Lluís Solà-Hernández

Fabien Claudel

Frédéric Maillard

Christian Beauger

Doped tin oxide aerogels as oxygen evolution reaction catalyst supports

Keywords: Tin dioxide, Aerogel, doping, Iridium Catalyst Support, Oxygen Evolution Reaction, PEM Water Electrolyzers

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

Introduction

Polymer Electrode Membrane Water Electrolysers (PEMWE) are attractive devices for hydrogen production from sustainable and clean energy sources such as solar or wind energy. [START_REF] Grigoriev | Pure hydrogen production by PEM electrolysis for hydrogen energy[END_REF][START_REF] Barbir | PEM electrolysis for production of hydrogen from renewable energy sources[END_REF] Materials challenges in these systems are huge, especially at the anode where the catalyst should be able to face high electrochemical potential (E > 1.6

V vs. the standard hydrogen electrode -SHE), highly-acidic environment, presence of oxygen and moderate temperature (< 80 °C), and to electrocatalyze in a sustainable manner the oxygen evolution reaction (OER).

Former studies have established that ruthenium oxide (RuO2) and iridium oxide (IrO2), two precious metals, are best electrocatalyzing the OER. [START_REF] Carmo | A comprehensive review on PEM water electrolysis[END_REF] However, they are presently used in the form of micrometre-sized particles, and typical anode loadings range between 2.0 and 4.0 mg cm -2 in present PEMWE anodes, hence a reduction of the Ir content would be highly desirable. 4,[START_REF] Reier | Electrocatalytic Oxygen Evolution Reaction (OER) on Ru, Ir, and Pt Catalysts: A Comparative Study of Nanoparticles and Bulk Materials[END_REF][START_REF] Danilovic | Using Surface Segregation To Design Stable Ru-Ir Oxides for the Oxygen Evolution Reaction in Acidic Environments[END_REF][START_REF] Antolini | Iridium As Catalyst and Cocatalyst for Oxygen Evolution/Reduction in Acidic Polymer Electrolyte Membrane Electrolyzers and Fuel Cells[END_REF] One possibility to decrease the catalyst loading consists in using nanometre-sized Ru or Ir catalysts supported on high surface area and electron conducting supports. [START_REF] Seitz | A highly active and stable IrOx/SrIrO3 catalyst for the oxygen evolution reaction[END_REF][START_REF] Nong | Oxide-Supported IrNiOx Core-Shell Particles as Efficient, Cost-Effective, and Stable Catalysts for Electrochemical Water Splitting[END_REF] High surface area carbon blacks (CBs) is a popular and widely used support material in electrochemistry, due to its low cost, high specific surface area and high electrical conductivity [START_REF] Dicks | The role of carbon in fuel cells[END_REF][START_REF] Antolini | Polymer supports for low-temperature fuel cell catalysts[END_REF][START_REF] Antolini | Graphene as a new carbon support for low-temperature fuel cell catalysts[END_REF] . However, CBs face corrosion issue at PEMWE anode potential, thus leading to sintering [START_REF] Oh | Polypyrrole-modified hydrophobic carbon nanotubes as promising electrocatalyst supports in polymer electrolyte membrane fuel cells[END_REF]14,[START_REF] Yu | Recent advances in activity and durability enhancement of Pt/C catalytic cathode in PEMFC: Part I. Physico-chemical and electronic interaction between Pt and carbon support, and activity enhancement of Pt/C catalyst[END_REF] and detachment of the noble-metal nanoparticles (NPs), and resulting in depreciated electrocatalytic performance. [START_REF] Oh | Corrosion resistance and sintering effect of carbon supports in polymer electrolyte membrane fuel cells[END_REF][START_REF] Oh | On-line mass spectrometry study of carbon corrosion in polymer electrolyte membrane fuel cells[END_REF][START_REF] Maass | Carbon support oxidation in PEM fuel cell cathodes[END_REF][START_REF] Claudel | Degradation Mechanisms of Oxygen Evolution Reaction Electrocatalysts: A Combined Identical-Location Transmission Electron Microscopy and X-ray Photoelectron Spectroscopy Study[END_REF] So far, carbon based materials displaying stability under the required potential for the OER have not been found. [START_REF] Antolini | Ceramic materials as supports for low-temperature fuel cell catalysts[END_REF][START_REF] Lu | Strongly Coupled Pd Nanotetrahedron/Tungsten Oxide Nanosheet Hybrids with Enhanced Catalytic Activity and Stability as Oxygen Reduction Electrocatalysts[END_REF][START_REF] Luo | Tin and Silicon Binary Oxide on the Carbon Support of a Pt Electrocatalyst with Enhanced Activity and Durability[END_REF] On the contrary, metal oxides feature excellent corrosion resistance and may exhibit strong interactions with the noble metal catalysts. [START_REF] Resasco | A model of metal-oxide support interaction for Rh on TiO2[END_REF][START_REF] Huang | Development of a Titanium Dioxide-Supported Platinum Catalyst with Ultrahigh Stability for Polymer Electrolyte Membrane Fuel Cell Applications[END_REF][START_REF] Liu | High Stability, High Activity Pt/ITO Oxygen Reduction Electrocatalysts[END_REF] In particular, titanium based oxides have received many attention due to their interesting properties. For example, Wang et al. obtained an improved Ir utilization compared to the commercial Ir-black by deposition of metallic Ir(0) nanoparticles (NPs) on Ti4O7 without any thermal treatment. [START_REF] Wang | Nanostructured Ir-supported on Ti4O7 as a cost-effective anode for proton exchange membrane (PEM) electrolyzers[END_REF] Chen and co-workers also demonstrated enhanced OER activity in acid media when using Ti4O7 as an Ir catalyst support. [START_REF] Chen | Development of Supported Bifunctional Electrocatalysts for Unitized Regenerative Fuel Cells[END_REF] Siracusano et al. also obtained better performances when depositing IrO2 electrocatalysts on Ti based suboxides. [START_REF] Siracusano | Preparation and characterization of titanium suboxides as conductive supports of IrO2 electrocatalysts for application in SPE electrolysers[END_REF] Tin oxide doped based materials have also shown promising stability under OER conditions, as for example Indium doped (ITO) [START_REF] Puthiyapura | Investigation of supported IrO2 as electrocatalyst for the oxygen evolution reaction in proton exchange membrane water electrolyser[END_REF] or Antimony doped (ATO). [START_REF] Kotz | Electrochemical waste water treatment using high overvoltage anodes. Part I: Physical and electrochemical properties of SnO2 anodes[END_REF][START_REF] Oh | Preparation of Mesoporous Sb-, F-, and In-Doped SnO2 Bulk Powder with High Surface Area for Use as Catalyst Supports in Electrolytic Cells[END_REF][START_REF] Vicent | Characterization and stability of doped SnO2 anodes[END_REF][START_REF] Puthiyapura | Physical and electrochemical evaluation of ATO supported IrO2 catalyst for proton exchange membrane water electrolyzer[END_REF] A highly active OER catalyst consisting of IrNiOx supported on mesoporous ATO has been developed by the Strasser's group. The authors reported improved OER activity and stability, which was accounted for by a Metal-Metal Oxide Support Interaction (MMOSI) between the amorphous IrOx catalyst and the supporting material.9 , [START_REF] Oh | Electrochemical Catalyst-Support Effects and Their Stabilizing Role for IrOx Nanoparticle Catalysts during the Oxygen Evolution Reaction[END_REF] Wang and co-workers [START_REF] Wang | Improving the activity and stability of Ir catalysts for PEM electrolyzer anodes by SnO2:Sb aerogel supports: does V addition play an active role in electrocatalysis?[END_REF] also reported enhanced OER activity and stability of Ir catalysts supported onto ATO aerogels. [START_REF] Ozouf | Niobium-and antimony-doped tin dioxide aerogels as new catalyst supports for PEM fuel cells[END_REF] However, Claudel et al. recently reported that stability of the doping element is a key issue for ATO to be implemented in PEMWE anode. [START_REF] Claudel | Degradation Mechanisms of Oxygen Evolution Reaction Electrocatalysts: A Combined Identical-Location Transmission Electron Microscopy and X-ray Photoelectron Spectroscopy Study[END_REF] Ta-doped SnO2 supports (TaTO) have been first used by Senoo et al. as a catalyst support at a proton-exchange membrane fuel cell (PEMFC) cathode. The authors demonstrated enhanced catalytic performance toward the oxygen reduction reaction (ORR) and improved corrosion resistance for Pt/SnO2:Nb vs. Pt/CB. [START_REF] Senoo | Cathodic performance and high potential durability of Ta-SnO2δ-supported Pt catalysts for PEFC cathodes[END_REF][START_REF] Fabbri | Boosting Pt oxygen reduction reaction activity by tuning the tin oxide support[END_REF] Fabbri et al. also demonstrated superior ORR activity of Pt NPs deposited on Ta-, Nb-and W-doped SnO2 relative to Pt/glassy carbon. [START_REF] Fabbri | Pt nanoparticles supported on Sb-doped SnO2 porous structures: developments and issues[END_REF] Uchida et al. reported improved utilization of Ir atoms in Ir/TaTO catalysts used at a PEMWE anode. [START_REF] Ohno | Effect of Electronic Conductivities of Iridium Oxide/Doped SnO2 Oxygen-Evolving Catalysts on the Polarization Properties in Proton Exchange Membrane Water ElectrolysisCatalysts[END_REF] Herein, SnO2 (TO), SnO2:Ta (TaTO) and SnO2:Sb (ATO) aerogels with three-dimensional texture, amenable morphology, and tuneable electronic conductivity (via doping) were synthesized, decorated with partially oxidized iridium oxide (IrOx) NPs and tested in simulated PEMWE electrolyser conditions.

Experimental

Synthesis and preparation of the alkoxide precursor (Sn(OiPr)4)

A tin metal alkoxide precursor (Sn(OiPr)4) was first prepared using the procedure described in Thomas et al. [START_REF] Thomas | Method for producing stannic tertiary alkoxide[END_REF] In brief, 3.7 mL of tin tetrachloride (SnCl4) were added into a 500 mL dry reactor flask under inert atmosphere with 50 mL of previously dried and degassed heptane. After 10 minutes of stirring at room temperature, a solution of 17 mL of diethylamine dissolved in 20 mL of dried heptane was added by using a syringe in order to keep the inert atmosphere, and then the reaction was kept under stirring for 2 hours. After that, a 17 mL solution of tertbutanol dissolved in 70 mL of dried heptane was added to the reactor and kept under stirring for 72 hours. The product was then filtered inside an inert atmosphere glove box and the filtrate was washed several times with heptane. 30 mL of dried isopropanol were added to the filtrate, and the reaction was kept under stirring and inert atmosphere for 24 hours more. Finally the solution was evaporated under vacuum in order to eliminate non desirable by-products and solvents, and the obtained alkoxide was re-diluted with the desired amount of isopropanol in order to obtain a solution of Sn(OiPr)4 at 10% mass in volume (total yield = 40%).

Aerogels preparation by the sol-gel route

Doped and un-doped TO aerogels with high surface area were synthetized by the sol-gel method, previously reported by our group, 37 using alkoxide precursors. Two solutions (A & B) were prepared. Solution A, consisting of tin isopropoxide (Sn(OiPr)4) dissolved in isopropanol and the corresponding amount of the antimony alkoxide precursor as doping element (Ta(OiPr)5 or (Sb(OiPr)3, Across Organics), were mixed under magnetic stirring. Solution B, consisting of nitric acid (Alfa Aesar, 2M) as the sol-gel reaction catalyst (Cat/Sn = 0.072) and water (H2O/Sn = 3.06 mol/mol) were diluted in isopropanol and mixed under magnetic stirring. Solution B was then slowly added into solution A and a gel formed after a few minutes. Then the gels were covered with 10 mL of isopropanol (to prevent drying), and aged for 72 hours at room temperature before being washed with isopropanol three times a day for three days. Once washed, the resulting gels were dried under CO2 in supercritical conditions (80 bars, 40 °C) to obtain amorphous aerogels. After calcination under air at 600°C, the ATO aerogels turned blue, while TaTO aerogels remained white.

Deposition of IrOx nanoparticles by chemical reduction

IrOx NPs were deposited onto the aerogel supports using the procedure previously described by Wang et al. [START_REF] Ozouf | Niobium-and antimony-doped tin dioxide aerogels as new catalyst supports for PEM fuel cells[END_REF] In brief: 1.17 g of cetyltrimethylammonium bromide (CTAB, Merck Millipore), 0.095 g of as-prepared TO, ATO or TaTO aerogel and 0.075 g of the Ir precursor (IrCl3, Sigma Aldrich) were dispersed in a 120 mL anhydrous ethanol solution (VWR Chemicals) and magnetically stirred for 4 hours under an argon (Ar) atmosphere. To reduce Ir 3+ ions, 0.114 g of sodium borohydride (NaBH4, Sigma Aldrich) were dissolved in 50 mL of anhydrous ethanol, and the resulting solution was added slowly to the first reaction suspension under vigorous stirring and Ar atmosphere. The reaction mixture was kept for 4 hours until the reduction reaction completed. Afterwards, the suspension containing Ir/TO-based support was centrifuged and rinsed with ample amounts of pure ethanol and deionized water to remove CTAB and the residual non-reduced IrCl3. Finally, the synthesized catalyst was dried overnight at 80°C under air atmosphere. The nominal Ir loading was 30 wt. %.

Materials Characterization

X-ray diffraction (XRD) analyses were performed using an X'Pert pro-Philips diffractometer (Cu Kα of λ = 1.5405 Å), operating at 45 kV and 30 mA. Data were collected from 20° to 90° in 2-θ mode with a Pixcel counter. The crystallites size was determined using the Debye-Scherrer method and the (111) diffraction peak.

The morphology of the synthesized aerogels was determined using a Supra 40 scanning electron microscope (SEM) equipped with a Gemini column, and operated at 3.00 kV. Samples were deposited onto adhesive conducting carbon tapes and coated with a 7 nm thick platinum layer, using a Quorum (Q150 T).

Transmission electron, microscopy (TEM) measurements were performed using a JEOL 2010 operated at 200 kV (point-to-point resolution of 0.19 nm).

Nitrogen sorption analyses were performed with a Micromeritics ASAP 2020. Before analyses, samples were degassed for 120 min at 100 °C and 10 μm Hg. For specific surface area determination, the Brunauer-Emmett-Teller (BET) model was applied. The pore size distributions were determined by applying the Barret-Joyner-Halenda (BJH) method to the desorption branch of the isotherms. The t-plot construction using Harkins-Jura correlation was used for microporosity assessment. We assumed that the calcined samples were mechanically strong enough to withstand the pressure applied during the measurement.

The bulk chemical compositions of the samples was determined by Energy Dispersive X-ray Spectroscopy (EDX) during SEM observations with a Philips XL30 operated at 15 kV.

Insights into the near-surface chemical composition were obtained by X-ray Photoelectron Spectroscopy (XPS) using a Thermo Scientific K-Alpha system. The spectrometer was equipped with a monochromatic Al Kα source and a low energy flood-gun for charge compensation. The ellipsoid spot size is about 350 µm x 700 µm.

The electronic conductivity of the synthesized aerogels was determined with a homemade conductivity cell, made up of two copper electrodes (S = 0.785 cm²) surrounded with a Teflon ring. Approximatively 100 mg of sample were introduced between these two electrodes (see Figure 1). A potentiostat was used to apply a current of 100, 150, 250 and 400 mA and the voltage was measured for each current. The homemade conductivity cell was placed in a press and measurements were made at room temperature with 1 ton of pressure. The conductivity (σ, S/cm) was calculated with the formula σ = (e.I/U.Selectrode) where "e" is the thickness of the sample (cm), "I" the applied current (mA), "Selectrode" the surface of the electrode (cm 2 ) and "U" the measured voltage (mV). The electrochemical measurements were performed with an Autolab PGSTAT20 potentiostat and a rotating disk electrode (RDE). Inks were prepared by mixing a given mass of the synthesized catalysts, a 5 wt. % Nafion ® solution, isopropanol and ultrapure water. An aliquot of 10 µL of the catalyst suspension was deposited on the working electrode (glassy carbon disk, geometric area: 0.196 cm²) by spin coating, targeting a final loading of 20 µgIr cm -²geom. The counter electrode was a Pt foil and the reference electrode a Reversible Hydrogen Electrode (RHE). The OER evaluation tests were performed in N2-saturated 0.05 M H2SO4 using a rotating speed of 1600 revolutions per minute (rpm). The sequence test described in Table 1 was used for electrochemical characterization and determination of the OER activity. The current obtained at 1.51 V (η = 0.28V) in the last sequence step, was normalized either by the mass of Ir initially deposited on the electrode (jmass) or by the anodic charge measured between 0.40 and 1.40 V vs. RHE, which is known to be proportional to the amount of Ir oxide (jspec). [START_REF] Reier | Molecular Insight in Structure and Activity of Highly Efficient, Low-Ir Ir-Ni Oxide Catalysts for Electrochemical Water Splitting (OER)[END_REF][START_REF] Ardizzone | Inner" and "outer" active surface of RuO2 electrodes[END_REF] 3. Results and discussion

Aerogels characterization

Antimony-doped (ATO), Tantalum-doped (TaTO) and un-doped (TO) tin oxide aerogels were prepared by the sol-gel route described in the Experimental Section.

Structure and morphology of aerogels

The morphology and the structure of the as-prepared aerogels were characterized by SEM, nitrogen sorption measurements and XRD. 2, the "a" and "c" lattice parameters of TaTO or ATO aerogels are unchanged compared to pure TO, suggesting no deformation of the rutile cell of TO. The XRD patterns displayed in Figure 2a and the crystallite sizes evaluated using the Debye-Scherer equation (Table 2)

nevertheless show that the doped aerogels are less crystalline and feature smaller crystallites than the pure TO material. SEM images displayed in Figure 2(c-e) show that aerogels feature an airy morphology lying on a three-dimensional network composed of interconnected primary particles with size comprised between 15 and 20 nm (Table 2). This morphology is similar to that of carbon and SnO2 based aerogels previously synthesized by our group. [START_REF] Ouattara-Brigaudet | Carbon Aerogels as Catalyst Supports and First Insights on Their Durability in Proton Exchange Membrane Fuel Cells[END_REF] The agglomeration of the primary particles leads to pores of different sizes: micropores (< 2 nm), mesopores (2 < size < 50 nm) and macropores (50 nm). The mean crystallite size and the mean primary particle size determined by SEM (150 particles were analysed using Image J) are displayed in Table 2. For the doped samples, we observed that the primary particle size systematically exceeds the crystallite size, suggesting that the aerogel is polycrystalline. Importantly, we also noticed that large and small particles co-exist inside the TaTO aerogel (Figure 2d). Local EDX analyses indicated that the large particles are depleted in Ta, and such domains are believed to be responsible of the higher degree of crystallinity of TaTO vs. ATO. In contrast, the crystallite and particle sizes are similar for the un-doped aerogels (Figure 2e), suggesting a monocrystalline material. Nitrogen sorption measurements were performed (Figure 2b and Table 3) to determine the textural properties of the synthesized aerogels. All measured isotherms indicated mesoporous materials, and this was confirmed by the specific surface area values (Table 3) and the pore size distributions (PSD, Figure 2b) calculated based on the BET and BJH models, respectively. The presence of mesoporosity is key to efficiently deposit IrOx NPs within the aerogel and thus ensure higher mass activity towards the OER. Figure 2b reveals a multimodal pore size distribution centred at 20 and 40 nm for all synthesized aerogels. However, due to smaller primary particles, higher specific surface area was measured for the doped aerogels.

Aerogel chemical composition

It is well-established that the electronic conductivity strongly depends on the nature and the content of the doping element as well as the homogeneity of doping [START_REF] Senoo | Cathodic performance and high potential durability of Ta-SnO2δ-supported Pt catalysts for PEFC cathodes[END_REF][START_REF] Cognard | Benefits and limitations of Pt nanoparticles supported on highly porous antimony-doped tin dioxide aerogel as alternative cathode material for proton-exchange membrane fuel cells[END_REF][START_REF] Cognard | Pt nanoparticles supported on niobium-doped tin dioxide: impact of the support morphology on Pt utilization and electrocatalytic activity[END_REF][START_REF] Cognard | Insights into the stability of Pt nanoparticles supported on antimony-doped tin oxide in different potential ranges[END_REF][START_REF] Ozouf | Sb-Doped SnO 2 Aerogels Based Catalysts for Proton Exchange Membrane Fuel Cells: Pt Deposition Routes, Electrocatalytic Activity and Durability[END_REF][START_REF] Beauger | Doped TiO2 aerogels as alternative catalyst supports for proton exchange membrane fuel cells: A comparative study of Nb, V and Ta dopants[END_REF]51 . During the synthesis of the TO-based aerogels, elements may segregate during the sol-gel process or during the calcination step. To determine the homogeneity of doping in ATO and TaTO aerogels, the bulk and the near-surface chemical compositions were determined by EDX and XPS, respectively. Some difficulties arised to quantify the Sb content by XPS, as the most intense peak of Sb (Sb 3d5) is located at the same binding energy than the O 1s peak (≈ 530 eV). To avoid interferences, the Sb content was determined using the less intense Sb 3d3 peak at ca. 540 eV (Figure 3). For determination of the near-surface composition of TaTO, the Ta 4d5 and Ta 4d3 peaks were used. According to EDX measurements (Table 4), the bulk concentration of the doping element was 12 at. % for ATO and 14 at. % for TaTO (nominal value 10 at. %). The near-surface Sb content in ATO measured by XPS was found notably higher than the bulk concentration (15 at. % vs 12 at. %), in agreement with former results. [START_REF] Senoo | Cathodic performance and high potential durability of Ta-SnO2δ-supported Pt catalysts for PEFC cathodes[END_REF] Similarly, the Ta concentration was found higher in the near-surface region of the aerogel than in the bulk (16 at.

% vs 14 at. %). Despite results obtained by EDX and XPS should be regarded semi-quantitative, we believe that the observed trend is significant. Surface and near-surface enrichment by the doping element can be attributed to interatomic diffusion during the calcination step. Different kinetics of hydrolysis or condensation of the used precursors, represent another possibility to account for these results. Segregation of the doping element represents a significant pitfall in terms of electronic conductivity and can be mitigated, for example by selecting other sol-gel precursors with similar hydrolysis/condensation kinetics than those of the Sn precursor.

Electronic conductivity measurements

As seen in Table 5, Ta is much less efficient than Sb to enhance the electronic conductivity of the TO aerogels.

Indeed, while a four orders of magnitude enhancement was reached with 12 at. % of Sb, a modest 3-fold enhancement of the electronic conductivity was achieved upon doping the TO aerogel with 14 at. % of Ta, in agreement with former reports in the literature. Despite optimal doping rates depend on the nature of the element, [START_REF] Fabbri | Pt nanoparticles supported on Sb-doped SnO2 porous structures: developments and issues[END_REF] we purposely kept similar values for ATO and TaTO so as not to impact the morphology of the support and allow a straightforward comparison between the different samples.

Table 5. Aerogels conductivity determined by resistance measurements obtained with an applied pressure of 1 ton. The electronic conductivity is also known to depend on both the charge carriers' concentration and their mobility, the latter being notably influenced by grain and domain boundaries. [START_REF] Cognard | Insights into the stability of Pt nanoparticles supported on antimony-doped tin oxide in different potential ranges[END_REF] Since ATO and TaTO samples synthesized in this study feature similar particle sizes, we believe that the density of grain boundaries is similar for both materials. Hence, the higher conductivity observed for ATO most likely results from a higher charge carrier's density, in agreement with former claim of Bruneaux et al. 52 This may be rationalized by considering different possible oxidation states for Sb. As already discussed [START_REF] Senoo | Cathodic performance and high potential durability of Ta-SnO2δ-supported Pt catalysts for PEFC cathodes[END_REF] , the valency of the doping element will determine the concentration of oxygen vacancies hence the electronic conductivity.

Sample Conductivity (S cm

IrOx/Aerogel characterization

IrOx NPs were deposited onto the different aerogels using a chemical route (see section 2.4 of Experimental for details). As revealed by Figure 4, these IrOx NPs feature a face-centred cubic structure. Because the synthesis was performed at low temperature and in air-free atmosphere, we did not observe typical reflections of IrO2. to the rutile structure of pure TO and green color ones correspond to Ir(0) pattern. [START_REF] Cognard | Benefits and limitations of Pt nanoparticles supported on highly porous antimony-doped tin dioxide aerogel as alternative cathode material for proton-exchange membrane fuel cells[END_REF]53 TEM images of unsupported and aerogel-supported IrOx NPs are displayed in Figure 5. The IrOx particle and crystallite sizes were similar for the aerogels-supported IrOx NPs, reflecting the fact that agglomerate-free single crystalline nanoparticles were deposited onto the TO-based aerogels (Table 6). On the contrary, the particle and crystallite size strongly differed for the un-supported IrOx NPs, providing evidence of a highly polycrystalline material.

EDX and XPS analyses were used to gain insights into the weight fraction (wt. %) and the valency of Ir atoms in each catalyst (Table 7). The Ir wt. % determined by EDX was close to the nominal value (30 wt.%), thus detection, the peak at 9.17 eV (IrLα emission) was selected, due to the overlapping of the peak at 1.98 eV with the Pt loading signal. x < 2) resulting from oxidation in air. The Ir4f peak was fitted using the parameters and the line shapes proposed by Freakley et al. 54 Three contributions were considered: metallic Ir(0), IrO2 and IrCl3. As shown in Table 7, the unsupported IrOx NPs featured a higher Ir metallic content than the aerogel-supported IrOx nanocatalysts. This higher metallic content mostly is a consequence of the larger particle size observed for this material. Regarding the deposited nanocatalysts over the TO aerogels, all IrOx NPs present similar particle and crystallite sizes (Table 6), and similar metallic and oxide contributions, suggesting a crystalline metallic core and an amorphous oxide layer on the surface.

Electrocatalytic activity towards the OER and stability in acidic electrolyte

Finally, the OER activity of the four prepared catalysts was assessed using the protocol described in the experimental section. Conditioning of the electrodes was first carried out by performing a series of 10 cyclic voltammogram (CV) between 0.0 and 1.6 V vs. RHE and another of 3 CV cycles between 1.0 and 1.6 V. A 24 hour chronopotentiometry at 1 mA cm -2 was used to determine the stability of each catalyst under OER conditions.

The results, displayed in Figure 7, show a 5-fold and nearly 10-fold enhancement of the mass activity towards the OER on the aerogel-supported IrOx NPs compared to unsupported IrOx NPs at 1.51 V and 1.58 V, respectively. We related this to the effect of the IrOx crystallite. Indeed, the size of the IrOx NPs is much smaller for supported than for unsupported catalysts, hence much higher density of sites is available to the reaction and faster OER kinetics result according to Butler-Volmer equation.The stability of the IrOx NPs was also enhanced by the presence of the aerogel support, as the lifespan of the materials was improved from 1 hour for unsupported IrOx NPs up to 17 hours for the IrOx/TO catalyst (Figure 7.c and Table 8). Again, this is due to the smaller IrOx NP size. Indeed, due to smaller density of catalytic sites, the unsupported IrOx NPS are subjected to a higher overpotential during the chronopotentiometry (in order to be able to provide the requested current density), hence the lower durability observed.

Finally, we noticed that despite the different electronic conductivity of their supports (Table 5), similar geometric, mass and specific activities towards the OER were measured for the three aerogel-supported IrOx catalysts. Even more surprising, IrOx/TO was found to be the best-performing material. These results may however be rationalized by considering that, in RDE configuration, a very thin film of catalyst covers a glassy carbon disk with high electron-conductivity. As supported IrOx/TO-based aerogel and carbon Fermi levels align upon contact, different OER activity are unlikely to translate when OER activities are determined in RDE configuration. However, the situation will be very different for ca. 150-200-fold thicker PEMWE anodes, in which the electronic conductance of TO-based aerogels will be determined by their intrinsic conductivity, the resistance of their contacts, and the volume density of the latter. In that frame, it was no surprise to note that the time during which a current density of 1 mA cm -2 could be sustained (a cut-off voltage of 2 V was used) was almost not dependent of the nature of aerogel (Table 8). We thus conclude that quantifying the impact of the nature of the TO-based aerogel (undoped vs. Sb or Ta doped SnO2) is hardly possible in RDE configuration.

Conclusion

In conclusion, we showed that supporting IrOx nanoparticles on tin oxide aerogels allows enhancing their OER activity and stability. Despite very different electronic conductivity, the OER mass and specific activities of IrOx nanoparticles deposited onto tin oxide-based aerogels were found similar in thin-film electrode configuration. This is in agreement with the recent results obtained by the group of professor Uchida on different types of tin dioxide support. On top of a better OER activity, all aerogel supported IrOx based catalysts also exhibited enhanced durability compared to unsupported IrOx nanoparticles.

Also, since the morphology of tin dioxide based aerogels (TO, ATO and TaTO) is very similar to that of previously developed carbon aerogels, it is reasonable to think that the same fluid management benefits can be expected in real PEMWE device than we did observe in PEMFC. Considering the promising results reported in this study, we now aim at assessing their performance and durability in membrane electrodes assembly (MEA)

where the impact of the support's electronic conductivity is key. Specific studies will also be carried out in order to optimize the doping element concentration as well as the Ir loading.

Figure 1 :

 1 Figure 1: Homemade conductivity cell picture assembled (left) and disassembled (right)

  Figure 2a shows the characteristic XRD peaks, (110), (101), (200), (211), (200), of the rutile structure of TO. No peak corresponding to Sb or Ta phases (SnO, or M, MO, M2O3, M2O5 with M = Sb or Ta) was found, indicating that the dopant atoms substituted Sn atoms or are inserted randomly in the rutile network. Moreover, as shown in Table

Figure 2 :

 2 Figure 2: X-ray diffraction patterns (a), pore size distribution (b), SEM images (Magnification of the images = 50000 X) and associated primary particle size distributions (c) for pure and doped SnO2 aerogels (Red bars in (a) correspond to the pattern of the rutile structure of tin oxide 45 JCPDS 14-1445).

Figure 3 .

 3 Figure 3. XPS spectra focusing on Sb3d (left) and Ta 3d5/3d3 (right)

Figure 4 .

 4 Figure 4. X-ray diffractograms of Ir NPs (black), Ir/TO (blue), Ir/ATO (dark red) and Ir/TaTO (yellow). Red bars correspond

Figure 5 .

 5 Figure 5. TEM images of (a) IrOx NPs, (b) IrOx/ TO, (c) IrOx/ ATO and (d) IrOx/TaTO and associated IrOx particle size distribution.

Figure 6 .

 6 Figure 6. Detailed Ir 4f spectra of (a) IrOx NPs, (b) IrOx/TO, (c) IrOx/ATO and (d) IrOx/TaTO catalysts, showing the contribution of each Ir species.

Figure 7 .

 7 Figure 7. Electrocatalytic performance and durability of the as-prepared catalysts: (a) geometric activity (b) mass activity (c) stability at 1 mA/cm² and (d) mass activity comparison at an overpotential of 0.28 V. All measurements were performed at 25°C in N2-saturated 0.05 M H2SO4 using a potential sweep rate of 5 mV s -1 and a rotation rate of 1600 rpm. The Ir loading was 20 µg cm -2 for all electrodes (3.9 µg Ir per RDE tip).

Table 1 .

 1 Sequence used for electrochemical characterizations

	Sequence	Starting Potential (V	Potential range	Sweep rate (mV s -1 )	N° Cycles	Purpose
	Number	vs. RHE)	(V vs. RHE)
	1	1.0	1.0 -1.6	5	3	OER pre-test
						Electrochemical
	2	OCP	0.0 -1.6	20	10	conditioning
						OER activity
	3	1.0	1.0 -1.6	5	3	evaluation
	4	Chronopotentiometry at 1 mA.cm -2 for 24 hours		Stability test

Table 2

 2

	. "a" and "c" lattice parameters and crystallite size determined by XRD and primary particles size (SEM) of doped
		and un-doped samples.
		a // c lattice	Crystallite size
	Sample			Primary particle size (nm)
		parameters (nm)	(nm)
	TO	4.7 // 3.2	23.0	22.1 ± 5.1
	ATO	4.7 // 3.2	5.1	16.3 ± 2.8
	TaTO	4.7 // 3.2	8.2	15.4 ± 4.1

Table 3 .

 3 Nitrogen sorption measurement results for doped and pure SnO2 aerogels.

		Specific surface	PSD	Pore volume	µ-pore volume
	Sample	area BET (m 2 g -1 )	(BJH, nm)	(BJH, cm 3 g -1 )	(BJH, cm 3 g -1 )
	TO	41.1	20 -30 -40	0.059	0.4 x 10 -2
	ATO	76.9	20 -25 -35	0.295	0.9 x 10 -2
	TaTO	74.6	20 -25 -40	0.293	0.1 x 10 -2

Table 4 .

 4 Bulk and near-surface chemical composition of the as-prepared aerogels determined by EDX and XPS analyses.

	Sample	Dopant/(Dopant + Sn)	Dopant/(Dopant + Sn)
		EDX (at.%)	XPS (at.%)
	TO	-	-
	ATO	11.8 ± 2.5	14.9 ± 0.1
	TaTO	14.0 ± 1.9	16.1 ± 0.4

Table 6 .

 6 Crystallite and particle sizes of unsupported and supported IrOx nanocatalysts.

	Sample	IrOx crystallite size	IrOx particle size
		(nm)	(nm)
	Unsupported IrOx	8.9	1.8 ± 0.4
	NPs
	IrOx/TO	1.6	1.8 ± 0.4
	IrOx/ATO	1.9	1.7 ± 0.4
	IrOx/TaTO	1.9	1.7 ± 0.3

Table 7 .

 7 Bulk and surface chemical composition of the IrOx/TO-based aerogels determined by EDX and XPS measurements.

		Ir wt.%	Ir wt.%	Ir(0) at.%	IrO2 at.%	IrClx at.%
	Sample
		(EDX)	(XPS)	60.8 eV (4f7/2)	61.6 eV (4f7/2)	62.9 eV (4f7/2)
	Unsupported	96.3 ± 2.4*	96.6 ± 2.5*	53.6 ± 5.9	42.6 ± 3.9	3.8 ± 1.4
	IrOx NPs
	IrOx/TO	30 ± 1.3	30 ± 0.5	41.2 ± 4.0	49.9 ± 5.6	8.9 ± 2.4
	IrOx/ATO	29 ± 1.5	22 ± 0.3	42.7 ± 1.9	50.4 ± 1.9	6.9 ± 2.2
	IrOx/TaTO	33 ± 2.0	32 ± 0.9	41.9 ± 2.6	49.6 ± 2.5	8.6 ± 1.4
	*complement to 100% with O and possibly Cl and/or C

Table 7

 7 also shows that the IrOx nanocatalysts are composed of both metallic Ir and Ir oxide in approximately the same amount. This was no surprise as nanocatalysts are prone to formation of an amorphous IrOx layer (with

Table 8 .

 8 Stability tests results for the synthesized catalysts (24 hour chronopotentiometry using a constant current density of 1 mA cm -2 and a cut-off voltage of 2.0 V.

	Sample	Durability (@ 1 mA cm -2 )
	Unsupported IrOx NPs	1.0 ± 0.5 h
	IrOx/TO	17.0 ± 2.5 h
	IrOx/ATO	16.0 ± 1.0 h
	IrOx/TaTO	14.5 ± 2.0 h

Abbott D, Lebedev D, Waltar K, Povia M, Nachtegaal M, Fabbri E, Copéret C, Schmidt TJ, Iridium Oxide for

ACKNOWLEDGMENTS

The authors wish to thank Pierre Ilbizian for supercritical drying, Frédéric Georgi for XPS analysis, Suzanne Jacomet for SEM/EDX analysis and Gabriel Monge for XRD. This work was funded by the European Union's H2020 Program within the Fuel Cells and Hydrogen 2 Joint Undertaking under grant agreement 779478 (FCH-JU project PRETZEL) and the French National Research Agency (ANR-17-CE05-0033 project MOISE). It was supported by Capenergies and Tenerrdis.

AUTHOR INFORMATION

Corresponding Authors lluis.sola-hernandez@mines-paristech.fr christian.beauger@mines-paristech.fr

Author Contributions

The manuscript was written through contributions of all authors.

Declarations of interest None


Thierry Tran

Céline Lafarge

Rémi Pradelles

Jean-Marie Perrier-Cornet

Nathalie Cayot

Camille Loupiac
email: camille.loupiac@agrosupdijon.fr

Effect of high hydrostatic pressure on the structure of the soluble protein fraction in Porphyridium cruentum extracts

Keywords: High hydrostatic pressure, B-phycoerythrin, spectroscopy, structure, protein, Porphyridium cruentum

High hydrostatic pressure (HHP) treatments are trending as "green" stabilization and extraction process. The extraction of B-phycoerythrin from microalgae is getting more and more interest due to its numerous potentialities in foods, cosmetics and medicine. Thus, the effects of high pressure on the structural characteristics of B-phycoerythrin extracted from Porphyridium cruentum are explored in this paper.

Spectrophotometric methods allowed to measure B-phycoerythrin content (UV-visible) and gave an indication on the protein structure (fluorescence). Micro-DSC analysis and electrophoresis complemented this structural investigation for all the protein fractions of P. cruentum extracts.

Applying high hydrostatic pressure treatments up to 300 MPa during five minutes had no significant effect on B-phycoerythrin content and structure in P. cruentum extracts.

Nevertheless, conformational changes of the protein are suggested by fluorescence yield decrease at 400 MPa, and protein aggregation of B-phycoerythrin, observed by Micro-DSC and electrophoresis, occurred at 500 MPa.

Industrial relevance

The HHP process is an emerging technology for the microbiological stability of various food matrices, including the proteins of microalgae as natural colorant. The target pressure to stabilize is around 400 MPa. High hydrostatic pressure can be used on P. cruentum extracts up to 300 MPa without any change in protein structure, as the threshold of protein aggregation is observed at 400 MPa. The observed changes of the proteins structure after applying HHP above 400 MPa can have a strong impact at macroscopic scale on the food matrices: increase of turbidity, change of texture, stability of emulsion.

Introduction

Porphyridium cruentum is a spherical unicellular red alga (Rhodophyta) without any organized cell wall [START_REF] Jones | Studies on the growth of the red alga Porphyridium cruentum[END_REF]. This microalga is photoautotrophic and is encapsulated in an exopolysaccharide sheath [START_REF] Arad | The potential of production of sulfated polysaccharides from Porphyridium[END_REF]. Photosynthesis is achieved by the lamella-shaped thylakoids that host the photosynthetic material [START_REF] Gantt | The ultrastructure of Porphyridium cruentum[END_REF].

P. cruentum hosts numerous pigment proteins, such as B-phycoerythrin (B-PE). This macromolecule is part of a bigger protein structure called phycobilisome that involves other proteins of the same family (phycobiliproteins) [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum: I. Isolation[END_REF][START_REF] Glazer | Phycobilisomes: structure and dynamics[END_REF].

P. cruentum phycobilisomes host three phycobiliproteins: allophycocyanin, phycocyanin and phycoerythrin. Allophycocyanin acts as a core to which are attached the other proteins arranged in rods. Those rods are composed of phycoerythrin in the extremity of the rods attached to the core and phycocyanin is located between allophycocyanin and phycoerythrin [START_REF] Glazer | Phycobilisomes: structure and dynamics[END_REF]. The amounts of phycobiliproteins of P. cruentum and their spectral properties reported in the literature are shown in Table 1. These proteins have gained importance in commercial applications in many industrial sectors (food, cosmetics, pharmaceutics and textile), as the demand of natural colorants is increasing. Because of their spectroscopic and biological properties, phycobiliproteins are fluorescent probes widely used in clinical diagnostics, flow cytometry and immunochemistry [START_REF] Sekar | Phycobiliproteins as a commodity: trends in applied research, patents and commercialization[END_REF].

The important B-phycoerythrin content of P. cruentum makes this microalga an ideal raw material for extraction and study of this pigment protein. In fact, commercial B-phycoerythrin is mainly used as fluorescent biomarker in medical sciences due to its safety aspect for human patients [START_REF] Glazer | Phycobiliproteins-a family of valuable, widely used fluorophores[END_REF][START_REF] Manirafasha | Phycobiliprotein: Potential microalgae derived pharmaceutical and biological reagent[END_REF]. Consequently, its natural fluorescence facilitates the investigation of the protein structure response to environmental conditions. The three-dimensional structure of phycoerythrin from P. cruentum has been determined [START_REF] Camara-Artigas | pH-dependent structural conformations of Bphycoerythrin from Porphyridium cruentum[END_REF][START_REF] Ficner | Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23-nm resolution and localization of the gamma subunit[END_REF]. All phycobiliproteins consist of two dissimilar subunits, α and β, of 17 kDa to 19 kDa respectively, depending on the species. These subunits are associated to form a heterodimeric complex called the (αβ) monomer, and sometimes an additional γ-subunit, which has been proposed to be located in the center of the hexamer formed by a trimer of the (αβ) monomer acting as a linker protein. The presence of this γ-subunit is the main structural difference between b-phycoerythrin and B-phycoerythrin. The chromophores (phycobilins) are openchain tetrapyrroles attached to thioether bonds to cysteine residues of the α and β subunits.

The spectral properties of bilins are determined not only by the chemical nature of the prosthetic groups, but are also influenced by their nearby proteins.

B-phycoerythrin stability towards different parameters has been studied by [START_REF] Munier | Physicochemical factors affecting the stability of two pigments: Rphycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum[END_REF] as unpurified in phosphate buffer resulting from a "crude extract" of P. cruentum and by [START_REF] González-Ramírez | Thermal and pH Stability of the B-Phycoerythrin from the Red Algae Porphyridium cruentum[END_REF] as purified in phosphate or potassium buffer. Both studies concluded that B-phycoerythrin's pH stability ranged between 4 and 10, making this pigment interesting for food application. At extremely low pH, the color darkened from pink to purple and tended to bleach at extremely high pH. The pH dependence of B-phycoerythrin has been studied and explained at the conformation structural level by [START_REF] Camara-Artigas | pH-dependent structural conformations of Bphycoerythrin from Porphyridium cruentum[END_REF].

Studies also pointed its important sensitivity to direct light exposure with a significant decrease in 545 nm absorbance after 4 h at 4°C from 33.57 µmol m -2 s -1 light intensity [START_REF] González-Ramírez | Thermal and pH Stability of the B-Phycoerythrin from the Red Algae Porphyridium cruentum[END_REF][START_REF] Munier | Physicochemical factors affecting the stability of two pigments: Rphycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum[END_REF]. Towards temperature, B-phycoerythrin remained stable between 0 and 40°C [START_REF] Munier | Physicochemical factors affecting the stability of two pigments: Rphycoerythrin of Grateloupia turuturu and B-phycoerythrin of Porphyridium cruentum[END_REF]. A study performed by circular dichroism on this protein revealed two irreversible structural transitions at 65°C and 85°C at pH4, with the latter being absent at pH7 and pH10. It has also been shown in this study that the melting temperature of the protein is 77.5°C ± 0.5 [START_REF] González-Ramírez | Thermal and pH Stability of the B-Phycoerythrin from the Red Algae Porphyridium cruentum[END_REF].

In this paper, we experimentally explored the properties of B-phycoerythrin under high hydrostatic pressure (HHP). Indeed, in recent years, HHP have attracted attention to food processing and preservation as an alternative to pasteurization [START_REF] Baptista | Inactivation of Staphylococcus aureus by high pressure processing: An overview[END_REF][START_REF] Gayán | Impact of high hydrostatic pressure on bacterial proteostasis[END_REF][START_REF] Perrier-Cornet | High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures[END_REF]. HHP is an efficient non-thermal physical method to cause modification in protein secondary structure. The fluid used to transmit the pressure is usually water. The advantages of HHP are reduced energy costs, a "green" process with no use of chemicals, and the avoiding of temperature-induced denaturation of active substances [START_REF] Huang | Advances in the extraction of natural ingredients by high pressure extraction technology[END_REF].

Only few studies have been performed on the stability of B-phycoerythrin under HHP. The impact of high hydrostatic pressure up to 300 MPa on the absorbance spectrum of a P. cruentum extract in phosphate buffer has been studied by [START_REF] Brody | Effect of pressure on the absorption spectra of phycobiliprotein and Porphyridium cruentum[END_REF]. A shift of phycocyanin absorbance peak from 620 nm to 634 nm was observed as well as a shift from 545 nm and 565 nm to 550 nm and 570 nm respectively for phycoerythrin absorbance peaks.

Additionally, they measured an absorbance increase at 550 nm at a rate of to 1.4.10 -1 MPa -1 and at 570 nm at a rate of 0.61.10 -4 bar -1 . The main hypothesis of the authors of this study to explain these changes in spectral properties as function of the high pressure is correlated to a configurational changes in the pigment-protein complex.

Therefore, the aim of the present study was to investigate, by using multiscale tools, the structural characteristics of B-phycoerythrin, in crude P cruentum extracts treated by HHP ranging from 0.1 MPa to 500 MPa.

Materials and methods

Materials

The marine Rhodophyta Porphyridium cruentum (1380-1C) was provided by the SAG Culture Collection (Göttingen, Germany). The microalga was grown by Microphyt (Baillargues, France) in 5,000 L photobioreactor (PBR) consisting in 1.2 km of glass tubes with co-circulation of liquid medium and CO2 enriched air (Patent No. WO2010109108 A1, 2010;[START_REF] Muller-Feuga | Appraisal of a horizontal two-phase flow photobioreactor for industrial production of delicate microalgae species[END_REF]. The PBR was set under a greenhouse in order to control temperature (between 22 and 28 °C) and the intensity of natural light using curtains. The pH set point of 7.5 was automatically controlled by CO2 injection and monitored by an inline Fermprobe F-235 pH probe from Broadley James (Silsoe, United Kingdom). Air was injected continuously at rate of 35 L min -1 . The culture medium used is a marine type medium, corresponding to a modified Hemerick's medium (Stein-Taylor, 1973). This medium was modified by addition of N and P up to 20 and 4 mmol L -1 respectively. Cultivation was conducted in semi-continuous conditions, thus maintaining the exponential growth phase during 85 days. Cells were harvested by bowl centrifugation at 6000 rpm for 60 min at room temperature (maximum reachable temperature of 35°C) using a KG 8006 from GEA (Oelde, Germany) and concentrated at the rate of around 10-13% dry weight. The biomass was frozen at -20°C in polyethylene bags, heat sealed, and stored at -20°C. The biomass was then dispatched in aliquots in airtight glass bottles at -18°C away from light. The total number freezing/thawing cycle is 2 with freezing at -18°C and thawing at room temperature.

Preparation of protein extracts

P. cruentum biomass was resuspended in 0.5M Tris HCl buffer pH 7 (Sigma Aldrich, Saint Louis, USA) at the rate of 3% (w/v) dry weight equivalent under magnetic stirring for one hour at room temperature away from direct light exposure. The suspension was then centrifuged (Sorvall RC6 (Thermofischer Scientific, Waltham, USA) with a SLC-3000 rotor) at 5000g for 20 minutes at 20°C. The extract corresponds to the supernatant, the pellet was discarded.

Application of high hydrostatic pressure

Homogenous samples of about 1.8 mL of P. cruentum extract were transferred to polyethylene bags, which were then heat-sealed and placed in a high pressure vessel. They were exposed to high pressures at 50, 100, 200, 300, 400 and 500 MPa at 20°C for 5 minutes with a compression rate of approximately 1.6 MPa s -1 . The untreated sample was used as control. The samples were made in triplicates for each pressure level.

The high-pressure treatments were performed in a high hydrostatic pressure vessel (Top Industrie S.A., France). A hand operated pressure pump (Nowaswiss, Switzerland) was used to obtain the pressure, using distilled water as pressure transmitting medium. A sheath Type K thermocouple (Top Industrie S.A., France) was passed though the upper plug, and used to measure and monitor the inner temperature of the vessel during the pressure treatment. The sample chamber pressure was measured using a pressure gauge (SEDEME, France). This system is the same as the one described and used in [START_REF] Perrier-Cornet | High-pressure inactivation of Saccharomyces cerevisiae and Lactobacillus plantarum at subzero temperatures[END_REF].

Characterization of proteins

Dry weight and protein content

The dry weight ratio of P. cruentum extract was determined using gravimetric method: 10 g of sample was kept 24 hours at 102°C in the oven and cooled down in a desiccator. The total nitrogen determination was performed using elementary analysis with a Dumas Nitrogen Analyzer NDA 701 (Velp Scientifica, Usmate, Italy). Dry samples were manually ground. Then, a known weight of sample was inserted in a stainless-steel capsule for mineralization by combustion. Total nitrogen content was converted to soluble protein content using a nitrogen-to-protein ratio of 6.34 ± 0.04, which was precisely determined for P. cruentum extracts by [START_REF] Safi | Influence of microalgae cell wall characteristics on protein extractability and determination of nitrogen-to-protein conversion factors[END_REF].

Three repetitions have been done by sample for the determination of dry weight and protein content.

UV-visible spectroscopy

The suspension was then centrifuged (541/R with F45-30-11 rotor from Eppendorf (Hambourg, Germany)) at 5000 g for 15 minutes at 4°C. The extract corresponds to the supernatant, the pellet was discarded.

The absorbance spectrum between 220 and 800 nm was measured using a Monaco UVmc1 from Safas (Monaco) and semi-micro UV-cuvettes Brand (Wertheim, Germany). Extract samples were diluted in Tris buffer 50 times to respect the Beer-Lambert law and each modality was measured in triplicate.

The quantification of B-phycoerythrin in aqueous extracts was calculated using the following equations firstly used by [START_REF] Bermejo | Recovery of pure B-phycoerythrin from the microalga Porphyridium cruentum[END_REF]:

[R-phycocyanin (R-PC)] (mg mL -1 ) = (OD620 -0.7*OD650)/7.38 (Equation 1)

[Allophycocyanin (APC)] (mg mL -1 ) = (OD650-0.19*OD620)/5.65 (Equation 2)

[B-phycoerythrin] (mg mL -1 ) = (OD565-2.8*[R-PC]-1.34*[APC]
)/12.7 (Equation 3) OD620, OD650 and OD565, correspond to the optical densities measured respectively at 620 nm, 650 nm and 565 nm. The B-phycoerythrin content was determined in mg mL -1 and was then converted in mg g -1 dry biomass.

It is important to mention that this quantification method reflects the ability of the native protein to emit at specific wavelengths and does not reflect the actual protein content.

Fluorescence emission measurements

Fluorescence emission spectra of B-phycoerythrin in P. cruentum extracts were measured using a Fluorolog 3 T (Horiba, Kyoto, Japan). The excitation wavelength was 545 nm and corresponded to the absorbance peak of B-phycoerythrin. Fluorescence emission was measured between 555 and 700 nm with a step value of 0.5 and a slit of 1 nm. One measurement was done per repetition, which is equivalent to 3 measurements per pressure level.

Electrophoresis in native conditions

Invitrogen Novex Wedge Well 4-12% Tris-Glycine Gel (Thermo Fischer Scientific, Waltham, USA) was used as migration gel with 0.3 % Tris pH 8.9 (CAS 77-86-1), 1.4 % glycine (CAS 56-40-6) buffer. Sample buffer was composed of 25 % glycerol (CAS 56-81-5), 12.5 % Tris HCl 0.5M pH 6.8 and 0.05 % bromophenol blue (CAS 115-39-9). All reagents were purchased at Sigma-Merck (Darmstadt, Germany). The NativeMark Unstained Protein Standard Protocol molecular weight scale (Thermo Fischer Scientific, Waltham, USA) was used at the rate of 10 µL per well. 5µL of 50% extract and 50% sample buffer were introduced in each well. 35mA was applied on a single gel. Revelation was performed applying Coomassie blue (CAS 6104-58-1) and then the gel was rinsed with water.

Electrophoresis gel image processing

The electrophoresis gel was scanned by a Chemidoc MP device (BioRad, Hercules, USA) and processes by the software Imagelab ver.5.1. (BioRad, Hercules, USA) with the following parameters: 30 s exposure time, "Red epi" illumination and 695/55 filter. The relative band intensity values were calculated relatively to the control sample (no HHP treatment corresponds to 100%).

Micro-differential scanning calorimetry (Micro-DSC)

A precise amount of extract of about 500 mg was introduced in a micro-DSC device Setaram MicroDSC III (Caluire-et-Cuire, France) with linear temperature profile of 0.5 K minute -1 from 25 °C to 100 °C. The data treatment software associated with the device and used for the peak integration is SETSOFT2000 v.1.2.

The parameters measured on the thermograms are: peak temperature (Tp), peak onset (Ton)

and peak offset (Toff) temperatures and peak enthalpy (through integration). One measurement was done per repetition, which is equivalent to 3 measurements per pressure level.

Statistical analysis

The statistical analysis of Micro-DSC data, principal component analysis (PCA) and hierarchic cluster analysis (HCA) were performed using the software Statistica v.8. by

StatSoft, Inc (Paris, France). The statistical analysis of other data was processed by XLStat v.14.0. by Addinsoft (Paris, France). Regardless of the software used, analysis of variance (ANOVA) and confidence intervals were performed to determine significant differences between the samples for a given parameter. Significance was established at p < 0.05. ANOVA showing significant differences lead to the use of Newman-Keuls pair test.

Results and discussion

Characterization of soluble protein extract

The crude P. cruentum extract was characterized by a pH value of 7.23 ± 0.10 and a dry weight of 8.25 ± 0.02 %. The nitrogen content of this dry weight was 7.49 % corresponding to a protein content of 158.6 mg g -1 dry weight using the nitrogen-to-protein conversion factor of 6.34.

The spectrum between 220 nm and 800 nm of the extract (Figure 1-A) showed a characteristic profile described for the first time by [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF]. Beside the 280 nm peak corresponding to UV-absorbing substances such as protein, the other peaks are typical markers of phycobiliproteins: the double peak at 545 nm and 565 nm along with the smaller peak at 320 nm are related to B-phycoerythrin, whereas the peak around 620 nm and the hardly distinguishable peak at 650 nm are linked respectively to R-phycocyanin and allophycocyanin. The spectrophotometric measurements allowed the calculation of Bphycoerythrin content of 66.11 ± 0.69 mg g -1 dry biomass and a purity index of 2.56 ± 0.04.

In order to investigate specifically the structure of B-phycoerythrin, the extract was submitted to an excitation wavelength of 545 nm, the fluorescence emission of B-phycoerythrin could be measured (Figure 1-B). This fluorescence emission spectrum was close to the one observed by [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF] with a peak around 575 nm.

The molecular weight of the different protein fractions of the crude extract was determined by electrophoresis in native conditions (Figure 2). In the control sample (well 1), several bands were visible before coloration: an originally blue band above 480 kDa, two originally pink intense bands around 242 kDa and a group of three originally pink bands ranging from less than 66 kDa to 146 kDa. The same electrophoretic profile has been described by [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF]. The pink bands were easily linked to B-phycoerythrin with the two intense bands around 242 kDa being identified as charge isomers of B-phycoerythrin and the group of bands of lower molecular weight representing the polydispersed forms of b-phycoerythrin (meaning dimers and tetramers). It is worth to mention that the main difference between Bphycoerythrin and b-phycoerythrin is the presence of a γ subunit of ca. 35 kDa in the B form, which explains its high molecular weight [START_REF] Glazer | Subunit structure and chromophore composition of rhodophytan phycoerythrins. Porphyridium cruentum B-phycoerythrin and bphycoerythrin[END_REF]. The blue band can be linked to R-phycocyanin and this has been confirmed by spectrophotometry [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF]. However, there is no explanation of why the R-phycocyanin band was present above 480 kDa whereas its molecular weight has been determined between 103 kDa and 125 kDa [START_REF] Gantt | Phycobilisomes of Porphyridium cruentum. Pigment analysis[END_REF][START_REF] Glazer | Characterization of R-phycocyanin. Chromophore content of R-phycocyanin and C-phycoerythrin[END_REF]. A similar observation was made by [START_REF] Ma | Characterization of the artificially covalent conjugate of B-phycoerythrin and R-phycocyanin and the phycobilisome from Porphyridium cruentum[END_REF] involving R-phycocyanin and B-phycoerythrin without making further comment. R-phycocyanin's position in native condition electrophoresis should result from a higher aggregation state. As a matter of fact, Rphycocyanin is known to assemble itself in hexamers under specific conditions [START_REF] Glazer | Phycobilisomes: structure and dynamics[END_REF], although the hexamer association is not enough to explain the molecular weight above 480 kDa.

Complementary to electrophoresis, micro-DSC measurements were carried out to investigate the thermodynamic behavior of the proteins in the extract. The thermogram of the control sample is presented in Figure 3. It reveals two main endothermic peaks. These peaks can be integrated to determine unfolding temperature and enthalpy of this unfolding process. For the control sample, we observed a first peak (peak 1) at 58.8 °C ± 0.9 (Ton = 53.8 ± 2.8 °C ; Toff = 64.3 ± 1.2 °C) with an enthalpy of 0,0433 ± 0,0155 J g -1 fresh sample, and a second peak (peak 2) at 85.8 °C ± 0.5 (Ton = 83.7 ± 1.5 °C ; Toff = 87.7 ± 0.7 °C) with an enthalpy of 0,0141 ± 0,0028 J g -1 fresh sample. A third minor peak could be considered at 90.2 °C ± 1.3 (Ton = 89.3 ± 1.1 °C; Toff = 91.4 ± 1.0 °C) with an enthalpy of 0.0021 ± 0.0016 J g -1 fresh sample. These results are reminiscent of the two main irreversible structural transitions at 65 °C and 85 °C revealed by [START_REF] González-Ramírez | Thermal and pH Stability of the B-Phycoerythrin from the Red Algae Porphyridium cruentum[END_REF] using circular dichroism with the two first endothermic peaks appearing at similar temperatures.

Effect of HHP on the soluble protein extract

The spectral properties of the P. cruentum extract treated by high hydrostatic pressure were measured to determine the evolution of B-phycoerythrin concentration and its fluorescence emission yield (Table 2).

The B-phycoerythrin concentration remained stable between 0.1 and 400 MPa treatment but underwent a significant loss (p = 0.007) at 500 MPa of ca. 4 mg g -1 dry biomass (6%) compared to the untreated extract. The fluorescence yield of B-phycoerythrin also remained stable between 0.1 and 300 MPa but showed a significant loss at 400 MPa (-6 % compared to control) that significantly increased at 500 MPa (-20 % compared to control) (p < 0.001).

Those impacts on fluorescence properties of B-phycoerythrin suggest that high pressure induced a conformation change of the protein from 400 MPa leading to its denaturation hence the concentration decrease at 500 MPa.

The observation of the electrophoresis in native conditions (Figure 2) showed that increasing high hydrostatic pressure treatment up to 300 MPa (wells 2 to 5) did not impact the electrophoretic profile. The 400 MPa treatment (well 6) induced the apparition of protein aggregates located at the bottom of the well originally wielding a pink color. Additionally, the b-phycoerythrin bands of lower molecular weight tended to decrease (value intensity of 98, Figure 2). With a 500 MPa treatment (well 7), the pink aggregates gained intensity and the Band b-phycoerythrin bands appeared even weaker (respectively, value intensity of 65 and 94, Figure 2). This result suggests that B-phycoerythrin is more sensitive to pressure than bphycoerythrin between 400 MPa and 500 MPa. Moreover, the bands intensity suggests opposite evolutions between the two forms with an increase of b-PE (value intensity from 97 to 108) and a decrease of B-PE band intensity (value intensity from 98 to 91) between 100

MPa and 300 MPa. The R-phycocyanin band is heavily faded at 500 MPa (if not completely absent) (Table 2).

These observations support the hypothesis that B-phycoerythrin and b-phycoerythrin reach a stability limit towards pressure from 400 MPa with a clear loss at 500 MPa by undergoing aggregation. This statement can be extended to R-phycocyanin at 500 MPa. The disappearance of bands could be related to the low initial concentration rather than intrinsic resistance to pressure. Therefore, it is not possible to conclude that R-phycocyanin showed higher sensitivity to pressure than B-or b-phycoerythrin because of the limit of sensitivity of the used methods.

Moving forwards to the impact of the effects of HHP, the micro-DSC data show no significant evolution of the peaks' summit, onset, offset, width nor enthalpy, except for peak 2 with a significant decrease in enthalpy at 500 MPa (p = 0.001). This suggests a potential link between peak 2 and B-phycoerythrin or R-phycocyanin.

As a way to sum up the effects induced by HHP treatment on B-phycoerythrin in P. cruentum extracts, a Principle Component Analysis (PCA) was performed (Figure 4). F1 is the main axis with 85.80 % proper value and F2 only having 9.66 %. As expected, samples are strongly distributed along the F1 axis with increasing pressure level from left to right (Figure 4-A).

The parameters are strongly correlated with the F1 axis: pressure and peak 3 enthalpy positively and B-PE content, fluorescence yield, peak 1 enthalpy and peak 2 enthalpy negatively. Increasing pressure levels in HHP treatments negatively impact the Bphycoerythrin content and its fluorescence yield. The PCA also highlights the high correlation between peak 2 and B-PE fluorescence yield (correlation value of 0.96, Table 3) which could strongly suggest that peak 2 is related to the structure of B-phycoerythrin. Peak 1 enthalpy is strongly negatively correlated to pressure (-0.98, Table 3) which may tie this parameter to native proteins, but not specifically B-phycoerythrin (correlation with B-PE content of 0.77 and with B-PE fluorescence yield of 0.70, Table 3). The groups formed according to hierarchic cluster analysis (Figure 4-B) separates the 500 MPa treatment from control, 300 and 400 MPa treatments, indicating that the effects induced by HHP clearly happen at 500

MPa. Nevertheless, the 400 MPa treatment constitutes a sub-group alone and differentiates itself from the control, confirming that modifications occurred from 400 MPa. The value of this threshold 400 MPa is in agreement with the previous study performed by [START_REF] Brody | Effect of pressure on the absorption spectra of phycobiliprotein and Porphyridium cruentum[END_REF] on P. cruentum and microalgae extract. Even if there is a lack of information in the literature concerning the effect of HHP treatment on proteins in food matrices, and on the mechanism of proteins unfolding under high pressure in presence of other ingredients, it is now well establish that a pressure between 400 and 600 MPa inactivates most of the food pathogens [START_REF] Baptista | Inactivation of Staphylococcus aureus by high pressure processing: An overview[END_REF].

Conclusion

The HHP process is an emerging technology for the microbiological stability of various food matrices, including the proteins of microalgae as natural colorant. The target pressure to stabilize the food product is above 400 MPa. The results of our study show that the structure of the proteins can be impacted at this level of HHP leading to aggregation at higher pressure.

In fact, five minutes HHP treatments up to 300 MPa had no significant effect either on Bphycoerythrin content and structure in P. cruentum extracts. Nevertheless, signs of conformational changes of the protein are suggested by fluorescence yield decrease from 400

MPa. This phenomenon might lead to protein aggregation of B-phycoerythrin occurring at 500 MPa. R-phycocyanin content was also clearly impacted at 500 MPa and might undergo the same denaturation path.

Previous studies aimed at characterizing model or isolated proteins under high pressure or after applying high pressure. In the present study, the investigation was done on a protein extract from microalgae with a mixture of proteins, and at a concentration relative of their future use as colorant in food or cosmetic applications.

Indeed, with this complex matrix, we observed the structural change with different methods (fluorescence, microcalorimetry, electrophoresis), which allows observing different levels of structure. It is now well established that protein functionalities are linked to their structure.

The observed changes of the proteins structure after applying HHP can thus have a strong impact at macroscopic scale on the food matrices: increase of turbidity, change of texture, stability of emulsion. For example, in the case of the use of microalgae soluble extracts as natural colorant in food or cosmetic matrices (such as drinks or emulsions), if high pressure process is used as an emerging technology to avoid any microbiological contamination, the molecular aggregation of the natural colorants which can happen at around 400 MPa can be the cause of other changes in interaction with the other food ingredients.

At molecular scale, further studies are needed to improve the understanding of the behavior of a mixture of proteins during high pressure processing and if possible at higher concentrations. Peak 2 enthalpy (J g -1 fresh sample) 0.0141 a nd nd nd 0.0101 a 0.0099 a 0.0041 b

  Figure captions

Figure 2 .Figure 3 .

 23 Figure 2. Migration of extracts obtained from Porphyridium cruentum biomass on

Figure

  Figure 4. (A) Results of PCA loading and (B) score plot of the different samples treated

  Figure 4 516

Table 3 :

 3 Correlation matrix based on PCA analysis. 527 Pressure [B-PE] B-PE Fluorescence Yield Peak 1 Enthalpy Peak 2 Enthalpy Peak 3 Enthalpy

	Pressure	1	-0.88	-0.82	-0.98	-0.91	0.69
	[B-PE]	-0.88	1	0.90	0.77	0.85	-0.67
	B-PE Fluorescence Yield -0.82	0.90	1	0.70	0.96	-0.92
	Peak 1 Enthalpy (J g -1 )	-0.98	0.77	0.70	1	0.84	-0.61
	Peak 2 Enthalpy (J g -1 )	-0.91	0.85	0.96	0.84	1	-0.93
	Peak 3 Enthalpy (J g -1 )	0.69	-0.67	-0.92	-0.61	-0.93	1

Acknowledgements

We thank Bernadette Rollin for her technical support.

The micro-DSC analysis was performed with equipment from technical platform "DIVVA" (Développement Innovation Vigne Vin Aliments), AgroSup Dijon -Université de Bourgogne Franche-Comté.

Funding

This work was supported by Conseil Régional de Bourgogne Franche Comté (PARI) and had no specific role in the conduct of the research or in the writing of this paper.