Vincent Escande

Chun Ho Lam

Claude Grison

Paul T Anastas

EcoMnOx, a Biosourced Catalyst for Selective Aerobic Oxidative Cleavage of Activated 1,2-Diols

Keywords: Heterogeneous catalysis, Manganese oxide, Glycol cleavage, Ecocatalysis, Phytoextraction, Metal recycling

A novel catalyst, EcoMnOx, was prepared from waste biomass of Mn-hyperaccumulating plants. The valorization of this Mn-rich biomass is an alternative to its costly usual disposing and provides a new source of Mn. Extracted metal ions, including Mn 2+ , were oxidized in mild conditions by H 2 O 2 /NaOH to afford EcoMnOx, as a polymetallic oxide material containing from 8.9 to 14.1 wt % Mn. Spectroscopic studies of this material revealed the presence of Mn layered mixed oxides, rich in Mn IV along with Mn III and Mn II species. EcoMnOx catalytic properties were assessed in the aerobic oxidative cleavage of 1,2-diols, under atmospheric pressure of O 2 or air. With only 10 mol % Mn, up to complete conversions were obtained on activated benzylic and allylic diols, with excellent selectivity toward aldehydes or ketones (98-99%). Moreover, because of its heterogeneous nature, the catalyst can be removed easily by filtration, and reapplied for a minimum of six successive runs without any loss of activity. Finally, E-factor analysis showed the EcoMnOx generates 4 to 17 times less waste compared to classical reagents such as NaIO 4 and Pb(OAc) 4 , respectively, highlighting the sustainable assets of this new heterogeneous catalyst.

INTRODUCTION

Sustaining global population growth is one of the major upcoming challenges. According to the United Nations, the world population is expected to rise rapidly from the current 7.5 to 11.2 billion by the end of this century. [START_REF]World Population Prospects: The 2015 Revision, Key Findings and Advance Tables[END_REF] Population growth and the consequent increased urbanization inevitably trigger a rise in demand of natural resources, such as energy, water, and minerals. Even with a shift toward sustainable energy production modes, building the necessary infrastructures (such as wind turbines or solar panels) will require high quantities of metals, ranging from base metals to rare earth elements, which will be sequestered for decades. At the same time, global population growth will lead to an increased demand in consumer goods, such as personal electronic devices, likely to exacerbate this forthcoming metals scarcity. As a result, base metals demand is currently increasing by 5% annually, such that metal production for the next 15 years will need to match that from the start of humanity up to the current time. 2 This growing need for metals is also often decoupled from local production, stressing the risks of geopolitical tensions. For example, it has been stated that European industries consume more than 20% of the global production of metals, whereas European mines produce only 1.5% of iron and aluminum. 2,3 Addressing future metal scarcity will suppose a necessary evolution of production and consumption habits, as well as a crucial improvement of current metals recycling rate. This can be dealt with the optimization of existing recycling processes, but also with the valorization of metal-rich wastes, unexploited until now.

Among these underestimated metal-rich wastes, [START_REF] Rawlings | The microbiology of biomining: development and optimization of mineral-oxidizing microbial consortia[END_REF][START_REF] Widmer | Global perspectives on e-waste[END_REF][START_REF] Wagner | Landfill mining: Case study of a successful metals recovery project[END_REF] biomass from phytoextraction crops is very promising. Phytoextraction is an emerging biotechnology using plants to remove contaminants, including metal pollutants from soils. [START_REF] Ansari | Management of environmental contaminants[END_REF][START_REF] Raskin | Phytoremediation of toxic metals: using plants to clean up the environment[END_REF][START_REF] Singh | Phytoremediation: an overview of metallic ion decontamination from soil[END_REF][START_REF] Grison | Combining phytoextraction and ecocatalysis: a novel concept for greener chemistry, an opportunity for remediation[END_REF] Specific plants, named "hyperaccumulators" can uptake and store unusually high amounts of metal ions from the soil, achieving progressive decontamination of metal-polluted sites, like former mining areas. After accumulation, biomass from hyperaccumulators is harvested to avoid a new release of metal pollutants after plant death. However, the resulting metal enriched biomass is usually treated as a toxic waste, which induces a supplementary cost upon its disposala practice that is detrimental to the implementation of this ecofriendly biotechnology. More importantly, disposing these valuable metals is even more regrettable, especially in view of the aforementioned metal shortages. [START_REF] Ernst | Phytoextraction of mine wastes -Options and impossibilities[END_REF][START_REF] Vigil | Is phytoremediation without biomass valorization sustainable? -Comparative LCA of landfilling vs. anaerobic co-digestion[END_REF] In order to overcome this situation, attempts of valorization of this metal-rich biomass have recently emerged. [START_REF] Van Der Ent | Farming for Metals in the Future?[END_REF] Ecocatalysis is a new strategy to valorize metal-rich biomass as a source of metal species for chemical catalysis. [START_REF] Grison | Ecocatalysis: A New Approach Toward Bioeconomy[END_REF][START_REF] Escande | Ecological catalysis and phytoextraction: Symbiosis for future[END_REF][START_REF] Clave | Ecological Recycling of a Bio-Based Catalyst for Cu Click Reaction: a New Strategy for a Greener Sustainable Catalysis[END_REF][START_REF] Clave | Ullmann reaction through ecocatalysis: insights from bioresource and synthetic potential[END_REF] As catalysis requires low amounts of metal (usually about a few mole percent) while promoting the synthesis of highly valuable compounds, the concept is potentially cost-effective enough to ensure its development on a large scale. Previous works have revealed the possibility of producing a manganese-rich material, starting from biomass of Mn-hyperaccumulating plants. The resulting material, named EcoMn, was used in organic synthesis directly as catalyst, or as source of Mn for producing more elaborate catalysts (Figure 1). [START_REF] Escande | Phytoextracted mining wastes for ecocatalysis: Eco-Mn, an efficient and eco-friendly plant-based catalyst for reductive amination of ketones[END_REF][START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H2O2/ NaHCO3: Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] Manganese displays appealing properties for catalysis, thanks to its multiple stable oxidation states and its low toxicity. [START_REF] Gerber | Carcinogenicity, mutagenicity and teratogenicity of manganese compounds[END_REF] Consequently, the use of manganese in industrial catalytic processes is expected to increase, as a substitute for precious metals or toxic elements. [START_REF] Browne | Manganese-Catalyzed Oxidation with Hydrogen Peroxide[END_REF][START_REF]In Economic Synthesis of Heterocycles: Zinc, Iron, Copper, Cobalt, Manganese and Nickel Catalysts[END_REF][23] Herein, we describe the synthesis of EcoMnOx, a new material rich in Mn oxides, which is made from EcoMn starting with Mn-hyperaccumulating plants. Study of the oxidizing properties of this material revealed excellent catalytic performances for aerobic oxidative cleavage of activated 1,2-diols. This reaction is usually performed with stoichiometric reagents, such as sodium periodate or lead tetraacetate, producing large amounts of toxic byproducts. Classical reaction conditions for oxidative cleavage of 1,2-diols are thus challenged by current standards of green chemistry. [START_REF] Anastas | Green Chemical Syntheses and Processes: Introduction[END_REF][START_REF] Anastas | Promoting Green Chemistry Initiatives[END_REF] Although catalytic alternatives have been proposed recently, they suffer from several drawbacks, as these catalysts are either based on precious metals or are homogeneous and not reusable. [START_REF] Schmidt | The Glycol Cleavage in Natural Product Synthesis: Reagent Classics and Recent Advances[END_REF][START_REF] Wong | Glycol Cleavage Reactions[END_REF] The heterogeneous nature of the EcoMnOx catalyst and its reuse are thus key advantages, combined to the use of O 2 or air as benign oxidant. This Mn catalyst prepared by valorization of waste biomass provides thus an efficient and sustainable alternative to previous methods of 1,2-diols oxidative cleavage.

EXPERIMENTAL SECTION

Materials and Methods. All commercially available chemicals were used as received. Solvents were purchased from Fisher Scientific and were of analytical grade; meso-hydrobenzoin (99%), (R,R)hydrobenzoin (99%), cis-1,2-cyclohexanediol (99%), 2,3-diphenyl-2,3butanediol (>90%), and benzopinacol (99%) were supplied by Sigma-Aldrich. Noncommercially available 1,2-diols were prepared by reduction of corresponding commercial diketones (purchased from Fisher Scientific), according to a known procedure. [START_REF] Anju | -Tetraarylporphycenes[END_REF] Characterization. ICP-AES measurements were performed using the PerkinElmer Optima 3000. Samples of 10 mg were digested and dissolved in 2% HNO 3 acid and were quantified against external standards. SEM analyses were performed using the Hitachi SU-70 field emission microscope. Samples were predried in vacuum overnight and were examined with a 10 kV bean power under ultrahigh vacuum without surface treatment. Energy-dispersive X-ray spectroscopy analysis (EDX) was performed using a Zeiss EVO HD15 microscope coupled with an Oxford X-MaxN EDX detector. XRPD measurements were performed on a Rigaku SmartLab X-ray diffractometer equipped with a Cu line-focus sealed tube and a NaI SC70 scintillation detector. Measurements were made with a 40 kV, 44 mA beam, with an incident slit of 2/3°and a length limiting slit of 5.00 mm, in the range 2θ from 10°to 90°locked couple scan type, a step size of 0.05°, and a scan speed of 10.33°/min. XPS analysis was performed using a ThermoScientific ESCALAB 250 instrument. Spectra were collected using a monochromatic Al X-ray source. A low energy electron flood and top-side contact were used for charge neutralization. Survey spectra were collected using a pass energy of 150 eV. Multiplex composition scans were acquired with 20 eV pass energy. FTIR spectra were recorded on a FTIR/Raman Thermo Nicolet 6700 spectrometer, in ATR mode. All dynamic light scattering (DLS) samples were suspended in methanol (1 mg/3 mL) and were sonicated for 5 min prior analysis. DLS was performed at an angle of 90°(ALV-5000, ALV-GmbH) using an incident laser of wavelength 532 nm (coherent). Thirty runs were recorded per sample, with a duration of 30 s per run. All DLS measurements were carried out at room temperature. Reaction products were characterized by 1 H/ [START_REF] Van Der Ent | Farming for Metals in the Future?[END_REF] C NMR spectroscopy and by GC, by comparison of their retention times with those of pure standards, and analytical data were in accordance with those previously published. NMR characterization was performed with an Agilent DD2 400 MHz NMR spectrometer. GC were recorded on a Shimadzu GC-2010 Plus, equipped with an Agilent J&W GC column, DB-5, 60 m × 0.25 mm × 0.25 μm, using He as carrier gas, with a flame ionization detector (FID). All analyses were performed including dodecane as internal standard.

Preparation of EcoMn. EcoMn was prepared from biomass of the Mn-hyperaccumulating plant Grevillea exul subsp. rubiginosa (Proteaceae), by calcination, acidic extraction then concentration. Grevillea exul subsp. rubiginosa is a native plant from New Caledonia, grown on mining sites of this subtropical Pacific island, for phytoextraction purposes. Details of the procedure and EcoMn characterization were reported in previous publications. [START_REF] Escande | Phytoextracted mining wastes for ecocatalysis: Eco-Mn, an efficient and eco-friendly plant-based catalyst for reductive amination of ketones[END_REF][START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H2O2/ NaHCO3: Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] Preparation of EcoMnOx. A solution of EcoMn (0.9 mmol Mn) was prepared by dissolution in 10 mL of deionized water. Under stirring at 400 rpm, 0.28 mL (2.7 mmol) of a 30% aqueous solution of H 2 O 2 were added in one portion. The resulting colorless solution was stirred for 5 min, then 2 mL of 19 M NaOH (38 mmol) solution were added in one portion, and a black precipitate appeared immediately. After rinsing the sides of the flask with 10 mL of deionized water, the black catalyst suspension was stirred at 800 rpm for 90 min. The resulting suspension was filtered through a glass microfiber filter (Whatman, 1.6 μm) before washing it with 3 portions of 100 mL deionized water, followed by one portion of 50 mL ethanol. The black resulting solid should not be fully left dry before the end of all rinses to preserve its activity. The black solid, labeled as EcoMnOx hereafter, was then collected and dried in air, in an oven at 105 °C, for 18 h.

Representative Procedure for Oxidative Cleavage of 1,2-Diols. In a typical reaction, a mixture of meso-hydrobenzoin (0.2 mmol), EcoMnOx (10 mol % Mn), and 1-butanol (1 mL) was added in a 7 mL flask, under a balloon filled with O 2 . The mixture was heated to 100 °C under stirring (400 rpm) for 1 h. After reaction, the mixture was cooled to room temperature, and the catalyst removed by filtration through PTFE membrane syringe filter (Millipore, 0.2 μm). Conversion and selectivity were determined by GC-FID, using dodecane as internal standard. Most reactions were repeated twice, and average values were given.

RESULTS AND DISCUSSION

Catalyst Preparation and Characterization. Synthesis of EcoMnOx was adapted from literature for preparing birnessitetype Mn oxides using Mn II salts. [START_REF] Feng | Transformation of manganese oxides from layered structures to tunnel structures[END_REF][START_REF] Feng | Synthesis of birnessitetype lithium manganese oxide[END_REF] In our case, the source of Mn II ions was EcoMn prepared from Mn-hyperaccumulating plants and described in our previous works. [START_REF] Escande | Phytoextracted mining wastes for ecocatalysis: Eco-Mn, an efficient and eco-friendly plant-based catalyst for reductive amination of ketones[END_REF][START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H2O2/ NaHCO3: Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] These precedent studies showed that EcoMn was mainly constituted of polymetallic chlorides. EcoMnOx was prepared by rapid oxidation of a solution of EcoMn in aqueous H 2 O 2 /NaOH, affording the new material as a precipitate, collected by filtration. Analysis of EcoMnOx by inductively coupled plasma atomic emission spectroscopy (ICP-AES) revealed that EcoMnOx was enriched in Mn (14.1 wt %) compared to starting EcoMn (6.7 wt %) (Table 1). This was due to EcoMnOx process of preparation, ensuring selective precipitation of insoluble oxides and hydroxides of Mn, Fe, Ca and Mg. Metal enrichment also resulted from substitution of Cl (in starting EcoMn) by O (in resulting EcoMnOx), of lower molar mass and double valence. Comparison of several batches of EcoMn and their corresponding EcoMnOx, prepared from Grevillea exul subsp. rubiginosa harvested in different locations, showed some variation in elemental content (e.g., Mn: 8.9-14.1 wt %), due to the natural origin of the biomass feedstock (Table S1). Unless specified, all the following analyses and reactions were performed with the batch of EcoMnOx described in Table 1.

X-ray photoelectron spectroscopy (XPS) confirmed the oxide nature of EcoMnOx (Figure S1). An indication of Mn oxidation state was given by Mn 2p 3/2 binding energy (642.8 eV, Figure 2), characteristic of Mn IV . [START_REF] Junta | Manganese (II) oxidation at mineral surfaces: A microscopic and spectroscopic study[END_REF] Mn 3s multiplet splitting supported this conclusion, while being less informative because of obscuration of the peak by the interfering Mg peak (Figure S2). X-ray powder diffraction (XRPD) was performed to get complementary insights on EcoMnOx composition, as shown in Figure 3. Among detected crystalline species, the presence of birnessite was confirmed. Birnessite is a Mn layered mixed oxide, mainly composed of Mn IV oxide, including also Mn III and Mn II species. [START_REF] Liu | Layered Manganese Oxides: Synthesis, Properties, and Applications[END_REF] As birnessite is often of low crystallinity, its weak signal does not typically reflect its actual proportion in the EcoMnOx catalyst. [START_REF] Post | Manganese oxide minerals: Crystal structures and economic and environmental significance[END_REF] Interestingly, other Mn oxides with different oxidation states such as CaMn 3 O 6 (Mn IV and Mn III ), [START_REF] Hadermann | Coupled cation and charge ordering in the CaMn3O6 tunnel structure[END_REF] groutite MnO(OH) (Mn III ), and Mn 3 O 4 (Mn III and Mn II ), were also detected. While the complex polymetallic nature of EcoMnOx did not allow quantification of each Mn oxidation state because of interferences with other ions like Fe 3+ , these previous analyses demonstrated that Mn IV was preponderant based on XPS analysis, along with some Mn III and Mn II species present. These observations are in accordance with the characteristics of layered Mn oxides such as birnessite, whose Mn average oxidation degree is typically between +3.4 and +3.99. [START_REF] Brock | A review of porous manganese oxide materials[END_REF] Fourier transform infrared (FTIR, Figure 4) spectroscopy confirmed the attribution of this layered oxide structure for EcoMnOx, with a characteristic absorption at 400 cm -1 . [START_REF] Potter | Tetravalent Manganese Oxides -Identification, Hydration, and Structural Relationships by Infrared-Spectroscopy[END_REF] Morphological analysis by scanning electron microscopy (SEM) of EcoMnOx revealed the presence of structures of various sizes, within the range of 10-100 μm (Figure 5a). These structures proved to be aggregates of smaller particles, as shown on Figure 5b. Dynamic light scattering (DLS) measurements were thus conducted after sonication of EcoMnOx, in order to fragment the aggregates, and revealed that average particle size was 103 ± 91 nm.

Catalyst Performance Evaluation. EcoMnOx was then tested as catalyst for oxidative cleavage of meso-hydrobenzoin as model substrate, under atmospheric pressure of O 2 . Solvent screening revealed that alcohols are the best solvents for the reaction (Table 2, entries 6-7). Previous works with manganese oxide reagents for 1,2-diols oxidative cleavage were only conducted with dichloromethane, in addition to requiring 40 equiv of Mn reagent toward substrate. [START_REF] Ohloff | Conversion of Vicinal Diols into Dicarbonyl-Compounds by Manganese-Dioxide[END_REF] As alcohols are among the greener solvents for organic synthesis, [START_REF] Henderson | Expanding GSK's solvent selection guide -embedding sustainability into solvent selection starting at medicinal chemistry[END_REF][START_REF] Prat | CHEM21 selection guide of classicaland less classical-solvents[END_REF] their use in this reaction is thus a valuable asset. With 10 mol % EcoMnOx (based on Mn content), almost complete conversion (98%) was obtained in 1 h, in 1-butanol allowing heating to 100 °C (Table 2, entry 7). A complete selectivity toward benzaldehyde was observed, with no traces of benzoic acid or other oxidation byproducts. No aldehyde disproportionation was noticed, even after prolonged reaction times.

Different atmospheres were also surveyed to assess the role of O 2 . Even if O 2 lead to shorter reaction times, oxidative cleavage of meso-hydrobenzoin was also possible under air, with the same conversion in 3 h (Table 3, entries 1-2). The absence of conversion under N 2 confirmed that O 2 acted as terminal oxidant in the reaction (Table 3, entry 3). H 2 O 2 was also tried as an alternative to O 2 for oxidative cleavage of 1,2-diols. Indeed, in a previous paper on alkene epoxidation with NaHCO 3 /H 2 O 2 catalyzed by EcoMn, some cleavage of the substrates was described, probably resulting of the hydrolysis of the epoxide, then oxidation of the resulting 1,2-diol. [START_REF] Escande | Switchable Alkene Epoxidation/Oxidative Cleavage with H2O2/ NaHCO3: Efficient Heterogeneous Catalysis Derived from Biosourced Eco-Mn[END_REF] However, when H 2 O 2 was used in the present methodology, a complete decomposition of H 2 O 2 was observed upon addition of the EcoMnOx catalyst, resulting in absence of conversion of mesohydrobenzoin (Table 3, entry 4). This decomposition can be attributed to the Mn oxides in EcoMnOx, known to decompose H 2 O 2 into H 2 O and O 2 . [START_REF] Do | Hydrogen peroxide decomposition on manganese oxide (pyrolusite): Kinetics, intermediates, and mechanism[END_REF] This result showed that the active species in EcoMnOx were different from those involved in the epoxidation/cleavage of alkenes catalyzed by EcoMn, which in a Reaction conditions: meso-hydrobenzoin 1a (42.9 mg, 0.2 mmol), EcoMnOx (10 mol % Mn), 1 mL of solvent, O 2 balloon, 1 h. b Conversion and selectivity were determined by GC using dodecane as internal standard. c Ratio of yield of aldehyde to conversion as a percentage.

the latter case were mainly Mn 2+ ions with probably only transient formation of higher oxidation states.

In another control experiment, a "synthetic EcoMnOx", prepared in the same conditions than the biosourced catalyst, but starting from only pure MnCl 2 •4H 2 O, gave an almost complete conversion (99%) with complete selectivity toward benzaldehyde (Table 3, entry 5). This result, combined to the absence of conversion without catalyst (Table 3, entry 6), confirmed that Mn oxides present in EcoMnOx were indeed the active species, and that other metal elements detected in EcoMnOx were only spectator elements. For this reason, even if some variation in elemental content was noted between several batches of EcoMnOx prepared from Grevillea exul subsp. rubiginosa harvested in different locations (Table S1), all the batches of EcoMnOx showed the same catalytic performances when used in 10 mol % Mn for oxidative cleavage of meso-hydrobenzoin.

As XPS and XRPD analyses have showed that Mn was mainly present under the oxidation state Mn IV in EcoMnOx, we tested MnO 2 and activated MnO 2 , both commercial, as catalysts in the reaction. While in both cases a complete selectivity toward benzaldehyde 2a was observed, the conversion was only 5% (Table 3, entries 7-8). While EcoMnOx, MnO 2 and activated MnO 2 have a common content in Mn IV oxide, previous studies have extensively showed that Mn IV oxides can exist under a wide variety of forms, with completely different catalytic activities. [START_REF] Liu | Layered Manganese Oxides: Synthesis, Properties, and Applications[END_REF] This could explain why EcoMnOx (and "synthetic EcoMnOx") was much more active than commercial MnO 2 , due to its unique structure, resulting of its particular preparation.

Upon optimizing reaction condition, we extended the substrate scope to explore the versatility of EcoMnOx as shown in Table 4. Like anti-1,2-diol 1a, the syn-1,2-diol 1b also cleaved efficiently with high selectivity (Table 4, entry 1), but in a slightly longer reaction time. Benzylic 1,2-diol 1c activated by electron-donating aromatic substituents was slightly more reactive than those substituted by electron-withdrawing groups such as 1d and 1e (Table 4, entries 2-4). However, cleavage of the latter ones was still performed with high conversion and in short reaction times. Among the diols surveyed, selectivity toward the corresponding aldehyde or ketone was always high (≥98%), and overoxidized products such as carboxylic acids were not detected. Interestingly, 1,2-diols with secondary hydroxyl groups showed a higher reactivity than the tertiary ones (Table 4, entry 5), probably because of steric effects in the last case, affecting interaction with the catalyst. However, tertiary diols bearing highly activating substituents, like benzopinacol 1g, were still cleaved with a complete conversion (Table 4, entry 6). Reaction with a less activated, mixed benzylic-aliphatic 1,2-diol 1h yielded relatively little products even at prolonged reaction time, which indicated the method was highly selective toward dibenzylic diols (Table 4, entry 7). This particular selectivity was confirmed by the absence of conversion of cis-1,2-cyclohexanediol 1i (Table 4, entry 8). High conversions (≥95%) were obtained with diverse activated 1,2-diols, like heterocyclic diol 1j (Table 4, entry 9) and allylic diol 1k (Table 4, entry 10), thereby extending the potential of the method. Since a difference in reaction times was observed between anti and syn diastereomers 1a and 1b respectively, a similar variation in reactivity should be probably noted for the pure diastereomers of the other substrates, used here as mixtures of variable syn/anti ratios.

In order to investigate the kinetic aspects of the reaction, time-dependent studies were conducted. A EcoMnOx-catalyzed kinetic experiment with meso-hydrobenzoin revealed a first order rate equation toward substrate, with a rate constant of 5.48 h -1 (Figure 6). When the catalyst was removed by filtration shortly after the beginning of the kinetic experiment and then the reaction continued with the filtrate, no further conversion was observed, confirming the heterogeneous nature of the catalytic species (Figure 7).

The heterogeneous nature of EcoMnOx enables catalyst recycling for multiple runs: for example, after oxidative cleavage of benzopinacol 1g, EcoMnOx was separated by centrifugation, washed with ethanol and then dried. The recovered catalyst was reapplied for an identical experiment. The procedure was repeated six successive times, and the catalyst showed no apparent loss of activity (Figure 8).

The characterization of another batch of EcoMnOx was performed after reaction, in order to assess the stability of the catalyst. The content in metal elements remained virtually unmodified before and after oxidative cleavage of mesohydrobenzoin (Table S2). Morphological analysis by SEM showed a constant size of aggregates before and after reaction (Figure S3). According to energy-dispersive X-ray (EDX) analysis, the oxygen content of EcoMnOx stayed the same (see Figure S4), which revealed that the oxide character of the material remained unmodified after reaction.

Finally, comparison with traditional reagents NaIO 4 and Pb(OAc) 4 highlighted the sustainability assets of this new method, especially for cleavage of activated 1,2-diols (Table 5). Not only the EcoMnOx-catalyzed version was performed in nontoxic 1-butanol, compared to classical dichloromethane and benzene, but the only byproduct formed with this method was water, whereas traditional reagents produce stoichiometric quantities of toxic wastes. In addition, the heterogeneous catalytic nature of EcoMnOx allowed its reuse in at least six successive runs without loss of activity. Finally, the combination of these characteristics was summarized in the E-factor value, assessing the ratio of amounts of waste formed by unit of product. [START_REF] Constable | Metrics to 'green' chemistry-which are the best?[END_REF] With a E-factor of 0.8 for meso-hydrobenzoin oxidative cleavage, the EcoMnOx-catalyzed method produced 4-17 times less waste than Pb(OAc) 4 and NaIO 4 , respectively.

CONCLUSION

In the face of metal shortages due to population growth and rapid urbanization, the EcoMn feedstock made from phytoextraction biomass serves as a novel, effective strategy to clean up and recycle Mn from mining waste. Through a simple process using readily available reagents, we turned this biosourced Mn feedstock into a new catalyst, EcoMnOx, that rivals the existing methodology for 1,2-diols oxidative cleavage. In mild reaction conditions, EcoMnOx catalyzed the aerobic oxidative cleavage of a range of activated 1,2-diols, with high conversion and selectivity. These promising results will motivate further mechanistic studies in order to determine the role of each Mn oxide species present in EcoMnOx, depending on their oxidation state and their possible cooperative catalytic effect. Considering the serious environmental concerns associated with some of the traditional reagents used for 1,2-diols oxidative cleavage, the heterogeneous nature of EcoMnOx catalyst, its possible reuse, and the formation of water as the sole byproduct while using O 2 or air as benign oxidants in a green solvent confer strong sustainable advantages to this new method.

ASSOCIATED CONTENT

Experimental details, XPS spectra, ICP-AES analyses, SEM images, EDX spectra, GC chromatograms, and E-factor calculations (PDF)

Figure 1 .

 1 Figure 1. EcoMn production as alternative valorization of Mn-rich biomass from phytoextraction crops.

Figure 2 .

 2 Figure 2. XPS Mn 2p peaks of EcoMnOx.

Figure 3 .

 3 Figure 3. XRPD pattern of crystalline species detected in EcoMnOx.

Figure 4 .

 4 Figure 4. FTIR spectrum of EcoMnOx.

Figure 5 .

 5 Figure 5. SEM images of EcoMnOx.

Table 4 .

 4 Scope of the EcoMnOx-Catalyzed Aerobic Oxidative Cleavage of 1,2-Diols a a Reaction conditions: substrate (0.2 mmol), EcoMnOx (10 mol % Mn), 1-butanol (1 mL), O 2 balloon, 100 °C. b Unless specified, the starting diols bearing two stereogenic centers were used as variable mixtures of diastereomers, with syn/anti ratio determined by 1 H NMR. c Determined by GC using dodecane as an internal standard. d Ratio of yield of aldehyde or ketone to conversion as a percentage. e Diol used in diastereomeric ratio syn/ anti 15:85.[START_REF] Uchiyama | Development of a Catalytic Electron Transfer System Mediated by Transition Metal Ate Complexes: Applicability and Tunability of Electron-Releasing Potential for Organic Transformations[END_REF] f Diol used in diastereomeric ratio syn/anti 26:74.[START_REF] Li | Pinacol Coupling of Aromatic Aldehydes and Ketones Using TiCl3-Al-EtOH Under Ultrasound Irradiation[END_REF] g Diol used in diastereomeric ratio syn/anti 29:71.[START_REF] Uchiyama | Development of a Catalytic Electron Transfer System Mediated by Transition Metal Ate Complexes: Applicability and Tunability of Electron-Releasing Potential for Organic Transformations[END_REF] h Diol used in diastereomeric ratio syn/anti 54:46. 41 i Diol used in diastereomeric ratio syn/anti 30:70.43,44 j Diol used in diastereomeric ratio syn/anti 42:58.[START_REF] Li | Pinacol Coupling of Aromatic Aldehydes and Ketones Using TiCl3-Al-EtOH Under Ultrasound Irradiation[END_REF] k Diol used in diastereomeric ratio syn/anti 52:48.[START_REF] Li | Pinacol Coupling of Aromatic Aldehydes and Ketones Using TiCl3-Al-EtOH Under Ultrasound Irradiation[END_REF]

Figure 6 .

 6 Figure 6. Kinetic study of the aerobic oxidative cleavage of mesohydrobenzoin, catalyzed by EcoMnOx. Reaction conditions: mesohydrobenzoin (0.2 mmol), EcoMnOx (10 mol % Mn), 1-butanol (1 mL), O 2 balloon, 100 °C. C(0) original concentration of substrate; C(t) concentration of substrate at time t.

Figure 7 .

 7 Figure 7. Effect of catalyst filtration on the aerobic oxidative cleavage of meso-hydrobenzoin. Reaction conditions: meso-hydrobenzoin (0.2 mmol), EcoMnOx (10 mol % Mn), 1-butanol (1 mL), O 2 balloon, 100 °C. After 0.08 h, EcoMnOx was removed by filtration then the reaction was continued with the filtrate. Yield of benzaldehyde was determined by GC using dodecane as an internal standard.

Figure 8 .

 8 Figure 8. Reuse of EcoMnOx catalyst in successive reaction runs.Reaction conditions: benzopinacol (0.2 mmol), EcoMnOx (10 mol % Mn), 1-butanol (1 mL), O 2 balloon, 100 °C, 1 h. After reaction, the catalyst was recovered by centrifugation, rinsed with 2 × 4 mL of ethanol and then dried in oven under air at 105 °C for 2 h. Yields were determined by GC using dodecane as an internal standard.

Table 1 .

 1 ICP-AES Analysis of EcoMnOx Compared to Starting EcoMn

	element wt % (±sd)	Fe	Mn	Ni	Ca	Mg	Al	Na	K
	EcoMn	0.9 (±0.008)	6.7 (±0.04)	0.04 (±0.0004)	10.4 (±0.2)	5.3 (±0.09)	0.2 (±0.02)	2.0 (±0.1)	4.3 (±0.008)
	EcoMnOx	0.3 (±0.006)	14.1 (±0.4)	nd a	28.2 (±0.2)	28.7 (±0.3)	nd a	0.5 (±0.09)	nd a

a Not detected.

Table 2 .

 2 Solvent Screening for EcoMnOx-Catalyzed Oxidative Cleavage of meso-Hydrobenzoin

	1	acetonitrile	78	43	>99
	2	ethyl acetate	78	35	>99
	3	toluene	78	55	>99
	4	water	78	53	>99
	5	acetic acid	78	47	>99
	6	ethanol	78	61	>99
	7	1-butanol	100	98	>99

a entry solvent T [°C] conversion 1a [%] b selectivity 2a [%] b,c

Table 3 .

 3 meso-Hydrobenzoin Oxidative Cleavage with Different Mn Oxide Catalysts and Atmospheres a Conversion and selectivity were determined by GC using dodecane as an internal standard. c Ratio of yield of aldehyde to conversion as a percentage. d Reaction performed in the presence of H 2 O 2 (30 wt % aqueous solution, 3.0 mmol). e Catalyst prepared according the same protocol than EcoMnOx, but with commercial MnCl 2 •4H 2 O.

	entry	catalyst	atmosphere	t [h]	conversion 1a [%] b	selectivity 2a [%] b,c
	1	EcoMnOx	O 2	1	98	>99
	2	EcoMnOx	air	3	98	>99
	3	EcoMnOx	N 2	1	2
	4 d	EcoMnOx	N 2	1	0
	5	≪synthetic≫ EcoMnOx e	O 2	1	99	>99
	6	none	O 2	1	0
	7	commercial MnO 2	O 2	1	5	>99
	8	commercial activated MnO 2	O 2	1	5	>99
	a Reaction conditions: meso-hydrobenzoin 1a (42.9 mg, 0.2 mmol),
	catalyst (10 mol % Mn), 1-butanol (1 mL), gas balloon, 100 °C.

b

Table 5 .

 5 Comparison of the EcoMnOx-Catalyzed Method with NaIO 4 and Pb(OAc) 4 , on the Basis of Sustainability Characteristics Calculated for 90% yield in typical reaction conditions from ref 46. c meso-Hydrobenzoin oxidative cleavage was used as model reaction for E-factor calculations with the different methods.and harvest of Mn-hyperaccumulating plants, as well as the New Caledonia authorities for supporting phytoextraction crops on mining sites. Dr. Stephen Golledge from University of Oregon is acknowledged for assistance with XPS analyzes. Dr. Yves-Marie Legrand from Institut Europeén des Membranes is acknowledged for assistance with SEM and EDX analyses. Special thanks to Dr. Sara Hashmi at the Facility for Light Scattering at Yale University for her assistance and instrumentation support.

		EcoMnOx/ O 2 a	NaIO 4 b	Pb(OAc) 4 b
	solvent typically used	1-butanol	dichloromethane	benzene
	wastes formed	water	NaIO 3	Pb(OAc) 2
	successive runs without	≥6 (6 runs	1	1
	loss of activity	tested)
	E-factor (E) c	0.8	13.9	3.2

a See the SI for details of reaction conditions and calculations. b

ACKNOWLEDGMENTS V.E. gratefully acknowledges support from the French National Center for Scientific Research (CNRS). C.H.L. would like to thank the Gaylord and Dorothy Foundation for the fellowship support. Dr. Cyril Poullain is acknowledged for the cultivation

AUTHOR INFORMATION

Corresponding Authors *E-mail: vincent.escande@cnrs.fr *E-mail: paul.anastas@yale.edu ORCID Vincent Escande: 0000-0003-3164-8128 Paul T. Anastas: 0000-0003-4777-5172

Notes

The authors declare no competing financial interest.


Javiera Toledo-Alarcón
email: javiera.toledo@pucv.cl

Lea Cabrol

David Jeison

Eric Trably

Jean-Philippe Steyer

Estela Tapia-Venegas

Impact of the microbial inoculum source on pre-treatment efficiency for fermentative H 2 production from glycerol

Keywords: Aeration Treatment, Biohydrogen, Biokinetic control, Dark fermentation, Microbial Community

Hydrogen (H 2 ) production by dark fermentation can be performed from a wide variety of microbial inoculum sources, which are generally pre-treated to eliminate the activity of H 2consuming species and/or enrich the microbial community with H 2 -producing bacteria.

This paper aims to study the impact of the microbial inoculum source on pre-treatment behavior, with a special focus on microbial community changes. Two inocula (aerobic and anaerobic sludge) and two pre-treatments (aeration and heat shock) were investigated using glycerol as substrate during a continuous operation. Our results show that the inoculum source significantly affected the pre-treatment efficiency. In aerobic sludge no pretreatment is necessary, while in anaerobic sludge the heat pre-treatment increased H 2 production but aeration caused unstable H 2 production. In addition, biokinetic control was key in Clostridium selection as dominant species in all microbial communities. Lower and *Manuscript (Revised, clean, unmarked, FINAL version) 2 unstable H 2 production were associated with a higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H 2 production in continuous systems and how the microbial community is affected. This provides key information for efficient selection of operating conditions for future applications.

INTRODUCTION

The growing environmental pollution of cities has motivated the search for new sources of clean and renewable energy. In this context hydrogen (H 2 ) appears as a great environment friendly alternative for transportation. Indeed, its combustion produces only water vapor instead of greenhouse gases, with a combustion efficiency 2.75 (122 kJ/g) times higher than traditional fuels and can also be easily converted into electricity in electric vehicle fuel cells [START_REF] Patel | Nanoparticles in Biological Hydrogen Production: An Overview[END_REF], [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF]. Green H 2 is considered a renewable energy since it is produced from renewable resources, such as organic matter by dark fermentation. This latter technology has been widely studied because of a high simplicity and the low operating and maintenance costs when compared to other biological H 2 production systems, such as photofermentation and biophotolysis. In addition, a wide variety of substrates and inocula can be used allowing the production of energy while treating waste [START_REF] Patel | Nanoparticles in Biological Hydrogen Production: An Overview[END_REF], [START_REF] Yin | Changes in microbial community during biohydrogen production using gamma irradiated sludge as inoculum[END_REF]- [START_REF] Azwar | Development of biohydrogen production by photobiological, fermentation and electrochemical processes: A review[END_REF]. Different types of waste and organic substrates have already been studied including simple sugars such as glucose and more complex organic matter such as organic industrial wastes [START_REF] Toledo-Alarcón | Basics of bio-hydrogen production by dark fermentation[END_REF]. A special interest has been focusing on crude glycerol, the main by-product of the biodiesel industry, as a low-cost feedstock [START_REF] Lo | Dark fermentative hydrogen production with crude glycerol from biodiesel industry using indigenous hydrogen-producing bacteria[END_REF]- [START_REF] Moscoviz | Electro-fermentation triggering population selection in mixed-culture glycerol fermentation[END_REF].

Dark fermentation H 2 production performances from glycerol are mostly dependent to the microbial physiological capacities. As microbial inoculum, strains of known H 2 -producing bacteria could be used in pure cultures, including facultative anaerobes as Klebsiella sp.

and Enterobacter sp. of the Enterobacteriaceae family, as well as the strict anaerobes Clostridium sp. of the Clostridiaceae family [START_REF] Ngo | High-yield biohydrogen production from biodiesel manufacturing waste by Thermotoga neapolitana[END_REF]- [START_REF] Papanikolaou | High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain[END_REF]. Mixed cultures coming from natural and engineered ecosystems such as soil, compost, anaerobic sludge and other anaerobic environments [START_REF] Temudo | Glycerol fermentation by (open) mixed cultures: A chemostat study[END_REF]- [START_REF] Seifert | Hydrogen generation from glycerol in batch fermentation process[END_REF] have also been used as inocula, with the advantage of providing better adaptation capacity in response to environmental stresses including substrate limitation and abrupt changes in pH and temperature [START_REF] Toledo-Alarcón | Basics of bio-hydrogen production by dark fermentation[END_REF], [START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF], [START_REF] Wang | Factors influencing fermentative hydrogen production: A review[END_REF]. The higher robustness of mixed cultures has been attributed to the diversity of the microbial community, enabling positive interspecies interactions such as syntrophy [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF], [START_REF] Kouzuma | Microbial interspecies interactions : recent findings in syntrophic consortia[END_REF]. Some mixed community members can also generate adverse effects on the system performance through negative interactions [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF]. The origin of the inoculum, its pre-treatment, the operating strategy of the reactors and the biokinetic control in continuous systems are of crucial importance to ensure H 2 -producer enrichment and achieve high and stable H 2 -production performance [START_REF] Ivanov | Microbiology of Environmental Engineering Systems BT -Environmental Biotechnology[END_REF].

Inocula pre-treatments seek to eliminate H 2 -consumers such as hydrogenotrophic methanogenic archaea and enrich the community with H 2 -producers [START_REF] Viana | Fermentative H2 production from residual glycerol: a review[END_REF], [START_REF] Saady | Homoacetogenesis during hydrogen production by mixed cultures dark fermentation: Unresolved challenge[END_REF]. Heat shock pre-treatment is the most used at lab scale for its efficiency in batch systems. Pre-treatment conditions are generally arbitrary and range from 50°C to 125°C and from 20 to 30 min [START_REF] Valdez-Vazquez | Semi-continuous solid substrate anaerobic reactors for H2 production from organic waste: Mesophilic versus thermophilic regime[END_REF]- [START_REF] Bundhoo | Effects of pre-treatment technologies on dark fermentative biohydrogen production: A review[END_REF]. In this case, the microbial community is enriched with spore-forming species such as the H 2 -producer Clostridium sp., resisting to high temperature [START_REF] Toledo-Alarcón | Basics of bio-hydrogen production by dark fermentation[END_REF], [START_REF] Bundhoo | Effects of pre-treatment technologies on dark fermentative biohydrogen production: A review[END_REF]. However, other non-spore-forming H 2 -producing species are also depleted such as Klebsiella sp., and Enterobacter sp. [START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF], [START_REF] Kumar | Impact of pH control and heat pre-treatment of seed inoculum in dark H2 fermentation: A feasibility report using mixed microalgae biomass as feedstock[END_REF]. Moreover, heat shock pre-treatment requires additional energy consumption, which is questionable in terms of economic and technical feasibility for a potential industrial application [START_REF] Kothari | A critical review on factors influencing fermentative hydrogen production[END_REF], [START_REF] Baghchehsaraee | The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures[END_REF]. Another less common pre-treatment is aeration, which enriches the inoculum with aerobic and facultative anaerobic H 2 -producers such as Klebsiella sp., but also eliminates other oxygen-intolerant H 2 -producing bacteria such as some Clostridium sp. [START_REF] Wang | Principle and application of different pretreatment methods for enriching hydrogen-producing bacteria from mixed cultures[END_REF]. Unlike heat shock pre-treatment, aeration could be performed in-situ as an industrially viable alternative to the common instability problems of continuous systems during H 2 production [START_REF] Castelló | Possible causes for the instability in the H2production from cheese whey in a CSTR[END_REF], [START_REF] Bakonyi | Review on the start-up experiences of continuous fermentative hydrogen producing bioreactors[END_REF].

This paper aims to study the combined effects of inoculum source and pre-treatment on continuous H 2 production efficiency from glycerol, with special focus on the dynamics of microbial communities. For this, two inocula (aerobic and anaerobic sludge) and two pretreatments (aeration and heat shock pre-treatment) were compared.

MATERIALS AND METHODS

Inocula source

Two mixed cultures were used as inoculum: (i) anaerobic sludge (13.1 gVSS.l -1 ) from a sludge stabilizing anaerobic reactor and (ii) aerobic sludge (15.8 gVSS.l -1 ) from an activated sludge reactor. Both were collected from the sewage treatment plant La Farfana located in Santiago, Chile.

Pre-treatments of inocula

Inocula were either used without pre-treatment (in control conditions), or prepared using two different pre-treatments prior to reactors inoculation (Table 1). A heat treatment (HT) was conducted at 105 °C for 2 h. Aeration (AT) was performed by bubbling air for 4 weeks at a rate providing oxygen saturation. Dissolved oxygen was monitored during these treatments, using a probe and a controller. During aeration, glucose was added as carbon source (10 g.L -1 ), as well as other nutrients detailed in Section 2.3 (patent N° 201402319, INAPI, Chile).

Experimental set-up

Six continuous stirred tank reactors (CSTR) were operated at different conditions, to compare the combined effects of two inocula (aerobic and anaerobic sludge) and two pretreatments (HT and AT) on continuous H 2 -production. In addition, a control (C) without pre-treatment was performed for each inoculum. Tested conditions are summarized in Table 1. Reactors had a useful volume of 2 L, and were operated at 12 hours of hydraulic retention time (HRT), pH 5.5 and 37 °C. The reactors were inoculated with 0.4 L of inoculum and then operated in batch mode for 24 hours before starting continuous operation. The reactors were operated continuously for at least 16 HRT. The cultivation medium was composed of 7.5±1.1 g.L -1 glycerol and others nutrients as follows (mg.l -1 ) 1 [START_REF] Silva-Illanes | Impact of hydraulic retention time (HRT) and pH on dark fermentative hydrogen production from glycerol[END_REF].

Analytical methods

An online MILLIGASCOUNTER® Type MGC-1 was utilized to continuously determine the volume of biogas produced. Biogas composition (H 2 , CO 2 , and CH 4 ) was daily measured by gas chromatography (Perkin Elmer Clarus 500, Hayesep Q 4m x 1/8''OD column, thermal conductivity detector). The concentration of ethanol, acetate, propionate and butyrate was daily measured by gas chromatography (Perkin Elmer Clarus 500, 60/80 Carbopack C column, flame ionization detector). The concentration of glycerol, formate and succinate was measured by HPLC with a refractive index detector (Biorad Aminex HPX-87H column, Bio-Rad laboratories, Hercules, CA -US). The biomass concentration was estimated using dry weight in terms of volatile suspended solids (VSS).

Microbial community analysis

For molecular biology analysis, 2 mL biomass samples were collected from original inocula, after pre-treatments and at the end of the continuous operation. Biomass samples were centrifuged, and the pellet was stored in 9 % NaCl at -20 °C. Total genomic DNA was extracted with the Power Soil DNA isolation kit (MoBio Laboratories, Carlsbad, CA, USA). The V3-V4 region of the bacterial 16s rRNA gene was PCR-amplified according Carmona-Martinez et al. (2015) [START_REF] Carmona-Martínez | Long-term continuous production of H2 in a microbial electrolysis cell (MEC) treating saline wastewater[END_REF]. The community composition was evaluated by sequencing using the MiSeq v3 chemistry (Illumina) with 2x300 bp paired-end reads at the GenoToul platform (http://www.genotoul.fr). Sequences were retrieved after demultiplexing, cleaning, clustering (97%) and affiliating sequences using Mothur [START_REF] Schloss | Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities[END_REF]. A total of 3216 operational taxonomic units (OTU) were found and then used for statistical analysis. Sequences have been submitted to GenBank with accession No. KX632952-KX636081.

Data analysis

Averages and standard deviations (±SD) of biomass production, H 2 yields and metabolites concentrations were calculated from daily measurements during continuous operation (for at least 16 HRT). H 2 yields was expressed in moles of H 2 produced by moles of glycerol consumed. Chemical oxygen demand (COD) mass balance was performed and the metabolites concentrations were expressed in %COD i.e. COD of metabolites produced by COD of glycerol consumed.

An ANOVA analysis was performed to evaluate significant differences in H 2 yields and biomass production between the conditions. Simpson diversity index was calculated to compare microbial diversity at the beginning and end of each condition. Principal component analysis was performed from (i) initial and final microbial community and, (ii) final microbial community and metabolic patterns. For the PCA were used the microbial community data with a relative abundance >5.0% in at least one sample. All statistical analyses were carried out with PAST 3.24 software (http://folk.uio.no/ohammer/past/).

RESULTS & DISCUSSION

Microbial communities in original and pre-treated inocula

The initial microbial community was analyzed in the original inocula (AI and AnI) and in the pre-treated inocula (AI-HTi, AnI-HTi, AI-ATi and AnI-ATi). Whereas the Simpson Diversity Index quantifies microbial diversity, where 1 represents infinite diversity and 0 represents no diversity. The original inocula have a high diversity with a Simpson Diversity Index of 0.98 and 0.92 for aerobic and anaerobic sludge, respectively. After any pretreatments the Simpson Diversity Index showed no great changes compared to the original inocula (Table 2). When comparing all communities, the highest similarity is observed between both original inocula AI and AnI, as shown on the PCA (Fig. 1).

At the phylum level, aerobic and anaerobic inocula were dominates by Bacteroidetes and Spirochaetae phyla, representing between 34.8% -40.4% and 20.3% -26.4% of bacterial community respectively. In particular, the most abundant families in aerobic sludge (AI)

were Spirochaetaceae (12.4%) and Rikenellaceae (10.3%), with OTU11 (8.2%) dominating. OTU11 had 91% of 16S rRNA sequence similarity with Rectinema cohabitans. In anaerobic sludge (AnI) the most abundant families were Rikenellaceae (18.8%) and WCHB1-69 (11.5%), with OTU5 (18.4%) and OTU6 (16.2%) dominating (Fig. 2). These two OTUs were related to Mucinivorans hirudinis (87% 16S rRNA sequence similarity with OTU5) and Eubacterium minutum (78% 16S rRNA sequence similarity with OTU6). Although these families have been reported as dominant in other initial microbial communities of H 2 -producing reactors [START_REF] Marshall | Electrosynthesis of commodity chemicals by an autotrophic microbial community[END_REF], [START_REF] Tapia-Venegas | Bio-hydrogen production during acidogenic fermentation in a multistage stirred tank reactor[END_REF], none of these dominant species in AI or AnI have been reported as H 2 -producing. Heat shock pre-treatment has a rather limited impact on total microbial community, as shown on the PCA in Fig. 1. After heat shock, the total abundance of the initially dominant Bacteroidetes and Spirochaetae phyla decreased in both inocula. By contrast, the relative abundance of Firmicutes and Proteobacteria increased, representing between 9.4 -18.5% and 29.2 -29.5% of the bacterial community respectively (Table 2). Proteobacteria became dominant in both inocula. However, at the family level, the same Rikenellaceae family as before the heat shock was maintained dominant in the anaerobic inoculum (AnI-HTi) (17.3%) and became dominant in the aerobic inoculum (AI-HTi) (8.2%), even if its relative abundance slightly decreased with respect to the original inocula. In anaerobic inoculum the same OTU5 (17.1%) and OTU6 (14.5%) remained dominant, while in aerobic inoculum OTU25 (5.3%) became dominant. The OTU25 had 95% of 16S rRNA sequence similarity with Desulfonatronobacter acetoxydans. As expected, the aerobic inoculum community was more evenly distributed than the anaerobic one, even after heat-treatment. Besides, heat treatment not only favored families with known spore-forming species such as Peptostreptococcaceae, but also families with non-spore forming species such as Desulfobacteraceae and Christensenellaceae [START_REF] Morotomi | Description of Christensenella minuta gen. nov., sp. nov., isolated from human faeces, which forms a distinct branch in the order Clostridiales, and proposal of Christensenellaceae fam. nov[END_REF], [START_REF] Rosenberg | The prokaryotes: Prokaryotic physiology and biochemistry[END_REF]. However, this finding is not unusual since other studies have reported that non-spore forming species can survive drastic treatments such as heat shock [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF]. Surprisingly, the heat treatment resulted in a very limited enrichment of the community with members of well-known H 2 -producing families such as Clostridiaceae or Enterobacteriaceae.

The aeration pre-treatment resulted in more drastic composition changes than the heat shock, and more divergent communities depending on the inoculum source, as shown on PCA in Fig. 1. Especially, aeration decreased the abundance of the initially dominant Bacteroidetes and Spirochaetae phyla, strongly increasing the relative abundance of Firmicutes and Proteobacteria representing between 32.2 -41.7% and 32.9 -41.7% of the bacterial community respectively (Table 2). After aeration, Clostridiaceae became the most abundant family in both aerobic (AI-ATi) and anaerobic (AnI-ATi) inocula, representing 27.1% and 18.6% of microbial community, respectively. The second most abundant family was Prevotellaceae (11.8%) and Flavobacteriaceae (12.1%) for aerobic and anaerobic inocula, respectively. Clostridiaceae and Prevotellaceae families have strict anaerobic species commonly found in H 2 producing systems, while Flavobacteriaceae is mainly composed of aerobic species not commonly found in H 2 -producing reactors [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF], [START_REF] Berbardet | Proposed minimal standards for describing new taxa of the family[END_REF].

Besides, in aerobic inoculum OTU3 (11.4%) and OTU2 (8.9%) were dominant, while OTU240 (8.9%) and OTU1 (8.8%) in anaerobic inoculum. These four OTUs were related to Clostridium butyricum (100% 16S rRNA sequence similarity with OTU3), Prevotella paludivivens (90% 16S rRNA sequence similarity with OTU2), Sphingobium yanoikuyae (100% 16S rRNA sequence similarity with OTU240) and Clostridium pasteurianum (98% 16S rRNA sequence similarity with OTU1).

Our results show that aerobic pre-treatment allows the selection of species with aerobic and facultative anaerobic metabolisms belonging to families such as Pseudomonadaceae and Moraxellaceae (Fig. 2). Especially, the increase of the Enterobacteriaceae family known for its facultative anaerobic H 2 producing members was observed [START_REF] Sinha | Role of formate hydrogen lyase complex in hydrogen production in facultative anaerobes[END_REF]. Surprisingly, the important presence of the Clostridiaceae family was also observed, whose members managed to remain and multiply despite theoretically lethal aeration conditions. This could show positive interactions, where non oxygen tolerant microorganisms could be protected by others through oxygen consumption during stressful conditions such as aeration.

Performance indicators during continuous H 2 production

Biomass production for all experiments did not differ significantly (ANOVA, p-value ≤0.05), with average growth yields reaching 6.0±2.1 g VSS .mol gly-consumed -1 (Table 3). All experiments produced H 2 with yields ranging from 0.29±0.10 to 0.55±0.08 mol H2 .mol gly- consumed -1 , except for the anaerobic sludge after aeration treatment (AnI-AT), which produced H 2 unsteadily (Table 3). In general, the H 2 yields obtained in this study are within the ranges reported in literature (0.05 to 0.58 mol H2 mol gly-consumed -1 ) for dark fermentation from glycerol using mixed cultures in continuous systems [START_REF] Temudo | Glycerol fermentation by (open) mixed cultures: A chemostat study[END_REF], [START_REF] Silva-Illanes | Impact of hydraulic retention time (HRT) and pH on dark fermentative hydrogen production from glycerol[END_REF], [START_REF] Tapia-Venegas | Biohydrogen production by dark fermentation: scaling-up and technologies integration for a sustainable system[END_REF]- [START_REF] González-Pajuelo | Metabolic engineering of Clostridium acetobutylicum for the industrial production of 1,3-propanediol from glycerol[END_REF]. Soluble metabolites produced concomitantly with H 2 are detailed in Comparing the two-original sludge (i.e., not pre-treated) in the control experiences (AI-C and AnI-C), a 72% higher H 2 yield was obtained along with 31.2% more butyrate and 47.2% less ethanol using aerobic sludge than using anaerobic sludge. This is consistent with literature where higher H 2 production is often associated with higher butyrate production [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF], [START_REF] Lee | Thermodynamic evaluation on H2 29 production in glucose fermentation[END_REF], [START_REF] Ghimire | A review on dark fermentative biohydrogen production from organic biomass : Process parameters and use of by-products[END_REF]. This demonstrates a better adaptability for H 2 production of untreated aerobic sludge compared to anaerobic sludge in a continuous system using glycerol as substrate. As already reported, untreated anaerobic sludge may require more time to adapt to glycerol [START_REF] Tapia-Venegas | Adaptation of acidogenic sludge to increasing glycerol concentrations for biohydrogen production[END_REF]. Besides, although no methane production was observed in any reactor, a part of H 2 could have been consumed by other H 2 -consuming microorganisms present in the untreated anaerobic sludge such as homoacetogens or hydrogenotrophic methanogenic archaea.

When inoculum pre-treatments were performed, different effects were observed depending on the inoculum sources. Within aerobic sludge experiments (AI-C, AI-HT, and AI-AT), the compared pre-treatments did not have any significant effect on H 2 yield (ANOVA, pvalue ≤0.05). On the contrary, within anaerobic sludge experiments, heat treatment (AnI-HT) increased H 2 -yields by 45% compared to control (AnI-C). In addition, the heat pretreatment resulted in two similar H 2 production systems (AnI-HT and AI-HT) with slightly different metabolite production but statistically equal H 2 yields (ANOVA, p-value ≤0.05), despite the inocula came from different sources. This shows the reproducibility and effectiveness of heat treatments, leaving evidence why has been widely reported in the literature to prepare different inocula for the H 2 production by dark fermentation [START_REF] Kumar | Impact of pH control and heat pre-treatment of seed inoculum in dark H2 fermentation: A feasibility report using mixed microalgae biomass as feedstock[END_REF], [START_REF] Ghimire | A review on dark fermentative biohydrogen production from organic biomass : Process parameters and use of by-products[END_REF],

[49]- [START_REF] Vasconcelos | Factors that affect bacterial ecology in hydrogen-producing anaerobic reactors[END_REF].

Unlike heat treatment, aerobic treatment on anaerobic sludge (AnI-AT) generated a negative effect respect to the control (AnI-C), causing unstable H 2 production during all operation days. Consequently, when comparing the behavior of both sludge when exposed to aerobic treatment, again aerobic sludge showed a better adaptability to H 2 production compared to anaerobic sludge.

In conclusion, and depending on the inoculum source, three effects of pre-treatment on H 2 production can be observed respect to the control: Positive effect (i.e. heat pre-treatment on anaerobic sludge), negative effect (i.e. aerobic pre-treatment on anaerobic sludge) and neutral effect (i.e. heat pre-treatment and aerobic pre-treatment on aerobic sludge).

Consistently, the inoculum source importance on the efficiency of the pre-treatment was already evidenced but in a study performed in batch mode operation using glucose, and comparing two pre-treatments: heat treatment and acidification [START_REF] Kawagoshi | Effect of inoculum conditioning on hydrogen fermentation and pH effect on bacterial community relevant to hydrogen production[END_REF].

Link between final microbial community and metabolic patterns during continuous H 2 production

DNA samples were collected at the end of the continuous operation to assess changes in the microbial community. As shown in Table 2, the Clostridiaceae family was most abundant in all conditions, with a relative abundance between 35.3% and 56.4% and was mainly represented by OTU1 and OTU3 (Fig. 2). OTU1 was dominant in AnI-C (44.7%), AI-HT (46.2%), AnI-HT (49.5%) and AI-AT (39.2%), while OTU3 in AI-C (28.5%) and AnI-AT (34.9%). The Prevotellaceae family was the second most abundant in AnI-C (25.6%), AI-HT (35.9%), AnI-HT (34.1%) and AI-AT (32.2%) reactors and was represented by OTU2 (Fig. 2). The second and third most abundant family in AI-C were Enterococaceae (27.1%)

and Porphyromonadaceae (20.8%) and were mainly represented by OTU7 (18.0%) and OTU8 (20.8%), respectively (Fig. 2). These two OTUs were related to Enterococcus gallinarum (99% 16S rRNA sequence similarity with OTU7) and Dysgonomonas mossii (100% 16S rRNA sequence similarity with OTU8). In AnI-AT, the second and third most abundant family were Enterobacteriaceae (25.4%) and Prevotellaceae (24.9%) and were mainly represented by OTU4 (22.4%) and OTU10 (17.5%), respectively. These two OTUs were related to Klebsiella aerogenes (99% 16S rRNA sequence similarity with OTU4) and Prevotella dentalis (90% 16S rRNA sequence similarity with OTU10). In AnI-C the Enterobacteriaceae (16.7%) family was the third most abundant and was mainly represented by OTU16 (16.4%). The OTU16 had 100% of 16S rRNA sequence similarity with Raoultella ornithinolytica.

Illustratively Fig. 3 shows a principal component analysis (PCA) performed from final microbial community at family level and metabolic patterns to observe the relations between them according to each experiment. The PCA shows that the control experiences (AI-C and AnI-C) are negatively related, probably due to the great impact of the inoculum origin on both the final microbial communities and reactor behavior. Particularly, AI-C is related to butyrate production and with Enterococaceae and Porphyromonadaceae families.

While, AnI-C is slightly related to acetate and ethanol production. The heat-treated reactors, independently of the inoculum (AI-HT and AnI-HT), were characterized by higher abundance of the Clostridiaceae and Prevotellaceae families along with the production of ethanol, acetate and butyrate, as is observed in Fig. 3. This is consistent with the literature, since some species of the Clostridiaceae family could present an acidogenic or solventogenic metabolism, associated to a higher H 2 production along with acetate-butyrate pathway and a lower H 2 production along with the production of alcohols such as ethanol, respectively [START_REF] Sarma | Microbial hydrogen production by bioconversion of crude glycerol: A review[END_REF]. Unlike heat pre-treatment, aerobic pre-treatment generated two slightly different microbial communities. Fig. 3 shows how AnI-AT is related to the Enterobacteriaceae family, while AI-AT is related to the succinate production.

In addition, Fig. 3 shows that microbial diversity is positively related to AnI-AT and negatively related to AI-HT and AnI-HT. The literature is not clear on how microbial diversity could affect H 2 production. Contradictorily, it has been reported that greater diversity may increase the possibilities of selecting H 2 -producing bacteria, but it may also increase competition among members of the microbial community, leading to a decrease in the H 2 production [START_REF] Baghchehsaraee | The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures[END_REF], [START_REF] Favaro | Effects of inoculum and indigenous microflora on hydrogen production from the organic fraction of municipal solid waste[END_REF], [START_REF] Marone | Hydrogen production from vegetable waste by bioaugmentation of indigenous fermentative communities[END_REF]. Our results show that the microbial community was considerably simplified and that the Simpson diversity index decreased by 15.8 to 33.0% compared to the initial inocula. In particular, heat shock pre-treatment reduced microbial diversity by 32.7±0.5%, while aeration decreased by 19.5±1.0% (Table 2). However, greater microbial diversity could be linked to lower H 2 production efficiency.

Combined effect of inoculum source and pre-treatments on microbial community

Fig. 1 shows a PCA performed from samples taken before inoculating the reactors and at the end of continuous operation. Three main groups are observed, in which the change of the microbial community from the original sludge, after pre-treatment and after continuous operation is clearly evidenced. In the first group (Fig. 1, on the right) the original sludge (AI and AnI) is associated with the sludge after heat pre-treatment (AI-HTi and AnI-HTi).

In turn, this group is associated with the most important families of their microbial community, i.e., Rikenellaceae, Spirochaetaceae and WCHB1-69. The second group (Fig. 1, top) includes sludge after aeration pre-treatment (AI-ATi and AnI-ATi) and are related to families that increased their relative abundance in at least one of these samples such as Sphingomonadaceae, Flavobacteriaceae and Pseudomonadaceae. While the third group (Fig. 1, left down) is composed of all the samples taken at the end of the continuous operation and are related to the families that dominated the final microbial communities in each case, i.e. Porphyromonadaceae, Enterococcaceae, Clostridiaceae, Prevotellaceae and

Enterobacteriaceae. In all cases, there is more similarity between reactor communities inoculated with different sludge exposed to same pre-treatment, suggesting that the pretreatment has more impact than the inoculum source on the total community structure.

Despite the pre-treatments performed and the inoculum origin, the selection pressure imposed by biokinetic control appears to be crucial in determining the dominant families of the H 2 -producing microbial community, particularly in the selection of Clostridiaceae family members. This is consistent with the literature, as members of this family are often selected during continuous H 2 production operated at low pH (values between 5.0 -6.0) and short HRT (<12 h) [START_REF] Palomo-Briones | Dark-fermentative biohydrogen pathways and microbial networks in continuous stirred tank reactors: Novel insights on their control[END_REF], [START_REF] Silva-Illanes | Impact of hydraulic retention time (HRT) and pH on dark fermentative hydrogen production from glycerol[END_REF], [START_REF] Rafrafi | Sub-dominant bacteria as keystone species in microbial communities producing bio-hydrogen[END_REF], [START_REF] Palomo-Briones | Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor[END_REF].

When considering the experiments that used untreated inoculum, it is observed that despite the impact of biokinetic control (as discussed above) on selection of Clostridiaceae family members, AI-C had a 72% higher H 2 yield than in AnI-C (Table 3). Among the dominant species of the AI-C microbial community is Dysgonomonas mossii (Fig. 2), a fermentative but not H 2 -producing bacteria [START_REF] Montpart | Hydrogen production in single chamber microbial electrolysis cells with different complex substrates[END_REF]- [60]. AI-C reach the maximum H 2 yield of this study, suggesting a positive interaction of Dysgonomonas mossii with the microbial community and especially with the known H 2 -producing bacteria. Unlike AI-C, all dominant families in the AnI-C microbial community (i.e., Clostridiaceae, Prevotellaceae, and Enterobacteriaceae) have known H 2 -producing members, but the low H 2 yield obtained in this experiment suggests the predominance of negative interactions in the microbial community. Therefore, the inoculum source plays a key role in determining the final microbial community when no pre-treatment is performed.

Comparing the metabolic patterns when a heat-treated inoculum (AI-HT and AnI-HT) was used, and especially the H 2 yields, no statistically significant differences are observed, although the inocula come from different sources. Surprisingly, the final microbial community of both is very similar, with Clostridium and Prevotella as dominant genus. The relative abundance of Clostridium at the end of these experiments was more than 50%, which was expected since the heat treatment objective is to enrich the microbial community with spore-forming species such as Clostridium. Contrary to our results, [START_REF] Baghchehsaraee | The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures[END_REF] obtained lower H 2 yield when using heat-treated aerobic sludge, attributed to a decrease in microbial diversity due to pre-treatment [START_REF] Baghchehsaraee | The effect of heat pretreatment temperature on fermentative hydrogen production using mixed cultures[END_REF]. However, they worked with glucose as substrate in batch mode operation and heat pre-treatment conditions were 65°, 80° or 95° for 30 min. Consequently, they are all important parameters affecting the microbial community composition.

Aeration as pre-treatment generated important differences in microbial communities and H 2 yields depending on the inoculum source (AI-AT and AnI-AT). The main difference was the relative abundance of Klebsiella aerogenes in AnI-AT, a known H 2 producing bacteria.

However our results show that it is negatively related to H 2 production suggesting a negative interaction with other known H 2 producers in the community, which are in the ratio 1.1:1.6:1.0 for Prevotella:Clostridium:Klebsiella, respectively [START_REF] Cabrol | Microbial ecology of fermentative hydrogen producing bioprocesses: Useful insights for driving the ecosystem function[END_REF]. In contrast to our results Silva-Illanes et al., (2017) evidenced the existence of positive interactions between all H 2 -producing bacteria present in the microbial community, which are in the ratio 1.2:5.4:1.0 for Prevotella:Clostridium:Klebsiella, respectively [START_REF] Silva-Illanes | Impact of hydraulic retention time (HRT) and pH on dark fermentative hydrogen production from glycerol[END_REF]. Consequently, the difference in the results is the relative abundance of H 2 producers and their ratio, while in our results the genera are in a ratio around 1.0, in Silva-Illanes et al., (2017) Clostridium is the most important being 5.4 times more abundant.

In conclusion it was shown that the inoculum source played a key role for H 2 production in continuous reactors. The inoculum source determines not only the metabolic patterns when using untreated sludge, but also affects the efficiency of the pre-treatments performed. A combined effect between pre-treatments and inoculum sources was evidenced by probably affecting the microbial interactions and final selection of the microbial community.

CONCLUSION

Inoculum source has a strong impact on the reactor behaviors when non-pretreated sludge is used, but also on pre-treatment efficiency.

Fig. 1 :

 1 Fig. 1: Principal component analysis (PCA) based on initial and final microbial

Fig. 2 :

 2 Fig. 2: Microbial community distribution based on OTU at the end of continuous

Fig. 3 :

 3 Fig. 3: Principal component analysis (PCA) based on metabolic patterns and final

Table 3 .

 3 Butyrate was the main metabolite in all experiments, reaching between 22.0±8.8% COD consumed and 39.7±21.5% COD consumed . Succinate production represented between 12.0±4.5% COD consumed and 16.1±5.6% COD

consumed in experiments that used heat-treated sludge (AI-HT and AnI-HT) and aerationtreated aerobic sludge (AI-AT), but was in less amount 2.5±1.2% COD consumed in the control experiments (AI-C and AnI-C) and in the experiment with unstable H 2 production (AnI-AT). Ethanol production represented less than 12.4±5.4% COD consumed in all experiments except in AI-AT reaching 27.3±10.1% COD consumed . Acetate and propionate were also detected in all experiments but at low concentrations (<5.7±2.0% COD consumed ) except in AnI-AT where acetate accumulated 13.2±9.4% COD consumed . Formate was also produced at very low concentrations (<2.9±4.4% COD consumed ), only in aerobic sludge experiments (AI-C, AI-HT and AI-AT). Overall, glycerol removal was between 74±22% COD consumed and 90±15% COD consumed .

Table 1 : Summary of experimental design

 1 Heat pre-treatment of anaerobic sludge increased H 2 yield, while aeration resulted in unstable H 2 production. Whereas when aerobic sludge is used no pre-treatment is necessary, as there are no statistically significant differences in H 2 yields when comparing all experiments, including control. In addition, biokinetic control was key in the Clostridium sp. selection as dominant in the microbial community of all assays. While, lower or intermittent H 2 production were associated with higher relative abundance of Enterobacteriaceae family members. Our results allow a better understanding of H 2 production in continuous systems, providing key information for an efficient selection of operating conditions for future industrial applications.Silva, "Evaluation of hydrogen and methane production from sugarcane vinasse in an anaerobic fluidized bed reactor," Int. J. Hydrogen Energy, vol. 40, no. 27, pp.

	8498-8509, 2015.

Table 2 : Simpson diversity index and microbial community composition at the family level, expressed as percentage of total community. DNA samples were collected from original inocula, after pre-treatments and after continuous operation. Only families with a relative abundance ≥5.0% in at least one sample are shown. AI, AnI, HT, AT and C represent aerobic inoculum, anaerobic inoculum, heat shock pre-treatment, aeration pre-treatment and control, respectively. The "i" at the end of the sample names refers to samples taken after pre-treatments. Family Original inocula After pre-treatment After continuous operation AI AnI AI-HTi AnI-HTi AI-ATi AnI-ATi AI-C AnI-C AI-HT AnI-HT AI-AT AnI-AT

 2

	Simpson diversity index	0.98	0.92	0.99	0.94	0.95	0.96	0.83	0.71	0.67	0.64	0.75	0.79
	Bacteroidetes
	Flavobacteriaceae	1.7	3.5	1.6	0.1	4.8	12.1	0.0	0.0	0.0	0.0	0.0	0.1
	Porphyromonadaceae	1.8	3.5	0.4	2.5	0.5	1.6	20.8	0.0	0.0	0.0	1.3	0.0
	Prevotellaceae	0.2	0.0	0.3	0.3	11.8	6.3	6.4	25.6	35.9	34.1	32.2	24.9
	Rikenellaceae	10.3	18.8	8.2	17.3	1.1	1.4	0.0	0.0	0.0	0.0	0.0	0.0
	WCHB1-69	5.0	11.5	1.1	1.6	0.5	0.2	0.0	0.0	0.0	0.0	0.0	0.0
	Others (<5.0%)	15.8	3.0	11.2	0.5	2.7	1.0	0.2	1.4	1.1	0.2	2.1	2.0
	Total	34.8

40.4 22.8 22.4 21.5 22.6 27.5 27.0 37.1 34.3 35.6 27.1

	Desulfobacteraceae	3.4	0.0	7.7	0.0	0.1	0.4	0.0	0.0	0.0	0.0	0.0	0.0
	Enterobacteriaceae	0.0	0.0	1.6	0.0	6.7	4.4	0.4	16.7	3.3	1.3	5.4	25.4
	Moraxellaceae	0.1	0.1	4.5	0.1	7.5	5.1	0.4	0.0	0.6	0.1	1.5	0.0
	Pseudomonadaceae	0.2	0.1	0.1	0.0	8.8	9.6	0.2	0.1	0.8	2.2	6.2	5.8
	Sphingomonadaceae	0.6	0.8	1.4	1.0	0.8	9.6	0.0	0.0	0.0	0.0	0.0	0.0
	Others (<5.0%)	11.1	11.7	9.2	23.4	6.1	7.1	0.3	1.1	4.3	3.9	0.3	0.6
	Total	19.5	16.0	29.5	29.2	32.9	41.7	1.3	18.0	9.1	7.8	13.3	32.0
	Spirochaetae
	Spirochaetaceae	12.4	1.4	6.5	1.2	1.0	0.2	0.0	0.0	0.1	0.0	0.0	0.0
	Unknown_Family	0.1	8.8	0.0	1.5	0.1	0.2	0.0	0.0	0.0	0.0	0.0	0.0
	Others (<5.0%)	7.8	16.3	1.8	14.6	0.9	0.7	0.0	0.0	0.0	0.0	0.0	0.0
	Total	20.3	26.4	8.3	17.4	2.0	1.1	0.0	0.0	0.1	0.0	0.0	0.0
	Others (<5.0%)	16.7	10.9	20.9	21.6	1.9	2.3	0.0	0.0	0.0	0.0	0.0	0.0
	Firmicutes
	Christensenellaceae	0.9	0.1	7.1	0.5	0.1	0.1	0.0	0.0	0.0	0.0	0.0	0.0
	Clostridiaceae	0.4	0.3	1.3	0.1	27.1	18.6	39.4	46.2	51.0	56.4	40.1	35.3
	Enterococcaceae	0.0	0.0	0.1	0.0	1.6	3.7	27.1	0.3	0.1	0.1	0.6	1.9
	Lachnospiraceae	0.0	0.0	0.1	0.1	2.5	4.6	0.2	1.0	0.1	0.1	5.3	2.2
	Peptostreptococcaceae	0.5	0.2	4.0	0.2	6.5	0.8	0.0	0.0	0.0	0.0	0.0	0.0
	Others (<5.0%)	6.8	5.5	6.0	8.5	3.8	4.6	4.5	7.5	2.4	1.3	5.1	1.5
	Total	8.8	6.3	18.5	9.4	41.7	32.3	71.2	55.0	53.7	57.9	51.1	40.9
	Proteobacteria
	Comamonadaceae	4.1	3.3	5.0	4.7	2.7	5.6	0.0	0.0	0.1	0.4	0.0	0.2

Table 3 : Performance indicators during continuous operation of H 2 producing reactors, including biomass yield, H 2 yield, and soluble metabolites production. Average values and standard deviations (±SD) were calculated from daily measurements during continuous operation.

 3 Metabolite distribution based on COD mass balance. %COD were calculated based on total glycerol consumed *During AnI-AT the H 2 production was unstable.

	Parameter	Unit			AI-C	AnI-C	AI-HT	AnI-HT	AI-AT	AnI-AT
	Biomass yield	g VSS mol gly-consumed	-1	5.8 (±2.1)	6.3 (±1.5)	7.0 (±3.1)	5.8 (±2.4)	5.3 (±2.0)	6.7 (±2.7)
	H 2 yield	mol H2 mol gly-consumed	-1 0.50 (±0.19) 0.29 (±0.10) 0.47 (±0.17) 0.42 (±0.08) 0.55 (±0.08)	*
	Ethanol	%COD			6.5 (±2.6)	12.3 (±5.1) 12.4 (±5.4)	3.0 (±1.3) 27.3 (±10.1) 9.5 (±6.4)
	Acetate	%COD			3.7 (±1.9)	5.7 (±2.0)	4.8 (±1.5)	4.1 (±1.6)	3.4 (±1.2)	13.2 (±9.4)
	Propionate	%COD			1.4 (±0.7)	3.2 (±1.5)	2.7 (±1.0)	1.4 (±0.6)	1.6 (±0.4)	5.2 (±3.5)
	Butyrate	%COD			32.8 (±11.1) 25.0 (±8.7) 22.0 (±8.8) 30.7 (±13.8) 23.4 (±8.2) 39.7 (±21.5)
	Succinate	%COD			1.9 (±0.8)	2.5 (±1.2)	12.0 (±4.5) 16.1 (±5.6) 15.5 (±5.9)	1.8 (±3.9)
	Formate	%COD			1.1 (±0.5)	-	1.8 (±0.7)	-	1.6 (±0.4)	2.9 (±4.4)
	Glycerol removal efficiency	%			90 (±15)	79 (±19)	78 (±20)	80 (±31)	87 (±16)	74 (±22)

ACKNOWLEDGEMENTS

We thank Pontificia Universidad Católica de Valparaíso (PUCV) for providing postdoctoral funding for J.T-A. This study was funded by GRAIL 613667 (KBBE-7PM) and ECOS-CONICYT program project N° C12E06.This work is dedicated to the memory of our beloved colleague, Prof. Gonzalo Ruiz-Filippi.


Sébastien Villotte
email: s.villotte@exeter.ac.uk

Christopher J Knüsel

Piers D Mitchell

Dominique Henry-Gambier

Probable carpometacarpal and tarsal coalition from Baousso da Torre Cave (Italy): Implications for burial selection during the Gravettian

Keywords:

Introduction

In the past, authors have recognised selective funerary practices during the Upper Palaeolithic according to sex, age-at-death, social status, pathological conditions, or possible family ties (e.g., [START_REF] Formicola | The triplex burial of Barma Grande (Grimaldi, Italy)[END_REF][START_REF] Formicola | From the Sunghir children to the Romito dwarf: aspects of the Upper Paleolithic funerary landscape[END_REF][START_REF] Mussi | Les vivants et les morts. Les sépultures du Paléolithique supérieur en Italie et leur interprétation[END_REF][START_REF] Alt | Twenty-five thousand-year-old triple burial from Dolní V estonice: an ice-age family?[END_REF][START_REF] Oliva | The Brno II Upper Palaeolithic burial[END_REF][START_REF] Henry-Gambier | La sépulture des enfants de Grimaldi (Baoussé-Roussé, Italie). Anthropologie et palethnologie funéraire des populations de la fin du Paléolithique supérieur[END_REF][START_REF] Henry-Gambier | Comportement des populations d'Europe au Gravettien: pratiques funéraires et interprétations[END_REF][START_REF] Pettitt | The living dead and the dead living: burials, figurines and social performance in the European Mid Upper Palaeolithic[END_REF]. In this short communication, we report three pathological conditions, which may be genetic in origin, observed in two Gravettian skeletons from the same cave, Baousso da Torre, in Italy. This discovery once again raises the issue of the nature of relationships among Upper Palaeolithic burials.

Materials

Baousso da Torre is one of the caves of the Balzi Rossi, a major Italian Palaeolithic locality on the Mediterranean coast. In 1873, Rivière found three single burials in this cave [START_REF] Rivière | Antiquité de l'homme dans les Alpes-Maritimes[END_REF]. Two of these skeletons were subsequently lost and re-discovered in 2008, more than 80 years later [START_REF] Villotte | The rediscovery of two Upper Palaeolithic skeletons from Baousso da Torre cave (Liguria-Italy)[END_REF]. The collagen content from BT1 and BT3, as well as for BT2 (curated in the Musée 'd'Archéologie Nationale, Paris), was too low to obtain reliable radiocarbon dates, but the archaeological context indicates that an Upper Palaeolithic date is assured and a Gravettian date (28e22 ka) for these skeletons is likely [START_REF] Mussi | On the chronology of the burials found in the Grimaldi Caves[END_REF][START_REF] Mussi | Earliest Italy: An Overview of the Italian Paleolithic and Mesolithic[END_REF][START_REF] Henry-Gambier | La sépulture des enfants de Grimaldi (Baoussé-Roussé, Italie). Anthropologie et palethnologie funéraire des populations de la fin du Paléolithique supérieur[END_REF][START_REF] Villotte | The rediscovery of two Upper Palaeolithic skeletons from Baousso da Torre cave (Liguria-Italy)[END_REF]. The preservation of the osseous remains of BT1, BT2, and BT3 is relatively good, but numerous bones are missing (Table 1). Sex and age-at-death of these three individuals were recently reassessed [START_REF] Villotte | The rediscovery of two Upper Palaeolithic skeletons from Baousso da Torre cave (Liguria-Italy)[END_REF]Villotte et al., in press). BT1 and BT2 are both males; BT1 is a young or mature individual (20e49 years old) and BT2 is a young adult (20e29 years old). BT3 is an unsexed adolescent. These new assessments confirm previously published estimations of sex [START_REF] Rivière | Antiquité de l'homme dans les Alpes-Maritimes[END_REF][START_REF] Verneau | Les grottes de Grimaldi (Baoussé-Roussé)[END_REF] and detail the ageat-death of the adults.

Results

Pathological changes in the hind foot and wrist were noted. The anterior process of the left calcaneus of BT1 is abnormally elongated (Figs. 1 and2) with a maximum height of 11.5 mm, a maximum breadth of 15 mm, and a maximum length of 8.5 mm (measured from the anterior articular surface for the cuboid bone). The anterior margin of this elongation is broken. In a normal calcaneus there is no finger-like protuberance here. The protuberance is oriented towards where the navicular bone would have been during life, although this bone has not survived. This individual also displays abnormal bossing of the dorsal part of the base of the right second metacarpal (Fig. 3). Pitting is present on the proximal face of this bossing. Unfortunately, the right calcaneus and the left second metacarpal, as well as the navicular and trapezoid of both sides, are missing. The feet and right hand of BT2 were not preserved, but the left hand is present. A carpal boss, formed by an abnormal dorsal bossing of the left trapezoid and the base of the left second metacarpal was also found in this individual (Fig. 3). Pitting, filled by sediment, is present on the proximal face of the bossing of the metacarpal base. The feet of BT3 are not pathological, but the hand bones are not preserved.

Discussion

Osseous modifications like those seen in BT1 and BT2 have previously been related to tarsal (e.g., [START_REF] Leonard | The inheritance of tarsal coalition and its relationship to spastic flat foot[END_REF] [START_REF] Nakamura | The ligament and skeletal anatomy of the second through fifth carpometacarpal joints and adjacent structures[END_REF][START_REF] Alemohammad | Incidence of carpal boss and osseous coalition: an anatomic study[END_REF] coalition. Coalition in the foot or wrist can be due to bone, cartilage, or fibrous tissue connecting two bones that would normally not be connected. Of the three, osseous coalition will be most easily seen in archaeological specimens, as the bony bar itself will be preserved. Cartilaginous and fibrous coalitions can be identified by changes to the shape of the surfaces, often associated with pitting, in the classic locations where coalitions tend to occur (Case and Burnett, in press).

Reported cases of calcaneonavicular coalition in archaeological material are mainly of the non-osseous type (Case and Burnett, in press). In these cases, the anterior border of affected calcanei is usually sharply angled and irregular, and some bones exhibit a finger-like protuberance at the lateral part of the lesion. This lateral tubular elongation of the anterior process of the calcaneus is called the "anteater nose sign" in radiology, and it is seen in patients with osseous or non-osseous calcaneonavicular coalition [START_REF] Fopma | Tarsal coalition[END_REF][START_REF] Chapman | The anteater nose sign[END_REF]. The anterior border of the left calcaneus of BT1 does not display any irregularly, except the tubular projection. Based on the size of the protuberance, and by analogy with observations from living patients and cadavers (Leonard, 1974: Figure 5;Fopma and Macnicol, 2002: Figure 4;Solomon et al., 2003: Figure 3;Lysack and Fenton, 2004: Figures 2e4), a diagnosis of a bony calcaneonavicular coalition for BT1 is most likely. Calcaneonavicular coalition is considered an uncommon condition today [START_REF] Fopma | Tarsal coalition[END_REF], but recent studies on skeletons (Case and Burnett, in press), cadavers [START_REF] Solomon | A dissection and computer tomograph study of tarsal coalitions in 100 cadaver feet[END_REF], and patients with foot pain [START_REF] Lysack | Variations in calcaneonavicular morphology demonstrated with radiography[END_REF][START_REF] Nalaboff | MRI of tarsal coalition: frequency, distribution, and innovative signs[END_REF] indicate prevalence rates between 1 and 9%. Calcaneonavicular coalition leads to a stiff flat foot that, in some individuals, is also painful. It arises as a congenital failure of segmentation of the primitive mesenchyme or, more rarely, as an acquired trait [START_REF] Fopma | Tarsal coalition[END_REF]. A secondary synostosis due to trauma, inflammatory arthropathy, neoplasm, or osteonecrosis is unlikely in the case of BT1 due to the lack of any other pathological change in the bones and joints. Thus, a diagnosis of a congenital calcaneonavicular coalition can be posited.

Patients with a symptomatic carpal boss complain of pain in the affected hand [START_REF] Alemohammad | Incidence of carpal boss and osseous coalition: an anatomic study[END_REF]. Degenerative osteophyte formation, trauma, microtrauma, and persisting os styloideum are among the suspected causes of the condition [START_REF] Carter | Carpal boss: a commonly overlooked deformity of the carpus[END_REF][START_REF] Curtiss | The hunchback carpal bone[END_REF][START_REF] Artz | The carpometacarpal boss[END_REF][START_REF] Conway | The carpal boss: an overview of radiographic evaluation[END_REF][START_REF] Maquirriain | Acute os styloideum injury in an elite athlete[END_REF]. Recently, an 18% incidence of a carpal boss associated with a partial carpometacarpal coalition of the second ray was found in a general cadaver population, and it was suggested that the carpal boss could be an incomplete (fibrous or fibrocartilaginous) congenital coalition [START_REF] Nakamura | The ligament and skeletal anatomy of the second through fifth carpometacarpal joints and adjacent structures[END_REF][START_REF] Alemohammad | Incidence of carpal boss and osseous coalition: an anatomic study[END_REF]. Although a bony protuberance located at the dorsum of the wrist can have different causes, the discovery of both a congenital calcaneonavicular coalition and a carpal boss in the same individual, as well as two individuals with a carpal boss in the same cave, support the hypothesis of a congenital partial carpometacarpal coalition for BT1 and BT2. The presence of pitting of the dorsal part of both bosses further reinforces this diagnosis. Another hypothesis is that the dorsal bossing represents a normal morphological variant of the second metacarpal during the Upper Palaeolithic. This seems less probable as this morphology has not been described previously for this time period.

Congenital calcaneonavicular coalition is thought to be an unifactorial disorder of autosomal dominant inheritance [START_REF] Wray | Hereditary transmission of congenital coalition of the calcaneus to the navicular[END_REF][START_REF] Leonard | The inheritance of tarsal coalition and its relationship to spastic flat foot[END_REF][START_REF] Fopma | Tarsal coalition[END_REF]. This report of the oldest known case to date e the previous one was dated to the Late Neolithic [START_REF] Silva | Non-osseous calcaneonavicular coalition in the Portuguese prehistoric population: report of two cases[END_REF] e indicates that similar genetic mutations were present in the Upper Palaeolithic gene pool. However, neither carpal nor carpometacarpal anomalies were recognised in modern patients with tarsal coalition, except in such conditions as symphalangism, phocomelia, or hemimelia [START_REF] Leonard | The inheritance of tarsal coalition and its relationship to spastic flat foot[END_REF]. These conditions are not present in the Baousso da Torre remains. Therefore, the mutation that produced segmentation Neurocranium (f), face (ff), mandible (f), cervical vertebrae (2d), ribs (ff), scapulae (f/f), clavicles (i/f), humeri (i/d), radii (f/i), ulnae (f/i), carpals (-/7i-f), metacarpals (-/5i-d), phalanges (5i/9i), coxal bones (-/d), femora (d/d), tibiae (d/f), fibulae (f/-), tali (i/-), phalanges (1i/-). BT3

Neurocranium (ff), face (ff), sacrum (ff), humeri (d/-), ulnae (f/-), coxal bones (-/ff), femora (f/d), patellae (-/i), tibiae (-/ff), tali (f/i), calcanei (d/f), other tarsals (1f/4i-d), metatarsals (2d-f/3i-f), phalanges (3i/5i-f).

The state of preservation of the remains is reported separately for bones from the right and the left sides, respectively. -: missing, i: intact; d: damaged; f: fragmented; ff: very fragmented (according to Alciati et al., 2005). defects for BT1 and BT2 could be different to the mutation present in the current gene pool. In this case, a gene with reduced penetrance is more likely because these conditions have not been observed in other near-contemporary Upper Palaeolithic burials. The hypothesis of close familial relationships among the buried dead during the Gravettian was previously formulated by [START_REF] Formicola | The triplex burial of Barma Grande (Grimaldi, Italy)[END_REF] for the triple burial of Barma Grande (near Baousso da Torre Cave) and by [START_REF] Alt | Twenty-five thousand-year-old triple burial from Dolní V estonice: an ice-age family?[END_REF] for the triple burial of Dolní V estonice, based on phenotypic similarities among individuals buried together. The presence of similar congenital disorders shared by two skeletons in Baousso da Torre Cave adds further support for this hypothesis. However, because these skeletons were found in separate burials and the time span between the two deaths is unknown (although the two burials are very close to one another and in the same archaeological layer), conclusions regarding the degree of relationship between BT1 and BT2 remain speculative.

Conclusions

Three pathological changes, perhaps due to an underlying genetic aetiology, were discovered in two skeletons from Baousso da Torre Cave (BT1 and BT2). This discovery suggests that these two adult males may have been genetically related, and strengthens the hypothesis of familial relationships among people buried together, or near to each other, during the Gravettian. It also highlights a potential 25,000-year-old genetic mutation that appears to have been present in prehistoric humans in Italy, but with a phenotype unknown in modern clinical studies. Such discoveries are clearly of considerable significance to our understanding of population biology during the course of human evolution and, potentially, as an aid to understanding the occurrence of these conditions today.

Figure 1 .

 1 Figure 1. Medial view of the left calcaneus of BT1. The arrow indicates the bony projection ("anteater nose sign") from the calcaneus to the navicular (which has not survived).

Figure 2 .

 2 Figure 2. Plantar view of the left calcaneus of BT1. Close-up of the anterior part of the bone showing the anterior articular surface (bottom of image) and the bony projection.

Table 1

 1 Inventory and state of preservation of the skeletal remains.

	Specimen	Skeletal elements present and state of preservation
	BT1	Neurocranium (ff), face (ff), thoracic vertebrae (1f), ribs (ff),
		scapulae (-/ff), humeri (f/d), radii (f/-), carpals (3i/-),
		metacarpals (3i-f/1f), phalanges (9i-d/2d), coxal bones (ff/-),
		femora (f/d), patellae (i/-), tibiae (d/f), calcanei (-/f), other
		tarsals (2i/1i), metatarsals (3i-d/3i-f), phalanges (2i/2i).
	BT2

  Please

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	11	176
	12	177
	13	178
	14	179
	15	180
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	69	234
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	71	236
	72	237
	73	238
	74	239
	75	240

cite this article in press as: Villotte, S., et al., Probable carpometacarpal and tarsal coalition from Baousso da Torre Cave (Italy): Implications for burial selection during the Gravettian, Journal of Human Evolution (2011), doi:10.1016/j.jhevol.2011.01.007

  Please cite this article in press as: Villotte, S., et al., Probable carpometacarpal and tarsal coalition from Baousso da Torre Cave (Italy): Implications for burial selection during the Gravettian, Journal of Human Evolution (2011), doi:10.1016/j.jhevol.2011.01.007 Please cite this article in press as: Villotte, S., et al., Probable carpometacarpal and tarsal coalition from Baousso da Torre Cave (Italy): Implications for burial selection during the Gravettian, Journal of Human Evolution (2011), doi:10.1016/j.jhevol.2011.01.007

	YJHEV1553_proof ■ 2 March 2011 ■ 3/4 YJHEV1553_proof ■ 2 March 2011 ■ 4/4
	241 371	306 384
	242 372	307 385
	243 373	308 386
	244 374	309 387
	245 375	310 388
	246 376	311 389
	247 377	312 390
	248 378	313 391
	249 379	314 392
	250 380	315 393
	251 381	316 394
	252 382	317 395
	253 383	318 396
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Acknowledgements S.V. and D.H.-G. thank T. Dechezleprêtre (Musée Lorrain, Nancy, France), and C. Schwab (Musée d'Archéologie Nationale, Saint Germain-en-Laye, France) who allowed them access to fossils. The authors thank Pr. S. Leigh, his assistant, and the anonymous reviewer for their useful comments. Grant sponsors: CNRS and ANR Guerope.