Florian ; De Bettignies
email: f.debettignies@sb-roscoff.fr

Patrick Dauby

François Thomas

Angélique Gobet

Ludovic Delage

Olivier Bohner

Stéphane Loisel

Dominique Davoult

DEGRADATION DYNAMICS AND PROCESSES ASSOCIATED WITH THE ACCUMULATION OF KELP FRAGMENTS: AN IN SITU EXPERIMENTAL APPROACH

Keywords: kelp, Macroalgae, Phaeophyceae Degradation, Metabolism, Composition, Bacteria, Organic Matter Kelp, Degradation, Metabolism, Composition, Bacteria, Organic Matter 1. INTRODUCTION

A high proportion of the kelp Laminaria hyperborea production is exported from kelp forests following seasonal storm events or natural annual old blades loss. Drift kelp is transported and can accumulate temporarily in benthic subtidal habitats. We investigated the degradation processes of L. hyperborea in a low subtidal (-8 m) sandy bottom ecosystem by setting up a sixmonth cage experiment to simulate drift kelp accumulation. During the degradation process, we compared changes in biomass, nutritional quality (C:N ratio), respiration, quantum efficiency of photosystem II (Fv/Fm) and chemical defence concentrations. We found that biomass decomposition started after 2 weeks and followed a classic negative exponential pattern. Half of the biomass had degraded after 8 weeks and only 16% of the initial biomass remained after 25 weeks. The degradation process seemed to reach a critical step after 11 weeks, with an increase in respiration rate and phlorotannin concentration. These results likely reflect an increase in bacterial activity and a weakening of the kelp cell wall. Surprisingly, the large fragments remained visually intact after 25 weeks of degradation, and photosystems were still responding correctly to light stimuli, indicating that photosynthesis appears to persist over time in fragments.

Reproductive tissues appeared after 4 months of degradation, showing a capacity to maintain the reproductive function. Our results indicate that degrading kelps degrade slowly and because they maintain their major functions (photosynthesis, reproduction, etc.) and accumulate on adjacent ecosystems may play a long-term ecological role in coastal ecosystem dynamics.

Degradation is a key process in the organic matter cycle and drives biogenic ecosystems in terrestrial and marine environments. Degradation includes the decomposition (biomass breakdown) of primary producers and litter, and leads to remineralisation driven by micro-and macroorganisms that colonise the organic matter [START_REF] Pandey | Litterfall, litter decomposition and nutrient dynamics in a subtropical natural oak forest and managed plantation in northeastern India[END_REF][START_REF] Polyakova | Impact of deciduous tree species on litterfall quality, decomposition rates and nutrient circulation in pine stands[END_REF][START_REF] Rees | Sustainable management of soil organic matter: based upon papers offered at a meeting held in Edinburgh in September 1999[END_REF]. Litterfall amounts and dynamics are closely related to the growth and productivity of source ecosystems [START_REF] Matala | Litterfall in relation to volume growth of trees: Analysis based on literature[END_REF][START_REF] Miller | Dynamics of nutrient cycling in plantation ecosystems[END_REF]. In terrestrial ecosystems, each plant species has a specific decomposition rate depending on its lignin constituents and nitrogen content [START_REF] Hemminga | Transport, deposition and in situ decay of seagrasses in a tropical mudflat area (Banc D'Arguin, Mauritania)[END_REF][START_REF] Wesemael | Soil organic matter accumulation, litter decomposition and humus forms under mediterranean-type forests in southern Tuscany, Italy[END_REF], but also depending on environmental conditions (litterfall moisture, temperature) [START_REF] Krishna | Litter decomposition in forest ecosystems: a review[END_REF]. In contrast to terrestrial systems, marine environments are highly dynamic and variable in terms of water motion and environmental conditions, especially in coastal systems. Despite this major difference, accumulations of seagrass [START_REF] Cardona | Meadows of the seagrass Posidonia oceanica are a significant source of organic matter for adjoining ecosystems[END_REF][START_REF] Pergent | Primary production, stocks and fluxes in the Mediterranean seagrass Posidonia oceanica[END_REF] and macroalgae, such as kelps, are observed on the seafloor from deep [START_REF] Filbee-Dexter | Detrital kelp subsidy supports high reproductive condition of deep-living sea urchins in a sedimentary basin[END_REF][START_REF] Vetter | Hotspots of benthic production[END_REF] to subtidal [START_REF] Tzetlin | Fauna associated with detached kelp in different types of subtidal habitats of the White Sea[END_REF] and intertidal habitats [START_REF] Bustamante | Maintenance of an Exceptional Intertidal Grazer Biomass in South Africa: Subsidy by Subtidal Kelps[END_REF].

Macroalgal accumulations are comparable to terrestrial litterfall, modifying the recipient substratum and being influenced by associated micro-and macroorganisms and environmental conditions.

Among macroalgae, kelps are major components of marine ecosystem functioning in temperate and sub-polar regions around the globe. Kelp forests play a key role in coastal environments as habitat-forming species, wave-energy dissipaters and major actors in the carbon cycle [START_REF] Christie | Species distribution and habitat exploitation of fauna associated with kelp (Laminaria hyperborea) along the Norwegian coast[END_REF][START_REF] Leclerc | Community, trophic structure and functioning in two contrasting Laminaria hyperborea forests[END_REF][START_REF] Teagle | The role of kelp species as biogenic habitat formers in coastal marine ecosystems[END_REF]. As highly productive primary producers, kelps assimilate carbon dioxide to produce organic matter via photosynthetic processes [START_REF] Bartsch | The genus Laminaria sensu lato : recent insights and developments[END_REF]. Kelps are fast growing producers that accumulate a high quantity of organic matter (>1 kg C.m².yr -1 , Krumhansl and Scheibling, 2012a). A high proportion of this biomass is exported during blade erosion, natural mortality or dislodgment related to strong hydrodynamic events (de [START_REF] De Bettignies | Contrasting mechanisms of dislodgement and erosion contribute to production of kelp detritus[END_REF]Krumhansl and Scheibling, 2012;[START_REF] Pessarrodona | Carbon assimilation and transfer through kelp forests in the NE Atlantic is diminished under a warmer ocean climate[END_REF]. Detrital material can drift across habitats and, when hydrodynamic conditions are suitable, accumulates in benthic habitats. Accumulation of kelp detritus can strongly influence the recipient trophic food web (Krumhansl and Scheibling, 2012b;[START_REF] Tzetlin | Fauna associated with detached kelp in different types of subtidal habitats of the White Sea[END_REF]. Globally, 82% of annual kelp production is exported, becoming detrital subsidies [START_REF] De Bettignies | Contrasting mechanisms of dislodgement and erosion contribute to production of kelp detritus[END_REF]Krumhansl and Scheibling, 2012a). Within kelp forests, detrital material contributes to the particulate organic matter (POM) pool and plays an important role in trophic food webs [START_REF] Leclerc | Trophic significance of kelps in kelp communities in Brittany (France) inferred from isotopic comparisons[END_REF].

Laminaria hyperborea (Laminariales) is a dominant kelp species in the North-East Atlantic, distributed from Portugal to Norway [START_REF] Steneck | Kelp forest ecosystems: biodiversity, stability, resilience and future[END_REF]. In Brittany, France, this species dominates subtidal rocky ecosystems from 0 to 30 m below chart datum. Growth of L. hyperborea sporophytes occurs in winter and spring, ceasing in July [START_REF] Lüning | Growth strategies of three Laminaria species (Phaeophyceae) inhabiting different depth zones in the sublittoral region of Helgoland (North Sea)[END_REF]. In spring, L. hyperborea retains the old blade from the previous year during the rapid growth of the new blade.

Between April and May, the old blade is shed from the fast-growing new blade. The release of the previous season's growth contributes significantly to the total detritus production [START_REF] Pessarrodona | Carbon assimilation and transfer through kelp forests in the NE Atlantic is diminished under a warmer ocean climate[END_REF]. L. hyperborea detritus represents a trophic resource that connects habitats, becoming increasingly accessible to consumers as it degrades [START_REF] Norderhaug | Trophic importance of Laminaria hyperborea to kelp forest consumers and the importance of bacterial degradation to food quality[END_REF].

In some areas, within kelp forest grazers may only consume a small fraction of kelp productivity [START_REF] Hereward | Seasonal variability in the population structure of a habitat-forming kelp and a conspicuous gastropod grazer: Do blue-rayed limpets (Patella pellucida) exert top-down pressure on Laminaria digitata populations?[END_REF], whereas in other systems, grazing by sea urchins is a major process structuring L. hyperborea forests [START_REF] Ling | Global regime shift dynamics of catastrophic sea urchin overgrazing[END_REF]. In Brittany, grazers appear to consume a very small fraction of the L. hyperborea production, with a high proportion being exported to adjacent ecosystems. Therefore, the degradation kinetics of L. hyperborea detritus and its impact on recipient ecosystems appear to be key elements in ecosystem functioning.

In the present study, we explored L. hyperborea decomposition dynamics and the degradation process in an in situ six-month experimental approach using litterfall cages to simulate drift kelp accumulation on a sandy bottom ecosystem. To investigate the kinetics of detritus biomass decomposition, we monitored multiple parameters such as carbon (C) and nitrogen (N) content, phlorotannin concentration, photosynthesis, respiration and abundance of cultivable algaassociated bacteria. We tested whether L. hyperborea degradation was a quick process, with total decomposition of biomass after several months. The parameters were thus expected to vary accordingly, with a decrease in the C:N ratio (often related to nutritional quality and palatability), a change in tissue phlorotannin concentration (related to stress and defence mechanisms) and a decrease in the major functions such as production, respiration and reproduction.

MATERIALS AND METHODS

Study site

The present study was conducted near Roscoff, in the Bay of Morlaix, along the north-western coast of Brittany, France. The bay is composed of numerous rocky reefs supporting high L. hyperborea biomass stock [START_REF] Gorman | Modeling kelp forest distribution and biomass along temperate rocky coastlines[END_REF], separated by large areas of fine and soft sediments that temporarily receive accumulations of drift kelp (personal observation). The experimental site (Guerhéon: 48°42'33.78" N, 003°57'12.36" W) was located in a semi-enclosed part of the bay, protected from the prevailing westerly winds, but exposed to north and easterly winds that occasionally occur during spring and summer anticyclonic conditions. The site substratum was characterised by a mixture of coarse sand and shell fragments at 4.5 m depth below chart datum (9 m average depth) and distant by ca. 100 m from rocky reefs covered with kelp forests.

Experimental design

We set up an in situ cage experiment to investigate the degradation dynamics of L. hyperborea on a subtidal sandy ecosystem (Figure 1). The study was conducted from 18 April to 5 October 2017 (5.5 months). The experiment was deployed in April to mimic the storm events that typically occur during spring and break up and dislodge part of the kelp population and increase the amount of detrital kelp deposits. In this experiment, we chose to simulate storm damage on kelps that are losing their old frond and studied the degradation process on the physiologically active new blades.

During scuba dives, we randomly collected whole adults of L. hyperborea with a 80-90 cm stipe length corresponding to 5-6 year-old kelps [START_REF] Sheppard | Studies on the Growth of Laminaria hyperborea (Gunn.) Fosl. and Laminaria ochroleuca (De La Pylaie) on the French Channel Coast[END_REF] without visible signs of degradation from a natural population on a rocky reef close to the experimental site. Kelp samples were kept in seawater and brought back to the harbour where they were processed within 2 hours. New blades were cut once in length and once in width. This method gave different fragment sizes similar to those found in the field. Fragments from two individuals were pooled and weighed with a digital spring scale (Amiaud Durer, 40 kg, ± 10 g) for a mean ± SD wet mass of 911 ± 105 g. Batches were gently packed and randomly allocated to custom-built numbered plastic litterfall cages (30 × 25 × 10 cm, 1 cm mesh) made of hard plastic mesh used in oyster farming. The quantity of algal material in the cage corresponded a 10 cm thick accumulation of kelp fragments with a three-dimensional structure, as previously observed during prospection dives. A total of 35 litterfall cages were prepared with new blades, kept in seawater and quickly immerged on the experimental site by divers. To compare the degradation kinetics between young and old blades, five litterfall cages were filled with old blade fragments following the same procedure. Cages were attached on four anchored chain lines arranged parallel to each other, forming a relatively continuous rectangle of accumulated fragments (Figure 1). We checked the cages throughout the experiment to ensure that they were not buried in sediment. At each sampling time (2, 4, 6, 11, 15, 20, 24 weeks), five replicates of new-blade litterfall cages were randomly collected. Additionally, one old-blade litterfall cage was randomly collected at five sampling dates (4, 6, 11, 15, 20 weeks). Cages were collected with caution using 1 mm mesh bags, for 3 of them incubated for in-situ respirometry measurements, brought to the surface, transported within site seawater to the laboratory for bacterial sampling before any other processing. Cages were then kept in the dark in a continuous-flow seawater tank with constant air bubbling until laboratory measurements (performed within 24 h).

Field measurements

Environmental parameters

An array of environmental sensors were deployed on the experimental site to measure temperature, light intensity and water motion over the six-month period. Temperature and light intensity were measured with the same sensor (HOBO Pendant Temperature/Light Weatherproof Pendant Data Logger 16K) attached on a plate fixed horizontally on a threaded rod at 20 cm from a concrete block. Relative water motion was measured with an accelerometer (HOBO Pendant G Data Logger) attached to a small dome-shaped buoy. The buoy was tethered to the seabed with a 0.75 m rope clamp on a different concrete block according to [START_REF] Bennett | Canopy interactions and physical stress gradients in subtidal communities[END_REF] design. These sensors were deployed at 3 m from the experimental system and a replicate set was deployed to offset any sensor failure. At each sampling time, sensors were changed or cleaned to prevent too much fouling on the rope or on the sensor. All sensors were programmed to take one measurement every five minutes. For each sampling time, the maximum, minimum and average values of the previous week were measured for temperature and relative water motion (RWM).

Only the daily average was used for light. For the RWM, only two channels were used (x, y) to account for horizontal acceleration (cf. H 2 O motion V2 design; [START_REF] Evans | A cost-effective technique for measuring relative water movement for studies of benthic organisms[END_REF]. The sensor was set to record acceleration (m.s -2 ). The RWM was standardised and measured as the vector sum for all pairwise recordings according to [START_REF] Evans | A cost-effective technique for measuring relative water movement for studies of benthic organisms[END_REF]) methodology.

In situ litterfall cage respiration rate

Litterfall cage respiration rates were measured underwater at the experimental site during each collection time on three of the five randomly collected new-blade litterfall cages. Three benthic chambers were set up in situ by scuba divers. The custom-built incubation chambers [START_REF] Ouisse | Comparative study of methodologies to measure in situ the intertidal benthic community metabolism during immersion[END_REF] were composed of a base to isolate the measured sample from the sediment, a Plexiglas cylinder and dome sealed by a clamp system enclosing a total volume of 26.2 L of seawater.

Chambers were assembled ensuring no air bubbles remained inside and seawater was mixed within the chambers with an autonomous stirrer. The three selected cages harvested in their mesh bags were allocated to one benthic chamber each. Cages were incubated in the benthic chambers covered by black tarpaulins to ensure darkness for 30 min. A sample of seawater was collected from each chamber by using 100 mL syringes at the beginning and at the end of the incubation (two-point method) and directly brought to the surface after sampling. Dissolved oxygen concentration was immediately measured on the boat using a portable multi-meter (HQ40d, Hach®, Loveland, CO, USA) coupled with a luminescent/optical dissolved oxygen probe (Intellical™ LDO101, Hach®, accuracy ± 0.2 mg.L -1 ). Litterfall cage respiration rate was estimated from the difference between initial and final oxygen (O 2 ) concentrations after being corrected for temperature change. Respiration was expressed as O 2 consumption in mg O 2 .cage - 1 .h -1 , to allow comparisons between litterfall cages over time. Although continuous O 2 measurement is recommended, measurement only at the beginning and at the end of the incubation is considered acceptable to obtain a correct measurement of community metabolism for short incubations [START_REF] Ouisse | Comparative study of methodologies to measure in situ the intertidal benthic community metabolism during immersion[END_REF].

Laboratory measurements

In the laboratory, the contents of each cage were placed in the corresponding 1 mm mesh bag, and kept in the dark in a large tank with running seawater. For each sample and measurement, bags were opened in the laboratory and directly brought back to the seawater tank after processing.

Cultivable bacteria counts

For each litterfall cage, 10 pieces of algal tissue were collected using a stainless steel punch (13 mm diameter) sterilised by flaming with 70% ethanol. Algal pieces were transferred to 20 mL of sterile saline solution (for 1 L: 24.7 g NaCl, 6.3 g MgSO 4 •7H 2 O, 4.6 g MgCl 2 •H 2 O, 0.7 g KCl), stored on ice and returned within 1 h to the laboratory for further processing. The 10 algal pieces from one cage were rinsed twice in 20 mL sterile saline solution and homogenised in a single batch for 1 min at 8000 rpm in 20 mL sterile saline solution using a T25 Ultra-Turrax blender, followed by 1 min of vortex agitation at maximum speed. Serial dilutions (from 10 -1 to 10 -4 ) of homogenates were prepared in sterile saline solution. Then, 100 µL of the dilutions were spreadplated in duplicate and incubated at 20°C for 7 days. Four media were used to screen cultivable bacteria presenting different functions. The Zobell agar medium (for 1 L: 5 g tryptone, 1 g yeast extract, 15 g agar in natural seawater), a highly nutritive medium, was used as a proxy of the total community of cultivable bacteria. Bacteria resisting high concentrations of iodine, one of the defence compounds released by kelps [START_REF] Verhaeghe | Microchemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation[END_REF], were screened on a Zobell agar medium supplemented with 100 mM KI. Bacteria were also grown on a minimum medium solidified with Phytagel (for 1 L: 24.7 g NaCl, 6.3 g MgSO 4 •7H 2 O, 4.6 g MgCl 2 •H 2 O, 2 g NH 4 Cl, 0.7 g KCl, 0.6 g CaCl 2 , 200 mg NaHCO 3 , 100 mg K 2 HPO 4 , 1X vitamin mix, 20 mg FeSO 4 •7 H 2 O, 20 g Phytagel) [START_REF] Thomas | Waterborne Signaling Primes the Expression of Elicitor-Induced Genes and Buffers the Oxidative Responses in the Brown Alga Laminaria digitata[END_REF] and supplemented with 4 g.L -1 alginate, a polysaccharide accounting for 10-45% of the dry mass of brown algal cell wall [START_REF] Kloareg | Structure of the Cell Walls of Marine Algae and Ecophysiological Functions of the Matrix Polysaccharides[END_REF] or 4 g.L -1 mannitol, one of the main carbon storage forms in brown algae [START_REF] Iwamoto | Salt-regulated mannitol metabolism in algae[END_REF]. (Technical) duplicate measurements were averaged and results were expressed as the number of colony-forming units per unit area of algal tissue (cfu.cm -2 ). The detection limit was 75 cfu.cm -2 . The proportion of iodine-resistant cultivable bacteria was estimated as the ratio of counts obtained on KI-supplemented Zobell agar compared to counts obtained on Zobell agar.

Phlorotannin assay

For each cage, 10 disks of 28 mm diameter were randomly cored on kelp fragments and stored at -20°C before processing. Phlorotannin extraction was performed using an method adapted from [START_REF] Li | Extraction and Identification of Phlorotannins from the Brown Alga, Sargassum fusiforme (Harvey) Setchell[END_REF] and [START_REF] Koivikko | High-performance liquid chromatographic analysis of phlorotannins from the brown algae Fucus vesiculosus[END_REF]. Frozen samples were stored overnight at -80°C and freeze-dried for 24 h. First, 100 mg of freeze-dried tissue were ground into a fine homogeneous powder under liquid nitrogen with a mortar and pestle and stored at -20°C until analyses. The extraction buffer consisted of a 30:70 ethanol:water mixture acidified with hydrochloric acid (pH = 2.6) to prevent oxidation. Extraction was performed at 25°C for 30 min with 0.5 mL of extraction buffer under constant agitation. The extract was centrifuged and the supernatant was retrieved and stored at -20°C. Total soluble phlorotannin quantification in the extracts was determined using a modified Folin-Ciocalteu method [START_REF] Van Alstyne | Phlorotannin allocation among tissues of northeastern pacific kelps and rockweeds[END_REF][START_REF] Zhang | A Simple 96-Well microplate method for estimation of total polyphenol content in seaweeds[END_REF]. Phloroglucinol (Sigma) was used as a standard. Extracts were diluted 2 to 10 times with extraction buffer for absorbance to be in the range of the standard curve. Absorbance measurements were performed using multiwall plates (Nunc UV-Star 96 wells) containing 10 µL of extract, 50 µL of Folin-Ciocalteu reagent (Sigma) and 40 µL of Na 2 CO 3 7.5 % (m/v). Plates were incubated in the dark at room temperature for 2 h. Absorbance was measured at λ = 750 nm on a spectrophotometric microplate reader (Safire²Tecan Multi-detection Microplate reader). For each sampling time, the cage was used as biological replicate (n = 5) and each sample was measured in two technical replicates. Technical (measurement) replicate values were averaged prior to statistical analyses. Each standard solution was run in triplicate. The concentration of soluble phlorotannins within kelp fragments was calculated according to the phloroglucinol standard curve and expressed in phloroglucinol equivalent in mg.g DM -1 (where DM is kelp dry mass).

Carbon-nitrogen content

For each cage, 10 disks of 28 mm diameter were randomly cored on kelp fragments and stored at -20°C until preparation and analysis. Algal samples were rinsed with freshwater to remove possible epiphytes and dried at 60°C during 48 h. Dried material was ground with a mortar and a pestle and powder samples were put in tin capsules before C:N analyses. Carbon and nitrogen content were measured using a Flash EA 1112 CHN analyser (ThermoFinningan) coupled with a Finningan Delta Plus mass spectrometer, via a Finnigan Con-Flo III interface. C:N data were expressed as a mass ratio.

Chlorophyll fluorescence measurements

Fluorescence was measured to evaluate the conservation of photosynthetic capacity of algal tissues. Blades were transported from the seawater tank to the laboratory inside a dark box filled with seawater. All measurements were carried out 24 h after collection between 4:00 pm and 6:00 pm to avoid differences in physiology related to daily cycles [START_REF] Edwards | Diurnal variation in relative photosynthetic performance in giant kelp Macrocystis pyrifera (Phaeophyceae, Laminariales) at different depths as estimated using PAM fluorometry[END_REF][START_REF] Gevaert | Photoinhibition and seasonal photosynthetic performance of the seaweed Laminaria saccharina during a simulated tidal cycle: chlorophyll fluorescence measurements and pigment analysis[END_REF]. Visually, the degradation state of blade fragments varied with size. Fragments were therefore separated in two groups according to their surface area: small (area < 25 cm²) and large (area > 50 cm²) fragments. The fluorescence signals were taken from the middle of the fragments for three small and three large fragments randomly selected from each cage. At each sampling time, measurements were taken for the five new-blade cages collected. Fragments were darkadapted for an additional 15 min using the Dark Leaf Clip Diving-LC (Walz) to which the optical fibre of the fluorometer was applied to perform the measurements. In vivo Chl. a fluorescence of photosystem II (PSII) was measured from each fragment using an underwater pulse amplitude modulated fluorometer (Diving-PAM; Heinz Walz, Effeltrich, Germany). The maximum quantum yield (Fv/Fm) of PSII, here used as a proxy of physiological capacity, was measured using a 0.8 s saturating light pulse of white light (2500 μmol photons.m -2 s -1 ). F v represents the variable fluorescence and corresponds to the difference between the minimum fluorescence yield (F 0 ) measured under weak red modulated light and the maximal fluorescence (F m ) measured following the short saturating light pulse.

Remaining biomass

The remaining fragments from each litterfall cage were air-dried for 1 minute and wet-weighed (wet mass, WM). Samples were then dried at 60°C for at least 48 h until reaching a constant mass before being reweighed (dry mass, DM). The remaining biomass was estimated by dividing the final WM by the initial WM measured at the beginning of the experiment and expressed in percentage of remaining biomass (%RB). To estimate the decomposition kinetics, we calculated a decomposition rate with a single exponential decay model commonly used in the literature [START_REF] Enríquez | Patterns in decomposition rates among photosynthetic organisms: the importance of detritus C: N: P content[END_REF][START_REF] Olson | Energy storage and the balance of producers and decomposers in ecological systems[END_REF]. The decomposition rate (k, in day -1 ) was calculated from the change of biomass (M) over time (t, in days) since the beginning of the experiment following the equation:

𝑀 𝑡 = 𝑀 0 𝑒 -𝑘𝑡
From this equation, the half-life of fragment detritus was measured (T 1/2 = k -1 .Ln(2), days). It gives a more intuitive description of detritus turnover times [START_REF] Enríquez | Patterns in decomposition rates among photosynthetic organisms: the importance of detritus C: N: P content[END_REF]. In our case, M 0 corresponds to the initial percentage of remaining biomass that is equal to 100. Additionally, before drying algae, frond surface was checked for reproductive tissue presence.

Data analysis

Phlorotannin concentrations and C:N contents were compared with a one-way ANOVA using degradation time as fixed factor. Post-hoc comparisons were applied using Tukey's honestly significant difference (HSD) tests when significant ANOVA results were found (p < 0.05).

Phlorotannin concentration data were square-root transformed prior to analysis to meet the assumptions of normality (Shapiro Wilk's test, α = 0.05) and homogeneity of variance (Levene's test, α = 0.05). Remaining biomass, litterfall cage respiration and bacterial count values were compared using a non-parametric one-way analysis of variance Kruskal-Wallis H-test with degradation time as fixed factor. This procedure was used for biomass data because assumptions of normality and homoscedasticity were not met even after log(x) and square-root transformations. For litterfall cage respiration and bacterial counts, the low number of replicates (n = 3) at each sampling time were not amenable to parametric analyses. Bonferroni adjustment and the low number of replicates precluded the detection of differences by post-hoc Dunn pairwise tests. Fv/Fm data within the same litterfall cage were averaged for small and large fragments prior to analysis, due to data dependency. Fv/Fm values were compared using a twoway ANOVA with degradation time and fragment size as fixed factors. Given that the response of the fragment size depended on degradation time, we chose a nested design. The assumption of homoscedasticity was met. Although the data did not meet the assumption of normal distribution of residuals (Shapiro Wilk's test, α = 0.05), we nonetheless selected this parametric analysis because the distribution of residuals was nearly normal in the histograms and the ANOVA is robust to small deviations from normality [START_REF] Schmider | Is It Really Robust?: Reinvestigating the Robustness of ANOVA Against Violations of the Normal Distribution Assumption[END_REF].

All statistical analyses and graphics were carried out using the freeware R statistical environment (R Core Team 2019).

RESULTS

Environmental parameters

During the experiment, temperature ranged from 11.7 to 17.8 °C reflecting seasonal fluctuation typically observed in the Bay of Morlaix. No lengthy strong storms were experienced during the experiment, indicating that there were no long periods of low light and high RWM between experimental times (Table 1).

Decomposition of biomass and tissue content

The biomass of new L. hyperborea blades decreased with degradation time (Kruskal-Wallis, H = 34.2, df = 7, p < 0.001). Biomass decomposition started after 2 weeks and showed negative exponential dynamics with rapid loss during the first 11 weeks and then slowing after 15 weeks (Figure 2a). After 24 weeks (5.5 months), the remaining biomass represented 16.5% and 3.8% of initial mass for new blades and old blades, respectively. The decomposition rate calculated with the non-linear regression model was three times higher for old blades (k = 0.0366 day -1 , p < 0.001, R raw 2 = 0.9682) compared with new blades (k = 0.0107 day -1 , p < 0.001, R 2 raw = 0.949), corresponding to a fragment degradation half-life (T 1/2 ) of 18.9 and 65.0 days, respectively (Figure 2b). Furthermore, after 20 and 24 weeks of degradation, reproductive tissues were observed on the surface of degrading new blades.

Tissue composition was significantly affected by degradation time (Figure 2c,2d). Phlorotannin concentration in degrading L. hyperborea fragments increased significantly with time (F 7, 32 = 30.492, p < 0.001). The concentration was two times higher after 11 weeks of degradation compared with any time point the first 6 weeks. This increase was significant (Tukey's HSD test, p < 0.05) except for the comparison between week 4 and 15 (p = 0.116, Figure 2c). The content in C:N (mass ratio) was significantly affected by degradation time (F 7, 32 = 6, p < 0.001), with the ratio increasing slightly from 4.6 after 2 weeks to a maximum of 7.3 after 15 weeks of degradation. The Post-hoc Tukey's HSD test showed only some differences between times within the first 6 weeks and after 11 weeks. No significant differences in C:N ratio were observed among sampling times after 11 weeks (Figure 2d).

Detritus metabolism

The litterfall cage respiration rate was significantly influenced by degradation time (Kruskal-Wallis, H = 15.406, df = 7, p < 0.05, Figure 2e). Respiration dramatically decreased by a factor of 3 after 2 weeks of degradation; thereafter the rate did not vary from 2 to 6 weeks. A gradual increase was measured starting from 11 weeks with a maximum rate after 20 and 24 weeks of degradation. At the end of the experiment, the respiration rate doubled relative to the rate observed between 2 and 6 weeks (Figure 2e).

The photosynthetic capacity (Fv/Fm) of degrading algae varied significantly across degradation time (F 7, 60 = 6.858, p < 0.001) and according to fragment size (F 7, 60 = 11.821, p < 0.001) (Figure 2f). Under optimal conditions, Fv/Fm values range from 0.7 to 0.8 for brown algae [START_REF] Bischof | Acclimation of Maximal Quantum Yield of Photosynthesis in the Brown Alga Alaria esculenta under High Light and UV Radiation[END_REF] and a reduction reflects a stress response in algae [START_REF] Pearson | Frayed at the edges: selective pressure and adaptive response to abiotic stressors are mismatched in low diversity edge populations[END_REF]. In large fragments, Fv/Fm remained stable (Tukey's HSD, p > 0.05) and high (between 0.70 and 0.76), reflecting satisfactory photosynthetic capacity, whereas the Fv/Fm of small fragments was affected over time. Especially after 15 weeks, Fv/Fm values decreased and were highly variable (mean ± SD; 0.43 ± 0.24, 0.32 ± 0.15, 0.59 ± 0.16 after 15, 20, 24 weeks, respectively, Figure 2f).

Fluctuations of cultivable bacteria during the degradation of L. hyperborea

Bacterial counts on Zobell agar, alginate-Phytagel and mannitol-Phytagel plates increased by 1-2 orders of magnitude in the first two weeks of the experiment (Figure 3), and thereafter remained stable until the end of the experiment. This variation over time was found to be statistically significant for counts on alginate-Phytagel (Kruskal-Wallis chi-squared = 11.697, df = 5, P = 0.039) and mannitol-Phytagel (Kruskal-Wallis chi-squared = 11.655, df = 5, P = 0.040) plates.

Furthermore, the proportion of iodine-resistant bacteria decreased with time (Kruskal-Wallis chisquared = 10.5, df = 4, P = 0.033, Fig. 3b), from 75% at the beginning of the experiment to 5% after 24 weeks.

DISCUSSION

Understanding the kinetic and the processes of kelp fragments degradation is essential regarding the quantity of organic matter exported within adjacent habitats by kelps forests. During our experiment, the degradation of kelp biomass followed a classic pattern of organic matter decomposition according to a simple negative exponential model, with high heterogeneity between young and old L. hyperborea blades. Old blades showed degradation kinetics that were three times faster than those of young blades. An additional study showed that stipes degrade slightly faster than old blades and four times faster (data not shown) than young blades (k = 0.0425 d -1 , compared to k = 0.0366 d -1 and k = 0.0107 d -1 for respectively old and young blades).

This shows that, depending on the export process (detachment of old blades, erosion or dislodgment during storms), fragments may have different residence times in coastal ecosystems.

The degradation of kelp organic matter thus follows two different types of patterns. The classic export due to the natural kelp life cycle results in a pulse of organic matter during spring and summer, and this tissue degrades relatively quickly. By contrast, material exported during storms is more variable, producing slowly degrading material. Considering these accumulations or drifting fragments as a transient habitat and resource for adjacent ecosystems, their influence in benthic systems can be significant for several months. Surprisingly, the L. hyperborea degradation rate appeared slow compared with other macroalgae that dominate coastal ecosystems. The measured degradation rate for young L. hyperborea blades is 10 to 20 times slower than fast growing ephemeral algal species such as Ulva sp. (k = 0.34 -0.51 d -1 ) or Gracilaria sp. (k = 0.5 d -1 ) [START_REF] Conover | Biomass decay rates and tissue nutrient loss in bloom and non-bloom-forming macroalgal species[END_REF], more than 5 times slower than intertidal canopy-forming species such as Fucus vesiculosus (k = 0.09 d -1 ) [START_REF] Conover | Biomass decay rates and tissue nutrient loss in bloom and non-bloom-forming macroalgal species[END_REF], about 3 times slower than other kelp species such as Macrocystis integrifolia for young blades (k = 0.032 d -1 ) [START_REF] Albright | In situ degradation of the kelps Macrocystis integrifolia and Nereocystis luetkeana in British Columbia coastal waters[END_REF] and similar to seagrass species ( k ≈ 0.01 d -1 ) [START_REF] Harrison | Detrital processing in seagrass systems: A review of factors affecting decay rates, remineralization and detritivory[END_REF][START_REF] Hemminga | Transport, deposition and in situ decay of seagrasses in a tropical mudflat area (Banc D'Arguin, Mauritania)[END_REF] and mangrove leaves (k= 0.011 d -1 ) [START_REF] Gladstone-Gallagher | Mangrove (Avicennia marina subsp. australasica) litter production and decomposition in a temperate estuary[END_REF]. In addition, degradation dynamics may be strongly affected by seasonal patterns and environmental conditions including changes in local hydrodynamics and temperature fluctuations. For example, physical mechanisms certainly fragment detritus more than that observed within our cages. Furthermore, our study covered the spring-summer period that corresponds to late spring storms. The lower temperatures during winter likely slow down the degradation process, but the high water motion during winter may speed up the process. For these reasons, winter and autumn degradation kinetics are not readily predictable. Further research is also needed to understand change of degradation dynamics depending on the type of recipient habitat.

In addition to the slow degradation dynamics, photosynthetic capacity was maintained for 5.5 months for large fragments and for 3.5 months for small fragments. However, this capacity appears to be highly variable depending on the degree of fragmentation and time of degradation.

Here, small fragments tended to lose their photosynthetic ability. These results have wide implications for coastal ecosystem functioning, because exported detritus may continue to carry out primary production and retain the energy and capacity to renew their tissues, potentially explaining their partial recalcitrance to degradation. In highly hydrodynamic systems such as the English Channel, this recalcitrance may increase the area of kelp forest influence and magnify the impact of kelp forests on coastal and deep-sea ecosystems. Furthermore, reproductive tissues were observed on degrading fragments during this six-month experiment (data not shown). The persistence of reproductive tissue in drift kelp may increase the dispersal capacity of L.

hyperborea, partly compensating for the naturally short dispersal ranges of spores (5-200 m) [START_REF] Fredriksen | Spore dispersal in Laminaria hyperborea (Laminariales,Phaeophyceae)[END_REF].

Respiration showed a rapid decline at the beginning of the experiment. This early change can be attributed to the capacity of kelps to modulate their metabolism depending on environmental conditions [START_REF] Kregting | The influence of water motion on the growth rate of the kelp Laminaria digitata[END_REF]. Respiration increased after 11 weeks, likely reflecting the metabolism of the holobiont complex formed by the degrading kelp and its associated microorganisms. Degrading kelp is rapidly colonised by bacteria such as those growing on alginate or mannitol, reaching an apparent maximum abundance as early as 2 weeks after fragmentation.

Kelps possess a unique defence metabolism, associated in particular with the production of toxic iodine compounds and phlorotannins [START_REF] Potin | Biotic interactions of marine algae[END_REF]. Iodine is highly concentrated in the peripheral cell layers, creating specific niches for associated bacteria during iodovolatilisation [START_REF] Küpper | Iodide accumulation provides kelp with an inorganic antioxidant impacting atmospheric chemistry[END_REF][START_REF] Verhaeghe | Microchemical imaging of iodine distribution in the brown alga Laminaria digitata suggests a new mechanism for its accumulation[END_REF]. Our preliminary results on iodine-tolerant bacteria suggest some changes in bacterial community composition. As L. hyperborea tissue is degrading, iodine from the alga may be released in the environment, making the associated specific niches disappear. Interestingly, the detected concentration of phlorotannins in degrading tissues doubled after 11 weeks. This result is counterintuitive and refutes the claim that kelps quickly initiate defence responses to minimise grazing pressure and stress soon after dislodgement [START_REF] Norderhaug | Importance of phlorotannin content and C : N ratio of Laminaria hyperborea in determining its palatability as food for consumers[END_REF]. Instead, this apparent increase in phlorotannins may reflect the early degradation stage of kelp tissue after 11 weeks. The degradation of cell walls by colonising bacteria may make phlorotannins more accessible and increase the extraction efficiency during phlorotannin concentration assays.

The C:N ratio also followed a counterintuitive trend and remained stable for 5.5 months. Previous studies have shown a decrease in the C:N ratio related to the degradation of kelps, reflecting an enrichment in nitrogen [START_REF] Norderhaug | Importance of phlorotannin content and C : N ratio of Laminaria hyperborea in determining its palatability as food for consumers[END_REF][START_REF] Sosik | Isotopic evidence and consequences of the role of microbes in macroalgae detritus-based food webs[END_REF]. Our results show the opposite trend, with no change or a slight increase in C:N as previously observed by [START_REF] Dethier | Degrading detritus: Changes in food quality of aging kelp tissue varies with species[END_REF]. We suggest that, in the present experiment, kelp tissues did not degrade enough to observe a decrease in C:N.

Our study allows a better understanding of in situ kelp degradation dynamics over the spring/summer period. We showed that drifting Laminaria hyperborea have a high capacity to resist degradation, maintaining photosynthesis function for 5.5 months depending on the degree of fragmentation. Confirming this pattern of degradation dynamics requires testing different environmental conditions (hydrodynamism, depth) and seasons in future studies. In natural conditions, accumulations or marine litterfall vary over time and fragments can drift between different habitats after being exported. Although these changes in environmental conditions during the degradation process complicate the understanding of the impact of kelp fragments on coastal ecosystems, there is no doubt that this pulse of organic matter can be used by recipient ecosystem food webs.

Figure 1 :

 1 Figure 1: (a) In situ experimental set-up on the seabed in the Bay of Morlaix (Guerhéon: 48°42'33.78" N, 003°57'12.36" W) at 9 m depth (on average). Panel (b) shows 40 custom-built plastic cages filled with 1 kg of fresh Laminaria hyperborea blades arranged to form an artificial accumulation of kelp fragments (c). (d) Benthic chamber incubations to estimate the holobiont respiration rates. Photographs: Wilfried Thomas, experimental schematic: Florian de Bettignies.

Figure 2 :

 2 Figure 2: Parameters of degradation dynamics showing the change in (a) remaining biomass, (b) biomass decomposition models for new-blades (black circles) and old-blades (grey triangles) cages, (c) phlorotannin content, (d) C:N composition, (e) holobiont respiration rate and (f) Fv/Fm values for large (dark grey) and small (light grey) fragments. Bars give means with standard deviations (n = 5, except for respiration n = 3). Measures were taken at seven sampling times (0, 2, 4, 6, 11, 15, 20, 24 weeks). All measures are for new blades, except in panel b.

Figure 3 :

 3 Figure 3: Cultivable bacterial counts (in number of colony-forming units per unit area of algal tissue: cfu.cm -2 ) on different growth media: (a) Zobell-agar, (b) KI-supplemented Zobell-agar, (c) alginate-Phytagel, (d) mannitol-Phytagel. Proportion of iodine-resistant bacteria was estimated as the ratio of counts obtained on KI-supplemented Zobell agar relative to the counts obtained on Zobell agar. Dots represent the raw data points from each cage replicate and the open diamonds represent the mean values.

Table 1 :

 1 Environmental parameters for each sampling time. Data provided are those measured the week before retrieval. Relative water motion data for week 20 are missing due to sensor failure.

	Light (lux)

Journal of Phycology

ACKNOWLEDGMENTS

We would like to thanks the marine operation staff of Roscoff Biological Station (Service Mer & Observation SBR), especially L. Levêque, Y. Fontana, W. Thomas, M. Camusat, N. Guidal and F. Le Ven for their help with scuba diving fieldwork and experimental set-up. We also thank Metabomer platform of Roscoff Biological Station (FR2424) for C:N sample analyses. This work benefited from the support of the Brittany Regional Council and the French Government through the National Research Agency with regards to the investment expenditure program IDEALG (reference: ANR-10-BTBR-04).


Andrei Y Khodakov
email: andrei.khodakov@univ-lille.fr

Vitaly V Ordomsky

Ana Palčić

Mengdie Cai

Vijayanand Subramanian

Yuan Luo

Valentin Valtchev

Simona Moldovan

Ovidiu Ersen

Assessment of metal sintering in the copper-zeolite hybrid catalyst for direct dimethyl ether synthesis using synchrotron-based X-ray absorption and diffraction

Keywords: syngas, dimethyl ether, hybrid catalyst, deactivation, zeolite, hydrogenation

Dimethyl ether is one of the most promising environmentally optimized alternatives to the conventional fossil fuels and an important platform molecule for chemical industry. Catalyst deactivation is one of the most important challenges of the single-step dimethyl ether synthesis from syngas. Because of the lack of direct characterization techniques working under harsh reaction conditions, the information about deactivation mechanisms of bifunctional Cu/ZSM-5 catalysts is rather contradictory. In this paper, a combination of synchrotron-based in-situ time-resolved X-ray diffraction and X-ray absorption spectroscopy operating under high pressure and temperature alongside with the conventional ex-situ characterization uncovered very rapid copper sintering occurring under realistic conditions of direct dimethyl ether synthesis. Copper sintering was strongly affected by the presence of water either produced by the reaction or co-fed to the reactor.

No copper oxidation was observed under a wide range of experimental conditions.

Introduction

Dimethyl ether (DME) is one of the most promising environmentally optimized alternatives to the conventional diesel fuels due to its high cetane index (> 55), low emission of CO, NO x and particulates and reduced noise [1,2,3,4]. DME, which is biodegradable in air, non-carcinogen and non-corrosive, can be also used as a substitute for liquefied petroleum gas (LPG). DME is also an important intermediate for the production of useful chemicals (i.e. methyl acetate and dimethyl sulphate) and petrochemicals (light olefins, BTX aromatics). From the viewpoint of CO 2 emissions, synthesis of DME from renewable feedstocks ("bio-DME") is particularly attractive. In the industry, DME synthesis traditionally proceeds as a two stages process: methanol synthesis from syngas followed by methanol dehydration. The maximum syngas conversion to methanol is limited by thermodynamics especially at high temperatures [5].

At least, three reactions are involved in DME synthesis: (i) Methanol synthesis: CO + 2H 2 → CH 3 OH. Several reports suggest that methanol synthesis proceeds via intermediate formation of CO 2 [6,7].

(ii) Dehydration of methanol to DME and water: 2CH 3 OH → CH 3 OCH 3 + H 2 O.

(iii) Water gas shift reaction (WGS), which could modify partial pressures in the reactor: CO +

H 2 O → CO 2 + H 2 .
Direct DME synthesis overcomes the thermodynamic constraints of the two-stage process and leads to higher DME productivity. In the direct DME synthesis, the reaction occurs in a single reactor under high pressures and temperatures over a bifunctional catalyst, which contains simultaneously active phases for carbon monoxide hydrogenation to methanol and methanol dehydration to DME. Methanol synthesis occurs on copper-based catalysts, while methanol dehydration involves acid catalysts. Copper metal nanoparticles associated with a promoter, i.e.

Zn, are usually considered as active phase for methanol synthesis. The advantages of the Cu/Zn system include low cost and high methanol selectivity. Copper-zinc oxide interactions are considered crucial for the state of Cu phase and thus for the catalytic activity [8,9,10]. Methanol dehydration occurs on an acid catalyst. Zeolite based catalysts have several advantages for methanol dehydration to DME. For instance, zeolite-type catalysts exhibit better resistance to steam relevant to more conventional alumina. Thus, zeolites with controlled acidity, in particular ZSM-5, have been employed for direct dehydration of methanol to DME [11,12].

Catalyst deactivation is currently the most important challenge of direct DME synthesis. It reduces the catalyst life-time, DME productivity and hinders implementation of this process on a larger industrial scale. Previous reports suggest that the deactivation of copper zeolite hybrid catalysts can be due to the following phenomena: copper sintering, copper oxidation and migration to the zeolite acid sites, coke deposition on both copper nanoparticles and zeolites or contamination by impurities in syngas [13,14,15,16,17,18]. Because of the lack of direct characterization techniques working under harsh reaction conditions (high temperature and pressure), the information about deactivation mechanisms occurring in bifunctional Cu/ZSM-5 catalysts is rather contradictory. Catalyst removal and transfer from the catalytic reactor to the characterization equipment can significantly modify the copper oxidation state and whole catalyst structure.

Synchrotron-based techniques such as X-ray diffraction (XRD) and XANES/EXAFS provide important direct in-situ information about the structure of heterogeneous catalysts. High brilliance of synchrotron source allows acquiring time-resolved in-situ XRD patterns and X-ray absorption spectra even for diluted and dispersed systems exposed to high gas pressure. Previously, deactivation phenomena in cobalt Fischer-Tropsch catalysts were investigated with a greater precision using in-situ synchrotron-based techniques [19,20,21] In the present work, the structure of copper-zeolite hybrid catalysts during activation and under realistic conditions of direct DME synthesis from syngas was investigated using a combination of in-situ time-resolved XRD and XANES/EXAFS in the specifically designed experimental set up operating under high pressures and temperatures. The in-situ data are discussed together with the results of conventional ex-situ characterization and catalytic tests.

Experimental

Catalyst preparation

The hybrid catalyst for direct DME synthesis was prepared by physically mixing the powders

(

Ex-situ catalyst characterization

The zeolite and hybrid catalysts have been characterized at different preparation steps by a wide range of ex-situ techniques. The BET surface area, pore volume and average pore diameter were determined by low temperature N 2 adsorption using a Micromeritics ASAP 2000 automated system.

The NH 3 -TPD curves were obtained in an Autochem 2910 equipment (Micromeritics) coupled with a quadrupole mass spectrometer (OmniStar from Balzers Instruments). Prior to ammonia adsorption, ca. 100 mg of the sample was pretreated at 500 °C for 60 min in He. The chemisorbed ammonia was then desorbed by heating from 50 °C to 1000 °C at a heating rate of 10 ˚C/min. The m/z = 16 signal in the coupled mass spectrometer was used to obtain the corresponding NH 3 desorption profiles.

The TEM analysis was carried out on a Jeol 2100F (field emission gun) microscope operating at 200 kV equipped with a probe corrector for the spherical aberrations.

The catalyst reducibility was examined by temperature-programmed reduction (TPR) performed in an AutoChem II 2920 apparatus (Micromeritics). The samples were reduced in a flow of 5 % H 2 /Ar flow (60 ml/min) and heated up to 900 °C with the ramping rate of 10 °C /min.

In-situ catalyst characterization

The in-situ time-resolved XRD and XANES/EXAFS data in the transmission mode were simultaneously measured at the BM01B beamline (ESRF, France) using a high-resolution diffractometer, a gas manifold system and a capillary reactor. The experimental setup was described in detail in previous reports [19,20,21]. The copper-zeolite hybrid catalyst (3-5 mg) was loaded in the quartz capillary (OD=1 mm, wall thickness=0.02 mm) and pressed from both sides with quartz wool. The setup has zero dead volume and allows maintaining constant very low gas velocities (~1-2 ncm 3 /min) indispensable for conducting experiments in the capillary cell reactor at high pressure. Before the in-situ tests, the catalysts were reduced in hydrogen flow at 290 °C under atmospheric pressure. The in-situ time-resolved XANES/EXAFS XRD patterns were recorded under gas flows at desired temperatures and pressures. The experimental conditions were the same as in the reactor set up used for evaluation of the catalytic performance. The measuring times of an XRD pattern or an XAS spectrum were respectively 10 min and 5 min. In the co-feeding experiments, water purged from air was added to the reactor with the rate of 0.5 cm 3 /h using a Harvard syringe pump. In this work, copper metal crystallite sizes were calculated from broadening in-situ XRD patterns. Note that for the metal particles larger than 1.5-2 nm, the EXAFS nearest neighbor metal coordination numbers are not sensitive to the sizes of metal clusters [22].

Catalytic tests

The conventional DME synthesis catalytic tests were carried out in a fixed-bed stainless-steel tubular reactor (d int =8 mm). Before reaction, the sample was reduced in hydrogen flow at 290 °C.

The catalytic tests were conducted at 260 °C, total pressure of 20 bar, H 2 /CO ratio in syngas of 2 and different gas-space velocities.

Results and discussion

Structure of catalyst precursors

CuO and ZnO diffraction peaks were observed in the XRD patterns of the calcined CZA catalyst.

No diffraction peaks of the Al 2 O 3 phase were present even at the aluminum content of 10 %. This suggests that aluminum oxide is present in the amorphous form. The TPR profile measured for the CZA catalyst is shown in Figure 1. The hydrogen consumption area observed between 200 and 330 °C was attributed to the reduction of CuO into metallic copper. The characterization results for the CZA methanol synthesis catalyst are consistent with previous reports [23,24,25]. The parent ZSM-5 zeolite showed the ammonia desorption at two temperatures: a low-temperature peak at 100-300 °C and a high-temperature peak at 400-600 °C corresponding respectively to the weaker and stronger acid sites. The total amount of acid sites was 993 μmol/g (Table 1). [17,18]. In order to shed light on the evolution of the catalyst structure during the catalyst activation in hydrogen and exposure to syngas, we conducted in-situ synchrotron based X-ray absorption and diffraction experiments.

In-situ X-ray absorption and X-ray diffraction characterization

The XANES spectra and EXAFS Fourier transform moduli of the CZA/ZSM-5 hybrid catalyst during its reduction are presented in Figure 3a and3b. The XANES spectrum of oxidized hybrid catalyst resembles the spectrum of bulk CuO. The XANES spectrum of CuO has two distinguishable features characteristic of Cu 2+ (3d 9 ) complexes with a small pre-edge absorption at 8978 eV and a shoulder at 8986 eV, which is assigned to the 1s-4p "shake down" transition [23,26]. The Fourier transform moduli of EXAFS for the calcined copper zeolite hybrid catalyst are also similar to the reference spectrum of crystalline CuO (Figure 3b). Broader and lower intense radial distribution peaks for the second and third CuCu coordination shells in the calcined catalysts indicate smaller sizes of CuO particles compared to bulk copper oxide and a more significant Debye-Waller factor. Heating the CZA/ZSM-5 hybrid catalyst in hydrogen flow results in significant modifications of the XANES spectra and Fourier transform moduli. The XANES spectra show a major decrease in the intensity of white line, which corresponds to the formation of copper metallic phase (Figure 3a). Both XANES and Fourier transform modulus suggest that after treatment in hydrogen, the copper local environment in the catalyst reduced at 160 °C gets very similar to that in metallic copper.

The conclusion about the important concentration of copper metallic phase in the reduced sample is also consistent with the in-situ XRD data. The XRD patterns of the oxidized hybrid catalyst show the peaks attributed to CuO, ZnO and ZSM-5 zeolite. After heating in hydrogen at temperatures higher than 180 °C, new intense peaks attributed to Cu fcc (Figure 4) appear. The catalyst reduced at 290 °C, clearly shows characteristic peaks of Cu fcc metallic phase along with the peaks of the ZSM-5 zeolite. Cu fcc is the only copper phase detected by XRD in the reduced catalysts. The results are in agreement with the catalyst XANES/EXAFS (Figure 3a andb) and TPR for the CZA catalyst (Figure 1) showing copper reduction occurring during temperature ramping in diluted hydrogen (5%H 2 /Ar) between 200 and 330 °C.

Figure 5 shows TEM images of the oxidized CZA/ZSM-5 catalysts. Copper oxide nanoparticles of a few nm were clearly observed. The copper oxide crystallite sizes in the calcined catalyst calculated from XRD peaks broadening were in the range of 5-7 nm, while in the freshly reduced counterparts, the copper metallic crystallites have diameters of 9-10 nm. The observed increase in copper particle sizes after reduction compared to the sizes of CuO crystallites in the calcined catalysts (5-7 nm) is consistent with previous studies [23]. This indicates copper sintering, which occurs already during copper reduction. Indeed, if no copper sintering happened during the catalyst reduction, calculation using molar volume of CuO and Cu suggests that the copper particle size after the reduction should decrease by 60 % but not increase. Importantly, no further modifications of the in-situ XRD patterns were observed after extended heating of CZA/ZSM-5 in hydrogen at 290 °C.

After activation in hydrogen, the following experiments were conducted with the copper-zeolite hybrid catalyst under the conditions of direct DME synthesis in the conventional fixed bed reactor and under total pressure of 20 bar.

After the reduction, the catalyst in the capillary (4 mg) was exposed to the syngas (H 2 /CO=2) at atmospheric pressure and 120 °C. Then the system was pressurized up to 20 bar and temperature of the catalyst was risen up from 120 °C to 260 °C. In the first set of experiments, the catalyst was exposed to syngas with high gas space velocities (GHSV=90 000 cm 3 /g cat h). Noteworthy, no modifications of the XANES spectra were observed during the exposure to H 2 /CO (Figure 6). The in-situ XANES spectra measured are almost identical. The XANES shapes are similar to reference copper foil indicating the presence of mainly metallic Cu in the sample at the reaction conditions.

This suggests that no copper oxidation occurs.

The in-situ XRD analysis (Figure 7a) provided important information about modification of copper dispersion under these reaction conditions. As expected, Cu (111) XRD peak slightly shifts to smaller 2 Theta angles as a function of temperature, which is attributed to the increase in unit cell parameter. Note that the Cu unit cell parameter changes with the temperature from 120 to 260 °C because of the thermal expansion [START_REF] Touloukian | Thermal Expansion: Metallic Elements and Alloys[END_REF] of copper nanoparticles. In addition, a progressive narrowing of XRD copper fcc (111) peaks was observed. Copper sintering could be the most probable reason of the narrowing of Cu (111) XRD peaks. The mean diameters of copper fcc crystallites calculated from XRD patterns using the Scherrer method are displayed in Figure 7b.

The copper crystallite size increases from 9 nm to 12 nm as the copper-zeolite hybrid catalyst was heated in the flow of syngas at 120-260 °C temperature range.

In the second set of experiments, the syngas before entering the quartz capillary containing the catalyst (3.5 mg) passed through a stainless steel pre-reactor filled with the same CZA-ZSM-5 catalyst (100 mg). The catalyst pre-bed allowed us obtaining much lower gas space velocity (GHSV) without reducing the total gas flow rate. This is achieved by setting an additional catalyst bed heated at the same temperature upstream of the quartz capillary. The gas space velocity calculated taking into account the total amount of the catalyst in the pre-reactor and quartz capillary was 3600 cm 3 /g cat •h, which is close to the conventional GHSV used in direct DME synthesis [17] No change was observed in XANES spectra (Figure 6). This result confirms that the copper still remains in metallic state. At the same time, XRD analysis shows narrowing of the patterns attributed to copper metallic phase (Figure 8a). XRD peaks broadening analysis suggests an increase in copper crystallite size to 16 nm during the first 4 h of the reaction (Figure 8b). The increase in copper particle size seems to be more significant in the experiments with lower than with higher gas space velocity (d Cu =17 nm versus d Cu =12 nm). One of the possible explanations of the larger copper nanoparticles in the experiments with lower gas space velocity could be due to the presence of water generated during the DME synthesis, which could accelerate copper sintering and lead to larger copper nanoparticles. Note however, that even at higher CO conversion the pressure of water in DME synthesis could be rather moderate, because of significant water gasshift reaction.

In order to confirm further the effects of water on copper sintering, additional experiments were conducted at higher water pressure. In these experiments, water was intentionally added to the catalytic reactor. The setup contained a stainless steel pre-reactor heated at 260°C and filled with CZA-ZSM-5 (100 mg). After conducting the in-situ test with dry syngas, water with the rate of 0.5 cm 3 /h was co-fed to the reactor. The water co-feeding corresponded to the following composition of the reactor feed gas (H 2 /CO/H 2 O=2/1/5). The pressure of the introduced water was 12 bar. Cofeeding with water leads to higher rate of water gas shift reaction and higher production of CO 2 .

The effect was studied in detail in our recent work [18]. Addition of water resulted in further noticeable narrowing of copper XRD patterns (Figure 8a). The calculated copper particles size in the presence of water increased to 20 nm (Figure 8b). Note that the copper crystallite sze was about 9 nm in the activated catalyst. Analysis of the in-situ XRD data obtained both at high and low gas space velocities and with water co-feeding points out that, the stability of copper nanoparticle could be strongly affected by water pressure. Higher water pressure results in more significant copper sintering. Surprisingly, the reacting atmosphere has almost immediate effect on the size of copper nanoparticles. This suggests that kinetics of coper sintering is very fast under the conditions of DME synthesis. Note that no copper sintering was observed in the DME synthesis experiments previously conducted under atmospheric pressure [START_REF] Kabir | [END_REF]. Indeed, conducting reaction at atmospheric pressure should significantly reduce the water pressure even at the same carbon monoxide conversion and thus the rate of copper sintering.

The in-situ results are also consistent with the ex-situ characterization of the spent catalyst conducted using high resolution TEM, which revealed significant copper sintering (Figures 9 and10). In the catalyst freshly activated in hydrogen, Cu-based nanoparticles, which are homogenously distributed on the plate-like structures with sizes of about 10 nm or inferior, were observed. The small ZnO platelets are locally covered by very thin layers (1-2 nm) of Cu; these layers do not extend over the entire external surface of the support grain. The yellow arrows points to nanoparticles from the grains. In the catalyst exposed to the DME synthesis reaction (Figure 10), the copper nanoparticles are significantly larger (superior to 20 nm) than for the initial specimen. No small nanoparticles or thin layers were identified on the supporting grains, most probably due to Cu migration under the reaction conditions.

Thus, copper sintering under the conditions of DME synthesis has been shown by both in-situ and ex-situ characterization. The most important finding obtained using the in-situ characterization is relevant to the kinetics of copper sintering. Our data indicate very fast kinetics of copper sintering.

The final size of copper nanoparticles is strongly affected by the presence of water.

In addition to rapid deactivation, the copper zeolite hybrid catalyst also exhibits longer-term deactivation for dozen hours on stream (Figure 2). Note that the deactivation of copper zeolite catalysts in direct DME synthesis may involve several mechanisms. Though copper sintering obviously contributes to the overall catalyst deactivation, the longer-term deactivation after 100 h of reaction (Figure 2) is strongly influenced by other phenomena. As suggested in the previous reports [17], these deactivation mechanisms may involve copper migration inside the zeolite with ion exchange with the zeolite hydroxyl groups. Coke deposition in the zeolite pore and over copper metal species [29] leading to the catalyst deactivation cannot be also excluded in particular, at longer reaction time.

Conclusion

The T=260 ˚C, 3600 cm 3 /g cat -1 h -1 , H 2 /CO=2, 20 bar)

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .

 123456 Figure 1. TPR profile of CZA catalyst

Figure 7 .

 7 Figure 7. Evolution of the Cu (111) XRD peak (a) and copper metal nanoparticle size calculated

Figure 8 .

 8 Figure 8. Evolution of the Cu (111) peak (a) and copper metal nanoparticle size calculated using

Figure 9 .Figure 10 .

 910 Figure 9. TEM images of CZA-ZSM5 after activation in hydrogen and before the catalytic reaction

  The catalytic data in direct DME synthesis over the hybrid CZA/ZSM-5 catalysts measured in a conventional catalytic setup are presented in Figure2. The reaction was conducted at 260 °C, total pressure of 20 bar. The DME selectivity was over 60%, while the selectivity to CO 2 was about 30 %. In direct DME synthesis, CO 2 is produced by the WGS reaction: CO+H 2 O=CO 2 + H

2

. The methanol and hydrocarbon selectivities (mostly methane) were about 4 %. Carbon monoxide conversion at different gas space velocities slowly decreased with time on stream. This suggests that Cu-zeolite hybrid catalyst exhibits noticeable deactivation. Note that the reaction selectivity is only slightly affected by the deactivation. The observed catalytic performance and catalyst deactivation are consistent with previous reports

Table 1 .

 1 in-situ synchrotron experiments performed during catalyst activation and under the realistic conditions of direct DME synthesis (high pressure, 260°C) in combination with the conventional ex-situ characterization unambiguously uncovered rapid copper sintering in the hybrid copperzeolite catalysts, while no copper oxidation was detected. Copper is always present in the metallic state under a wide range of reaction conditions. Copper sintering, which proceeds with a very high rate, leads to larger particles in the presence of more significant amounts of water. The in-situ data are consistent with the results of ex-situ characterization of spent catalysts. Copper sintering could be one of key factors responsible for the rapid short-term deactivation of copper zeolite hybrid catalysts during direct DME synthesis from syngas. Physico-chemical characteristics of the parent ZSM-5 zeolite

	29 H. Zhang, W. Li, W. Xiao, International Journal of Chemical Reactor Engineering
	10(2012) A82.

Acknowledgements

The authors are thankful to Dr. W. van Beek for the help with synchrotron experiments. The support of the French National Research (CATSYN-BIOFUELS project, ANR-12-BS07-0029) is gratefully acknowledged. M. C. thanks China Scholarship Council for the PhD stipend. The European Synchrotron Radiation Facility is acknowledged for providing synchrotron beam time.