led to lower glyceraldehyde yields than pure WO3 catalysts (but still much higher than those for pure TiO2-based materials), and overall faster kinetics. Therefore, further studies could optimize the composition and synthesis procedure of the photocatalyst for practical purposes.

Conclusions

In this study, we demonstrated, for the first time in literature, the high performance of WO3based catalysts for the selective photooxidation of glycerol towards glyceraldehyde. By comparing the activity of pure TiO2 (Anatase, Rutile and P25anatase/rutile) and pure WO3

(prepared by a simple hydrothermal method), we can conclude that: i) TiO2 catalysts allowed for a higher overall conversion of glycerol, where the selectivity to glyceraldehyde was hindered by its further photooxidation, and ii) WO3 catalysts exhibit slower kinetics for glycerol oxidation, but show an outstanding selectivity and yield of glyceraldehyde. While the activity of TiO2 was attributed to electronic effects (better separation of photogenerated charge carriers), the high glyceraldehyde selectivity of WO3 was linked to its enhanced acidity, which selectively activates C-O bonds in glycerol, and facilitates the desorption of glyceraldehyde (avoiding its further photooxidation). Hence, a commercial WO3/TiO2 material (DTW5) was proven to combine the best features of both semiconductors, with high levels of glycerol conversion and selectivity to glyceraldehyde. This study opens therefore a new field dealing with the development of advanced photocatalytic materials for an advanced valorization of glycerol into high added-value products. Tables captions:

Figure 5 Figure 5

 55 Figure 5 Figure 5 depicts the spectra of pyridine chemisorbed at 100 o C. The bands obtained at 1448

Figure 1 .Figure 2 .Figure 3 .Figure 4 .

 1234 Figure 1. X-ray diffraction (XRD) patterns of commercial catalysts (Anatase, Rutile, P25 and DTW5) and as-prepared WO3

Figure 5 .

 5 Figure 5. pyridine-DRIFTS spectra of P25, DTW5 and WO3. B and L denote Brönsted and Lewis acid sites, respectively.

Figure 6 .

 6 Figure 6. Glycerol conversion over commercial catalysts (Anatase, Rutile, P25 and DTW5) and as-prepared WO3. Reaction conditions: Room temperature, Glycerol initial concentration: 1.10 mmol L -1 , Volume of glycerol solution: 30 mL, UV irradiation intensity of 5.60 mW cm 2

Figure 7 . 2 Figure 8 . 2 Figure 9 . 2 Figure 10 .

 72829210 Figure 7. Evolution of products with time over commercial catalysts (Anatase, Rutile, P25 and DTW5) and as-prepared WO3. Reaction conditions: Room temperature, Glycerol initial concentration: 1.10 mmol L -1 , Volume of glycerol solution: 30 mL, UV irradiation intensity of 5.60 mW cm -2

Fig. 1 .Fig. 2 .

 12 Fig. 1. X-ray diffraction (XRD) patterns of commercial catalysts (Anatase, Rutile, P25 and DTW5) and as-prepared WO3

Fig. 3 .Fig. 4 .Fig. 5 .

 345 Fig. 3. (a) UV-Vis diffuse reflectance spectroscopy (DRS) spectra and (b) Tauc plot for band gap determination for commercial catalysts (Anatase, Rutile, P25 and DTW5) and asprepared WO3

Fig. 6 .

 6 Fig. 6. Glycerol conversion over commercial catalysts (Anatase, Rutile, P25 and DTW5) and as-prepared WO3. Reaction conditions: Room temperature, Glycerol initial concentration: 1.10 mmol L -1 , Volume of glycerol solution: 30 mL, UV irradiation intensity of 5.60 mW cm 2

Fig. 7 . 2 Fig. 8 . 2 Fig. 9 . 2 Fig. 10 .

 72829210 Fig. 7. Evolution of products with time over commercial catalysts (Anatase, Rutile, P25 and DTW5) and as-prepared WO3. Reaction conditions: Room temperature, Glycerol initial concentration: 1.10 mmol L -1 , Volume of glycerol solution: 30 mL, UV irradiation intensity of 5.60 mW cm -2

Table 1 .

 1 BET specific surface area, pore volume, pore size, crystallinity and average crystallite size of the photocatalysts

Table 2 .

 2 Comparison of Lewis, Brönsted and total acid sites of P25, DTW5 and WO3,

	obtained by pyridine-DRIFTS

Table 3 .

 3 Comparison of glyceraldehyde Yield and Selectivity for all the photocatalysts at similar levels of glycerol conversion

	Scheme captions:
	Scheme 1. A possible path for photocatalytic glycerol oxidation

Table 1 .

 1 BET specific surface area, pore volume, pore size, crystallinity and average crystallite size of the photocatalysts

	Sample	SBET (m 2 g -1 )	Porous volume (cm 3 g -1 )	Mean Pore Size (nm)	Crystallinity a (%)	Crystal size a (nm)
	Anatase	92	0.43	15.2	82.0	17
	Rutile	85	0.39	16.9	80.8	16
						23 (Anatase)
	P25	63	0.17	10.5	81.6
						33 (Rutile)
	DTW5	78	0.31	13.8	84.9	20 (Anatase)
	WO3	33	0.07	13.3	48.6	17

a Calculated from XRD spectra

Table 2 .

 2 Comparison of Lewis, Brönsted and total acid sites of P25, DTW5 and WO3,

		obtained by pyridine-DRIFTS
		µmol Py • g -1 • m -2 Cat.
	Catalyst	Lewis Brönsted	Total	L/B
	P25	0.87	0.21	1.08	4.2
	DTW5	0.95	0.50	1.45	1.9
	WO3	0.64	3.33	3.97	0.2

Table 3 .

 3 Comparison of glyceraldehyde Yield and Selectivity for all the photocatalysts at similar levels of glycerol conversion

	Sample	Glycerol conversion (%)	Yield of glyceraldehyde (%)	Selectivity of glyceraldehyde (%)
	Anatase	43	2	4
	Rutile	44	/	/
	P25	47	3	5
	DTW5	43	8	19
	WO3	44	13	29
	Homogeneous TiO2 [27]	35	5	13
	P25 [28]	36	5	13
	Lab-made TiO2 [53]	97	5	5
	Black TiO2 [54]	92	3	3

Acknowledgment

The authors would like to thank the CSC (CHINA SCHOLARSHIP COUNCIL) for the Jie Yu Ph.D. grant (CSC No. 201906740016). The authors also thank L. Burel (HRTEM) Y. Aizac (PXRD) and P. Mascunan (BET measurements) of IRCELYON.


Luís H De Oliveira

Pollyana Trigueiro

Baptiste Rigaud

Edson C Da Silva-Filho

Josy A Osajima

Maria G Fonseca

Jean-François Lambert

Thomas Georgelin

Maguy Jaber
email: maguy.jaber@upmc.fr

When RNA meets montmorillonite: influence of the pH and divalent cations

Keywords: Ribonucleic acid, Clay minerals, Montmorillonite, Prebiotic chemistry

A key question for the investigation of the origins of life is to understand the interaction between complex organic molecules and minerals. In this general frame, the present study investigated the nature of the interactions at the molecular level between a ribonucleic acid and ion-exchanged montmorillonites. We observed that the formation of RNA/clay mineral complexes was strongly pH-dependent. In a sufficiently acidic medium, the RNA molecules were intercalated in the interlayer space, in a flat-lying conformation, with a large contribution of electrostatic interactions that may be complemented by hydrogen bonds. The secondary structure of the RNA strands was strongly affected. The presence of different cations such as Na + , Ca 2+ , Mg 2+ , and Sr 2+ influenced the adsorption of organic molecules.

Apparently, metal cations directly took part in the formation of bridges between the negative charges on the mineral surface and the phosphate groups of the biomolecule.

Introduction

Interactions between biomolecules and clay minerals are key for different fields like separations processes and hybrid nanomaterials. Among clay minerals, montmorillonites belong to the group of smectites with a 2:1 layer structure, negative surface charges (due to isomorphic substitutions), counterbalanced by cations present in the interlayer space, high internal surface area, cation exchange capacity and reactive groups on their surfaces, as well as the ability to confine molecules in the interlayer space [START_REF] Jaber | Synthesis, characterization and applications of 2:1 phyllosilicates and organophyllosilicates: Contribution of fluoride to study the octahedral sheet[END_REF][START_REF] Wu | Influence of interlayer cations on organic intercalation of montmorillonite[END_REF][START_REF] Escamilla-Roa | A DFT study of the adsorption of glycine in the interlayer space of montmorillonite[END_REF]. These intrinsic properties make them possible "hosts" for biomolecules such as RNA [START_REF] James Cleaves | Mineral-organic interfacial processes: Potential roles in the origins of life[END_REF][START_REF] Ruiz-Mirazo | Prebiotic systems chemistry: New perspectives for the origins of life[END_REF].

The mechanisms of nitrogen bases, nucleotides and RNA/montmorillonite interactions have been reviewed and both electrostatic interactions and hydrogen bonding were found to play a role [START_REF] Yu | Adsorption of proteins and nucleic acids on clay minerals and their interactions: A review[END_REF]. Some studies suggested the participation of the interlayer cations as a "bridge" for interaction between the negatively charged clay surface and the phosphate of the RNA, which also bears a negative charge [START_REF] Franchi | Cations as mediators of the adsorption of nucleic acids on clay surfaces in prebiotic environments[END_REF][START_REF] Pedreira-Segade | Effects of salinity on the adsorption of nucleotides onto phyllosilicates[END_REF][START_REF] Hao | Transition Metals Enhance the Adsorption of Nucleotides onto Clays: Implications for the Origin of Life[END_REF]. Alkaline earth metal cations have been observed to strongly enhance the adsorption of single-strand (but not double-strand) RNA to montmorillonite [START_REF] Gujjari | Factors affecting the association of single-and double-stranded RNAs with montmorillonite nanoclays[END_REF]. Some studies suggest the likely presence of these metallic cations in rocks and in the primitive ocean [START_REF] Eder | Influence of alkali-and alkaline-earth-metal cations on the "salt-induced peptide formation" reaction[END_REF][START_REF] Izawa | Composition and evolution of the early oceans: Evidence from the Tagish Lake meteorite[END_REF]. They are important for the folding, structure and function of RNA, especially the divalent ion Mg 2+ [START_REF] Pyle | Metal ions in the structure and function of RNA[END_REF][START_REF] Leonarski | Mg2+ ions: Do they bind to nucleobase nitrogens?[END_REF], and their involvement has been suggested in the stabilization and/or activation of biological macromolecules [START_REF] Black | Biological coordination chemistry of magnesium, sodium, and potassium ions. Protein and nucleotide binding sites[END_REF]. Villafañe-Barajas et al. have also underlined the importance of added cations for the adsorption of nucleotides, the monomers of RNA; and molecular modelling studies [START_REF] Carrascoza Mayén | Prebiotic Soup Components Trapped in Montmorillonite Nanoclay Form New Molecules: Car-Parrinello Ab Initio Simulations[END_REF] suggest that they could play a significant role in the reactivity of adsorbed organic molecules.

pH is a key parameter that controls the adsorption of biomolecules, since acido-basic speciation determines the electrostatic interaction between RNA and the surface [START_REF] Theng | Clay-polymer interactions: Summary and perspectives[END_REF][START_REF] Lawless | pH Profile of the Adsorption of Nucleotides onto Montmorillonite.1. Selected Homoionic Clays[END_REF]. Some studies in recent years have addressed the interaction of nitrogen bases [START_REF] Benetoli | Adsorption of nucleic acid bases on clays: An investigation using Langmuir and Freundlich isotherms and FT-IR spectroscopy[END_REF][START_REF] Hashizume | Adsorption of adenine, cytosine, uracil, ribose, and phosphate by Mg-exchanged montmorillonite[END_REF][START_REF] Carneiro | Adsorption of Adenine, Cytosine, Thymine, and Uracil on Sulfide-Modified Montmorillonite: FT-IR, Mössbauer and EPR Spectroscopy and X-Ray Diffractometry Studies[END_REF], nucleotides [START_REF] Feuillie | Adsorption of nucleotides onto Fe-Mg-Al rich swelling clays[END_REF][START_REF] Pedreira-Segade | Adsorption of nucleotides onto ferromagnesian phyllosilicates: Significance for the origin of life[END_REF][START_REF] Hao | Transition Metals Enhance the Adsorption of Nucleotides onto Clays: Implications for the Origin of Life[END_REF] and less often nucleosides [START_REF] Fornaro | Binding of Nucleic Acid Components to the Serpentinite-Hosted Hydrothermal Mineral Brucite[END_REF] with montmorillonite, varying the pH and the nature of metal cations, and trying to elucidate the types of interactions between these molecules and montmorillonite. However, the interactions of polymeric RNA with the surface of clay minerals remain understudied, especially at the molecular level. In this perspective, the aim of this study was to evaluate the adsorption of RNA (ribonucleic acid diethylaminoethanol salt) on montmorillonite in order to contribute to the understanding of a bigger puzzle, the origin of life. The organic-inorganic interactions were investigated at the molecular level and the influence of different parameters such as nature of the interlayer cation (Na + , Ca 2+ , Mg 2+ , and Sr 2+ ) and the pH was evaluated.

Materials and Methods

Chemicals

Ribonucleic acid diethylaminoethanol salt (diethylethanolammonium; ribonucleic acid derived from Torula yeast, CAS 63231-63-0, Sigma-Aldrich), hydrofluoric acid (HF, 40% w/w, Fluka), sodium acetate (CH3COONa, 99%, Sigma-Aldrich), magnesium acetate ((CH3COO)2Mg. 4H2O, 99%, Sigma-Aldrich), Al2O3 (98%, Pural-Sasol), and SiO2 (99.8%, Aerosil 130, Evonik Industries). The others chemicals were purchased from Sigma-Aldrich (analytical grade). All chemicals were used without any previous purification.

Synthesis of montmorillonite

The sodium montmorillonite (Na-Mt) used in this work was synthesized according to the procedure described previously (Jaber and Miehé-Brendlé, 2005;[START_REF] Jaber | Synthesis, characterization and applications of 2:1 phyllosilicates and organophyllosilicates: Contribution of fluoride to study the octahedral sheet[END_REF]Trigueiro et al., 2018bTrigueiro et al., , 2018a) ) and had the ideal formula per half unit cell Na0.2[Si4Al1.8Mg0.2O10(OH,F)2]. The reagents were mixed in the following order: 196.860 g of deionized water, 2.410 g of hydrofluoric acid, 0.990 g of sodium acetate, 2.587 g of magnesium acetate, 3.325 g of alumina and 7.249 g of silica. The obtained hydrogel was aged under stirring at room temperature for 2 h and then was autoclaved at 220°C for 72 h. The autoclave was cooled to room temperature and the product was thoroughly washed with distilled water and centrifuged. Finally, the synthesized Na-Mt was dried at 50°C for 72 h.

The synthesis conditions had previously been optimized for the crystallization of pure montmorillonite. The Al/Mg ratio influenced the preferential location of Al 3+ in the tetrahedral sheets. The initial pH of the hydrogel, around 5, determined the di-or trioctahedral character of the obtained phyllosilicate and the Si/F ratio as well as the synthesis time influenced the crystallinity of pure montmorillonite [START_REF] Reinholdt | Fluorine Route Synthesis of Montmorillonites Containing Mg or Zn and Characterization by XRD, Thermal Analysis, MAS NMR, and EXAFS Spectroscopy[END_REF][START_REF] Reinholdt | Synthesis and characterization of montmorillonite-type phyllosilicates in a fluoride medium[END_REF].

Ion exchange reaction

The ion exchange of Na-Mt was performed according to previously published procedures [START_REF] Lepoitevin | BSA and lysozyme adsorption on homoionic montmorillonite: Influence of the interlayer cation[END_REF]. Na-Mt was suspended in 1 mol L -1 of CaCl2.2H2O, MgCl2.6H2O or Sr(NO3)2 solutions for 24 h under magnetic stirring at 25°C. Then, the obtained materials were centrifuged, washed in deionized water and dried at 50°C. The same process was repeated twice. Finally, the solids were washed and dried in the same conditions.

The exchanged montmorillonites will be designated as Ca-Mt, Mg-Mt, and Sr-Mt.

Adsorption of RNA salt on montmorillonite

The adsorption of RNA on the exchanged montmorillonites was performed according to a procedure published previously [START_REF] Greaves | The adsorption of nucleic acids by montmorillonite[END_REF] with some modifications.

Initially, the clay mineral samples (Na-Mt, Ca-Mt, Mg-Mt and Sr-Mt) were dispersed in water at a 1.25 mg mL -1 concentration over a period of 4 h under magnetic stirring at 25°C. In all cases, the natural pH of all clay mineral dispersions was around 5.0. Then, 1 mg mL -1 of RNA solution (pH 6.8) was slowly added. The RNA/Mt ratio was 2:1 w/w for all systems. After this step, the pH of all systems was between 6.2-6.5 without pH adjustment. The system was kept under magnetic stirring for 16 h at 25°C. The same procedure was also performed for all systems at pH 2.9-3.1, adjusted with 0.5 mol L -1 of the HCl solution. The resulting samples were centrifuged, washed with deionized water and dried at 50°C overnight. As a control, Na-Mt was maintained at pH 3.0 without RNA in the same conditions and analyzed after the reaction.

Characterizations

X-ray diffraction (XRD): powder X-ray diffraction was carried out on a Shimadzu XRD-6000 model diffractometer equipped with a monochromatic CuKα (λ = 0.15406 nm) source operating at 2 kVA, 40 kV, and 30 mA. The diffraction patterns were recorded from 3 to 80° with a scan rate of 0.5° min -1 in the same conditions as for previous works [START_REF] França | Monitoring diclofenac adsorption by organophilic alkylpyridinium bentonites[END_REF][START_REF] Da Silva | What happens when chitosan meets bentonite under microwave-assisted conditions? Clay-based hybrid nanocomposites for dye adsorption[END_REF].

Scanning electron microscopy (SEM):

Scanning electron microscopy was performed using a FEI/Thermo Fischer, model Quanta FEG 250 with cannon per field and an acceleration voltage of 1 to 30 kV. Samples were fixed on carbon tape and coated with gold in a Quorum model Q150R sputter-coater for 35 s at 20 mA by a plasma generated under an argon atmosphere.

Fourier transform infrared spectroscopy: the FTIR spectra of samples -dispersed in KBr pellets containing approximately 1% (w/w) of the sample -were recorded using a Shimadzu PESTIGE -21 model spectrometer over the 4000 -400 cm -1 range at a resolution of 4 cm -1 and with 30 scans for each run.

Thermal analyses (TG-DTG): Thermogravimetric analyses and derivate thermogravimetry were performed using equipment from TA Instruments SDT Q600 model.

The samples were heated from 20 to 1000°C in an alumina crucible with a heating rate of 10°C min -1 under nitrogen flow.

Solid state nuclear magnetic resonance: 31 P, 13 C, and 27 Al MAS NMR spectra were obtained on a Bruker Avance III spectrometer equipped with a 4 mm H-X probe, operating at Larmor frequencies of 500.17 MHz ( 1 H), 202.47 MHz ( 31 P), 125.77 MHz ( 13 C) and 130.33 MHz ( 27 Al) based on previous studies [START_REF] Guillermin | New pigments based on carminic acid and smectites: A molecular investigation[END_REF][START_REF] Rodrigues | Confinement and Time Immemorial: Prebiotic Synthesis of Nucleotides on a Porous Mineral Nanoreactor[END_REF][START_REF] Cavalcanti | Inorganic-organic hybrid pigments based on carminic acid and clay minerals[END_REF]. Chemical shifts were calibrated using triphenylphosphine (19.64 ppm) for 31 P, the carboxyl signal of adamantane (38.52 ppm) for 13 C, and AlNO3 (0 ppm) for 27 Al as external standards. All spectra were recorded with a spinning rate of 14 kHz. The 13 C Cross-Polarization spectra were acquired with a ramp-CP contact time of 2 ms and a 1.5 s recycle delay and with a 1 H decoupling spinal 64. The acquisition time was 40 ms. The number of scans to obtain the spectra varied depending on the S/N obtained for each sample.

Spectra were processed with a zero-filling factor of 2 and with an exponential decay corresponding to a 50 Hz line broadening in the transformed spectra. Only spectra with the same line broadening are directly compared. 27 Al single-pulse spectra using a 30° pulse were recorded with 100 kHz spectral width and with a recycle delay of 500. 31 P experiments using a 90° pulse were acquired with a recycle delay of 5 s and with a 1 H decoupling spinal 64.

Results

XRD diffraction

XRD patterns of the sodium and exchanged montmorillonites and resulting RNA-Mt hybrids are presented in Figure 1. A reflection at 6.77° (2θ), corresponding to a d001-value of 1.30 nm, was observed for Na-Mt. Other reflections were observed at 2θ = 19.91°, 35.31°, 54.71° and 62.13° and were indexed to (110) (0.445 nm), (200) (0.253 nm), (210) (0.167 nm) and (060) (0.149 nm) reticular planes, respectively [START_REF] Jaber | Selectivities in Adsorption and Peptidic Condensation in the (Arginine and Glutamic Acid)/Montmorillonite Clay System[END_REF]Trigueiro et al., 2018a).

The formation of the Na-Mt was monitored by SEM (Figure SM-1). Well-formed particles in typical plates morphology were observed without the presence of other phases.

For all ion-exchanged samples, the (001) reflections shifted up to 1.56 nm.

Figure 1:XRD patterns of (i) Na-Mt, Ca-Mt, Mg-Mt, and Sr-Mt montmorillonites and of the RNA@cations-Mt hybrids prepared at (ii) pH 6.2-6.5 and (iii) pH 2.9-3.1.

After adsorption, the observed pH of the solutions varied between 6.2-6.5 and 2.9-3.1, for systems without and with pH adjustment. Figure 1(ii) depicts the XRD patterns of RNA@cations-Mt at pH 6.2-6.5 and 1(iii) pH 2.9-3.1. At pH 6.2-6.5, the d001-value is 1.37 nm for all samples. At pH 2.9-3.1, in Figure 1(iii), the (001) reflection shifted to higher basal distances: d001-value= 2.03 nm, 1.87 nm, 1.89 nm, and 1.85 nm in RNA@Na-Mt, RNA@Ca-Mt, RNA@Mg-Mt and RNA@Sr-Mt, respectively, corresponding to interlayer spacings in the 0.9 nm range. In contrast, XRD (Figure SM-2) of the Na-Mt maintained in an acidic medium (pH 3) without the presence of RNA molecules presented the same reflections as the original sample. In particular, the d001 value stayed at 1.30 nm and did not show any broadening, suggesting that no alteration in long-range order or formation of exfoliated phase occurred in the conditions of the synthesis.

Infrared spectroscopy

FTIR spectra of the solids before and after RNA adsorption are presented in Figure 2.

Irrespective of the compensating cation, the spectra were similar with absorptions at 3638 cm - 1 and 3400 cm -1 (broad) assigned to OH stretching of structural OH and water, respectively [START_REF] Bizovská | Montmorillonite modified with unconventional surfactants from the series of octylammonium-based cations: Structural characterization and hydration properties[END_REF]. Bands at 1640 and 922 cm -1 were attributed to water deformation, and to the OH bending bands in Al2OH, respectively [START_REF] Madejová | IR Spectra of Clay Minerals[END_REF].

Absorptions at 1120 cm -1 and 1046 cm -1 were assigned to Si-O-Si asymmetric and symmetric stretchings, respectively, in the silicate matrix [START_REF] Bizovská | Montmorillonite modified with unconventional surfactants from the series of octylammonium-based cations: Structural characterization and hydration properties[END_REF]. Two bands at 790 cm -1 and 622 cm -1 were associated with Al-O-Si bending and to Al-O out-of-plane and Si-O vibrations [START_REF] Madejová | IR Spectra of Clay Minerals[END_REF]. Furthermore, Al-O-Si and Si-O-Si bending modes were observed at 528 and 471 cm -1 , respectively [START_REF] Pentrák | Alteration of fine fraction of bentonite from Kopernica (Slovakia) under acid treatment: A combined XRD, FTIR, MAS NMR and AES study[END_REF]. If amorphous silica was present, it would be detected by absorption bands at 800 -810 cm -1 [START_REF] Costa | Infrared and thermogravimetric study of high pressure consolidation in alkoxide silica gel powders[END_REF][START_REF] Pentrák | Near-IR study of water adsorption on acid-treated montmorillonite[END_REF][START_REF] Santos | Acid-leached mixed vermiculites obtained by treatment with nitric acid[END_REF]; they were not observed in present case. Figure 2. FTIR spectra for (i) Na-Mt, Ca-Mt, Mg-Mt and Sr-Mt montmorillonites, and spectra in range 2200 -400 cm -1 for RNA and RNA@cations-Mt hybrids obtained at (ii) pH 6.2-6.5 and (iii) pH 2.9-3.1.

Figure 2ii and iii shows FTIR spectra in the 400-2000 cm -1 range of RNA and solids after RNA adsorption. FTIR spectra in the complete 400-4000 cm -1 range are presented in Figures SM-2i and ii. The spectrum of RNA presents a band at 3387 cm -1 (Figure SM-2i and ii) associated with the N-H stretch and one at 2954 cm -1 due to the C-H stretch [START_REF] Tsuboi | Application of Infrared Spectroscopy to Structure Studies of Nucleic Acids[END_REF]. The bands at 1690, 1644 and 1602 cm -1 were associated with the C=O vibration on purine and pyrimidines [START_REF] Tsuboi | Application of Infrared Spectroscopy to Structure Studies of Nucleic Acids[END_REF][START_REF] Banyay | A library of IR bands of nucleic acids in solution[END_REF]. The absorption band at 1473 cm -1 has been attributed to ring vibrations of the uracil base [START_REF] Kumar | Zn2+-induced folding of RNA to produce honeycomb-like RNA-mediated fluorescing Zn2+/PbSe nanostructures[END_REF] and the one at 1400 cm -1 was attributed to in-plane C-O-H deformation mode, at 2' in the ribose ring [START_REF] Tsuboi | Application of Infrared Spectroscopy to Structure Studies of Nucleic Acids[END_REF][START_REF] Banyay | A library of IR bands of nucleic acids in solution[END_REF]. The bands at 1240 and 1080 cm -1 were attributed to asymmetrical and symmetrical PO2 -stretch, respectively [START_REF] Tsuboi | Application of Infrared Spectroscopy to Structure Studies of Nucleic Acids[END_REF][START_REF] Benedetti | Determination of the relative amount of nucleic acids and proteins in leukemic and normal lymphocytes by means of fourier transform infrared microspectroscopy[END_REF] and the band at 1118 cm -1 to the C-O stretch in ribose [START_REF] Benedetti | Determination of the relative amount of nucleic acids and proteins in leukemic and normal lymphocytes by means of fourier transform infrared microspectroscopy[END_REF]. Finally, the band at 967 cm -1 is an unidentified ribose-phosphate main chain vibrations, and that at 811 cm -1 is a sugar marker of complex origin [START_REF] Tsuboi | Application of Infrared Spectroscopy to Structure Studies of Nucleic Acids[END_REF][START_REF] Banyay | A library of IR bands of nucleic acids in solution[END_REF].

The spectra of the solids after RNA adsorption in the 400-2000 cm -1 range are depicted in Figures 2ii andiii. At pH 6.2-6.5 (Figure 2ii), similar new bands were observed in the FTIR spectra of the solids, both at the same position as in the spectra of RNA: 1473 cm -1 (ring vibrations) and 1400 cm -1 (in-plane C-O-H deformation of ribose). Other diagnostic bands of RNA may be present as shoulders, but are hidden by the strong absorption of the clay mineral lattice in the 900-1200 cm -1 region.

Infrared spectra of the hybrids obtained in an acidic medium (Figure 2iii) showed different additional bands at 1693 cm -1 (C=O stretching in nucleobases). The band of PO2 -, observed at 1240 cm -1 in RNA spectra (Figure 3), shifted to 1227 cm -1 in the RNA@cations-Mt.

Bathochromic shifts of the bands attributed to the structural OH of the clay mineral lattice (cations-Mt: 3638 cm -1 , RNA@cations-Mt at pH 6.2-6.5: 3632 cm -1 and RNA@cations-Mt at pH 2.9-3.1: 3630 cm -1 ), were observed (see Supplementary materials 2.9-3.1 are shown in Figure 3i and ii. All precursor samples presented characteristic chemical shifts at 3.9 and 69.1 ppm. Another chemical shift was observed in both spectra at 55 ppm.

The same chemical shifts were observed in the RNA@cations-Mt samples.

Figure 3. 27 Al NMR spectra of the (i) exchanged montmorillonites and (ii) RNA@Mt hybrids obtained at pH 2.9-3.1.

The 31 P spectrum of RNA is shown in Figure 4i. A broad, asymmetric signal is observed with a maximum at +1.4 ppm. For the RNA@cations-Mt samples, a strong new component appeared at -9.3 to -10.6 ppm, i.e., 8 to 9 ppm upfield from the signal in pristine RNA.

Figure 4. 31 P NMR spectra of RNA (i) and hybrids RNA@Na-Mt (ii), RNA@Ca-Mt (iii), RNA@Mg-Mt (iv) and RNA@Sr-Mt (v) in pH 2.9-3.1. 14 13 C NMR spectra of the RNA and the hybrids obtained at pH 2.9-3.1 are depicted in Figure 5 and values of chemical shifts and respective attributions are summarized in Table 1.

The observed NMR signals for the RNA@Mt hybrids were similar.

The signals at 60 -90 ppm were assigned to carbons in the ribose moiety [START_REF] Chang | Solid-State Carbon-13 Nuclear Magnetic Resonance Study of Ribonucleosides and Ribonucleic Acid[END_REF][START_REF] Rodrigues | Confinement and Time Immemorial: Prebiotic Synthesis of Nucleotides on a Porous Mineral Nanoreactor[END_REF]. Signals in the 100 -170 ppm range were attributed to the nucleotides bases [START_REF] Chang | Solid-State Carbon-13 Nuclear Magnetic Resonance Study of Ribonucleosides and Ribonucleic Acid[END_REF][START_REF] Fujiwara | 13C-13C and 13C-15N Dipolar Correlation NMR of Uniformly Labeled Organic Solids for the Complete Assignment of Their 13C and 15N Signals: An Application to Adenosine[END_REF]. In particular, the signals of carbon positions G4 and A4 were located at 150.3 ppm, and were assigned to C=C groups in both nucleotides bases. This signal was also present in the RNA@Mt hybrids, but shifted to 151.5 ppm. The signal at 165.4 ppm associated with the carbon at position 4 in cytosine (-(NH2) C=N) group, shifted to 166.4, 166.6, 166.4, and 166.5 ppm, in the RNA@Na-Mt, RNA@Ca-Mt, RNA@Mg-Mt, and RNA@Sr-Mt, respectively. The other well-individualized signals did not shift as much.

Figure 5. 13 C NMR spectra of the RNA and RNA@Mt hybrids obtained at pH 2.9-3.1 (The symbol C' represents the carbons present in the ribose group and U5, for example, represents the carbon 5 in the uracil nitrogen base).

Table 1. 13 C chemical shifts of RNA and RNA@clay hybrids in pH 2.9-3.1*. [START_REF] Lapper | A Carbon-13 Nuclear Magnetic Resonance Study of the Conformations of 3′,5′-Cyclic Nucleotides[END_REF]. C' refers the carbons of the ribose group; A4, C4, U4 and G4 for example, refer to carbon 4 in the adenine, cytosine, uracil, and guanine nitrogen base, respectively.

Thermogravimetry

TG and DTG curves of the solids before and after RNA adsorption at pH 2.9-3.1 are presented in Figure SM-3, and summarized in Table SM1. It was hoped that the amount of intercalated RNA could be estimated by integrating the weight losses after 200°C and correcting for the weight loss of the raw montmorillonite clay. Some thermal events attributable to the degradation of RNA are indeed apparent between 200 and 460°C, but organic matter elimination is not complete even at 1000°C, so that the amounts tabulated are to be interpreted as lower bounds. They appear to be only little sensitive to the nature of the compensation cation, or even to its charge.

Discussion

XRD diffraction

The XRD patterns of the sodium montmorillonite confirm the expected dioctahedral character of the sample according to the 2θ = 62.13° reflection, indexed (060) plane; in other words, they indicate the predominance of trivalent cations in the octahedral sheet of Na-Mt [START_REF] Moore | X-Ray Diffraction and the Identification and Analysis of Clay Minerals[END_REF][START_REF] Reinholdt | Fluorine Route Synthesis of Montmorillonites Containing Mg or Zn and Characterization by XRD, Thermal Analysis, MAS NMR, and EXAFS Spectroscopy[END_REF][START_REF] Reinholdt | Synthesis and characterization of montmorillonite-type phyllosilicates in a fluoride medium[END_REF]. For the exchanged samples, the observation of the (001) reflections at 1.56 nm for all samples are compatible with a two-layer hydrate in the interlayer, as compared to a one-layer hydrate in the starting material [START_REF] Cases | Mechanism of adsorption and desorption of water vapor by homoionic montmorillonite. 3. The Mg 2+ ,Ca 2+ ,Sr 2+ and Ba 2+ exchanged forms[END_REF][START_REF] Cavalcante | Organophilization of a Brazilian Mg-montmorillonite without prior sodium activation[END_REF][START_REF] Chang | Mechanism of tyramine adsorption on Camontmorillonite[END_REF].

After RNA adsorption, there is no intercalation of bulky species in the interlayer space in the pH 6.2-6.5 systems, as the d001 still corresponds to a two-water layer hydrate. The possibility of intercalation strongly depends on the electrostatic interactions between the clay mineral layers (negatively charged) and the molecule to be intercalated. Individual nucleotides exhibit two or three acid-basic couples due to the protonation/deprotonation of nucleobase nitrogens and of the bridging phosphate P-OH [START_REF] Martin | Nucleoside Sites for Transition Metal Ion Binding[END_REF][START_REF] Feuillie | Adsorption of nucleotides onto Fe-Mg-Al rich swelling clays[END_REF]. Deprotonation of the heterocycle NH group is possible for GMP and UMP nucleotides (pKa = 9.4 to 9.5) but this equilibrium is not active in our pH conditions. The nucleobases may be protonated in the cases of AMP (N1, pKa = 3.84), CMP (N3, pKa = 4.33) and GMP (N7, pKa = 2.48) [START_REF] Feuillie | Attachment of ribonucleotides on αalumina as a function of ph, ionic strength, and surface loading[END_REF]. This part of the nucleotides would then be mostly neutral at pH 6.2 to 6.5, while at pH 2.9 to 3.1, the majority of AMP and CMP groups, and a minority of the GMP (20 to 30%) would be protonated. Finally, the phosphate group has a pKa of the order of 0.5 and is always deprotonated in our conditions.

Thus, in a neutral medium, each nucleotide residue would bear a net negative charge of -1 and the intercalation of the biopolymer would be electrostatically disfavored because of repulsive interactions with the negative clay mineral layers, since in all pH conditions, montmorillonite has a negative charge [START_REF] Saka | The effects of electrolyte concentration, ion species and pH on the zeta potential and electrokinetic charge density of montmorillonite[END_REF][START_REF] Yukselen | Artificial neural networks approach for zeta potential of Montmorillonite in the presence of different cations[END_REF][START_REF] Liu | Stability of Na-montmorillonite suspension in the presence of different cations and valences[END_REF][START_REF] Şans | Contribution of cations and layer charges in the smectite structure on zeta potential of Ca-bentonites[END_REF][START_REF] Guillermin | New pigments based on carminic acid and smectites: A molecular investigation[END_REF]. It is therefore likely that RNA molecules at pH 6.2-6.5 are adsorbed on the external surface and edges of the clay mineral samples [START_REF] Mignon | Theoretical study of the adsorption of RNA/DNA bases on the external surfaces of Na+-montmorillonite[END_REF][START_REF] Mignon | Theoretical study of the adsorption of DNA bases on the acidic external surface of montmorillonite[END_REF]. In addition, the cations (Na + , Ca 2+ , Mg 2+ and Sr 2+ ) may be contributing to adsorption, by creating "ion bridges" between the negative surface of montmorillonite and the phosphate groups present in RNA. A similar mechanism was invoked for the adsorption of DNA in montmorillonite in the presence of different cations [START_REF] Sheng | Metal cation saturation on montmorillonites facilitates the adsorption of DNA via cation bridging[END_REF].

For the system at pH 2.9-3.1, higher basal reflections were observed for all samples, indicating the intercalation of a rather large biomolecule in the interlayer space of the clay mineral. The occurrence of intercalation is associated with the partial protonation of the nitrogen bases in the RNA nucleotides at pH values lower than their pKa. With the pKa values listed above, and supposing that the four possible nucleotide monomers have similar abundances in the RNA chain, it is easy to calculate that at pH 2.9-3.1, the chain bears a charge that is reduced by over one half with respect to pH 6.2-6.5 -about 0.48 negative charges per monomer. In these conditions, electrostatic repulsion between the RNA chain and the negatively charged surface is reduced, permitting intercalation. These interpretations are compatible with other studies in RNA adsorption [START_REF] Greaves | The adsorption of nucleic acids by montmorillonite[END_REF][START_REF] Jelavić | Prebiotic RNA polymerisation: energetics of nucleotide adsorption and polymerisation on clay mineral surfaces[END_REF] and also studies about the adsorption of the nitrogen bases [START_REF] Carneiro | Adsorption of Adenine, Cytosine, Thymine, and Uracil on Sulfide-Modified Montmorillonite: FT-IR, Mössbauer and EPR Spectroscopy and X-Ray Diffractometry Studies[END_REF] and nucleotides (Pedreira-Segade et al., 2016) on montmorillonite. Similar results were observed in the RNA intercalation in montmorillonite in an acidic medium (pH 5 to 3.5) [START_REF] Greaves | The adsorption of nucleic acids by montmorillonite[END_REF]. Other studies observed no displacement of the basal plane in torula yeast RNA/Mg-smectite [START_REF] Viennet | Influence of the nature of the gas phase on the degradation of RNA during fossilization processes[END_REF] and DNA/ion-exchanged Mt (Li + , Na + , Mg 2+ , Ca 2+ , Fe 3+ and Al 3+ ) Mt [START_REF] Xie | Charge-Dependent Regulation in DNA Adsorption on 2D Clay Minerals[END_REF]., but they were not conducted at low pH.

It is obvious that what is intercalated is not the intact torula yeast RNA, which has an intricate ternary structure and exists in solution as a Y-shaped molecule [START_REF] Tripathi | Oxovanadium complex as potential nucleic acid binder[END_REF] with a 30 nm gyration radius [START_REF] Manzano | Quantitative study of RNA transmission through ultrafiltration membranes[END_REF]. Indeed, a double strand of nucleic acid has a diameter of about 26 Å, and consequently, even a single strand lying in the interlayer with its nucleobases perpendicular to the clay layers would give an interlayer spacing of about 13 Å, significantly higher than the values we observe. This is why in the structural sketch of intercalated RNA we propose in Figure 6, the RNA strand is represented with the bases lying flat in the interlayer. This would involve a strong degree of RNA deformation; therefore, it is likely that the whole RNA molecule does not enter the interlayer.

It is proposed that only fragments of the RNA chain are intercalated, while the rest of the molecule may be adsorbed on the surface or edges of the Mt. A similar adsorption mechanism has been documented for other biomacromolecules in Mt, such as enzymes [START_REF] Sanjay | Enhanced pH and thermal stabilities of invertase immobilized on montmorillonite K-10[END_REF][START_REF] Gopinath | Enzymes immobilized on montmorillonite K 10: Effect of adsorption and grafting on the surface properties and the enzyme activity[END_REF].

Infrared spectroscopy

At both pHs, new bands were observed in the FTIR spectra of samples after the absorption of RNA. For example, at pH 6.2-6.5, bands assigned to ring vibrations and inplane C-O-H deformation of ribose were observed. At pH 2.9-3.1, new bands assigned to C=O and PO2 -stretching were apparent. Furthermore, changes in the position of the PO2 - band were observed. This shift is probably associated with distortions in the RNA molecule, possibly caused by the interactions of cations with phosphate groups [START_REF] Duguid | Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd, and Cd[END_REF][START_REF] Andrushchenko | Infrared absorption detection of metal iondeoxyguanosine monophosphate binding: Experimental and theoretical study[END_REF][START_REF] Pedreira-Segade | Effects of salinity on the adsorption of nucleotides onto phyllosilicates[END_REF]. It is likely that the clay/RNA interaction simultaneously includes several contributions from different local interactions such as H-bonds. It may be relevant from this point of view that the stretching vibration of the clay layer structural O-H shifts by a few cm -1 , which may be indicative of their implication in H-bonding [START_REF] Schnetzer | Impact of Intrinsic Structural Properties on the Hydration of 2:1 Layer Silicates[END_REF], but a more precise study would be needed to identify Hbonds with confidence.

27 Al, 13 C and 31 P solid state NMR 27 Al NMR spectra in all precursor samples presented characteristic chemical shifts at 3.9 and 69.1 ppm related to hexacoordinated (Al 6-c) and tetracoordinated (Al 4-c) aluminum located in octahedral and tetrahedral sheets of the Mt, respectively [START_REF] Cadars | New insights into the molecular structures, compositions, and cation distributions in synthetic and natural montmorillonite clays[END_REF]Marsh et al., 2018). Another chemical shift corresponding to the Al 4-c environment was observed in both spectra at 55 ppm and can be attributed to small impurities of amorphous aluminosilicate, also observed in the literature for synthetic [START_REF] Cadars | New insights into the molecular structures, compositions, and cation distributions in synthetic and natural montmorillonite clays[END_REF] and natural [START_REF] Garg | Thermal activation of a pure montmorillonite clay and its reactivity in cementitious systems[END_REF] montmorillonites. In the RNA@cations-Mt samples (Figure 3ii), no change was observed with respect to cations-Mt, indicating the absence of interaction between the biomolecule and Al sites in the Mt.

As regards the 31 P spectrum of RNA, Figure 4i, there are surprisingly few literature results available for comparison. Solution studies of short RNA sequences [START_REF] Legault | 31P Chemical Shift as a Probe of Structural Motifs in RNA[END_REF][START_REF] Maderia | Metal Interactions with a GAAA RNA Tetraloop Characterized by 31 P NMR and Phosphorothioate Substitutions †[END_REF] revealed a series of narrow peaks in the (+2.0, -2.0) ppm range corresponding to all different phosphodiester bridges in the RNA chain. In the solid state, the loss of resolution may produce a signal such as the one we are observing. For the RNA@cations-Mt samples, a strong new component appeared. This finding is quite unexpected since the shift is comparable to the chemical shift difference separating the Q 0 signal in monophosphate from the Q 1 in diphosphate. The formation of diphosphate seems unlikely however, as it would require a breakdown and rearrangement of the RNA structure.

Another possibility is that the new signal could correspond to the effect of the adsorption interaction on the bridging phosphate groups. Among likely interactions, only coordinative bonds to metal cations would be expected to result in such shifts. Intuitively, however, they would also be expected to induce a decrease in electron density in the phosphate moiety, and therefore a downfield shift (increase in chemical shift values) -the opposite of the effect observed here. The already quoted study by [START_REF] Maderia | Metal Interactions with a GAAA RNA Tetraloop Characterized by 31 P NMR and Phosphorothioate Substitutions †[END_REF] indeed reports shifts of several ppm when modified hairpin RNA is contacted with Cd 2+ or Mg 2+ cations. For different RNA structures, they can be either upfield or downfield. The authors could not provide any clear explanation for the upfield shifts; they supposed that the affected phosphate groups did not directly coordinate to the cations, but were indirectly affected through changes in RNA conformations. We will not speculate any further on the reasons for the new signal observed here, except to say that it reveals a strong RNA/clay mineral interaction. One can also note that in at least three of the four samples, there are indications of a third, even more upfield shifted component in the 31 P spectrum; attempts at signal deconvolution necessitated the introduction of a component at -14 to -17 ppm. The hypothesis of strong deformations of the RNA chain is in line with the structural picture deduced from XRD.

The 13 C NMR spectra show chemical shifts values that are practically the same for the different montmorillonites. This suggests that the nucleotide bases, mainly adenine and cytosine, can be protonated in an acidic medium (pH 2.9-3.1). These results are in keeping with calculations based on the pKa values. They corroborate the FTIR and XRD results, indicating that the protonation of these moieties induces hydrogen bonds and electrostatic interactions with the montmorillonite surface.

Figure 6 sketches a model of the RNA-clay composites that is compatible with the information from XRD, IR and 13 C and 31 P NMR.

Conclusions

We have investigated the interaction of an RNA-derived molecule with synthetic montmorillonites having different cations in their interlayer space. The systems were prepared in both neutral and acidic media.

The interaction of the biomolecule with montmorillonites was pH-dependent: the intercalation of RNA in the interlayer space of montmorillonite was largely controlled by electrostatic interactions. At close to neutral pH, the RNA molecules have a significant negative charge and are prevented from entering the interlayer space by electrostatic repulsion from the clay mineral layers. It is likely that RNA is adsorbed on edge sites, maybe with compensating cations acting as ions bridges.

At lower pH values, extensive intercalation occurs, as evidenced by the value of basal spacing observed in XRD. Indeed, the nucleobases are then expected to be partly protonated, resulting in significantly lower negative charges on the RNA molecules. The driving force for intercalation could include contributions of localized electrostatic interactions (between the positively charged aromatic nuclei and the negatively charged clay mineral layers), probably accompanied by H-bonding, and also of coordinative bonding with the interlayer cations. Some authors have suggested that at pH close to 7, van der Waals interactions between the montmorillonite layers and the purine and pyrimidine bases of the nucleotides may also be acting [START_REF] Kawamura | Clay catalysis of oligonucleotide formation: Kinetics of the reaction of the 5'-phosphorimidazolides of nucleotides with the non-basic heterocycles uracil and hypoxanthine[END_REF][START_REF] Ferris | Montmorillonite-catalysed formation of RNA oligomers: The possible role of catalysis in the origins of life[END_REF]. Indeed, both FTIR and 13 C NMR indicate small but significant modifications of the ring carbon atoms local environment. FTIR also shows a rather important shift of the phosphate band. Taken together, these results suggest that both groups are involved in the interaction mechanism. 31 P NMR shows a surprisingly strong effect of adsorption on the phosphates, but the direction of the shift does not seem compatible with a coordination to the electropositive compensating cations. Rather, it might be indicative of important configuration changes induced by the intercalation of the RNA backbone into a constrained space.

These results indicate that RNA oligomers and polymers may have interacted effectively with clay minerals, such as montmorillonite, in an acidic medium on the primitive Earth. Such an RNA/montmorillonite interaction may have stabilized and protected RNA molecules from an environment of likely hostile conditions on the primitive earth, such as high UV radiation.

Fig

  Fig. SM-3i and ii).

Figure 6 .

 6 Figure 6. Illustrative scheme for proposed interactions between fragment of RNA and montmorillonite in neutral and acidic medium. 395

Acknowledgements

The authors thank CAPES and CNPq (grants 310921-2017-1 and 431727/2016-3) for the financial support.