Xavier Bellanger

Raphaël Schneider

Clément Dezanet

Boussad Arroua

Lavinia Balan

Patrick Billard

Christophe Merlin

Zn2+ leakage and photo-induced reactive oxidative species do not explain the full toxicity of ZnO core Quantum Dots

Keywords: Metal oxide nanoparticles, ZnO Quantum Dots, Nanotoxicity, Photo-induced Reactive oxygen species, Cell surface damages

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

dramatically in terms of ROS-generated damages on biomolecules. We propose that direct interactions between NPs and bacterial cell surfaces prime over any kind of intracellular damages to explain the ZnO QDs toxicity on whole bacterial cells.

INTRODUCTION

Late developments in nanotechnologies are experiencing such progresses that they led to the engineering of a plethora of nanoparticles (NPs) with unique properties already fulfilling various tasks in modern life as they massively enter in industrial processes and consumer products [START_REF] Charitidis | Manufacturing nanomaterials: from research to industry[END_REF][START_REF] Stark | Industrial applications of nanoparticles[END_REF]. Among them, metal oxide NPs, such as zinc oxide (ZnO) NPs, are currently focusing considerable attentions due to their high potential for applications such as catalysis, sensing and environmental remediation [START_REF] Wang | Zinc oxide nanostructures: growth, properties and applications[END_REF][START_REF] Moussa | ZnO rods/reduced graphene oxide composites prepared via a solvothermal reaction for efficient sunlight-driven photocatalysis[END_REF][START_REF] Liu | 3D hierarchically porous ZnO structures and their functionalization by Au nanoparticles for gas sensors[END_REF][START_REF] Khin | A review on nanomaterials for environmental remediation[END_REF]. Nevertheless, the outstanding properties associated to the nanoscale of these particles has led to the rapid development of innovative technologies that surpasses the full understanding of the adverse effects of nanomaterials when health and environmental impacts are considered. However, gaining a wide public acceptance for such NPs necessarily implies a deep knowledge and a fine control of their toxicity in order to limit the risks for human and environmental health.

Regarding metal oxide toxicity, a plethora of reports are regularly opposing the production of reactive oxygen species (ROS), the release of metal cations and even direct interaction between the NPs and the cell surface, a long standing debate that reflects the complexity of the underlying toxicity mechanisms involved [START_REF] Li | Mechanism of photogenerated reactive oxygen species and correlation with the antibacterial properties of engineered metal-oxide nanoparticles[END_REF][START_REF] Ma | Impact of solar UV radiation on toxicity of ZnO nanoparticles through photocatalytic reactive oxygen species (ROS) generation and photo-induced dissolution[END_REF][START_REF] Prasanna | Insight into the mechanism of antibacterial activity of ZnO: Surface defects mediated reactive oxygen species even in the dark[END_REF][START_REF] Djurišić | Toxicity of metal oxide nanoparticles: mechanisms, characterization, and avoiding experimental artefacts[END_REF][START_REF] Ying | Metal Oxide Nanomaterial QNAR Models: available structural descriptors and understanding of toxicity mechanisms[END_REF][START_REF] Leung | Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of MgO nanoparticles towards Escherichia coli[END_REF][START_REF] Feris | Electrostatic interactions affect nanoparticle-mediated toxicity to gram-negative bacterium Pseudomonas aeruginosa PAO1[END_REF][START_REF] Bellanger | Stability and toxicity of ZnO quantum dots: interplay between nanoparticles and bacteria[END_REF][START_REF] Ying | Metal Oxide Nanomaterial QNAR Models: available structural descriptors and understanding of toxicity mechanisms[END_REF]. The toxicity of ZnO NPs may be partially attributed to the dissolution of the nanocrystals, which depends on pH, on the size of the particles and on the medium composition, and the associated release of Zn 2+ ions may enter the cells and damage cellular components such as proteins and DNA [START_REF] Lemire | Antimicrobial activity of metals: mechanisms, molecular targets and applications[END_REF][START_REF] Song | Role of the dissolved zinc ion and reactive oxygen species in cytotoxicity of ZnO nanoparticles[END_REF][START_REF] Li | Toxicity of ZnO Nanoparticles to Escherichia coli: mechanism and the influence of medium components[END_REF].

Recent reports highlight also the key role played by the ROS generated by ZnO NPs under light irradiation and even in the dark [START_REF] Ma | Impact of solar UV radiation on toxicity of ZnO nanoparticles through photocatalytic reactive oxygen species (ROS) generation and photo-induced dissolution[END_REF][START_REF] Prasanna | Insight into the mechanism of antibacterial activity of ZnO: Surface defects mediated reactive oxygen species even in the dark[END_REF][START_REF] Applerot | Enhanced antibacterial activity of nanocrystalline ZnO due to increased ROS-mediated cell injury[END_REF][START_REF] Wang | Quantitative analysis of reactive oxygen species photogenerated on metal oxide nanoparticles and their bacteria toxicity: the role of superoxide radicals[END_REF][START_REF] Jiang | Light-induced assembly of metal nanoparticles on ZnO enhances the generation of charge carriers, reactive oxygen species, and antibacterial activity[END_REF][START_REF] Mao | Photo-inspired antibacterial activity and wound healing acceleration by hydrogel embedded with Ag/Ag@AgCl/ZnO nanostructures[END_REF][START_REF] Moussa | Trace amounts of Cu²⁺ ions influence ROS production and cytotoxicity of ZnO quantum dots[END_REF]. ZnO is a semiconductor with a bandgap of 3.37 eV in the bulk state and is able to be activated by wavelengths equal of below 368 nm. that cause membrane damages by lipid peroxidation [START_REF] Dutta | Studies on antibacterial activity of ZnO nanoparticles by ROS induced lipid peroxidation[END_REF]. Moreover, these membrane damages favor the internalization of ZnO NPs and lead to cell death. Noteworthy is also that these photo-generated ROS not only oxidize chemical and biological species located in the vicinity of the NPs but may also affect the ligands at the periphery of the NPs and cause NPs aggregation and/or dissolution [START_REF] Ma | Photochemical instability of thiol-capped CdTe quantum dots in aqueous solution and living cells: process and mechanism[END_REF][START_REF] Kauffer | Incidence of the core composition on the stability, the ROS production and the toxicity of CdSe quantum dots[END_REF]. Although the photoxicity of ZnO NPs is directly related to the level of ROS produced, it also depends on the proximity between the source of ROS and the cells, and thus the level of interaction between the cells and the NP for which the surface chemistry is a key parameter [START_REF] Arakha | The effects of interfacial potential on antimicrobial propensity of ZnO nanoparticle[END_REF]. Clearly, future developments of metal oxidebased nanotechnologies will require a better understanding of the different sources of toxicity and their relative contribution, in order to engineer NPs of controlled and predictable toxicity.

In this study, we investigated the relationship between the surface chemistry of ZnO core QDs and the toxicity of the nanoparticles against Escherichia coli using whole cell assays. Although surprising, different ZnO core QDs exhibiting different levels of reactivity did not differ significantly in terms of toxicity when assessed using whole cell luminescent biosensors. This led us to develop a molecular-based assay where DNA degradation was used as surrogate to evaluate the level of ROS-generated damages on biomolecules. This approach allows pointing out different levels of deleterious effects that were otherwise undetectable when using whole cell toxicity assays.

MATERIALS AND METHODS

Synthesis of ZnO core QDs and evaluation of their stability

3-Aminopropyltrimethoxysilane

(APTMS), (3-glycidyloxypropyl)trimethoxysilane (GLYMO) and the sulfobetaine zwitterionic (SBZ) ligand were used to disperse oleatecapped ZnO QDs in water (Fig. 1). The synthetic protocols used to prepare ZnO@APTMS, ZnO@GLYMO, ZnO@SBZ QDs are detailed in the Supporting Information. ZnO QDs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and UV-visible and fluorescence spectroscopy. The stability of the ZnO QDs with respect to dissolution was assessed by measuring the amount of leaked Zn 2+ (ICP-OES) recovered from the dialysate of an 8-10 kD dialysis device (Float-A-Lyzer G2; Spectra/Por), see Supporting information for details [START_REF] Bellanger | Stability and toxicity of ZnO quantum dots: interplay between nanoparticles and bacteria[END_REF].

Evaluation of QDs-generated ROS

The amount of water and cannot be transposed directly into a possible toxicity level against bacteria in culture media as production of ROS and their availability are influenced by the surrounding biological medium. Nevertheless, comparative assays made using the ZnO@APTMS QDs showed that photogenerated-ROS detected in MOPS culture medium were about 30% of the value measured in water (Supporting information, section 2.4), which remains far from being negligible.

Evaluation of ZnO QDs toxicity

Toxicity tests on live bacterial cells were based on bioluminescence extinction assays of the E. coli strain MG1655(pUCD07) as described elsewhere [START_REF] Bellanger | Stability and toxicity of ZnO quantum dots: interplay between nanoparticles and bacteria[END_REF] (see Supporting information).

Typically, the luminescent bacterium was cultivated in a MOPS mineral medium known to limit metal chelation, and supplemented with gluconate as sole carbon source. Cultures were exposed to a set of ZnO QDs, with or without exposure of a full spectrum neon lighting (18W T8 "Solar Reptil Sun" from JBL, illuminance ca. 9 klux). The extinction of the bacterial luminescence was recorded on plate reader and normalized to the biomass (OD600).

Evaluation of QD-generated stresses

Exposure to free Zn 2+ , oxidative stress or DNA damaging stress, were assessed using whole cell biosensor assays with dedicated E. coli strains. These consist of genetically modified bacteria specifically engineered to emit a bioluminescent signal when the stress is perceived by the live bacteria (transcriptional fusions of regulated promoters to lux reporter genes).

Strains construction and use are detailed in the Supporting information.

Assessing QDs reactivity against DNA

Photo-induced damage to DNA assays consisted in exposing 500 ng of a well-defined pBELX plasmid DNA [START_REF] Bellanger | Natural microbial communities supporting the transfer of the IncP-1β plasmid pB10 exhibit a higher initial content of plasmids from the same incompatibility group[END_REF], for 10 min, to known concentrations of ZnO QDs, with or without exposure to a full spectrum neon lighting as described for the toxicity test. The exposed DNA was further purified using a QIAquick® PCR purification kit (Qiagen) and used as template for the quantification of unaltered molecules by qPCR (Supporting information). DNA treated similarly with free Zn 2+ did not show significant damage nor recovery limitation.

RESULTS AND DISCUSSION

ZnO QDs synthesis and characterization

Oleic acid-capped ZnO QDs were prepared via a sol-gel process according to our previous reports [ref]. To disperse the hydrophobic NPs in aqueous solution, a ligand exchange was conducted using 3-aminopropyltrimethoxysilane (APTMS), (3glycidyloxypropyl)trimethoxysilane (GLYMO) or with a sulfobetaine zwitterionic (SBZ) ligand (Fig. 1) in order to engineer NPs presenting different level of photo-induced ROS production, as well as different surface chemistries enabling to develop different types of interactions with bacterial cells (Supporting Information).

The size and the shape of ZnO QDs were characterized by TEM (Fig. 2a-c). The micrographs show that the dots were relatively monodispersed and the average size of the spherical/ellipsoidal NPs is of ca. 4-5 nm. The clear lattice fringes and the selected area electron diffraction patterns (SAED) (insets of Fig. 2a-c) indicate the good crystallinity of ZnO QDs. This was further confirmed by the XRD patterns of ZnO QDs in which the diffraction peaks could well be indexed to the wurtzite structure of ZnO (space group P6 3 mc, JCPDS No 36-1451) (Fig. 3). The broad peaks observed for all samples further confirm the small size of ZnO NPs.

The UV-visible absorption spectra of ZnO QDs are shown in Fig. 2d-f

1240/λ 1/2 = a + b/D 2 -c/D
where λ 1/2 is the wavelength corresponding to an absorption of 50% to that of the excitonic peak and a,b and c are parameters for ZnO QDs with diameters in the 2.5-6.5 nm range, the diameters D of ZnO@APTMS, GLYMO and SBZ QDs were estimated to be xx, yy, and zz nm, respectively, values in good agreement with those determined by TEM. Finally, due to the intrinsic defects present in ZnO QDs prepared by a sol-gel method, the photoluminescence (PL) spectra only exhibit deep-level visible emission centered at 557, 565 and 559 nm for of ZnO@APTMS, GLYMO and SBZ QDs, respectively (Fig. 2d-f). These emissions originate from an electronic transition from a level close to the conduction band edge to a defect associated trap state (oxygen vacancies, interstitial zinc sites or donoracceptor pairs).The PL quantum yields of ZnO@APTMS, GLYMO and SBZ QDs in water were determined to be 21, 25 and 17%. ZnO@APTMS, (e) ZnO@GLYMO and (f) ZnO@SBZ QDs (the excitation wavelength is 330 nm).

Surface chemistry-dependent ROS production of ZnO QDs

In this study, the toxicity of NPs-generated ROS was explored on the bacterial system, using a set of ZnO core QDs. Four nanometer-sized ZnO core QDs were functionalized with either 3-aminopropyltrimethoxysilane (APTMS), (3-glycidyloxypropyl)trimethoxysilane (GLYMO) or with a sulfobetaine zwitterionic (SBZ) ligand (Fig. 1) in order to engineer NPs presenting different level of photo-induced ROS production, as well as different surface chemistries enabling to develop different types of interactions with bacterial cells (Supporting information, section 1). The three QDs exhibited different levels of photo-induced ROS production (Fig. 1), therefore highlighting the importance of their surface chemistry on their photoreactivity and possibly their toxicity if ROS production is to be concerned.

Fig. 4.

Structure and photo-reactivity of the ZnO core QDs. ZnO@APTMS, ZnO@GLYMO, and ZnO@SBZ QDs are all silane-based nanoparticles differing only by their surface functionalization (amino groups, epoxides and a zwitterion surface coating respectively). The ROS production following 1 h photoactivation was measured in water using common procedures, and normalized to a same initial QD concentration for comparing ROS production by the three nanoparticles (Supporting information, section 3).

Toxicity assessment of ZnO QDs on whole bacterial cells

The toxicity of the three ZnO QDs was evaluated on the E. coli strain MG1655(pUCD607) using a bioluminescence extinction assay. A first set of experiments, carried out in the dark, showed that the QDs exhibit similar toxicities, with Minimum Inhibitory Concentration (MIC) values ranging from 1.8×10 -4 to 2.9×10 -4 MZnEq (molar concentration of zinc equivalent). Noteworthy is that ZnO QDs are ca. twice less toxic than their equivalent content in Zn 2+ as seen with a ZnCl 2 control (MIC ≈ 1×10 -4 M; Fig. 5). With this respect, a dialysis experiment carried out using ZnO@APTMS QDs at concentration slightly lower than MIC (ca. 8×10 -5 MZnEq) demonstrates that the concentration of free Zn 2+ released does not exceed the "No Observable Effect Concentration" (NOEC) obtained for ZnCl 2 (ca. 1×10 -5 M). This therefore rules out the possibility that the toxic effects observed were solely due to the release of Zn 2+ originating from the dissolution of QDs or Zn-containing adsorbed complexes (Supporting information). In a second set of experiments, the QDs toxicity against E. coli was investigated under simulated sunlight exposure. Despite a marked difference regarding their photo-reactivity in terms of ROS production (Fig. 1), no significant difference in toxicity on E. coli was observed between the three QDs, where MIC values were similar to those observed in the dark (Fig. 2, Supporting information Fig. S2). Despite stable MIC values, the bioluminescence profiles appeared to evolve with the illumination time. If part of it could be attributed to the photo-sensitivity of bioluminescence production, as demonstrated in Fig. 2D, the NOECs are clearly displaced towards lower concentrations in a QD-dependent manner. It should be observed that according to Fig. 2C, the ZnO@GLYMO QDs, displaying the lowest NOEC value after two hours of illumination, are also the higher hydroxyl radical producers. ZnO@APTMS ZnO@GLYMO ZnO@SBZ ZnCl 2 ZnO@APTMS ZnO@GLYMO ZnO@SBZ ZnCl 2 ZnO@APTMS ZnO@GLYMO ZnO@SBZ 100% 143% 58%

ZnO@GLYMO, and ZnO@SBZ QDs maintained in the dark for 2 hours (A), maintained in the dark for 110 min and then exposed to a 10 min illumination (B), or illuminated for 2 h (C). Control assays without ZnCl 2 or ZnO QDs amendment displayed a light sensitivity depending of the exposure time (D). Further comparisons are available in the Supporting information. Error bars represent minimum and maximum values of two independent sets of experiments for (A), (B), (C), and standard deviation (n=8) for (D).

Stress assessment using whole cell biosensor assays

When considering the results of the bioluminescence-based toxicity tests, it clearly appears that (i) the three ZnO QDs are less toxic than their Zn content, and (ii) the levels of Zn 2+ leaking from the NPs are so low (Supporting information Fig. S1) that QDs dissolution cannot be considered as a significant source of toxicity by itself. If Zn 2+ leakage is of limited incidence, it is tempting to propose ROS production as an alternative source of toxicity.

However, the stable MIC values observed regardless of the illumination conditions would rather be in favor of a non-ROS-based toxicity. An elucidation of this dilemma was attempted using a set of specific E. coli whole cell biosensors for Zn 2+ , oxidative stress, and DNA damages (Supporting information, section 6), in order to identify which cell stress dominates when bacterial cells are exposed to ZnO QDs. The use of such biosensors non-ambiguously demonstrated that only Zn 2+ ion leakage was perceived by the bacteria (Supporting information Fig. S3C andS3D), although Zn 2+ concentration was too low to explain the QD toxicity by itself. In addition, even if the three ZnO QDs produce ROS, no oxidative stress response was observed using a dedicated biosensor (Supporting information Fig. S3A). All together, the relative stability of the NPs and the absence of photo-induced toxicity tend to show that neither Zn 2+ leakage nor ROS production are responsible for the QDs toxicity. This necessarily implies an alternative source of toxicity, which is not without reminding previous suggestions regarding the deleterious effect of direct cell-NPs interactions [START_REF] Djurišić | Toxicity of metal oxide nanoparticles: mechanisms, characterization, and avoiding experimental artefacts[END_REF][START_REF] Leung | Mechanisms of antibacterial activity of MgO: non-ROS mediated toxicity of MgO nanoparticles towards Escherichia coli[END_REF][START_REF] Feris | Electrostatic interactions affect nanoparticle-mediated toxicity to gram-negative bacterium Pseudomonas aeruginosa PAO1[END_REF]. Still, the absence of ROS-induced stress response in the whole cell biosensors may be surprising as ROS production could be measured for all QDs (Fig. 1). Since ROS are produced extracellularly, cell impairments caused by the oxidative damages of bacterial surface structures are likely to occur before cells could trigger any detectable stress response.

Alternatively, the non-ROS non-Zn 2+ toxicity mentioned before may dominate the QDs toxicity, which leads to strong cell damages that preclude subsequent oxidative stress response to be triggered. At this stage, the level of damages generated by photo-induced ROS remains an open question.

Setting up a DNA degradation assay to assess QD photo-induced reactivity

Apart from being limited to settings allowing physiological conditions, the use of live material for toxicity testing requires that cells should be able to respond before being too damaged. On the principle, this could be overcome if the analytical procedures focus on the alteration of biological material by itself rather than the alteration of the cell functioning. In the next series of experiments, DNA was used as a surrogate biomolecule to estimate the QDs injuriousness at elevated concentrations (to prevent high dissolution rate), and in nonphysiological medium (to limit ROS scavenging if any). Five hundred ng fractions of H 2 Odissolved DNA (plasmid pBELX) were independently exposed for 10 min to each of the QDs at concentrations ranging from 1.5 to 1.8 ×10-3 MZnEq, in the dark or under light irradiation.

An agarose gel electrophoresis of the mixtures clearly showed that in the dark, the QDs had no effect on DNA integrity (Fig. 3A). Only in the case of the ZnO@APTMS QDs, both a showed that, in the dark, ZnO@APTMS and ZnO@SBZ QDs poorly altered pBELX DNA (ca. 75% integrity) while only 4% of undamaged DNA could be recovered after exposure to ZnO@GLYMO. A 10 min light irradiation resulted in dramatic effects on DNA integrity, with ca. 0.4-0.5% of unaltered pBELX recovered after exposure to ZnO@APTMS and ZnO@SBZ QDs, and as low as 0.02% recovery in the case of the ZnO@GLYMO QDs.

These experiments clearly indicate that (i) despite being undetected in whole cell-based toxicity tests, ZnO QDs could cause severe photo-induced damages on biomolecules, and (ii) the levels of photo-induced damages observed partially follow the levels of ROS generated by the various NPs, with ZnO@GLYMO being both the more reactive and the more damaging QDs.

CONCLUSIONS

In conclusion, using a set of differently functionalized ZnO QDs, NPs generating various levels of photo-induced ROS could be engineered. Although the highest ROS producers QDs

Fig. 1 .

 1 Fig. 1. Structures of the APTMS, GLYMO and SBZ ligands used to disperse ZnO QDs in water.

Fig. 2 .

 2 Fig. 2. TEM images of (a) ZnO@APTMS, (b) ZnO@GLYMO and (c) ZnO@SBZ QDs (the insets are the SAED patterns). UV-visible absorption and PL emission spectra of (d)

Fig. 3 .

 3 Fig. 3. XRD patterns of ZnO QDs functionalized with APTMS, GLYMO or SBZ.

Fig. 2 .

 2 Fig. 2. Effect of light irradiation on ZnO core QD toxicity. Bioluminescence extinction curves of strain E. coli MG1655(pUCD607) exposed to ZnCl 2 , ZnO@APTMS,

  plasmid band reduction and a fluorescent smear above the plasmid band indicate a strong interaction between the DNA and the QDs leading to a gel retardation. Conversely, a 10 min light irradiation had a dramatic effect on DNA integrity where fluorescent smears below the normal plasmid band size indicate random DNA fragmentation and thus severe DNA damages. The levels of DNA alteration were further measured by quantitative PCR (qPCR), considering that a full DNA integrity is a prerequisite for a proper PCR-amplification to proceed. Analyses of DNA integrity by qPCR, after purification of the QDs-exposed DNA,

Fig. 3 .

 3 Fig. 3. Photo-induced DNA damages by ZnO QDs. a, Alteration of pBELX plasmid DNA22(3169 bp) upon exposure to ZnO@APTMS (A), ZnO@SBZ (S), and ZnO@GLYMO (G) for 10 min with or without light irradiation (full spectrum white light, 9 klux illuminence) and the mixture was run on a 1% agarose gel in TAE buffer. DNA was stained with ethidium bromide and "2log DNA ladder" (New England Biolabs®) was used as DNA molecular weight marker. An image analysis (Image Lab™ Software, Gel Doc™, Bio-Rad) shows that the total amount of the fluorescence is conserved between lane except for from lanes #13, #14, #15, where only 40% to 10% of the fluorescence is kept, indicating a pure loss of DNA material. b, Quantification of pBELX DNA integrity following a 10 min exposure to ZnO@APTMS (A), ZnO@SBZ (S), and ZnO@GLYMO (G) at 1.610-3, 1.510-3, and 1.810-3 MZnEq respectively, with or without light irradiation. DNA integrity was estimated by qPCR and is given as a fraction (% recovery) of what obtained for untreated DNA controls. Error bars represent standard deviations of 6 values (2 independent experiments with triplicate qPCR assays each).

  GLYMO) were the most damaging towards DNA, the levels of photo-induced damages did not fully correlate with the ROS production, indicating that alternative phenomena such as QD-biomolecule interactions might also contribute to the overall NP reactivity. However, if all ZnO QDs were undoubtedly very photo-reactive towards a surrogate biomolecule (DNA), no clear photo-induced toxicity could be observed in whole bacterial cells toxicity assays. This likely illustrates the barrier effect exerted by numerous biomolecules present on cell surfaces that prevent the ROS produced to reach critical cell functions. With this respect, it is worth mentioning that, contrary to eukaryotic cells, bacteria do not practice endocytosis and QDs have to cross several external barriers before reaching vital functions, suggesting that only closely delivered ROS could be effective. While the ROS did not appear to be directly involved in the cell toxicity observed for the studied ZnO QDs, it was quite surprising to observe that the free Zn 2+ leaked by the QDs did not reach the critical level where cell functioning could be impaired. Considering that both ROS production and Zn 2+ leaks are insufficient to impair cell functioning, a third source of toxicity has to be considered. Taking into account the nanoscale dimensions of the particles, direct and deleterious interactions of the NPs with important extracellular/periplasmic cell components (e.g. respiratory chain, transporters) could well be a plausible explanation to explore. Whatever the toxicity mechanisms involved, this study emphasizes the fact that toxicity tests carried out on live bacterial cells are likely inappropriate for evaluating NP generated-ROS deleterious effects. For that reason, the DNA-damaging test we propose here advantageously offers (i) the possibility of quantifying photo-induced damages on a biomolecule without making use of live cells therefore avoiding interfering parameters such as NP internalization or bio-generated ROS, (ii) to work in non-physiological conditions, e.g. in water, thus avoiding ROS scavenging for instance, and (iii) to work at NP concentrations well over the toxic level if necessary.ACKNOWLEDGMENTThis work was supported by the program CESA of the French National Research Agency (ANR) (Project "NanoZnOTox"; ANR-11-CESA-0004ANR). Additional support was gained from the Hydreos competitiveness cluster. The authors wish to thank Hervé Marmier (LIEC, Université de Lorraine) for ICP-OES analyses, Ghouti Medjhadi (IJL, University de Lorraine) for XRD analyses and Mathieu Lhuire for technical assistance.

  The associated oxido-reduction reactions occurring with O 2 and H 2 O at the surface of ZnO

	NPs generate hydroxyl ( • OH), superoxide (O 2	•-) and hydroperoxyl (HO 2	• ) radicals and H 2 O 2

  and the excitonic peaks are nearly located at the same wavelength (ca. 336 nm), further confirming that ZnO QDs functionalized by APTMS, GLYMO or SBZ exhibit the same size. The excitonic absorption of bulk ZnO appears at ca. 380 nm (3.26 eV), value significantly higher than those determined for ZnO QDs, indicating the spatial confinement of the charge carriers in these nanocrystals. Using the experimental formula [ref]:

APPENDIX A. SUPPLEMENTARY DATA

Supplementary data associated with this article can be found, in the online version.


G Sdanghi

J Dillet

S Didierjean

V Fierro

G Maranzana

Feasibility of hydrogen compression in an electrochemical system: focus on water transport mechanisms

Keywords: hydrogen electrochemical compression, polymer electrolyte, water management

In this study, the behavior of an electrochemical hydrogen compressor (EHC) able to compress hydrogen up to 32 bar was investigated. The current density distribution along the EHC was measured using a segmented cell. It was found to decrease from 0.75 A cm -2 to 0.65 A cm -2 along the EHC when using Nafion 117 and when high relative humidity hydrogen was fed to the EHC (at 0.66 A cm -2 and 0.36 V). This drop corresponds to a decrease of the relative humidity of the hydrogen flow from 90 to 55% along the gas channels at the anode side, evidencing the local dehydration of the PEM due to the unbalanced water transport across the membrane. As a consequence, the membrane resistance increases, thus a higher voltage must be supplied to the EHC in order to maintain good performances, decreasing the overall efficiency. On the other hand, unstable operating conditions were observed when using hydrogen with a low relative humidity.

A pseudo-2D model was developed along with experimental studies to estimate the physical parameters enhancing the efficiency of the EHC. A higher efficiency was obtained when using a thinner membrane than Nafion 117 (53% vs. 37%, at 60°C and 0.66 A cm -2 ).

Introduction

Compression is nowadays the most recommended method to store hydrogen for automotive applications [1] as it allows an increase of the hydrogen volumetric energy density, e.g. from 0.01079 MJ L -1 at 1 bar and 15°C to 4.6 MJ L -1 at 700 bar and 15°C. For applications demanding high flow rates and high discharge pressures, mechanical compressors, such as the piston compressors and diaphragm compressors [2,3], are employed. Even though these technologies are widely used worldwide, they exhibit several drawbacks, as: (i) many moving components during operation; (ii) noise and vibration; (iii) possibility of contaminating hydrogen because of the use of lubricating oils; (iv) operational and maintenance difficulties; and (v) large dimensions.

In this frame, the electrochemical hydrogen compressor (EHC) seems to be a valid alternative for compression up to 1000 bar [START_REF]HyET Hydrogen | HyET Hydrogen[END_REF], as it provides pure hydrogen in a silent and practical system, with a lower capital cost and energy requirements compared to a mechanical compressor [START_REF] Fishel | High Temp. Polym. Electrolyte Membr. Fuel Cells[END_REF].

In an EHC, hydrogen at atmospheric pressure is fed to the anode of an electrochemical cell where the oxidation reaction occurs, thus hydrogen splits into protons and electrons while electric energy is provided to the system. Protons cross a solid polymer electrolyte membrane (PEM) towards the cathode where protons reduction takes place producing high-pressure hydrogen. Grigoriev et al. [START_REF] Grigoriev | [END_REF] showed that it is possible to compress hydrogen from atmospheric pressure up to 48 bar in a single EHC by applying a cell voltage of 140 mV and at 0.2 A cm -2 , with a corresponding energy consumption of around 0.3 kWh Nm -3 . Nevertheless, it is quite hard to achieve very high pressures in an EHC because of the occurring hydrogen permeation from the cathode to the anode side [7]. Rohland et al. [8] showed that the hydrogen permeation linearly increases with the pressure gradient. Moreover, they found that the higher the cell temperature the higher the hydrogen back diffusion: a diffusion rate of around 2 mA cm -2 was obtained at 24°C, whereas 6 mA cm -2 were obtained at 70°C and 15 bar. Grigoriev et al. [START_REF] Grigoriev | Proc Fundam. Dev. Fuel Cells 2008 Conf[END_REF] carried out a numerical investigation to estimate the maximum output pressure reachable by a single EHC. They found that high output pressures are achievable at low temperatures mainly because of the lower hydrogen permeation.

The EHC was found to be more economical and convenient than a mechanical compressor, as the capital expenditure for an EHC is around $170-1700 kg -1 day -1 compared to $2300 kg -1 day - 1 of a hydrogen reciprocating compressor [START_REF] Sdanghi | [END_REF]. Moton et al. [START_REF] Moton | Proc. ASME 2014 12th Int. Conf. Fuel Cell Sci. Eng. Technol[END_REF] carried out a deep technoeconomic analysis of an EHC, finding that the cost of both the EHC and the balance of plant (BOP) increases in a non-linear way with the outlet pressure in order to withstand the higher mechanical loads and leaks. Furthermore, they found that the membrane-electrode-assembly (MEA) contributes the most to the cost increase, as it covers 45% of the total costs. Wu et al. [START_REF] Wu | [END_REF] investigated the possibility of using non-fluorinated membranes with the aim of reducing costs, but this leads to a decrease in efficiency compared to Nafion-based membranes. For all these reasons, several authors found that an outlet pressure as high as 150 bar is recommended in order to reach satisfying efficiencies at acceptable costs [START_REF] Fishel | High Temp. Polym. Electrolyte Membr. Fuel Cells[END_REF]8,[START_REF] Moton | Proc. ASME 2014 12th Int. Conf. Fuel Cell Sci. Eng. Technol[END_REF]. Nevertheless, the abovementioned mechanical limitations can be overcome by connecting several cells in series: in this way, the pressure of 700 bar demanded for hydrogen automotive applications can be reached with a minimum of 4 stages of single cell compressors [START_REF] Najdi | Proc 3rd Int. Conf. Adv. Comput. Tools Eng. Appl. ACTEA[END_REF]. Such a solution allows reaching even higher pressures and efficiencies of 90% for small hydrogen flowrates [START_REF] Bouwman | [END_REF]16].

Water management is one of the key factors affecting the performance of an EHC. Alike in the PEM fuel cells (PEMFCs), the membrane has to be hydrated in order to enhance its proton conductivity [17]. However, water is not a reaction product in an EHC, thus it needs to be fed along with hydrogen in order to preserve the optimal hydration degree of the membrane. Casati et al. [18] showed that better control of the membrane hydration degree can be reached by adopting tensiostatic operating conditions instead of galvanostatic ones. Onda et al. [19] found that both the relative humidity of the hydrogen flow and the current density distribution decrease along the channel direction during operation. This behavior was found to be directly related to the water transport across the membrane. In fact, the unbalanced contribution of the two main water transport mechanisms across the membrane, i.e. the electro-osmotic drag and the water back diffusion, can lead to the local dehydration of the membrane, which entails the increase of the local electric resistance. The main consequence of these operating heterogeneities could be a dramatic increase in the voltage to supply, which would reduce the overall efficiency of the system.

In this study, the local behavior of an EHC was investigated through both an experimental and a numerical approach. A segmented cell was used to perform a distributed performance analysis of the EHC. This diagnostic tool allows measuring the current density at specific segments of the MEA, which are electrically independent of the others [20]. In this way, the distribution of both the current densities and the electric resistances along the gas channel direction can be obtained, evidencing the local heterogeneities arising during operation. The segmented cell technology has been already used for investigating the PEM fuel cell durability [21][22][23] as well as that of PEM electrolysis cells [24], but it was never used for an EHC, as far as we know. The effect of the current density supplied to the system, the humidity of the feed gas and the membrane thickness on the local current density distribution was evaluated and discussed.

A pseudo-2D model was developed and validated by comparison to the obtained experimental data. This model takes into account both the overall mass and energy balances occurring in the EHC and the heterogeneities in the relative humidity along the gas distribution channels. The model also allowed investigating the effect of the aforementioned operating parameters on the EHC performance, in order to optimize the system and enhance its overall efficiency.

A single 30 x 1 cm 2 cell segmented into 20 electrically-insulated parts of 1.5 cm 2 each was used, a detailed description is given elsewhere [25,26]. All segments were connected after the measurement system so that they operated at identical voltage. The local current densities were measured using shunt resistors (5mΩ), with an uncertainty of ± 1 mA. The schema of the EHC along with the BOP is shown in Figure 1. An external power supply of nominal capacity 1500 W was used. The EHC temperature was set by using a hot water circulating system mechanized in the support plates and driven by a platinum Pt100 probe. Hydrogen was fed to the cell after bubbling it through water for humidification. All the gas lines were thermostated at 75°C in order to prevent water condensation and purged with nitrogen before each experiment.

Humidified hydrogen was fed into the anode side through 5 parallel channels of 0.1 cm x 0.1 cm x 30 cm, mechanized on the support plates, and the hydrogen flow was adjusted by means of mass flow controllers. Unreacted hydrogen was collected at the anode outlet and its humidity was measured by a probe Vaisala HUMICAP ® humidity and temperature transmitter HMT330, also thermostated at 75°C. At the cathode, the inlet was closed and a condenser unit was placed at the outlet to remove excess water from the high-pressure hydrogen flow. Downward the condenser unit, a pressure regulator Brooks ® SLA5820s regulated the discharge pressure of hydrogen.

The MEAs were supplied by Ion Power. Two kinds of Nafion membranes were tested: (i) Nafion XI of thickness 25 µm and (ii) Nafion 117 of thickness 175 µm. Gas diffusion layers (GDLs) Sigracet® (thickness 240 µm) and a gold-coated titanium porous transfer layer (PTL) with a pore size of 5 µm and thickness 1 mm were used at the cathode and at the anode side, respectively. The use of a thick PTL instead of carbon papers on the anode side was essential to withstand the important pressure gradient over the cell. (vi) high-pressure hydrogen formation at the cathode; (vii) hydrogen and water transport across the GDL towards the distribution channels; and (viii) heat transfer across the PTL, the MEA and the GDL. All these phenomena take place in the perpendicular direction to the MEA. In addition, a significant variation of water and hydrogen concentrations could be observed while the binary mixture flows along the gas channels, thus a one-dimensional (1D) model is not able to accurately describe the transport phenomena occurring in an EHC.

For this reason, a pseudo-2D model was developed, which revealed to be more computationally efficient compared to 2D and 3D models [27] . A schema illustrating the principles of the pseudo-2D model is shown in Figure 2. The transport phenomena were investigated in both directions,

x and y, i.e. the perpendicular direction to the MEA and the along-the-channels direction respectively. The EHC was divided into k control volumes; hence the mass and energy balances along the x-direction were solved for each control volume. The hydrogen and water concentrations in the gas channel, cH2 chan (k) and cH2O chan (k), were both evaluated through the mass transport equations in the y-direction, hence they were used as spatially boundary conditions for mass and energy balances along the x-direction. Thus, the only coupling between the two directions is achieved through boundary conditions.

In developing the pseudo-2D model, several simplifying assumptions were used: i. Both hydrogen and water vapor were assumed to behave as ideal gases since the highest pressure and temperature reached by the experimental investigation were 32 bar and 60°C, respectively. Indeed, the highest compressibility factor reached at these operating conditions, determined by using the REFPROP-7 software, is 1.02. Thus, the deviation from the ideal behaviour is negligible;

ii. The partial pressure of water at the cathode side was assumed to be equal to the vapor pressure saturation at the operating temperature due to the high-pressure conditions. Indeed, the saturation pressure of water at 60°C is about 0.2 bar (calculated by the following Eq. ( 4)), whereas the total pressure at the cathode side is 32 bar. This means that, the highest molar fraction of vapour water achievable herein is < 0.006. Thus the presence of liquid water was assumed at the cathode of the EHC in order to observe the total mass balance;

iii. Catalyst layers were assumed to act as thin interfaces where the electrochemical reactions occur without resistance to the mass or heat transfer [27].

In the following sections, every single part of the model is introduced and discussed.

Hydrogen and water transport along the gas channels

The mass transport along the y-direction allows finding the hydrogen and water concentrations at the anode gas channels for each control volume k. These concentrations will be used as boundary conditions for the balances in the x-direction.

The hydrogen flow rate changes because of both hydrogen consumption on the anode and the permeation of the dissolved hydrogen molecules through the membrane, which is favored by the pressure gradient existing between the cathode and the anode side. Hence, the hydrogen flow rate generally decreases along the y-direction because of its consumption given by Faraday's law. The hydrogen flow rate 𝑛Ḣ 2 along the anode gas channels is given by:

{ 𝑑𝑛̇𝐻 2 𝑑𝑦 = -𝑝 [ 𝑖(𝑦) 2𝐹 -𝑁 ̇𝐻2 ,𝑝𝑒𝑟𝑚 ] 𝑛̇𝐻 2, (𝑦=0) = 𝑆 𝐴 𝐼 2𝐹 (1)
where p is the width of the active area, i(y) is the local current density, F is the Faraday constant (96485 C mol -1 ), S is the stoichiometric ratio, A is the active area, I is the supplied current density to the EHC and 𝑁 ̇𝐻2 ,𝑝𝑒𝑟𝑚 is the hydrogen molar flux permeating across the membrane.

We used the expression proposed by Schalenbach et al. [28] to describe the temperature dependence of 𝑁 ̇𝐻2 ,𝑝𝑒𝑟𝑚 :

𝑁 ̇𝐻2 ,𝑝𝑒𝑟𝑚 = 𝜀 0 𝑒𝑥𝑝 ( -𝐸 𝜀 𝑘 𝐵 𝑇 ) ( 𝑃 𝐻 2 𝑐𝑎𝑡 -𝑃 𝐻 2 𝑎𝑛 𝑥 𝑚𝑒𝑚𝑏 ) (2)
The permeation flux depends on the gas permeability, on the difference of hydrogen partial pressure at the cathode, PH2 cat , and the anode, PH2 an , and on the membrane thickness, xmemb. kB is the Boltzmann constant (1.38•10 -23 J K -1 ), whereas the value of the constants ε0 and Eε were taken from the literature [29] and are listed in Table 1.

On the other hand, water concentration varies along the y-direction because of the mass transport across both the PTL and the membrane. The water flow rate, 𝑛̇𝐻 2 𝑂 , along the gas channels is given by:

{ 𝑑𝑛Ḣ 2 𝑂 𝑑𝑦 = 𝑝𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 𝑛̇𝐻 2 𝑂 (𝑦=0) = 𝑆 𝐴 𝐼 2𝐹 𝑅𝐻 𝑐ℎ𝑎𝑛,𝑖𝑛 𝑝 𝑠𝑎𝑡 (𝑇) 𝑃 𝑎𝑛 -𝑅𝐻 𝑐ℎ𝑎𝑛,𝑖𝑛 𝑝 𝑠𝑎𝑡 (𝑇) (3)
where 𝑛̇𝐻 2 𝑂 is the water flow rate along the distribution channels, 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 is the water molar flux across the PTL, RHchan,in is the relative humidity of hydrogen at the gas channels inlet, psat(T) and Pan are the saturation pressure and the total pressure on the anode side, respectively. psat(T) was evaluated by using the Rankine's formula [30]:

𝑝 𝑠𝑎𝑡 (𝑇) = 𝑃 0 exp (13.669 - 5096.23 𝑇 + 273.15 ) ( 4
)
where P0 is the standard pressure.

Eq. ( 3) shows that drying of the hydrogen flow inwards the EHC is possible when there is an important water flow from the anode to the cathode; in contrast, the hydrogen flow is humidified when water flows is directed from the cathode to the anode side. Thus, the evolution of the relative humidity along the gas channels, RHchan, can be evaluated by the following expression:

𝑅𝐻 𝑐ℎ𝑎𝑛 (𝑦) = 𝑛̇𝐻 2 𝑂 𝑛̇𝐻 2 𝑂 + 𝑛Ḣ 2 𝑃 𝑎𝑛 𝑝 𝑠𝑎𝑡 (𝑇) (5)

Hydrogen and water transport across the PTL

The Stefan-Maxwell equation was used to predict hydrogen and water transport by convection and diffusion across the PTL. The Stefan-Maxwell equation is a generalization of the Fick's Law, and it is commonly used to describe the multicomponent convective and diffusive transports in a porous medium, and it was well demonstrated for a uniform velocity profile. The integrated form of Stefan-Maxwell equation [31] for the specific case of a binary flow of hydrogen and water is reported:

𝑦 𝐻 2 = (𝐻𝑅 𝑐ℎ𝑎𝑛 𝑃 𝑠𝑎𝑡 𝑃 𝑎𝑛 - 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 𝑁 ̇𝐻2 𝑃𝑇𝐿 + 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 ) * 𝑒𝑥𝑝 ( (𝑁 ̇𝐻2 𝑃𝑇𝐿 + 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 ) * 𝑥 𝑃𝑇𝐿 𝑐𝒟 𝐻 2 ,𝐻 2 0 𝑒𝑓𝑓 ) - 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 𝑁 ̇𝐻2 𝑃𝑇𝐿 + 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 ( 6
)
where 𝑦 𝐻 2 PTL is the molar fraction of hydrogen in the PTL, 𝑁 ̇𝐻2 PTL and 𝑁 ̇𝐻2 𝑂 PTL are the molar flux of hydrogen and water across the PTL respectively, c is the global molar concentration of the hydrogen/water mixture, 𝒟 𝐻 2 ,𝐻 2 0 eff is the effective diffusion coefficient and xPTL is the PTL thickness. 𝒟 𝐻 2 ,𝐻 2 0 eff was used instead of the intrinsic diffusion coefficient, 𝒟 𝐻 2 ,𝐻 2 0 since the first one takes into account the PTL porosity [START_REF] Grigoriev | Proc Fundam. Dev. Fuel Cells 2008 Conf[END_REF], which affects the mass transport across the diffusion layer. Furthermore, the effect of temperature and pressure on 𝒟 𝐻 2 ,𝐻 2 0 eff was considered as well [32] trough:

𝒟 𝐻 2 ,𝐻 2 0 𝑒𝑓𝑓 (𝑇, 𝑃) = 𝒟 𝐻 2 ,𝐻 2 0 0 (𝑇 0 , 𝑃 0 ) 𝑃 0 𝑃 ( 𝑇 𝑇 0 ) 3/2 • 𝜀 𝑃𝑇𝐿 3/2 (7)
where 𝒟 𝐻 2 ,𝐻 2 0 0 is the diffusion coefficient evaluated at a specific temperature, T 0 = 303 K, and pressure, P 0 = 1 bar [33] and 𝜀 is the porosity of the PTL. Eq. (7) shows that the higher the system temperature and 𝜀 the higher the 𝒟 𝐻 2 ,𝐻 2 0 eff .

Water transport across the membrane

In order to ensure optimum compression performances, a good hydration level of the membrane is required, since its protonic conductivity is enhanced when the membrane is saturated with water. Hence, protons cross the membrane in the hydrated form: H2ζ+1Oζ + , where ζ is the electro-osmosis coefficient. Membrane hydration is a key factor for water management in an EHC, and it can be evaluated by using the equation proposed by Springer and Zawodzinski [34]:

𝜆 = 0.043 + 17.81𝑎 -39.85𝑎 2 + 36𝑎 3 ( 8
)
where a is the water activity and λ is the membrane water content, defined as the number of water molecules per sulfonic acid site. a can be defined by the ratio partial water pressure/vapor water pressure, at the same temperature. Eq. ( 8) was derived for the Nafion 117 membrane at 30°C, but it has been applied also to temperatures as high as 60°C [35,36]. According to the assumption 2, we considered that the cathode side is saturated with water and then a = 1 and λ =14 as boundary conditions at the membrane-cathode interface. Actually, Schroder paradox [37] states that λ can be higher than 14, therefore the final value depends on both the pretreatment process and the mechanical constraints.

Water transport across the membrane occurs via two main mechanisms: electro-osmosis and diffusion. The electro-osmosis flux, from anode to cathode, takes place when water molecules are dragged across the membrane by the protons because of the electrostatic attraction. On the other hand, water diffusion, from cathode to anode, occurs as a consequence of the water concentration gradient across the membrane. In the present model, the global water flux across the membrane, 𝑁 ̇𝐻2 𝑂 𝑚𝑒𝑚𝑏 , was supposed to be positive when directed from the anode to the cathode, and it can be evaluated by using the following equation:

𝑁 ̇𝐻2 𝑂 𝑚𝑒𝑚𝑏 = 𝜁 𝑖(𝑦) 𝐹 -𝐷 𝐻 2 𝑂 𝑚𝑒𝑚𝑏 𝜌 𝑑𝑟𝑦 𝐸𝑊 𝑑𝜆 𝑑𝑥 ( 9
)
where ζ is the electro-osmotic drag coefficient, 𝑖(𝑦) is le local current density, ρdry is the dry membrane density, EW is the membrane equivalent weight and DH2O memb is the diffusion coefficient of water across the membrane .

ζ, defined as the number of water molecules dragged per proton, was considered as constant in the present work. On the other hand, the variation of DH2O memb with respect to both λ and T was modeled by using the following expression from [38]:

𝒟 𝐻 2 𝑂 𝑚𝑒𝑚𝑏 = (5.1 -0.66𝜆 + 5.2 10 -2 𝜆 2 -1.3 10 -3 𝜆 3 ) ⋅ 10 -6 𝑒𝑥𝑝 [2416 ( 1 303 - 1 𝑇 )] (10)
According to these assumptions, the following equation for 𝑁 ̇𝐻2

where λcat and λan are the membrane water content at the cathode and at the anode side, respectively, and xmemb is the membrane thickness.

Charge

The voltage to be supplied to the EHC, E, is given by the following equation [START_REF] Sdanghi | [END_REF]:

𝐸 = 𝑅𝑇 2𝐹 𝑙𝑛 ( 𝑃 𝑐𝑎𝑡 𝑃 𝑎𝑛 ) + 𝑅𝑇 2𝛼 0,𝑎𝑛 𝐹 𝑙𝑛 ( 𝑖 𝑖 0,𝑎𝑛 ) + 𝑅𝑇 2𝛼 0,𝑐𝑎𝑡 𝐹 𝑙𝑛 ( 𝑖 𝑖 0,𝑐𝑎𝑡 ) + 𝑅 𝑒𝑙 𝑖 (12)
where α0,an and α0,cat are the charge transfer coefficient for the hydrogen oxidation and reduction, respectively; and, i0,an and i0,cat are the exchange current density for the anode and the cathode reaction, respectively. Rel incorporates: (i) the protonic resistance of the membrane, 𝑅 𝑚𝑒𝑚𝑏 ;

(ii) the ionic and electronic resistance of the electrodes; and (iii) the contacts and cell hardware resistances. The first term of Eq. ( 12) is the Nernst voltage, which indicates the minimum voltage to be applied to the system in order to achieve the desired compression ratio. The second and the third terms are the anode and the cathode overpotentials, which can be defined as the potential losses due to the kinetics of the reaction. Finally, the last term of Eq. ( 12) indicates the Ohmic losses, which are mainly governed by 𝑅 𝑚𝑒𝑚𝑏 and is roughly given by the membrane thickness/ membrane protonic conductivity ratio, xmemb/𝜎memb. The following expression for 𝜎memb was used [34]:

𝜎 𝑚𝑒𝑚𝑏 = (5.139𝜆 -3.26) • 10 -3 𝑒𝑥𝑝 (1263 ( 1 303 - 1 𝑇 )) (13)
Eq. ( 13) clearly shows that the higher λ the higher its 𝜎memb. Thus, high water content is necessary in order to decrease the protonic resistance of the membrane. Eq. ( 13) is valid for λ > 0.63 (which gives σmemb > 0). For λ < 0.63, σmemb was set to 10 -10 S cm -1 and not to zero to prevent numerical difficulty.

Precisely, the global 𝑅 𝑚𝑒𝑚𝑏 is given by the following expression:

𝑅 𝑚𝑒𝑚𝑏 = ∫ 𝑑𝑥 𝜎(𝜆(𝑥), 𝑇) 𝑥 𝑚𝑒𝑚𝑏 0 (14)
To solve the previous definite integral, the trapezoidal rule was used, which gives an average value between the protonic resistance evaluated at the anode and that evaluated at the cathode:

𝑅 𝑚𝑒𝑚𝑏 = 𝑥 𝑚𝑒𝑚𝑏 2 ( 1 𝜎(𝜆 𝑐𝑎𝑡 , 𝑇 𝑐𝑎𝑡 ) + 1 𝜎(𝜆 𝑎𝑛 , 𝑇 𝑎𝑛 ) ) (15)

Heat transfer and temperature evolution

The one-dimensional heat transfer in the through-plane direction was described using the analogy with an electrical circuit, and composed by the thermal resistances and heat sources or sinks (Figure 3).

The following three heat sources were considered:

i. Heat conduction: the heat conduction occurring across the PTL, the membrane and the GDL was modeled by using the Fourier law. Thus, a thermal resistance was introduced for each layer and it was defined as the layer thickness/ thermal conductivity ratio.

ii. Water condensation/evaporation: water vapor is present along the PTL, liquid water is present along the membrane and the GDL contains both liquid and vapor water. Thus, sorption/desorption phenomena may occur on the membrane layer interfaces with PTL and GDL. Water condensation occurs on the anode/membrane interface and the amount of released heat can be obtained by the product between the water flow across the membrane (Eq. ( 10)) and the heat of sorption, which can be assimilated to the latent heat of water vaporization [39]. However, evaporation was not considered on the cathode/membrane interface because high pressure almost avoids water vaporization, only a small amount of water could vaporize. Therefore, saturation conditions all over the cathode side were assumed.

iii. Joule effect: heat is released in the membrane because of its resistance, which is the main factor causing the increase of the temperature along the membrane.

The contribution of the activation overpotential to the heat production was not taken into account in our model, as it is negligible in an EHC. Indeed, both the hydrogen oxidation and reduction reactions are highly reversible [13]. Hence, we assumed that the temperature gradients were only due to the above-stated heat sources, as both support plates are initially at the same temperature on the low and on the high-pressure compartments.

Figure 3: Equivalent electric circuit for the heat transfer along the x direction

Implementation of the numerical method

The coupled equations of the above-presented pseudo-2D model were solved by using the Matlab software. Table 1 contains all the parameters used for the numerical simulations. Most of the parameters are related to the cell geometry and to the dimensions of every single component, i.e. the PTL, the membrane and the GDL. The thermal conductivity of the PTL was determined from the measurement of the heat capacity, using a calorimeter (Setaram® dSc3),

and from the measurement of the thermal diffusivity, using the flash method (equipment) [START_REF] De Giovanni | Thermal Properties Measurement of Materials[END_REF].

A porosity of 28% was determined for the PTL, taking into account the density of the bulk and of the porous titanium. The parameters for which reliable estimates were available in the literature were set and also listed in Table 1, e.g. the thermal conductivity of the membrane and the parameters related to the hydrogen permeation across the membrane. On the other hand, parameters that could not be reliably estimated and were difficult to be directly measured were obtained by fit of data taking into account both the boundary conditions and the experimental results. It is the case of the electro-osmotic drag coefficient ζ, which was estimated by considering both the measured cell voltage and the RH of the hydrogen flow measured at the anode outlet. For all the simulations, a value of ζ around 0.9 has been adjusted, in agreement with experimental data from literature [START_REF] Zawodzinski | [END_REF]. From [43] Diffusion coefficient / cm 2 s -1 DH2,H2O 0 Membrane equivalent weight / kg mol -1 EW 1.1 [START_REF] Ramousse | [END_REF] Membrane thermal conductivity / W cm

Overview of the EHC performances

Our results confirmed that the isothermal compression of hydrogen can be achieved by using an EHC. Figure 4 shows the predicted average temperature of the anode, the membrane and the cathode, as a function of the supplied current density. The temperature difference between the membrane and the supporting plates, which were kept at 60°C, was lower than 0.3°C for low supplied current densities and never higher than 1°C. Thus the EHC system ensured an isothermal hydrogen compression, which is one of its most important advantages over any other hydrogen compression system available nowadays. Isothermal compression requires low compression work at very high pressure contrarily to an adiabatic process [START_REF] Çengel | Thermodynamics: An Engineering Approach[END_REF], thus high efficiency can be reached.

The theoretical compression work, Wth, of an EHC can be written by considering the compression as isothermal without any voltage losses [START_REF] Çengel | Thermodynamics: An Engineering Approach[END_REF]:

𝑊 𝑡ℎ = 𝑅𝑇 𝑀 𝑙𝑜𝑔 ( 𝑃 𝑐 𝑃 𝑎 ) (16)
Eq. ( 16) can be rewritten by using ENernst as previously shown in Eq. ( 12):

𝑊 𝑡ℎ = 𝐸 𝑁𝑒𝑟𝑛𝑠𝑡 • 𝐼 𝑀 𝐼 2𝐹 (17)
where M is the molar mass of hydrogen. Thus, the efficiency of an EHC can be evaluated by considering the real compression work, Wreal, which takes into account the increase of both the cell voltage, due to the overpotentials and the ohmic resistance, and the current density due to the hydrogen permeation across the membrane (Iloss):

𝜂 = 𝑊 𝑡ℎ 𝑊 𝑟𝑒𝑎𝑙 = 𝐸 𝑁𝑒𝑟𝑛𝑠𝑡 𝐸 ⋅ 𝐼 -𝐼 𝑙𝑜𝑠𝑠 𝐼 (18)
According to this definition, compression efficiency up to 60% can be reached at low pressure (< 100 bar) with an operating current density, I, of around 1 A cm -2 and Iloss = 0.04 A cm -2 .

However, the membrane thickness severely affects the EHC compression efficiency. A compression efficiency of 37% (3.55 kWh kg -1 ) was calculated considering Nafion 117, whereas 53% (2.5 kWh kg -1 ) was calculated considering a thinner membrane as Nafion XL, at the same operating conditions (T = 60°C, I = 0.33 A cm -2 , RHchan,in = 90% and Pc = 32 bar).

The efficiency achievable by using an EHC was proved to be higher than that generally reached by using a hydrogen mechanical compressor [START_REF] Sdanghi | [END_REF].

Effect of the current density applied to the EHC

Figure 5 shows the current density distribution over the EHC depending on the applied current density. The current density distribution remained uniform until the applied current density was fixed at 0.66 A cm -2 . At this value, the local current density was higher at the anode inlet than at the anode outlet (0.75 A cm -2 vs. 0.65 A cm -2 ) due to the unbalanced water flux across the membrane. In fact, a non-uniform water concentration profile was set along the membrane by the more important contribution of the electro-osmotic flux from the anode to the cathode with respect to the water back diffusion flux, in full agreement with Eq. ( 9). Indeed, the higher the current density applied to the system the higher the contribution of the water transport by electro-osmosis, which produced the decrease of the water concentration along the gas channel.

Hence, the contribution of the water back diffusion became important close to the anode outlet, stabilizing the current density distribution and the hydration level of the membrane. On the other hand, when a lower current density was applied to the EHC (0.16 A cm -2 or 0.33 A cm -2 ), a quite homogeneous distribution of the local current densities was obtained probably due to the equilibrium between the electro-osmotic and water diffusion flux all over the EHC.

The developed pseudo-2D model shown in Section 3 also proved that the stability of the current density strictly depends on the local water content of the membrane. Our model succeded in describing the decrease of the current density along the EHC at 0.66 A cm -2 as well as its stability when supplying a low current density, as shown in Figure 5. Indeed, the sum of squared residuals was lower than 10 -6 in all the cases. Table 2 shows that the higher I the higher E according to Eq. ( 12), and some measured and predicted physical quantities when considering the three current densities used in Figure 5 are also listed. The model gave a good prediction of the relative humidity at the anode outlet.

Indeed, it predicted a RH equal to 55% when supplying 0.66 A cm -2 , whereas the value measured by the humidity probe was 56%. The RH predicted for the case of I = 0.33 A cm -2 was 5% lower than that measured experimentally. Moreover, both the experimental and predicted values of the RH at the anode outlet and the calculated λan confirm that a significant dehydration of the membrane occurred when increasing the current density. Indeed, λan was found to be equal to 3.95 when supplying 0.66 A cm -2 , whereas 11.16 was obtained in the case of I = 0.16 A cm -2 , meaning that the average water content on the anode is significantly low at relatively high current densities. The occurred dehydration is the reason of the increase in the total resistance of the EHC, by more than 50% when increasing the current density from 0.16 to 0.66 A cm -2 . Hydrogen pumped / NL h -1 (experiment) 2.6 5.2 10.3

Efficiency / % (experiment) 56% 37% 14%

Figure 6 shows both the experimental cell voltages and the predicted polarization curve describing the I-V relationship in the EHC. The cell voltage increased sharply with the global current density due to the significant increase in the total resistance of the EHC at high current density, as previously described and in agreement with Eq. ( 14).

Figure 7 shows the predicted evolution of the relative humidity along both the gas channel and the anode-membrane interface for two different current densities, 0.33 and 0.66 A cm -2 . At 0.66 A cm -2 , the decrease of RH is more significant along the gas channel than along the anode, both reaching the same value close to the EHC outlet due to the equilibration of the electro-osmosis and the water diffusion flux. In addition, the RH at the EHC inlet is much higher at the gas channel than at the anode. At 0.33 A cm -2 , an opposite behavior is observable. Both behaviors are due to the resistance to the water transfer exhibited by the PTL. In fact, the PTL used in the present study comprised very small pores, with an average diameter of around 3 μm. Whereas small pores hinder water diffusion, the presence of large pores is harmful to the membrane, which is then poorly mechanically supported. Thus, an optimum value of the pore diameters exists, ensuring both an efficient mass transfer and good mechanical support.

The dramatic increase in the total resistance of the EHC caused a significant decrease in the overall efficiency. A global efficiency around 14% was calculated by our model when 0.66 A cm -2 was supplied to the system, whereas 37% was obtained at 0.33 A cm -2 and 56% at 0.16 A cm -2 (see Table 2). Low current densities allow obtaining higher global efficiencies but the hydrogen flow rate at high-pressure is also reduced from 10.3 to 2.6 NL h -1 at 0.66 and 0.16 A cm -2 , respectively. Therefore, a trade-off between the amount of high-pressure hydrogen produced and its efficiency is necessary when evaluating the performance of an EHC system.

Effect of the relative humidity of the inlet hydrogen flow

The effect of the hydrogen relative humidity at the inlet of the cell on the local current density distribution and on the resistance is presented in Figure 8 for RHchan,in = 30% and 90%.

For RHchan,in = 30%, the local current density was around 0.2 A cm -2 close to the anode inlet and it slightly increased up to 0.23 A cm -2 in the first 7 centimeters; it sharply increased in the middle of the EHC becoming stable at around 0.43 A cm -2 at 23 cm from the anode inlet. This evolution is directly related to a decrease in the total resistance of the EHC, which was evaluated by the I-V curves measured for each segment (see Figure 9). Indeed, the total resistance of the EHC decreased from 0.8 Ω cm 2 to 0.4 Ω cm 2 along the whole system, with a remarkable reduction of 50%. It can be deduced that gradual hydration of the membrane occurred, contrarily to the behaviour observed in the previous Subsection 4.2. Indeed, the water flux due to the water back diffusion was higher than the electro-osmotic one at the first 10 cm of the EHC because of the dry inlet conditions herein. This led to a net water flux from the cathode to the anode which "consumed" water at the cathode and gave rise to unstable performances that cannot be explained by our modeling assumptions, which proved to give good predictions of experimental data once stationary conditions are reached. Liquid water may be supplied on the high-pressure side in order to prevent the cathode dehydration thus balancing the water transport across the membrane when low humidity hydrogen is fed to the EHC. However, this solution would clearly make the system more complex because it requires pumping water at high pressures.

For RHchan,in = 90%, the current density distribution along the EHC was quite uniform due to the fact that the membrane was uniformly hydrated. Nevertheless, the homogenous current density distribution shown in Figure 8 was obtained at relatively low current density, 0.33 A cm -2 .

Effect of the membrane thickness

Figure 10 shows the experimental and predicted current density distribution along the EHC for two membranes: (i) Nafion XL -thickness 30 μm and Nafion 117 -thickness 175 μm. When using Nafion XL, a slight increase of the current density was observed in the first segments of the cell while the current density slight decreased close to the anode outlet. The numerical simulation predicted that the behavior of both membranes should be identical, i.e., the local current density distribution is supposed to be quite stable all over the system since water should be uniformly transported across the membrane. The discrepancy between the predicted and experimental Nafion XL behavior could be explained by both the non-uniform clamping force, which led to non-uniform contact resistance, and the tightness differences between the extremities and the central part of the EHC. Indeed, the relative contribution of the contact resistance is generally higher when a thin membrane is used.

The membrane thickness significantly affects the hydrogen permeation induced by the pressure difference between the anode and the cathode. The permeation flux of hydrogen through the membrane was measured by supplying nitrogen to the anode side meanwhile high-pressure hydrogen was present on the cathode side. Once hydrogen permeated through the membrane, the oxidation reaction took place at the anode. Hence, the hydrogen permeation flux was correlated to an equivalent current density through the Faraday's Law (Iloss). A permeation current density around 5.22 mA cm -2 was obtained when using Nafion 117, whereas 45.31 mA cm -2 was obtained in the case of Nafion XL. Thus, the thicker is the membrane the lower is the hydrogen permeation, in agreement with Eq. ( 2). According to these results, Nafion 117 should be preferred to Nafion XL, since an important hydrogen permeation flux makes decreasing the compression efficiency (in agreement with Eq. ( 18)). Nevertheless, the efficiency loss associated with permeation through the membrane is negligible when compared to that associated with the proton resistance of the membrane for a pressure gradient of 32 bar. The advantage is clearly on the side of the thin membrane. In fact, a cell voltage of 0.137 V was supplied by using Nafion 117, whereas around 0.06 V were supplied by using Nafion XL when the global current density was 0.33 A cm -2 (T = 60°C, RHchan,in = 90%, Pc = 32 bar). In good agreement, Figure 11 shows that a linear I-V relationship and high current density were achieved by using Nafion XL while worse performances were obtained by using Nafion 117.

Conclusion

In this study, the feasibility of hydrogen compression in an electrochemical system was proved.

We got insight on the importance of water management to obtain high efficiencies by using an electrochemical hydrogen compressor (EHC) with a single 30 cm 2 cell segmented into 20 parts.

In this system, a pressure as high as 32 bar was achieved, and the compression process proved to be isothermal. The effects of the current density supplied to the system, the relative humidity of the inlet hydrogen and the membrane thickness on the performances of an EHC were studied.

The current density distribution along the EHC strictly depended on the current density supplied to the system when high relative humidity hydrogen was fed to the cell (RHchan,in = 90%). A decrease of the current density distribution from 0.75 A cm -2 to 0.65 A cm -2 was observed when supplying a global current density of 0.66 A cm -2 to the EHC, due to the membrane dehydration.

Indeed, the relative humidity was found to decrease from 90% to 55% along the gas channels at the anode side .

The developed pseudo-2D model also proved that the stability of the current density distribution strictly depends on the local water content of the membrane. Our model gave a good prediction of both the current density distribution and the relative humidity at the anode outlet for different global current density supplied to the EHC. Nevertheless, unstable operating conditions were obtained when hydrogen with a low relative humidity was fed to the EHC. At these conditions, the EHC "consumed" water at the cathode side, giving unstable performance which could not be predicted by our numerical model.

Slight differences in water transport across the membrane were observed by using different membrane thickness. However, a lower cell voltage can be supplied to the EHC when a thinner membrane is used, increasing the compression efficiency. At 0.33 A cm -2 , the specific energy consumption was around 3.5 kWh kg -1 (37% efficiency), when using a 175 μm thickness membrane, compared to 2.5 kWh kg -1 (53% efficiency), when using a 30 μm thickness membrane. Therefore, we showed that EHC systems could be highly competitive to the traditional hydrogen mechanical compressors (efficiency lower than 50%) in the near future.

List of symbols

Figure 1 : 3 Modeling

 13 Figure 1: Schema of the experimental system used (the segmented EHC is in the central part

Figure 2 :

 2 Figure 2: Graphical description of the pseudo-2D model

Figure 4 :

 4 Figure 4: Average temperature of the anode, the membrane and the cathode of an EHC as a function of the supplied current densities (Nafion 117, RHchan,in = 90% and Pc = 32 bar)

Figure 5 :

 5 Figure 5: Distribution of the local current densities in an EHC as a function of the distance from the anode inlet when considering Nafion 117 for different supplied current densities (T = 60°C, RHchan,in = 90% and Pc = 32 bar )

Figure 6 :Figure 7 -

 67 Figure 6: Experimental and predicted average polarization curves when considering Nafion 117

Figure 8 :Figure 9 :

 89 Figure 8: Experimental distribution of the current density in an EHC for different values of RHchan,in (Nafion 117, T = 60°C, I = 0.33 A cm -2 and Pc = 32 bar)

Figure 10 :Figure 11 :

 1011 Figure 10: Distribution of the current density in an EHC for different membrane thickness (T = 60°C, I = 0.5 A cm -2 , RHchan,in = 90% and Pc = 32 bar)

A Active area / cm 2 α0 2 c 3 𝒟 𝐻 2 , 1 𝒟 𝐻 2 𝑚𝑒𝑚𝑏 1 E 1 i 2 i0 2 I 2 Iloss 1 λ 1 p 2 𝑎𝑛 2 𝑎𝑛 3 R 1 Rel 2 Rmemb 2 RHchan 1 T

 22321211222112231221 Water activity in the membrane / -Charge transfer coefficient / A cm -Total molar concentration in the PTL / mol cm -𝐻 2 𝑂 Binary diffusion coefficient in the PTL / cm 2 s -Water diffusion coefficient across the membrane / cm 2 s -EHC voltage / V Eε H2 permeation activation energy / J EW Equivalent weight membrane / kg mol -1 ε0 H2 permeation pre-factor / mol cm -1 s -1 bar -1 εPTL PTL porosity / % ζ Electro-osmotic drag coefficient / -F Faraday's constant / C mol -Local current density / A cm -Exchange current density / A cm -Supplied current density / A cm -Permeation current density / A cm -2 kB Boltzmann's constant / J K -Water content /λan Water content at the anode /λcat Water content at the cathode / -𝑛̇𝐻 2 H2 molar flow along the gas channel / mol s -1 𝑛̇𝐻 2 𝑂 H2O molar flow along the gas channel / mol s -1 𝑁 ̇𝐻2 𝑃𝑇𝐿 H2 molar flux across the PTL / mol cm -2 s -1 𝑁 ̇𝐻2 𝑂 𝑃𝑇𝐿 H2O molar flux across the PTL / mol cm -2 s -1 𝑁 ̇𝐻2 𝑂 𝑚𝑒𝑚𝑏 H2O molar flux across the membrane / mol cm -2 s -1 𝑁 ̇𝐻2,𝑝𝑒𝑟𝑚 H2 permeation flux across the membrane / mol cm -2 s -Width of active area / cm Pan Anode pressure / bar Pcat Cathode pressure / bar 𝑃 𝐻 Hydrogen partial pressure at anode / bar 𝑃 𝐻 Hydrogen partial pressure at cathode / bar psat(T) Vapor pressure / bar ρdry Density of dry membrane / kg cm -Gas constant / J kg -1 K -Total electric resistance of EHC / Ω cm Protonic resistance of the membrane / Ω cm Relative humidity gas flow along the gas channel / % RHchan,in Relative humidity gas flow at the gas channel inlet / % S Stoichiometry /σmemb Membrane proton conductivity / S cm -Temperature / °C xmemb Membrane thickness / cm xPTL PTL thickness / cm

Table 1 -

 1 Parameters used in the present numerical model

	Parameter / units	symbol	value	source
	Activation Energy -Hydrogen Permeation / J	Eε	4.28•10 -20	[29]
	Active area / cm 2	A	30	This study
	Active area width / cm	p	1	This study
	Anode pressure / bar	Pa	1	This study
	Cathode pressure / bar	Pc	32	This study
	Dry membrane density / kg cm -3	ρdry	2•10 -3

Table 2 -

 2 Experimental and predicted parameters in an EHC for three different current densities (Nafion 117, T = 60°C, RHchan,in = 90% and Pc = 32 bar )

	Parameter values as a function of I / A cm -2
	0.16	0.33	0.66

Acknowledgments

This study was partly supported by the French PIA project "Lorraine Université d'Excellence", reference ANR-15-IDEX-04-LUE and TALiSMAN project, funded by FEDER (2019-000214).