Pablo Padilla
email: pablo.padilla@uliege.be

Anthony Herrel

Mathieu Denoël

May future climate change promote the invasion of the marsh frog? An integrative thermo-physiological study

Keywords: Invasive species, Ectotherm, Temperature, Thermal tolerance, Performance curve

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

Introduction

Human-induced climate change [START_REF] Cattiaux | Projected increase in diurnal and interdiurnal variations of European summer temperatures[END_REF][START_REF] Simmons | A reassessment of temperature variations and trends from global reanalyses and monthly surface climatological datasets[END_REF]IPCC 2022) puts pressure on living organisms, pushing them to their physiological limits [START_REF] Parmesan | A globally coherent fingerprint of climate change impacts across natural systems[END_REF][START_REF] Warren | The projected effect on insects, vertebrates, and plants of limiting global warming to 1.5 °C rather than 2 °C[END_REF]. Indeed, temperature affects the rates of chemical reactions within an organism and consequently their physiology [START_REF] Tattersall | Coping with thermal challenges: physiological adaptations to environmental temperatures[END_REF]. Their response to temperature is described by reaction norms that typically have a parabolic shape [START_REF] Cossins | Temperature biology of animals[END_REF][START_REF] Angilletta | Thermal adaptation: a theoretical and empirical synthesis[END_REF]) (Fig. 1). Among Metazoa, ectothermy is the most common thermoregulatory mechanism, and because ectotherms rely mostly on environmental temperature, many species are expected to be impacted by climate change [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF][START_REF] Sinclair | Can we predict ectotherm responses to climate change using thermal performance curves and body temperatures?[END_REF][START_REF] Lotze | Global ensemble projections reveal trophic amplification of ocean biomass declines with climate change[END_REF][START_REF] Sánchez-Bayo | Worldwide decline of the entomofauna: a review of its drivers[END_REF]. Regardless of their geographical origin, both tropical and temperate species are at risk from global warming [START_REF] Deutsch | Impacts of climate warming on terrestrial ectotherms across latitude[END_REF]) and of the predicted stronger temperature variations [START_REF] Paaijmans | Temperature variation makes ectotherms more sensitive to climate change[END_REF][START_REF] Vasseur | Increased temperature variation poses a greater risk to species than climate warming[END_REF][START_REF] Dillon | Life in the frequency domain: the biological impacts of changes in climate variability at multiple time scales[END_REF]. This is especially the case for amphibians which are already among the most threatened vertebrates [START_REF] Stuart | Status and trends of amphibian declines and extinctions worldwide[END_REF]IUCN 2022) and for which climatic change has been invoked as a direct driver of population extinction [START_REF] Araújo | Climate warming and the decline of amphibians and reptiles in Europe[END_REF][START_REF] Wake | Are we in the midst of the sixth mass extinction? A view from the world of amphibians[END_REF][START_REF] Blaustein | Direct and indirect effects of climate change on amphibian populations[END_REF]). Other drivers of population decline such as invasive species may interact with climate change and precipitate the extinction of threatened populations [START_REF] Pounds | Widespread amphibian extinctions from epidemic disease driven by global warming[END_REF][START_REF] Mcmenamin | Climatic change and wetland desiccation cause amphibian decline in Yellowstone National Park[END_REF].

In many cases invasive species can maintain an activity over a broad range of temperatures and as such may be less impacted or even benefit from the ongoing climatic crisis [START_REF] Huang | Does global warming increase establishment rates of invasive alien species? a centurial time series analysis[END_REF][START_REF] Zerebecki | Temperature tolerance and stress proteins as mechanisms of invasive species success[END_REF][START_REF] Kelley | The role thermal physiology plays in species invasion[END_REF]. [START_REF] Bennett | Evolution of the control of body temperature: is warmer better?[END_REF] hypothesized that warmer temperatures maximize locomotor capacity which may benefit dispersal in invasive species. Consequently, the interaction between invasive species and climate change may result in unprecedented risks to native ecosystems [START_REF] Rahel | Assessing the effects of climate change on aquatic invasive species[END_REF][START_REF] Mainka | Climate change and invasive species: double jeopardy[END_REF]. Despite the general vulnerability of amphibians to temperature change, some introduced amphibians are able to withstand and quickly adapt to new environmental conditions [START_REF] Vimercati | Rapid adaptive response to a Mediterranean environment reduces phenotypic mismatch in a recent amphibian invader[END_REF][START_REF] Mittan | Rapid adaptation to cold in the invasive cane toad Rhinella marina[END_REF][START_REF] Araspin | Rapid shifts in the temperature dependence of locomotor performance in an invasive frog, Xenopus laevis, implications for conservation[END_REF]. African clawed frogs (Xenopus laevis) and bullfrogs (Lithobates catesbeianus), for example, are two species that have been introduced worldwide (IUCN 2015(IUCN , 2020)). [START_REF] Andersen | Predicting global climatic suitability for the four most invasive anuran species using ecological niche factor analysis[END_REF] recently predicted suitable niches on all continents except Antarctica for both species. [START_REF] Johovic | A potential threat to amphibians in the European Natura 2000 network: forecasting the distribution of the American bullfrog Lithobates catesbeianus[END_REF] also showed that climate change would benefit the spread of bullfrogs. However, most of the model predictions for invasive species are based on occurrence data only. Empirical measurements of not only the thermal limits but also thermal preferences and physiological response to temperature of invasive species need to be gathered and then used to improve these predictions [START_REF] Ginal | More time for aliens? Performance shifts lead to increased activity time budgets propelling invasion success[END_REF]). Some traits have been recognized to be associated with the invasive potential of a species [START_REF] Sakai | The population biology of invasive species[END_REF][START_REF] Allen | Fast life history traits promote invasion success in amphibians and reptiles[END_REF]. Range expansion, for example, is a stage during invasion that benefits from greater dispersal capacity and survival. Stamina may play an important role during dispersal and is driven by the capacity to maintain prolonged muscular effort and an efficient metabolism. On the other hand, a quick reaction time and explosive locomotor performance may allow a better escape from predators and increase prey capture success. To establish and disperse, invaders also need to overcome abiotic ecological filters, such as temperature [START_REF] Olyarnik | Ecological factors affecting community invisibility[END_REF]. Indeed, global surface temperature is continuously increasing and a global warming of 2 °C will be exceeded in the 21 st century (IPCC 2022). A broad thermal performance breadth would then help invaders to maintain their performance and potentially fitness. By consequence, global warming will result in a shift in environmental temperature that may maximize locomotor performance (Fig. 1). These two adaptations (i.e. large thermal tolerance range and broad thermal performance breadth) combined may allow activity and habitat use of invasive species to be less constrained by daily and annual variations in environmental temperature in addition to increasing their dispersal efficiency and invasive potential.

In the present paper, we performed a multi-trait study using, as our model species, marsh frogs. The Pelophylax genus is widely distributed in the Palearctic [START_REF] Lymberakis | Mitochondrial phylogeography of Rana (Pelophylax) populations in the Eastern Mediterranean region[END_REF][START_REF] Dufresnes | Hybridogenesis in water frogs[END_REF], with multiple introductions out of their native range particularly in Western Europe, such as Belgium [START_REF] Holsbeek | Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (Pelophylax esculentus complex)[END_REF], Switzerland [START_REF] Dubey | Multiple origins of invasive and "native" water frogs (Pelophylax spp.) in Switzerland[END_REF] or France [START_REF] Doniol-Valcroze | Discovery of a Pelophylax saharicus (Anura, Ranidae) population in Southern France: A new potentially invasive species of water frogs in Europe[END_REF]). These have resulted in the establishment of invasive populations (Dufresnes et al. 2017b). The major threats reported from their invasion is their potential hybridization with native species of the genus [START_REF] Holsbeek | Potential impact of genome exclusion by alien species in the hybridogenetic water frogs (Pelophylax esculentus complex)[END_REF] as well as predation on native species [START_REF] Pille | Predation pressure of invasive marsh frogs: a threat to native amphibians?[END_REF]. The invasive range of these animals has increased over the last decades with new areas being rapidly colonized such as the Larzac plateau in southern France where Pelophylax frogs were introduced after being historically absent (Dufresnes et al. 2017a;[START_REF] Denoël | High habitat invasibility unveils the invasiveness potential of water frogs[END_REF][START_REF] Duret | Efficiency of aquatic PIT-tag telemetry, a powerful tool to improve monitoring and detection of marked individuals in pond environments[END_REF]. It has been suggested that recent warmer conditions may have enhanced their invasion (Dufresnes et al. 2017a), but this remains untested to date. The Larzac plateau has seen its temperature increased over the last years [START_REF] Ali | Cross-chapter paper 4: Mediterranean region[END_REF]. Climate models predict that warming will be more rapid in the region compared to the global average [START_REF] Kjellström | European climate change at global mean temperature increases of 1.5 and 2 °C above pre-industrial conditions as simulated by the EURO-CORDEX regional climate models[END_REF]. If this increase in temperature does indeed benefit the invasive capacity of these frogs, then this may have major consequences to the native ecosystem.

Specifically, the goals of this study were to evaluate whether warm temperatures such as those arising due to climate change could favour invasive marsh frogs. We expect marsh frogs to show: (i) a broad thermal tolerance providing them with a large period of activity; (ii) a preference for high environmental temperatures that may be higher than the current environmental temperatures; (iii) locomotor capacities that are maximised at high temperatures, i.e. higher than the current environmental temperatures. Lastly, sex differences in thermal traits are not often investigated whereas they may be expected due to the inherent differences in physiology and behaviour between males and females [START_REF] Bodensteiner | Thermal adaptation revisited: How conserved are thermal traits of reptiles and amphibians?[END_REF]. We therefore took the opportunity of this study to test for an effect of sex in each thermal trait previously mentioned.

Materials and methods

Study organisms

Adult marsh frogs (Pelophylax ridibundus) were caught in August 2020 (Critical thermal limits: n = 40, 20 for critical minimum and 20 for critical maximum with 10 females and 10 males in each; Stamina: n = 21, 8 females and 13 males; Jump force: n = 25, 11 females and 14 males) and April 2022 (Preferred temperature: n = 29, 11 females and 18 males) at the border of ponds in Larzac (43°51'N -3°21'E, Hérault, France). Previous molecular analysis confirmed the taxonomic assignment and the invasive status of the marsh frogs in the studied population (Dufresnes et al. 2017a;M. Pabijan, pers. comm.). All individuals were PIT-tagged (Biolog-ID, 134.2 KHz) to allow individual identification. The tag was inserted dorsally under the skin [START_REF] Christy | The efficacy of using Passive Integrated Transponder (PIT) tags without anaesthetic in free-living frogs[END_REF]. The snout-vent length (SVL; from the tip of the snout to the end of cloaca) was measured with callipers after gently laying each frog on a flat surface (mean ± SE = 68.23 ± 1.91 mm). Each frog was weighed (mean ± SE = 30.97 ± 2.61 g) using a digital scale (Ohaus, Brooklyn, NY, USA; precision 0.1 g). Sex was identified by the presence of a vocal sac and nuptial pads on the males' thumbs and later verified through dissection and inspection of the reproductive organs.

Transportation and housing

Frogs were brought inside large coolers filled with 3cm of water to the Function and Evolution (FUNEVOL) laboratory at the Muséum National d'Histoire Naturelle of Paris, France. Frogs were maintained singly in 5L tanks (21.4 x 36.5 x 16.5 cm; water depth: 5 cm) with holes in the lid. Tanks were inclined by placing a 10 cm wood block under one of the sides creating a dry area that occupied one third of the tank. Animals rested for the first week to allow them to acclimate to laboratory conditions. Water in the tanks was renewed weekly. Two adult crickets (Acheta domestica) were given twice weekly and additionally after each trial. Night and day periods were set to 12 hours each. The room temperature was set at 24 °C.

Field temperatures

Air temperatures were recorded each 6 minutes and obtained from a meteorological station situated at 6 km from the site from January 2018 to January 2019 [START_REF] Larvor | MeteoNet, an open reference weather dataset by METEO FRANCE[END_REF]; Table 1).

Water frogs spend a large part of their time at the water-land interface and take shelters in the bottom of ponds, therefore, we incorporated water temperatures with automatic measures each 30 minutes from March 2019 to March 2020 with a submersible temperature logger (HOBO U20L-01) placed at a depth of 1.5m in a pond at the center of the invaded area (Table 1).

Critical temperatures (CT)

Each frog was placed individually inside an incubator (Aqualytic-LIEBHERR, TC 256 G/256L/2-40 °C) with light in a 5L plastic box (21.4 x 36.5 x 16.5 cm), with 5 mm of water depth to prevent dehydration. Twenty animals (10 males, 10 females) were used to measure minimal critical temperatures and twenty others (10 males, 10 females) were used for maximal critical temperatures to avoid too much stress on the same animals. To establish critical minimum temperature (CTmin), the incubator was set at 12 °C and the frogs were kept at that temperature for one hour. Next, the temperature was decreased at a rate of cooling of 2°C each 30 minutes until reaching 6°C when we switched to a rate of 1°C each 30 minutes (Fig. 2a). For critical maximal temperature (CTmax), frogs were placed in a container placed inside a large cooler filled with water heated by a water heater. Experiments started at the acclimation temperature of 24 °C after which the temperature was increased with a rate of warming of 2°C each 30 minutes until reaching 34°C at which point we switched to a rate of 1°C each 30 minutes (Fig. 2a). For both minimal and maximal critical temperatures, the temperature was increased or decreased by 0.5 °C every 30 minutes whenever animals showed slower righting response (Fig. 2a). Even if the onset of spasms has been considered by some researchers as a more precise measurement of thermal tolerance [START_REF] Lutterschmidt | The critical thermal maximum: data to support the onset of spasms as the definitive end point[END_REF], we used the loss of righting response instead to avoid excessive stress [START_REF] Taylor | The thermal ecology and physiology of reptiles and amphibians: a user's guide[END_REF]. The lack of a righting response was tested by giving the animal two seconds to recover a normal upright position after having been placed on their back. When the frog was no longer able to right itself during this short time period, its internal body temperature was measured using a thermocouple inserted inside the cloaca (APPA51 thermometer K-type, Appa technology corp, resolution 0.1°C).

Preferred temperature (Tpref)

The experimental setup consisted of five individual lanes (190 x 20 x 30 cm each) within a wood arena covered with cork. The gradient ranged from approximately 14.5 °C on the side cooled by ice packs to approximately 42 °C on the side heated by light bulbs (60 watts, Repti Basking Lamp Spot ZooMed, SL2-60) (Fig. 2b, Fig. S1). The room temperature was set to 24 °C and the average temperature in the lanes was 24 ± 1.73 (SE) °C. Each lane was humidified on its whole length before the trial with a spray bottle filled with tap water. Animals were not fed for three days before trials to avoid digestion effect on thermoregulation [START_REF] Gatten | Effect of nutritional status on the preferred body temperature of the turtles Pseudemys scripta and Terrapene ornata[END_REF]). One frog was set inside each lane and was left free to thermoregulate for one hour. After one hour we measured the body temperature of each frog and calculated the mean across all individuals which we considered as an estimate of the population thermal preference. Each frog was recorded twice separated by three days. As we did not find significant difference between each recording (paired t-test: t28 = -0.56, p = 0.57, first recording: mean ± SE = 21.79 ± 0.68 °C, second recording: 22.27 ± 0.66 °C), we used the second recording to avoid potential bias coming from their initial discovery and exploration of the experimental setup. The temperature of the animals was assessed using a thermal camera (FLIR-E6xt 2.1L) and thermal image analysis software (FLIR Tools, version 6.4.18039.1003). Thermal pictures were taken with an incidence angle close to 0°C and at a distance of one meter and an emissivity of 0.97 (Fig. S2).

Temperature was also verified by a thermocouple (APPA51 thermometer K-type, Appa technology corp) inserted in the cloaca at the end of the trial. Since there were no significant differences of the methodology (i.e. thermal camera vs thermocouple measurements) on the measures of body temperatures (linear mixed model with method, sex and SVL as covariates on body temperature; methods: estimate = -0.8, CI = -1.62 to 0.03, p = 0.06 ; sex: estimate = 0.76, CI = -1.59 to 3.11, p = 0.52; SVL: estimate = -0.08, CI = -0.29 to 0.12, p = 0.42), we decided to use the thermal camera values because of its instantaneity and to avoid delays created by the cloacal measurements due to the capturing and handling of the animals.

Terrestrial exertion

Frogs were stimulated by hand to move back and forth across a linear track (200 x 40 x 50 cm ; graduated each 10 cm) with a humidified cork bottom providing grip. The time and the distance that frogs were able to continuously move until exhaustion were measured (Herrel and Bonneaud 2012a, b) (Fig. 2c). Time was measured using a stopwatch and distance was based on the number of graduations crossed by the frogs during movement. From these variables, we calculated a composite variable referred to henceforth as mean speed. Exhaustion was defined by the lack of a righting response determined by giving the animal two seconds to recover a normal position after having been placed on its back. After the trial, body temperature was verified using a thermocouple. Frogs were then placed back in their maintenance boxes, given food, and not used for other observations for one week. Trials were repeated twice at each temperature, and the maximum time and distance at each temperature were retained for each individual. To evaluate how exertion changes as a function of temperature [START_REF] Angilletta | Thermal adaptation: a theoretical and empirical synthesis[END_REF] our observations were repeated in random order. Because the thermal optimum for exertion was not clear after testing animals at four temperatures (9,14, 19, 24°C), we ran additional exertion tests at two additional temperatures (4 and 29 °C) providing a better fit of our models.

Jump forces

Maximal jump forces were measured using a piezo-electric force platform (20 x 10 cm, Kistler Squirrel force plate, ± 0.1 N; see [START_REF] Herrel | Jumping performance in the highly aquatic frog, Xenopus tropicalis: sexspecific relationships between morphology and performance[END_REF]) (Fig. 2d). The platform was connected to a charge amplifier (Kistler -Charge Amplifier type, 9865) and forces were recorded for 60 seconds at 500 Hz using the BioWare software (Kistler). A cork surface was glued to the force plate to provide a better grip. Frogs were placed individually on the force plate, allowed to rest a few seconds, and then induced to jump by approaching them. Peak force data were extracted using the Kistler BioWare software and the total resultant force (vector sum of the X-, Y-and Z-forces) was calculated. Three sessions of at least 3 jumps within each session were recorded and only the single best jump was retained and used in the analyses. Tests at four temperatures (14, 24, 28, 33 °C) were enough to determine the thermal optimum. Prior to each test, frogs were placed individually for 3 hours at the desired temperature in an incubator in a box with some water to prevent dehydration.

Statistical analyses

For both one-way analyses of co-variance (ANCOVAs) and generalized additive mixed model (GAMM), model validation was performed by visualising the residuals which had a homogeneous, normal distribution and by plotting the response variable on fitted values. All analyses were done using R version 4.1.2 (R Core Team 2019).

Mass and snout vent length (SVL) were significantly correlated (t19 = 12.01, R = 0.94; p < 0.01). We therefore used snout-vent length as our covariate in the analyses because mass is dependent on the daily food intake and excretion. Snout-vent length did not differ between males and females (t19 = 0.64, p = 0.52).

ANCOVas were run for CTmin and CTmax to determine the effect of sex on body temperature after controlling for SVL. Another ANCOVA was run with the effect of sex on the preferred temperature with SVL as covariate.

Because temperature often has a non-linear effect on biological processes [START_REF] Huey | Evolution of thermal sensitivity of ectotherm performance[END_REF][START_REF] Bulté | Cautionary notes on the descriptive analysis of performance curves in reptiles[END_REF], we fitted a GAMM [START_REF] Wood | Fast stable restricted maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models[END_REF] for each performance trait. GAMM can fit complex non-linear relationships using a smoother function on sections of the data [START_REF] Wood | Generalized Additive Models: An Introduction with R[END_REF]. They are therefore good tools to estimate thermal performance curves (TPC) and were previously used in the study of reptiles [START_REF] Vickers | Using GAMM to examine inter-individual heterogeneity in thermal performance curves for Natrix natrix indicates bet hedging strategy by mothers[END_REF][START_REF] Cecchetto | Running performance with emphasis on low temperatures in a Patagonian lizard, Liolaemus lineomaculatus[END_REF]. Effective degrees of freedom (edf) were used to quantify the strength of non-linearity. An edf of one being a linear effect, an edf between one and two was considered as a weak non-linear effect and an edf greater than two was a highly non-linear relationship [START_REF] Zuur | Mixed effects models and extensions in ecology with R[END_REF]). In each model, SVL was used as covariate; temperature as a fixed effect dependent on a smooth function and sex was added as a fixed factor. Because the same frogs were measured at each temperature, we added individual identity as a random term [START_REF] Wood | Generalized Additive Models: An Introduction with R[END_REF]. For temperature, we used a smoothing parameter (k) that avoids both over smoothing and overfitting biologically incoherent variation. This was based on the models having the highest R-squared values and verifications from the function of the "mgcv" R package (v1.8-35; Wood 2011). F-statistics were reported with the model degrees of freedom (mdf), calculated by adding up the degrees of freedom of each term, and the residual degrees of freedom (rdf) calculated as the difference between the number of observations (n) and the mdf. To create the thermal performance curves, we used the predictions from the statistical models. The thermal optimum and the thermal performance breadth, i.e. the range of temperatures over which an animal can maintain 95, 80 and 50% of its maximal performance, were extracted from the model predictions [START_REF] Taylor | The thermal ecology and physiology of reptiles and amphibians: a user's guide[END_REF]. Note that positive and negative predicted values at 2.8°C and 38.4°C can seem biologically impossible from our values observed at each CT, however these predictions only indicate predicted CTs at colder/warmer temperature (Fig. 4,5).

Results

Critical temperature

The studied marsh frogs showed a CTmin (mean ± SE) of 2.80 ± 0.21 °C, a CTmax of 38.40 ± 0.09 °C and, an average thermal range (CTmax -CTmin) of 35.60 ± 0.23° C (Fig. 3). There was no significant effect of sex (ANCOVA; F1,17 = 0.13, p = 0.91) or SVL (F1,17 = 0.09, p = 0.76) for CTmin. However, for CTmax the effect of sex was significant (F1,17 = 7.49, p = 0.01) with males having a slight but significantly higher upper thermal tolerance (0.44 °C) than females (Fig. 3). Snout-vent length had no significant effect on CTmax (F1,17 = 2.08, p = 0.16).

Preferred temperature

After thermoregulating for an hour, marsh frogs showed an average body temperature of 21.8 ± 3.71 °C. There was no significant effect of either sex (F1, 26 = 0.12, p = 0.73) or SVL (F1, 26 = 0.19, p = 0.67).

Stamina

Based on the adjusted R-squared values (GAMM), temperature, SVL and sex together explained 82.8% of variation in the distance travelled by the frogs before exhaustion.

Temperature had a significant highly non-linear effect on the distance travelled before exhaustion (edf = 3.91 ; F20.27, 147.77 = 185.23, p < 0.01; Fig. 4a). Predictions from the model fit estimated a thermal optimum (Topt) at 24.32 °C and frogs were able to maintain more than 95, 80 and 50 % of their performance over a range of respectively 11.58, 20.58, and 28.06 °C (Fig. 4a). Snout-vent length showed a significant highly non-linear effect on the distance travelled before exhaustion (edf = 4.67 ; F20.27, 147.77 = 3.55, p < 0.01). There was no significant effect of sex (t21 = 1.32, p = 0.18).

Based on the adjusted R-squared values (GAMM), temperature, SVL and sex together explained 66.4% of variation in time spent moving by the frogs before exhaustion. Temperature showed a significant highly non-linear effect on time spent moving before exhaustion (edf = 3.92 ; F15.02, 152.98 = 78.10, p < 0.01; Fig. 4b). Predictions from the model fit estimated a thermal optimum at 11.70 °C and frogs were able to maintain more than 95, 80 and 50 % of their performance over a range of 4.82, 10.09, and 24.52 °C, respectively (Fig. 4b). There were no significant effects of SVL (F15.02, 152.98 = 1.15, p = 0.28) and sex (t21 = -0.10, p = 0.91).

Based on the adjusted R-squared values (GAMM), temperature, SVL and sex together explained 88.1% of the variation in average speed of the frogs. Temperature showed a significant highly non-linear effect on average speed during the endurance trial (edf = 3.96 ;F16.23,151.76 = 272.75,p < 0.01;Fig. 4c). Predictions from the model fit estimated a thermal optimum at 27.20 °C and frogs were able to maintain more than 95, 80 and 50 % of their performance over a range of 4.97, 10.58, and 21.88 °C, respectively (Fig. 4c). Snout-vent length showed a significant (F16.23, 151.76 = 1.83, p = 0.17) positive linear effect (edf = 1). There was no significant of sex (t21 = 0.75, p = 0.45).

Jump force

Based on the adjusted R-squared values (GAMM), temperature, SVL, and sex together explained 89.2% of variation in jump force of the frogs. Temperature showed a significant (F21.73, 128.26 = 237.37, p < 0.01; Fig. 5) highly non-linear relationship (edf = 3.97) with jump force. Predictions from the model fit estimated a thermal optimum at 29.77 °C and frogs were able to maintain more than 95, 80 and 50 % of their performance over a range of 4. 53,10.16,and 26.35 °C,respectively (Fig. 5). Snout-vent-length showed a significant effect (F21.73, 128.26 = 5.94, p < 0.01) that was highly non-linear (edf = 2.67). However, no significant effects were detected for sex (t25 = 1.08, p = 0.28).

Discussion

Marsh frogs showed a thermal tolerance that covers the entire annual variation of bottom pond temperatures and 76.5% of the annual variation in air temperature, with colder temperature being the only limit (Fig. 6, Table 1). It is important to point out that air temperature is a representation of the environmental temperature in shaded area and that solar radiation can generate surface temperatures far exceeding observed air temperatures under the shade. These results suggest that warmer temperatures may allow invasive marsh frogs to have an increased margin of locomotor activity particularly in shaded conditions or during night time.

Nonetheless, even if their upper thermal limit is 5.2°C greater than the current maximal air temperature recorded (Fig. 6), extreme thermal events may limit their fitness before reaching this critical limit. It is also interesting to note that under laboratory conditions marsh frogs try to maintain a body temperature (Tpref) that is higher than the average temperature they can experience in shaded areas (Fig. 6, Table 1). The same applies for their thermal performance breadths on distance jumped before exhaustion, average speed, and jump force (Fig. 6, Table 1). Our data suggest that warmer air temperature may allow them to better spread across shadier habitats and also to colonize previously unfavourable ponds while continuing to inhabit more exposed ponds [START_REF] Denoël | High habitat invasibility unveils the invasiveness potential of water frogs[END_REF]) due to their ability to bask or dive into the pond to thermoregulate. Lastly, regarding variation between males and females, a slight difference was observed in CTmax but not for other traits, suggesting a similar thermal biology for the two sexes.

Marsh frogs, an overlooked invasive species with a broad thermal tolerance

Despite the extent of their invasion over a large part of Western Europe, the invasive potential of the marsh frog is underestimated [START_REF] Pille | Predation pressure of invasive marsh frogs: a threat to native amphibians?[END_REF]). Here we show that their thermal tolerance extends to over 35 °C with an upper thermal limit beyond the maximum water and air temperatures (in shadow) in their natural environment. Similar tolerance ranges were found in the three most invasive amphibians: Rhinella marina [START_REF] Johnson | Thermal relations and daily variation in the thermal tolerance in Bufo marinus[END_REF], Xenopus laevis [START_REF] Araspin | Rapid shifts in the temperature dependence of locomotor performance in an invasive frog, Xenopus laevis, implications for conservation[END_REF] and Lithobates catesbeianus [START_REF] Lotshaw | Temperature adaptation and effects of thermal acclimation in Rana sylvatica and Rana catesbeiana[END_REF]) (Suppl. Table S1). The first two species showed thermal tolerance range that are respectively 9% and 37% narrower than the marsh frogs (Suppl. Table S1). The thermal tolerance of Lithobates catesbeianus may, however, appear wider than that of marsh frogs (38.55 °C, Suppl. Table S1), yet was measured using the lower lethal temperature instead of the loss of righting response. [START_REF] Spellerberg | Temperature tolerances of Southeast Australian reptiles examined in relation to reptile thermoregulatory behaviour and distribution[END_REF] evaluated both CTmin using the loss of righting response and the lethal minimal temperature on ten species of reptiles and the mean gap (± SD) between each measurement was 8.4 ± 2.5 °C. Overall, a broad thermal tolerance may allow invasive ectotherms to maintain a long period of activity year-round, in addition to being able to face daily variations in temperature as well as temperature extremes. We can expect from predicted warming that the environmental temperatures may remain within the thermal tolerance range of invasive ectotherms with a high upper thermal tolerance which may lead to phenological changes [START_REF] Sheridan | Shifts in frog size and phenology: Testing predictions of climate change on a widespread anuran using data from prior to rapid climate warming[END_REF].

However, the lack of knowledge on the thermal tolerance of native amphibians is a limitation preventing a mechanistic understanding of their respective success. Some data on palmate newts from the studied area highlight that they will have a lower reproductive fitness in warmer temperatures [START_REF] Galloy | Detrimental effect of temperature increase on the fitness of an amphibian (Lissotriton helveticus)[END_REF] and that droughts could impact population sizes in natural populations [START_REF] Denoël | Seasonal variation of morph ratio in facultatively paedomorphic populations of the palmate newt Triturus helveticus[END_REF]. Moreover, even in the scenario of thermal adaptation of some native species, the increased invasion of habitats due to the high thermal tolerance of marsh frogs would likely lead to multiple situations of habitat overlap with the native species and a consequent increase in predation risk [START_REF] Pille | Predation pressure of invasive marsh frogs: a threat to native amphibians?[END_REF].

Is warmer better?

A recent study [START_REF] Denoël | High habitat invasibility unveils the invasiveness potential of water frogs[END_REF]) highlighted the invasiveness potential of this species and, more specifically, revealed that sun exposure was one of the pond features associated with their occurrence. Marsh frogs prefer habitats with a high sun exposition to bask under the current climatic conditions. Our data show that marsh frogs try to maintain a body temperature higher than the average air temperature found in a shaded environment. Niche shifts have already been observed at a larger scale in invasive amphibians [START_REF] Rödder | Global realized niche divergence in the African clawed frog Xenopus laevis[END_REF][START_REF] Tingley | Realized niche shift during a global biological invasion[END_REF] and our results suggest that warmer climates may facilitate the invasibility of habitats cooler than open landscapes, such as forests, and therefore increasing their overlap with native amphibians in these environments.

Each performance trait showed, similarly, a thermal optimum at a temperature that was higher than the mean air temperature in addition to showing broad thermal performance breadths. Warmer climatic conditions may shift the environmental temperature to conditions that will maximize most performance traits of this frog species and therefore its invasion potential. Furthermore, broad performance breadths indicate that this species is able to maintain performance despite daily variations in temperature. This is particularly the case for dispersal, as suggested by the distance moved before exhaustion, where 80% of its maximum capacity covers 58% of the frog's absolute thermal tolerance (Fig. 6). Araspin and co-authors (2020) similarly measured the thermal performance breadth at 80% for exertion (mean ± SE = 8.25 ± 0.4 °C) in African clawed frogs, Xenopus laevis, a worldwide invasive species that was introduce in Western France and that shares the habitat with introduced populations of marsh frogs. Yet this range is 60% narrower, unveiling again the wide performance breadth and invasive potential of marsh frogs.

Thermal performance differs across traits

Given that an increase in temperature accelerates chemical reaction rates [START_REF] Tattersall | Coping with thermal challenges: physiological adaptations to environmental temperatures[END_REF] which in turn drive the thermal dependence of muscle contraction [START_REF] Bennett | Thermal dependence of muscle function[END_REF], it is not surprising that all performance traits were significantly affected by temperature. Average speed during the endurance trial and jump force increase with temperature to reach their optimum at high temperatures and finally drop off near the critical maximal limit of the animal, typical for burst performance traits [START_REF] Batty | The effect of temperature on the burst swimming performance of fish larvae[END_REF][START_REF] Pinch | Effects of temperature and slope on the sprint speed and stamina of the eastern fence lizard, Sceloporus undulatus[END_REF][START_REF] Araspin | Rapid shifts in the temperature dependence of locomotor performance in an invasive frog, Xenopus laevis, implications for conservation[END_REF]. Both traits follow the response of chemical reaction rates to an increase in temperature predicting that locomotor capacity is optimized at high temperatures that are not often reached in a shaded environment (Fig. 6). These two traits are essential for predator escape or prey capture, both relevant to the survival of invasive species. Time and distance before exhaustion show, however, different thermal responses compared to one another, and also different from what was observed for burst performance.

Because endurance capacity is dependent on oxygen uptake and the metabolic rate of the organism, we expected their thermal performance curves to follow the response observed for enzymatic reactions. James and co-authors [START_REF] James | Warmer is better: thermal sensitivity of both maximal and sustained power output in the iliotibialis muscle isolated from adult Xenopus tropicalis[END_REF]) also showed that higher temperatures maximize the performance at the muscle level for both burst performance and endurance. Interestingly time and distance before exhaustion were not maximized at high temperatures. This difference across traits needs to be quantified and used in future predictions. Mechanistic models, for example, often use data on a single trait which does not reflect the overall thermal performance of an organism [START_REF] Van Damme | The thermal dependence of feeding behaviour, food consumption and gut-passage time in the lizard Lacerta vivipara Jacquin[END_REF][START_REF] Kellermann | Comparing thermal performance curves across traits: how consistent are they?[END_REF].

The time spent in movement is maximized at lower temperatures and declines towards the critical maximal limit. This trait being maximal at lower temperature may not reflect the dispersal of the frog, because longer time spent in movement does not mean more distance covered per se. This result seems mostly explained by slower responses and shorter jumps, but this requires further study. Lastly, the distance covered before exhaustion increases from low temperatures to quickly show a broad plateau. This low sensitivity to temperature is reflected through the shape of its thermal performance curve (Fig. 4a,6), which is typically not observed in other studies on the thermal sensitivity of locomotion [START_REF] Bennett | Thermal dependence of locomotor capacity[END_REF]Angilletta 2001;[START_REF] Araspin | Rapid shifts in the temperature dependence of locomotor performance in an invasive frog, Xenopus laevis, implications for conservation[END_REF]. This thermal response suggests that the distance covered before exhaustion, at least in invasive marsh frogs, is likely determined by a number of physiological factors that each cover different parts of the overall thermal range. In contrast to jump force which is mostly determined by muscle contractile properties, stamina is indeed also driven by the functioning of the cardiovascular and the respiratory systems.

Conclusions

Integrating multiple-trait measures with environmental variables enhances our understanding of the thermal dependence of invasive ectotherms [START_REF] Ryan | Competing native and invasive Anolis lizards exhibit thermal preference plasticity in opposite directions[END_REF][START_REF] Young | How do the physiological traits of a lizard change during its invasion of an oceanic island?[END_REF]. Broad thermal tolerance and thermal preferences with optima of performance at temperatures higher than the air temperature of their environment were observed for the invasive marsh frogs studied here. As previously suggested [START_REF] Hellmann | Five potential consequences of climate change for invasive species[END_REF]), all steps of the invasion process can benefit from an increase of temperature if it leads to more frequent temperatures within their thermal performance breadth and closer to their thermal preference.

Our results support this hypothesis with warmer temperatures favouring jumping capacity as well as the distance moved, and the average speed. This can allow animals to cover more distance which can facilitate their spread, including across shaded habitats. Dufresnes and coauthors (2017a) questioned whether the rapid warming of the last two decades may have helped invasive Pelophylax to successfully spread and establish in the region. Given that the annual mean air temperature in the region has been increasing by more than 0.05 °C each year since 1980 (field meteorological data), we suggest that climate change has indeed favoured the spread of this invasive species, and will continue to do so given the current predictions of a mean warming in the range of 0.9 to 5.6 °C by the end of the 20 th century [START_REF] Ali | Cross-chapter paper 4: Mediterranean region[END_REF].

Fig. 1 Theorical representation of a shift of annual temperature distribution (histogram) that better fits a performance curve of an invasive ectotherm. The shift has been increased for readability. The curve, in purple, is bounded by the critical thermal minimum (CTmin, blue triangle), and the critical thermal maximum (CTmax, red triangle). The range of temperature between these bounds defines the thermal tolerance where the organism can maintain activity (horizontal grey dashed lines). The temperature associated with the maximum value of performance defines the thermal optimum (Topt, red). Finally, the range of temperatures where performance is greater or equal to a desired level (commonly 80% or 95%) defines a performance breadth (Tpb, horizontal black dashed line; [START_REF] Angilletta | Is physiological performance optimized by thermoregulatory behavior?: a case study of the eastern fence lizard, Sceloporus undulatus[END_REF].

Fig. 2 :

 2 Fig. 2: Graphical summary of each set of measurements made during the study on marsh frogs. (a) Critical thermal limits assessed with a thermocouple inserted inside the cloaca; (b) Preferred temperature estimated after one hour within a thermal gradient; (c) Stamina was estimated through the distance and the time travelled until exhaustion; (d) Jump force was measured using a force plate.

Fig. 3 :

 3 Fig. 3: Thermal tolerance in males and females of the invasive marsh frog. Critical thermal minimum (CTmin) is shown on the upper panel and the critical thermal maximum (CTmax) on the lower panel. A white diamond indicates the mean value and the middle line within the boxplot the median. Females are shown in purple and males in green. To allow a better visualisation of individuals the Y axis scale was rescaled for CTmax.

Fig. 4 :

 4 Fig. 4: Thermal performance curves predicted from GAMM models for (a) the distance travelled before exhaustion, (b) the time spent moving before exhaustion, and (c) the average "speed" (i.e. distance divided by time before exhaustion) in invasive marsh frogs. Thermal optimum (Topt) and thermal performance breadths (Tpb) at 50, 80 and 95% of the maximal performance are displayed. The uncertainty of the estimate is displayed through a 95% grey confidence band. Data is shown as dots for each individual. Temperature is shown by the color gradient.

Fig. 5 :

 5 Fig. 5: Thermal performance curve predicted from GAMM models on jump force in invasive marsh frogs. Thermal optimum (Topt) and thermal performance breadths (Tpb) at 50, 80 and 95% of the maximal performance are displayed. Uncertainty of the estimate is displayed through a 95% grey confidence band. Data is shown as dots for each individual. Temperature is shown

Fig. 6 :

 6 Fig. 6: Graphical summary of the thermal response of the invasive marsh frog (Pelophylax ridibundus), to the air temperature during their activity (grey) or during the annual variation (beige). The dotted horizontal red line indicates the 80% thermal performance breadth. The dashed vertical red line indicates the preferred temperature (Tpref).

Table 1 :

 1 Environmental temperature annually and relative to marsh frog period of activity in Larzac, France.

	Temperature (°C)		mean ± SE	min	max
	Annual	Air Bottom of the pond 10.73 ± 0.03 °C 5.34 °C 11.17 ± 0.02 °C -13.5 °C 33.2 °C 21.37 °C
	During period of activity	Air	15.67 ± 0.03 °C -5.6 °C	33.2 °C
	(early spring to late summer)	Bottom of the pond 13.47 ± 0.04 °C 6.97 °C	21.37 °C

Acknowledgements

We would like to thank F. Pille for his help in fieldwork, H. Clamouze for his help in conceiving experimental setups, the landowners and municipalities for allowing access to their ponds, and the Institut Géographique National for providing maps through the INSPIRE program. Animals were captured under a permit from the Direction Régionale de l'Environnement, de l'Aménagement et du Logement (Hérault). MD is a Research Director at Fonds de la Recherche Scientifique -FNRS; PP is a PhD student at Fonds pour la Formation à la Recherche dans l'Industrie et dans l'Agriculture (FRIA).

manuscript; MD and AH administrated research grants;

Funding This study benefited from the Fonds de la Recherche Scientifique-FNRS by a PDR grant number T.0070.19 and mobility grants; a grant for a laboratory internship from the Royal Belgian Zoological Society; a mobility grant from the University of Liège.

Availability of data and materials

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Code availability

The code used for statistical analysis was written in R and is available from the corresponding author on reasonable request.

Author contribution statement PP, AH and MD conceived the study; PP acquired the data, did the statistical analysis, and wrote the first draft of the manuscript; MD and AH administrated research grants; All authors contributed equally to the drafts and gave final approval for publication.

Declarations

Conflict of interest The authors declare that they have no conflict of interest.

Ethical approval All applicable institutional and/or national guidelines for the care and use of animals were followed.

Consent to participate Not applicable.

Consent for publication All authors gave consent.


Dr Lama Omar

Dr Thomas Onfroy

Prof Stéphane Daniele

Dr Noémie Perret
email: noemie.perret@ircelyon.univ-lyon1.fr

Copper zinc aluminate synthesized by sol-gel method with polyacrylic acid as catalysts for glycerol hydrogenolysis

Keywords: Glycerol, sol-gel processes, copper, polyacrylic acid, zinc aluminate

This study describes the use of novel Cu/ZnO/ZnAl 2 O 4 /PAAH (PAAH = polyacrylic acid) hybrid nanomaterials for glycerol hydrogenolysis. Their elaboration by sol-gel process either in one step or in two steps allowed to modulate their structural properties in terms of specific surface area, Lewis acidity, sizes and dispersion of copper nanoparticles. The presence of PAAH and aluminium during the synthesis particularly showed benefits in terms of dispersion and metal-support interaction. The characterization of the catalyst surfaces in composition and size of the copper nanoparticles allowed to establish structure-reactivity relationships. Their optimization allowed to reach remarkable conversions up to 96% with selectivity towards 1,2propanediol of 83%. A recyclability study also showed a possible reuse of these catalysts after a reactivation step.

Introduction

The valorization of glycerol, co-produced during the production of biodiesel, is still a major challenge. [START_REF]Glycerol: A Versatile Renewable Feedstock for the Chemical Industry[END_REF] 1,2-propanediol is an important commodity chemical with a wide range of applications, including antifreeze additive, de-icing fluid, and polyester resins. [START_REF]Glycerol: A Versatile Renewable Feedstock for the Chemical Industry[END_REF] The synthesis of this later compound from glycerol has been widely studied, but the development of a cost-effective process remains difficult. [START_REF] Checa | [END_REF] A number of noble (e.g. Ru, Rh, Pt) [3][4][5] and non-noble (e.g. Cu, Ni) [6] metal catalysts have been tested for the hydrogenolysis of glycerol. Catalysts based on noble metals usually exhibit high activity but low selectivity towards 1,2propanediol, due to C-C bond cleavage. [3] A review of the literature indicates that the most promising catalysts are copper-based, and many studies have focused on their design [START_REF] Checa | [END_REF][6][7][8] . Different supports have been employed, including ZnO, [9][10][11][12][13] Al 2 O 3 , [13][14][15][16] CeO 2 , [17] TiO 2 , [13,14] SiO 2 [13,[18][19][20][21] and some aluminosilicates, [15] ZrO 2 , [13,[22][23][24] and MgO [13,22,[25][26][27][28] . There is a general consensus that bifunctional catalysts with metallic Cu sites as well as (Brønsted and/or Lewis) acidic sites favor the selective formation of propanediols. Thus, the support plays a crucial role in the catalytic performance. Different studies have shown that Cu/ZnO catalysts exhibit high selectivity towards 1,2-propanediol in the range of 77% to 100%, however the conversion generally remains below 50%. [9][10][11][29][30][31][32] Additionally, Cu/ZnO catalysts usually undergo strong deactivation when used in water, due to metal leaching, loss of surface area and sintering of ZnO and Cu nanoparticles. [12,32,33] It is worth noting that Cu/ZnO/Al 2 O 3 may exhibit improved catalytic properties for the conversion of glycerol, especially in terms of stability, due to the presence of aluminum. [34,35] For the hydrogenolysis of glycerol, Cu-Zn and Cu-Zn-Al systems have principally been synthesized by the traditional impregnation and co-precipitation methods. [9][10][11][12]31,34,[36][37][38][39] Other studies report the use of solid state fusion [40] or evaporation-induced self-assembly [41] . It is known that the synthesis method can affect the physico-chemical properties of catalysts. It was shown that the precipitating agents in co-precipitation and solid-state fusion followed by precipitation methods affected the Cu crystallite size and the strength of (Brønsted and Lewis) acid sites. [40] Of particular relevance to this study is the work of P. Claus et al. [30] who reported higher glycerol conversion (46% vs. 17%) for Cu/ZnO prepared by an oxalate gel method than the one synthesized by co-precipitation method due to their higher copper surface area (30 vs. 17 m 2 g -1 Cu ). After incorporation of Al, [42] 76% yield towards 1,2propanediol was obtained employing Cu-ZnO-Al 2 O 3 with high catalyst (72 m 2 g -1 catalyst ) and copper (117 m 2 g -1 Cu ) surface area. We reported in a first study, the synthesis, by sol-gel method, of ZnO support with high surface area (up to 120 m 2 g -1 ). After addition of Cu nanoparticles, these catalysts showed much higher conversion than when employing commercial ZnO (70% vs. 25%). There is only one report in the literature of the hydrogenolysis of glycerol over Cu-Zn-Al systems with high surface area synthesized by solgel method (175 m 2 g -1 ). [35] High yield towards propylene glycol (≈ 90%) was obtained in a continuous-flow reactor for ca. 5 h, then the catalyst strongly deactivated due to coke formation along with Cu sintering. The catalyst was prepared following a multi-steps method with aluminium isopropoxide, copper nitrate, zinc nitrate, ethylene glycol, nitric acid and ammonia.

The preparation of supported metal catalysts by sol-gel processing provides solid with distinct catalytic properties. [43] Sol-gel synthesis generates materials with high surface area and homogeneous distribution of active species. Preparations can be made in one step and additional elements can easily be added. Finally, catalysts made by sol-gel method have been shown to exhibit improved tolerance to thermal treatment [44] and metal sintering [45] for other reactions of interest.

In this work, we developed a one pot sol-gel method with trimethylaluminium, diethylzinc, copper nitrate and polyacrylic acid for the synthesis of Cu-Zn-Al catalysts. We then investigated the catalytic action of these systems in the hydrogenolysis of glycerol directed at the selective formation of 1,2-propanediol. Finally, the impact of sol-gel method on the physico-chemical properties and the active sites of these systems were examined.

Results and discussion

Synthesis and characterization of the catalysts

The syntheses of the different catalysts differed only i) in the composition of the medium and ii) the method of synthesis of the Cu/ZnAl x O y solid, starting from hydrolysis of zinc and aluminium alkyl metal precursors. In one case, deposition-precipitation of Cu nitrate with urea were conducted after sol-gel synthesis and isolation of the ZnAl x O y support (two steps synthesis -S2) and in the other case the synthesis of the support and deposition-precipitation of Cu nitrate were carried out in the same medium (one step synthesis -S1), following Figure 1.

Figure 1. Schematization of the two synthetic routes studied

Synthesis and characterization of the catalysts synthesized by one step method

The aim of this study was to investigate whether a one-step route (simultaneous presence of PAAH and Cu 2+ during the elaboration of ZnAl x O y ) could result in materials with stable Al/Zn ratio, high copper dispersion, high specific surface area and efficient catalytic properties. The physico-chemical characteristics of these catalysts will be assessed and correlated to the performances in the hydrogenolysis of glycerol. A series of catalysts were synthesized by one step sol-gel method. Different contents of Cu (4-25 wt%), Zn (15-63 wt%) and Al (0-23 wt%) (Table S1, entries 1-6), and Cu/Zn and Al/Zn ratios in the range of 0.08-1.18 and 0.47-3.68 (Table 1), respectively, were studied in order to evaluate the optimal composition. Interestingly, all experimental Cu values were close to the nominal values (5, 10, 20 or 25 wt%) (entries 1-6, Table 1). The low carbon (< 0.4 wt%) and nitrogen (< 0.05 wt%) contents of the catalysts confirmed that the calcination step at 400 °C was sufficient to remove the polyacrylic acid present during the syntheses, in agreement with our previously reported results on ZnO and Cu/ZnO synthesized by sol-gel method. [46] As a representative example, the XRD diffractograms associated with the synthesis process of 25Cu-16Al-S1 after hydrolysis, calcination and reduction are presented in Figure 2. After hydrolysis, the structure is mainly amorphous. The peaks at 36.4°, 42.3° and 61.3° can be attributed to cubic Cu 2 O (, Pn-3m, PDF 00-005-0667) with a mean crystallite size of 34 nm. After calcination at 400 °C, the material presents mainly a crystalline monoclinic CuO structure (, C2/c, PDF 00-045-0937) with the main peaks at 35.3° and 38.4°. The peaks at 31.8° and 36.3° are associated with hexagonal ZnO (, P63mc, PDF 01-079-0205). After reduction at 350 °C, CuO is reduced to cubic Cu (, Fm-3m, PDF 04-13-9963), with peaks at 43.3°, 50.4 and 74.1°. ZnO is still present, but some crystalline cubic ZnAl 2 O 4 (, Fd-3m, PDF 04-014-1594) can also be observed, with the main contribution at 31.2° and 36.8°.

Figure 2. XRD diffractograms associated with 25Cu-16Al-S1 i) after hydrolysis, ii) after calcination at 400 °C and iii) reduction at 350 °C and passivation. Symbols refer to peaks

associated with () Cu 2 O (Pn-3m, PDF 00-005-0667), () ZnO (P63mc, PDF 01-079-0205), () CuO structure (C2/c, PDF 00-045-0937), () ZnAl 2 O 4 (Fd-3m, PDF 04-014-1594) and () Cu (Fm-3m, PDF 04-13-9963).

The XRD diffractograms associated with the catalysts synthesized in one step (after reduction and passivation) are included in Figure 3. The Cu mean crystallite sizes of Cu-Zn x Al y O are in the range of 4-13 nm (Table S1), for all the catalysts synthesized by sol-gel method with polyacrylic acid. The peaks associated with CuO are not detected, which suggest full reduction of the copper phase. The presence of Cu 3 Zn has previously been reported for Cu/ZnO [47] however we did not observe it for any of the catalysts. The lattice parameters associated with Cu were in the range of 3.614-3.625 Å, which is consistent with values for pure copper metallic phase reported in database PDF4+ (a = 3.607-3.625 Å). The crystallite size increases from 4 to 13 nm when the Cu content increases from ca. 4 to 25 wt%. The presence of Al has a strong impact on the crystallinity of the samples. Indeed, the diffractogram associated with Cu/ZnO (21Cu-S1) exhibits sharp ZnO peaks, leading to d ZnO around 20 nm, associated with large crystallites of Cu (d Cu = 16 nm, Table S1). While in presence of Al, the crystallite size of ZnO decreases (from 5 to 2 nm) when Al content ranges from 10 to 24 % and Al/Zn ratio increases from ca. 0.5 to 3.7 (Figure 4, Table S1). The peaks associated with ZnAl 2 O 4 are small which suggest that the phase is predominantly amorphous. The formation of CuAl 2 O 4 could not be ruled out as this phase exhibit a diffraction pattern similar to ZnAl 2 O 4 , however this phase is usually unstable below 600 °C. [48] Amorphous Al 2 O 3 might also be present at high Al/Zn ratio. However, after calcination of a sample at 600 °C, Al 2 O 3 was still not detectable and only crystalline ZnAl 2 O 4 was observed in higher quantity (XRD not shown). When the synthesis was conducted without polyacrylic acid (11Cu-11Al-S1-P, Figure 3), large crystallites of Cu (d Cu = 36 nm) and ZnO (d ZnO = 13 nm) were formed which demonstrates the beneficial effect of polyacrylic acid during the sol-gel method. The catalyst without Al (21Cu-S1) exhibit low surface area (ca. 24 m 2 g -1 , Table 1). This result agrees with our previous study on ZnO synthesized by sol-gel method. [46] Indeed, despite high surface area after synthesis (up to 91 m 2 g -1 ), the removal of polyacrylic acid after thermal treatment at 400 °C generated a collapse of the mesosphere associated with a low surface area. However, after incorporation of aluminum, surface areas up to 265 m 2 g -1 were obtained despite the thermal treatment (Table 1). This value is higher than those previously reported in the literature for Cu-Zn-Al synthesized by co-precipitation (29-187 m 2 g -1 ), [34][35][36]38] evaporation-induced self-assembly (79-225 m 2 g -1 ), [41] oxalate gel co-precipitation (72-113 m 2 g -1 ), [49,50] or sol-gel method (175 m 2 g -1 ), [35] tested for the hydrogenolysis of glycerol.

The morphology of the samples was assessed by SEM. Pictures associated with the representative materials 11Cu-11Al-S1 and 18Cu-24Al-S1 are shown in Figure 5. The later sample, which displays a larger specific surface, is accompanied by a rougher structure (Figure 5b). The stoichiometries of the samples determined by EDX were in good agreement with the ICP analyses and confirmed a good homogeneity of the samples (Table S2). In order to assess the metal-oxide interaction, TGA analysis under 3% v/v H 2 /Ar flow coupled to MS analysis were conducted on the materials after calcination. The profile associated with 21Cu-S1 is presented in Figure 6. The mass spectrum of the evolved gas was recorded and the only signals observed were at m/z = 17 and 18, which correspond to the formation of water caused by reduction of CuO to Cu. The peak at 219 °C observed with the MS is consistent with the mass loss and the endothermic signal of the heat flow. The temperature of reduction of the materials are included in Table 1, and the TGA profiles are presented in Figure S1. The temperatures of reductions (230-250 °C) are consistent with the values reported in the literature for Cu-Zn-Al systems (250-285 °C). [35,41,42] For these systems, the temperature of reduction depends mainly on the Cu content, the Cu crystallite size and the metal-oxide interaction which is linked to the method of preparation, the structure and morphology of the oxides, and the Al/Zn content. [35,41,48,50] It seems that at similar Al content (11Al-S1; entries 1-3 Table 1), the temperature of reduction increases with Cu content and particle size. The lower temperature of reduction for 21Cu-S1 suggest that when the nanoparticles of copper are dispersed on ZnO, the interaction between Cu and the support is weak. In presence of Al, Cu particles might be dispersed on the amorphous ZnAl 2 O 4 phase and the interaction is stronger.

The specific Cu surface area of the catalysts were determined based on N 2 O chemisorption. After reduction in situ at 350 °C under H 2 , the analyses were conducted at 90 °C. The gas flow is switched to N 2 O/Ar/He and the MS signals of He, Ar, N 2 O and N 2 are recorded. A representative example of the experiment is presented in Figure S2. N 2 O is consumed and N 2 is formed due to the oxidation of Cu atoms at the surface of nanoparticles. The slight increase in temperature, from 90.0 °C to 90.6 °C, prove that this process is exothermic. No N 2 O was chemisorbed during a second passage of N 2 O/Ar/He which confirms the complete oxidation during the first run. The specific Cu surface area (Cu exp , m 2 Cu g -1 Cu , Table S1) and the concentration of accessible Cu sites (Cu s , µmol g -1 , Table 1) of the materials were then determined based on N 2 O chemisorbed, with a stoichiometry of Cu s :O estimated to be 2:1. [51] Mean particles sizes can be calculated from N 2 O chemisorption (d Cu , N 2 O) and are reported in Table S1. For all the catalysts with Al, the particles sizes are consistent with the crystallite sizes obtained by XRD (Table S1). However, for the catalyst without Al, there is some noticeable difference. This suggest that there is agglomeration of the Cu crystallites, hence a decrease in metal dispersion (due to the low BET surface area) along with a drop in active sites (i.e. concentration of accessible Cu sites). FTIR analysis using pyridine as an established probe molecule was employed to investigate the surface acidity. The interaction of the nitrogen lone pair of the pyridine with Lewis acid sites on alumina and aluminates is associated with  8a and  19b bands whom the positions are in the range 1580-1625 cm -1 and 1445-1455 cm -1 , respectively, where a higher frequency reflects stronger acidity. [52,53] The IR spectra of 18Cu-24Al-S1, 11Cu-11Al-S1 and 21Cu-S1 following outgas at 150 °C and their Lewis acid site concentrations are presented in Figure 7. The IR spectra following outgas at 200, 250, and 300 °C are shown in Figure S3. According to Figure 7a, the three catalysts exhibit two principal peaks at 1613 (± 2) cm -1 and 1450 (± 3) cm -1 . After desorption at higher temperature (Figure S3), a higher frequency band at 1620 (± 2) cm -1 becomes predominant. The position of the first one is associated with relatively weak Lewis acidity associated to pyridine coordinated mainly to Zn 2+ in zinc aluminate structure. [54] However, Pyridine coordination to Zn 2+ in ZnO or Al 3+ sites cannot be ruled out. Indeed, in a first hand, pyridine adsorption on ZnO induce a band at 1605-1610 cm - 1 which fully disappears after desorption at 130 °C. [55,56] In addition, the sample Cu/ZnO ( 21Cu-S1) present a broad band at 1615 cm -1 . In an other hand, in our desorption condition, pyridine on coordinatively unsaturated Al 3+ in octahedral sites should give a band at 1614-1617 cm -1 [57][58][59] i.e. close to the position observed after evacuation at temperature higher than 150 °C. In the specific case of 18Cu-24Al-S1, Figure 7a evidenced a band at 1622 cm -1 that can be attributed to pyridine adsorbed on strong Lewis acid sites (i.e. coordinatively unsaturated Al 3+ in tetrahedral sites). Finally, Figure 7a does not evidence the presence of Brønsted acid sites (characterized by a band at ca. 1540 cm -1 ) which is consistent with previous reports on ZnAl 2 O 4 . [54,60] Quantification of Lewis acid sites (Figure 7b) shows an increase of the total acid site concentration in the order 18Cu-24Al-S1 > 11Cu-11Al-S1 > 21Cu-S1. Therefore, the number of acid sites on the catalysts is directly linked to the Al content and formation of ZnAl 2 O 4 . While the acidity of Cu-ZnO [12,33,36] and Cu-Zn-Al [34] catalysts have been assessed by temperature-programmed desorption of ammonia, we could not find any other report in the literature dealing with the hydrogenolysis of glycerol that provides measure of acidity of Cu-Zn-Al catalysts by FTIR analysis with pyridine.

Synthesis and characterization of the catalysts synthesized by two steps method

For comparison, two supports with two different Al contents were synthesized by sol-gel method in two steps. The Al and Zn contents of the supports were consistent with the theoretical loadings and Al/Zn ratios of 0.5 and 2.5 were obtained (Table 2 and Table S1). For low Al content, 11Al support exhibits a ZnO crystalline phase (Figure 8-a1), associated with a relatively low surface area (71 m 2 g -1 , Table 2). As the support did not undergo any thermal treatment after synthesis, an amorphous zinc aluminate phase might also be present. At higher Al content, 22Al is amorphous (Figure 8-b1) and the material displays a much higher surface area (223 m 2 g -1 , Table 2). The copper was then added by depositionprecipitation with urea, followed by calcination, reduction and passivation. The XRD diffractograms (Figure 8) showed that the catalyst 26Cu/11Al-S2 exhibit relatively dispersed Cu particles (d Cu = 9 nm, Table S1) on a support composed of crystalline ZnO. The zinc aluminate must be principally amorphous, as the peaks associated with crystalline ZnAl 2 O 4 () are small (Figure 8-a2). The metal contents of the catalysts were measured by ICP and compared to the ones of the support after calcination at 400 °C, i.e. after similar heat treatment. The results included in Table S1 show a major difference between 11Al and 26Cu/11Al-S2, since the final Zn content was lower than the expected one (37% vs. 48%). This suggest that ZnO is not really stable in the conditions employed for the depositionprecipitation (water, pH ≈ 8, 60 °C) and dissolved during the synthesis. This phenomenon was confirmed by ICP analysis of the solution after filtration which showed the presence of Zn 2+ . This demonstrates the beneficial effect of the one pot sol-gel method in presence of polyacrylic acid, for the catalysts with low Al contents. This problem does not occur for 16Cu/22Al-S2 which exhibits the expected metal loading (Zn, Al, Cu). This could be attributed to the sole presence of amorphous ZnAl 2 O 4 which is more stable in aqueous phase. After thermal treatment, the catalyst 16Cu/22Al-S2 exhibits a crystalline ZnAl 2 O 4 phase. The surface area, T reduction and Cu s (Table 2) are in the range of the values obtained for Cu/ZnAl x O y -S1 synthesized in one pot. However, the particle size obtained by N 2 O chemisorption are slightly higher than those obtained by XRD, suggesting a lower dispersion of the Cu nanoparticles. It is worth noting that compared to syntheses without Al which gave catalysts with specific surface areas of 20-30 m² g -1 , [46] the addition of aluminium significantly increased those of the support and the final catalyst. Representative SEM images of the catalysts are shown in Figure S4. The catalyst 16Cu/22Al-S2 exhibits platelet-like structure, while 26Cu/11Al-S2 presents agglomerates of flake-like particles. The hydrogenolysis of glycerol was studied over all the catalysts in a batch reactor at 200 °C, under 30 bar of H 2 . The temporal evolution of the conversion of glycerol observed over 24 h, with the catalysts synthesized by S1 method are included in Figure 9. Those associated with the other catalysts are shown in Figure S5. Glycerol was converted to 1,2-propanediol as major product and ethylene glycol as secondary product (Scheme S1). 1,3-propanediol was not detected, suggesting a higher reactivity of the primary than secondary hydroxyl group, which might be due to less steric hindrance. [61] The further hydrogenolysis of the diols to monoalcohols (i.e. propanol, ethanol) did not occur. The conversion and the selectivity to 1,2propanediol (S PDO ) after 24 h are included in Table 3. For all the reactions, the carbon balance was always above 95%. The selectivity towards 1,2-propanediol is high (≥ 95%) at low conversion (≤ 20%) and decreases slowly (down to ≈ 80%) with increasing conversion, in favor of ethylene glycol. The selectivity is directly correlated to the conversion (Figure 10) and does not depend on the catalysts or time.

Hydrogenolysis of glycerol over Cu/ZnAl x O y

Figure 10. Selectivity towards 1,2-propanediol () and ethylene glycol () as function of glycerol conversion, obtained over all the catalysts (after 24 h). Aqueous solution of glycerol (100 mL of 0.23 M glycerol in water; 500 mg of catalyst, 200 °C and 30 bar of H 2 ).

It is well accepted that on Cu catalysts (Cu/ZnO [11] , Cu/ZrO 2 [23,24] , Cu/Al 2 O 3 , Cu/AlO(OH) [62] , Cu/ZnO/Al 2 O 3 [63] ) the dehydration of glycerol on acid sites can generate the acetol which is then hydrogenated on the metallic sites to 1,2-propanediol. This pathway has been shown to be favored in presence of Lewis acid sites originating mainly from the supports, [14,38,62] but also from metal sites [64,65] . On the contrary, Brønsted acid sites favor the formation of 1,3-propanediol and lower alcohols. [64,66,67] It is well known that the presence of water can partially hydroxylate the surface. [68] The formation of OH groups poison the Lewis acid sites and inhibit dehydration reactions. [69] Moreover it has also been reported on Al 2 O 3 that water can promote the formation of (Al,O) Lewis acid-base pair; thus, increasing the basicity of neighboring oxygens can enhance the catalytic activity. [70,71] However, we did not observe any 1,3-propanediol which suggests that this phenomenon hardly occurs. The formation of ethylene glycol is believed to occur through dehydrogenation of glycerol to glyceraldehyde, followed by direct decarbonylation. [72] The formation of ethylene glycol through retro-aldol condensation of glyceraldehyde followed by hydrogenation has also been reported, [72] however it would be associated with the formation of methanol, which was not observed under our conditions.

The performances of the catalysts may be documented by different values: The amount of glycerol converted in 24 h, per mol of Cu (Cu efficiency, n gly /n Cu ) or per mass of catalyst (productivity, n gly /m catalyst , mmol gly g -1 ) and the initial rates (V 0, mmol gly g Cu -1 h -1 ) are included in Table S3. The dehydration of glycerol is favored on Lewis acid sites and generates the acetol, as a first step. We did not observe any acetol intermediate during the reaction when working under H 2 which suggest that the dehydration is not the limiting step; one reaction was conducted with Cu-ZnO under Ar in order to confirm the formation of acetol as intermediate. Moreover, there is no clear correlation between the productivity and the Al content, i.e. Lewis acidity, suggesting partial blockage of Lewis acid sites due to the water. The results obtained over the catalysts synthesized by S1 show that the conversion increases, from 21% to 96%, when increasing the Cu loading, from 4 to 25% (Table 3, entry 1 to 5). This suggest that the activity is correlated to the metallic sites, which favor the hydrogenation step. Despite the increase in conversion with the Cu loading, the Cu efficiency varied between 5 and 20 mol gly mol Cu -1 and the initial rates ranged from 4 to 28 mmol gly g Cu -1 h -1 , i.e. there is no clear trend associated with Cu efficiency and initial rates (Table S3). For example, without Al, the catalyst 21Cu-S1 (entry 6) is far less active than 18Cu-24Al-S1 with similar Cu loading (entry 4). This suggest that the Cu loading is not the only parameter contributing to the activity. The conversion observed for 11Cu-11Al-S1-P (entry 7) is much lower (45%) than for 11Cu-11Al-S1 (entry 3, 74%), which proves the crucial role of polyacrylic acid during the synthesis.

We then focused on the productivity of the catalyst (n gly /m catalyst ) in function of the concentration of accessible Cu sites (Cu s ) where a clear correlation is observed (Figure 11). This is consistent with previous literature on the hydrogenolysis of glycerol to 1,2propanediol over Cu/ZnO [9,37] , Cu/SiO 2 [18,19] and Cu/ZrO 2 [23] . The concentration of active sites is correlated to the Cu loading and particle size. This also explains the lower conversion observed with 21Cu-S1 as the main particle size obtained by N 2 O chemisorption is large (46 nm). The beneficial effect of Al seems to be associated with the larger surface area of the catalysts and the stronger metal-oxide interaction, which favor a better dispersion of the copper, i.e. an increase of the concentration of accessible metallic sites. Similarly, in the absence of polyacrylic acid during the synthesis, large particles of Cu were obtained, which generated a low conversion. One catalyst does not fit perfectly the trend, namely 4Cu-11Al-S1. Indeed, while its initial rate is quite high, it seems to deactivate quickly, so the productivity of the catalyst (n gly /m catalyst ) is low. This might be due to the weak metal-oxide interaction observed with this catalyst (i.e. low temperature of reduction, Table 1) and small crystallite size which favor sintering and problem of stability during the reaction. It is interesting to note that the two catalysts synthesized in two steps fits perfectly the trend (Figure 11). 26Cu/11Al-S2 exhibits similar Cu efficiency and productivity as 25Cu-16AlS1. To conclude, the activity of the catalysts is directly linked to the concentration of accessible Cu active sites. Moreover, the presence of polyacrylic and Al is crucial for the formation of ZnAl 2 O 4 along with small Cu particles, which favor the conversion of glycerol to 1,2-propanediol.

Our productivity results obtained are competitive compared to the best literature values reported for non-noble metal catalysts. The difficulty in the interpretation of these data lies in the variation of the parameters, such as pressure, temperature and the lack of quantification of active sites for some studies. However it is worth noting that the literature focused on the hydrogenolysis of aqueous glycerol over Cu supported on oxides report conversion below 80%, when working under similar reaction conditions (T = 180-200 °C, P H2 = 20-80 bar, batch reactor). [73] Moreover, our best catalyst generated yields up to 80 % of 1-2-propanediol, which is similar to the highest values reported for other Cu-Zn-Al systems (76-85 %). [41,42,50]

Stability and recyclability of the catalysts

The stability of catalyst is a crucial criterion for industrial application. For the hydrogenolysis of glycerol in liquid phase, it has been shown that deactivation of non-noble catalysts usually occurs and a number of factors have been proposed. For Cu catalysts, the deactivation has been principally attributed to metal sintering. [12,19,23,30,33,38,41,42] This phenomenon is usually linked to a change in morphology of the solid from spherical particles to lamellar or rod-like structure, associated with an increase in crystallite size and a decrease in surface area and pore volumes of the material. [12,19,33,34] It has also been shown that the use of water as solvent emphasis particles agglomeration. [33,42] The catalysts exhibiting the highest Cu efficiency (11Cu-11Al-S1) and productivity (25Cu-16Al-S1, 26Cu/11Al-S2) were characterized after reaction and compared to 21Cu-S1. Figure S6 presents the XRD diffractograms associated with these catalysts after 24 h reaction. In agreement with the literature, all the catalysts exhibit larger Cu crystallite size (Table S4). Moreover, the three catalysts present the formation a new phase corresponding to monoclinic Zn 5 (CO 3 ) 2 (OH) 6 , with a characteristic peak at 12.9°. We previously demonstrated for Cu/ZnO [46] that this carbonic phase appeared only at high conversion (> 60 %) and was associated with the formation of products resulting from C-C bond cleavage. This phase was not observed for 21Cu-S1 due to the lower conversion (40 %). SEM images of 11Cu-11Al-S1 (Figure 12) shows a change in morphology with the formation of a lamellar structure due to the formation of the hydrozincite phase. It is worth noting that this change of structure of the solids is also associated with a drastic increase in surface area after reaction for Cu/Zn x Al y O-S1 (from 80-100 m 2 g -1 to ca. 145 m 2 g -1 ) while it remained stable for Cu/Zn x Al y O-S2 (ca. 110 m 2 g -1 ) and Cu/ZnO (ca. 25 m 2 g -1 ; Table S4). The solutions after 24 h reaction were analyzed by ICP in order to assess any metal leaching. In line with the literature, [19,34,38] no Cu or Al were lost during the hydrogenolysis of glycerol, however small quantities of Zn were found (10-15 % wt.; Table S4). The stability of ZnO under hydrothermal conditions is limited, so it might solubilize and reprecipitate, which might favor the formation of carbonates species such as Zn 5 (CO 3 ) 2 (OH) 6 . The recyclability of 25Cu-16Al-S1 was assessed over 4 consecutive runs. Test 2 was conducted without regeneration, i.e. the catalyst was reused directly after filtration and drying at 80 °C. Unfortunately, the catalyst was far less active (Figure 13). This result suggests that the presence of Zn 5 (CO 3 ) 2 (OH) 6 must be detrimental for the activity. It could also be due to the formation of copper oxide during the recovery of the catalyst. In the literature, [41,42] the stability of Cu-Zn-Al systems are usually assessed after regeneration. Therefore, the catalyst was then reduced under H 2 before test 3 and before test 4. It is interesting to note that the surface area and the main Cu crystallite size remained stable from the second run (Table 4) and no Zn was lost (< 1 % wt.). The conversion of glycerol and selectivity to 1,2-propanediol remained stable after the 4° runs (Figure 13).

Conclusion

In this study, we have elaborated Cu-Zn-Al systems based on sol-gel synthesis in presence of polyacrylic acid. This simple method can be conducted in one step. Catalysts with really high surface area, up to 265 m 2 g -1 , were obtained. The presence of aluminum and polyacrylic acid during syntheses favored the formation of small and well dispersed Cu nanoparticles, with a mean size in the range of 4 to 13 nm. The acidity of the catalysts was assessed by FTIR analysis using pyridine. The catalysts exhibit Lewis acidity where the number of acid sites is correlated to the Al content. The hydrogenolysis of glycerol was studied in a batch reactor at 200 °C, under 30 bar of H 2 . Glycerol was converted to 1,2-propanediol as major product and ethylene glycol as secondary product. Conversions of glycerol up to 96% were observed, with selectivity towards 1,2-propanediol around 80-90%. We demonstrated that the productivity of the catalyst was directly linked to the number of Cu active sites. Our results showed that the activity is correlated to the metallic sites, which favor the hydrogenation step and that the catalysts can be recycled several times after reactivation under H 2 .

Experimental Section

Reagents

ZnEt 2 (15%wt in toluene), [Al(CH 3 ) 3 ] 2 (97%), Cu(NO 3 ) 2 .3H 2 O (99.9%), glycerol (99.5%), urea (99%), ethanol (99.5%) and aqueous solutions of polyacrylic acid (50 wt%) of molecular weight of 2000 g mol -1 were purchased from Merck and used as received.

Catalysts preparation

Two steps synthesis of Cu/ZnAl x O y

Synthesis of ZnAl x O y by Sol-Gel process (XAl)

200 mL of an aqueous solution of polyacrylic acid (PAAH, molecular weight of 2000 g mol -1 ) at 0.63 wt% was placed in a Schlenk flask of 500 mL. The solution was stirred at 1000 rpm under Ar. The flask was immerged in an ice bath for 30 min in order to reach the 0 °C desired temperature. In another Schlenk flask of 50 mL, between 3 and 12 mL of [Al(CH 3 ) 3 ] 2 (in regards to x) was added to 14.4 mL of ZnEt 2 solution in toluene under argon. The mixture of [Al(CH 3 ) 3 ] 2 and ZnEt 2 was then transferred slowly (under argon) to the aqueous solution of polyacrylic acid. Immediately after the transfer, ethane and methane gases were generated and evacuated by the bubbler connected to the 500 mL flask and a white precipitate appeared. After 1 h of stirring, the suspension was centrifuged at 8000 rpm for 30 min. 90 mL of ethanol was added to the wet powder obtained after centrifugation and the mixture was kept under stirring at room temperature for 30 min. Once again, the suspension was centrifuged at 8000 rpm for 30 min. The resulting white powder was dried in an oven at 80 °C for 20 h. The samples will be labelled XAl, with X = Al content (wt%).

Synthesis of Cu/ZnAl x O y by deposition-precipitation process with urea (ACu/XAl-S2)

1.8 g of the support (XAl) was suspended in 150 mL of distilled water, in a 500 mL flask and 4 g of urea (66 mmol) and the appropriate volume of an aqueous copper nitrate solution [Cu(NO 3 ) 2 •3H 2 O, 0.157 M] were added to the support. The appropriate amount of distilled water was added to obtain a final Cu 2+ concentration of 20.8 mM. The suspension was heated at 1 °C min -1 to 60 °C and hold for 3 h. The resulting solids were filtered and washed with 100 mL of distilled water. After drying at 100 °C for 20 h, the solids were calcined at 400 °C (5 °C min -1 ; hold for 4 h) in a furnace. They were then reduced in a cell under 50 mL min -1 of H 2 at 3 °C min -1 up to 350 °C and hold for 1 h, and finally passivated for 30 min under 50 mL min -1 of 1% v/v O 2 /N 2 at room temperature. The samples will be labelled ACu/XAl-S2, with A = Cu content (wt%), X = Al content (wt%), and S2 referring to the two steps synthesis.

One step Sol-Gel synthesis of Cu/ZnAl x O y (ACu-XAl-S1)

The same procedure as above (Synthesis of ZnAl x O y by Sol-Gel process) was followed except that 4 g of urea (66 mmol) and the appropriate volume of an aqueous copper nitrate solution [Cu(NO 3 ) 2 •3H 2 O, 0.157 M] were added from the start in the aqueous solution of polyacrylic acid. After stirring at 0 °C for 1 h, the solution was heated at 1 °C min -1 to 60 °C and hold for 3 h. The resulting precipitates were filtered and washed with 100 mL of distilled water. After drying at 100 °C for 20 h, the solids were calcined at 400 °C (5 °C min -1 ; hold for 4 h) in a furnace. They were then reduced in a cell under 50 mL min -1 of H 2 at 3 °C min -1 up to 350 °C and hold for 1 h, and finally passivated for 30 min under 50 mL min -1 of 1% v/v O 2 /N 2 at room temperature. The samples will be labelled ACu-XAl-S1, with A = Cu content (wt%), X = Al content (wt%) and S1 referring to the one step synthesis. For comparison, one sample was synthesized without polyacrylic acid, and labelled ACu-XAl-S1-P.

Characterization of catalysts

Powder X-ray diffraction patterns (XRD) of the samples were recorded using a Bruker D8 Advance Diffractometer equipped with a nickel filter, a copper tube (λKα (Cu) = 1.54184 Å) and a multi-channel fast detector (LynxEye 192 channels an active length of 2.947 °). The samples were scanned at 0.04 ° s -1 over the range 20 ≤ 2θ ≤ 80°. Phase identification was achieved using HighScore Plus and the JCPDS-ICDD-PDF4+ database. Mean crystallite sizes (± 0.5 nm) and lattice parameters (± 0.005 Å) were obtained by performing Rietveld refinements using Topas 5.

The Cu, Zn and Al contents (wt%) were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES) by using an ACTIVA instrument (HORIBA Jobin Yvon). Before analysis, the solid samples were mineralized in an acid mixture and heated at 250-300 °C. Elemental analysis of light elements (CHN) was performed on an analyzer Thermo Fisher Flash 2000. The values are reported with an absolute accuracy of ± 0.1 %.

The surface areas of the samples (± 5 m 2 g -1 ) were determined with the BET method from N 2 physisorption at -196 °C using an ASAP 2020 Micromeritics apparatus. Before the measurements, the materials were outgassed at 150 °C for 2 h under vacuum (10 -4 mbar). The specific surface area was calculated from the obtained isotherms for P/P° between 0.05 to 0.25. The values are reported with an absolute accuracy of ± 5 m 2 g -1 .

The thermogravimetric analyses (TGA) were performed on a SETARAM LABSYS instrument in the 25-650 °C temperature range, on ca. 20 mg of sample, with a heating rate of 5 °C min -1 , under air flow (50 mL min -1 ). This technique was used to determine the optimal temperature of calcination. Simultaneous thermogravimetric and differential thermal analyses (TG-DTA) of the samples were conducted on a SETARAM SETSYS Evolution 12 thermoanalyzer coupled via a heated (ca. 150 °C) capillary with a Pfeiffer OmniStar quadrupole mass spectrometer (MS). For TG-DTA-MS studies, ca. 20 mg of samples were heated from room temperature to 750 °C at a constant rate of 5 °C min -1 under 3% v/v H 2 /Ar (50 mL min - 1 ).

In order to reveal morphology of the materials, SEM images were obtained using ZEISS MERLIN COMPACT VP microscope with low-vacuum detector (SE) and Oxford 50 mm 2 EDX detector (12 analyses were conducted for each sample).

The specific Cu surface area (Cu exp , ± 2 m 2 Cu g -1 Cu ), the mean particles sizes (d Cu, N2O , ± 0.5 nm) and the concentration of accessible Cu sites (Cu s , ± 3 μmol Cu g -1 ) of the materials were determined based on N 2 O chemisorption. Quantitative N 2 O adsorption measurements were made with a homemade analytical setup allowing to follow the change in the composition of the gas mixture at the outlet of a quartz micro reactor ( 1.5 mL) containing the pretreated catalyst (weight range of 0.2-0.3 g, deposited on quartz wool) by using a quadrupole mass spectrometer (Inficon, Transpector CPM). The temperature was recorded via a stainless-steel K type thermocoax (= 0.25 mm) inserted in the sample. The catalyst was first activated in situ under H 2 (100 mL min -1 ) at 350 °C and cooled down to 90 °C. The gas flow was then switched to He (100 mL min -1 ) for 10 min. The adsorption/desorption of N 2 O at T = 90 °C on the copper containing catalyst was studied according to the following switches: He (100 mL min -1 )  2% N 2 O/2% Ar/He (100 mL min -1 ) providing the total amount of adsorbed N 2 O species and N 2 production. The flow (100 mL min -1 ) was switched to He, then 2% N 2 O/2% Ar/He, then back to He in order to verify the absence of weakly adsorbed species and attest of the saturation of the sites. Previous to analyses, gas mixtures of known compositions were used for the calibration of the MS and the ascertainment of the linearity of the response of the gases used in the present study: N 2 O, N 2 and Ar. N 2 O chemisorbed (n N2Ochem , mol) correspond to the amount of N 2 O consumed which is calculated based on the molar fraction of N 2 O at the inlet and outlet of the reactor. The molar flow rate of Ar serves as reference.

The concentration of accessible Cu sites (Cu s , expressed in mol Cu g -1 ) were estimated based on N 2 O chemisorbed, with a stoichiometry of Cu s :O estimated to be 2:1 using the following equation: [51] [eq. ( 1)] The specific Cu surface area (Cu exp , expressed in m 2 Cu g -1 Cu ) of the materials were then determined based on N 2 O chemisorbed, the mass of Cu in the sample (m Cu , g), N A the Avogadro's number, and the copper surface density (ρ s = 1.46  10 19 atoms m 2 ) according to: [START_REF] Bergeret | Particle Size Dispersion Measurements in Handbook of Heterogeneous Catalysis[END_REF] [eq. ( 2)] The mean particles sizes (d Cu, N2O , expressed in nm) were calculated based on eq. ( 3) assuming spherical particles: [eq. ( 3)] with ρ Cu the Cu metal density (8.94 g cm -3 ) and Cu exp expressed in m 2 Cu g -1 Cu .

Analysis of the acidic properties of Cu/ZnAl x O y samples was performed by adsorption of pyridine follow by infrared spectroscopy. Before analysis, samples were pressed at ~1 ton cm - 2 into thin wafers of 5 to 8 mg cm -2 and placed inside the IR cell. Before pyridine adsorption/desorption experiments, the wafers were treated under H 2 (5%)/Ar flow (30 mL min -1 ) at 350°C (ramp: 5 °C min -1 ) for 1 h and then outgassed under secondary vacuum at 350°C for 1 h. Wafers were then contacted at 150°C with gaseous pyridine (approximately 133 Pa) via a separate cell containing liquid pyridine. The spectra were then recorded following desorption from 150 to 300 °C with a Bruker Vector 22 spectrometer (resolution 4 cm -1 , 64 scans). The reported spectra were obtained after subtraction of the spectrum recorded before pyridine adsorption and normalization at 10 mg cm -2 . The amount of Lewis acidic centers titrated by pyridine was obtained using a molar absorption coefficient value of ε = 1.71 cm µmol -1 for the ν 19b vibration of coordinated pyridine (Py-L) at ~1450 cm -1 . [START_REF] Zholobenko | [END_REF]

Catalytic tests

The glycerol hydrogenolysis was performed using a 300 mL reactor autoclave. Typically, 100 mL of glycerol aqueous solution (2.14 wt%) and 0.5 g of catalyst were loaded into the reactor. After sealing, the autoclave was purged three times with Ar. Then the heating was turned on to reach 200 °C, and the stirring was started. When the temperature was stable, 30 bar of hydrogen was added into the reactor (total pressure of the reactor = 46 bar).

The operating conditions were chosen to ensure negligible mass transport limitation. The stirring was set at 1000 rpm in order to avoid interphase diffusion limitation. Isothermal conditions (± 1 °C) were ensured by working in diluted solution. The H 2 content in a aqueous solution under our reaction conditions is around 0.067 mol L -1 based on Henry's law constant [76] , hence the initial molar fraction X H2 / glycerol is ca. 30%. H 2 was periodically added during the reaction in order to maintain the pressure constant and an excess of H 2 . Liquid samples were collected periodically during the reaction (up to 24 h) and analyzed by gas chromatography (Shimadzu GC-2010 with a AOC-20s Auto Sampler, column ZB-FFAP 30 m × 0.32 mm × 0.25 μm, N 2 as carrier gas, FID detector). The concentrations of the products were determined after calibration. At the end of the reaction, the autoclave was cooled down, the pressure was released, and the suspension was collected and filtered.

The conversion of glycerol as well as the selectivity and yield were calculated based on the concentration of the substrate at time 0 and t, in liquid products. The carbon selectivity to a desired product was based on the concentration of product and substrate at time t, and the number of carbon atoms. [46] Repeated reactions delivered conversion reproducible within ± 3%. The selectivity is given with an absolute accuracy of ± 3%. The initial reaction rate (V 0 , ± 0.3 mmol gly g -1 Cu h -1 ) was calculated based on the slope of the linear part of the curve (conversion = f(t)) at low conversion (< 40%).

Table of contents

Novel Sol-gel method for Cu / Zinc Aluminate as catalyst for glycerol hydrogenolysis. The presence of polyacrylic acid and aluminium during the synthesis improved the catalytic properties. Conversions up to 96% with selectivity towards 1,2-propanediol of 83% were obtained.

Figure 3 .

 3 Figure 3. XRD diffractograms associated with 4Cu-11Al-S1, 9Cu-11Al-S1, 11Cu-11Al-S1, 18Cu-24Al-S1, 25Cu-16Al-S1, 21Cu-S1 and 11Cu-11Al-S1-P. Symbols () refer to Cu (Fm-3m, PDF 04-13-9963). The other peaks are associated with ZnO and ZnAl 2 O 4 .

Figure 4 .

 4 Figure 4. Effect of Al content on the main crystallite size of ZnO, for samples synthesized by S1 method.

Figure 5 .

 5 Figure 5. Representative SEM images associated with (a) 11Cu-11Al-S1 and (b) 18Cu-24Al-S1.

Figure 6 .

 6 Figure 6. Evolution of mass (%), temperature (°C) and heat flow (a.u.) during TGA (5 °C min -1 ) under 3% v/v H 2 /Ar and associated H 2 O MS signal (m/z = 18) for 21Cu-S1.

Figure 7 .

 7 Figure 7. (a) FTIR spectra of pyridine adsorbed following outgas treatment at 150 °C and (b) total amount of Lewis sites after desorption at 150 °C, 200 °C, 250 °C, 300 °C, for 18Cu-24Al-S1, 11Cu-11Al-S1, and 21Cu-S1.

Figure 8 .

 8 Figure 8. XRD diffractograms associated with (a1) 11Al, (a2) 26Cu/11Al-S2, (b1) 22Al and (b2) 16Cu/22Al-S2. Symbols refer to peaks associated with () ZnO (P63mc, PDF 01-079-0205), () ZnAl 2 O 4 (Fd-3m, PDF 04-014-1594) and () Cu (Fm-3m, PDF 04-13-9963).

Figure 9 .

 9 Figure 9. Temporal evolution of the conversion of glycerol (%) for the catalysts synthesized by S1 method: () 25Cu-16Al-S1 ()18Cu-24Al-S1 () 11Cu-11Al-S1 () 9Cu-11Al-S1 ()21Cu-S1 () 4Cu-11Al-S1. Aqueous solution of glycerol (100 mL of 0.23 M glycerol in water; 500 mg of catalyst, 200 °C and 30 bar of H 2 ).

Figure 11 .

 11 Figure 11. Productivity of the catalysts (n gly / m catalyst ) in function of the total number of active sites, for the catalysts synthesized by S1 () and S2 () methods. Aqueous solution of glycerol (100 mL of 0.23 M glycerol in water; 500 mg of catalyst, 200 °C and 30 bar of H 2 ).

Figure 12 .

 12 Figure 12. Representative SEM image associated with 11Cu-11Al-S1 after reaction.

Figure 13 .

 13 Figure 13. Recyclability of 25Cu-16Al-S1. The catalyst was re-activated under H 2 at 350 °C before test 3 and test 4.

Table 1 .

 1 Cu/ZnAl x O y -S1 and Cu/ZnO-S1 synthesized by one step sol-gel method: main textural and structural properties

	Entry	Catalysts	n Cu /

n Zn a n Al /n Zn a Cu s (µmol Cu g -1 ) b

  Based on ICP analysis; b based on N 2 O chemisorption; c based on N 2 physisorption; d based on TGA analysis under H 2

						Surface area (m 2 g -1 ) c	T reduction (°C) d
	1	4Cu-11Al-S1	0.08	0.47	136	143	230
	2	9Cu-11Al-S1	0.18	0.49	196	154	246
	3	11Cu-11Al-S1	0.23	0.56	216	76	250
	4	18Cu-24Al-S1	1.18	3.68	328	265	249
	5	25Cu-16Al-S1	0.89	1.39	382	97	246
	6	21Cu-S1	0.32	-	72	24	219
	7	11Cu-11Al-S1-P	0.24	0.56	-	64	-

a

Table 2 .

 2 Main textural and structural properties of Cu/ZnAl x O y synthesized in two steps

Catalysts n Cu /n Zn a n Al /n Zn a Cu s (µmol Cu g -1 ) b Surface area (m 2 g -1 ) c T reduction (°C) d

	11Al	-	0.51	-	71	-
	26Cu/11Al-S2	0.72	0.75	322	111	250
	22Al	-	2.47	-	223	-
	16Cu/22Al-S2	0.76	2.41	210	167	250
	a Based on ICP analysis; b based on N 2 O chemisorption; c based on N 2 physisorption; d based on TGA analysis
	under H 2

Table 3 .

 3 Conversion and selectivity to 1,2-propanediol for the hydrogenolysis of glycerol

	Entry	Catalysts	Conversion	S PDO (%)
			(%)
	1	4Cu-11Al-S1	21	97
	2	9Cu-11Al-S1	52	86
	3	11Cu-11Al-S1	74	83
	4	18Cu-24Al-S1	79	85
	5	25Cu-16Al-S1	96	83
	6	21Cu-S1	40	89
	7	11Cu-11Al-S1-P	45	91
	8	26Cu/11Al-S2	93	78
	9	16Cu/22Al-S2	44	91
	Results after 24 h, with 500 mg of catalyst; 100 mL of 0.23 M
	glycerol in water; 200 °C and 30 bar of H 2

Table 4 .

 4 Surface area and d Cu for 25Cu-16Al-S1 after syntheses and after recycling

	Catalyst	Surface area
		(m 2

g -1 ) a d Cu (nm) b

	Fresh	97	12
	Test 1	142	18
	Test 2	149	18
	Test 3 c	150	20
	Test 4 c	155	21
	a based on N 2 physisorption, b based on XRD analysis c The catalyst was re-activated under H 2 at 350 °C before test 3
	and test 4

Acknowledgments

L.O. acknowledges the Lebanese association Daw for the financial support. The authors thank in particular Thibaut Cornier and the members of the IRCELYON scientific platforms

IRTECH for their assistance in the characterization of catalysts. Thanks to Christelle Fabrer-Boulé and the Centre Technologique des Microstructures (CTµ) for the SEM analyses.

Conflict of Interest

The authors declare no conflict of interest.