Steve Belon
email: steve.belon@cea.fr

Benjamin Erzar

Elodie Kaeshammer

Lionel Borne

Influence of the shape of RDX grains on the creation of hot spots by mesoscale modeling

CEA-Gramat studies the sensitivity of energetic materials to enhance their security and reliability. The conditions leading to the initiation of an explosive must be understood to control its sensitivity. According to the hot spots theory, the shock initiation of heterogeneous explosives is related to their microstructure: the shock interacts with the heterogeneities of the microstructure (pores and inclusions, morphology of grains and fragments, debonding, etc.) and creates local deposits of energy. To describe these hot spots, energetic materials have to be modeled at a scale allowing the discretization of their microstructure: the mesoscale.

Micro-computed tomographies of energetic materials are done at CEA-Gramat and analyzed to build geometric models used in finite element simulations. Two kinds of models are studied: -Real models are directly built on the real microstructures extracted from micro-computed tomographies.

-Virtual models are based on the same microstructures but simplified to study independently the effects of microstructural parameters (granulometry, porosity, filler content…) on the creation of hot spots.

Compositions based on different kind of RDX particles in an inert binder are studied through numerical simulation. The influence of particle shape on the inert shock response is investigated at the mesoscale. Local heterogeneities of pressure and temperature fields appear intimately related to the morphological properties of the microstructures. Particles with sharp edges create more hot spots than spherical particles.

Introduction

The shock-initiation of heterogeneous solid explosives is related to the formation of hot spots. Hot spots depict localized elevation of pressure and temperature in the material. They are due to the interaction of the propagating shock with the heterogeneities of the material microstructure. According to the size, intensity and density of hot spots, they will either extinguish or be the starting point of chemical reactions. Knowing the formation of hot spots is thus of primary importance to handle the initiation of solid explosives.

Numerous studies concerning the formation of hot spots have been published; see for instance [START_REF] Field | Hot-spot Ignition Mechanisms for Explosives and Propellants[END_REF][START_REF] Amstrong | Investigation of Hot Spot Characteristics in Energetic Crystals[END_REF]. Several mechanisms have been identified to be a potential source of hot spots, including void collapse, plastic deformation, fracture of grains, friction, debonding [START_REF] Rai | Mesoscale Simulation of Reactive Pressed Energetic Materials under Shock Loading[END_REF][START_REF] Labarbera | Heterogeneous Thermo-Mechanical Behavior and Hot Spot Formation in RDX-Estane Energetic Aggregates[END_REF][START_REF] Arora | Modelling the Damage and Deformation Process in a Plastic Bonded Explosive Microstructure under Tension using the Finite Element Method[END_REF][START_REF] Keyhani | Energy Dissipation in Polymer-Bonded Explosives with various Levels of Constituent Plasticity and Internal Friction[END_REF]. Each of these mechanisms may play a role in the appearance of hot spots, according to the kind of energetic material or the kind of loading considered. A better knowledge of the effects of each of these mechanisms allows a better control of the initiation of solid explosives, whether for intentional or accidental initiation. All these mechanisms have in common to be strongly dependent on the microstructure of the energetic material. Thus, the initiation of heterogeneous explosives should be studied at a scale taking into account their microstructural parameters.

The energetic material studied in this work is a plastic-bonded explosive (PBX) made of 70 % in weight of RDX and 30 % in weight of wax. The considered loading is an impact of a rigid projectile at 1200 m/s, corresponding to the initiation threshold of the RDX/Wax composition. The scenario studied in this work thus corresponds with an accidental kind of initiation. The microstructural parameter examined in this paper is the effect of the shape of RDX grains on the appearance of hot spots. Influence of the shape of RDX crystals on the shock-sensitivity of PBXs has been observed experimentally; see for instance van der Steen et al. [START_REF] Van Der Steen | Influence of RDX Crystal Shape on the Shock Sensitivity of PBXs[END_REF] who noticed a greater sensitivity threshold of spherical particles than irregularly shaped particles. The objective of the present work is to show the influence of the RDX crystal shape on the propagating shock locally, thanks to mesoscale numerical modeling.

Materials

The energetic materials studied in this work are RDX/Wax compositions produced by the french-german research Institute of Saint-Louis (ISL). They are composed of 70 % in weight of RDX particles and 30 % in weight of wax. The RDX grains used in these compositions are subjected to two treatments to become less sensitive than common grade RDX particles. This process, presented at Figure 1, is a trademark of ISL [START_REF] Borne | Particle of Explosive of Low-Sensitivity to Shock and Associated Treatment Process, US Patent 20060272755A1[END_REF]. The first treatment consists in a recrystallization of commercial grade RS-RDX particles furnished by Eurenco. Most of crystal internal defects, including porosities and micro-cracks, disappear during the recrystallization. The resulting particles have less than 0.05 % in volume of internal voids. The shape of the particles is also modified by the recrystallization process as they present sharp edges, whereas the RS-RDX particles are rather spherical. Recrystallized particles are called "raw VI-RDX". The second step is a surface treatment process aiming at removing the sharp edges created by the recrystallization. The resulting particles are spherical and called "VI-RDX".

Figure 1: Steps of the formation of VI-RDX crystals

ISL produced RDX/Wax compositions using either raw VI-RDX or VI-RDX particles. They characterized the shock-sensitivity threshold of these compositions by impact experiments [START_REF] Corbel | Micro-computed tomography for an accurate microstructure imaging of ISL formulations and correlation with experimental data[END_REF]. Their results are presented at Figure 2. They found that the sensitivity threshold of the raw VI-RDX composition (6.2 GPa) is lower than the pressure threshold of the VI-RDX composition (7.2 GPa). The only difference between both compositions is the shape of the RDX particles. All other microstructural parameters are identical: weight and size distributions of RDX grains, rate of porosity, etc. Thus, the difference in the sensitivity threshold of both compositions under shock-loading should be explained by the effect of the RDX crystal shape on the propagating shock.

Figure 2: Results of ISL small projectile impact experiments for several formulations

Methods

To understand the effect of the shape of the raw VI-RDX and VI-RDX grains on the propagating shock we model the microstructure of both compositions at the mesoscale. First, we model RDX crystals with 2D geometrical forms: the angular raw VI-RDX particle is represented by a square and the spherical VI-RDX grain is represented by a disc. These models contain only one grain in the domain to study the interaction of the propagating shock and the grain without disturbance coming from other grains. Secondly, we model both raw VI-RDX and VI-RDX compositions with 3D realistic microstructures to quantify the appearance of hot spots in presence of many reflective shocks.

Our models run with the eulerian OURANOS solver, developed at the CEA, using a BBC integration scheme. RDX is modeled with a Birch-Murnaghan equation of state and a Johnson-Cook behavior taking into account a linear thermal softening. RDX is treated as an inert: the chemical initiation is not modeled. The wax material is also modeled with a Birch-Murnaghan equation of state. Wax is considered purely hydrostatic as the melting temperature of this binder is low (350 K at ambient pressure). The shock loading represents a steel projectile continuously moving at the speed of 1200 m/s. This is modeled by an inflow boundary. Reflective boundary conditions, similar to rigid walls, are on the top and bottom sides of the domain. The 2D models span on 900x900 µm² with a constant mesh size of 1 µm, totalizing 810,000 elements. The raw VI-RDX particle is represented by a square with a side of 230 µm. The VI-RDX grain is represented by a disc with a diameter of 260 µm. The surfaces of the square and the disc are identical, so that both models are comparable. The simulation time of the 2D model is 200 ns, corresponding to the time necessary for the shock to propagate along the whole domain. Materials and boundary conditions of the 3D models are identical to those of the 2D models. The 3D models span on 1x1x2.5 mm³ with a constant mesh size of 3.33 µm, totalizing 67.5 million cells. The simulation time of the 3D model is 600 ns.

To compare the shock-sensitivity of the different configurations we have to quantify the appearance of hot spots. There is no standard definition of a hot spot. Several criteria have been suggested to characterize the existence of hot spots; see for instance [START_REF] Walker | Derivation of the P 2 T Detonation Criterion[END_REF][START_REF] Barua | Ignition Criterion for Heterogeneous Energetic Materials based on Hotspot Size-Temperature Threshold[END_REF][START_REF] Sen | Multi-scale Shock-to-detonation Simulation of Pressed Energetic Material: A meso-informed ignition and growth model[END_REF]. They are commonly based on a pressure, temperature or energetic threshold. We choose a hot spot criterion resting on a temperature threshold: the melting temperature of RDX crystal. At ambient pressure RDX grains melt at 500 K. We assume an evolution of the melting temperature with density following a Lindemann type law. The loading considered in the present work leads to a melting temperature around 600 K for RDX crystals.

Results of the 2D models

In 2D, we model RDX grains with geometrical forms to simplify the observation of the interaction between the propagating shock and the grain. Angular raw VI-RDX particles are represented by a square and spherical VI-RDX particles are modeled with a disc. The time sequences of temperature of these configurations are presented at Figure 3. The orientation of the square to the propagating shock varies from 0 to 45 degrees to observe if any difference occurs in the interaction of the shock with the grain.

We notice that the first interaction of the lead shock with the grain (t = 95 ns) is not sufficient to reach the hot spot criterion. As the shock passes, a Mach stem forms at the top and bottom sides of the disc and the square oriented at 22.5 and 45 degrees (t = 110 ns). These Mach stems are the places where hot spots appear. The Mach stems keep heating the same surfaces of the grains as their triple points move along the lead front (t = 125 ns). The square particle oriented at 0 degree does not produce Mach stems. In this configuration the temperature never reaches the hot spot threshold. In the absence of microstructural defects inside the grains (porosities, cracks, etc.) no hot spot appears inside the particle (t = 140 ns). These results are similar to those obtained by Menikoff [START_REF] Menikoff | Hot spot formation from shock reflections[END_REF] who studied numerically the formation of hot spots by shock reflections in nitromethane charged with glass beads.

Results of the 3D models

In 3D we are interested in studying the effect of shock reflections inside a real microstructure on the formation of hot spots. Thus, we model real grains of raw VI-RDX and VI-RDX in a large domain respecting the 70/30 weight ratio between charges and binder. The intra-granular and extra-granular porosities of these compositions are especially low. Yet, no porosity is included to be sure that the only parameter influencing the shock propagation is the RDX crystal shape. Besides removing any porosity, the microstructures are simplified by using only one grain of each kind. In other words, we select one real grain of raw VI-RDX, representative of the mean microstructural properties of this kind of grain, and build a composition made of a hundred copies of this grain. The same process is done with a mean VI-RDX particle to obtain a composition made of a hundred copies of the particle. This simplification of the microstructure avoids the effects of the variation of particle size and shape inside one kind of composition, which is not the goal of the present work. Figure 4 and Figure 5 depict the selected real grains, their finite element models and the representative microstructures made of these particles. To compare results obtained on both compositions we focus on the hot spots extracted from these models. Figure 6 depicts the time evolution of hot spot density for both microstructures. In the raw VI-RDX material hot spots start to appear from the beginning of the simulation whereas for the VI-RDX material hot spots start to form at a third of the simulation. The rate of appearance of hot spots is slightly greater for the raw VI-RDX than the VI-RDX composition. At the end of the simulation there is 50 % more hot spots in the microstructure built with angular raw VI-RDX particles than in the microstructure built with spherical VI-RDX particles.

Discussion

The models used to perform the present comparison of the shock-sensitivity of angular and spherical RDX crystals suffer of several limitations. They are lacking of mechanisms like thermal conduction, grains fracture, plastic heating, etc. The quantification of hot spots is not accurate as we sum the cells reaching a temperature threshold taking into account neither the lighting duration nor the merging of hot spots. Moreover the microstructures used in this study are free of all other kinds of defects like porosities, which have been identified to be a source of hot spots.

Nevertheless this work highlights some mechanisms involved in the creation of hot spots. The formation of a Mach stem at the surface of the explosive particles seems of primary importance for the initiation of a single crystal as only the configurations where a Mach stem forms reach the hot spot criterion.

In the 3D realistic microstructures it is hard to say if hot spots appear from a Mach stem similarly to what occurs in 2D models. The difference in the amount of measured hot spots between angular and spherical crystals is sufficiently large to claim that the shape of explosive particles by itself is an important parameter for the shock-sensitivity of heterogeneous energetic materials.

Conclusion

Influence of RDX crystal shape on the formation of hot spots in a shocked plastic-bonded explosive is studied by numerical modeling at the mesoscale. This work is based on two RDX/Wax compositions produced by ISL. One contains RDX crystals presenting sharp edges. The other contains only rounded particles. Two kinds of models are used to compare the effects of angular and spherical grains. 2D models made of only one grain in a wax binder are used to study the interaction between the propagating shock and the grain. Grains are assumed free of internal defects and modeled with simple geometrical forms. In these conditions hot spots only appear on the sides of the grain where a Mach stem forms. This location is related to the shape of the grain and to the orientation of the grain with the leading shock front. 3D models made with a hundred of grains are used to study the formation of hot spots in a real material microstructure subject to many shock reflections. Shapes of real explosive grains are used in the 3D models. Other microstructural parameters are not considered, especially porosity. The composition made of angular RDX crystals creates twice more hot spots than the material made of spherical RDX particles.

Figure 3 :

 3 Figure 3: Time sequences of temperature for the VI-RDX particle (disc) and the raw VI-RDX particle (square oriented at 0, 22.5 and 45 degree)

Figure 4 :Figure 5 :Figure 7

 457 Figure 4: The raw VI-RDX crystal modeled to build the microstructure

Figure 6 :

 6 Figure 6: Hot spot densities of raw VI-RDX and VI-RDX compositions

Figure 7 :

 7 Figure 7: Time sequence of temperature for both raw VI-RDX and VI-RDX compositions

Acknowledgements

This work is part of a collaboration between ISL and CEA-Gramat. It is supported by the French General Directorate for Armament (DGA) of the Ministry of the Armed Forces.


Santiago Rodríguez-Valderrama

Carlos Escamilla-Alvarado

Jean-Pierre Magnin

Pasiano Rivas-García

Idania Valdez-Vázquez

Elvira Ríos-Leal

ScDr. Carlos Escamilla Alvarado

Batch biohydrogen production from dilute acid hydrolyzates of fruitsand-vegetables wastes and corn stover as co-substrates

Keywords: dark fermentation, dilute acid, factorial experimental design, hydrolyzates, overliming

Batch biohydrogen production from dilute acid hydrolyzates of fruitsand-vegetables wastes and corn stover as co-substrates

Introduction

Dark fermentation biohydrogen production has become a promising technology as it may be used to process various types of organic wastes (solid or liquid). It does not require luminous energy, it has high hydrogen production rates in comparison to other biological methods and it may be carried out at non-sterile conditions [START_REF] Nissilä | Dark fermentative hydrogen production from lignocellulosic hydrolyzates-A review[END_REF]. Lignocellulosic wastes have been highly esteemed for their abundance and bioprocessing potential [2]. Corn stover (CS) is a worldwide important lignocellulosic waste; high amounts are produced in countries such as Mexico where corn production reaches 21.1 million tons yearly along with the production of up to 38 million tons of corn cobs and CS [START_REF] Sagarpa | Planeación agrícola nacional 2017-2030[END_REF][START_REF] Valdez-Vazquez | Distribution and potential of bioenergy resources from agricultural activities in Mexico[END_REF].

Due to the highly stable and organized microstructure of its lignocellulosic components, pretreatments are required to reduce its recalcitrance and the cellulose crystallinity to facilitate the carbohydrates depolymerization [START_REF] Gonzales | Effect of severity on dilute acid pretreatment of lignocellulosic biomass and the following hydrogen fermentation[END_REF]. Cellulose and hemicellulose can be broken-down to its monoand disaccharides constituents by dilute acid hydrolysis. Yet the resultant sugar concentration depends on the operational conditions (i.e. reaction time, temperature, acid concentration) and type of feedstock [START_REF] Nissilä | Dark fermentative hydrogen production from lignocellulosic hydrolyzates-A review[END_REF][START_REF] Fangkum | Biohydrogen production from sugarcane bagasse hydrolysate by elephant dung: Effects of initial pH and substrate concentration[END_REF]. Moreover, acid hydrolyzates influence dark fermentation by many factors, such as the type and concentration of inoculum, sugars and inhibitors concentration, temperature, and pH [START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF]. An excessive loading of carbohydrates may hinder the biohydrogen due to the excessive production of organic acids that lower the fermentation pH and promote solventogenic lactic-acid oriented metabolism [2]. Therefore, the determination of an adequate inoculum to substrate ratio (ISR), which relates the amounts of inoculum fed to the bioreactor in comparison to the substrate loadings, is important to foster biohydrogen production. Moreover, ISR may help reduce the inhibitory characteristic of acid hydrolysis. On the other hand, at higher proportion of substrate (e.g. 1:6 ISR), hydrogen production has been inhibited due to increase of osmotic pressure that affects microbial growth [START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF], and the excessive production and accumulation of volatile fatty acids (VFA) that cause pH decrease.

CS dilute acid hydrolyzates are mainly composed of xylose since its hemicellulose content present easier hydrolysis [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF]. Indeed, this represents a challenge for the microbial metabolism preference of hexoses over pentoses. An opportunity to increase the hexoses content would be to combine the acid hydrolysis pretreatment with that of a substrate with easier degradability, such as the fruits-and-vegetables wastes (FVW) another carbohydrates rich lignocellulosic material [START_REF] Razaghi | Hydrolysis treatments of fruit and vegetable waste for production of biofuel precursors[END_REF]. Indeed, combination of substrates has been previously assayed through the co-digestion processes. These are characterized by the simultaneous processing of an incompletely degraded and microbiologically rich substrate (e.g. sewage sludge, pig manure, cow manure, waste activated sludge) and a carbohydrate rich substrate (e.g. sugarcane bagasse, rice straw, corn stover, corn stalk, wheat straw, cassava stillage) [START_REF] Wang | Enhanced fermentative hydrogen production from cassava stillage by codigestion: The effects of different co-substrates[END_REF]. Co-digestion has been resorted for its advantages, such as dilution of toxic compounds, improvement of nutrients balance (carbohydrates/proteins) [START_REF] Wang | Biohydrogen and methane production by co-digestion of cassava stillage and excess sludge under thermophilic condition[END_REF] and microbial synergies [START_REF] Wang | Enhanced fermentative hydrogen production from cassava stillage by codigestion: The effects of different co-substrates[END_REF]. Recently, co-digestion of vegetable and food wastes with cow manure has been associated to process stability, but not necessarily with improved biomethane production [START_REF] Miramontes-Martínez | Semi-continuous anaerobic co-digestion of vegetable waste and cow manure: a study of process stabilization[END_REF].

Even though lignocellulosic biomasses have been successfully co-digested with microbial rich sludges or manures for hydrogen production (e.g. cassava stillage and food waste) [START_REF] Wang | Enhanced fermentative hydrogen production from cassava stillage by codigestion: The effects of different co-substrates[END_REF][START_REF] Kim | Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge[END_REF], the co-processing of the CS and FVW has not been previously assayed elsewhere. Researching this kind of alternatives would be useful in the development of future biorefineries by avoiding monosubstrates implicit hurdles, such as seasonality and lack of nutrients, and improving supply chain management through the reduction of delivery costs and harmful impacts related to climate change and fossil depletion [START_REF] Sultana | Optimal configuration and combination of multiple lignocellulosic biomass feedstocks delivery to a biorefinery[END_REF][START_REF] Borrion | Environmental life cycle assessment of bioethanol production from wheat straw[END_REF].

In this work, the main aim was to evaluate how the mix ratio of FVW and CS affected the production of hydrolyzates used in biohydrogen production. Specifically, design of experiments were developed for insightful determination of the effects of acid catalyst on reducing sugars production, and thereafter the effects of reducing sugars concentration and the inoculum to substrate ratio on biohydrogen production. As it is becoming of utter importance to assess the economics of bioenergetics (hydrogen, methane, bioethanol), an economic prospection at the laboratory level was also developed.

Material and Methods

Feedstocks

The CS feedstock obtained from Cuencamé, Durango, Mexico, was dried at 85 °C during 24 h. Subsequently, dried CS was grinded to a particle size of 180 µm using a manual mill.

The characterization of the CS was made following methods previously reported [2]: pH 7.54, total solids (TS) 94.59%, volatile solids (VS) 89.78%dry basis and ashes 10.22%dry basis. The main composition of the CS in dry basis (db) was 33.25%db cellulose, 24.35%db hemicellulose, 24.74%db lignin, 3.25%db protein and 10.19%db extractives. The elemental composition of CS was C-43.84%, H-15.74%, O-39.98% and N-0.44%.

The FVW were collected from a local cafeteria (Facultad de Ciencias Químicas, Universidad Autónoma de Nuevo León, Nuevo León, México). FVW were shredded (Hamilton Beach, Food Processor) and dried at 85 °C during 24 h, and grinded to a particle size of 180 µm using a mortar. FVW consisted of (wet weight basis): papaya peels (16.94%), squash peels (15.57%), potato peels (17.67%), spinach stems (20.26%); parsley stems (6.71%), cucumber peels (5.72%); melon peels (10.20%) and apple residues (6.92%). FVW presented pH 5.52, total solids 10.19%, volatile solids 87.66%db and 12.34%db of ashes. Further characterization of this material showed as composition of 12.8%db cellulose, 23.4%db hemicellulose, 10.26%db lignin, 12.63%db protein and 38.11%db extractives. The elemental composition of FVW was C-51.69%, H-3.43%, O-42.69% and N-2.19%.

Dilute acid hydrolysis pretreatment

Dilute acid hydrolysis was evaluated using a 5  2 general factorial experimental design [START_REF] Montgomery | Design and analysis of experiments[END_REF]. The factors were the FVW to CS ratio (0:1, 1:3, 1:1, 3:1, 1:0 FVW:CSdb) and the type of acid catalyst (HCl or H2SO4, 0.5% in volumetric basis). The acid hydrolysis was carried out by triplicate in a block digestor (DRB 200, HACH, U.S.A.); the co-substrates preparations were put into HACH tubes at 6.6% of total solid content and the reaction volume was 5 mL. The hydrolysis time and temperature were set at 120 min and 120 °C [START_REF] Kumar | Kinetic studies of two-stage sulphuric acid hydrolysis of sugarcane bagasse[END_REF][START_REF] Zheng | Dilute acid pretreatment and fermentation of sugar beet pulp to ethanol[END_REF]. After the hydrolysis pretreatment, solid residues were removed by centrifugation at 10 000 g, 10 min. Liquid hydrolyzates (LH) were used for sugar analysis (reducing and monomeric sugars) and for the determination of degradation products (furfural; 5-hydroxymethyl-furfural, HMF; total phenolic compounds, TPC; formic acid; acetic acid; propionic acid; succinic acid; lactic acid) according to the method reported by Muñoz-Páez et al [START_REF] Muñoz-Páez | Distinct effects of furfural, hydroxymethylfurfural and its mixtures on dark fermentation hydrogen production and microbial structure of a mixed culture[END_REF].

After obtaining the LH, these were processed to remove inhibitory compounds (i.e. HMF, furfural, TPC). For that, the pH was adjusted to pH 10 by adding powder Ca(OH)2 and continuous stirring for an hour. The formed precipitate was separated by centrifugation at 10 000 g, 15 min; supernatants were decanted and neutralized to pH 7 by HCl 6 M addition [START_REF] Chang | Hydrogen production by the anaerobic fermentation from acid hydrolyzed rice straw hydrolysate[END_REF].

Dark fermentation of LH

Hydrogenogenic inoculum was obtained from an anaerobic digester fed with FVW at 30 days of hydraulic retention time, started-up according to the Poggi-Varaldo et al. [START_REF] Poggi-Varaldo | Solid substrate anaerobic codigestion of paper mill sludge, biosolids, and municipal solid waste[END_REF]. To inhibit methaneproducing microflora, the anaerobic sludge was heat-shock treated in boiling water at 96 °C for 2 h [START_REF] Rodríguez-Valderrama | Evaluation of feeding strategies in upflow anaerobic sludge bed reactor for hydrogenogenesis at psychrophilic temperature[END_REF].

The effects of inoculum to substrate ratio (ISR = g VSinoculum g VSsubstrate -1 ) and initial reducing sugars concentration (CRS,i ) on the hydrogen production were evaluated using a 3 2 factorial design.

The levels for ISR and CRS,i were 0.8, 1.0, 1.2 and 10, 13, 16 g L -1 , respectively. Batch dark fermentation experiments were carried out by duplicate in 120 mL serum bottles with 70 mL of working volume. The CRS,i was adjusted by dilution of concentrated LH (ca. 21.13 g L -1 ) according to the experimental design. The fermentation was supplemented with 0.4 mL of 200 fold concentrated mineral medium [START_REF] Rodríguez-Valderrama | Evaluation of feeding strategies in upflow anaerobic sludge bed reactor for hydrogenogenesis at psychrophilic temperature[END_REF]. After inoculation, each bottle was flushed during 3 min with nitrogen to promote anaerobic conditions and sealed with a rubber stopper and aluminum rings. The serum bottles were incubated at 35 °C in a controlled temperature incubator with orbital agitation (150 rpm).

Analytical methods

The pH of co-subtrates was determined according to proceeding described by NMX-AA-25-1984 [START_REF]Protección al ambiente-Contaminación del sueloresiduos sólidos-determinación del pH-Método potenciométrico[END_REF]; 5 g of CS or FVW were diluted with 25 ml of distilled water, stirred for 10 min, and finally measure using a pH-meter (Conductronic PC45, Mexico). Total solids (TS), volatile solids (VS) and ashes were measured according standard methods [START_REF] Wef | Standard methods for the examination of water and wastewater[END_REF]. Cellulose and insoluble lignin analysis were determined by gravimetric method according to AOAC [START_REF]Official methods of analysis[END_REF]. The hemicellulose amount was determined by difference between holocellulose and cellulose content. Holocellulose was determined by lignin oxidation using NaClO according to AOAC modified proceeding [START_REF] Escamilla-Alvarado | Saccharification of fermented residues as integral part in a conceptual hydrogenproducing biorefinery[END_REF].

Extractives were measured by water extraction in a water bath at 60°C during 24 h [START_REF] Sluiter | Determination of extractives in biomass[END_REF]. Elemental quantities of carbon, hydrogen, oxygen and nitrogen were determined by organic elemental analyzer (Thermo Scientific Flash 2000, U.S.A.).

The content of total reducing sugars (RS) in LH was determined by 3,5-dinitrosalicylic acid method (dextrose as the standard) [START_REF] Miller | Use of dinitrosalicylic acid reagent for determination of reducing sugar[END_REF]. Monomeric sugars were quantified by high performance liquid chromatography (LDC Analytical, U.S.A.) with a Rezex RHM-Monosacharide (300mm x 7.8 mm) column and a refractive index detector (Varian Prostar, U.S.A.) with H2O as mobile phase.

The column temperature and mobile phase flow rate were 65 °C and 0.6 mL min -1 , respectively; glucose, xylose and arabinose were used as standards.

The content of total phenolic compounds (TPC) was determined by colorimetric method using Folin Ciocalteu reactive (tannic acid as standard) proposed by Blainski et al. [START_REF] Blainski | Application and analysis of the Folin Ciocalteu method for the determination of the total phenolic content from Limonium Brasiliense L[END_REF]. The subproducts of the acid hydrolysis (furfural, HMF, formic acid, acetic acid, propionic acid, succinic acid, and lactic acid) were determined against standards bought from Sigma Aldrich. These compounds were analyzed using a gas chromatograph (Varian CP 3380, U.S.A.) equipped with a column ZB-FFAP (15m x 0.53 x 1 μm) and flame ionization detector (FID). The temperatures of the injector and the detector were 230 and 280 °C, respectively. The column was first heated to 90 °C for 3 min, then the temperature was raised gradually to 200 °C with the step rate of 20 °C min -1 , the temperature was set at 200°C for 3 min and finally raised to 250 °C at 30 °C min -1 , which was then kept for 4 min. Calibration curves were previously developed using pure components.

Hydrogen gas content was determined using a gas chromatograph (Thermo Scientific Trace 1310, U.S.A.) equipped with a molecular sieve column (TG-BOND Msieve 5A, 30 m x 0.33 mm) and thermal conductivity detector (TCD). The temperatures of the oven, injector and detector were 100 °C, 150 °C and 200°C, respectively. Nitrogen gas was used as carrier gas with a flow rate of 3 mL min -1 . The amount of total gas produced was determined by acid-brine displacement method.

Volatile fatty acids (i.e acetic acid, propionic acid, butyric acid) and solvent (ethanol) composition in the liquid phase were determined by using a gas chromatograph (Thermo Scientific Trace 1310, U.S.A) as described in our previous publication [START_REF] Rodríguez-Valderrama | Evaluation of feeding strategies in upflow anaerobic sludge bed reactor for hydrogenogenesis at psychrophilic temperature[END_REF] .

Calculations and statistical analysis

Hydrolysis potential (HP, g mol -1 H3O + ) was considered as the quotient of RS concentration, CRS in g L -1 , and the theoretical hydronium concentration of the acid catalyst, CH3O + as mol H3O + L -1 , as described in Eq. 1. According to the concentration 0.5% volumetric basis for H2SO4 and HCl used for the hydrolysis experiments, their respective theoretical hydronium ion concentrations were 0.193 and 0.447 mol H3O + L -1 .

3

RS HO C HP C + = (1)
Reducing sugars production yield, YRS (%), was calculated comparing the CRS and the total carbohydrates content in the co-substrates (Eq. 2):

( )

100 RS r RS H CS cs FVW FVW CV Y m H C H C  =    +  ( 2
)
where Vr is the hydrolysis volume (L), mH is the co-substrates mass (g), HCS and HFVW are the total carbohydrates content (considered as the sum of cellulose, hemicellulose and extractives) in CS and FVW in dry basis (g g -1 ), respectively, whereas CCS and CFVW are the CS and FVW mass concentration in each co-substrate preparation (g g -1 ), respectively.

The hydrogen cumulative hydrogen production, H(t) (mL H2), was fitted by the modified Gompertz equation (Eq. 3), to estimate maximum cumulative H2 production, Hmax (mL H2), maximum H2 production rate, Rmax, (mL H2 h -1 ), and lag time,  (h) [START_REF] Zwietering | Modeling of the bacterial growth curve[END_REF]:

( ) max ( ) exp -exp - 1 max max R e H t H t H           =  +           (3)
where is t is any time (h) and e is 2.718.

The hydrogen molar yield in terms of glucose equivalents consumed (YH2 mol H2 mol -1 glucose)

was determined according Eq. 4:

( ) ( ) 2 cos ( ) max 1000 o f glu e H R RS RS M H MW Y V C C V  =  -   (4)
where VR is the fermentation volume (L), CRS,i and CRS,f are the initial and final RS concentrations (g L -1 ), respectively, MWglucose is the glucose molar weight (180.16 g mol -1 ), VM is the molar volume at standard reference conditions (22.4 L mol H2 -1 ) and 1000 is a volume conversion factor (mL L -1 ).

Analysis of variance (ANOVA) was performed for the experimental designs in the hydrolyzates and hydrogen production, using Design-Expert 6.0 (Design-Ease Inc. Co., Minneapolis, USA).

The main effects of the factors evaluated in the experimental designs were calculated according to Montgomery [START_REF] Montgomery | Design and analysis of experiments[END_REF]. In the dilute acid hydrolysis experimental design, reducing sugars yield (YRS), HMF concentration, furfural concentration and TPC were selected to evaluate the main effects, whereas for the dark fermentation experimental design the maximum cumulative hydrogen production (Hmax) and hydrogen molar yield (YH2) were selected. The standard error of the experimental designs was calculated from the square root of the mean square of the error divided by the number of experimental repetitions.

Economic prospection

The economic prospection accounted the expenses from the main operation stages, and the substances and compounds required for the process. The prospection considered the processing of 1 kg preparation of 3:1 FVW:CS, including the size reduction, acid hydrolysis and dark fermentation. The size reduction accounted the shredding of FVW in a food processer whereas CS was milled in a vibratory mill. Afterwards, the co-substrates mixture was acid hydrolyzed using HCl (0.5% volume basis, 2 h, 120 °C). The LH were recovered by centrifugation (10 000 g, 10 min) and were overlimed with Ca(OH)2 (1 h, room temperature). After centrifugation (ibid.), LH were neutralized using HCl (6 M). The LH were fed to a bioreactor with hydrogenogenic inoculum (35 °C). The same mineral composition as in the experiments was considered. Hydrogen content in biogas was 50.1% and hydrogen yield was 1.91 mol H2 mol -1 glucose.

Collection costs of the substrates were retrieved from literature. Thompson and Tyner [START_REF] Thompson | Corn stover for bioenergy production: Cost estimates and farmer supply response[END_REF] calculated the collection, baling and transport costs of CS at farms. For the cost of collection and transport of FVW, an average was obtained after the works of Yepes et al. [START_REF] Yepes | Agroindustrial waste valorization-fruits-in-Medellin and the south of Valle de Aburrá, Colombia[END_REF] and Mattsson et al. [START_REF] Mattsson | Waste of fresh fruit and vegetables at retailers in Sweden -Measuring and calculation of mass, economic cost and climate impact[END_REF], who evaluated the management for FVW valorization in production fields and cities, respectively. Operation costs for the processing of FVW and CS, and hydrogen production, were calculated from energy balances and the Mexican electricity rates at industry [34]. Water cost was taken from Mexican National Water Commission [START_REF] Conagua | Tarifas de agua[END_REF]. Chemicals for acid hydrolysis, overliming and mineral medium were taken from different market sources.

The energy calculations from shredding, milling and overliming were obtained as previously described [START_REF] Rodríguez-Valderrama | Enfoque de biorrefinería para la producción de hidrógeno y metano a partir de residuos orgánicos[END_REF]. The energy consumption of acid hydrolysis was determined according to Mafe et al. [START_REF] Mafe | Development of an estimation model for the evaluation of the energy requirement of dilute acid pretreatments of biomass[END_REF]. The energy balance in dark fermentation reactor was carried out as described by Lübken et al. [START_REF] Lübken | Modelling the energy balance of an anaerobic digester fed with cattle manure and renewable energy crops[END_REF]. The total process revenue was determined from the market price of H2 and CO2 [START_REF] Han | Biohydrogen production from waste bread in a continuous stirred tank reactor: A techno-economic analysis[END_REF] and their productivities.

Results and Discussion

Diluted acid hydrolysis pretreatment

The highest concentrations of RS were obtained at 1:0 FVW:CS ratio, being 19.32 and 27.32 g L -1 for H2SO4 and HCl, respectively (Fig. 1A), probably as a consequence of a number of factors such as: a greater digestibility of FVW over CS, heterogeneous composition of FVW, lower lignin content and lower degradation temperature [START_REF] Razaghi | Hydrolysis treatments of fruit and vegetable waste for production of biofuel precursors[END_REF]. Regarding the co-substrate preparations, the maximum RS were 24.69 and 17.99 g L -1 for HCl and H2SO4, respectively, obtained from the 3:1 FVW:CS ratio.

PLEASE INSERT FIGURE 1

The HCl as acid catalyst presented higher RS production than H2SO4 at each co-substrate ratio, probably influenced by its 2.32 times higher concentration of hydronium ion than H2SO4 treatments.

However, when observing the HP (Fig. 1B), H2SO4 was better than HCl in achieving a HP of 100.33 g mol -1 H3O + at 1:0 FVW:CS ratio, versus 61.13 g mol -1 H3O + for HCl at same ratio. Hilpmann et al. [START_REF] Hilpmann | Acid hydrolysis of xylan[END_REF] observed that hydronium concentration is one of the several factors that have influence on the efficiency of the acid hydrolysis of xylan. They found that hydronium concentrations lower than 0.1 M presented yields below 86%, whereas hydronium concentrations higher than 0.1 M promoted yields close to 100%. It is worth noticing that due to the lower corrosivity of H2SO4 compared to HCl or HNO3, it has been the most used catalyst in hydrolysis processes of lignocellulosic wastes [START_REF] Hutomo | The effect of sulfuric and hydrochloric acid on cellulose degradation from pod husk cacao[END_REF][START_REF] Argun | Glucose and 5-hydroxymethylfurfural production from cellulosic waste by sequential alkaline and acid hydrolysis[END_REF].

The main effects analysis (Fig. 2) showed that the maximum YRS was observed at 1:0 FVW:CS ratio, which corroborated the ease of degradation of FVW and also demonstrated a correlation amidst the contents of FVW and YRS (Fig. 2A). Regarding the type of acid catalysts influence on YRS, HCl was superior over H2SO4 (Fig 2B ), thus the combination of high contents of FVW and HCl is advisable to promote high YRS. The ANOVA (Table S1) of YRS showed that the model, the co-substrate ratios, the acid catalyst and their interaction were significant (p < 0.0053). The R 2 and R 2 adj were 0.8411 and 0.7696, respectively.

PLEASE INSERT FIGURE 2

Regarding the production and distribution of monosaccharides, noticeable differences were observed amidst the type of catalyst as shown in Fig. 3. When using HCl as catalyst (Fig. 3A), maximal concentrations of glucose were obtained at 3:1 FVW:CS ratio (10.02 g L -1 ), and at 1:3 FVW:CS for xylose (8.41 g L -1 ). No particular trends were appreciated after H2SO4 treatment (Fig. 3B). In the experiments with HCl, it was observed that the ratios with higher CS content presented higher xylose concentrations, whereas those ratios with higher FVW presented higher glucose concentrations. This difference is interesting and has also been reported elsewhere. For instance, Zu et al. [START_REF] Zu | Pretreatment of corn stover for sugar production using dilute hydrochloric acid followed by lime[END_REF] evaluated the CS hydrolysis with HCl as acid catalyst and found that xylose yield was also higher than that of glucose (20.44 and 1.82 g per 100 graw material, respectively) at 120 °C, 40 min and 1% HCl. Similarly, Cao et al. [START_REF] Cao | Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16[END_REF] found higher xylose concentrations than glucose (6.25 and 1.68 g L -1 , respectively) when assessing the CS acid hydrolysis with H2SO4 at 121 °C, 105 min, and 0.25% H2SO4, ascribing this phenomenon to the easier hemicellulose solubilization than cellulose by acid catalyst in this particular substrate.

PLEASE INSERT FIGURE 3

An undesirable trait about the acid hydrolysis processes is the concomitant degradation of hydrolyzed carbohydrates into compounds such as furfural and HMF, and the lignin degradation into acetic acid and phenolic compounds, which altogether act as inhibitors to microbial growth [START_REF] Gonzales | Effect of severity on dilute acid pretreatment of lignocellulosic biomass and the following hydrogen fermentation[END_REF][START_REF] Hutomo | The effect of sulfuric and hydrochloric acid on cellulose degradation from pod husk cacao[END_REF]. In our experiments, there were significant differences (p < 0.0085) of both the acid catalysts and the FVW:CS ratio on HMF, furfural and TPC production (Table S2, Table S3 and Table S4, respectively). Fig. 4 shows that for HMF and TPC, their concentrations were higher for HCl and as FVW increased its proportion. Regarding furfural, HCl also produced higher concentrations than H2SO4, although FVW effect was not as evident as in HMF and TPC showing a maximum at 1:3 and a minimum at 3:1 FVW:CS ratios. These observations have some relation to that observed for YRS, and the respective explanation is also applicable to this case: FVW is a substrate much easier to degrade, and HCl provides higher H + concentration than H2SO4 when both substances are assayed at the same volumetric concentration.

PLEASE INSERT FIGURE 4

The concentrations of HMF, furfural and TPC in our experiments (Table 1) were lower than the inhibitory concentrations (<1 g L -1 ) reported for dark fermentation of mixed cultures, except for the hydrolyzates at 1:0 FVW:CS ratio and HCl [START_REF] Quéméneur | Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures[END_REF]. Moreover, when two or more inhibitors are present in the substrate medium, inhibition concentrations may be considerably lower, yet not consistent data has been found amidst literature. For instance, Kumar et al. [START_REF] Kumar | Effects of 5-hydromethylfurfural, levulinic acid and formic acid, pretreatment byproducts of biomass, on fermentative H2 production from glucose and galactose[END_REF] found that at 0.69 g HMF L - 1 and 12 g formic acid L -1 concentrations, microbial growth and hydrogen production were inhibited.

On the other hand, Zheng et al. [START_REF] Zheng | Dilute acid pretreatment and fermentation of sugar beet pulp to ethanol[END_REF] reported that up to 4 g L -1 of HMF or furfural and 6 g acetic acid L -1 inhibited microbial metabolism. On their behalf, Muñoz-Páez et al. [START_REF] Muñoz-Páez | Distinct effects of furfural, hydroxymethylfurfural and its mixtures on dark fermentation hydrogen production and microbial structure of a mixed culture[END_REF] observed that individual concentrations of furfural and HMF up to 1 and 0.09 g L -1 , respectively, were even beneficial for biohydrogen production; yet when concomitantly present, these have inhibited 10% the hydrogen production even at low concentrations as 0.10 and 0.02 g L -1 for furfural and HMF, respectively. In general, the most important inhibitor has been suggested to be HMF, and it should be considered that concentrations above 1 g L -1 in hydrolyzates have reduced the yield in hydrogen bioproduction [START_REF] Gonzales | Effect of severity on dilute acid pretreatment of lignocellulosic biomass and the following hydrogen fermentation[END_REF]. Indeed, except for one of our assays, such value was not reached, pointing out that our hydrolyzates would be suitable for dark fermentation or anaerobic digestion [START_REF] Quéméneur | Inhibition of fermentative hydrogen production by lignocellulose-derived compounds in mixed cultures[END_REF].

PLEASE INSERT TABLE 1

It is important to note that for both HCl and H2SO4 at 1:0 FVW:CS assays (Fig. 3), no xylose content was detected, most likely due to its conversion into furfural and subsequent transformation into formic, lactic and succinic acids [START_REF] Möller | Hydrothermal production of furfural from xylose and xylan as model compounds for hemicelluloses[END_REF]. Indeed, the sum of these latter compounds was around 9.5 g L -1 , a concentration comparable to that of xylose at FVW:CS ratio of 1:3.

Since the acid hydrolysis of 3:1 FVW:CS with HCl catalyst (Fig. 3) showed a balanced distribution of glucose, xylose and arabinose, as well as high RS concentration, dark fermentation was developed in the next section using the hydrolyzates from such conditions. Despite the low concentration of inhibitors, the liming treatment was applied to the hydrolyzates to avoid possible interactions of any kind during the hydrogen production. The overliming treatment decreased both the reducing sugars by 11.71% (24.00 to 21.19 g L -1 ) and the TPC by 26.4% (1.06 to 0.78 g L -1 ) in 26.41%. This diminution of RS was comparable with the 9.1% reduction observed by Chang et al. [START_REF] Chang | Hydrogen production by the anaerobic fermentation from acid hydrolyzed rice straw hydrolysate[END_REF] when rice husk HCl hydrolyzates were overlimed. The removals of TPC and HMF over 20% are according to expected, whereas it is common that some organic acids might not be reduced by overliming [START_REF] Larsson | Comparison of different methods for the detoxification of lignocellulose hydrolyzates of Spruce[END_REF].

Dark fermentation of LH

The highest Hmax (212 mL H2) and Rmax (8.83 mL H2 h -1 ) were obtained for ISR 1.2 and CRS,i, of 13 g L -1 (Fig. 5, Table 2). Comparing the cumulative hydrogen production against literature is intricate due to the different reported operation conditions. For mixed cultures, Datar et al. [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF] demonstrated a cumulative hydrogen volume of 4138 mL when using CS hydrolyzates (10 g L -1 ) in dark fermentation by anaerobic heat-treated sludge (105 °C, 2h) in a 1250 mL CSTR. Their volumetric hydrogen productivity, 3310 mL H2 L -1 reactor was 13% higher than our best result of 2933 mL H2 L - 1 reactor at ISR 1.2 and 13 g RS L -1 (Table 3). However, in their experiments the longer lag time obtained, 38 h, was ascribed to the presence of inhibitory compounds such as HMF and furfural obtained after extreme hydrolysis conditions (acid steam-explosion 1.2% H2SO4, 200 °C, 1 min).

The longest lag time registered in our experiments was 22.5 h for the assay with the lowest inoculum load (ISR 0.8) and the highest substrate amounts (RS 16 g L -1 ), which could have been an indicative of a slower adaptation of hydrogen-microbial producers to substrate [START_REF] Chang | Hydrogen production by the anaerobic fermentation from acid hydrolyzed rice straw hydrolysate[END_REF].

PLEASE INSERT FIGURE 5 PLEASE INSERT TABLE 2 PLEASE INSERT TABLE 3
The ANOVA (Table S5 and Table S6) of this experiment showed that CRS,i had significant effects on both Hmax and YH2, whereas the ISR did not present significant effects on neither response variables in the studied operation range. The main effects of the CRS,i on the Hmax (Fig. 6A) demonstrated that 10 g L -1 presented the lowest average hydrogen production (183 mL H2), whereas using 13 or 16 g L -1 presented higher values (ca. 202 mL H2). Oppositely, the main effects on YH2 (Fig. 6B) were higher at CRS,i 10 g L -1 (2.2 mol H2 mol glucose -1 ) than at 13 and 16 g L -1 (1.8 and 1.9 mol H2 mol glucose -1 , respectively). This was in accordance to the reducing sugars consumption (Table 3): at CRS,i of 10 and 13 g L -1 the RS consumption was higher than 94.9%, whereas at 16 g L -1 , it was close to 81%, except for the experiment with ISR 0.8 that presented ca. 70% consumption, indicating the possible inhibition by substrate or metabolites. Therefore, in terms of Hmax and YH2, It may be drawn that CRS,i of 10 and 13 g L -1 may be the best options for conducting dark fermentation of hydrolyzates. Fangkum et al. [START_REF] Fangkum | Biohydrogen production from sugarcane bagasse hydrolysate by elephant dung: Effects of initial pH and substrate concentration[END_REF] reported an increase in cumulated hydrogen production (42 to 92 mL H2) when the initial concentration of fermentable sugars increased from 5 to 10 g L -1 ; yet when initial concentration of sugars exceeded 20 g L -1 , cumulated hydrogen production decreased mainly due to the accumulation of organic fatty acids, which was reflected as a drop in pH, causing bacterial growth inhibition. In our experiments, the final pH dropped as CRS,I increased, being the lowest of 5.17 when using RS 16 g L -1 at ISR 0.8 (Table 3).

PLEASE INSERT FIGURE 6

Comparing our highest molar yields of 1.9-2.3 mol H2 mol glucose -1 amidst the range 0.8-1.2 ISR (Table 3) to literature (Table 4), ours were higher than some using pure cultures as in the case of Pattra et al. [START_REF] Pattra | Bio-hydrogen production from the fermentation of sugarcane bagasse hydrolysate by Clostridium butyricum[END_REF] who reported 1.73 mol H2 mol glucose -1 by Clostridium butyricum when fermenting sugarcane acid hydrolyzates at higher concentration (20 g L -1 ); or as in the case of Cao et al. [START_REF] Cao | Acid hydrolysis of corn stover for biohydrogen production using Thermoanaerobacterium thermosaccharolyticum W16[END_REF] who obtained 2.24 mol H2 mol glucose -1 by Thermoanaerobacterium thermosaccharolyticum W16 fermenting 12 g L -1 of sugars from corn hydrolyzates at thermophilic temperatures (60 °C). Using microbial consortia, Zhang et al. [START_REF] Zhang | Fermentative hydrogen production from corn stover hydrolyzate by two typical seed sludges : Effect of temperature[END_REF] obtained low hydrogen yields (0.35 mol H2 mol glucose -1 ) with an anaerobic granular sludge at 37 °C, whereas at 55 °C the hydrogen yield was increased to 1.39 mol H2 mol glucose -1 . The main difference from our experiments against those listed in Table 4 was our noticeably higher ISR. Indeed, the next highest ISR, 0.4, was that from Datar et al. [START_REF] Datar | Hydrogen production from the fermentation of corn stover biomass pretreated with a steam-explosion process[END_REF], who indeed obtained the highest YH2. Our results have demonstrated that it was feasible to hydrolyze preparations of FVW and CS, and to use the sugars released to achieve competent batch hydrogen production by microbial consortia.

PLEASE INSERT TABLE 4

Regarding the metabolites produced, in all the experiments butyrate was the most abundant, followed by acetate and lastly by propionate. High butyrate concentrations are indicative of the predominance of the butyrogenic pathway in this dark fermentation hydrogen production [START_REF] Gonzales | Effect of severity on dilute acid pretreatment of lignocellulosic biomass and the following hydrogen fermentation[END_REF]. On the other hand, low propionate concentrations is advantageous since it may indicate low presence of propionogenic microorganisms that use H2 as electron donor [START_REF] Bundhoo | Inhibition of dark fermentative bio-hydrogen production: A review[END_REF]. Low ethanol concentrations are attributed to a low partial pressure effect due to the frequent hydrogen depressurization. Since a high variation in the partial pressure may change the route of metabolites production (i.e.

solvents such as ethanol, acetone or butanol instead of VFA) [START_REF] Wong | A review of sustainable hydrogen production using seed sludge via dark fermentation[END_REF][START_REF] Hernández | Use of coffee mucilage as a new substrate for hydrogen production in anaerobic co-digestion with swine manure[END_REF].

Economic prospection

A net benefit of 0.009 USD per kg of co-substrates (wet basis) processed was observed (Table 5). The total costs were 0.029 USD/kg (including energy, water and chemicals), whereas the revenue for H2 and CO2 produced was 0.038 USD/kg. The costs were distributed as 54% related to FVW and CS supply, 26% due to substances and compounds, and the remaining 20% to electricity consumption. From this analysis, it is evident that waste biomass long before considered to be low cost [START_REF] Poggi-Varaldo | Biohydrogen, biomethane and bioelectricity as crucial components of biorefinery of organic wastes: a review[END_REF] is not necessarily so. Different alternatives have been implemented to reduce such related production costs, such as government funding and subsidies [START_REF] Han | Biohydrogen production from waste bread in a continuous stirred tank reactor: A techno-economic analysis[END_REF].

The unit costs for H2 and CO2 considering a revenue-based allocation were 2.05 and 0.3 USD/m 3 , respectively. The market price of H2 and CO2 are 2.7 and 0.3 USD/m 3 , which indicates the profitability in hydrogen production of 24%. Indeed, hydrogen cost was comparable to other works in literature using different substrates. For instance, Han et al. [START_REF] Han | Techno-economic analysis of a novel bioprocess combining solid state fermentation and dark fermentation for H2 production from food waste[END_REF] reported a cost of 2.29 USD/m 3 H2 from the techno-economic analysis of hydrogen production from food waste by integrated solid state fermentation and dark fermentation. The plant capacity was proposed to be 10 ton/d, and yield was 52.4 mL H2/g food waste. Using bread waste, Han et al. [START_REF] Han | Biohydrogen production from waste bread in a continuous stirred tank reactor: A techno-economic analysis[END_REF] reported a production cost of 1.34 USD/m 3 for the continuous hydrogen production in a 2 ton/d CSTR.

The production of hydrogen from hydrolyzates of FVW and CS has shown a promissory alternative for the development of biorefinery models. Still, as hydrogen production finds alternatives of production from different substrates, biorefinery configurations and operation conditions, it is becoming necessary to improve economic evaluations. We envisage areas of opportunity that will have consequences both for revenues and costs. For instance, the process profitability can be improved if the sub-products as organic acids and solvents from dark fermentation are further processed (e.g. hydrogen production by the photofermentation or methane production by anaerobic digestion) or marketed, as in the case of un-solubilized biomass that could be sold as biofertilizer. On the other hand, equipment, construction, and other operating costs such gas compression, salaries and taxes will increase production costs [START_REF] Han | Techno-economic evaluation of a combined bioprocess for fermentative hydrogen production from food waste[END_REF].

PLEASE INSERT TABLE 5

Conclusion

The main conclusions of this work were several and noticeworthy. They arranged according to the principal sections of the research, as follows:

i) Hydrolysis. HCl as acid catalyst was superior than H2SO4 in terms of amount and quality of hydrolyzates, and the 3:1 FVW:CS ratio using HCl resulted in the highest production of reducing sugars in a balanced distribution of the monosaccharides glucose, xylose and arabinose.

ii) Dark fermentation. The FVW:CS sugar-rich hydrolyzates were efficiently fermented.

Moreover, the initial concentration of reducing sugars had important and significant effects both on maximum hydrogen production and yield. H2 production was not dependent by inoculum to substrate ratio in the range 0.8 to 1.2 g VSinoculum g VSsubstrate -

1

. Concentrations of 10 and 13 g L -1 are suitable for conducting competent dark fermentation of hydrolyzates.

iii) Economic prospection. Hydrogen production presented economic benefits, such as net revenues of 0.009 USD per kg of co-substrates (wet basis) processed and H2 unit cost below its market price, resulting in 24 % profitability of hydrogen production. The main costs were associated to FVW and CS supply.

Finally, the production of hydrogen from hydrolyzates of FVW and CS, as alternative to monosubstrates fermentation, has shown a promissory option for the development of biorefinery models.

CH3O + theoretical hydronium concentration of the acid catalyst (mol H3O

LIST OF TABLES

Table 1. Secondary products concentration from acid hydrolysis of fruits-and-vegetables wastes to corn stover ratios, FVW:CS.

Table 2. Parameters of the modified Gompertz equation of the factorial design 3 2 from the HCl treatment at assayed reducing sugars initial concentrations, CRS,i, and inoculum to substrate ratios, ISR.

Table 3. Secondary metabolites and response variables of hydrogen production of HCl hydrolyzates, at assayed reducing sugars initial concentrations, CRS,i, and inoculum to substrate ratios, ISR.

Table 4. Hydrogen production by dark fermentation of organic wastes.

Table 5. Economic prospection of hydrogen production from FVW:CS (3:1 ratio) at lab scale.

Table 1. Secondary products concentration from acid hydrolysis of fruits-and-vegetables wastes to corn stover ratios, FVW:CS.

Notes: FVW:CS of fruits-and-vegetables wastes to corn stover ratios; HMF, Hydroxymethyl-furfural; TPC, Total phenolic compounds.

Fig. 2 .

 2 Fig.2. Main effects on reducing sugars production yield, YRS, by (A) fruits-and-vegetables wastes to corn stover ratios, FVW:CS, and (B) acid catalysts, HCl or H2SO4.

Fig. 3 .

 3 Fig.3. Monomeric sugars production at different fruits-and-vegetables wastes to corn stover ratios, FVW:CS, by acid hydrolysis using (A) HCl, and (B) H2SO4.

Fig. 4 .

 4 Fig. 4. Main effects by fruits-and-vegetables wastes to corn stover ratios, FVW:CS, and acid catalysts, HCl or H2SO4 on the production of HMF (A and B), furfural (C and D), and TPC (E and F).

Fig. 5 .

 5 Fig. 5. Cumulative hydrogen production by dark fermentation of overlimed HCl hydrolyzates from FVW:CS preparation at ISR equal to: 1.2 (A), 1.0 (B), and 0.8 (C). Reducing sugars initial concentration, CRS,i, were 10 (triangle), 13 (circle), and 16 g L -1 (square).

Fig. 6 .

 6 Fig.6. Main effects by the reducing sugars initial concentration, CRS,i, on the (A) maximum cumulative hydrogen production, Hmax, and (B) the hydrogen molar yield, YH2.

Fig. 1 .

 1 Fig. 1. Influence of fruits-and-vegetables wastes to corn stover ratios, FVW:CS, and acid catalysts, HCl or H2SO4, on (A) reducing sugars production, RS, and (B) hydrolysis potential, HP.

Fig. 2 .Fig. 3 .Fig. 4 .

 234 Fig. 2. Main effects on reducing sugars production yield, YRS, by (A) fruits-and-vegetables wastes to corn stover ratios, FVW:CS, and (B) acid catalysts, HCl or H2SO4.

Fig. 5 .Fig. 6 .

 56 Fig. 5. Cumulative hydrogen production by dark fermentation of overlimed HCl hydrolyzates from FVW:CS preparation at ISR equal to: 1.2 (A), 1.0 (B), and 0.8 (C). Reducing sugars initial concentration (CRS,i) were 10 (triangle), 13 (circle), and 16 g L -1(square)

  Fig.1. Influence of fruits-and-vegetables wastes to corn stover ratios, FVW:CS, and acid catalysts, HCl or H2SO4, on (A) reducing sugars production, RS, and (B) hydrolysis potential, HP.

	LIST OF FIGURES	+ L -1 )
	db	dry basis
	FVW	fruits-and-vegetables wastes
	Hcs	carbohydrates content in corn stover (g g -1 db)
	HFVW	carbohydrates content in FVW (g g -1 db)
	HMF	hydroxymethyl-furfural
	H(t)	cumulative hydrogen production at time 't' (mL H2)
	Hmax	maximum cumulative hydrogen production (mL H2)
	HP	hydrolysis potential (g mol -1 H3O + )
	ISR	inoculum substrate ratio
	LH	liquid hydrolyzates
	mH	co-substrates mass (g)
	MWglucose	glucose molar weight (g mol -1 )
	Rmax	maximum hydrogen production rate (mL H2 h -1 )
	RS	reducing sugars
	t	time (h)
	TPC	total phenolic compounds
	VFA	volatile fatty acids
	VS	volatile solids
	Vr	hydrolysis volume (L)
	VR	fermentation volume (L)
	YH2	hydrogen molar yield (mol H2 mol -1 glucose)
	YRS	reducing sugars yield (%)
	Greek symbols
		lag time (h)

Table 2 . Parameters of the modified Gompertz equation of the factorial design 3 2 from the HCl treatment at assayed reducing sugars initial concentrations, CRS,i, and inoculum to substrate ratios, ISR.

 2

	Assay	Real values	Coded values	Hmax	Rmax	λ
		CRS,i (g L -1 )	ISR	CRS,i	ISR	(mL H2)	(mL H2 h -1 )	(h)
	1	10	1.2	-1	1	180	6.70	16.5
	2	13	1.2	0	1	212	8.83	15.4
	3	16	1.2	1	1	208	6.92	17.0
	4	10	1.0	-1	0	180	6.19	15.2
	5	13	1.0	0	0	201	6.13	16.1
	6	16	1.0	1	0	203	7.61	17.3
	7	10	0.8	-1	-1	196	7.03	16.6
	8	13	0.8	0	-1	200	6.34	16.1
	9	16	0.8	1	-1	201	7.52	22.5

Note: For all experiments, R 2 was greater than 0.99; Hmax, maximum cumulative hydrogen production; Rmax, maximum hydrogen production rate; , lag time (h).

Table 3 . Secondary metabolites and response variables of hydrogen production of HCl hydrolyzates, at assayed reducing sugars initial concentrations, CRS,i, and inoculum to substrate ratios, ISR.

 3

Table 4 . Hydrogen production by dark fermentation of organic wastes.

 4 : a Experimental data; CS, corn stover; FVW, fruits and vegetables wastes; Hmax, maximum cumulative hydrogen production; ISR, inoculum to substrate ratio; Pv, volumetric productivity (hydrogen production/oper); Rmax, maximum hydrogen production rate; T, temperature (°C); t, time (h); Vo, operational volume (mL); YH2, hydrogen molar yield; , lag time (h).

	Inoculum	Substrate	Operational	ISR	Hydrogen	Hydrogen molar	Ref
		(initial	conditions		production and	yield
		concentration)			Gompertz kinetic	(YH2) and
					parameters	volumetric
						productivity (Pv)
	Thermoanaerobact erium	Corn stover acid hydrolyzates	Vo= 50 mL T=60 °C	0.0123	Hmax=165 mL Rmax=5.51 mL h -1	2.24 mol H2 mol glucose -1	[44]
	thermosaccharolyt	(11.84 g L -1 )	t= 30 h		λ=4 h	Pv=3305 mL L -1
	icum W16
	Clostridium butyricum	Sugarcane bagasse	Vo= 70 mL T=37 °C	0.001	Hmax=180 mL Rmax=3 mL h -1	1.73 mol H2 mol glucose -1	[49]
		hydrolyzates	t= 60 h		λ=5 h	Pv=2571.43 mL L -
		(20 g L -1 )				1
	Anaerobic sludge	Food waste and	Vo= 415 mL	0.041	Hmax=359 mL	0.98 mol H2 mol	[13]
		sewage sludge	T=35 °C		Rmax=14.93 mL h -1	glucose -1
		(15.5 g L -1 )	t=216 h		λ=6.48 h	Pv=796.39 mL L -1
	Anaerobic sludge	Corn stover steam-explosion	Vo= 1250 mL T=35 °C	0.4	Hmax= 4138 mL Rmax=206 mL h -1	2.42 mol H2 mol glucose -1	[8]
		(10.98 g L -1 )	t=78 h		λ=38 h	Pv=3310 mL L -1
	Activated sludge	Corn stover acid hydrolyzates	Vo= 50 mL T=37 °C	0.032	Hmax= 14 mL Rmax=0.22 mL h -1	0.44 mol H2 mol glucose -1	[50]
		(5 g L -1 )	t= 60 h		λ=15.3 h	Pv= 275 mL L -1
	Granular anaerobic sludge	Corn stover acid hydrolyzates	Vo= 50 mL T=37 °C	0.192	Hmax= 11 mL Rmax=0.19 mL h -1	0.35 mol H2 mol glucose -1	[56]
		(5 g L -1 )	t= 60 h		λ=15.4 h	Pv= 223.6 mL L -1
	Anaerobic sludge	Organic fraction of municipal	Vo= 500 g T=55 °C	0.068	Hmax a = 331 mL Rmax a =0.70 mL h -1	0.29 mol H2 mol glucose -1	[2]
		solid waste	t= 470 h		λ a =0 h	Pv= 662 mL g -1
		(150 g kgr -1 )
	Anaerobic sludge	FVW:CS acid	Vo= 70 mL	1.2	Hmax a = 212 mL	1.91 mol H2 mol	This
		hydrolyzates at	T=35 °C		Rmax a =8.83 mL h -1	glucose -1	work
		3:1 ratio	t= 87 h		λ a =15.35 h	Pv= 2933 mL L -1
		(13 g L -1 )

Notes

Table 5 . Economic prospection of hydrogen production from FVW:CS (3:1 ratio) at lab scale.

 5

	Stage	Component	Price	Quantity	Unit	Price (USD) Ref
			(USD/kg)
	Raw materials	CS	0.088	0.035	kg	-0.003	[31]
		FVW	0.013	0.965	kg	-0.013	[32,33]
	Co-substrates size	Electricity	0.014	0.089	MJ	-0.001	[34]
	reduction
	Acid hydrolysis	HCl (30%)	0.029	0.039	kg	-0.001	[57]
		Water	0.0006	1.079	kg	-0.001	[35]
		Electricity	0.014	0.322	MJ	-0.004	[34]
	Mechanical	Electricity	0.014	0.012	MJ	-0.002	[34]
	separation
	Overliming	Ca(OH)2	0.12	0.013	kg	-0.002	[58]
	pretreatment	Electricity	0.014	0.009	MJ	-0.0001	[34]
	Dark fermentation	NH4Cl	0.11	0.003	kg	-0.0004	[59]
		Buffer media 0.3	0.008	kg	-0.0023	[60]
		Water	0.0006	0.016	kg	-0.00001	[35]
		ZnCl2	0.9	0.0002	kg	-0.0002	[61]
		MgCl2•6H2O	0.1	0.0003	kg	-0.00003	[62]
		MnCl2•4H2O	2	0.00004	kg	-0.00007	[63]
		FeCl3•6H2O	2	0.00009	kg	-0.00017	[64]
		CuCl2•2H2O	2.5	0.00001	kg	-0.00003	[65]
		NiCl2•6H2O	3	0.0003	kg	-0.001	[66]
		H2	0.0027	13.353	L	0.036	[39]
		CO2	0.0003	6.663	L	0.002	[39]
	Total system cost					-0.029
	Total revenue					0.038
	Total Net					0.009

Acknowledgements

The authors express their gratitude to Dr. Eduardo Soto Regalado and Dr. Felipe Cerino Córdova for supporting the project (02-106534-PST-15/123), and to Dr. Refugio Bernardo García Reyes, Dr. Luis H. Alvarez, Chem. Eng. Gustavo Gerardo Medina Mendoza and Damián Isaac Peña-Ramírez for their support in analysis determination. Santiago Rodríguez-Valderrama thanks Consejo Nacional de Ciencia y Tecnología (Conacyt) for the Scholarship No. 714579.

Abbreviations

CS corn stover

CCS corn stover concentration in acid hydrolysis (g g -1 dry base) CFVW fruits-and-vegetables wastes concentration in acid hydrolysis (g g -1 dry base)

CRS

reducing sugars concentration in acid hydrolysis (g L -1 ) CRS,i reducing sugars initial concentration in dark fermentation (g L -1 ) CRS,f reducing sugars final concentration in dark fermentation (g L -1 )