Ruben Teixeira Franco

Pierre Buffière
email: pierre.buffiere@insa-lyon.fr

Rémy Bayard
email: remy.bayard@insa-lyon.fr

Teixeira Rúben
email: ruben.teixeirafranco@insa-lyon.fr

Pierre Franco

Rémy Buffière

Bayard

Co-ensiling of cattle

Keywords: Cattle manure, Additives, Storage, Ensiling, Anaerobic digestion, Methane potential ADL, acid detergent lignin, BMP, biochemical methane potential, BU, butyric acid, CEL, cellulose, COD, chemical oxygen demand, FA, formic acid, HEM, hemicellulose, LA, lactic acid, LAB, lactic acid bacteria, LIG, lignin, NDF, neutral detergent fiber, NH3-N, ammonia nitrogen, TKN, total Kjeldahl nitrogen, TS, total solids, VS, volatile solids, WSC, water soluble carbohydrates, VFA, volatile fatty acids

Introduction

Cattle manure is a major input for agricultural biogas plants, especially in countries with intensive livestock systems. In France there are currently 267 of these installations [START_REF]Les avis de l'ADEME: Méthanisation[END_REF] and nearly 87 Mt of cattle manure annually produced that may be mobilized for anaerobic digestion (AD) in the near future [START_REF] Degueurce | Fumiers de bovins, une ressource à fort potentiel pour la filière de méthanisation en France ?[END_REF]. However, there are still several constraints slowing down the valorization of cattle manure. This includes the storage requirements and the consequences on the methane potential of cattle manure that it may entail. On the one hand, biogas plants should operate continuously and feedstock should be available throughout the year. Yet, cattle manure, such other agricultural wastes and crops, undergoes production fluctuations, which periodically leads to the need of storage during long periods.

The main issues related to the storage of cattle manure are that the current field-scale practices are not optimized for energy production purposes and scientific studies about this subject are scarce. Cattle manure is typically stored outdoors in contact of air, in order to carry out a simple and low-cost operation. This has several drawbacks: it leads to leachate generation, ammonia and odor emissions [START_REF] Berg | Mitigation of methane emissions during manure storage[END_REF] and energy losses. Recently, Teixeira Franco et al. [START_REF] Franco | Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?[END_REF] evidenced that up to 74% of cattle manure's biochemical methane potential (BMP) may be lost during 4 months of open-air storage.

Nevertheless, the authors showed that anaerobic storage, or ensiling, may limit energy losses to a maximum of 46% after 4 months.

Traditionally used as fodder conservation method, ensiling is a biochemical process that mainly rely on anaerobic conditions and biological acidification of the feedstock [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Franco | Ensiling for biogas production: Critical parameters. A review[END_REF]. This biomass acidification consists in lowering the pH around 4.0 by organic acid production (especially lactic acid), which will impede further significant fermentative activity. The main reactions involved in carbon degradation during ensiling are represented in Figure 1. While applied to specific crops, ensiling leads to full BMP conservation, even after 1 year [START_REF] Herrmann | Effects of ensiling, silage additives and storage period on methane formation of biogas crops[END_REF]. However, fresh cattle manure does not satisfy the chemical requirements for an efficient ensiling, whether due to its high moisture, low water soluble carbohydrates (WSC) content, strong basic buffering capacity or endogenous microflora. For instance, without WSC or another source of easily fermentable sugars, hetero or homo-lactic fermentation cannot occur and lactic acid cannot be rapidly produced in a significant extent, so as to abruptly lower the pH value and inhibit further bacterial activity.

To the best of our knowledge, few authors reported work on cattle manure storage.

Teixeira Franco et al. [START_REF] Franco | Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?[END_REF] have approached the optimization of cattle manure silage before biogas production. The authors evidenced that co-ensiling with wheat straw may enhance methane conservation of cattle manure to more than 86% after 4 months. The decrease of moisture content and the alteration of buffering properties should be in the origin of this improvement. On the one hand, considering the actual market value of bio-methane, the additional revenue for this higher energy conservation rate (vs. openair storage) was estimated as 24-26 €/t per 4-months batch. On the other hand, the expected cost increase by silo construction is around 57 €/t. This shows that improved energy conservation should clearly overcome in less than a year the additional cost of silo construction if an efficient ensiling of cattle manure is carried out [START_REF] Franco | Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?[END_REF]. However, since few authors have worked on cattle manure storage and since there may exist a variability among biochemical properties of different manures, e.g. concerning the bacterial populations [START_REF] Manyi-Loh | An Overview of the Control of Bacterial Pathogens in Cattle Manure[END_REF][START_REF] Marañón | Anaerobic mesophilic treatment of cattle manure in an upflow anaerobic sludge blanket reactor with prior pasteurization[END_REF], these conclusions may not be generalized for all types of manure so far.

The use of additives is considered as a resourceful method to ensure a good silage quality. Fermentation stimulants and inhibitors are two of the most important silage additives categories among the wide range of biological and chemical products commercially available. The role of fermentation stimulants is to encourage lactic fermentation either by supply of substrate (e.g. fermentable sugars), lactic acid bacteria (LAB) or enzymes [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. In contrast, inhibitors (acids) are also used to prevent biological activity of the degrading microorganisms by lowering the pH of the feedstock. These two types of silage additives are serving as a basis for most of ensiling research in recent years [START_REF] Herrmann | Effects of ensiling, silage additives and storage period on methane formation of biogas crops[END_REF][START_REF] Haag | Effects of ensiling treatments on lactic acid production and supplementary methane formation of maize and amaranth -An advanced green biorefining approach[END_REF][START_REF] Herrmann | Improving aerobic stability and biogas production of maize silage using silage additives[END_REF][START_REF] Lehtomäki | Biogas Production from Energy Crops and Crop Residues[END_REF][START_REF] Mceniry | Grass for biogas production: The impact of silage fermentation characteristics on methane yield in two contrasting biomethane potential test systems[END_REF][START_REF] Neureiter | Effect of silage preparation on methane yields from whole crop maize silages[END_REF][START_REF] Pakarinen | Storing energy crops for methane production: Effects of solids content and biological additive[END_REF][START_REF] Pakarinen | Evaluation of preservation methods for improving biogas production and enzymatic conversion yields of annual crops[END_REF][START_REF] Vervaeren | Biological ensilage additives as pretreatment for maize to increase the biogas production[END_REF]. Nevertheless, none of these co-storage studies was performed with cattle manure.

In order to assess the effects of additives on the ensiling of cattle manure with straw, laboratory experiments were conducted for up to 4 months. A fermentation stimulant (glucose) and an inhibitor (formic acid) were used as model molecules of co-substrates for ensiling. It is important to underline that glucose was used as a representative molecule of simple sugars that are conventionally consumed during the first steps of ensiling, such as other non-structural compounds (sucrose, fructose, xylose, starch, etc.).

In real applications, glucose should be replaced by a substrate containing easily accessible sugars such as maize, sugar beet pulp or another fermentable sugar-rich substrate. A similar reasoning applies in the case of formic acid, by using low-pH wastes as the one assessed by Lianhua et al. [START_REF] Lianhua | Low-cost additive improved silage quality and anaerobic digestion performance of napiergrass[END_REF]. Besides the evaluation of BMP during storage, the monitoring of fermentative profiles, organic matter losses and potential gas emissions were included in our work. Our objective is to contribute to the improvement of cattle manure management before energy production, which will have a major impact on the ammonia emissions and on the methane yield of agricultural biogas plants.

Material and methods

Feedstocks

Fresh cattle manure (10% of total solids (TS) content) was collected on June 1 st , 2016 from an agricultural site in the Rhône-Alpes region of France (Gaec Béreyziat, Les Teppes, 01340 Béréziat, France) and it was stored at 4 °C before further use. A mixture of fresh cattle manure and wheat straw was prepared at the laboratory and it was tested for ensiling. Wheat straw with 0.10 m maximum length was mixed with cattle manure in order to achieve a final TS content of 19%. The TS content of cattle manure with straw was chosen according to the recommendations of Teixeira Franco et al. [START_REF] Franco | Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?[END_REF] for the optimization of cattle manure storage.

Besides the control assay (i.e. ensiling without silage additives), the following additives were added to cattle manure with straw at the beginning of each experiments (Table 1): formic acid, 8.2% of volatile solids (VS) content; glucose, 16.6%VS and; glucose, 40.2%VS. The amount of each additives was selected through a short-term preliminary screening performed at the laboratory, in which the impact of the additive on the pH evolution was examined [START_REF] Leroux | Recherche d'additif de conservation à l'ensilage de déchets agricoles avant méthanisation[END_REF].

Experimental approach

Laboratory trials were performed in 3.5 L airtight round plastic storage drums. In order to enable biogas output and at the same time minimizing headspace, silos were filled up to 2.55 L with raw material at packing density of 0.7 kg/L, the remaining volume being filled with gravel, using a geotextile membrane to separate it from biomass. Proper plastic lid and rubber ring were used for silo sealing and its airtightness was reinforced with silicone sealant. Then, silos were weighed and placed in a controlled-temperature room at 25±2 °C. Storage duration varied between 7, 15, 30 and 120 days. A total of 16 (4 operating conditions x 4 storage durations) ensiling assays were performed.

Chemical analysis

For each sample time, one silo (per tested condition) was sacrificed. It was opened and weighed. Then, biomass was homogenized and two samples were taken, which followed the experimental procedure illustrated in Figure 2. The first sample was used for direct analyses on the crude material and the other one was mixed with water in order to get two fractions: a water-soluble phase and a particulate phase. This leaching test was performed with a 10:1 water/TS ratio during 2 h under constant bottle rotation. Phase separation was achieved by centrifugation (5000 G; 10 min) followed by 0.7 µm particle size filtration. Finally, the particulate phase was dried at 70 °C until constant weight and ground at 2 mm theoretical length. Crude material/water-soluble and particulate samples were stored at 4 °C and -20 °C, respectively, until analysis.

Crude material was analyzed for its TS/VS content and BMP. For the water-soluble phase, besides TS/VS content, pH, WSC, volatile fatty acids (VFA), chemical oxygen demand (COD), total Kjeldahl nitrogen (TKN) and ammonia nitrogen (NH3-N) fractions were determined. Particulate solid was analyzed for its TS/VS, COD, TKN and cell wall constituents. Feedstocks were not analyzed for COD, TKN and NH3-N content.

TS was measured by oven drying at 105 °C during 24 h and VS was subsequently burned for 2 h at 550 °C. Since TS/VS contents are underestimated due to the loss of volatile compounds during the drying tests [START_REF] Kreuger | Ensiling of crops for biogas production: effects on methane yield and total solids determination[END_REF], the data were corrected according to the volatilization coefficients at 100 °C suggested by Porter and Murray [START_REF] Porter | The volatility of components of grass silage on oven drying and the inter-relationship between dry-matter content estimated by different analytical methods[END_REF]. pH was measured by a Consort C3020 device with a SP10B pH-electrode. WSC, lactic acid and formic acid contents were determined with high performance liquid chromatography (LC Module 1 plus, Waters) equipped with a Supelcogel™ C-610H column (300 x 7.8 mm, Sigma-Aldrich), both refractive index (RID) and UV detectors and operating with H3PO4 0.1%v as solvent (flow rate of 0.5 mL/min). WSC content was estimated as the sum of glucose, xylose, galactose, mannose, arabinose and cellobiose and was determined using the UV detector (210 nm). Lactic acid and formic acid contents were obtained with the RID detector. Acetic, propionic, butyric, valeric and caproic acids content were analyzed by gas chromatography (Shimadzu Corp.) equipped with a HP-FFAP fused silica capillary column (30 m x 0.25 mm, Agilent Technologies), a flame ionization detector and using H2 as carrier gas. Total VFA was calculated as the sum of lactic, formic, acetic, propionic, butyric, valeric and caproic acids. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were analyzed through Van Soest and Wine [START_REF] Van Soest | Use of detergents in the analysis of fibrous feeds IV. Determination of plant cell-wall constituents[END_REF] modified extractions method, based on FD U44-162 standard [START_REF] Afnor | Amendements organiques et supports de culture -Caractérisation de la matière organique par fractionnement biochimique et estimation de sa stabilité biologique[END_REF]. Hemicellulose content was calculated as NDF minus ADF; cellulose as ADF minus ADL and; lignin as equal to ADL. TKN and NH3-N were determined through the procedure described in the NF EN 25663 standard [START_REF] Afnor | 25663: Qualité de l'eau -Dosage de l'azote Kjeldahl -Méthode après minéralisation au sélénium[END_REF]. COD of watersoluble phase was determined through the colorimetric HACH procedure (method 8000). COD of particulate phase was measured by a Walkley and Black [START_REF] Walkley | An examination of the Degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method[END_REF] modified method, based on the NF ISO 14235 international standard [START_REF]ISO 14235: Soil quality -Determination of organic carbon by sulfochromic oxidation[END_REF].

The interest of our experimental procedure is to assess the composition and BMP based on the initial mass of product used, since the weight loss is measured. The results for the chemical analysis will thus be presented in two ways: based on %VSadded or %VSoriginal.

VSadded relates to the organic matter of the sample analyzed. The results based on VSoriginal take into account the loss of volatile solids during storage and allow the study of the results based on the VS of the initial material.

Biochemical methane potential tests

Batch anaerobic digestion tests were conducted in a temperate room at 35 °C using 2L glass reactors. Vessels were filled with 5 gVS of sample, inoculum in way to keep a substrate/inoculum VS ratio of 0.5 and a certain volume of a mineral solution to achieve 60% of the total volume of the vessel. The inoculum used (TS 2.3-3.3%wt; VS 1.5-2.2%wt) was a digested sludge originating from the wastewater treatment plant of La Feyssine, Lyon, France. The sludge used met the criteria for a good inoculum quality (e.g. pH, VFA, NH4 + ) suggested by the international task group on the harmonization of BMP protocols [START_REF] Holliger | Towards a standardization of biomethane potential tests[END_REF]. The mineral solution, which contains essential elements to microbial growth and also gives the solution a buffer able to control any pH adjustments, was prepared according to the recommendations of ISO 11734

international standard [START_REF]Water quality -Evaluation of the "ultimate" anaerobic biodegradability of organic compounds in digested sludge -Method by measurement of the biogas production[END_REF]. Once filled, reactors were purged with a N2/CO2 mixture (80/20%v) for about 5 minutes, sealed and equilibrated at 35 °C. Blanks with only inoculum and mineral solution were performed for each batch series in order to correct the BMP from residual methane production of the inoculum. All tests were performed in triplicates.

Biogas production was determined by pressure measurement using a Digitron precision manometer. Biogas was released when the pressure exceeded 1200 hPa. Gas composition was analyzed using an Agilent 3000 micro gas chromatography with thermal conductivity detector (GC-TCD). Molsieve 5A (14 m length; pore size: 5 Å)

and PoraPlot A (10 m length; 0.320 mm ID) columns were used as stationary phases for GC-TCD, with Argon and Helium as carrier gases, respectively. Biogas production and composition were analyzed at least 8 times during the incubation and BMP was considered achieved when daily vessel overpressure of controls equalized the sample ones. The BMP tests followed the recommendations provided by Holliger et al. [START_REF] Holliger | Towards a standardization of biomethane potential tests[END_REF].

Results and discussion

Feedstock characterization

Chemical properties of feedstocks are gathered in Table 1 and Table 2. Considering the pH value (8.3) and the TS content (19.2%) of fresh cattle manure with wheat straw, AD microflora should have proliferated during storage. However, energy sources of raw material were mostly structural carbohydrates. Indeed, cellulose and hemicellulose contents of cattle manure were estimated as 36.1 and 30.6% of organic matter, respectively, while WSC fraction was negligible. This means that biomass acidification during storage should be preceded by hydrolysis of polymers, as in biogas production process [START_REF] Batstone | The IWA Anaerobic Digestion Model No 1 (ADM1)[END_REF]. Since hydrolysis reactions take several weeks to occur in substantial extent under ensiling conditions [START_REF] Franco | Optimizing storage of a catch crop before biogas production: impact of ensiling and wilting under unsuitable weather conditions[END_REF], fermentation of sugars will occur slowly. In the meantime, hydrogen-consuming reactions may have occurred, together with proteins mineralization and subsequent NH4 + /NH3 equilibrium. Besides, it was detected a rather high VFA content in cattle manure before storage. The fact that high pH and VFA content co-existed in the raw material evidenced that cattle manure had strong basic buffer compounds. Generally speaking, the biochemical properties of cattle manure make it not suitable for ensiling, due to the absence of easily fermentable sugars and the elevated pH.

A proportional increase of TS content was observed with the use of co-substrates. This was expected since additives were used as pure compounds and not in solution. Since the activity of silage bacteria is at least in part vulnerable to changes in TS [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Neal | A model of the anaerobic phase of ensiling[END_REF][START_REF] Pitt | A quantitative model of the ensilage process in lactate silages[END_REF],

this may have influenced the ensiling process. In the case of formic acid addition, the pH of the feedstock was affected as well. Indeed, it decreased to 3.5, which is a considerable lower value than the ones recommended by Kalač [START_REF] Kalač | The required characteristics of ensiled crops used as a feedstock for biogas production: a review[END_REF]

Effects of storage method on biomass preservation

Fermentation profiles

Anaerobic storage of cattle manure with wheat straw addition only (Control) did not promote a biomass acidification in the long-term, Table 2. In the first 7 days of storage, pH of control decreased to 7.1. Then, it constantly increased until the end of the storage period, reaching a value of 8.4 after 4 months. This can be explained by the evolution of VFA concentrations along ensiling duration. Indeed, there was a slight VFA accumulation in cattle manure during the first days of storage, mainly due to the short production of butyric acid. In contrast, the acetate initially present in fresh cattle manure was almost totally lost during the first month of storage. This evidences that the methanogenic activity was already present at the beginning of storage and became predominant between 7 and 15 days of storage, which impeded biomass stabilization.

This was confirmed by one-off composition measures of the gas produced during these experiments (results not shown): CH4/CO2 ratio was already 0.26 after 1 day and 0.61 after 10 days of storage. Furthermore, since cattle manure was successfully ensiled with straw in our previous study [START_REF] Franco | Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?[END_REF], this suggested that methanogens concentration may vary from case to case. In fact, it is known that bacterial populations can be influenced by the characteristics of manure [START_REF] Manyi-Loh | An Overview of the Control of Bacterial Pathogens in Cattle Manure[END_REF] which, according to Marañón et al. [START_REF] Marañón | Anaerobic mesophilic treatment of cattle manure in an upflow anaerobic sludge blanket reactor with prior pasteurization[END_REF], may depend on the type of cattle, animal's diet, as well as, on the time of the year. In any case, silage stabilization was not achieved for control assay and only trace concentrations of organic acids were detected after 4 months of storage.

In addition, structural carbohydrates content regularly decreased for control silage. After 4 months of storage, hemicellulose and cellulose concentrations were 14.6 and 18.7% of original VS, respectively. This represented a degradation of around 50% of the total original hemicellulosic and cellulosic compounds. Since no accumulation of WSC nor VFA was recorded during the same period, major organic and energy losses have occurred. Regarding organic matter, losses ascend to 14.8% after 30 days and 42.0% at the end of the storage. An average of 0.35% of organic matter was lost per day during this period.

Co-ensiling with formic acid improved the conservation of cattle manure, especially for short periods of storage. During the first 30 days, formic acid was fully conserved and pH persisted below 4.0. In parallel, VFA content increased, which was supposed to be partially caused by lactic acid production. This should be a result of acid hydrolysis of hemicelluloses by the existing organic acids in silage [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Dewar | The hydrolysis of grass hemicelluloses during ensilage[END_REF]. As a matter of fact, hemicellulose content of formic acid silage decreased to 29.1%VSoriginal after 30 days, while cellulose fraction was stable. However, between 30 and 120 days of ensiling the fermentation profiles reversed and both formic and lactic acids were consumed. At the same time, other organic acids were produced, notably acetate and butyrate.

Consequently, VFA content decreased from 11.9%VSadded after 30 days to 9.1%VSadded at the end of storage. Due to that and to the fact that the VFA produced were weaker acids than formate and lactate, the pH value increased to 6.9 after 4 months of ensiling.

One hypothesis that can be suggested for the fermentation shift is that the low pH of silage was only delaying the growth of methanogens. This would explain the slow pH increase in the first month of storage. Then, as pH increased, microbial growth became important and formic acid was further degraded into biogas, which also had an impact on the pH. Consequently, lactic acid must have been converted, e.g., into butyric acid by clostridial fermentation, which is a typical mechanism in poor quality silage [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF].

Furthermore, the increased pH should have allowed the enzymatic hydrolysis of structural polymers for prolonged silage periods [START_REF] Dewar | The hydrolysis of grass hemicelluloses during ensilage[END_REF]; and the subsequent fermentation into VFA. This is corroborated by the accentuated decline of structural carbohydrates that was verified in the last 90 days of storage. Indeed, after 4 months of ensiling, hemicellulose content decreased to 23.2% of original VS. Finally, the two different phases of fermentation had an impact on the conservation of organic matter. After 1 month, VS losses were lower than 1%, which ascended to 13.4% at the end of the storage.

Co-ensiling of cattle manure with glucose underwent a strong acidification in the early days of storage. Indeed, pH value of silage was 4.0 for both Glucose 4% and Glucose 10% conditions after 7 days, which was due to the conversion of WSC into lactic acid (Table 2). This shows that the concentration of LAB in the original raw material was significant. Consequently, the lack of easily fermentable sugars probably blocked the conservation of cattle manure through the ensiling process of control trials. Silage acidification slowed down LAB activity but it was not completely stopped. For this reason, WSC continued to be consumed during the remaining ensiling period. After 15 days of storage, lactic acid concentration was around 15%VSadded for both co-silages with glucose addition. After this period, two distinct fermentation pathways were observed for the experiments with glucose. On the one hand, for Glucose 10% condition, WSC was in large excess and lactic fermentation persisted until the end of the storage. As a result, after 4 months, pH value of Glucose 10% silage was 3.7 and its lactate concentration rose to nearly 20%VSadded. On the other hand, WSC of Glucose 4% assay was extinguished between 15 and 30 days of ensiling. Coupled to that, there was a degradation of lactic acid and a significant increase of butyric acid content during this period. This suggests that the acid pH value (4.1) was unable to avoid clostridial activity since there was no more available substrate for lactate production. This clostridial proliferation led to a secondary fermentation that was mainly based on lactic acid consumption for the production of butyrate, carbon dioxide and hydrogen [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF].

Between 30 and 120 days, clostridial fermentation led to full degradation of lactic acid on Glucose 4% silage. Besides butyric acid, other VFA were found, such as caproic and valeric acids (results not shown). Moreover, after 4 months of storage, the pH value of Glucose 4% increased to 5.4. This is explained by the fact that butyrate is a weaker acid that lactate and because this reactional mechanism involves the consumption of two moles of lactic acid for the production of one mole of butyric acid.

Additionally, co-ensiling of cattle manure with glucose enhanced the preservation of hemicellulosic and cellulosic compounds. Regarding Glucose 4% silage, degradation of structural carbohydrates was not significant. For Glucose 10%, around 27% of the sum of initial hemicellulose and cellulose fractions were missing. This degradation should be linked with an acid hydrolysis by the VFA produced during ensiling, as already mentioned by other authors [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Dewar | The hydrolysis of grass hemicelluloses during ensilage[END_REF]. This was not observed in the condition Glucose 4%, since the pH was higher. Nevertheless, the degradation of the structural carbohydrates for Glucose 10% did not led to energy losses since biomass was extremely well conserved under acidic conditions. Therefore the hydrolysis products were likely conserved either under the form of non-structural oligomers, simple sugars or organic acids. Consequently, VS losses were limited to 9.0% and 4.6% after 4 months for Glucose 4% and Glucose 10% assays, respectively. The poor conservation of organic matter for Glucose 4% silage should be connected to the secondary fermentation.

Chemical oxygen demand balance

Effects of ensiling and co-substrates on the COD of crude material and its water-soluble and particulate fractions are exposed in Figure 3. The evolution along ensiling time is related to VSoriginal in order to account for biomass weight loss in the COD balance.

The most significant results were obtained for the ensiling of cattle manure without additives. Before storage, around 93% of the total COD was in the particulate phase. This is linked to earlier statements, evidencing that in cattle manure the majority of the energy sources were hardly bio-accessible. Second, there was a clear decline of particulate COD during the experiments, which is explained by the loss of structural carbohydrates. Furthermore, no accumulation of their degradation products in the watersoluble COD was observed. Thus, global COD in the control (without additives) decreased with ensiling time, which surely led to large methane potential losses.

The use of co-substrates for the ensiling of cattle manure improved the conservation of COD. For this reason, the fluctuations of its composition along duration were minimal, which made it difficult to establish long-term trends in co-ensiling. Regarding formic acid assays, despite the important VS losses recorded for prolonged durations, COD of crude material was stable until 120 days of storage. This can be in part explained by the fact that formic acid has low COD content (0.35 gO2/g), so that its degradation did not cause significant COD decrease. Furthermore, there was an important solubilization of particulate COD in the last 3 months of ensiling. This should correspond to the hydrolysis of structural carbohydrates and subsequent conversion into VFA, as it was seen for the fermentation profiles (subsection 3.2.1). In addition to the solubilization effect and the potential kinetic gain in AD conditions, this may have caused an amplification of carbohydrates accessibility. Error bars represent the standard deviation of triplicates.

(A) (B) (C) (D)
Ensiling of cattle manure with glucose addition had little impact on COD conservation (Figure 3). For Glucose 4%, both particulate and water-soluble fractions of COD were stable until the end of the 4 months. This was expected for the particulate phase, since fibers content did not suffer any significant degradation during storage. However, a degradation of soluble COD should have been recorded between 30 and 120 days of storage, since clostridial fermentation led to hydrogen gas formation. The reasons for this inconsistency remain unclear. Concerning Glucose 10% condition, there was a persistent solubilization of particulate COD along the experiment. This can be attributed to the acid hydrolysis of hemicelluloses by the VFA produced during ensiling.

Total Kjeldahl nitrogen balance

Anaerobic storage of cattle manure with straw (Control) led to constant TKN loss during the experiments, Figure 4. This can be explained through the analysis of TKN fractions along the storage period. On the one hand, there was a decrease of organic nitrogen in both particulate and water-soluble phases after 4 months of about 12% and 39%, respectively. This loss represented 19% of the total organic nitrogen. This suggests that nitrogen mineralization occurred through two successive mechanisms typically found in anaerobic conditions [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Batstone | The IWA Anaerobic Digestion Model No 1 (ADM1)[END_REF]: hydrolysis of proteins into amino acids and subsequent fermentation into ammonia nitrogen. Therefore, NH3-N concentration should increase under such conditions. However, for control assays ammonia content of cattle manure declined with storage time. This can be explained by the fact that pH interruptedly increased to values near the pKa (9.25) of NH4 + /NH3. Therefore, although ammonia was produced, its chemical equilibrium led to ammonia losses through gas emissions. After 4 months, around 23% of original TKN was lost for control experiment. If we hypothesize that the TKN was lost by ammonia emissions, around 8.2LNH3/kgVSoriginal were expected to be released into the atmosphere during the 4 months of control experiments.

Regarding the formic acid tests, 11% of particulate TKN content was degraded in the 4 months of storage. This may be linked with acid hydrolysis in the first 30 days; and due to the proteolytic clostridial activity (for higher pH values) in the last 3 months of ensiling. Nevertheless, since pH was always below 7.0, the mineralized nitrogen was partially conserved as water-soluble ammonia. At the end of formic acid assays, less than 10% of original TKN was lost into the atmosphere. Error bars represent the standard deviation of triplicates.

(A) (B) (C) (D)
For both conditions of cattle manure with glucose addition, no decrease of TKN was recorded until 4 months of ensiling. Yet, as for fermentation profiles, TKN composition of the two glucose experiments evolved differently during storage. In the case of 0.0% 0.5% Glucose 10%, there was no degradation of particular TKN and NH3-N was constant.

This shows that silage was extremely stable with glucose addition and that no secondary fermentation occurred. For Glucose 4%, the same trend was observed in the early days of ensiling. However, in the last 3 months there was a decrease of around 18% concerning the particulate TKN and an increase of around 39% for the soluble TKN.

This indicates that proliferation of proteolytic clostridia occurred for prolonged storage duration. Hence, the amount of co-substrate used in Glucose 4% was not enough to fully stabilize long-term silage of cattle manure with straw.

Effects of storage method on methane potential

Initial conditions

Before storage BMP was 229±8 LSTP/kgVSadded for fresh cattle manure, 250±8

LSTP/kgVSadded for cattle manure + Formic acid, 239±26 LSTP/kgVSadded for cattle manure + Glucose 4% and 276±8 LSTP/kgVSadded for cattle manure + Glucose 10%.

Formic acid and glucose had a clear impact on the BMP of the feedstock. The effect of Glucose 4% addition was not significant (within the BMP accuracy limits). Considering the theoretical BMP of glucose (373 LSTP/kgVSadded) and the amount of co-substrate used, a BMP of 287 LSTP/kgVSadded was expected for Glucose 10% before storage.

Furthermore, glucose contribution to the total BMP should be around 52% in this case.

This evidences that glucose added was fully degradable under anaerobic conditions and that it was responsible for the initial BMP value increase of Glucose 10% mixture. In opposite, the additional methane production of Formic acid feedstock cannot be attributed to the co-substrate, due to its low BMP value (122 LSTP/kgVSadded). In fact, the theoretical BMP of this feedstock mixture should be around 221 LSTP/kgVSadded with a 5% contribution of formic acid for these value. Therefore, it can be pointed out an improvement of the biochemical accessibility of cattle manure after being treated with formic acid (before ensiling). This might be caused by an increase of accessible surface area of biomass, hemicellulose removal and alteration of lignin structure, which are typical features of dilute acid pretreatments for biomass [START_REF] Mosier | Features of promising technologies for pretreatment of lignocellulosic biomass[END_REF]. However, it is worth mentioning that these treatments are performed at high temperatures, which was not the case in this study. 5. In the first 7 days of control assays, both methane potential based on VSadded and VSoriginal were stable. This evidences that neither the energy content nor the biochemical accessibility of cattle manure with straw changed during short-term ensiling, which should be linked with the small fermentation activity and pH decrease that occurred in this period. However, for longer storage durations, the BMP of control was significantly damaged. Indeed, after 30 and 120 days of ensiling, 24% and 67% of original BMP was lost, respectively. This obviously was a consequence of the extensive degradation of structural carbohydrates (without any VFA accumulation), due to the high pH levels.

Co-ensiling with formic acid limited the harmful impact of storage on the methane potential of the raw material. In this case, BMP values were 192-250 LSTP/kgVSadded and 189-250 LSTP/kgVSoriginal during the 4 months. The major variation of methane potential for formic acid silage occurred in the first 7 days of storage. In this period, around 21% of both BMP related on VSadded and VSoriginal was lost. It is important to mention that the energy conservation rates of co-ensiling conditions are related to the initial BMP of the mixture manure/additive and not only to fresh cattle manure. As previously discussed for formic acid assays, organic matter losses were negligible and no noticeable modification of chemical characteristics was observed during the first week. Therefore, this indicates that formic acid addition had an impact on the biodegradability of the biomass in the first days of storage. After that, BMP based on VSoriginal remained constant and global energy losses were limited to 25% at the end of the 4 months. Yet, BMP related to VSadded increased by 14% in the last 3 months of storage. Two conclusions can be deduced from these results. First, there was an increment of biochemical accessibility, which can be attributed to the production of VFA from cell wall constituents. In fact, structural carbohydrates are not fully biodegradable in mesophilic AD conditions [START_REF] Ghosh | Hemicellulose conversion by anaerobic digestion[END_REF]. Therefore, their hydrolysis and fermentation during ensiling may explain the BMP increase in a VSadded basis. Second, this gain of accessibility countered the formic acid loss in the last 90 days, so that BMP related to VSoriginal persisted unaltered until the end of the storage.

(A) (B) BMP was observed until the end of the 4 months. An eventual positive impact on the methane potential could be seen (probably due to the hemicellulose hydrolysis and fermentation), but it was not significant within the precision limits. In contrast, in the remaining 90 days of ensiling, BMP of Glucose 4% progressed differently. For this condition, 13% of the methane potential related to VSoriginal was lost after 4 months.

This BMP damage can be explained by the secondary fermentation that led to lactic acid degradation and nitrogen mineralization.

Conclusions

Ensiling of cattle manure with straw at 19%TS led to extensive methane potential losses. The use of co-substrates enhanced biomass and energy conservation during ensiling. In particular, co-ensiling with glucose (100 g/kg of feedstock) encouraged an extensive lactate production. This allowed biomass stabilization, suppressed ammonia emissions and led to full preservation of methane potential after 4 months. Therefore, co-ensiling with a high easily fermentable sugar content substrate appears be the most resourceful method to optimize cattle manure preservation before biogas production. At full scale application this may include a starch-rich residue, or any other sugar-rich agro product.

Figure 1 -

 1 Figure 1 -Simplified diagram of carbon degradation during ensiling, including microorganisms, substrates and reactions. Efficient energy conservation will occur if the two last reactions are inhibited, especially the methanogenesis.

Figure 2 -

 2 Figure 2 -Flowchart of the experimental procedure, including phase separation and

Figure 3 -

 3 Figure 3 -Evolution of COD related to VSoriginal along ensiling period for: (A) Control; (B) Formic acid; (C) Glucose 4%; (D) Glucose 10%. Feedstocks were not analyzed.

Figure 4 -

 4 Figure 4 -Evolution of TKN related to VSoriginal along ensiling period for: (A) Control; (B) Formic acid; (C) Glucose 4%; (D) Glucose 10%. Feedstocks were not analyzed.

Figure 5 -

 5 Figure 5 -BMP evolution of crude material over storage duration. (A): methane potential based on VSadded; (B): methane potential based on VSoriginal, therefore considering storage losses. BMP for 15 days of ensiling was not determined. Error bars represent the standard deviation of triplicates.

Table 1 -

 1 Ensiling conditions and treatments applied to cattle manure with wheat straw (% of total sample weight)

	Ensiling condition	Molecule	Additive	Content	TS content
	Control	-		-	19.2±0.1
	Formic acid	Formic Acid		2.0	20.4±0.1
	Glucose 4%	Glucose		4.0	22.3±0.5
	Glucose 10%	Glucose		10.0	24.9±1.4

Table 2 -

 2 Chemical properties of biomass over ensiling duration (%VSadded/original).

	for lactate silages

a LA stands for Lactic Acid, AA for Acetic Acid, BA for Butyric Acid, FA for Formic Acid, HEM for Hemicellulose, CEL for Cellulose and, LIG for Lignin; b results based on %VSadded; c results based on %VSoriginal

Acknowledgements

Rúben Teixeira Franco held a doctoral fellowship from the Rhône-Alpes region. This work has been undertaken within the SAM project (Stockage Avant Méthanisation -Storage Before AD) funded by ADEME (# 1506C0038). The authors thank the DEEP laboratory team, including David Lebouil, Hervé Perier-Camby, Nathalie Dumont and Richard Poncet for the given support during the tests. We are grateful to Franck Barra for his permanent availability for discussion and raw material supply. Mathilde Hardier and SUEZ are also acknowledged for the inoculum provided for the BMP tests.


Ruben Teixeira Franco

Pierre Buffière
email: pierre.buffiere@insa-lyon.fr

Rémy Bayard
email: remy.bayard@insa-lyon.fr

Teixeira Rúben
email: ruben.teixeirafranco@insa-lyon.fr

Pierre Franco

Rémy Buffière

Bayard

Optimizing agricultural wastes storage before anaerobic digestion: impact of ensiling on methane potential of lignocellulosic biomass

Keywords: Agricultural wastes, Lignocellulosic biomass, Ensiling, Anaerobic digestion, Biochemical methane potential

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Over the last few years, the interest in energy production through anaerobic digestion (AD) of biomass has been increasing due to environmental concerns and political incentives. This renewable energy technology, which is based on the degradation of complex organic matter into a mixture called biogas (mainly composed of CH4 and CO2), has today more than 17000 operational plants over Europe [1]. Notwithstanding, AD sector is still known for having a quite fragile financial health. Therefore, in the future a focus on energy efficiency of biogas plants will be crucial. This optimization should concern not only the digester, but also the downstream and upstream systems, i.e. the biomass production and its end use.

Biomass storage before AD is an operation that can be potentially optimized. Nowadays, the diversification of biogas sources is quite wide, since energy can be recovered from almost all types of organic waste, fodder or catch/energy crops. Despite the need for a continuous supply of biogas throughout the year, some of these agricultural wastes or crops are seasonally produced. This aspect leads to storage requirements, which in some cases can be of extended durations. Among the preservation technologies that exist today, ensiling appears to be the logical choice to storage biomass before AD, since it might minimize weight and energy losses.

Ensiling provides a four-step biochemical process based on bacterial fermentation to prevent further degradation. After filling and sealing the silo, an initial aerobic period will occur, in which the oxygen trapped in the system will be consumed for biomass respiration. Once oxygen has been consumed, microorganisms capable of anaerobic growth will compete for available matter and, in suitable conditions, the pH will decrease to approximately 4. Then, maintaining anaerobic conditions and a relatively low pH, minimal enzymatic and microbial activity will occur until feed-out. After unloading the silo for bio-digester feeding, biomass enters once again into aerobic environment. Thereupon, aerobic microorganisms are reactivated, which may spoil the silage.

Given the diversification of critical parameters that influence ensiling and the types of biomass that may need to be stored before AD, ensiling research is still in its early days. Indeed, a recent work [START_REF] Franco | Ensiling for biogas production: critical parameters[END_REF], which reviews the critical parameters on ensiling for biogas, suggests that current research is mostly undergone with easily biodegradable biomass. Consequently, other types of feedstock, such as agricultural wastes are being left behind. In order to fill this gap, we have studied a real case of lignocellulosic agriculture waste during six months, using different storage approaches. Furthermore, the originality of this work lies in the multi-phase study of methane potential, which allows a deeper understanding about its evolution during storage.

Materials and methods

Raw materials

The substrate used was a sample of a catch crop harvested on 27 May 2015 and used as fodder and agricultural biogas plant feedstock in the Rhône-Alpes region of France (Gaec Béreyziat, Les Teppes, 01340 Béréziat, France). Biomass was composed by a mixture of triticale (50% of seed mixture), peas (30% of seed mixture), vicia (10% of seed mixture) and fodder radish (10% of seed mixture) and was chopped at 4cm maximum length at harvesting. Samples were stored at 4°C before further use.

Storage approach

Storage assays were conducted at laboratory scale to evaluate the impact of ensiling and its dry matter content over time. To evaluate ensiling impact, an aerobic control storage test was also performed. To assess different dry matter content a 6-h wilting was performed outdoors. Good weather conditions were recorded during wilting period: no rainfall occurred and the temperature ranged between 17-24°C. All storage tests were performed in 3.5L airtight round plastic storage drums modified to allow the gas to escape. In order to enable the output of gas produced and at the same time minimizing headspace, silos were filled up to 2.55L with raw material at packing density of 0.7kg/L, the remaining volume being filled with gravel, using a geotextile membrane to separate it from biomass. Silo sealing was different depending on the storage method tested. For ensiling assays, proper plastic lid and rubber ring were used and its airtightness was reinforced with silicone sealant. For aerobic storage purposes no cover was used and silo was left air-open. Once sealed, silos were stored in a temperate room at 25°C for a defined period of days. Storage duration varied between 3, 14, 30, 90 and 180 days.

Chemical analysis

At the end of storage duration, reactors were open and weighed, biomass was homogenized and two samples were taken. One was used for direct analyses on the crude material and other one was leached with water to obtain and analyze watersoluble and particulate phases. Leaching was performed with a 10:1 water/dry mater ratio during 2h under constant bottle rotation. Phase separation was achieved by centrifugation followed by 0.7µm particle size filtration. Finally, particulate was dried at 70°C until constant weight and ground at 2mm theoretical length. Crude material/water-soluble and particulate samples were stored at 4°C and -20°C, respectively, until use.

Chemical analysis performed depended on the sample studied. Crude material was analyzed for its Total Solids (TS), Volatile Solids (VS) content and biochemical methane potential (BMP). For the water-soluble phase, besides TS/VS content and BMP, pH, water-soluble carbohydrates (WSC), volatile fatty acids (VFA), ammoniacal nitrogen (NH3-N) and total Kjeldahl nitrogen (TKN) contents were determined. Particulate solid was analyzed for its TS/VS, TKN and cell wall constituents.

TS was measured by oven drying at 105°C during 24h and VS was subsequently determined by combustion in a muffle oven for 2h at 550°C. Since TS/VS contents are underestimated due to volatile compounds loss lost during procedure [START_REF] Kreuger | Ensiling of crops for biogas production: effects on methane yield and total solids determination[END_REF], values were corrected using an equation suggested by [START_REF] Porter | The volatility of components of grass silage on oven drying and the inter-relationship between drymatter content estimated by different analytical methods[END_REF] [START_REF] Porter | The volatility of components of grass silage on oven drying and the inter-relationship between drymatter content estimated by different analytical methods[END_REF]. pH was measured by a Consort C3020 device with a SP10B pH-electrode. VFA and WSC contents were determined with high performance liquid chromatography (LC Module 1 plus, Waters) equipped with a Supelcogel™ C-610H column (300 x 7.8 mm, Sigma-Aldrich), both refractive index (RID) and UV detectors and operating with H3PO4 0.1%v as solvent (flow rate of 0.5mL/min). WSC content was estimated as the sum of glucose, xylose, galactose, mannose, arabinose and cellobiose and was determined using the UV detector (210nm). Total VFA was calculated as the sum of lactic, formic, acetic, propionic, butyric, valeric and caproic acid contents obtained with the RID detector. Cell wall constituentscellulose (CEL), hemicellulose (HEM) and lignin (LIG)were analyzed through Van Soest modified extractions method [START_REF] Van Soest | Use of detergents in the analysis of fibrous feeds IV. Determination of plant cell-wall constituents[END_REF], based on XP U44-162 French standard. TKN and NH3-N were determined through the procedure described in the NF EN 25663 French standard.

Most of the results for the chemical analysis will be presented in at least one of two ways: based on %VSadded or %VSoriginal. VSadded relates to the organic matter of the sample analyzed. On the other hand, results based on VSoriginal take into account the loss of volatile solids during storage and allows to study the evolution of the results based on the VS of the raw material.

Biochemical methane potential tests

Batch anaerobic digestion tests were performed for crude material and water-soluble phase samples, which enabled to follow the evolution of BMP of each chemical compartment during storage.

Tests were conducted in a temperate room at 35°C using glass vessels of 2L for crude material and 0.1L for water-soluble phase. Vessels were filled with about 5g of sample VS, inoculum in way to keep a substrate/inoculum VS ratio of 0.3-0.8, and a certain volume of a mineral solution to achieve 60% of the total volume of the vessel. The inoculum used (TS 2.4-2.9%wt; VS 1.7-2.0%wt) was a digested sludge originating from the wastewater treatment plant of La Feyssine, Lyon, France. The mineral solution, which contains essential elements to microbial growth and also gives the solution a buffer able to control any pH adjustments, was prepared according to the recommendations of ISO 11734:1995 standard. Once filled, reactors were purged with a N2/CO2 mixture (80/20 %v) for about 5 minutes and then sealed and equilibrated at 35°C.

In order to subtract the volume of methane produced by the inoculum from the one originated from the sample, controls with only inoculum and mineral solution were performed for each batch series. All tests were performed in triplicates.

Biogas production was followed by the overpressure generated inside of the reactors using a Digitron precision manometer, being its composition determined using an Agilent 3000 micro gas chromatography with thermal conductivity detector (GC-TCD). Molsieve 5A (14 m length; pore size: 5 Å) and PoraPlotA (10m length; 0.320 mm ID) columns were used as stationary phases for GC-TCD, with Argon and Helium as carrier gases, respectively. Biogas production and composition were analyzed at least 10 times during batch tests and BMP was considered achieved when daily vessel overpressure of controls equalized the sample ones.

Results and discussion

Fresh material chemical characteristics

Feedstock used for storage purposes, which is characterized in Table 1, can be briefly described as hardly biodegradable biomass for ensiling. Two different reasons can support this fact: -Negligible presence of water-soluble carbohydrates (WSC) in raw materials. Indeed, it is known that silage will be favored by high lactic acid bacteria (LAB) activity, which will acidify the biomass and lead to better BMP preservation. Since LAB ferment naturally occurring sugars in the crop to produce a mixture of acids [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF], LAB activity and pH decrease will be directly linked to WSC content of raw material. Therefore, in absence of WSC or any other accessible source of sugars, a slow decrease of pH and a poorly ensiled material must be expected.

-Hard accessible (hemi-) cellulose content, due to a strong lignin protection. Cellulose and hemicellulose are the main sources of energy in AD, since they can represent more than half of biomass organic matter and they can be directly fermented in sugars upon hydrolysis. Even if it has already been shown that at least hemicellulose can in part be accessed and degraded during ensiling [START_REF] Pakarinen | Evaluation of preservation methods for improving biogas production and enzymatic conversion yields of annual crops[END_REF], this must always be preceded by lignin depolymerization or structure modification. This step may require prolonged storage durations to take place. Thereupon, with high lignin content crops and considering ensiling soft operating conditions, where temperature or fungi effect may be negligible, hemicellulose will not be readily accessible in large quantities to promptly acidify and stabilize silage. Regarding wilting impact, a 6h period allowed the increase of TS content from 18% to 23%. Besides moisture decrease, wilting favored a decrease of total VFA content in biomass. This should be related either with their evaporation or degradation and may have an important impact on biomass energy content. Finally, wilting should not impact the fibers content due to its short dry period and low temperature. Differences found in results, especially for hemicellulose, may be explained by the weak accuracy attributed to the fibers analysis when testing not perfectly homogenous samples.

Impact of storage on biomass fermentation

Effects of storage conditions and duration on silage quality are summarized in Table 2.

Aerobic storage (TS18%A) led to silage spoilage from only 3 days of storage. Since LAB never managed to impose itself and control fermentation, pH remained in neutral zone. Then, with open air environment, aerobic microorganisms were able to freely proliferate. This aerobic activity resulted in several organic losses: more than 10% after 3 days, reaching almost 75% after 180 days. Part of these losses were related to structural carbohydrates degradation, since more than 85% of cellulose content was lost after 180 days of storage. Constant deterioration of NTK content along storage time also indicates huge losses and modifications of organic matter for aerobic conditions. Concerning ensiling, non-negligible VS losses occurred, especially during the first month of storage. Wilting (TS23%E) was not effective on preventing organic matter deterioration, leading to around 20% of VS losses after 6 months of storage. Unlike, ensiled fresh material (TS18%E) restricted organic matter losses on 10% for the same storage duration. Organic matter losses can be in part explained by restricted acidification of biomass. Indeed, it is well established that if a stable, low pH silage is not achieved, clostridial activity will be encouraged and a secondary fermentation will occur, resulting in organic matter and energy losses [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. For both ensiling conditions, the necessary acidity for efficient silage, the so-called critical pH value, was never achieved (4.10-4.35 for the TS range used [START_REF] Kalač | The required characteristics of ensiled crops used as a feedstock for biogas production: a review[END_REF]). This suggests a major importance of the content of WSC or another source of readily available carbohydrates on the first moments of ensiling. Their presence or not will define the quantity of available substrate for LAB activity and then the degree and rate of acidification and biomass stabilization. Moreover, higher VS losses of wilted material may be related to a quite slower acidification than fresh biomass storage. In fact, wilted material took more than a month to get a low stable pH while fresh biomass took only 3 days to achieve a pH around 5. Therefore, it can be suggested that the faster the acidification will be, the lower the subsequent biodegradation and VS losses.

To get a deeper knowledge about the acidification mechanisms and their relation with the organic matter losses, an evaluation of the major fermentation products should be made. In these tests, similar patterns over storage time were recorded for both ensiling conditions. In the early days of ensiling, fermentation was controlled by LAB, as mainly lactic and acetic acid were produced. The presence of both compounds suggest equally homoand heterofermentative LAB activity. Then, somewhere between 3 and 15 days, lactic acid began to be consumed and butyrate to be produced. This indicates that secondary fermentation occurred trough undesired clostridial activity. In fact, clostridial fermentation is mainly based on sugars and lactic acid consumption as energy source via similar pathways, producing not only butyric acid but also carbon dioxide and hydrogen [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. This will result on VS losses and more important energy losses, since hydrogen will escape to the environment. Furthermore, secondary fermentation is associated with losses of acidity, as it can be confirmed by pH increase from 3 to 15 days of ensiling. This is explained by the fact that butyric acid is a much weaker acid than lactic acid and since only one mole of butyrate is produced from two moles of lactate [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF].

Increasing NH3-N contents along storage were observed for ensiling. This can be explained by proteolytic clostridia activity, which will be favored by perturbations on silage acidification. The proteolytic clostridia can selectively ferment amino acids and amines mainly into ammonia and carbon dioxide. Besides nutrients spoilage, proteolytic clostridia may have an impact on energy losses. In fact, although ammonia should be present in the form of soluble ammonium ions at current pH and temperature values, its liquid-gas equilibrium may allow a continuous slight fraction to pass in the gas phase and that subsequently to escape into the environment. This idea can be corroborated while analyzing TKN evolution along ensiling duration. This element shows a clear decline over storage time when related to the original VS, reaching around 25% of relative losses after 6 months. This shows that part of organic nitrogen or ammonia fraction was lost into the atmosphere, which may result in BMP losses.

From 15 days of storage and until the end of the tests, butyric acid and ammonia-nitrogen increase, and acetic acid remained stable, which strongly suggest a clostridial activity.

Regarding moisture impact on ensiling evolution, results showed that fermentation was not more restricted for all microorganisms in wilted crop. Indeed, while lactic acid production was less extensive for higher TS ensiled biomass, butyric acid contents and subsequently saccharolytic clostridial activity were higher. Concerning proteolytic clostridia activity, no difference was found depending on the ensiling condition. In contrast, several authors [START_REF] Borreani | Fermentative profiles of field pea (Pisum sativum), faba bean (Vicia faba) and white lupin (Lupinus albus) silages as affected by wilting and inoculation[END_REF][START_REF] Nash | Partial wilting of grass crops for silage. 2. Experimental Silages[END_REF][START_REF] Mahmoud | Factors affecting the microbial and chemical composition of silage: IV. Effect of wilting on maize silage[END_REF][START_REF] Wilkinson | A laboratory evaluation of comfrey (Symphytum officinale L.) as a forage crop for ensilage[END_REF] have shown that higher TS contents delay bacterial growth, especially clostridia due to their particularly sensitivity to water content, leading to a more restricted and well succeeded fermentation. These contradictory results may be explained through differences on readily biodegradable carbohydrates availability in the beginning of ensiling among the tests. For instance, Borreani et al. [START_REF] Borreani | Fermentative profiles of field pea (Pisum sativum), faba bean (Vicia faba) and white lupin (Lupinus albus) silages as affected by wilting and inoculation[END_REF] tested crops with high WSC contents, meaning that even if lower LAB activity was recorded, high carbohydrates content allowed a strong biomass acidification. At the same time, in our tests, as very low WSC content was present, LAB activity was dependent on the activity of other microorganisms, such as enzymes that allow, for instance, hemicellulose hydrolysis. Since it seems fair to assume that these microorganisms will also have more restricted activity at higher TS contents, less substrate will be available to LAB. Thereupon, acidification will be less effective and conditions will be more suitable for clostridia proliferation. In brief, these results suggest that, for the TS range studied, WSC availability will be more vital to avoid clostridial activity than wilting.

As a final point, despite the lack of WSC in raw materials, biomass is acidified in both ensiling conditions, reaching up to 16% of VFA in the organic biomass. The evolution of carbohydrate sources over storage duration give us hints about the origin of substrates that were acidified. Looking at the evolution of the WSC, there is always a glimmer of these compounds along silage, indicating that it was continuously produced and consumed. With regard to the structural carbohydrates in biomass, there is a degradation tendency of not only the hemicellulose but also cellulose over the 6 months of ensiling. This suggests that in case of absence of WSC, anaerobic microorganisms will get access to sources of structural carbohydrates by hydrolysis even when they are protected by high lignin content. However, in such cases the acidification will be less effective, due to difficulties of accessibility of these compounds and the time required for their partial hydrolysis. Finally, breakdown of cellulose was much more evident in the wilted silage, explaining the source of the largest losses VS during storage. storage (TS18%A) (Figure 1). Increasing values of BMP based on VSadded for silages suggest that ensiling may enhance bio-chemical accessibility of biomass. It can also be observed that remaining organic matter of TS18%E have higher methane potentials than other conditions regardless of storage duration.

Since BMP based on VSadded follow similar trends of VS losses according to the storage conditions, methane potentials based on the original organic matter were even more divergent among the studied conditions (Figure 1). This reflects the major importance of VS loss in determining the preservation of BMP, as it was stated in a previous work [START_REF] Pakarinen | Storing energy crops for methane production: Effects of solids content and biological additive[END_REF]. Therefore, while considering VS losses during storage, results evidence a major advantage in using ensiled fresh material than wilted silage or open air storage. Part of TS23%E BMP losses seem to be related with the wilting process itself, once before storage wilted biomass had already less 13% of BMP than the fresh one. Moreover, even after 6 months of ensiling, 18%TS biomass presented similar BMP values based on VSoriginal to the raw material (291-279 L/kgVSoriginal before and after 6 months of ensiling, respectively). Therefore, it can be stated that, even if a poorly ensilable biomass is used, conservation of its whole energy content for prolonged storages can be achieved if suitable ensiling conditions are used. The reason for that may reside on the fact that gains in bio-chemical accessibility will compensate organic matter losses during storage.

Solid/liquid fractions

BMP multi-phase analysis over storage duration (Figure 2) present distinct results depending on the storage method used. For open-air storage, particulate phase of BMP was constantly predominant, representing 89-96% of the total methane potential. This may be explained by the fact that water-soluble phase is only a transition step until VS be completely degraded under aerobic environment. Concerning ensiling, there is a clear trend of increasing water-soluble BMP phase during storage. Both ensiling condition exhibit the same behavior, with the water-soluble phase to range, depending on the storage duration, between 7-38% of the total BMP for fresh ensiled material and 12-40% for wilted silage. This suggest positive and negative aspects. On one hand, this means that the more easily biodegradable fraction will increase over storage, which normally will have a positive effect on methane production kinetics. As a result, for prolonged storage, optimization of AD retention time for this feedstock may be achieved. On the other hand, the increasing probability of BMP losses through effluent production over storage duration can be pointed as one drawback. Indeed, authors agree that liquid effluent or seepage losses will happen when biomass is ensiled too wet, due to high silo compaction rates [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Egg | Feedstock storage, handling and processing[END_REF]. However, when wilting is harmful to the crop as in the present tests, higher TS content may not be the solution for this issue. Without a doubt, it may be preferable to ensile biomass with low TS content, with one single condition: that potential liquid effluent is recovered and used as feedstock for AD. In the latter one, dry matter and energy losses may occur if effluent is not promptly introduced in AD digester. To avoid that, additional installation for effluent collection and its transportation until the digester should be anticipated.

Conclusions

Ensiling was proven to be the logical choice for biomass preservation before biogas production. Lack of accessible sources of carbohydrates hindered acidification, allowing undesired secondary fermentation, which led to non-negligible organic matter losses. This outcome had more impact for higher dry matter biomass, suggesting that wilting is an ineffective technique for this type of crop. Despite the use of a poorly ensilable biomass, fresh raw material at lower VS content conserved its methane potential along 6 months of ensiling. This suggests that for this condition, gains in bio-chemical accessibility will neutralize organic matter losses during storage. Finally, water-soluble phase of methane potential will increase with the storage duration. Although this may favor methane production kinetics, higher losses of energy content may occur in case of liquid seepage. Therefore, effluent production should be avoided whenever possible or collected and immediately added to the anaerobic digester.

3. 3 .Figure 1 -

 31 Figure 1 -Global BMP evolution over storage duration. A: methane potential based on VSadded; B: methane potential based on VSoriginal, therefore considering storage losses. TS18%E represents the biomass ensiling with original TS content; TS23%E concerns the ensiling of 6h-wilted biomass and; TS18%A symbolizes the open-air storage of the original raw material

Figure 2 -

 2 Figure 2 -BMP multi-phase analysis for the different conditions along storage duration. TS18%E represents the biomass ensiling with original TS content; TS23%E concerns the ensiling of 6h-wilted biomass and; TS18%A symbolizes the open-air storage of the original raw material

Table 1 -

 1 Chemical composition of initial and wilted biomass

		Initial	6h-wilted
	pH	7.20	7.23
	TS a	18.2	23.4
	VS a	16.2	21.0
	WSC b	0.15	0.15
	VFA b	4.0	2.1
	HEM b	13.2	16.4
	CEL b	40.2	39.6
	LIG b	18.2	16.8
	Results presented as a % of total sample weight; b %VS

Table 2 -

 2 Chemical characteristics of biomass over storage time (%VSadded/original unless otherwise specified) TS18%E represents the biomass ensiling with original TS content; TS23%E concerns the ensiling of 6h-wilted biomass and; TS18%A symbolizes the open-air storage of the original raw material b LA stands for Lactic Acid, AA for Acetic Acid and BA for Butyric Acid; c results based on %VSadded; d results based on %VSoriginal

	Condition a	Storage duration (days)	VS losses	pH	LA c	AA c	BA c	Chemical characteristics b VFA c WSC c NH3-N (%TKN)	TKN d	HEM d	CEL d	LIG d
		3	0.4	5.02	4.6	3.8	0.0	14.1	0.5	10.7	2.8	13.7	36.1	10.2
		15	0.8	5.22	2.5	4.2	3.1	14.0	0.4	23.2	2.0	12.6	35.3	7.5
	TS18%E	30	5.5	5.37	0.1	5.9	4.4	15.6	0.2	37.7	2.4	11.5	35.8	7.6
		90	8.5	5.41	0.1	4.9	5.4	14.9	0.2	44.1	2.3	8.7	30.7	10.5
		180	9.8	5.25	0.0	5.6	6.2	16.5	0.1	46.0	2.2	10.8	33.9	10.5
		3	0.7	5.87	2.8	3.8	0.0	9.5	0.2	10.0	3.2	16.6	34.4	12.5
		15	7.4	5.97	0.7	4.9	3.1	11.4	0.2	25.8	2.7	13.4	31.2	10.5
	TS23%E	30	21.7	5.54	0.1	8.8	3.5	14.5	0.1	38.0	2.4	11.3	26.7	8.8
		90	20.0	5.22	0.0	9.3	3.7	16.1	0.1	42.1	2.4	7.7	25.2	10.0
		180	19.8	5.39	0.0	8.9	4.0	17.2	0.0	46.3	2.2	11.1	26.5	9.7
		3	14.1	6.66	1.2	2.4	0.0	6.5	0.2	6.5	2.2	14.3	32.3	14.7
		15	32.4	8.08	0.0	1.1	0.7	2.2	0.1	20.7	2.3	13.1	26.3	10.6
	TS18%A	30	44.3	8.18	0.0	2.5	0.9	5.6	0.0	25.0	1.8	8.7	21.0	8.9
		90	68.8	9.12	0.0	0.1	0.0	0.3	0.1	1.7	0.9	3.5	8.9	8.4
		180	78.6	10.1	0.0	0.0	0.0	0.1	0.2	1.7	0.7	2.1	4.6	5.5

a

Acknowledgements

Rúben Teixeira Franco held a doctoral fellowship from the Rhône-Alpes region. The authors thank the DEEP laboratory team, including David Lebouil, Hervé Perier-Camby, Nathalie Dumont and Richard Poncet for the given support during the tests. We are grateful to Franck Barra for his permanent availability for discussion and raw material supply. Mathilde Hardier and SUEZ are also acknowledge for the inoculum provided for the BMP tests.


Ruben Teixeira Franco

Pierre Buffière
email: pierre.buffiere@insa-lyon.fr

Rémy Bayard
email: remy.bayard@insa-lyon.fr

Teixeira Rúben
email: ruben.teixeirafranco@insa-lyon.fr

Pierre Franco

Rémy Buffière

Bayard

How to preserve the energy potential of organic residues during storage? Focus on anaerobic digestion

Keywords: Cattle manure, Catch crop, Storage, Ensiling, Anaerobic digestion, Methane potential

This work presents a set of long-term storage assays at laboratory scale for two different types of catch crops and cattle manures. Comparison between the impact of ensiling and open-air storage techniques on organic matter and energy preservation was performed. Effects of co-ensiling with cattle manure and several additives on silage quality were assessed as well. Aerobic storage led to methane potential losses of more than 80% after 3 months of storage for catch crop assays and around 74% for cattle manure after 4 months. Higher energy recovery rates were obtained after ensiling, strongly depending on the nature of the organic residue used. For both catch crops, at least 96% of methane potential was preserved after 3 months. In contrast, single-handedly cattle manure lost 46% of its methane potential during long-term ensiling. Conservation of cattle manure was successfully enhanced through co-ensiling with fermentation stimulants and inhibitors. The best storage performance was obtained while combining cattle manure with wheat straw and glucose at high concentrations, for which methane potential was fully conserved even after 4 months of co-ensiling. These results highlight a major advantage in using ensiling rather than open-air storage for these organic residues. Moreover, the use of precursors of organic acids as cosubstrates improved silage quality of non-adapted biomass for ensiling, such as cattle manure. This work contributes to the optimization of biomass preservation before anaerobic digestion, which will have a major impact on the methane yield of agricultural biogas plants.

Introduction

Anaerobic digestion (AD) is accepted worldwide as a promising energy production technology for a green and sustainable future. This process is based on the biochemical conversion of complex organic matter under anaerobic conditions to a gaseous mixture mainly composed of CH4 and CO2, called biogas. Today, there are more than 17 000 operational plants over Europe [1] producing a biogas capable of generate heat, electricity or vehicle fuel.

Since energy can be recovered from nearly all types of organic matter, there is a wide diversity of feedstock that is used for biogas production. However, contrary to the need for a continuous supply of biogas throughout the year, some of these agricultural residues or crops are seasonally produced. Therefore, seasonal raw materials have to be stored for long periods before AD, which may have a strong impact on the energy potential of biomass.

Open air-storage and ensiling are two methods commonly used for biomass conservation before AD. The first one is mostly applied for agricultural wastes, due to the simplicity and low cost of the operation. However, open-air storage facilities are important source of ammonia and odor emissions [START_REF] Berg | Mitigation of methane emissions during manure storage[END_REF] and should lead to substantial energy losses. These drawbacks can be mitigated if an efficient ensiling is carried out. According to Herrmann et al. [START_REF] Herrmann | Effects of ensiling, silage additives and storage period on methane formation of biogas crops[END_REF], ensiling lead to full conservation of biochemical methane potential (BMP) of specific crops even after 1 year.

Ensiling is an acidification-based process typically divided in four phases [START_REF] Egg | Feedstock storage, handling and processing, Biomass and Bioenergy[END_REF][START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Franco | Ensiling for biogas production: Critical parameters. A review[END_REF][START_REF] Weinberg | Engineering aspects of ensiling[END_REF]. Right after filling and sealing the silo, biomass respiration occurs due to the presence of oxygen trapped in the system. Once oxygen has been depleted, ensiling passes to an anaerobic fermentation phase. If the conditions are suitable, lactic acid bacteria (LAB) will produce lactate from water-soluble carbohydrates (WSC) for several weeks, decreasing the pH to around 4.0. Maintaining anaerobic conditions and a relatively low pH, minimal enzymatic and microbial activity occur until feedout. After unloading the silo for AD feeding, biomass enters once again into aerobic environment, which may spoil the silage.

Although the fact that ensiling is pointed out as the logical choice to store wet biomass, to the best of our knowledge, there are currently no studies aiming the comparison between the impacts of open-air storage and ensiling on the conservation of the energy potential of different raw materials. Moreover, ensiling is neither been applied nor studied by researchers for some agricultural wastes as manure, mainly due to its unsuitable chemical characteristics for ensiling.

This work brings together a compilation of longterm storage assays at laboratory scale for two different types of catch crops and cattle manures. First, a comparison between open-air storage and ensiling was established during at least 3 months at laboratory scale. Furthermore, long-term assays of co-ensiling of cattle manure with several additives were performed. Finally, this work contributes to the optimization of biomass preservation before AD, which will have a major impact on the methane yield of agricultural biogas plants.

Materials and methods

Feedstocks

Two different types of catch crop and fresh cattle manure, which were collected on an agricultural site in the Rhône-Alpes region of France (Gaec Béreyziat, Les Teppes, 01340 Béréziat, France), were used as raw materials. Catch crop 1 (or "winter" catch crop -CC1) was a mixture of triticale, peas, vicia and fodder radish, and it was chopped at 4 cm maximum length at harvesting. Catch crop 2 (or "summer" catch crop -CC2) was a mixture of sunflower, sorghum, peas, vicia and Trifolium alexandrinum, and it was chopped to theoretical particle size of 8 mm before use. Cattle manure 1 (CM1) and Cattle manure 2 (CM2) were collected from the same site but on different seasons of the year. Samples were stored at 4°C before further use.

Catch crop 1, Catch crop 2 and Cattle manure 1 were tested for both open-air storage and ensiling. Besides that, co-ensiling assays were performed for both cattle manures with the following treatments: -Cattle manure 1: wheat straw (CM1+WS) -Cattle manure 2: wheat straw (CM2+WS); wheat straw and formic acid (20g/kg) (CM2+WS+FA); wheat straw and glucose (100g/kg) (CM2+WS+G)

Description of feedstocks used in storage experiments are summarized in Table 1.

Storage approach

Laboratory trials were performed in 3.5 L airtight round plastic storage drums. In order to enable the output of the gas produced and at the same time minimizing headspace, silos were filled up to 2.55 L with raw material at packing density of 0.7 kg/L, the remaining volume being filled with gravel, using a geotextile membrane to separate it from biomass.

Silo sealing was different depending on the storage method tested. For ensiling assays, proper plastic lid and rubber ring were used and its airtightness was reinforced with silicone sealant. For aerobic storage purposes no cover was used and silo was left airopen. Then, silos were weighed and placed in a controlled-temperature room at 25±2 °C. Storage duration was 3 months for catch crop assays and 4 months while using cattle manure.

Chemical analysis

At the end of storage, reactors were opened and weighed, biomass was homogenized and two samples were taken. The first one was used for direct analyses on the crude material: total solids (TS) content, volatile solids (VS) content and biochemical methane potential (BMP). The other one was mixed with water in order to measure the pH. This leaching test was performed with a 10:1 water/dry mater ratio during 2 h under constant bottle rotation. Identical sampling procedure and analysis were performed for biomass prior to storage. TS content was measured by oven drying at 105 °C during 24 h and VS was subsequently burned for 2 h at 550 °C. Since TS/VS contents are underestimated due to the loss of volatile compounds during the drying tests [START_REF] Kreuger | Ensiling of crops for biogas production: effects on methane yield and total solids determination[END_REF], the measures were corrected using the volatilization coefficients at 100 °C suggested by Porter and Murray [START_REF] Porter | The volatility of components of grass silage on oven drying and the inter-relationship between drymatter content estimated by different analytical methods[END_REF]. pH was measured by a Consort C3020 device with a SP10B pH-electrode. BMP tests were conducted in a temperate room at 35 °C using glass vessels of 2 L. Vessels were filled with 5 g VS of sample, inoculum in way to keep a substrate/inoculum VS ratio of 0.5 and a certain volume of a mineral solution to achieve 60% of the total volume of the vessel. The inoculum used (TS 2.3-3.3%wt; VS 1.5-2.2%wt) was a digested sludge originating from the wastewater treatment plant of La Feyssine, Lyon, France. The mineral solution, which contains essential elements to microbial growth and also gives the solution a buffer able to control any pH adjustments, was prepared according to the recommendations of ISO 11734:1995 standard. Once filled, reactors were purged with a N2/CO2 mixture (80/20%v) for about 5 minutes, sealed and equilibrated at 35 °C. Blanks with only inoculum and mineral solution were performed for each batch series in order to correct the BMP from residual methane production of the inoculum. All tests were performed in triplicates. Biogas production was determined by pressure measurement using a Digitron precision manometer. Biogas was released when the pressure exceeded 1200 hPa. Gas composition was analyzed using an Agilent 3000 micro gas chromatography with thermal conductivity detector (GC-TCD). Molsieve 5A (14 m length; pore size: 5 Å) and PoraPlot A (10 m length; 0.320 mm ID) columns were used as stationary phases for GC-TCD, with Argon and Helium as carrier gases, respectively. Biogas production and composition were analyzed at least 7 times during the incubation and BMP was considered achieved when daily vessel overpressure of controls equalized the sample ones. The BMP tests followed the recommendations provided by Holliger et al. [START_REF] Holliger | Towards a standardization of biomethane potential tests[END_REF].

Results and discussion

Effects of storage method

pH values

Lower pH values were obtained using ensiling as storage method, Fig. 1. This was especially marked for catch crop silages. Indeed, Catch crop 1 had pH values of 5.4 and 9.1 after 3 months of ensiling and aerobic storage, respectively. In parallel, pH of Catch crop 2 after storage period was 5.6 for ensiling and 9.6 for aerobic treatment. This suggests that catch crops passed through substantial fermentation and accumulation of organic acids during ensiling. This was expected, since these raw materials typically have high content of easily accessible carbohydrates, which are used as substrates for LAB fermentation.

However, pH values of silages were somewhat higher than those obtained by other authors for efficient lactate silages [START_REF] Herrmann | Effects of ensiling, silage additives and storage period on methane formation of biogas crops[END_REF][START_REF] Borreani | Fermentative profiles of field pea (Pisum sativum), faba bean (Vicia faba) and white lupin (Lupinus albus) silages as affected by wilting and inoculation[END_REF][START_REF] Desta | Ensiling Characteristics, structural and nonstructural carbohydrate composition and Enzymatic Digestibility of Napier grass Ensiled with Additives[END_REF][START_REF] Haag | Effects of ensiling treatments on lactic acid production and supplementary methane formation of maize and amaranth -An advanced green biorefining approach[END_REF][START_REF] Herrmann | Improving aerobic stability and biogas production of maize silage using silage additives[END_REF][START_REF] Kung | The Effect of Lactobacillus buchneri and Other Additives on the Fermentation and Aerobic Stability of Barley Silage[END_REF][START_REF] Lehtomäki | Biogas Production from Energy Crops and Crop Residues[END_REF][START_REF] Menardo | Effect of Conservation Time and the Addition of Lactic Acid Bacteria on the Biogas and Methane Production of Corn Stalk Silage[END_REF][START_REF] Weinberg | Effects of storage period on the composition of whole crop wheat and corn silages[END_REF][START_REF] Liu | Effect of harvest date on Arundo donax L. (giant reed) composition, ensilage performance, and enzymatic digestibility[END_REF][START_REF] Mceniry | Grass for biogas production: The impact of silage fermentation characteristics on methane yield in two contrasting biomethane potential test systems[END_REF][START_REF] Zheng | Effects of ensilage on storage and enzymatic degradability of sugar beet pulp[END_REF][START_REF] Zheng | Integrating sugar beet pulp storage, hydrolysis and fermentation for fuel ethanol production[END_REF][START_REF] Pakarinen | Evaluation of preservation methods for improving biogas production and enzymatic conversion yields of annual crops[END_REF]. Therefore, secondary fermentation should have occurred for catch crop silages through undesired clostridial activity.

Saccharolytic clostridial fermentation is mainly based on sugars and lactic acid consumption as energy source via similar pathways, producing not only butyric acid but also carbon dioxide and hydrogen [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. This will lead to VS and energy losses. Moreover, clostridial activity is associated with losses of acidity. This is explained by the fact that butyric acid is a much weaker acid than lactic acid and since only one mole of butyrate is produced from two moles of lactate [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. Low TS content of catch crop (Table 1) should have boosted secondary fermentation. Indeed, clostridia are known to be particularly sensitive to water availability and require wet conditions for active development [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. According to Borreani et al. [START_REF] Borreani | Fermentative profiles of field pea (Pisum sativum), faba bean (Vicia faba) and white lupin (Lupinus albus) silages as affected by wilting and inoculation[END_REF] clostridial fermentation exponentially decrease as TS content increase, being negligible from 30% of total solids. In our work, TS content of feedstock was quite below this value, so that clostridia activity was not inhibited.

Regarding storage of Cattle manure 1, pH values of biomass were 8.4 and 9.8 after 4 months of ensiling and aerobic treatment, respectively. The fact that pH of silage was above neutrality indicates that there was no significant accumulation of organic acids. Absence of WSC and high content of strong basic buffer components in raw material can explain the lack of acidity of cattle manure silage.

Finally, pH values of open-air stored biomass (9.1-9.8) were always higher than the respective raw material (6.4-7.9results not shown). Therefore, hydrogen-consuming reaction must have occurred, which evidences that aerobic microbial population spoiled biomass.

. Organic matter and energy conservation

The effects of open-air storage and ensiling on the conservation of biomass are summarized in Fig. 2.

For Catch crop 1 the use of ensiling instead of open-air storage reduced organic matter losses from around 69% to 10%. In the case of Catch crop 2, 68% of VS was lost during aerobic storage, which was reduced to 26% for ensiling. This discrepancy between methods was even more pronounced for the energy conservation during storage. On the one hand, after 3 months of storage in presence of air, both catch crops lost more than 80% of their original BMP. This demonstrates the harmful effect of an aerobic environment not only on the energy content of the remaining biomass but also on its biochemical accessibility. One the other hand, at least 96% of catch crop's original BMP was conserved after ensiling. Therefore, low TS content of feedstock did not impede the full conservation of its energy content for prolonged periods of ensiling. This allow two different considerations. First, it is conceivable that clostridial activity did not led to substantial energetic damage. In contrast, even if BMP damage and instability was caused by secondary fermentation, this was overcome by gains in biochemical accessibility during ensiling. In fact, according to several authors [START_REF] Desta | Ensiling Characteristics, structural and nonstructural carbohydrate composition and Enzymatic Digestibility of Napier grass Ensiled with Additives[END_REF][START_REF] Zheng | Effects of ensilage on storage and enzymatic degradability of sugar beet pulp[END_REF][START_REF] Chen | Ensiling agricultural residues for bioethanol production[END_REF], a partial degradation of (hemi-) cellulosic compounds occurs during ensiling. Since these polymers are not be fully biodegradable in mesophilic AD [START_REF] Ghosh | Hemicellulose conversion by anaerobic digestion[END_REF], their hydrolysis may induce a BMP increase.

Even though confined storage improved cattle manure preservation, substantial losses still occurred. Indeed, after 4 months, organic matter losses only decreased from 53% to 40% and BMP losses from 74% to 46% in ensiling rather than outdoor storage. Ensiling inefficiency for cattle manure should be related with the lack of acidification during the storage period. Since acid conditions were not achieved, silage did not escape from the pH range where damaging microorganism (e.g. methanogens) were active. Furthermore cattle manure use in this tests had low TS content (Table 1), which should have increased the microbial activity. Therefore, cattle manure was not anaerobically stabilized and high VS and energy losses occurred through biogas production during storage.

Effects of a co-ensiling approach

Ensiling of cattle manure with several co-substrates was investigated at laboratory scale in order to optimize energy conservation of the raw material during prolonged periods of storage. In the previous assays, two of the major issues related with cattle manure preservation were: low TS content and; absence of organic substrates like WSC for LAB fermentation. Wheat straw addition was used to decrease water availability of cattle manure. Formic acid and Glucose were tested as direct and indirect sources of acidification. These two latter compounds are high added-value products and they were tested only as molecule models of a real co-substrate. For instance, the use of glucose may be replaced in field application by a substrate with high WSC content, such as molasses.

pH values

The impact of co-substrates addition on the pH value of cattle manure before and after 4 months of ensiling is presented in Fig. 3.

Ensiling

Open-air Fig. 3 -pH value before and after 4 months of co-ensiling assays. CM1+WS stands for Cattle manure 1 with wheat straw; CM2+WS for Cattle manure 2 with wheat straw; CM2+WS+FA for Cattle manure 2 with wheat straw and formic acid; CM2+WS+G for Cattle manure 2 with wheat straw and glucose Wheat straw addition had different impacts on the fermentation of each Cattle manure. Regarding cattle manure 1 (CM1+WS), pH value decreased from 8.3 before storage to 5.5 after 4 months of coensiling. This indicates that fermentation occurred and that there was an accumulation of organic acids in co-silage. Yet, wheat straw was mainly composed of fibers and so, its addition should not have contributed to the increase of WSC content in feedstock. Therefore, silage acidification must have been a result of wheat straw impact on TS increase and, consequently, on the limitation of methanogen activity during anaerobic storage. Furthermore, it is suggested that wheat straw addition has reduced the buffer capacity of the raw material at neutral pH value, which facilitated acidification.

In opposite, pH value of Cattle manure 2 was stable during co-ensiling with wheat straw (8.3-8.4). This evidences that straw was not an infallible asset for the conservation all types of Cattle manure though ensiling. In fact, bacterial populations can be influenced by the characteristics of manure [START_REF] Manyi-Loh | An Overview of the Control of Bacterial Pathogens in Cattle Manure[END_REF], which according to Marañón et al. [START_REF] Marañón | Anaerobic mesophilic treatment of cattle manure in an upflow anaerobic sludge blanket reactor with prior pasteurization[END_REF] may depend on the type of cattle, animal's diet, as well as, on the time of the year. In our experiments, Cattle manure 1 was collected on March 2016, while Cattle manure 2 was from June 2016. Thus, it is possible that, for instance, the activity of methanogen population was predominant in Cattle manure 2, or at least in higher concentration than in Cattle manure 1. In that case, even if organic acids were still produced, they were quickly converted into biogas, hindering biomass acidification and preservation.

Lower pH was observed either for formic acid or glucose addition to CM2+WS after storage. For the formic acid condition, pH was 3.5 before storage and it increased to 6.9 at the end of 4 months of ensiling. The partial loss of acidity demonstrates that formic acid addition was not enough to inhibit fermentation and its consumption.

Co-ensiling of Cattle manure 2 with both glucose and wheat straw led to extensive acidification of biomass. Indeed, pH value was 7.9 for CM2+WS+G feedstock and deceased to 3.7 after 4 months of storage. The fact that the pH was that low indicates that lactic acid was produced in large quantities in the early days of ensiling and that microbial activity was stopped, thus preventing clostridial fermentation. Moreover, this shows that fresh cattle manure had a strong LAB population. Therefore, one may conclude that lack of WSC in raw material was the major barrier for the conservation of cattle manure through the ensiling process.

Organic matter and energy conservation

Results presented in Fig. 4 evidence the strong correlation between biomass acidification and both VS and BMP preservation during anaerobic storage.

Wheat straw addition to Cattle manure 1 enhanced ensiling performance: 92% and 98% of its original organic matter and energy content (BMP) were conserved after 4 months. These were remarkable results for a feedstock that is not naturally adapted for ensiling conditions. Furthermore, this indicates that a pH value around 5.5 may be enough to prevent substantial methanogenic activity for long-term storage of cattle manure (with around 20% of TS content).

Unlike, severe losses occurred for Cattle manure 2 with only wheat straw addition. For this condition, 42% and 67% of its original organic matter and BMP were lost after 4 months, respectively. This evidences the important activity of degrading microorganisms for neutral pH conditions in ensiling medium. Hence, wheat straw addition cannot be seen as a full guarantee to great biomass and energy conservation during long-term ensiling for all types of cattle manure.

Silage quality of Cattle manure 2 was improved with the use of direct and indirect sources of acidification. Regarding formic acid assays (CM2+WS+A), biomass conservation was enhanced to 87% and 75% of the initial VS and BMP content, respectively. However there were still nonnegligible losses for this condition. This should be linked the loss of acidity during ensiling and probable formic acid consumption previously discussed. In parallel, co-ensiling of cattle manure with both glucose and wheat straw showed outstanding results. As a matter of fact, 95% of original VS was preserved and no loss of BMP was observed until the end of the 4 months. This demonstrates that optimal conservation of energy content of cattle manure during storage may be achieved through co-ensiling with a substrate containing high concentration of available carbohydrates and strong TS content.

Conclusions

Energy losses during storage of organic residues were limited through ensiling. Even though, the efficiency of anaerobic storage strongly relied on chemical characteristics of each raw material. On the one hand, full conservation of methane potential occurred for ensiling of catch crops during 3 months. On the other hand, ensiling of single-handedly cattle manure led to 46% loss of its original energetic content after 4 months. Lack of easily available substrate for fermentation, high water availability and non-adapted buffering capacity were some of cattle manure potential features that hindered biomass preservation. For this latter feedstock, our work also revealed that the use of additives may prevent biomass spoilage and lead to full energetic conservation during long-term ensiling. Finally, coensiling with a substrate containing high concentration of available carbohydrates appears to be the most resourceful method to optimize cattle manure storage before biogas production. These outcomes may contribute to enhance economics of agricultural biogas plants.

Fig. 1 -

 1 Fig. 1 -pH value after 3 months of storage for Catch crop 1 / Catch crop 2 and 4 months for Cattle manure 1

Fig. 2 -

 2 Fig. 2 -Organic matter (A) and BMP (B) losses after 3 months of storage for Catch crop 1 / Catch crop 2 and 4 months for Cattle manure 1. BMP losses are related to the differences between values on a VSoriginal basis, therefore taking into account storage loss

Fig. 4 -

 4 Fig.4-BMP and organic matter preservation of co-ensiled cattle manure after 4 months. CM1+WS stands for Cattle manure 1 with wheat straw; CM2+WS for Cattle manure 2 with wheat straw; CM2+WS+FA for Cattle manure 2 with wheat straw and formic acid; CM2+WS+G for Cattle manure 2 with wheat straw and glucose. BMP balance takes into account storage losses

Table 1 -

 1 Storage conditions and treatments applied to feedstocks (% of total sample weight)

	Condition	Raw material	Co-substrate a	Storage method Open-air Ensiling	TS (%) b	VS (%) b
	CC1	Catch crop 1	-	Yes	Yes	18.2 ± 0.3 16.2 ± 0.4
	CC2	Catch crop 2	-	Yes	Yes	10.1 ± 0.1	8.4 ± 0.2
	CM1	Cattle manure 1	-	Yes	Yes	12.8 ± 0.1 10.2 ± 0.1
	CM1+WS	Cattle manure 1	Wheat Straw	-	Yes	18.8 ± 0.4 15.9 ± 0.3
	CM2+WS	Cattle manure 2	Wheat Straw	-	Yes	19.

2 ± 0.1 16.6 ± 0.1 CM2+WS+FA Cattle manure 2 Wheat Straw + Formic Acid (2%) -Yes 20.4 ± 0.1 18.0 ± 0.1 CM2+WS+G Cattle manure 2 Wheat Straw + Glucose (10%) -Yes 24.9 ± 1.4 22.6 ± 2.8

Acknowledgements

Rúben Teixeira Franco held a doctoral fellowship from the Rhône-Alpes region. This work has been undertaken within the SAM project (Stockage Avant Méthanisation -Storage Before AD) funded by ADEME (# 1506C0038). The authors thank the DEEP laboratory team, including David Lebouil, Hervé Perier-Camby, Nathalie Dumont and Richard Poncet for the given support during the tests. We are grateful to Franck Barra for his permanent availability for discussion and raw material supply. Mathilde Hardier and SUEZ are also acknowledged for the inoculum provided for the BMP tests.