Gisele Ferreira De Lima Andreani

Maria Regina

Martins Triques

Daniel Rodrigo Leiva

Virginie Roche

Regina Kátia

Cardoso

Toshimi Tomaz

Walter Jose Ishikawa

Alberto Botta

Jorge Moreira
email: moreira@ufscar.br

Maria Regina Martins Triques

Kátia Regina Cardoso

Tomaz Toshimi Ishikawa

Walter José Botta

Alberto Moreira

Jorge Jr

Hydrogen storage properties of 2 Mg-Fe mixtures processed by hot extrusion: Effect of ram speeds

Keywords: hydrogen storage, Mg hydrides, hot-extrusion, complex hydrides

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

Introduction

Since the 1990s, the scientific community has been interested in the development of a hydrogen economy based on such gas as an energy carrier [START_REF] Sakintuna | Metal hydride materials for solid hydrogen storage[END_REF][START_REF] Schulz | Recent developments in the applications of nanocrystalline materials to hydrogen Technologies[END_REF][START_REF] Shao | Progress and Trends in Magnesium-Based Materials for Energy-Storage Research: A Review[END_REF]. Mg and Mg-based composites are attractive materials for hydrogen storage.

The high hydrogen mass capacity of MgH 2 (7.6 wt.%) and Mg 2 FeH 6 (5.5 wt.%), their reversibility, and low cost make them promising for energy storage applications [START_REF] Sakintuna | Metal hydride materials for solid hydrogen storage[END_REF][START_REF] Shao | Progress and Trends in Magnesium-Based Materials for Energy-Storage Research: A Review[END_REF]. However, the high stability (desorption temperatures), the slow H-sorption kinetics and the weak resistance to air contaminants, which quickly degrade the H-sorption properties, limit the practical application of these hydrides [START_REF] Sakintuna | Metal hydride materials for solid hydrogen storage[END_REF].

It is known that nanostructured hydrogen storage materials produced by high-energy ball milling (HEBM) or mechanical alloying (MA) present better hydrogen sorption kinetics. The increased surface area and the structural defects, created by those processing routes, lower the activation energy barriers [START_REF] Schulz | Recent developments in the applications of nanocrystalline materials to hydrogen Technologies[END_REF][START_REF] Zaluski | Nanocrystalline metal hydrides[END_REF][START_REF] Zaluska | Nanocrystalline magnesium for hydrogen storage[END_REF][START_REF] Castro | Improving Hsorption in MgH2 powders by addition of nanoparticles of transition metal fluoride catalysts and mechanical alloying[END_REF].

With additions of catalysts, it is possible to produce a synergic effect between surface area, structural defects, and catalyst effect , thus further improving hydrogenation properties. Addition of Fe as a catalyst can decrease the decomposition temperature of MgH 2 by more than 100 o C, by reducing the activation energy for hydride decomposition. Additionally, iron can prevent the agglomeration of Mg and also act as Mg 2 FeH 6 former [START_REF] De Lima | Hydrogen Sorption Properties of the Complex Hydride Mg2FeH6 Consolidated by HPT[END_REF][START_REF] Lima | Hydrogen storage properties of 2Mg-Fe after the combined processes of hot extrusion and cold rolling[END_REF][START_REF] Lima-Andreani | Characterization of hydrogen storage properties of Mg-Fe-CNT composites prepared by ball milling, hot-extrusion and severe plastic deformation methods[END_REF][START_REF] Antiqueira | Severe Plastic Deformation and Additive Distribution in Mg-Fe to Improve Hydrogen Storage Properties[END_REF][START_REF] Edalati | New nanostructured phases with reversible hydrogen storage capability in immiscible magnesium-zirconium system produced by high-pressure torsion[END_REF][START_REF] Márquez | Hydrogen storage in MgH2LaNi5 composites prepared by cold rolling under inert atmosphere[END_REF][START_REF] Alsabawi | The effect of ball-milling gas environment on the sorption kinetics of MgH2 with/without additives for hydrogen storage[END_REF][START_REF] Tian | Mg-based composites for enhanced hydrogen storage performance[END_REF][START_REF] Yahya | Improvement of hydrogen storage properties in MgH2 catalysed by K2NbF7[END_REF][START_REF] Thongtan | Reversible hydrogen sorption and kinetics of hydrogen storage tank based on MgH2 modified by TiF4 and activated carbon[END_REF][START_REF] Youn | Enhancement of hydrogen sorption properties of MgH2 with a MgF2 catalyst[END_REF][START_REF] Alsabawi | Kinetic enhancement of the sorption properties of MgH2 with the additive titanium isopropoxide[END_REF][START_REF] Kumar | Development of MgLiB based advanced material for onboard hydrogen storage solution[END_REF][START_REF] Yavari | Improvement in H-sorption kinetics of MgH2 powders by using Fe nanoparticles generated by reactive FeF3 addition[END_REF][START_REF] Bogdanovic | Thermodynamics and dynamics of the Mg-Fe-H system and its potential for thermochemical thermal energy storage[END_REF][START_REF] Huot | Direct synthesis of Mg2FeH6 by mechanical alloying[END_REF][START_REF] Gennari | Synthesis of Mg2FeH6 by reactive mechanical alloying: formation and decomposition properties[END_REF][START_REF] Li | Controlled mechano-chemical synthesis of nanostructured ternary[END_REF][START_REF] Raman | Investigations on the synthesis, structural and microstructural characterization of Mg-based K2PtCl6 type (Mg2FeH6) hydrogen storage material prepared by mechanical alloying[END_REF][START_REF] Huot | Preparation of the hydrides Mg2FeH6 and Mg2CoH5 by mechanical alloying followed by sintering[END_REF][START_REF] Castro | Effect of the nature of the starting materials on the formation of Mg2FeH6[END_REF][START_REF] Herrich | Synthesis and decomposition of Mg2FeH6 prepared by reactive milling[END_REF]. However, there are several drawbacks in using powders for hydrogen storage, including the occurrence of surface contamination, the potential fire risk, and health concerns.

As an alternative, powder consolidation methods, including Severe Plastic Deformation (SPD) techniques, cold rolling and extrusion have been recently employed to develop hydrogen storage materials [START_REF] Leiva | Nanostructured MgH2 prepared by cold rolling and cold forging[END_REF][START_REF] Floriano | Nanostructured MgH2 Obtained by Cold Rolling Combined with Short-time High-energy Ball Milling[END_REF][START_REF] Floriano | Cold rolling of MgH2 powders containing different additives[END_REF][START_REF] Floriano | MgH2-based nanocomposites prepared by short-time high energy ball milling followed by cold rolling: A new processing route[END_REF][START_REF] De Lima | Hydrogen Sorption Properties of the Complex Hydride Mg2FeH6 Consolidated by HPT[END_REF][12][START_REF] Skripnyuk | The effect of equal channel angular pressing on hydrogen storage properties of a eutectic Mg-Ni alloy[END_REF][START_REF] Skripnyuk | The effect of ball milling and equal channel angular pressing on the hydrogen absorption/desorption properties of Mg-4.95 wt% Zn-0.71 wt% Zr (ZK60) alloy[END_REF][START_REF] Leiva | Magnesium-Nickel Alloy for Hydrogen Storage Produced by Melt Spinning Followed by Cold Rolling[END_REF][START_REF] Soyama | Hydrogen storage in heavily deformed ZK60 alloy modified with 2.5 wt.% Mm addition[END_REF][START_REF] Lima | Hydrogen storage properties of 2Mg-Fe after the combined processes of hot extrusion and cold rolling[END_REF][START_REF] Lima-Andreani | Characterization of hydrogen storage properties of Mg-Fe-CNT composites prepared by ball milling, hot-extrusion and severe plastic deformation methods[END_REF][START_REF] Antiqueira | Severe Plastic Deformation and Additive Distribution in Mg-Fe to Improve Hydrogen Storage Properties[END_REF][START_REF] Popilevsky | Hydrogenation-induced microstructure evolution in as cast and severely deformed Mg-10 wt.% Ni alloy[END_REF][START_REF] Hongo | Significance of grain boundaries and stacking faults on hydrogen storage properties of Mg2Ni intermetallics processed by high-pressure torsion[END_REF][START_REF] Edalati | Formation of metastable phases in magnesium-titanium system by high-pressure torsion and their hydrogen storage performance[END_REF][START_REF] Edalati | New nanostructured phases with reversible hydrogen storage capability in immiscible magnesium-zirconium system produced by high-pressure torsion[END_REF][START_REF] Márquez | Hydrogen storage in MgH2LaNi5 composites prepared by cold rolling under inert atmosphere[END_REF]. In general, these techniques produce a microstructure with a high density of defects (for instance, dislocations) due to a high level of deformation, grain refinement (when starting from bulks) that may lead to nanostructures, and bulks that are safer, easier to manipulate, and more air-resistant than powders.

Lima et al. [START_REF] Lima | Microstructural characterization, and hydrogenation study of extruded MgFe alloy[END_REF][START_REF] Lima | 2Mg-Fe alloys processed by hot-extrusion. Influence of processing temperature and the presence of MgO and MgH2 on hydrogenation sorption properties[END_REF][START_REF] Cerutti | 2Mg-Fe and 2Mg-Fe+5%C mixtures processed by hot extrusion. Influence of carbon on hydrogen sorption properties[END_REF] have reported successful results in the production of bulk 2MgFe composites by hot-extrusion, by SPD, and by using a combination of hotextrusion and SPD processes. By using hot-extrusion, they found excellent hydrogenation properties similar to as acquired with either MA or SPD processes. Such features were attributed to the better air resistance of bulks, the porosity (since powders were consolidated by hot-extrusion), and the pinning effect of Mg grain boundaries by the Fe particulate, which can keep the nanoscale level during hot working.

In previous works [START_REF] De Lima | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion. Influence of the extrusion ratio[END_REF][START_REF] Jr | Correlation between hydrogen storage properties and texture induced in magnesium through ECAP and cold rolling[END_REF][START_REF] De Lima-Andreani | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion at different temperatures[END_REF], we have investigated the influence of the extrusion ratio and temperature used during powder consolidation processing. In such investigations, it was verified that the smaller extrusion ratio [START_REF] De Lima | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion. Influence of the extrusion ratio[END_REF], the higher the porosity and, consequently, the better the hydrogenation properties. Also, when processing 2Mg-Fe mixtures at 300 °C, the bulk samples presented the best synergetic combination of grain sizes and desirable texture along (002) plan for Mg, thus resulting in fastest kinetic of H-absorption without any incubation time [START_REF] Jr | Correlation between hydrogen storage properties and texture induced in magnesium through ECAP and cold rolling[END_REF][START_REF] De Lima-Andreani | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion at different temperatures[END_REF].

In this work, results are presented concerning the influence of the ram speed during hot-extrusion of 2Mg-Fe mixtures on the microstructure and its consequence on the hydrogenation properties.

Experimental

Pure powders of Mg (-20+100 mesh, 99.98%, Alfa Aesar) and Fe (-22 mesh, 99.998%, Alfa Aesar) were mixed by high energy ball milling in a stoichiometry 2Mg-Fe (at.%). The milling was performed in a Fritsch P7planetary ball mill under an argon atmosphere, at 600 rpm, using a ball-to-powder ratio of 40:1. Powders were milled for four hours, after which they were sieved in an 80 mesh sieve. Samples (~1g) from the powder mixture were cold-pressed into pre-forms, which were then hot-extruded in a conventional Universal Testing Machine, EMIC Ltda DL-60.000, using the best conditions established in previous work: extrusion ratio of 3/1 [START_REF] De Lima | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion. Influence of the extrusion ratio[END_REF], at an extrusion temperature of 300°C [START_REF] Jr | Correlation between hydrogen storage properties and texture induced in magnesium through ECAP and cold rolling[END_REF][START_REF] De Lima-Andreani | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion at different temperatures[END_REF]. Ram speeds were 1, 5, and 10 mm/min. Extruded samples were named according to their processing speeds; and so hereafter they will be referred to as V1, V5, and V10, respectively for 1, 5, and 10 mm/min. Bulk samples were hydrogenated in a Sievert's type apparatus to perform sample activation, to acquire absorption kinetics' curves, and to determine incubation times. For such, the hydrogen pressure was kept at 1.5 MPa for 20 h at the temperature of 400 ºC, aiming to obtain the Mg 2 FeH 6 complex hydride with the maximum capacity (5.5% of H 2 ) and to favor the hydrogenation of such bulk samples. Mg powders were also used for comparison.

Thermal analyzes using differential scanning calorimetry (DSC) and thermogravimetry (TG), were performed in a NETZSCH STA 449C were used to evaluate the thermodynamic stability of hydrides, capacities and desorption kinetics of H 2 , which were compared to commercial MgH 2 powders.

The percentage by weight of H 2 was calculated using the procedure suggested by Varin et al. [START_REF] Varin | Nanomaterials for Solid State Hydrogen Storage[END_REF], as follows. Assuming the hydrogen as an ideal gas, the Volumetric Method was employed to achieve H 2 wt% applying the following equations [START_REF] Varin | Nanomaterials for Solid State Hydrogen Storage[END_REF]:

wt. % H = m × 100 m ⁄ Eq (1)
m = m + m Eq(2) m = 2.016 × ∆P × V R × T ⁄ Eq (3)
In Equation (1), wt. % H 2 is the weight percent of molecular hydrogen, m H is the absorbed hydrogen in molecular mass determined by utilizing the number of gas moles (∆n=((∆P×V)⁄((R×T) ))). In Equation (2), m s is the mass of metal (mass of 2MgFe mixture). Therefore, m H + m s is the mass of hydride (m . In Equation ( 3), R is the universal gas constant (R = 8.314472 J mol-1 K-1), V (= V h + V r ) is the total volume, V r is the calibrated volume at the reservoir temperature, Vh is the calibrated volume of the holder at the furnace temperature, T (K) denotes the H-absorption temperature inside of the furnace, and ∆P is the pressure range of hydrogen gas in the system during hydrogen absorption.

Information regarding the structure and phases' identification after each processing step were obtained by X-ray diffraction (XRD) using monochromatic CuKα radiation with an angular pass of 0.032 o in a Rigaku DMAX diffractometer, in which was used a C-monochromator to avoid fluorescence of Fe. The Rietveld method was employed to refine diffraction patterns using the Maud software [START_REF] Lutterotti | Quantitative Analysis of Silicate Glass in Ceramic Materials by the Rietveld Method[END_REF][START_REF] Lutterotti | MAUD -Materials Analysis Using Diffraction[END_REF].

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were conducted to characterize the microstructure and morphology of samples using respectively an FEI INSPECT S50 microscope, and an FEI CM-120 microscope operated at 120 kV.

Extruded samples were cut from the cross-section of each billet (perpendicular to the pressing direction) and embedded in resin, after which they were prepared by usual metallographic methods, including multistep grinding employing SiC abrasive paper (320, 400, 600, 800, 1200 and 2000 grit). At the final step of preparation, they were polished utilizing 2500-grit SiC paper and, finally, with 0.02-µm colloidal silica suspension, and diamond polishing paste. Such samples were used to be observed in the SEM using electron backscattered (BSE) mode to emphasize the chemical contrast.

Powders for TEM observations were placed in an ethanol suspension that was ultrasonicated to disperse powder agglomerates. A droplet of this suspension was poured on a 3mm in diameter copper grid coated with carbon film. Thin foil TEM samples were prepared from the cross-section of the as-extruded pieces.

Small flat pieces were cut from billets with diamond wheel, being carefully ground up to 50-70 µm thick and punched in 3.0 mm diameter disks with an appropriate device. Finally, thin areas were obtained by ion-milling using a Gatan precision ion polishing system (model 691) with beam energy of 5keV under an argon atmosphere. Both kinds of samples were hydrogenated for TEM observation.

Mg and Mg-based composites and their hydrides are humidity and oxygensensitive. However, to ascertain their resistance to such an atmosphere, samples used in this work were stored in the air.

Results and discussions

Figure 1 shows the XRD patterns of the extruded samples (V1, V5, and V10). As expected, only α-Mg and α-Fe phases were indexed in all of them. Broad and low-intensity peaks are observed in all the diffractograms, indicating the presence of refined grains after the extrusions. Table 1 shows the average crystallite sizes estimated by the Rietveld method. In general, one may observe that in all the extruded samples, the crystallites were larger than in the starting powders, which grew with the increase of the ram speed [START_REF] De Lima | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion. Influence of the extrusion ratio[END_REF][START_REF] Jr | Correlation between hydrogen storage properties and texture induced in magnesium through ECAP and cold rolling[END_REF][START_REF] De Lima-Andreani | Hydrogen storage properties of 2Mg-Fe mixtures processed by hot extrusion at different temperatures[END_REF]. Such an increase of crystallite size indicates that adiabatic heating is taking place during extrusion.

When mechanical stress is applied, a considerable amount of mechanical work per unit volume (∫σdε) is converted into heat. In all the mechanical working operations there is the occurrence of deformation heating, which increases with the product of stress (σ) and strain (ε), and σ increases with the strain rate (ram speed). As the time of extrusion is inversely proportional to the ram speed, when the ram speed (strain rate) increases, the time to remove heat is reduced, and such conditions approach the adiabatic ones. Therefore, the higher the ram speed, the higher the temperature increase, and the larger the resulting crystallite size. As expected, all extruded samples had slower absorption kinetics than the 2MgFe powders, which can be explained by the differences in surface areas, which is larger for the powders. However, powders reached saturation after almost 10h, while sample V1 reached it after ~15h. Samples V5 and V10 would take more time to reach their maximum capacity. The behavior presented by extruded samples, not excluding the catalytic effect of Fe, is probably due to three crucial factors: (1) smaller surface area because extruded samples are in the bulk form, (2) lack of porosity that tends to decrease with the increase of ram speed (Fig. 2), and (3) grain size (Table 1).

However, one may observe that factors ( 2) and ( 3) may be prevailing because kinetics are slowed down, and capacities are decreased following precisely the increasing sequence of ram speeds (decreasing porosity) and the increase of grain sizes, as presented in Figure 2 and Table 1, respectively. However, even explaining the different capacities and kinetics between extruded samples, the above argumentation does not justify why the hydrogen capacity of powders was smaller than for conditions V1 and V5. These results can be better understood by evaluating the XRD patterns for samples after hydrogenation, as shown in Figure 4. and, in a small percentage, in the powders. However, comparing relative amounts of iron between powders and conditions V1 and V5, one may observe that remaining iron in the powders is more significant than in these two other conditions. If one considers that also there was residual Mg in the powders, it is possible to affirm that the conversion of 2MgFe into Mg 2 FeH 6 was smaller in the powders than for conditions V1 and V5. This fact may be explained by the worst distribution of iron in the powders when compared to extruded samples, as seen in Figure 2. In other words, Fe agglomeration in specific regions in the powders hampered the complete transformation, thus reducing the powders' capacity. However, it is remarkable that none of the conditions completed the conversion to the complex hydride. This fact could also be related to the size and distribution of Fe agglomerates, which, even being better distributed in extruded samples, continued to be agglomerated with large particle sizes in addition to decreased porosity in such samples. As the diffusion of Fe in Mg is very slow at the hydrogenation temperature (the diffusion coefficient at 400 o C is about 10 -18 m 2 .s -1

[80], probably the conversion was kept around those large particles and in smaller ones as presented in Figure 5.

It is also noteworthy that the maximum capacity, even considering the presence of MgH 2 , was only 4.8 wt%, reached by condition V1. It must be borne in mind that the expected capacity for the complex hydride should be 5.5 wt%. The reason for this behavior may be explained by analyzing Equations (1-3) under the light of results presented in Table 2. If one pay attention in Table 2, it will possible, as stated before, to observe that there was no complete conversion of the mixture 2MgFe into Mg 2 FeH 6 for all conditions, remaining a considerable and crescent amount of Fe following the sequence of V1, V2, powders and V10, and the presence of Mg in the powders and condition V10. Therefore, one may infer that as Equations (1-3) calculate the H2 wt % considering the total mass and as materials were not totally hydrogenated, the total mass of hydrogen will be decreased.

Contrarily to all other samples, Mg powders were sensible to oxidation and presented an incubation time of about 4 hours. Such oxidation also slowed down its kinetics and decreased its maximum capacity during the hydrogenation, which, after 43 hours, was of only ~5.4 wt%. The absence of incubation time in any other condition than in Mg powders means that oxidation did not affect hydrogenation, probably due to the catalytic effect of iron in 2MgFe samples and reduced surface area in extruded ones. For extruded samples, these temperatures were very close to each other.

However, they were smaller and the same for V1 and V5, 270 o C (onset) and 326 o C (desorption peak). From these observations, it is clear that Fe has a strong kinetic effect in the desorption of the hydrides present. However, the general unanimity is that the Mg 2 FeH 6 stability does not vary markedly from that of β-MgH 2 (-74.4 kJ mol -1 H 2 [START_REF] Batalović | Transition metal doping of Mg2FeH6-a DFT insight into synthesis and electronic structure[END_REF]).

By analyzing the TG data (Fig. 7b), one may observe that the starting desorption temperatures follow the same behavior seen in the DSC curves of From these kinetics results, it is clear that, as stated before, Fe has a strong kinetic effect in the desorption when comparing with powders of commercial MgH 2 .

The behaviors described above can be explained in the same way as previously done for the absorption, i.e., mainly the effects related to porosity (Fig. 2) and grain sizes (Table 1) and, of course, the catalytic effect of Fe that was present in all the samples. In this case, even though the differences between V1 and V5 were small, the speed of 1 mm/min was the best processing speed, since no significant grain growth of Mg and Fe has occurred and the porosity was the largest compared to all of the extruded conditions studied. Finally, but not less relevant, Figure 7d presents the XRD results of asdesorbed samples. By comparing these results it the ones from Figure 1, it will be possible to observe that, after dehydrogenation, as expected, the samples return to their initial state, i.e., Mg and Fe are kept in the same ratio.

Summary

We have evaluated the changes in microstructure and the effect on hydrogen sorption behavior in 2Mg-Fe samples compacted by hot-extrusion using different ram speeds. The extrusion ram speed that produced samples with better hydrogenation properties was of 1 mm/min. The grain size increases and the porosity decreases with ram speed increasing, thus worsening the hydrogenation properties. Grain growth was not as expressive, remaining in the range of few nanometers, probably due to the pinning effect of nanometric Fe. During extrusion, Fe was redistributed along with consolidated samples but was kept agglomerated in the powders, thus decreasing powders capacity when compared with the bestextruded conditions. For all studied conditions, there was residual Fe, besides helping to pin grain boundaries during hydrogen absorption and desorption processes also help by acting as a catalyst in both cases. However, as expected,

Figure1-

  Figure1 -XRD patterns of the mixture of 2Mg-Fe, extruded at 300 o C: (a) V1; (b)

Figure 2

 2 Figure 2 presents BSE-SEM images of extruded samples in comparison

Figure 3

 3 Figure 3 shows curves of the first hydrogen absorption performed in a Sieverts-type apparatus at 400 o C under 15 bar of H 2 , for samples extruded at

Figure 2 -

 2 Figure 2 -BSE-SEM images of extruded samples and powders. (a) extruded at V1,

Figure 3 -

 3 Figure 3 -Curves of the first hydrogen absorption performed in a Sieverts-type apparatus at 400 o C under 15 bar H 2 , for samples V1, V5, and V10, compared to

Figure 4 -

 4 Figure 4 -XRD patterns of hydrogenated powders and extruded samples of 2Mg-

Figure 5

 5 Figure 5 present bright field images obtained by TEM for hydrogenated

Figure 5 -

 5 Figure 5 -BF-TEM images of the hydrogenated samples: (a) 2MgFe powder. (b)

Figure 6 -

 6 Figure 6 -Indexation of diffraction patterns shown in the insets of Figure 5.

Figure

  Figure 7a shows DSC results for hydrogenated samples V1, V5, and V10, in

Figure 7a .

 7a Figure 7a. Also, the desorbed mass follows the same behavior of the absorption as

Figure 7 -

 7 Figure 7 -Thermal analyzes of hydrogenated mixtures of 2Mg-Fe extruded under

  powders presented better absorption kinetics and saturated almost 5 hours before the sample extruded at 1mm/min (V1). During desorption, powders and bulks extruded at 1 (V1) and 5 (V5) mm/min presented the same kinetics and similar desorption temperatures, which were about 150 o C smaller than for powders of MgH 2 . Samples with good absorption properties fully desorbed in less than 8 min at 350 o C, while commercial MgH2 would take much more than 40 min for complete desorption.

Table 1 -

 1 Average crystallite size (nm ± 5 nm), estimated by the Rietveld method. Goodness of fitting (GOF= Rw/Rexp), satisfactory ≤ 1.3Although both Mg and Fe crystallite sizes have increased, Fe crystallites increased less. Iron has to diffuse through moving Mg grain boundaries. Once its diffusion coefficient in Mg is minimal (10 -16 m 2 /s[START_REF] Pavlinov | Self-Diffusion in Calcium and Diffuse of Barely Soluble Impurities in Magnesium and Calcium[END_REF]), the growth rate of Fe crystallites is limited. On the other hand, such a very small crystallite size has very high boundary energy, which is beneficial for pinning Mg grain boundaries, thus limiting also their growth.

	Phase	as-milled	V1	V5 V10
		[50-52]
	Mg	37	43	69	106
	Fe	39	41	48	56
	Rw/Rexp*	1.03	1.01 1.09 1.09

*

Table 2 -

 2 Calculated distribution of Mg, Fe, MgH 2, and Mg 2 FeH 6 for the various samples using Rietveld refinement.

	Condition Mg (%) Fe (%) MgH2 (%) Mg 2 FeH 6 (%)
	V1	0	22.25	8.47	69.29
	V5	0	23.30	5.92	70.78
	V10	17.09	41.31	9.51	32.09
	Powders	3.01	29.52	6.02	61.45

Acknowledgments

The authors would like to thank FAPESP (2014/21237-1) and CNPq (Universal 460471/2014-7) for financial support. AMJJ would like to acknowledge the CNPq (Brazil) under grant #301429/2017-0. KRC would like to acknowledge the FAPESP (Brazil) under grant # 18/09548-2


Thi-Phuong-Thao Pham

Didier Raoult

Matthieu Million
email: matthieumillion@gmail.com

IGF1 levels in children with Severe Acute Malnutrition after Nutritional Recovery: a Good Predictor for Children's Long-term Health Status

The use of ready-to-use therapeutic foods (RUTF) have successfully reduced mortality in children with severe acute malnutrition (SAM), but the incomplete restoration including growth retardation (or stunting) and persistent immaturity of gut microbiota [START_REF] Subramanian | Persistent gut microbiota immaturity in malnourished Bangladeshi children[END_REF] and the long-term consequences such as increased risk of developing obesity, type 2 diabetes, cardiopathies (2) remain a major challenge for children's health worldwide.

The metabolomics study of Bourdon C,et al. (2) reported in EBioMedicine has characterized the metabolite profiles of SAM survivors from the "ChroSAM cohort" (3) and showed that children who survived from early childhood SAM had a similar profile to control groups 7 years after treatment. This is the first quantitative metabolomics study of long-term consequences in SAM survivors. They indicated that early childhood SAM could mainly impact on growth and muscle mass, rather than inducing persistent metabolic derangement.

In addition, Bourdon et al. [START_REF] Bourdon | Metabolomics in plasma of Malawian children 7 years after surviving severe acute malnutrition: "ChroSAM" a cohort study[END_REF] also marked the association between stunting and low levels of circulating IGF1(Insulin-like Growth Factor 1). The stunted SAM survivors had even lower circulating IGF1 levels than other stunted children from similar low socioeconomic communities [START_REF] Bourdon | Metabolomics in plasma of Malawian children 7 years after surviving severe acute malnutrition: "ChroSAM" a cohort study[END_REF]. The findings are consistent with those of previous studies that observed the low levels of IGF1 in hospitalized children suffering from protein and energy malnutrition; the values were even lower in marasmus than in kwashiorkor children [START_REF] Kouanda | Insulin Growth Factor-I in Protein-Energy Malnutrition during Rehabilitation in Two Nutritional Rehabilitation Centres in Burkina Faso[END_REF]. After the nutritional rehabilitation, IGF1 increased significantly in both children with kwashiorkor and marasmus but the values were still lower than in healthy children 14 days post-treatment (4). Moreover, low levels of IGF1 in early life is known to be associated with late risk of NCDs (Non-Communicable Diseases), particularly diabetes (2). It has recently been shown that IGF1 was modulated by microbiota, especially gut resident-bacteria-derived SCFAs (acetate, butyrate and propionate) [START_REF] Yan | Gut Microbiota and IGF-1[END_REF]. Indeed, numerous bacterial species can produce SCFAs such as Akkermansia muciniphila, Faecalibacterium prausnitzii, Ruminococcus spp., Roseburia spp., Eubacterium spp. This suggests that these bacteria could be useful in the treatment of SAM as potential probiotics [START_REF] Douglas | Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism[END_REF]. Strikingly, loss of fecal butyrate has been associated with mortality among children with SAM suggesting the global collapse of gut microbiota mutualism in such a situation [START_REF] Attia | Mortality in children with complicated severe acute malnutrition is related to intestinal and systemic inflammation: an observational cohort study[END_REF].

Several studies, including ours [START_REF] Subramanian | Persistent gut microbiota immaturity in malnourished Bangladeshi children[END_REF][START_REF] Douglas | Formation of short chain fatty acids by the gut microbiota and their impact on human metabolism[END_REF][START_REF] Million | Increased Gut Redox and Depletion of Anaerobic and Methanogenic Prokaryotes in Severe Acute Malnutrition[END_REF][START_REF] Tidjani Alou | Gut Bacteria Missing in Severe Acute Malnutrition, Can We Identify Potential Probiotics by Culturomics?[END_REF][START_REF] Pham | Gut Microbiota Alteration is Characterized by a Proteobacteria and Fusobacteria Bloom in Kwashiorkor and a Bacteroidetes Paucity in Marasmus[END_REF] have demonstrated the link between microbiota alteration (including bacterial diversity depletion, increased gut redox, a massive invasion of pathogens and the missing of benefit taxa) with SAM. Undoubtedly, diet components have a strong influence in shaping the gut microbiota and conversely, the gut microbiota contributes to nutrients uptake. Thus, the transient or persisting alteration of gut microbiota during and perhaps after SAM may induce persisting low levels of IGF1, and the physical growth retardation (or stunting), even after nutritional recovery. However, the mechanisms by which microbiota induces IGF-1 production and whether microbiotamediated IGF-1 production is dependent on growth hormone is still under investigation [START_REF] Yan | Gut Microbiota and IGF-1[END_REF].

In the study, Bourdon et al. [START_REF] Bourdon | Metabolomics in plasma of Malawian children 7 years after surviving severe acute malnutrition: "ChroSAM" a cohort study[END_REF] choose the control group as children from similar low socio-economic communities, who were also stunted and have a history of poor health. In relation to the selection of control children, further sub-group analysis or exploration of interactions could be limited. Another limitation of the study is the lack of birth weight record which is also known as a risk factor for developing NCDs in later life.

Overall, Bourdon et al.'s exhaustive work through the period of 7 years follow-up children after admission, the minimal loss-to-follow-up, and the use of both sibling and community controls make this study particularly interesting. Their results not only provided a more comprehensive view of metabolic changes in long-term SAM survivors but also generated new directions for effective treatment response and for preventive medicine (potential marker IGF1 and its relationship with gut microbiota). Elucidating this relationship may contribute to the improvement of long-term care of SAM survivors.

Disclosure

The authors declared no conflicts of interest.


Florian Moureaux

Philippe Stevens

Gwenaëlle Toussaint

Marian Chatenet
email: marian.chatenet@grenoble-inp.fr

A low cost, durable and active electrode for oxygen evolution reaction in concentrated alkaline environments

Introduction

One cannot ignore the deleterious issues of global warming and the means that mankind can use to try to attenuate emissions of greenhouse gases that are at its origin [1]. Fossil fuels should no longer be extensively extracted and should wherever possible be substituted with a clean energy vector: electricity produced from renewable energy (of solar, wind or water origin) is an obvious solution, and is undergoing rapid growth. However, injecting large amounts of intermittent renewable electricity may destabilize the electrical grid if it is not consumed at the same rate that it is produced. One solution to this electrical engineering issue would be to efficiently store renewable electricity when it is less needed, so that the grid can return benefit from this storage for peaks in electricity demand [START_REF] Moseley | Electrochemical Energy Storage for Renewable Sources and Grid Balancing[END_REF]. There are many technological solutions to store electrical energy; one of the most widespread and most efficient of them is pumped hydro, but one cannot implement it everywhere and most sites where it could prove viable are already equipped.

Electrochemical storage in batteries is another well-studied option [START_REF] Tarascon | [END_REF]4], e.g. in Zn-air batteries not using any noble catalysts [5], which could provide a solution for short to medium storage times, typically between 15 minutes and 4 hours. For longer storage times or for large amounts of electricity, power-to-hydrogen could be a solution, at least for use in conjunction with the other storage means mentioned above.

Hydrogen is a clean energy carrier when its production does not involve fossil fuels, which is where water electrolysis has an important role to play [6,[START_REF] Mittelsteadt | Electrochemical Energy Storage for Renewable Sources and Grid Balancing[END_REF]. Although molecular hydrogen (H2) is usually the product of interest in water electrolysis, the research community soon realized that it is the other product of water splitting, molecular oxygen (O2) that limits the reaction, owing to the very sluggish oxygen evolution reaction (OER) on most electrocatalysts. In addition, the experimental conditions experienced by OER anodes in water electrolyzers are extremely aggressive, resulting in severe durability issues. Today, the majority of the research in the world is focused on water electrolyzers using proton-exchange membranes (PEM), which exhibit high initial performance [START_REF] Carmo | [END_REF][START_REF] Tunold | Materials for electrocatalysis of oxygen evolution process in PEM water electrolysis cells[END_REF]. However, the acidic environment of the PEM makes the use Pt-group metal (PGM) electrocatalysts [START_REF] Carmo | [END_REF][10][11][12][13][14][15] quasi-mandatory, these elements being of very limited abundance, and of questionable stability [16][17][18][19][20]. Most non-PGM electrocatalysts [21][22][23][24][25][26][27][28][29][30][31][32] are more abundant and therefore more desirable, but these materials are generally less active and durable than PGMs in acidic environments. For this reason, alkaline water electrolysis is a subject of rising interest, as it might be the only water electrolysis technology with sufficient electrical performances that is compatible with a widespread deployment. Alkaline water electrolysis is not a new technology, as it is the historical large-scale means of "clean" hydrogen production [23,[33][34][START_REF] Ursúa | Proceedings of the IEEE[END_REF][START_REF] Mergel | Hydrogen production by water electrolysis: A review of the current status and future trends[END_REF]. Nickel-based oxides [START_REF] Dionigi | [END_REF][38][39] (either used "alone" or in combination with a suitable support [40]) or cobalt-based oxides [41][42][43][44][45] are often considered amongst the best electrode materials for alkaline OER [27,[46][47][48]. One widely-used strategy to prepare stable OER electrocatalysts is to deposit a thin "active layer" of these metals/oxides on the surface of an electronically conductive substrate, e.g. carbon [38,47], conducting oxides [START_REF] Tunold | Materials for electrocatalysis of oxygen evolution process in PEM water electrolysis cells[END_REF][49][50][51][52][53][54][55] or steels [56][57][58][59]. Although electrocatalysts prepared on such electrodes can be very active, their long-term durability is rarely reported and is open to question, in particular in strong bases: except from Li's work [60], dealing with Janus Ni-Co-P nanowires, which were demonstrated for 3000 h in water electrolysis, durability studies of the literature usually do not exceed a few 10 or at best a few 100 of h in operation for such electrocatalysts. This has been demonstrated, for example for carbon surfaces [61][62][63][64], carbon-supported electrocatalysts [65][66][67][68][69][70] or metal and their oxides [18,19]. In addition, when stainless steel (hereafter denoted as "SS") is used as a conducting substrate only, the many transition metals that make them up turn out to be active for the (alkaline) OER, and they are not put to beneficial use. This is why some reports now focus on the use of stainless steels as "simple and easy" materials for the alkaline OER. In this case, the active (and stable) layer of the OER electrode originate from the components of the bulk material. This active layer plays the role of an "active passivation layer", i.e. a layer that exhibits antagonistic properties of high electrocatalytic activity together with high stability towards corrosion through passivation. A very good example in this direction is described in the previous work of the authors, where 316L SS was activated in situ by a simple long-term OER operation in concentrated LiOH electrolyte, producing a very active and stable surface (stability was demonstrated for 3000 h in OER operation) to promote the positive reaction of water splitting in 5 M LiOH (as a third charging electrode in an aqueous Li-air battery) [71]. Other examples have emerged in the literature, in particular from Schafer's group in Germany [72][73][74][75][76] and others [77], as recalled in a recent review [78]. One of the very important characteristic of stainless steel electrodes activated in situ by "simple (long-term) operation" in the electrolyte of operation, is their "self-healing ability", which accounts for their long-term durability. Because the active layer is made from controlled corrosion of the underlying bulk alloy (which also protects it), it is capable of re-forming later on if, by accident, the active layer is detached (for example under the mechanical pressure of harsh gas evolution) [71,77]. Another solution is to perform an ex situ treatment to generate a tailored surface layer that is prone to catalyze the reaction of interest. The group of Schafer made significant progresses in this direction for OER electrocatalysis [72,74,75], soon followed by others [79], the strategy being also used for alternative applications, e.g. to design supercapacitors [80] or Liion battery [81] electrodes.

Following the work on activated 316L SS electrodes for OER in concentrated LiOH electrolytes [71], the present paper investigates the same type of materials, but for use in KOH electrolyte.

The main objective of this contribution is to extend the concepts to more widely-used electrolytes and in particular to assess whether these electrodes could be useful in an industrial alkaline water electrolysis process, most of which use aqueous KOH electrolyte (at least prior the introduction of anion-exchange membranes in these technologies [82]), or as third electrode in aqueous metal-air batteries (e.g. Zn-air). In addition, knowing that in the previous work of the authors [71], the in situ activation in 5 M LiOH(aq) lasted ca. 250 h, which is not practical, a procedure for a fast ex situ activation of the 316L SS electrode in 5 M LiOH is proposed. The soobtained materials were benchmarked to home-made CoOx deposits, known to be very active for the OER [41,45,56,83,84]. Finally, because accelerated activation in LiOH electrolytes may not be practical, a procedure where the 316L SS surfaces were activated in KOH was analyzed to check whether such electrodes present the same morphology, composition and activity as those activated in LiOH electrolytes. All the experimental findings (activity reached and stability of the performances) will be supported by thorough physicochemical characterizations, namely scanning electron microscopy (SEM), X-ray diffraction (XRD) and Raman spectrometry, so as to draw conclusions about composition-structure/activity-durability relationships.

Experimental

Most of the experimental procedures are derived from previous works [71,[START_REF] Moureaux | Etude des réactions mettant en jeu l'oxygène dans un système électrochimique lithium-air aqueux rechargeable électriquement[END_REF] and the readers are directed to these documents for more details. Specific to this study are the preparation and characterizations of cobalt oxides deposits on 316L stainless steel substrate. Two types of CoOx deposition technics were performed.

The electrochemical deposition of CoOx were prepared from 0.3 M CoSO4 aqueous electrolyte at T = 50°C, by applying a cathodic current density of j = -1 mA cm -2 (geometric) during t = 1500 s on the desired 316L substrate, which should (in theory) yield a deposit thickness of ca. 500 nm of cobalt. The layer obtained was then thermally annealed (under air) using a 1°C min -1 ramp from T = 25°C to T = 400°C, followed by a 2 h step at T = 400°C and then a cooling ramp at -1°C min -1 down to T = 25°C.

Sol-gel deposition of CoOx was also experimented by following a procedure developed by Singh et al. [START_REF] Singh | [END_REF]. More specifically, the precursors of the oxides layer were blended in stoichiometric amounts (ca. 0.1 M CoSO4 + 0.2 M LiOH), resulting in a Co(OH)2 precipitate. The latter was filtered, abundantly rinsed with ultrapure water (18.2 MΩ cm, < 3 ppb total organic carbon, Millipore ® ) and dried for 24 h in room conditions. The powder obtained was then dissolved in acetic acid to produce a gel of Co(CH3OO)2, which was painted on the 316L substrate, and finally thermally-annealed under air for t = 30 min at T = 350°C to convert the cobalt acetate to Co3O4.

In both cases, and also for the bare 316L substrate, the CoOx/316L surfaces obtained were thoroughly characterized by scanning electron microscopy (SEM, HIROX SH-1500 SEM) coupled with elemental X-Ray energy dispersive spectrometry (X-EDS, XFLASH 410-H semiconductor Silicon Drift Detector, controlled with Esprit ® V1.8), X-Ray diffraction (XRD, PANalytical X'Pert Pro MPD operating at 45 kV and 40 mA -Bragg-Brentano configuration, Cu Kα radiation), Raman spectroscopy (Renishaw ® Raman microscope, operating with an argon LASER at 540 nm) and X-Ray photoelectron spectroscopy (XPS, performed on a XR3E2 apparatus (Vacuum Generator) using a Mg Kα source (1253.6 eV) and a hemispherical analyzer at constant pass energy of 30.0 eV).

The materials were also tested for their electrochemical activity and durability. The electrochemical characterizations were performed in a three-electrode electrochemical cell as previously described [71]. Although experimentally, the reference electrode used was a mercurymercury oxide electrode (Hg-HgO, 1 M KOH), all the data presented herein are expressed versus the reversible hydrogen electrode (RHE), so that the pH effect is corrected for the three aqueous alkaline electrolytes characterized here: 1 M KOH, 5 M KOH and 5 M LiOH. Given that measuring the pH of strongly alkaline solutions is not straightforward, the pH values determined by Pound et al. [87] (Table 1) were used. From these, the potential values experimentally measured versus (Hg-HgO, 1 M KOH) were converted on the RHE.

Results and discussion

Preamble: instability of electrodes prepared by electrodeposition of a foreign layer at the surface of a SS 316L electrode and interest of "self-activated" electrodes

The results shown in Figure 1 suggests that hetero-deposits of CoOx may not be stable on the long-term for the OER. In particular, the highly-active CoOx deposits investigated here are severely modified by long-term oxygen evolution in 5 M LiOH electrolyte. Two processes are at stake: (i) the very oxidizing conditions experienced during OER promote the "corrosion" of the CoOx deposit and (ii) the gas evolution which induces the mechanical disintegration of the layer from its support. Such mechanical disintegration occurs in particular when the interaction of the active layer with its support is not sufficiently strong, which is obvious for the sol-gel CoOx deposit in Figure 1. Once the layer has been completely destroyed, only the substrate remains and the OER activity decreases (at least until the 316L SS substrate self-activates upon operation, which takes time, as detailed below). In addition, the deep modifications of the electrodeposited CoOx electrode surface (Figure 1B,C) raises the question of the relevance of tightly controlling the initial electrode composition and texture as it will likely be (severely) modified (if not destroyed) in usage, with no hope of its re-formation in operation.

12 from [START_REF] Moureaux | Etude des réactions mettant en jeu l'oxygène dans un système électrochimique lithium-air aqueux rechargeable électriquement[END_REF] with permission from Univ. Grenoble-Alpes.

The authors believe that these problems will come upon any porous active deposit used in gas evolution reactions (in particular the OER). In addition, the stability of the layers formed "ex situ" (and using elements that do not enter the composition of the bulk substrate) at the surface of steels for operation in water spitting has previously been questioned [78]. The answer may come from the formation of a stable, homogenous and adherent catalytic layer on the surface of steel electrodes by a direct corrosion/passivation mechanism of this surface during OER, as previously demonstrated. This strategy was successfully adopted by the authors for the OER in concentrated

LiOH electrolyte [71,[START_REF] Moureaux | Etude des réactions mettant en jeu l'oxygène dans un système électrochimique lithium-air aqueux rechargeable électriquement[END_REF] (Figure 2). In that study, remarkable stability and OER activity was demonstrated. However, the activation of the 316L SS electrode in LiOH electrolyte was very long (ca. 250 h), which is a clear limitation for a practical application. Besides, these remarkable OER performances were obtained in 5 M LiOH electrolyte, and it would be interesting to check whether these materials are also active and durable for operation in KOH electrolyte, for a possible application in water electrolysis, or in rechargeable aqueous metal-air batteries (e.g. Znair). Results in this direction are presented in the next sections. (B) After the initial in situ "activation", the performances maintain for very long term (the variations in current density over time originated by the gradual failure of the reference and/or counter-electrode; as a result, the electrolyte (E) and counter-electrode (CE) had to be periodically replaced). Adapted from [71], with permission from Elsevier.

Ex situ accelerated activation of a 316L SS electrode

The formation of a Ni-rich "active layer" that results in a high OER activity but which also protects the underlying 316L SS electrode (Figure 2) is explained by the following reaction mechanism during the "early" stages (i.e. the first 250 h or OER operation) of the process [71,[START_REF] Moureaux | Etude des réactions mettant en jeu l'oxygène dans un système électrochimique lithium-air aqueux rechargeable électriquement[END_REF]: the first step is an anodic dissolution of the 316L metals (forming mainly nickel, iron and chromium cations, the base elements of 316L SS) followed by their possible precipitation owing to their low solubility in the 5 M LiOH electrolyte. In particular, the different solubility values of Cr, Fe and Ni cations produce the peculiar composition of this layer (82.5 at% Ni, 10 at% Fe, 7.5 at% Cr, i.e. very different from the base composition of bulk 316L SS: ca. 10-14 at% Ni, ca. 70 at% Fe, ca. 16-18.5 at% Cr). As a consequence, the Ni-rich layer so-obtained possesses the remarkable combined properties of passivation, semi-conductivity and electrochemical OER activity [71,88], the latter likely originating from a so-called hypo-hyper d interbonding effect put forth by Jaksic et al. [89] and then Micoud et al. [START_REF] Micoud | Influence d'un support MOx (M = W, Ti) sur les propriétés électrocatalytiques de nanoparticules de platine[END_REF]. As such, the passive film is capable of protecting the underlying OER electrode in the course of the continuous OER operation, which explains its very moderate thickness even after 3000+ hours of operation in 5 M LiOH (below 50 nm) [71]. On the contrary, the fact that this layer protects the underlying 316L SS raises questions about the reasons why such a layer forms. At that stage, the authors postulate that it is the initial characterization steps (see Figure 2A) that led to the formation of this layer. As such, it is anticipated that alternating the potential of the 316L electrode between "low" and "high" values in a similar electrolyte (5 M LiOH) would lead to the same surface structure/composition of the electrode, and therefore to the same electrochemical properties (activity and robustness).

This study aims at first to accelerate the formation of the Ni-rich passive (to limit corrosion) and active (to enable fast OER) film at the surface of the 316L electrode. Being admitted that it is the alternation of the electrode potential between "low" and "high" potential values (during the characterization steps of Figure 2A) that led to the active layer, the protocol of ex situ accelerated activation was built as follows.

- The rationale of these alternations of "high" / "low" potential values was to favor the electrochemical dissolution/chemical precipitation of the metal cations of the 316L electrode at high potential and to reduce the surface species at low potential, to re-enable some dissolution of the electrode and to build in this manner a "thick-enough" (and therefore stable-enough protection of the underlying bulk 316L material) active layer.

After such accelerated activation, the 316L SS electrodes were firstly tested for the OER in 5 M

LiOH electrolyte, so as to compare their activity and stability with regards to the electrodes activated in situ (see Figure 2) in a time-consuming procedure (ca. 250 h). Figure 3 This stable OER activity of the ex situ activated SS electrodes can be linked to its surface morphology, which is rather homogeneous, compact and near-free of defects (Figure 5C,D); in particular, no cracks or surface heterogeneities are seen, which differs from the observations made for an in situ activated electrode (Figure 5A,B). This better integrity of the Ni-rich film formed by ex situ accelerated activation accounts for the better stability of its electrochemical response compared to in situ activated electrodes (Figure 3). In addition, the fact that close to full performances can be reached in less than 9 hours (the duration of the accelerated activation treatment), i.e. 30 times faster than an in situ activation in the same electrolyte, holds great interest for a practical application. electrode is very homogeneous and near-free of defects. Adapted from [START_REF] Moureaux | Etude des réactions mettant en jeu l'oxygène dans un système électrochimique lithium-air aqueux rechargeable électriquement[END_REF] with permission from Univ. Grenoble-Alpes.

OER performances in KOH electrolytes of 316L SS electrodes activated ex situ in 5 M

LiOH

The electrochemical performances of 316L SS electrodes activated ex situ in 5 M LiOH by the accelerated procedure described above were characterized afterwards in KOH electrolytes, in revolution rate had no effect of on the OER current density monitored in the range 0 < j < 1 A cm -2 , indicating that mass-transport limitations are not at stake in the present experimental conditions, even at this reasonably high current density (unlike feared in other publications [46,47] for j > 0.01 A cm -2 ).

The results shown in Figure 6 demonstrate that the 316L SS electrodes activated ex situ by the accelerated procedure in 5 M LiOH electrolyte are also very active for OER in KOH electrolytes; the best performances are reached in 1 M KOH, likely owing to (i) the larger oxygen solubility in this electrolyte compared to the others (the solubility of O2 severely decreases at increasing pH [START_REF] Chatenet | [END_REF]92], leading to larger masking of the electrode by O2 bubbles) and to (ii) the larger water activity in this solution than in the 5 M concentrated hydroxide electrolytes [87]; the performances in 5 M KOH are also significantly larger than in 5 M LiOH. The fact that Li + species decrease the OER activity of nickel oxides electrodes, which is why LiOH is a popular electrolyte additive to enhance the cyclability and performances of positive NiOOH electrodes in alkaline batteries [93,94], cannot be neglected and could account for these observations. This effect can be explained by the ability of small Li + cations to substitute protons in hydrated forms of metal-oxides, thereby blocking redox transitions and impeding electrocatalytic activity. This was particularly demonstrated for MnOx/C oxygen reduction electrocatalysts: in LiOH electrolyte, the insertion of Li + into the MnOx lattice stabilizes both the Mn atoms at the oxidation state 3 and the oxygen groups at the carbon surface, which prevents their role of redox mediating species and further blocks the catalytic process, eventually producing increased ORR overpotential values when compared to the activity of the same electrocatalysts in KOH electrolytes [95]. and 2.9 cm -1 ), which signals an increase of the strain on the chemical bonds. This excess strain likely originates from the insertion of Li + in the crystalline lattice of Ni-oxides, and the authors speculate that this causes the largely decreased OER activity of the ex situ activated 316L SS electrodes in 5 M LiOH compared to KOH, as was also reported for the activity of MnOx/Cbased ORR electrocatalysts [95], which was depreciated when operated in LiOH electrolytes. In KOH electrolyte, the remarkable activity of the ex situ activated 316L SS electrodes for OER in these conditions is highlighted in Table 2. When the performances are benchmarked to nonnoble-metal OER electrocatalysts (Table 3), it is evident that a properly-activated 316L electrode is very active and competes with state-of-the-art (non-noble) OER materials of the literature. The present electrode is as active or more active than most of these materials, except for a mixed cobalt-lanthanum oxide, for which the OER overvoltage is decreased by ca. η = 0.1 V at j = 100 mA cm -2 geometric [22]. Nevertheless, the latter material, elaborated in the form of a thin-film onto a foreign substrate (nickel in that case), would likely experience the durability issues mentioned in preamble of the present study for hetero-deposits (see section 3.1 and Figure 1), issues which should not be at stake for a material with a "self-prepared" active layer like the activated 316L SS electrode [78]. In other words, if this "self-prepared" active layer, made from the very component of the 316L SS bulk substrate, degrades upon operation (e.g. from mechanical detachment due to harsh O2 bubbles evolution), it is likely that it will be capable of forming again (there is still plenty of the active material in the bulk of the underlying 316L SS material to form new layers). In the literature, to the best of the author's knowledge, only a few paper have presented stability data in OER operation beyond 1000 h; Table 3 for example presents the activity of Janus Ni-Co-P nanowires that have been demonstrated for 3000 h of OER (and of HER) in 1 M KOH (similar durability as for our 316L SS electrode). The results show that these materials fail to meet the activity of our stainless steel electrodes in similar operating conditions, demonstrating that the activated 316L SS electrode exhibits a unique combination of high OER activity and long-term durability. Now, the 316L SS electrodes tested so far have been activated in 5 M LiOH electrolyte and one would expect a different activity compared to electrodes activated in KOH electrolyte, because Li + cations likely insert into the NiOx-rich surface layer formed on the bulk 316L SS electrode in the former case, while K + are inserted in the latter (Figure 7). To get around this bias, 316L electrodes were activated in KOH electrolytes and then tested for the OER.

Electrolyte

Tafel slope / mV dec -1 Electrode potential / V vs. RHE Overvoltage / V @ j = 10 mA cm -2 @ j = 100 mA cm -2

In situ activation of 316L SS electrodes in KOH electrolytes and their OER performances in KOH electrolytes

Polarizing of a non-activated 316L SS electrode in 5 M KOH (Figure S1A in supporting information) shows comparable behavior to that in 5 M LiOH: the OER current density progressively increases for the first ca. 50 h and then plateaus, as a result of a surface-enrichment in nickel oxides during the first 250 h of test (Figure S1B). This film of nickel oxides has a rather rough texture (Figure S1C), an amorphous nature (Figure S2) and is composed of mixed oxides of Ni, Fe and Cr (Figure S3). The elemental analyses show that there is very little difference (in terms of composition in transition metal cations) in the active surface layer formed in KOH electrolyte (Table S1) compared to that formed in LiOH electrolyte [71].

The OER performances obtained after in situ activation in 5 M KOH (ca. j = 350 mA cm -2 at E = 1.63 V vs. RHE, η = 0.4 V, see Figure S1) can be compared to those obtained for the 316L electrode activated ex situ in 5 M LiOH and tested in 5 M KOH (ca. j = 550 mA cm -2 at E = 1.63

V vs. RHE, η = 0.4 V) (see Figure 6). These data demonstrate that the ex situ activation in 5 M

LiOH by the accelerated procedure is very efficient to prepare highly-active 316L electrodes for the OER, whatever the electrolyte composition of the medium in which the OER is performed.

The next section will study whether electrodes prepared in an accelerated manner by ex situ activation in KOH are also very active for OER operation in LiOH electrolytes.

OER performances in 5 M LiOH of 316L SS electrodes activated ex situ in 5 M KOH

316L SS electrode activated ex situ in 5 M KOH before long-term operation in 5 M LiOH (Figure S4) exhibit initial performances which are roughly twice larger than those for a 316L SS electrode activated in situ or ex situ in 5 M LiOH (see Figure 3). Based on Figure 7 and Table S1, these superior performances upon ex situ activation in 5 M KOH, may be accounted for by the presence of K + in the hydrated oxide layers (instead of Li + ), an enrichment of iron in the oxide layer and a larger active surface area (more details are available in supporting information). However, the catalytic layer formed by ex situ activation in 5 M KOH is not stable under long-term OER operation in 5 M LiOH electrolyte (Figure S4A); the surface charge related to the NiII/NiIII transition increases (Figure S4B) until the performances reached become comparable to those obtained after in situ activation in 5 M LiOH (see Figure 1 and Figure 3). In other words, the catalytic layer formed ex situ in 5 M KOH spontaneously and gradually evolves towards a different (and less active) state upon operation in LiOH electrolytes. This further confirms that only electrodes activated in the same environment than their environment of usage have stable performances in operation, even if activation in KOH yields larger initial performances of OER, whatever the medium of OER operation.

Ex situ activation of 316L SS electrodes in KOH or LiOH electrolytes and their OER performances in KOH electrolytes

A 316L stainless steel electrode ex situ-activated in 5 M KOH using the accelerated procedure described in section 3.2 exhibits equivalent behavior as that obtained after (much longer) in situ activation (Figure S5): the OER is stable over 1000+ hours of test. However, the electrode activated ex situ in 5 M LiOH is less active than that activated in 5 M KOH: activation in KOH is always preferable in terms of OER activity, whatever the electrolyte of final usage, probably because Li + species are rather-irreversibly inserted in the NiOx layer, on the contrary to K + .

Conclusions

This paper demonstrates that it is possible to activate a 316L stainless steel electrode for OER without using hetero-elements and without making deposits on the surface of the electrode. The activation step is not only possible by spontaneous (usually slow) evolution of the surface composition/structure in the medium of OER operation, but it can be accelerated by using a properly-designed procedure (e.g. succession of potential steps of "oxidation" and "reduction" of the surface). The present approach fundamentally differs from that usually used in electrocatalysis, where compounds of tailored composition/structure/texture are added/deposited at a conductive substrate, their initial activity being optimal but their long-term stability not being guaranteed in the harsh operating conditions of an OER electrode (mechanical stress due to bubbles + highly-oxidizing conditions). In the approach detailed herein, the catalytic surface is created from the bulk components of the stainless steel electrode in experimental conditions that are close to those of usage, which is inherently more favorable to the long-term stability of the prepared surfaces.

It has been observed that KOH(aq) is a better electrolyte for activation than LiOH(aq), whatever the final alkaline electrolyte used, but activating in the final electrolyte could be more practical and shall give the same results in the (very) long run. Besides, the OER performances reached in alkaline electrolytes for the materials activated by an ex situ accelerated procedure compare well to those obtained for the same materials activated in situ, the latter being a much more timeconsuming procedure. In particular, the performance of activated 316L electrodes in KOH electrolytes compare favorably to those of state-of-the-art OER materials of the literature in similar environments, the latter being prepared by depositing active layers at the surface of a conducting substrate. More importantly, the 316L electrodes did not show significant degradation in performance and surface over a few 100s of hours of OER operation, which should be highlighted given the very large current densities experienced (a few 100s of mA cm - 2 ).

This suggests that stainless steel electrodes could prove to be good OER materials for use in alkaline water electrolyzers, even if this needs to be confirmed for longer duration of operation, larger active areas and non-flat surfaces (e.g. mesh, felts or grids). As a result, this paper opens the way to the development of highly-active and low-cost OER materials for the production of hydrogen (and in particular for the oxygen evolution reaction), but also as charging electrode in KOH-based metal-air batteries.

ASSOCIATED CONTENT

There is no associated content for this contribution.

Figure 1 :

 1 Figure 1: (A) Time-dependent performance of OER electrodes consisting of CoOx deposits

Figure 2 :

 2 Figure 2: (A) Cycling voltamperograms monitored for a 316L SS electrode in 5 M LiOH at T =

--

  Step 1: a sequence of 10 min polarization at E = 1.53 V vs. RHE (a potential above the domain of formation of NiIII, see Figure2A) followed by 5 min at E = 0.93 V vs. RHE (a potential below the domain of formation of NiII, see Figure2A), overall repeated twice; Step 2: two voltamperometric cycles at v = 5 mV s -1 performed in the range 0.93 < E < 1.93 V vs. RHE; the potential is therefore, again, scanned above/below the NiII/NiIII redox transition; The sequences of Step 1 and then Step 2 was repeated 12 times, the entire protocol lasting overall 8 h 40 min.

  clearly demonstrates that the activity of 316L SS electrodes activated ex situ by this accelerated procedure is similar to that ultimately reached by the electrodes activated in situ. Interestingly, these high performances are reached much faster than the 200 h of operation required for the in situ activated electrodes. One should note however that the activity of the ex situ activated electrode slightly increases in the first 50 h of the OER durability test, probably owing to a stabilization and/or thickening of the surface film during the initial period of the OER operation.

Figure 3 :

 3 Figure 3: Comparison of the OER performances in 5 M LiOH at T = 25°C of the 316L SS

Figure 4 :

 4 Figure 4: Comparison of the surface charge relative to the NiII/NiIII redox couple as a function

Figure 5 :

 5 Figure 5: Representative micrographs of the surface of the 316L SS electrode activated (A, B) in

  possible application of these electrodes in low-cost and durable alkaline water electrolysis or KOH-based metal-air batteries. The experiments were performed in a rotating disk configuration (the geometric area of the activated 316L SS electrode was ca. 0.2 cm²); the RDE

Figure 6 :

 6 Figure 6: Representative OER voltamperograms measured at 316L electrodes ex situ activated

Figure 7 :

 7 Figure 7: Plots of X-Ray Photoelectron spectroscopy (XPS) of the surface of activated 316L SS

Table 2 :

 2 Kinetic parameters of the OER measured in 5 M LiOH, 1 M KOH and 5 M KOH electrolytes, on the 316L SS electrodes ex situ activated by the accelerated procedure in 5 M LiOH electrolyte.

Table 1 :

 1 values of the pH of the electrolyte solutions considered in the study, as determined by Pound et al.[87].

	Electrolyte	pH	ERHE vs. EHg/HgO -1 M KOH
	5 M LiOH	14.5	0.95
	5 M KOH	15.3	1.00
	1 M KOH	13.9	0.92

Marian Chatenet: Marian.Chatenet@grenoble-inp.fr; personal webpage : http://lepmi.grenobleinp.fr/personnels/m-chatenet-marian--429692.kjsp; team webpage: http://lepmi.grenobleinp.fr/team-eip/interfacial-electrochemistry-and-processes-team-eip--754099.kjsp ACKNOWLEDGEMENT This work was performed within the framework of the Centre of Excellence of Multifunctional Architectured Materials "CEMAM" n• AN-10-LABX-44-01. MC thanks the French Institut Universitaire de France "IUF" for its support and Stéphane Coindeau, Thierry Encinas, Frédéric Charlot and Grégory Berthomé (from the Consortium des Moyens Technologiques Communs (CMTC) of Grenoble-INP) for the XRD, FEG-SEM and XPS characterizations, as well as Michel Mermoux for the Raman analyses.

AUTHOR INFORMATION

Florian Moureaux: moureaux.florian@hotmail.fr Philippe Stevens: philippe.stevens@edf.fr Gwénaëlle Toussaint: gwenaelle.toussaint@edf.fr


Marie Hocquart

Hortense Drouet

Paul Levet

Didier Raoult

Philippe Parola

Carole Eldin
email: carole.eldin@gmail.com

Levet Paul

Cellulitis of the face associated with SENLAT caused by Rickettsia slovaca detected by qPCR on scalp eschar swab sample: An unusual case report and review of literature

Keywords: R. slovaca, SENLAT syndrome, Periorbital Cellulitis

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

Introduction

Tick-borne rickettsioses are infectious diseases caused by obligate intracellular Gramnegative bacteria of the spotted fever group (SFG), belonging to the genus Rickettsia in the order Rickettsiales. Hard ticks may act as vectors, reservoirs, and/or amplifiers of SFG rickettsiae [START_REF] Delord | Rickettsioses and Q fever in travelers (2004-2013)[END_REF]. The pathogenic role of Rickettsia slovaca was first demonstrated in 1997 in a patient who presented with a single inoculation lesion of the scalp and enlarged cervical lymph nodes after being bitten by a Dermacentor tick [START_REF] Raoult | Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks[END_REF]. The acronym TIBOLA (tick-borne lymphadenopathy) was proposed because of painful lymphadenopathy next to the region of the tick bite. Later, a similar clinical entity was described and named DEBONEL (Dermacentor-borne necrosis erythema lymphadenopathy) to emphasize the role of the Dermacentor ticks. Finally, the name SENLAT (scalp eschar and neck lymphadenopathy after tick bite) was suggested to collectively describe this entity because, although R. slovaca is frequently involved, there have been reports describing other bacteria, such as R. raoultii, R. sibirica mongolitimonae, R. massiliae, Bartonella henselae, Francisella tularensis. [START_REF] Angelakis | Scalp eschar and neck lymphadenopathy caused by Bartonella henselae after Tick Bite[END_REF][START_REF] Dubourg | Scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes[END_REF]. "Candidatus Rickettsia rioja", Coxiella burnetii and Borrelia burgdorferi sensu lato are possible etiological agents due to indirect molecular evidence in ticks involved in SENLAT cases [START_REF] Dubourg | Scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes[END_REF].

However, in some cases (20-25%), the causative agent remains undetermined suggesting that other microorganisms might be involved [START_REF] Dubourg | Scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes[END_REF].

Rickettsia slovaca, the most frequent reported agent of SENLAT, is transmitted by ticks of the genus Dermacentor and especially by D. marginatus (Supplementary Figure 1) or D. reticulatus. The geographical distribution of R. slovaca most likely corresponds to the geographical distribution of these ticks in Europe and North Africa [START_REF] Dubourg | Scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes[END_REF].

Rickettsia slovaca has also been found in D. marginatus ticks in the Kurgan region (Ural) of Russia and in Georgia, and in 6.5% of D. silvarum ticks in China. Human cases have not been reported in Asia [START_REF] Parola | Update on tick-borne rickettsioses around the world: a geographic approach[END_REF]. Infection is most frequent among women (67 to 100%) and children under 12 years old (41 to 43%). The median incubation period is of 5 to 10 days (range, 1 to 15 days). In Europe, infections occur most frequently from March to May and from September to November, which corresponds to the periods of greatest activity of Dermacentor adult ticks [START_REF] Parola | Update on tick-borne rickettsioses around the world: a geographic approach[END_REF]. Rickettsia slovaca has also been reported as an agent of fever of unknown origin (Botelho-Nevers and [START_REF] Botelho-Nevers | Fever of unknown origin due to rickettsioses[END_REF] among travelers returning from Mongolia, Italy and Corsica [START_REF] Delord | Rickettsioses and Q fever in travelers (2004-2013)[END_REF].

Clinical manifestations of SENLAT include inoculation lesion at the upper half of the body, mainly on scalp in humans, probably because Dermacentor ticks are used to parasitize long-haired animals like horses and wild boar [START_REF] Parola | Rickettsia slovaca and R. raoultii in tick-borne Rickettsioses[END_REF]. The inoculation lesion is often centered by the tick bite with an erythema of at least 5mm that encircles the lesion and second cervical or occipital lymphadenopathies, satellite of the eschar, which may be large, inflammatory and painful [START_REF] Parola | Rickettsia slovaca and R. raoultii in tick-borne Rickettsioses[END_REF]. These two elements may be associated with fever, rash, localized alopecia, headache, asthenia, sweats, arthralgia and myalgia (Dubourg et al., 2013).

We describe an unusual case of SENLAT associated with a cellulitis of the face diagnosed by qPCR on a scalp eschar swab and review the literature about this entity and the diagnosis by qPCR on eschar swabs.

The Case

A 70-year-old woman with medical history of hypothyroidism was admitted in our department in Marseille on April 2017 with a periorbital cellulitis associated with a scalp eschar and cervical lymphadenopathies. The patient reported a walk in the forest about ten days ago with the apparition, a few days later, of odynophagia, bilateral cervical lymphadenopathies and lesion of the scalp, without fever. Her family doctor thought she had tonsillitis and prescribed amoxicillin. Subsequently, a large erythema and edema of the face appeared, so that she consulted to our unit. At physical examination we recorded a scalp eschar on the left side of the head, an erythema and edema at the forehead with rapid extension to periorbital, retro auricular and cervical areas with left-sided lymphadenopathies (Figure 1). Laboratory tests were normal except a slight increase in the C-reactive-protein (15mg/l). Cervical and face tomodensitometry showed a cellulitis without fascia involvement or abscess. Empirical antibiotherapy with amoxicillin-clavulanic acid and clindamycin had been started before at the emergency unit and we added oral doxycycline (200mg/daily) for ten days because of the presence of the inoculation eschar on the scalp. A dry sterile swab sample was collected from the inoculation eschar. The swab, while being rotated vigorously, was directed to the base of the eschar at a 50°-60° angle for 5-6 times [START_REF] Mouffok | Diagnosis of rickettsioses from eschar swab samples, Algeria[END_REF]. qPCR on scalp eschar swab targeting a fragment of the gltA gene, as previously described [START_REF] Aubry | Bacterial agents in 248 ticks removed from people from 2002 to 2013[END_REF], was positive for Rickettsia sp. and sequencing of a fragment of the ompA gene [START_REF] Aubry | Bacterial agents in 248 ticks removed from people from 2002 to 2013[END_REF] identified Rickettsia slovaca. Culture of the scalp swab sample on human embryonic lung cells using shell vial methods as previously reported [START_REF] Raoult | Spotless rickettsiosis caused by Rickettsia slovaca and associated with Dermacentor ticks[END_REF] were negative. Serum sample were analysed using indirect Immunofluorescence Assay (IFA) against two spotted fever group rickettsial antigens (R.conorii conorii, Rickettsia felis) and a typhus group antigen (R.typhi). The IFA test during the acute phase was considered positive if antibody titres were > 1:128 for immunoglobulin G (IgG) and > 1:64 for immunoglobulin M (IgM) for the spotted fever group, and > 1:64 for IgG and > 1:32 for typhus group, as previously described (Hayet et al., 2018). These serological testings were negative in the acute phase serum sample and in a convalescent serum sample one month after the resolution of symptoms. The patient completely recovered after ten days of doxycycline except for a small cicatricial alopecia at the site of the eschar (Figure 1).

Discussion

In 2009, Parola et al. described facial edema without cellulitis as a new clinical feature of SENLAT because it was found in 19% of cases of R. slovaca and 40% of R. raoultii infections [START_REF] Parola | Rickettsia slovaca and R. raoultii in tick-borne Rickettsioses[END_REF]. A literature search revealed only one previous reference reporting a case of SENLAT associated with a similar cellulitis of the face. It was a nine-yearold French girl with an acute hemifacial edema and erythema requalified as TIBOLA in 2011 [START_REF] Gaston | Dermohypodermitis on the face revealing TIBOLA[END_REF]. The removal of two ticks in the scalp had occurred 10 days earlier. The edema appeared suddenly in a context of fever at 39.5 °C, headache and vertigo. The clinical examination revealed an erysipeloid placard of the left hemiface, associated with a homolateral jugulo-carotid inflammatory adenitis. At the same time, the examination of the scalp revealed two necrotic eschars surrounded by an erythematous halo. The diagnosis was done by seroconversion for Rickettsia fifteen days later [START_REF] Gaston | Dermohypodermitis on the face revealing TIBOLA[END_REF].

The microbiological methods available for the diagnosis of tick-borne rickettsioses are serology, culture and PCR. Serology is the most widespread method for the diagnosis of tickborne rickettsioses, but seroconversion occurs only 15 to 21 days after the beginning of the symptoms [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF]. Moreover, even when using IFA, which is the reference method, cross reactions are frequent and the specific serological sensitivity for R. slovaca is low (about 12%), probably due to a limited locoregional dissemination of the infection [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF], as illustrated by our case, where even convalescent serum sample was negative. Culture is fastidious and requires cell cultures that are only performed in specialized BSL 3 laboratories. On the other hand, molecular techniques (qPCR targeting the gltA gene for Rickettsia group and species-specific qPCR) are useful given their good sensitivity at the beginning of the infection, especially on eschar samples [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF]. qPCR on a swab sample of the eschar requires the use of a sterile swab to obtain a sample from inside the eschar, simply by inserting the swab into the eschar and rotating it to the basis of the eschar.

The material obtained via swabbing can be then processed for nucleic acid extraction and subsequent PCR analysis [START_REF] Luce-Fedrow | Strategies for detecting rickettsiae and diagnosing rickettsial diseases[END_REF]. It has a sensitivity of 85 to 90% and a specificity close of 100% [START_REF] Bechah | Identification of rickettsial infections by using cutaneous swab specimens and PCR[END_REF][START_REF] Mouffok | Diagnosis of rickettsioses from eschar swab samples, Algeria[END_REF][START_REF] Renvoise | Widespread use of real-time PCR for rickettsial diagnosis[END_REF][START_REF] Wang | Diagnosis of Queensland tick typhus and African tick bite fever by PCR of lesion swabs[END_REF]. These performances are almost comparable to the ones of qPCR on eschar cutaneous biopsy (sensitivity >95% and specificity >98%) [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF]. qPCR on eschar swab samples has been used with success around the world for detection of R. parkeri, R. conorii, R. sibirica mongolitimonae, R. africae, R. australis, R. felis and O. tsutsugamushi (Table 1). To date, only 2 references reported the detection of R. slovaca by qPCR on a swab eschar sample [START_REF] Bechah | Identification of rickettsial infections by using cutaneous swab specimens and PCR[END_REF][START_REF] Dubourg | Scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes[END_REF]. Swabbing an eschar has a lot of advantages because it is a noninvasive, simple, rapid and painless technique that can be easily performed without risk, especially in children. This test can be used at the bedside or in an outpatient clinic and could be useful for epidemiologic, surveillance and clinical studies even although health care workers are not able to perform biopsies [START_REF] Le | Use of eschar swabbing for the molecular diagnosis and genotyping of Orientia tsutsugamushi causing scrub typhus in Quang Nam province, Vietnam[END_REF][START_REF] Luce-Fedrow | Strategies for detecting rickettsiae and diagnosing rickettsial diseases[END_REF][START_REF] Renvoise | Widespread use of real-time PCR for rickettsial diagnosis[END_REF]. In a recent study, opinions of health care providers and patients were evaluated and they both preferred collecting swab samples over biopsy samples (46 vs. 5 and 57 vs. 2, respectively; p = 0.0001) [START_REF] Mouffok | Diagnosis of rickettsioses from eschar swab samples, Algeria[END_REF].

In a clinical setting, the treatment of choice for SFG rickettsia is doxycycline [START_REF] Botelho-Nevers | Treatment of Rickettsia spp. infections: a review[END_REF], including in children for whom its safety has been demonstrated, notably on the absence of dental damage [START_REF] Todd | No visible dental staining in children treated with doxycycline for suspected Rocky Mountain Spotted Fever[END_REF][START_REF] Volovitz | Absence of tooth staining with doxycycline treatment in young children[END_REF]. There is no data in the literature to determine the exact duration of treatment, which is related to the clinical response and is classically of 7 to 14 days [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF], or at least 3 days after apyrexia [START_REF] Renvoise | An update on rickettsiosis][END_REF]. The complete resolution of symptoms is long but antibiotic treatment can reduce the duration of symptoms from 62 to 50 days [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF]. Sequelar alopecia at the tick bite site, as observed in our patient, is reported in 20 to 51% of cases [START_REF] Foissac | Update on SENLAT syndrome: scalp eschar and neck lymph adenopathy after a tick bite[END_REF].

Figures legends Figure 1

 1 Figures legends

Table 1 :

 1 Agents of Rickettsia spp. detected by qPCR on eschar swab Fever, eschar on the scalp, tender local lymphadenopathy, and generalized rash with maculopapular and vesicular components.Case 4: Fever, eschar on the torso, tender local lymphadenopathy, and generalized sparse rash characterized by papular and vesiculopustular lesions.

			Positive
	Agents	Authors		Clinical presentation/ Localization of eschar
			SWAB
				Fever, chills, night sweats, a diffuse maculopapular rash, headache, myalgia, neck stiffness,
	R. parkeri	Myers et al., 2013	2
				arthralgia, and malaise.
		Bechah et al., 2011	25	Fever, eschar and generalized maculopapular rash.
	R. conorii
		Khrouf et al., 2016	12	Acute fever and cutaneous rashes and/or eschars.
		Mouffok et al., 2011	2	NS
	R. sibirica mongolitimonae	Bechah et al., 2011	2	NS
		Solary et al., 2014	1	Fever, 2 eschars on the lower right eyelid, periorbital edema, cervical lymphadenopathies.
	R. africae	Bechah et al., 2011	2	NS
		Socolovschi et al., 2012	4	Fever, eschar, rash, lymph-adenopathy
	R. australis	Bechah et al., 2011	1	NS

Funding source

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Rickettsia are strict intracellular bacteria and conventional microbiological techniques cannot be used to evaluate the efficacy of antibiotics. Cell culture techniques are used to test in vitro susceptibility of Rickettsia to antibiotics by determining the minimum inhibitory concentrations but can be performed only in specialized laboratories [START_REF] Renvoise | An update on rickettsiosis][END_REF].

However, beta-lactams, aminoglycosides and cotrimoxazole are ineffective on rickettsia [START_REF] Renvoise | An update on rickettsiosis][END_REF]. As a consequence, in a patient with a cellulitis, the presence of an eschar and lymphadenopathies should prompt clinicians to add doxycycline, because beta lactam are inefficient on SENLAT-associated cellulitis.

Conflict of interest

All the authors have no conflict of interest to disclose

Aknowledgements

None

Supplementary Figure

Supplementary Figure 1

Dermacentor marginatus ticks: female (Panel a) and male (Panel b).