Dr R Lucas
email: romain.lucas@unilim.fr

Keywords: Manufacturing, Consolidation, Hybrid, Ceramic-matrix composites (CMCs)

ZrC/SiC composites were fabricated using Spark Plasma Sintering (SPS) combined with the Polymer-Derived Ceramics (PDC) route. The originality of this work, consisted in the direct ceramization/sintering of a ZrC/polymer mixture without any previous crosslinking steps. The presence of a polymer in our mixtures required an optimization of the thermomechanical parameters of SPS in order to promote the polymer-to-ceramic conversion prior to the densification. The optimized SPS conditions were composed of a dwell temperature of 700 °C for 10 minutes followed by a soaking temperature of 1700 °C for 5 min under an applied load of 75 MPa. A fine and homogenous phase distribution was achieved at relatively low temperature (1700 °C) compared to

Introduction

In application areas such as aeronautics, aerospace, or nuclear, the use of materials operating under extreme environmental conditions is required [START_REF] Paton | Materials for advanced space propulsion systems[END_REF][START_REF] Borrelli | Thermo-structural behaviour of an UHTC made nose cap of a reentry vehicle[END_REF][START_REF] Xie | Thermal stress analysis of the FGLCS in hypersonic vehicles: Their application to fuel injection struts in scramjets[END_REF].

They include the family of carbides, which are non-oxide ceramics with a melting temperature higher than 2000 ° C and good mechanical properties [START_REF] Kral | Critical review on the elastic properties of transition metal carbides, nitrides and carbonitrides[END_REF]. Zirconium carbide is one of these carbides with a good refractoriness and interesting mechanical properties at high temperatures. However, it has a low oxidation resistance, which limits its use in the presence of oxygen at high temperature [START_REF] Shimada | Oxidation Kinetics of Zirconium Carbide at Relatively Low Temperatures[END_REF].

To overcome this problem, the use of composite materials based on silicon carbide is a promising route. Indeed, silicon carbide has, in addition to its thermomechanical properties close to those of ZrC, a passive oxidation resistance at high temperature, which limits the degradation of the material [START_REF] Pizon | Oxidation behavior of spark plasma sintered ZrC-SiC composites obtained from the polymer-derived ceramics route[END_REF].

Thus, the combination of these two materials should generate high-performance ceramics.

For this, different approaches have been used to incorporate an SiC phase into the ZrC matrix in order to achieve well homogenous ZrC/SiC composites with the desired properties. Mixing and sintering of carbide powders is a commonly method to manufacture such composites. However, the densification of these carbides requires high sintering temperatures and pressure-assisted techniques to achieve dense bodies. For example, Zhao et al. [START_REF] Zhao | Pressureless sintering of ZrC-based ceramics by enhancing powder sinterability[END_REF] have reached relative densities of 86.6% for ZrC and 90.8% for ZrC/20vol% SiC by pressureless sintering at 1900 °C combined with high-energy ball milling. The increase of the sintering temperature to 2100°C has improved the relative densities of ZrC and ZrC/20vol% SiC to 98.4% and 96.7% respectively.

In this way, research has focused on the use of field-assisted sintering techniques such as Spark Plasma Sintering (SPS) to improve the densification without resorting to high sintering temperatures. Starting from ZrC and SiC powders, a fully dense (99% relative density) ZrC/SiC (10 vol%) composite can be obtained by SPS under vacuum at 1850 °C for 5 min and a uniaxial pressure of 40 MPa [START_REF] Sagdic | Mechanical Properties of Spark Plasma Sintered ZrC-SiC Composites[END_REF]. Despite the densification improvement of the composite by SPS, utilizing carbide powders as starting materials did not offer the possibility to control the shaping of materials as well as their chemical and microstructural homogeneity. Indeed, the latter may enhance the mechanical properties and the oxidation resistance of the composites.

In this aim, a promising route to manufacture ZrC/SiC composites known as the Polymer-derived Ceramics (PDC) is increasingly explored [START_REF] Bao | Different strategies for the synthesis of silicon carbide-silicon nitride composites from preceramic polymers[END_REF]. Indeed, the use of liquid preceramic polymers opens a new way to produce advanced multicomponent ceramics by multiple techniques such as Precursor-Infiltration-and-Pyrolysis technique (PIP) [START_REF] Chen | Effects of polymer derived SiC interphase on the properties of C/ZrC composites[END_REF][START_REF] Jones | Polymer derived ceramic matrix composites[END_REF] or by mixing a polycarbosilane as a source of SiC phase with carbide powder [START_REF] Lucas | Elaboration of ZrC-SiC composites by spark plasma sintering using polymerderived ceramics[END_REF]. Nonetheless, the latter, requires a crosslinking step, which may induce an increase of the oxygen amount in the structure.

In this work, well homogenous ZrC/SiC composites were prepared using an SPS technique and the PDC route.

A first part of the work consisted in controlling the quality of the synthesized ZrC powders in terms of chemical composition and particle size. The second part dealt with the mixing of these synthesized ZrC powders and a commercial preceramic polymer as SiC precursor. The peculiarity of our method lies in the ceramization/sintering of the prepared mixtures directly by SPS without prior cross-linking. The optimization of sintering parameters was studied also and related to the final relative densities and microstructures of the composites.

Experimental procedure

Raw chemicals and powders

For the synthesis of ZrC powders, commercial zirconia (Alfa Aesar, Germanypurity 99.5%) and amorphous carbon black (Prolabo, France -purity 99.25%) were used as starting precursors. For the SiC phase, a commercially available preceramic polymer, the Allylhydridopolycarbosilane SMP10 (Starfire Systems Inc., USA) was utilized as received. It is a clear amber liquid with a theoretical molecular formula [SiH (CH2CH=CH2) CH2]0.1n [SiH2CH2]0.9n and a viscosity between 0.04 and 0.1 Pa.s at 25 °C.

Synthesis of ZrC powders and ball-milling process

The carbothermal reduction of zirconia has been employed to synthesize zirconium oxycarbide powders following the synthesis protocol presented in a previous paper [START_REF] Gendre | Synthesis of zirconium oxycarbide (ZrCxOy) powders: Influence of stoichiometry on densification kinetics during spark plasma sintering and on mechanical properties[END_REF]. Assuming a complete occupation of the octahedral sites of the fcc lattice of zirconium by carbon and oxygen, i.e. the sum of (C/Zr) and (O/Zr) is equal to 1; the carboreduction reaction can be expressed as follows:

ZrO2 (s) + (1+2x) C (s) ⇒ ZrCxO1-x (s) + (1+x) CO (g)
The carbon black and the commercial zirconia were mixed in mass proportions (77.96 wt% ZrO2 and 22.04 wt% C) and then heat-treated at 1750 °C for 8 hours in a graphite resistance furnace (oven V.A.S., Suresnes, France) in an argon flow (30 L.h -1 ) [START_REF] Gendre | Synthesis of zirconium oxycarbide (ZrCxOy) powders: Influence of stoichiometry on densification kinetics during spark plasma sintering and on mechanical properties[END_REF]. The obtained ZrC powders were agglomerated and therefore, required a ball-milling step to reduce their size and thus, improve the sinterability and the powder impregnation by the polymer.

Among the various ball-milling techniques, attrition milling of carbides seemed to give promising results [START_REF] Seo | Preparation of highly dispersed ultra-fine ZrC by combination of carbothermal reduction of ball-milled ZrO2 and C mixture and bead milling[END_REF]. For that, the synthesized powders were put in a container among with ZrO2 beads (Netzsch, Germany, Ø = 0.8 mm) and 200 mL of distilled water. The ball to powder ratio (BPR) was fixed at 10:1 in weight. The suspension was then stirred at 2000 rpm for one hour to obtain strong shearing forces and then dried in an oven (110 ° C). Once the desired granulometry is achieved, the powders were mixed with the commercial polymer in order to prepare the ZrC/polymer systems.

Preparation of the mixture (ZrC/polymer) and densification

To obtain the ZrC/polymer mixture, different proportions of ZrC/(0-20wt% SiC) were tested. The liquid preceramic polymer was directly mixed with the milled ZrC powders in toluene solvent, and then stirred for 24 hours at room temperature. At the end, the mixtures were dried in a rotary evaporator. The whole process is presented in Fig. 1.

After the evaporation, the composite systems were poured into a graphite die with an inner diameter of 13 mm, and finally densified under vacuum following an optimized treatment by Spark Plasma Sintering (Syntex, Japan, Dr.Sinter 825).

Characterization

Synthesized ZrC powders

The global oxygen and carbon content were measured by Instrumental Gas Analysis (IGA) using EMIA 820V for carbon and EMGA 620W for oxygen. For each analysis, CO(g) and CO2(g) released after the combustion of the powders were quantified by IR measurements. These measurements were performed in an alumina or a graphite crucible for the carbon and oxygen analyses, respectively. The calibration was achieved by steel standards containing well known carbon and oxygen contents. Zirconium content (mass percentage) was calculated taking into account the mass percentage of oxygen and the carbon obtained experimentally and using the principle of mass conservation for a composition ZrCxOy (Zr(wt%) = 100 -C(wt%) -O(wt%)). The oxycarbide powders were analyzed by X-ray diffraction with CuKα1,2 and radiations using LYNXEYE XE-T detector (D8 advance, Bruker, Wissembourg, France) for angles (2θ) ranging between 20° and 120° (step: 0.02°, step time: 0.3 s) to identify the crystallized phases. Scanning electron microscopy (JSM -IT300 LV, Japon) and transmission electron microscopy (JEOL -2100F, Tokyo, Japan) characterizations were carried out to observe the morphology of the synthesized oxycarbide powders. The size distribution of the powder before and after attrition milling was determined by laser granulometry (Horiba LA950, Kyoto, Japan).

Polymer (SMP-10)

Elemental analyses (C, H, O) of the polymer were investigated using a Flash EA 1112 (Thermo Finnigan). The structural characterizations of the polymer were carried out using different techniques. Attenuated Total Reflectance infra-Red spectroscopy (Nicolet IS10, Thermo Scientific, logiciel Omnic), in transmittance mode (4000-400 cm -1 ) was performed. 29 Si liquid-state NMR spectra were recorded at 99 MHz on a Bruker Advance 500 spectrometer with a number of scans of 12288 and a relaxation time of 5 seconds. 29 Si Solid-state NMR was used to characterize the pyrolyzed polymer (1400 °C). The spectra were recorded at 139 MHz on a Bruker Avance 700 spectrometer with a number of scans of 456 and a relaxation time of 120 seconds. For both analyzes, chemical shifts were referenced to tetramethylsilane (δ = 0 ppm). The thermal behavior was studied by a thermogravimetric analyzer (STA 449F3-Netzsch) coupled with a mass spectrometer (Omnistar, Balzers Instrument) following these experimental conditions: maximum temperature: 1400 °C, argon flow: 20 mL.min -1 , heating rate: 10 °C.min -1 .

Mixture (ZrC/polymer)

X-ray photoelectron spectroscopy (XPS, Kratos Axis Ultra DLD spectrometer) was used to highlight the presence of the polymer on the surface. A monochromatic AlKα source (15 mA, 15 kV) was utilized. All binding energy scales were recalibrated so that the C1s peak arising from surface contamination is placed at 285.0 eV. The pass energy was 20 and 160 eV for the high-resolution spectra.

Densified composites

Phase identification and distribution of the composites were analyzed by XRD and SEM following the same parameters applied to the synthesized ZrC powders.

Densities were determined using the Archimedes method with deionized water.

Apparent elastic constants (Young's modulus, shear modulus) were determined by ultrasonic method using 10MHz transductors working in reflexion mode (WC37-10 and SW37 10,Ultran,State College, USA) on 2 mm thick samples.

Vickers hardness was determined by microhardness tester (Micromet 6040, Buehler, USA) under a load of 100 g and dwell time of 10 s. the synthesized powders did not show any excess of carbon. The particles displayed an agglomeration tendency forming aggregates with a diameter ranging from 400 to 700 nm. Moreover, SEM characterization confirmed a coalescence of the particles (fig. 3.b). The presence of these agglomerates could be detrimental to the sintering of the composites. Indeed, a preferential sintering of the agglomerates (which may contain porosity) instead of elementary particles is likely to limit the densification of the composites. The objective was therefore to reduce the size of these agglomerates in order to improve the sinterability and the powder impregnation by the polymer. Among the various ball-milling techniques, attrition was performed following an optimized milling cycle. According to fig. 3.c, raw ZrC powders had a monomodal size distribution centered at 3.5 µm. After the milling, the average diameter of the agglomerates decreased to reach 1.7 µm. As a result of strong shearing forces applied by the micrometric milling beads, the optimized milling cycle proved to be efficient in dispersing the initial agglomerates into smaller agglomerated particles with a monomodal size distribution centered at 1.7 µm. After controlling the quality of the synthesized ZrC powders in terms of chemical composition and particle size, the thermal behavior and the chemical composition of the commercial polymer (SMP-10) were studied. This thermal behavior will be taken into account to optimize the sintering of the hybrid systems.

Structural characterizations and thermal behavior of the polymer

The structure/composition of the polymer influences the chemical composition of the final ceramic and therefore its physico-chemical properties [START_REF] Colombo | Polymer-Derived Ceramics: 40 Years of Research and Innovation in Advanced Ceramics[END_REF]. To elucidate the SMP-10 composition, elemental analysis was used. It can be seen from Tab.1 that the experimental composition is almost free of oxygen (< 0.05wt%) with a chemical formula near to the theoretical formula presented in literature (SiC1.33H4.4) [START_REF] Kaur | Pressureless fabrication of dense monolithic SiC ceramics from a polycarbosilane[END_REF]. Once the chemical composition and the structure of raw SMP-10 (fig. S1 and fig. S2a) were verified, it was necessary to investigate its thermal behavior. For this, SMP-10 was analyzed by TGA/MS at 1400 °C. TGA/MS curves showed three domains of mass loss (fig. 4). The first step started at 100 °C and indicated a mass loss of 10%. Li et al. [START_REF] Li | Effect of the polycarbosilane structure on its final ceramic yield[END_REF] stated out that at this temperature range, the evaporation of small oligomers is considered to be the main reason for weight loss. This assumption was confirmed by the MS spectra, which did not show any fragmentation of molecules (H2 or CH4). The second step began at 400 °C and displayed a mass loss of 14%, caused by the volatilization of fragments such as m/z = 2 (H2) and m/z = 16 (CH4). These fragments were detected by Li et al. [START_REF] Li | Effect of the polycarbosilane structure on its final ceramic yield[END_REF] and Yu et al. [START_REF] Yu | Template-Free Synthesis of Porous Fe3O4/SiOC(H) Nanocomposites with Enhanced Catalytic Activity[END_REF], and attributed to the dehydrocoupling reactions and decomposition of the organic groups, which indicate the beginning of the polymer-to-ceramic conversion. At the beginning of 600 °C, a low mass loss (3%) was observed accompanied also with the volatilization of m/z = 2 (H2), m/z = 16 (CH4) and also fragment with higher molecular weight m/z = 44 (C3H8). This feature represented the final stage of the polymer-to-ceramic conversion.

Beyond 800 °C, no significant mass loss was recorded, indicating a complete polymer-to-ceramic conversion with a ceramic yield of 73%. It should be noted that these gaseous species represent a critical parameter to be considered during the sintering optimization. Indeed, microstructural defects may be generated by the release of gaseous species during sintering. Furthermore, the heat treated SMP-10 were characterized by solid-state NMR 29

Morphological and chemical characterizations of the mixtures

ZrC/polymer

Once the powder was synthesized and the polymer characterized, both were mixed according to the protocol presented previously. The aim of this step of the process was to prepare powder/polymer mixtures where the polymer would be well dispersed in the mixture and would homogeneously coat the ZrC particles. This could favor the homogeneity of the final material. Indeed, after the evaporation, two textures were obtained (fig. S3). The compositions were calculated as a function of the equivalent mass percentage of SiC obtained after the heat treatment of the polymer. The compositions with a wt%SiC less than 10% had a pulverulent aspect while the composites with a wt%SiC higher than 10% displayed a viscous aspect (fig. S3). The samples with more than 10wt% of SiC were unsuitable for being sintered by SPS because of the leakage of matter when applying pressure. During this work, we concentrated only on the study of ZrC/(10wt%SiC) composition.

First, TEM analyses were used to investigate the morphology of zirconium carbide powder and the mixture ZrC/polymer at the nanometric scale. According to the images, ZrC particles presented a thin layer at the surface with a thickness of 3 nm (fig. 5.a). This layer is generally present in carbides and can be attributed to oxides phases [START_REF] Réjasse | Influence of chemical composition on mechanical properties of spark plasma sintered boron carbide monoliths[END_REF]. In fig. 5.b, a second amorphous layer appeared with a thickness ranging from 5 to 10 nm, likely due to the commercial polymer SMP-10.

To determine the surface composition of the mixture, a further XPS study was performed to define the nature of the bonds present on the surface. Among the detected elements (C, O, Si, Zr), the spectral zones of silicon and zirconium were presented.

For ZrC powders (fig. 6.a), the deconvolution of the Zr3d peak showed four contributions. The two lowest binding energy peaks (179.51 eV, 181.94 eV) can be attributed to Zr-C bonds and can be assigned to zirconium carbide, while the two highest bending energy peaks (182.59 eV, 185.02 eV) correspond to Zr-O bonds and can be attributed to amorphous zirconia. These results are consistent with the work of Chu et al. [START_REF] Chu | Carbothermal synthesis of ZrC powders using a combustion synthesis precursor[END_REF] and Lucas et al. [START_REF] Lucas | A simple route for organic covalent grafting onto zirconium carbide particles[END_REF], and confirm the presence of an oxide layer onto the synthesized powders surface. The presence of the polymer was confirmed from the Si2p spectrum (fig. 6.b). The deconvolution of the Si2p peak indicated two contributions at 101.46 and 102.59 eV. These peaks were located slightly far from the binding energy usually obtained from Si-C bonds in stoichiometric silicon carbide (100.5 eV) [START_REF] Busiakiewicz | Surface chemical composition of SiC-cored nanowires investigated at room and elevated temperatures in ultra-high vacuum[END_REF] and were assigned to Si-C bonds that belong to an Ox-Si-Cy environments (fig. 7.b). These results were in good agreement with XPS analyses of previous works [START_REF] Li | Silicate glass coated microchannels through a phase conversion process for glass-like electrokinetic performance[END_REF] where an hydrolyzed AHPCS film was analyzed. It is worth adding that since our polymer did not have any oxygen inside its structure (Tab.1), the presence of Ox-Si-Cy environments may be related to a possible reaction between either the zirconia layer or the adsorbed water and the hydrogenosilane functions of the polymer [START_REF] Osei-Agyemang | Periodic DFT and Atomistic Thermodynamic Modeling of Reactivity of H2, O2, and H2O Molecules on Bare and Oxygen Modified ZrC (100) Surface[END_REF].

Densification of the mixtures ZrC/polymer

Due to the presence of polymer in our mixtures, our optimization was firstly focused on the determination of a temperature that will promote the conversion of the polymer prior to sintering. Relying on the TGA results, which showed a mass loss up to 800 °C (fig. 4), three temperatures were chosen, i.e. 600,700 and 800 °C. During sintering, the temperature was measured by the pyrometer on the die surface, which is largely underestimates the actual temperature of the sample [START_REF] Antou | Spark plasma sintering of zirconium carbide and oxycarbide: Finite element modeling of current density, temperature, and stress distributions[END_REF]. This optimization was carried out using a fixed dwell time of 10 minutes and a pressure of 25 MPa. The latter was then increased at the end of the dwell time to reach 75 MPa. SPS conditions for the sintering step were then fixed with dwell time of 5 min, a heating rate of 200 °C/min and a pressure of 75 MPa, under vacuum. The maximum temperature was fixed at 1950 °C according to anterior studies [START_REF] Lucas | Elaboration of ZrC-SiC composites by spark plasma sintering using polymerderived ceramics[END_REF].

The evolution of the gas pressure in the SPS chamber and of the associated thermal treatment as a function of the time was plotted (fig. 7). Three different degassing steps, characterized by a peak in the pressure, were highlighted depending on the dwell temperature. The first peaks presented a high gas release at a temperature lower than the pyrometer detection (<573°C). This was due to the release of gaseous species caused during the conversion of the polymer into ceramics. The first step showed a less pronouncing degassing for the test conducted at 600 °C. The second step, which occurred when the heating up restarted, may be caused by the release of gaseous species resulting from the first step and that have condensed on the coldest areas of the SPS system, or it can be associated with the end of organic-inorganic conversion. It is also worth mentioning that the degassing at 600 °C was higher indicating that the release of gaseous species might still occur at this stage. Finally, the third step may correspond to an in-situ carboreduction of the oxide layer, which was often observed in the sintering of carbides [START_REF] Georges | Approche du frittage SPS de céramiques fines de carbure de bore : rôle des poudres initiales et de la mise en forme[END_REF].

As a result of this first optimization, different microstructures were obtained. The three microstructures presented a well homogenous distribution of the SiC phase into the ZrC matrix (fig. 8). Nevertheless, residual porosity was present in the microstructures. Indeed, the use of a dwell temperature of 600 °C (fig. 8.a) was not sufficient to promote a complete departure of volatile fragments. It is possible that some organic species remained trapped in the structure, and left as heating up restarted (fig. 8.a). This was consistent with the degassing curve at 600 °C, which showed a higher gas release at the beginning of step 2. Nevertheless, the increase of the applied pressure until 75 MPa at the beginning of step 2 may cause entrapment of gas from the organic species, and thus, generating porosity after sintering. A dwell temperature of 800 °C displayed a more porous microstructure (fig. 8.c). Indeed, the higher dwell temperature would cause a brutal removal of organic species generating microstructural defects with larger porosity (fig. 8.c). These differences were also observed by looking at the open porosity and the density (tab.3). Interrupted tests, before heating up restarts, were also performed at the three temperatures in order to analyze the morphology of the mixtures after the conversion step (fig. S5). For either manually interrupted or completed tests, the conversion at 800 °C led to higher open porosities (tab.3).

In addition, the impact on the density clearly identified the dwell temperature of 700 °C as the best choice in the conversion route from organic to ceramic. It should also be noted that the pellets obtained with this dwell temperature broke during polishing, unlike the ones sintered using 600 and 700 °C dwell temperature, respectively. Finally, the microstructure obtained using a conversion temperature of 700 °C displayed a good compromise between porosity and homogeneity (fig. 8.b and fig. S5). Indeed, the latter showed less porosity compared with the two other microstructures. It seemed that the use of a conversion temperature of 700 ° C promoted the total conversion of the polymer while avoiding the formation of microstructural defects generated by a brutal departure of organic species. For this, this conversion temperature was selected for the following tests.

Once the dwell temperature for the polymer to ceramic conversion was figured out, the optimization of the maximum sintering temperature was required in order to promote the removal of the remained porosity. For this, interrupting tests were performed from room temperature to 1950 °C in order to investigate the evolution of the microstructure during sintering. Each test was preceded by a dwell temperature of 700 °C for 10 minutes to promote the polymer to ceramic conversion prior to sintering.

The evolution of the displacement and the temperature as a function of time as well as the evolution of the microstructure at different steps of the sintering were studied (fig. 9). At room temperature, the microstructure showed ZrC particles coated with polymer. It was then converted during the first dwell time, and appeared to maintain the particles linked to each other (800 °C). It was also noticed that the particle size of ZrC (Dm< 1µm) did not change during the conversion of the polymer and the pellet was cohesive after the interrupted test.

The increase of the displacement was detected right before the heating restarted up. It was associated to the increase of the pressure from 25 to 75 MPa. By the beginning of the sintering around 1550 °C (confirmed by the increase of the displacement), the open porosity started to decrease. These first three temperatures (25, 800 and 1550 °C) represented the first step of sintering, which was manifested by the rearrangement and bridging between particles. Right after, the densification (increasing of the displacement) and the elimination of the open porosity occurred. Indeed, the open porosity decreased from 24 to 0.7% between 1550 and 1700 °C, respectively. The microstructure at this temperature showed a fine and homogenous phase distribution. By increasing the temperature until the end of the thermal treatment (1950 °C), step 3 of sintering occurred. Indeed, it was manifested by a coarsening of the SiC phase (black particles) and a growth of porosity (especially closed porosity), which was detrimental to densification [START_REF] German | Coarsening in Sintering: Grain Shape Distribution, Grain Size Distribution, and Grain Growth Kinetics in Solid-Pore Systems[END_REF]. Following these results, it seemed that the use of the optimized dwell temperature (700 °C) followed by a sintering at a maximum temperature of 1700 °C (without dwell time) offered a good compromise between obtaining a dense microstructure with a fine and homogeneous distribution of phases (fig. 10). The estimated size of SiC domains is centered at 300 nm (estimated by Image J tool, fig. 10.b).

Microstructure and mechanical properties of the sintered composites

After the optimization of the sintering cycle, the influence of SiC on the mechanical properties of ZrC were studied. For that, three samples of ZrC/ (0-10wt%SiC) were sintered using the optimized sintering cycle with a dwell time of 5 minutes at 1700 °C. undetectable open porosity and a relative density of 97% was obtained. As a result, it appears that increasing the SiC content improve the densification. The relative density increased from 93.4 to 97.1% for a ZrC monolith and a ZrC / 10wt% SiC composite, respectively. The improvement of the densification may be due to the homogenous distribution of SiC particles at grain boundaries and thus inhibiting grain growth of ZrC. It is possible also that the use of the preceramic polymer has a positive impact on the densification by improving rearrangement between particles during the first step of sintering. Regarding the mechanical properties, the addition of the SiC retained the elastic constants of the ZrC monolith. For a composition of ZrC /10wt%SiC, the elastic constants reported by Lucas et al. [START_REF] Lucas | Elaboration of ZrC-SiC composites by spark plasma sintering using polymerderived ceramics[END_REF] are pretty close to the one obtained in this study. For the Vickers hardness, the reported values were quite close to those reported by Sagdic et al. [START_REF] Sagdic | Mechanical Properties of Spark Plasma Sintered ZrC-SiC Composites[END_REF] but lower than the Vickers hardness of both monolith ZrC and SiC (Hv > 20 GPa) [START_REF] Ho | Handbook of Refractory Carbides and Nitrides: Properties, Characteristics, Processing and Applications[END_REF].

Conclusion

During this work, a promising route was employed to manufacture ZrC/SiC composites using PDC route and Spark Plasma Sintering. The originality of this work consists in the direct ceramization/sintering of a ZrC/polymer mixture without any previous crosslinking steps. For that, a control of the quality of the synthesized powders (chemical composition and particle size) and an optimization of the sintering parameters were required. The original process used in this study gave access to fabricate composites with a well fine and homogenous phase distribution and good mechanical properties at relatively low temperature (1700 °C) compared to the high temperature required for the sintering of this refractory composites. The influence of the addition of SiC (up to 10wt%) was studied and related to the final relative densities and mechanical properties of the composites. The main following conclusions can be issued from the obtained results:

-TEM analysis has demonstrated the presence of two layers on the surface of the mixture, one associated with the oxide layer and the other with the preceramic polymer. This would be beneficial for a potential fabrication of core-shell systems;

-The optimization of sintering parameters revealed that a dwell time of 10 minutes at 700 °C was essential to promote a conversion of the polymer into ceramics, followed by a treatment at 1700 °C for 5 minutes to fully densify the composites; 20 -The addition of 10 wt%SiC increased the relative density from 93.4 to 97.1% and a well homogenous phase distribution with fine SiC domains (300 nm) was achieved; -Finally, it has been shown that the addition of the SiC slightly increase the elastic constants of the ZrC monolith and does not affect its Vickers hardness. The apparent Young modulus, shear modulus and Vickers hardness were 402 GPa, 166 GPa, and 15.9 GPa respectively for the ZrC/ 10wt%SiC.

Samples

Elemental analysis Calculated composition C (wt%) O (wt%) H (wt%) Zr (wt%) Si (wt%)

Synthesized ZrC 1 10.4 ± 0.2 1.5 ± 0.

1

  Preparation of ZrC/SiC composites by using polymer-derived ceramics and spark plasma sintering Hatim LAADOUA 1 ; Nicolas PRADEILLES 1 ; Romain LUCAS 1 *; Sylvie FOUCAUD 1 ; William John CLEGG 2

  Si and XRD. The NMR spectra (fig.S2.b) showed a single chemical shift at -15 ppm, which reflected the single environment of the silicon in the cubic form of silicon carbide (β-SiC) [20]. To complete this analysis, the XRD diagram confirmed the presence of broad peaks associated to a nanocristallized β-SiC phase (fig.2.b).

  On the one hand, from fig.11.b and tab.2, the addition of 5wt%SiC did not affect the densification of the ZrC monolith (fig.11.a) despite a decrease in the open porosity. On the other hand, the addition of 10wt%SiC (fig.11.c) offered a well homogenous and fine microstructure of SiC phases. Indeed, a sample with an

Fig. 1 .Fig. 2 .Fig. 3 . 10 Fig. 5 .Fig. 6 .Fig. 7 .Fig. 9 .Fig. 11 .

 12310567911 Fig.1. General process for the preparation of ZrC/SiC

Fig. 1 .Fig. 2 .Fig. 3 . 10 Fig. 5 .Fig. 6 .Fig. 7 .Fig. 9 .Fig. 11 .

 12310567911 Fig.1. General process for the preparation of ZrC/SiC

Samples Interrupted tests Completed tests Open porosity (%) Density (g/cm 3 ) Open porosity (%) Density (g/cm 3 ) Relative density (%) Conversion at 600 °C 33

  : measured by Flash EA 1112 Zr (wt%) and Si (wt%) : calculated using the principle of mass conservation Tab.1. Elemental analysis of the synthesized ZrC powders and the raw SMP-10 Tab. 3: Open porosity and density measurements of ZrC/10wt% SiC samples obtained after the interrupted and complete tests, respectively

			2	-	88.1	-	ZrC 0.90 ± 0.02 O 0.10 ± 0,01
	SMP-10 2	33.1 ± 0.1	< 0.05	8.7 ± 0.1 -	58.2	SiC 1.33 ± 0.01 H 4.17 ± 0,06
	1 : measured by IGA
	Samples	Open porosity	Young's modulus	Shear modulus	Vickers hardness
			(%)	(GPa)	(GPa)	(GPa)
	100 ZrC		4	375 ± 2	155 ± 1	16.4 ± 1
	ZrC / 5wt% SiC		0.3	401 ± 2	168 ± 1	16.3 ± 1
	ZrC / 10wt% SiC	0	402 ± 2	166 ± 1	15.9 ± 1
	Tab.2. Mechanical properties of the sintered samples
				3.70	< 1	6.09	97
	Conversion at 700 °C	30	3.81	< 1	6.21	99
	Conversion at 800 °C	36	3.72	2		6.08	97

2

Results and discussion

Chemical composition and morphology of ZrC powders

Firstly, XRD analysis (fig. 2.a) highlighted the presence of the characteristic peaks of a single-phased XRD pattern that can be indexed to zirconium carbide (PDF file 00-035-0784). Residual zirconia or carbon black were not detected, which confirmed a complete reaction between these two reagents. It was therefore necessary to determine accurately the chemical composition of the synthesized powders.

The samples were globally analyzed by elemental analyses. The results reported in Tab.1 stated out a composition of ZrC0.90O0.10 for the synthesized powders with a good correlation between the oxygen and carbon amounts. Zirconium carbide being a sub-stoichiometric carbon compound, it has a solubility limit between ZrC0.55 and ZrC0.98. In addition, the size of the oxygen being close to that of carbon, it can be introduced into the crystal lattice of the carbide forming a solid solution ZrCxOy. The results obtained in this study were in good agreement with the elemental analysis of Réjasse et al. [START_REF] Réjasse | Experimental investigation and thermodynamic evaluation of the C-O-Zr ternary system[END_REF] for the same composition. The authors explained that the amount of detected oxygen may come from the oxygen incorporated into the zirconium oxycarbide crystal lattice since crystallized zirconia was not detected by XRD. The amount of carbon may represent the contribution of both structural carbon incorporated within the oxycarbide lattice and the unreacted free carbon.

The authors suggested also that TEM is a powerful tool to investigate the presence of free carbon in the oxycarbide powders. Indeed, due to the molar mass of carbon, a slight molar excess leads to a large volume proportion, which is easily detected by TEM observations. Referring to the TEM image (fig. 3.a),


Florian Paillet
email: florian.paillet@hotmail.fr

Carole Barrau

Renaud Escudié

Eric Trably

Inhibition by the ionic strength of hydrogen production from the Organic Fraction of Municipal Solid Waste

Keywords: Biohydrogen, Dark fermentation, Total ammoniacal nitrogen, Ionic strength

Composition of the Organic Fraction of Municipal Solid Waste (OFMSW) in organic compounds and inorganic ions is highly variable and might impact the microbial activity in dark fermentation processes. In this study, the effect of the total amount of inorganic ions on fermentative hydrogen production was investigated. Batch experiments were carried out at pH 6 and under a temperature of 37°C. A freshly reconstituted organic fraction of municipal solid waste (OFMSW) was used as model substrate. At low concentrations in ammonium or chloride ions (2.9 -5.1 g.L -1 , respectively), the hydrogen yield reached a maximum of 40.8±0.5. mLH2.gVS -1 and 25.1±5.6 mLH2.gVS -1 . In contrast, at high total ionic concentrations of ammonium and chloride (11.1 -35.5 g.L -1 respectively), a strong inhibition of the fermentative microbial activity and more particularly hydrogen production, was observed. When considering the ionic strength of each ion, the effects of ammonia, chloride or a mixture of different ions (Na + , K + , H + , Li + , NH4 + , Mn 2+ , NH4 + , Mg 2+ , Cl -, PO4 3-, Br -, I -, SO4 2-) showed very similar inhibitory trends regardless the type of ion or the composition of the ionic mixture. A threshold inhibitory value of the ionic strength was estimated at 0.75±0.13 M with a substantial impact on the fermentative activity from 0.81±0.12 M, with hydrogen yields of 18.1±3.3 and 6.2±4.1 mLH2.gVS -1 , respectively.

Introduction

Nowadays, fossil fuels are the main resources in primary energy, and their usages have important effects on global warming. Due to a rapid decrease of their stocks and a constant increase of the energy demand, it is now necessary to find new alternatives that are not only sustainable and environment-friendly but also widely available. In this context, organic biomasses, including the organic fraction of municipal solid waste (OFMSW) are well adapted resources. In 2012, Hoornweg and Bhada-Tata [START_REF] Hoornweg | What a Waste: A Global Review of Solid Waste Management[END_REF] reported that urban cities generated about 1.3 billion tons of solid waste per year worldwide. This amount is expected to increase up to 2.2 billion tons in 2025. In addition, the organic fraction of municipal solid waste (OFMSW) has a high calorific value making the OFMSW highly suitable for bioenergy production [START_REF] Alibardi | Composition variability of the organic fraction of municipal solid waste and effects on hydrogen and methane production potentials[END_REF][START_REF] Fantozzi | Anaerobic digestion of mechanically treated OFMSW: Experimental data on biogas/methane production and residues characterization[END_REF]. Historically, methane has been the main gas biologically produced from OFMSW [START_REF] Fantozzi | Anaerobic digestion of mechanically treated OFMSW: Experimental data on biogas/methane production and residues characterization[END_REF], due to the maturity of the technology in terms of performances and conversion efficiency with advanced automation and modelling [START_REF] Mata-Alvarez | Anaerobic digestion of organic solid wastes. An overview of research achievements and perspectives[END_REF]. Indeed, Anaerobic Digestion (AD) is a well-established biological process used to convert organic matter into a biogas that can further used as renewable energy. AD-based waste treatment plants are promoted by the European Parliament [START_REF]Directive 2006/12/EC of the European Parliament and of the Council of 5 April 2006 on waste[END_REF] to use "the waste as a source of energy". In more recent years, hydrogen production by dark fermentation has gained an increasing interest since H2 is considered as an important energy carrier for next-generation technologies towards decarbonation of the industrial sector [START_REF] Chinellato | Biohydrogen production from food waste in batch and semi-continuous conditions: Evaluation of a two-phase approach with digestate recirculation for pH control[END_REF]. Indeed, H2 has a higher heat of combustion (119.93 kJ.g -1 ) than methane (50.02 kJ.g -1 ) [START_REF] Pasupuleti | Single-stage fermentation process for high-value biohythane production with the treatment of distillery spent-wash[END_REF] and water is the only endproduct generated by its combustion or its use in fuel cells [START_REF] Kotay | Biohydrogen as a renewable energy resource-Prospects and potentials[END_REF]. Hydrogen can also be used as a fuel for vehicles and as a reactant for many chemical reactions (crack hydrocarbons, sulfur or nitrogen compounds removing) [START_REF] Das | Hydrogen production by biological processes: a survey of literature[END_REF]. The biological production of hydrogen by dark fermentation is an intermediate step of AD and could be implemented in AD-based plants treating OFMSW. During the dark fermentation process, hydrolytic bacteria convert complex organic substrate into monomers which can be easily assimilated by fermentative hydrogen producing bacteria (HPB), such as Clostridium sp. [START_REF] Kannah | Biohydrogen production from rice straw: Effect of combinative pretreatment, modelling assessment and energy balance consideration[END_REF][START_REF] Chatellard | The type of carbohydrates specifically selects microbial community structures and fermentation patterns[END_REF]. Many studies have already shown the possibility to produce hydrogen from OFMSW, by using mixed culture in continuous stirred tank or batch reactor, for a H2 yield ranging from 20 to 70.1 mLH2.gVS -1 [START_REF] Kannah | Biohydrogen production from rice straw: Effect of combinative pretreatment, modelling assessment and energy balance consideration[END_REF][START_REF] Romero Aguilar | Effect of HRT on hydrogen production and organic matter solubilization in acidogenic anaerobic digestion of OFMSW[END_REF]. In AD treatment plants, hydrogen and methane can be both produced by decoupling dark fermentation and methanogenesis in a two-stage AD process [START_REF] Cavinato | Optimization of two-phase thermophilic anaerobic digestion of biowaste for hydrogen and methane production through reject water recirculation[END_REF]. By separating hydrolysis/acidogenesis and methanogenesis, each process can be optimized with adequate operating conditions for improving the conversion of OFMSW into energy [START_REF] Blonskaja | Use of two-stage anaerobic treatment for distillery waste[END_REF]. In the first stage, 7.5 to 15% of the energy contained in the organic substrate can be converted into hydrogen and co-metabolites, such as volatile fatty acids (VFAs) [START_REF] Hallenbeck | Advances in fermentative biohydrogen production: the way forward?[END_REF]. The remaining organic matter and the VFAs can then be converted into methane in the second stage in order to transform a maximum of anaerobically biodegradable organic matter into energy.

However, hydrogen accumulation in a dark fermenter is often limited by the presence of hydrogen-consuming anaerobes such as methanogens [START_REF] Lay | Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose[END_REF] or the accumulation of inhibitors such as VFAs, chloride and total ammonia nitrogen (TAN) [START_REF] Wang | Inhibitory effect of ethanol, acetic acid, propionic acid and butyric acid on fermentative hydrogen production[END_REF][START_REF] Zhang | Effects of VFAs concentration on biohydrogen production with clostridium bifermentans 3AT-ma[END_REF]. When using OFMSW as organic substrate, ammonia nitrogen and chloride ions are often found in high concentrations [START_REF] Bakonyi | Leachate valorization in anaerobic biosystems: Towards the realization of wasteto-energy concept via biohydrogen, biogas and bioelectrochemical processes[END_REF] due to, respectively, the decomposition of proteins and amino acids [START_REF] Ramsay | Protein degradation during anaerobic wastewater treatment: Derivation of stoichiometry[END_REF] and the addition of sodium chloride (salt) during food preparation. Ammonia nitrogen, defined as the sum of ammoniac gas (NH3) and ammonium ions (NH4 + ), was already reported as an inhibitor of the microbial activity, especially under its non ionic form (NH3) [START_REF] Pan | Effect of ammonia on biohydrogen production from food waste via anaerobic fermentation[END_REF]. Ammonia inhibition has also been suggested on fermentative hydrogen production, but the effects are not yet well described. As an illustration, Salerno et al. [START_REF] Salerno | Inhibition of biohydrogen production by ammonia[END_REF] showed a negative effect on hydrogen production at ammonia nitrogen concentrations higher than 1.6 gN.L -1 , i.e. 0.4 mgNH3.L -1 , in a continuous reactor. In contrast, a neutral effect on hydrogen yields in batch test was observed in the same study, even at higher concentration of ammonia nitrogen (1.3 and 13 mgNH3.L -1 ). Cavinato et al. [START_REF] Cavinato | Bio-hythane production from food waste by dark fermentation coupled with anaerobic digestion process: A long-term pilot scale experience[END_REF] reported an inhibitory effect on hydrogen production with a change on metabolic pathways when ammonia nitrogen concentration increased from 0.97 to 1.98 gN.L -1 in a two stage process treating food waste.

Halophilic conditions can also be detrimental to hydrogen production in particular in OFMSW rich in chloride ions. Pierra et al. [START_REF] Pierra | Fermentative hydrogen production under moderate halophilic conditions[END_REF] showed inhibition of the hydrogen production at NaCl concentrations higher than 9 gNaCl.L -1 in the medium, using glucose as substrate.

To better understand the effect of these ions on the microbial activity in dark fermentation, the impact of ion addition on hydrogen production from OFMSW was investigated by decoupling the effect of a specific ion and the global ionic strength of the medium. The aim of this study was therefore (a) to evaluate the individual impact of ammonia and chloride ion concentrations on hydrogen production, and (b) to investigate the impact of the total ionic strength on the fermentative metabolism with different types of ions.

Materials and Methods

Feedstock and inoculum preparation

A reconstituted organic fraction of MSW was freshly prepared and its composition was representative of the average composition of OFMSW collected in France on a yearly basis, according to the national characterization of MSW (MODECOM TM , 1993). The proportions of each component are described in Table 1. Meat, rice, potatoes and coffee grounds were first cooked and mixed with yogurt and bread.

Garden waste, paper and cardboards were shredded and sieved at 1 cm. Total solid (TS) and volatile solid (VS) contents of the reconstituted OFMSW were 0.74±0.01 gTS.g -1 and 0.63±0.01 gVS.g -1 , respectively.

Microbial anaerobic inoculum corresponded to a sample of an anaerobic lagoon treating leachates from methanogenic storage cells in a real MSW landfill (TRIFYL). The initial pH was 7.64, with a TS and VS concentration of 20.5±0.3 gTS.L -1 and 7.4±0.2 gVS.L -1 , respectively. Since the leachate initially contained a high quantity of ammonium ions (4.02±0.79 gN.L -1 ), a stripping pretreatment was carried out prior to experimentation.

Stripping corresponded to a flush of the liquid phase of the leachate with air during 3 hours at pH 9, at a temperature of 90°C. Nitrogen removal efficiency was 95 %. Prior stripping, the microbial community was removed by centrifugation (8000 rpm during 30 min) to not be affected by the stripping treatment of the leachate. At the end of the stripping treatment, the temperature was stabilized at 25°C, the pH adjusted at 6 and the microbial community was reintroduced.

Experimental procedure

The batch reaction was carried out in a 500 mL flask with a working volume of 400 mL.

Each experiment was carried out at 37°C. Culture medium contained 9.25 gVS of OFMSW inoculated with 136 g of stripping-pretreated leachate diluted with 249.4 g of water to reach a S/X (substrate/biomass) ratio of 10 (on VS basis) [START_REF] Cappai | Effect of inoculum to substrate ratio (isr) on hydrogen production trhough dark fermentation of food waste[END_REF]. pH was adjusted at 6 with NaOH (1 M). Batch reactors were then flushed with N2 before experiment to remove oxygen traces. For each experiment, different solutions of ions were added to obtain a large range of ionic concentrations in the reactors, as described in the next section. Samples of 2 mL were collected and stored at -20°C at the beginning and the end of each experiment for metabolite and microbial analyses. A total of 152 batch assays was performed, with triplicates for each condition tested.

Impact of ammonium and chloride ions on hydrogen production

The first part of the study was carried out with three different types of ionic solutions. The first experiment consisted in adding NH4Cl only (Ammonium-Chloride solution), under a concentration ranging from 0 to 18.5 gN.L -1 for ammonia ions and from 0 to 42 gCl -.L -1 for chloride ions. In the second experiment, a mixture of different anions coupled to NH4 + (NH4Cl, NH4H2PO4, (NH4)2SO4) was added to avoid the effect of chloride ion. The final concentration in ammonium ions ranged from 0 to 22.8 gN.L -1 (Ammonium-X solution).

Finally, in the last experiment, a mixture of different cations coupled to Cl -(CaCl2, KCl, NaCl) (Chloride-X solution) was evaluated at a concentration range of 0 -40 gCl -.L -1 .

Impact of ionic strength on hydrogen production

The second part of the study was carried out to evaluate the impact of the total ionic strength regardless the type of ions and minimize the individual concentration. Two different solutions composed of seven and six components were used, corresponding to the main ions found in OFMSW leachate [START_REF] Bakonyi | Leachate valorization in anaerobic biosystems: Towards the realization of wasteto-energy concept via biohydrogen, biogas and bioelectrochemical processes[END_REF] : solution M1 (NaCl, KH2PO4, LiBr, KI, (NH4)2SO4, MnCl2, MgSO4) and solution M2 (NaCl, KH2PO4, LiBr, KI, (NH4)2SO4, LiCl).

A range of concentration from 0 to 1.9 M in term of ionic strength was tested. For M1 and M2, each components were added at the same concentrations depending of the fixed ionic strength (i.e. for M1 at ionic strength of 0.161 M, a concentration of 0.016 mol.L -1 of each component was added meaning a concentration of 0.9, 2.2, 1.4, 2.7, 2.1, 3.2 and 4.0 g.L -1

for NaCl, KH2PO4, LiBr, KI, (NH4)2SO4, MnCl2, MgSO4 respectively). In this mixture, ammonium and chloride concentrations did not exceed 1.4 gN.L -1 and 8.4 gCl -.L -1 (i.e. 0.08 and 0.24 M respectively).

Analytical methods

Gas pressure and composition were periodically measured (every four hours) with an automated multiplexed micro-gas chromatograph (μGC R3000, SRA instrument), as described by Cazier et al. [START_REF] Cazier | Biomass hydrolysis inhibition at high hydrogen partial pressure in solid-state anaerobic digestion[END_REF]. Cumulated hydrogen was estimated by automatic and periodic gas pressure measurement.

Volatile fatty acids (VFAs) were measured at the beginning and at the end of each experiment. Individual VFA concentrations were determined with a gas chromatograph Perkin Clarus 580 with an Elite-FFAP crossbond ® carbowax ® 15 m column connected to a flame ionization detector at 280°C and N2 at 6mL.min -1 as carrier gas, as previously described by Motte et al. [START_REF] Motte | Morphological structures of wheat straw strongly impacts its anaerobic digestion[END_REF].

For microbial analyses, samples were collected at the end of each experiment. DNA was extracted and purified with the Fast DNA SPIN kit for soil in accordance with manufacturer's instructions (MP Biomedicals). DNA quantity and purity were assessed by spectrophotometry (Infinite NanoQuant M200, Tecan). The V4 and V5 regions of the 16S rRNA genes were then amplified and sequenced by Illumina MiSeq (get.genotoul.fr) and sequences were analysed as described elsewhere [START_REF] Moscoviz | Consistent 1,3-propanediol production from glycerol in mixed culture fermentation over a wide range of pH[END_REF].

Data analysis and calculation

A modified Gompertz equation based model was fitted to the cumulative hydrogen production curve to assess the three following kinetic parameters: the hydrogen yield, the maximal hydrogen production rate and the lag phase [START_REF] Moscoviz | Consistent 1,3-propanediol production from glycerol in mixed culture fermentation over a wide range of pH[END_REF].

H(t)=H exp{-exp[Re/H (λ-t)+1]} (1)
where H(t) is cumulative hydrogen amount (mL) at time t, H is the hydrogen potential (mL), R is maximal hydrogen production rate (mL.h -1 ), λ is the lag-phase and t the time (hours).

The ionic strength was calculated according to the dimensional equation as described by Solomon [START_REF] Solomon | The Definition and Unit of Ionic Strength[END_REF].

I= (1/2) × ∑ (Ci Zi 2 ) ( 2
)
where I is the ionic strength of a solution (mol.l -1 ), Ci the concentration of each individual ion (mol.l -1 ) and Zi the charge state of each ion.

Results and discussion

Effect of ammonium and chloride ions on hydrogen production

In all experiments, no methane production was observed confirming that initial heat shock pretreatment of the inoculum was efficient to suppress methanogens. Hydrogen production from OFMSW was first evaluated at different concentrations of NH4Cl (Ammonium-Chloride) from 0 to 18.5 gN.L -1 (Fig. 1(a)). Overall, apart from one sample (containing 2.0 gN.L -1 ), a good fitting of the data to the Gompertz model was observed on the hydrogen response (details are presented in supplementary material).

In the control where no ion was added, the hydrogen yield reached 22.9±0.1 mLH2.gVS -1 with a lag phase of 5.9±0.1 hours and a productivity of 3.29±0.12 mLH2.gVS -1 .h -1 .

Consistently, Favaro et al. [START_REF] Favaro | Effects of inoculum and indigenous microflora on hydrogen production from the organic fraction of municipal solid waste[END_REF] reported a similar hydrogen yield of 23.4±2.9 mLH2.gVS -1 using real OFMSW in a reactor operated at mesophilic temperature with a pH buffered at 7.

However, this value is lower than the yields reported by Pan et al. [START_REF] Pan | Effect of food to microorganism ratio on biohydrogen production from food waste via anaerobic fermentation[END_REF] or Elbeshbishy et al.

[32] who used batch reactors with no heat shock treatment or pH regulation (39 and 40 mLH2.gVS -1 respectively). In these study, more fermentescible food waste was used as substrate which probably explains these differences in hydrogen production. As shown in Fig. 1, when NH4Cl was added at low concentration (from 1.4 to 3.5 gN.L -1 ) and in comparison with the control, a slight but significant increase of 8 % (p-value < 0.001) was observed with an average yield of 24.9±2.5 mLH2.gVS -1 . The lag phase was also shortened to 5.2±0.5 hours. A further increase of the ammonia nitrogen concentration between 3.5 and 8.6 gN.L -1 led to a slight decrease of the hydrogen production (p-value < 0.001) to reach a yield of 17.8±1.5 mLH2.gVS -1 at 8.6 gN.L -1 . For concentrations higher than 11.1 gN.L -1 , a substantial decrease of the hydrogen yield down to 1.9±2.8 mLH2.gVS -1 , and a significant increase of the hydrogen lag phase up to 44.9±9.3 hours were observed. Finally, hydrogen was not detected for concentrations above 15.9 gN.L -1 suggesting a strong impact of the NH4Cl concentration on hydrogen-producing microbial activity.

Thereafter, different forms of ammonia nitrogen (Ammonium-X) were added (Fig. (1a)) to avoid the effect of the anion. Similarly, a positive effect on hydrogen production was observed for concentrations up to 7.1 gN.L -1 , with a maximum at 2.9 gN.L -1 (40.8±0.5.

mLH2.gVS -1 ). However, the lag phase was longer (12.8±4.7 hours) and the productivity lower (1.65±0.39 mLH2.gVS -1 .h -1 ) than in the control. At higher values (10.2 gN.L -1 ,), the hydrogen yield decreased to a value lower than in the control (20.1±3 mLH2.gVS -1 ). The decrease of hydrogen yield from 10.2 gN.L -1 was concomitant with an increase of the lag phase (28.9±9.6 hours) and a decrease of the hydrogen productivity (0.39±0.08 mLH2.gVS - 1 .h -1 ), suggesting an impact on the microbial activity. At higher concentrations, a drastic drop of the hydrogen production was observed with a trend similar to the experiments supplemented with Ammonium-Chloride. These observations support the fact that the ions added to the medium had a strong impact on the microbial activity for concentrations above a threshold value of 8.6 gN.L -1 for the Ammonium-Chloride solutions, and 10.2 gN.L -1 for Ammonium-X solutions. The relative standard deviation was calculated on the hydrogen yield to evaluate the reproducibility of the triplicates (supplementary material). For NH4Cl experiments, 57 % of the triplicates had a relative standard deviation below 10 %, while 90 % of the triplicates were below 20 %, showing a high reproducibility. For Ammonium-X, 25 % of the samples had a relative standard deviation below 10 %, and 63 % below 20 %.

During this experiment, higher variability between the triplicates was observed, in particular when ammonia nitrogen was added at low concentration. Interestingly variability decreased with the increase of ammonia nitrogen concentration with a good reproducibility of the impact of ammonia-chloride addition on hydrogen production. Similar experiments were then performed by adding a mixture of CaCl2, KCl, NaCl (Chloride-X) to assess the impact of chloride ions (Fig. (1b)). The trend of the inhibition curve was statistically similar (p-value < 0.05) to the experiment where chloride ion was added in the form of NH4Cl only (Ammonium-Chloride experiments). Indeed, hydrogen production was not affected between 0 to 10.2 gCl -.L -1 (24.8±4.5 mLH2.gVS -1 ) with an average lag phase and a productivity of 9.9±2.1 hours and 1.56 mLH2.gVS -1 .h -1 , respectively. From 20 gCl -.L -1 to 40 gCl -.L -1 hydrogen production decreased and a total inhibition was observed at 35.5 gCl -.L -1 . By assessing the relative standard deviation of the Chloride-X experiments, 100% of the triplicates had a relative standard deviation below 20 %, showing a high reproducibility between the triplicates.

When combined to the results of the experiments carried out with Ammonium-Chloride solutions, it was concluded a strong inhibition of both ammonia nitrogen and chloride ions on hydrogen pathway. Several studies already reported that the non-ionic form of ammonia nitrogen (NH3) can act as an inhibitor on the hydrogen production due to its high permeability to bacterial cell membrane [START_REF] Müller | Ammonium toxicity in bacteria[END_REF]. In our study, a strong inhibitory effect was observed at an ammonia concentration of 8.6 gN.L -1 , equivalent to 11 mgNH3.L -1 .

Comparatively, Salerno et al. [START_REF] Salerno | Inhibition of biohydrogen production by ammonia[END_REF] reported that the maximum hydrogen production rate decreased from 56 mLH2.h -1 at 2 gN.L -1 to 16 mLH2.h -1 at 10 gN.L -1 (i.e. 13 mgNH3.L -1 ), respectively, in a batch reactor operated at pH 6.2, supplement with NH4Cl and fed with glucose. However, no impact on hydrogen yield was observed (1.0±0.04 molH2.molglucose -1 , i.e. 120 mLH2.gVS -1 ). Wang et al. [START_REF] Wang | Effect of ammonia concentration on fermentative hydrogen production by mixed cultures[END_REF] showed an inhibitory effect on hydrogen production at a concentration of ammonia nitrogen of 5 gN.L -1 (64 mgNH3.L -1 ) using glucose as substrate, with an initial pH of 7 and a reactor operated at mesophilic temperature.

In addition, Pan et al. [START_REF] Pan | Effect of ammonia on biohydrogen production from food waste via anaerobic fermentation[END_REF] showed an improvement of the hydrogen yield at concentrations of ammonia nitrogen as low as 1.5 gN.L -1 and 3.5 gN.L -1 , in a batch reactor using food waste as substrate. In the present study, similar improvement was observed at 3.5 gN.L -1

showing the benefic effect of ammonia nitrogen on microbial activity. When different forms of ammonium (Ammonium-X) were added, a higher positive effect on hydrogen production was observed suggesting that the molecules added (NH4H2PO4, (NH4)2SO4)

were probably lacking in the OFMSW. Such observation confirms the positive effect of phosphorus and nitrogen on hydrogen producing bacteria activity at low concentration as already shown by Wang and Wan [START_REF] Wang | Factors influencing fermentative hydrogen production: A review[END_REF].

In comparison, no positive effect of chloride ion addition was observed in this study. Such negative impact on hydrogen production of the Chloride-X solutions is consistent with the literature where chloride ions are described as strong inhibitors of the microbial activity in anaerobic bioreactors. Lefebvre et al. [START_REF] Lefebvre | Impact of increasing NaCl concentrations on the performance and community composition of two anaerobic reactors[END_REF] showed an inhibitory effect in anaerobic digestion at a concentration of NaCl of 20.0 g.L -1 (i.e. a chloride concentration of 12.1 gCl - .L -1 ), using ethanol as carbon source. In fermentation, Pierra et al. [START_REF] Pierra | Fermentative hydrogen production under moderate halophilic conditions[END_REF] observed a decrease of the hydrogen production at NaCl concentrations as low as 9 gNaCl.L -1 (i.e. 5.4 gCl -.L -1 ).

In comparison, our study showed an inhibitory effect on hydrogen production from 10.2 gCl -.L -1 .

In conclusion, the comparison of the three experiments clearly shows that ammonia and chloride ions had the same inhibition trend in terms of hydrogen yield, hydrogen lag phase and hydrogen productivity. Indeed the hydrogen yield decreased of about 92% and 89% for the Ammonium-Chloride and Ammonium-X experiments respectively, when increasing the ammonium concentration from [0-5] gN.L -1 to >10 gN.L -1 . A very similar trend was observed with the chloride ions, with a decrease of the hydrogen yield by 89% from the low [0-10] gCl -.L -1 to the high concentrations (up to 20 gCl -.L -1 ) of chloride, in the Chloride-X experiments.

Consistently, an increase of the ammonium and chloride ion concentration led to an increase of the lag phase for all the tested solutions. A very similar impact was shown on the hydrogen productivity, regardless the type of ion added, with a maximum of 3.04±0.71, 1.48±0.52 and 1.56±0.81 mLH2.gVS -1 .h -1 observed at low concentrations for Ammonium-Chloride, Ammonium-X and Chloride-X solutions, respectively, i.e. within the range of [0-5] gN.L -1 and [0-10] gCl -.L -1 .

These results suggest that a global metabolic inhibition could have occurred and might be responsible of the negative impact on microbial fermentation, independently of the nature of the ion.

Impact of the ionic strength on dark fermentation

Impact of the ionic strength on hydrogen production

In light of these results, an ionic strength index was calculated to evaluate the impact of the total ionic content rather than the effect of individual ion on the fermentative activity. The total ionic strength was estimated based on each individual ionic concentration and their number of charges. By revisiting the data, the impact of the ionic strength on hydrogen production is shown in Fig. 2, for Ammonium-Chloride, Ammonium-X and Chloride-X experiments.

Figure 2: Effect of ionic strength on biohydrogen production in dark fermentation reactor for Ammonium-Chloride (O), Ammonium-X ( ), Chloride-X (Δ) experiments and the control (•)

As noticed previously, an improvement of the hydrogen production was observed at low ionic strength, for the Ammonium-Chloride, Ammonium-X and Chloride-X experiments.

Such slight positive effect confirms the benefic effect of the molecules added on hydrogen production at low concentration of ammonium. A similar inhibitory trend was observed in all the experiments according to the value of the ionic strength. A threshold value of this O Ammonium-Chloride Ammonium-X Δ Chloride-X inhibition was estimated at 0.75±0.13 M, for an average H2 yield of 18.1±3.3 mLH2.gVS -1 , whatever the ions considered. The inhibition was significant at ionic strengths higher than 0.81±0.12 M with an average H2 yield of 6.2±4.1 mLH2.gVS -1 . Total inhibition of the hydrogen production was then observed at an ionic strength of 1.04, 1.63 and 1.73 M for Ammonium-Chloride, Ammonium-X and Chloride-X experiments, respectively, suggesting here an effect of the type of ions.

To better understand the impact of the ions regardless their nature, the total ionic strength was modified by changing the composition of the ionic solutions and considering several anions and cations, as follows: solution M1 was composed of Na + , K + , H + , Li + , NH4 + , Cl -, PO4 3-, Br -, I -, SO4 2-, while solution M2 was composed of Na + , K + , H + , Li + , NH4 + , Mn 2+ , Mg 2+ , Cl -, PO4 3-, Br -, I -, SO4 2-(Fig. 3). By increasing the number of ion types, the concentration of each individual element remained low (< 0.18 M) while the ionic strength increased from 0.18 to 1.9 M. As shown in Fig. 3, the trend was similar than previously observed with an improvement of the hydrogen production at the lowest ionic strengths of 0.09 and 0.25 M with an average yield of 26.5±6.1 and 25.0±0.7 mLH2.gVS -1 for M1 and M2, respectively. Inhibition of the hydrogen production was significant after values of ionic strength of 0.57 and 0.67 M for the M1 and M2 solutions, which is similar to the previous observations made with Ammonium-Chloride, Ammonium-X and Chloride-X solutions (0.75±0.13 M).

No hydrogen production was observed for ionic strength higher than 1.56 and 1.66 M for M1 and M2, respectively. The concentration of ammonia nitrogen and chloride ions at 1.56 M for M1 and at 1.66 M for M2 were 1.7 and 1.1 gN.L -1 and 13.1 and 6.5 gCl -.L -1 , respectively. Interestingly, these concentrations in individual components corresponded to the concentrations found at the highest performances of hydrogen production in the Ammonium-Chloride experiments. These observations clearly suggest that hydrogen inhibition was not due to the individual concentration in ammonium and chloride ions, but to the overall ionic strength.

Impact of ionic strength on by-products production

Furthermore, soluble fermentation co-products were analyzed at the end of the batch tests to estimate the impact of the ionic solutions on the global fermentation activity. Total amount of metabolic co-products was expressed in mgCOD.gVS -1 (Table 2).

In the control experiment, i.e. without addition of ions, the main accumulated metabolites were acetate and butyrate at a concentration of 104.2±8.3 mgCOD.gVS -1 (2.5±0.2 gCOD.L - 1 ) and 133.3±8.3 mgCOD.gVS -1 (3.2±0.2 gCOD.L -1 ) representing 36 % and 46 % of the total COD, respectively.

Theoretically acetate and butyrate are correlated to a high hydrogen yield. Indeed, two moles of acetate and one mole of butyrate leads to four and two mole of hydrogen respectively (Eq. 3 and 4) [START_REF] Hawkes | Continuous dark fermentative hydrogen production by mesophilic microflora: Principles and progress[END_REF].

(

Many studies show these metabolites as major end product during dark fermentation reaction [START_REF] Wang | Effect of temperature on fermentative hydrogen production by mixed cultures[END_REF][START_REF] Pierra | Fermentative hydrogen production under moderate halophilic conditions[END_REF]. Cavinato et al. [START_REF] Cavinato | Bio-hythane production from food waste by dark fermentation coupled with anaerobic digestion process: A long-term pilot scale experience[END_REF] reported the same range of acetate and butyrate concentration in a thermophilic dark fermentation using food waste (3.29±1.64 and 4.32±1.48 gCOD.L -1 respectively) which is slightly higher than our results due to thermophilic application which is known to improve the microbial activity [START_REF] Wang | Effect of temperature on fermentative hydrogen production by mixed cultures[END_REF]. These results suggest that the conditions in the control were favorable for hydrogen producing pathways. Indeed, the maximum H2 yield is related to acetate as by-product. However, from an experimental point of view, high H2 yields are usually associated to butyrate in mixed culture [START_REF] Hawkes | Sustainable fermentative hydrogen production: Challenges for process optimisation[END_REF]. Overall, the increase of ionic strength led to a decrease of the total accumulation in metabolites regardless the solutions added. Concerning the Ammonium-Chloride experiments, from 0.25 to 1.02 M, acetate and butyrate concentrations drastically dropped to nearly no production with concomitant decrease of hydrogen production. Propionate concentration reached a maximum value of 14.6±2.9 mgCOD.gVS -1 at 0.25 M, and decreased to 1.7±0.4 mgCOD.gVS -1 at 1.02 M. Valerate and caproate were only present in trace amounts up to 0.59 M and were not detected at higher ionic strengths. A similar trend was observed for Ammonium-X and M2 solutions, with a decrease of the metabolite production according to the increase of the ionic strength. At low ionic strength of 0.28 and 0.24 M, a maximal production of metabolites of 189±69 and 198±13 mgCOD.gVS -1 , respectively, were observed. The total metabolite concentration reached a low value (100±30 and 60±10 mgCOD.gVS -1 ) at higher ionic strengths of 1.02 and 1.23 M, respectively. Similar trends were observed for the Chloride-X experiments with a maximum of metabolite production (407±80 mgCOD.gVS -1 ) at low ionic strength (0.35 M) with acetate and butyrate as main metabolites (145±12 and 212±29 mgCOD.gVS -1 respectively). A decrease of the total concentration was observed with a low metabolite production of 61±47 mgCOD.gVS -1 at 1.71 M.

The consumption of hydrogen by homoacetogenesis to produce acetate was estimated and is presented in Table 2. The acetate produced by homoacetogenesis can be estimated by the following two equations (Eq. 5 and 6), as already described by Arooj et al. [START_REF] Arooj | Continuous biohydrogen production in a CSTR using starch as a substrate[END_REF] :

(5)

Where Total HAc is the acetate concentration; HAca and Hacb are the acetate concentration from the H2-producing (HACa) and H2-consuming (HACb) pathways; HBu the butyrate concentration and HPr the propionate concentration.

Indeed, hydrogen yield is theoretically associated to the stoichiometry of 2 mol of hydrogen generated per mole of acetate and butyrate accumulated, while propionate consumes 1 mol of hydrogen. Eq. ( 5) considers the production of acetate from two distinct pathways corresponding to H2-producing (HACa) and H2-consuming (HACb) homoacetogenic pathway. Considering Eq.( 5) and Eq. ( 6), the amount of acetate produced by homoacetogenesis was estimated according to molar balances and the results are presented in Table 2. The estimated amount of acetate produced by homoacetogenesis pathway in the control represented about 39.9% of the total COD. Consistently, Castelló et al. [START_REF] Castelló | Possible causes for the instability in the H2production from cheese whey in a CSTR[END_REF] reported that 33 to 57% of the acetate was produced by homoacetogenesis using a continuous stirred tank reactor fed with cheese whey. As already reported by Saady [START_REF] Saady | Homoacetogenesis during hydrogen production by mixed cultures dark fermentation: Unresolved challenge[END_REF],

homoacetogenesis is associated to fast growing strict anaerobes forming spores such as Clostridium sp. that can consume between 11% and 43% of the hydrogen produced in batch. In our experiments, the homoacetogenesis ratio is stable regardless the ionic strength concentration. The percentage of acetate issued from the homoacetogenesis pathway was estimated at 36.4±5.3, 25.1±3.3, 44.1±6 and 39.8±7.5% for Ammonium-Chloride, Ammonium-X, Chloride-X and M2 solutions whatever the ionic strength. Therefore, the decrease of hydrogen performances according to the increasing ionic strengths was not attributed to an increase of hydrogen consumption by homoacetogenic bacteria. Only the Ammonium-X solution showed a lower amount (25.1±3.3%) resulting from a high hydrogen yield observed at low ionic strength (41.1±0.8. mLH2.gVS -1 at 0.31±0.08 mol.L -1 ).

Overall, the analyses of metabolites showed the same impact of the ionic strength than previously observed with hydrogen production, i.e. a global decrease of the metabolite production when the concentration of ions in the medium increased, regardless the solutions added. Further increase of the ionic strength drastically affected the acetate and butyrate pathways which are the two metabolic pathways generating hydrogen [START_REF] Hawkes | Continuous dark fermentative hydrogen production by mesophilic microflora: Principles and progress[END_REF]. Similar observations were reported by Cavinato et al. [START_REF] Cavinato | Bio-hythane production from food waste by dark fermentation coupled with anaerobic digestion process: A long-term pilot scale experience[END_REF] who showed a decrease by 72 % of the acetate and butyrate concentrations when a nitrogen accumulated from 970 to 1976 mgN.L - 1 in dark fermentation, using municipal solid waste as substrate. Zheng et al. [START_REF] Zheng | Influence of NaCl on Hydrogen Production from Glucose by Anaerobic Cultures[END_REF] observed a similar decrease of the butyrate and H2 yields by adding NaCl from 0 to 500 mM and using glucose as substrate. In addition, our results showed that the increase of ionic strength did not change the metabolic pathways since acetate and butyrate remain the main metabolites in each experiments, with no accumulation of lactate or other by-products that can be produced during microbial stress [START_REF] Noike | Inhibition of hydrogen fermentation of organic wastes by lactic acid bacteria[END_REF]. Our observations rather suggest that the global microbial activity was strongly impacted by the increase of the ionic strength.

Impact of the ionic strength on the microbial community

Microbial characterization using phylogenetic affiliation of bacterial 16S rDNA was performed on the Ammonium-Chloride experiment, as model experiment, at different ionic strengths. The results are presented in Table 3. Only sequence having affiliations above 98% of similarity with sequences of known species are presented. [START_REF] Liu | The effect of pH on the production of biohydrogen by clostridia: Thermodynamic and metabolic considerations[END_REF] reported high hydrogen production using Clostridium butyricum in a fermentative batch reactor at pH 6 with glucose as substrate (1.83 mmol/mmolglucose). The ability of Enterococcus saccharolyticus to hydrolyze cellobiose and produce hydrogen was previously described [START_REF] Adav | Functional consortium for hydrogen production from cellobiose: Concentration-to-extinction approach[END_REF]. These observations are consistent with our experiment where freshly reconstituted OFMSW was composed of 51.8 % of paper and cardboard. Clostridium tertium was found in lower abundance in the experiment, and was also described as hydrogen producing bacteria [START_REF] Sivagurunathan | Batch fermentative hydrogen production by enriched mixed culture: Combination strategy and their microbial composition[END_REF]. The overall microbial community characterization suggests that the environment at low ionic strength was ideal for the development of hydrogen-producing bacteria.

A neutral impact of the ionic strength was shown at low concentration (from 0.25 to 0.53 M) on the microbial community composition, which is consistent with macroscopic observations of high hydrogen production.

At intermediate ionic strength (0.63 and 0.72 M), a modification of the microbial community was observed with a decrease in abundance of Clostridium butyricum (7 and 0% respectively) and an increase in unclassified Clostridiales (27 and 44 % respectively).

This correlates with the variability of hydrogen yields found in this range of ionic strength suggesting that the population structure was disturbed. At higher ionic strength (above 0.72 M), the dominant Clostridium butyricum, Clostridium tertium and Enterococcus saccharolyticus were not detected and the relative abundance in unclassified Clostridiales drastically decreased to 10%, 7% and 12% at 0.95, 1.02, 1.13 mol.L -1 , respectively. This observation is consistent with an absence of hydrogen production in these experiments. At high ionic strength, the presence of other microbial populations was detected such as Pseudomonas pertucinogena, Marinocpirillum minutulum and Atopostipes suicloacalis which are not associated with hydrogen production. Therefore, the increase of ionic strength seemed to have highly impacted hydrogen producing bacteria that are not particularly adapted to high ionic environments. As reported by Van Niel et al. [START_REF] Van Niel | Substrate and product inhibition of hydrogen production by the extreme thermophile, Caldicellulosiruptor saccharolyticus[END_REF], an accumulation of ions in the medium (above 175 mM) can cause a cell lysis in Clostridium saccharolyticus. More generally, high concentrations of ions cause high osmolarity across the bacterial that can lead to an inhibition of the microbial activity, as shown with

Clostridium butyricum [START_REF] Szymanowska-Powałowska | The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1[END_REF].

Conclusion

This study provides new aspects on fermentative hydrogen production using freshly reconstituted OFMSW. During dark fermentation, the presence of ions (chloride or ammonia) at low concentrations did not impact hydrogen performances and was even beneficial with the best performances found when 2.9 gN.L -1 was added (40.8±0.5.

mLH2.gVS -1 ). However, at higher concentration of ions and whatever the ions added, a very similar trend of inhibition on hydrogen production was observed. For all experiments, the inhibition started after a similar threshold value of ionic strength (0.75±0.13 M) whatever the ions considered from 18.1±3.3 mLH2.gVS -1 to 6.2±4.1 mLH2.gVS -1 at 0.81±0.12 M. Such inhibition was also observed on the overall microbial activity with a decrease of metabolite production. Moreover, the accumulation of ions caused a stressful environment as shown by microbial community changes with a particular impact on dominant H2-producing bacteria (Clostridium butyricum, Clostridium tertium and Enterococcus saccharolyticus). In the context of process upscaling and implementation of this technology for waste energy recovery, the ionic strength should be carefully monitored to avoid reaching the critical threshold value and thus, reduce the overall efficiency of the process.

Figure 1 :

 1 Figure 1: Hydrogen yields estimated by Gompertz model in batch reactors operated at (a) increasing total ammonia nitrogen concentrations in the forms of [NH4Cl] only (O) or [NH4Cl, NH4H2PO4, (NH4)2SO4] ( ) and (b) increasing chloride concentrations in the forms of [NH4Cl] only (O) or [CaCl2, KCl, and NaCl] (Δ), control (•).

Figure 3 :

 3 Figure 3: Effect of ionic strength on biohydrogen production for M1: NaCl, KH2PO4, LiBr, KI, (NH4)2SO4, MnCl2.4H2O, MgSO4.7H2O (◊) and M2: NaCl, KH2PO4, LiBr, KI, (NH4)2SO4, LiCl (◊) experiments.

Table 1 :

 1 Average composition of the reconstituted OFMSW. Data are expressed in % of wet weight (% w/w).

	Category	Components	% w/w
		Meat	7.0
		Coffee grounds	3.9
	Food waste	Rice Potatoes	4.3 20.9
		Bread	5.1
		Yogurt	2.0
	Garden waste	Grass	5.0
	Papers	Papers	35.1
	Cardboards	Cardboards	16.7

Table 2 :

 2 Final concentration of acetate, propionate, butyrate, valerate and caproate during all the experiments (Ammonium-Chloride, Ammonium-X and Chloride-X) at different ionic strength and estimation of the homoacetogenic activity in all experiments. For technical reasons, M1 solutions precipitates in the liquid fraction made the analyses no possible without damaging the analyzer.

	Experiments

Table 3 :

 3 Phylogenetic affiliation of bacterial 16S rDNA from experiments Ammonium-Chloride at different ionic strength (mol.L -1 ) using BLAST algorithm.

	Closely related to	Sequence similarity (%)	Relative abundance of species in each ecosystem (%) 0.25 0.35 0.53 0.63 0.72 0.95 1.02 1.13
	Clostridium butyricum	100	32	44	26	7	-	-	-	-
	Clostridium tertium	99	3	3	4	6	9	-	-	-
	Enterococcus saccharolyticus	99	32	23	48	39	16	-	-	-
	Enterococcus faecalis	100	-	7	5	5	15	11	-	-
	Pseudomonas caeni	99	5	5	4	5	2	2	1	1
	Pseudomonas pertucinogena	99	-	-	-	-	-	20	18	22
	Marinocpirillum minutulum	100	-	-	-	-	-	11	3	3
	Atopostipes suicloacalis	99	-	-	-	-	-	-	28	1
	Others (Clostridiales NC)		10	3	5	27	44	10	7	12
	Others (Oceanospirillales NC)		-	-	-	-	-	10	12	15
	Others (NC)		12	15	8	11	14	36	38	56

At low ionic strength (0.25, 0.35 and 0.53 M) the most abundant bacteria were assigned to known hydrogen producing bacteria, i.e. Clostridium butyricum and Enterococcus saccharolyticus.

Liu et al.

Acknowledgements

The authors wish to thank Celia Campan who performed the M1 and M2 experiments and undertook the chemical analyses.