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 storage of a

Keywords: Wilting, Storage, Ensiling, Anaerobic digestion, Methane potential , acid detergent fiber, ADL, acid detergent lignin, BA, butyric acid, BMP, biochemical methane potential, CEL, cellulose, 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

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

Introduction

Seasonality of crops and other agricultural wastes used for biogas production is at the origin of storage requirements before anaerobic digestion (AD). Since biogas production through AD should operate continuously throughout the year, seasonal biomass feedstock need to be preserved for prolonged periods, which may reach up to 6 months in some cases.

Among the current preservation methods, ensiling emerges as a logical choice to store wet biomass for energy production purposes. Based on biochemical processes, ensiling provides an anaerobic environment suitable for bacterial fermentation, which leads to biomass acidification, limiting further degradation and energy losses.

Together with the nature of the feedstock, air presence, storage duration or the use of additives, the control of the moisture content is one of the key parameters for the success of feedstock ensiling before AD [START_REF] Franco | Ensiling for biogas production: Critical parameters. A review[END_REF]. Indeed, it is commonly shared that low moisture contents slow down bacterial growth, leading to a more restricted fermentation and better silage preservation [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[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] 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]. Furthermore, silages with at least 25% of total solids content (TS) may prevent leachate formation during ensiling [START_REF] Bastiman | Factors affecting silage effluent production[END_REF][START_REF] Sutter | Problem of waste effluent from silage making and feeding of silage[END_REF][START_REF] Zimmer | The influence of pre-wilting on nutrient losses, particularly on the formation of gas, Tagungsberichte Der[END_REF], avoiding potential energy losses due to seepage.

Field wilting (i.e. natural air-drying of crops after harvesting) prior to ensiling is the most common method to achieve higher TS contents for biomass crops when moisture content is excessively high. Besides being inexpensive, it may enable water evaporation with little effect on the remaining chemical characteristics if short wilting duration (around 6h) is used [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] 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] Carpintero | The effect of some pretreatments on proteolysis during the ensiling of herhage[END_REF]. However, wilting effectiveness is strongly dependent on weather conditions of the harvest site. In fact, natural environmental conditions such as solar radiation, vapor pressure, rainfall and eventually wind speed are correlated with the drying rate of biomass [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. Consequently, when unfavorable weather conditions are present at harvesting, prolonged wilting durations must be used to obtain the desired TS content. In such cases, it is suggested that other chemical changes beyond water evaporation occur, which might influence ensiling conservation as well. For instance, Carpintero et al. [START_REF] Carpintero | The effect of some pretreatments on proteolysis during the ensiling of herhage[END_REF] observed an impact of 48h wilting on the water-soluble carbohydrates (WSC) and ammonia nitrogen (NH3-N) content of ryegrass-clover.

Likewise, Dawson et al. [START_REF] Dawson | The effects of wilting grass before ensiling on silage intake[END_REF] studied field wilting durations of 28 and 52h for perennial ryegrass and reported an impact of wilting on silage chemical characteristics, particularly on the pH and the buffering capacity. However, results on the impact of prolonged wilting durations on the conservation of the biochemical methane potential (BMP) during ensiling are still scarce and inconclusive. To our best knowledge, only Pakarinen et al. [START_REF] Pakarinen | Storing energy crops for methane production: Effects of solids content and biological additive[END_REF] have tested this parameter and observed better BMP conservation during ryegrass ensiling after 48h drying, while an opposite effect was obtained for grass silage.

The objective of this study was thus to compare ensiling and open-air storage as methods for preservation of a catch crop before biogas production. For ensiling, the aim was to optimize the management practices of the feedstock when unfavorable weather conditions are present at harvesting. Both fermentative profiles and BMP were monitored at different stages of preservation, which enabled a detailed analysis of the phenomena involved during storage.

Material and methods

Feedstock

Raw material used was a sample of a catch crop harvested on 9 November 2015 from an agricultural site in the Rhône-Alpes region of France (Gaec Béreyziat, Les Teppes, 01340 Béréziat, France). Biomass was composed by a mixture of sunflower, sorghum, peas, Vicia sp. and Trifolium alexandrinum.

Wilting process

Storage preparation procedure is summarized in Figure 1. After harvesting, part of the fresh biomass (10.1%TS) was ensiled and the remaining fraction was wilted. Nonsuitable weather conditions were recorded during wilting: daily average temperatures ranged between 7.7-14.9°C; extremely cloudy sky along with fog patches. After 72h of wilting, the dried sample had 14.1% of TS content. At this point, part of the 72h-wilted biomass was ensiled and the remaining amount was moved to an air-ventilated stove at 35°C, in order to be able to test a larger range of moisture content in the ensiling assays.

After a total of 96h of wilting, the last fraction of biomass (38.7%TS) was removed from the stove, equilibrated to room temperature and then ensiled. Fresh and wilted biomass were chopped to theoretical particle size of 8mm with a rotary shear shredder immediately before storage. This particle size was chosen taking into account the recommendations of Herrmann et al. [START_REF] Herrmann | Particle size reduction during harvesting of crop feedstock for biogas production I: effects on ensiling process and methane yields[END_REF][START_REF] Herrmann | Particle size reduction during harvesting of crop feedstock for biogas production II: effects on energy balance, greenhouse gas emissions and profitability[END_REF] for a compromise between high methane conservation rates and low chopping costs. Biomass was manually mixed between each step of storage preparation and samples used for storage assays were taken in representative quantity of raw material, i.e. 10%.

Storage approach

Storage assays were conducted at laboratory scale to monitor the impact of ensiling and prolonged wilting on biomass conservation. To evaluate ensiling impact, an aerobic control storage test was also performed for the 96h-wilted sample. 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 the silo was left air-open. Once sealed, the silos were weighed and placed in a temperate room at 25±2°C for a defined period. Storage duration varied between 7, 14, 28 and 98 days. A total of 16 (4 operating conditions x 4 storage durations) laboratory silos were used.

Chemical analysis

Chemical analysis procedure after sampling is illustrated in Figure 2. For each tested duration, one silo (per tested condition) was opened and weighed, biomass was homogenized and two samples were taken. One 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/dry mater ratio during 2h under constant bottle rotation. Phase separation was achieved by centrifugation followed by 0.7µm particle size filtration. Finally, the particulate phase 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.

Crude material was analyzed for its TS content, volatile solids (VS) content and BMP.

For the water-soluble phase, besides TS/VS content, pH, WSC, volatile fatty acids (VFA), NH3-N and total Kjeldahl nitrogen (TKN) contents were determined. Particulate solid was analyzed for its TS/VS, TKN and cell wall constituents content. TS was measured by oven drying at 105°C during 24h and VS was subsequently burned for 2h 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 equation 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. 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 acids content obtained with the RID detector. 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 XP U44-162 French standard. Cellulose content (CEL) was calculated as NDF minus ADF, hemicellulose (HEM) as ADF minus ADL and lignin (LIG) as approximatively equal to ADL.TKN and NH3-N were determined through the procedure described in the NF EN 25663 French standard.

The interest of our experimental procedure is, first, to be able to compare the results of composition and BMP to the initial mass of product used since the weight loss is monitored. 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 allows the study of the results based on the VS of the initial material.

Biochemical methane potential tests

Batch anaerobic digestion tests were performed for crude material samples. Tests were conducted in a temperate room at 35°C using glass vessels of 2L. Vessels were filled with at least 5gVS 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: 2.6-3.0%wt; VS: 1.8-2.0%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 pressure measurement using a Digitron precision manometer. Biogas was released when the pressure exceeded 1200 hPa.

Biogas composition was determined 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 batch tests 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

Raw materials characterization

Results of chemical analysis on fresh and wilted biomass before storage are summarized in Table 1. Fresh catch crop was characterized by a low level of TS (10.1%) and high WSC content in the organic matter (12.0%VS). These values are important to understand ensiling fermentation, since silage quality will strongly rely on TS and WSC content of feedstock [START_REF] Franco | Ensiling for biogas production: Critical parameters. A review[END_REF]. High WSC level of fresh material should enable a prompt acidification of silage by lactic acid bacteria (LAB) fermentation. However, secondary fermentation will be favored at low TS content if extremely acid environments are not reached. This should preclude biomass stabilization and lead to substantial VS losses.

To avoid the risk of having unstable silages due to low TS content, wilting was performed before storage. As mentioned earlier, natural drying of biomass had a minor effect on the moisture content of feedstock. TS content only increased from 10.1% to 14.1% of total biomass after 72h-wilting. Drying ineffectiveness should be closely linked to the poor weather conditions recorded during wilting process.

At the same time, TS content of 96h-wilted biomass increased to 38.7%, due to the use of an air-ventilated stove at 35°C on the last 24h of drying. Besides, 96h-wilting had a substantial impact on WSC, leading to degradation of more than half of the initial content. This suggest that some drying conditions, including high temperatures and wind speed (which may occur in summer), may lead to important organic matter losses before storage.

Wilting duration also had an impact on pH values: it decreased from 6.4 to 6.2 after 72h-wilting and to 5.8 after 96h-wilting. Nevertheless, the reason for this remained unclear, as VFA contents were stable and almost negligible during wilting. Regarding fibers constituents, prolonged periods of wilting had a negative impact on cellulose preservation. Finally, some fluctuations were recorded on NH3-N and TKN fractions, but no correlation was found between their content and wilting duration.

Storage impact on chemical characteristics

A major advantage is evidenced in using ensiling as biomass conservation method rather than open-air storage, Table 2. Regarding aerobic treatment, significant organic matter degradation occurred from the first week of storage: 21.9% and 68.8% of original VS content was lost after 7 and 98 days, respectively. This was a result of degradation of both non-structural and structural carbohydrates and organic nitrogen compounds present in the raw material. In fact, more than 99% of the original WSC content had been consumed after only 7 days of open-air storage. This led to organic matter losses, as no accumulation of VFA was recorded. For the structural carbohydrates, the degradation took place all along storage in aerobic environment as can be observed in Figure 3, resulting after 98 days in 78% and 47% losses of original hemicellulose and cellulose contents, respectively. Degradation of organic nitrogen was evidenced by TKN decrease from 5.1 to 3.8%VSoriginal and ammonia nitrogen increase from 4.5 to 46.8%TKN until 28 days of aerobic storage. Moreover, between 28 and 98 days of storage, the NH3-N and TKN contents dropped dramatically, resulting in around 85% ammonia loss during this period. This showed that, besides organic matter degradation, undesired ammonia emissions into the atmosphere are favored by open-air storage. This should be a result of the impact of a continuous pH increase for aerobic environment, from 5.8 at start to 9.6 after 98 days of storage, on the NH3-NH4 + equilibrium reaction and NH3 stripping. Additionally, fungi development was detected throughout aerobic storage. This suggests that microbial degradation of organic matter took place via an aerobic metabolism producing CO2 and microbial biomass.

In contrast, during ensiling, biomass is mainly subject to microorganism capable of anaerobic proliferation. For the same raw material used in open-air storage (96h-wilted biomass), ensiling enabled acidification, resulting in pH values between 4.9 after 7 days and 4.4 at the end of storage. A continuous production of lactic acid from available carbohydrates sources during ensiling was behind it. Lactic acid content varied from 7.7%VSadded after 7 days and 11.6%VSadded after 98 days of storage. Butyric acid production was negligible during ensiling of 96-h wilted biomass. Consequently, silage was stabilized by escaping from the pH range where spoiling anaerobic microorganisms are active. This limited VS losses to only 5% of the ones obtained in aerobic environment after 98 days.

Nevertheless, results showed that biomass stabilization through ensiling strongly depended on the TS content. For both fresh and 72h-wilted biomass (low TS contents) faster acidification occurred and after 7 days of ensiling pH was already at 4.0 and 4.2, respectively. This was a result of a more extensive LAB activity for lower TS contents, as lactic acid content was 18.9%VSadded after 7 days for these crops. However, from 14 days of ensiling, low TS crops revealed to be instable and fermentation profiles reverse direction. In fact, lactic acid content of fresh ensiled biomass dropped from 23.1%VSadded at 14 days to 0.5% VSadded and 0%VSadded after 28 and 98 days, respectively. Similarly in 72h-wilted silage, lactic acid content decreased from 17.9%VSadded at 14 days to 8.1%VSadded after 28 days and to nothing at the end of storage. In the same period of time, butyric acid started to be produced in both low TS silages. Final butyrate contents of 8.3%VSadded for fresh biomass and 5.5% for 72hwilted silage were obtained. The degradation of lactic acid and the concomitant production of butyric acid at low TS content indicates that secondary fermentation occurred 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].

Besides the unstable behavior, this will also 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]. Indeed, the pH of fresh ensiled biomass increased from 4.2 at 14 days to 5.6 at the end of storage. Likewise, 72h-wilted silage presented a huge increase of pH from 4.4 at 14 days to 7.0 at 98 days of storage. Major impact of clostridial fermentation on VS losses was also observed at low TS. After 98 days of ensiling, cumulative organic matter losses of fresh and 72h-wilted biomass reached 25.8% and 16.8%, respectively.

The instability of higher moisture crops was also illustrated by an increasing ammonia nitrogen content during ensiling. NH3-N fraction varied from 12.3%TKN at 14 days to 40.2%TKN at the end of fresh biomass ensiling. Similarly, NH3-N content of 72hwilted silage increased from 15.3%TKN to 37.0%TKN in the same period of storage. Proteolytic clostridial activity, which is favored by disturbances on silage acidification, should be in the origin of this increase. This type of 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 if an alkaline pH is reached. Since unstable silage never exceeded a pH value of 7.0, only a small fraction of NH3-N should be lost into the atmosphere. This is corroborated by the fact that there is no clear decline trend of TKN content during the tests. However, attention must be paid to this issue if pH values of poor silages tends to go towards the alkalinity for even more prolonged storage durations.

Concerning the substrates used by microorganisms for acidification, the results suggested that substances other than WSC are the original source for organic acids production. This is based on the fact that original WSC content of fresh biomass is largely inferior to the total VFA generated during ensiling. Taking the example of fresh ensiled biomass, 28.2%VSadded (26.0%VSoriginalresult not shown) of total VFA were produced after 14 days of storage, from a material with 12.0%VS of original WSC.

Additional information about the source of acidification products and phenomena can be found in the VS balance presented in Figure 3. Indeed, it can be observed that a great shift on VS structure occurred during the first 7 days of ensiling. This corresponds to the period of time where the major part of acidification took place. The major modification was the increase of water-soluble phase of VS, which varied from 13-24%VS before storage to 42-46%VSoriginal after 7 days of ensiling. This was an outcome of the degradation both non-structural and structural fractions of particulate phase. In fact, besides the reduction of Water+NDF soluble phase of particulate, there was a meaningful breakdown of (hemi-) cellulosic compounds during the first week of ensiling. Lignocellulosic degradation during early acidification may be a result of endogenous enzyme activity. Indeed, while studying grass hemicelluloses ensiling, Dewar et al. [START_REF] Dewar | The hydrolysis of grass hemicelluloses during ensilage[END_REF] reported that enzyme activity may contribute to additional sugar availability for fermentation. These additional substrate released by the particulate phase must have been a useful support to VFA production in the first week of ensiling.

From 7 days of storage VS structure of biomass evolved according to the stability of silage. For low TS crops (especially for non-wilted biomass), particulate phase of VS continued to be degraded, since pH level turned back into bacterial and enzymatic activity range. For 92h-wilted silage, minor further increase of soluble fraction was recorded. However, we observed a slow but continuous breakdown of lignocellulose carbohydrates for this condition. Since at lower pH levels enzyme activity is considerably reduced, this should be a result of acid hydrolysis by VFA produced during fermentation [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Dewar | The hydrolysis of grass hemicelluloses during ensilage[END_REF]. After 98 days of ensiling for fresh biomass, cumulative breakdowns of 83% and 64% of hemicellulose and cellulose were detected, respectively. For 76h-wilted silage, losses reached 50% for hemicellulose and 32% for cellulose. Finally, for the same ensiling duration 92h-wilted biomass lost 57% of hemicellulose and 30% of cellulose. These results show a greater extent of cell wall breakdown than previously reported results. For instance, Pakarinen et al. [START_REF] Pakarinen | Evaluation of preservation methods for improving biogas production and enzymatic conversion yields of annual crops[END_REF] and Menardo et al. [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] found a slight decrease of hemicellulose content during ensiling, while cellulose was not significantly degraded. In turn, other authors observed both hemicellulose and cellulose degradation with ensiling, but always below 10% of the original content [START_REF] Chen | Ensiling agricultural residues for bioethanol production[END_REF][START_REF] Zheng | Effects of ensilage on storage and enzymatic degradability of sugar beet pulp[END_REF][START_REF] Desta | Ensiling Characteristics, structural and nonstructural carbohydrate composition and Enzymatic Digestibility of Napier grass Ensiled with Additives[END_REF]. Discrepancy between these results may be partially explained through differences in cell wall accessibility. Firstly, higher particle size was used in part of literature [START_REF] Pakarinen | Evaluation of preservation methods for improving biogas production and enzymatic conversion yields of annual crops[END_REF][START_REF] Chen | Ensiling agricultural residues for bioethanol production[END_REF][START_REF] Desta | Ensiling Characteristics, structural and nonstructural carbohydrate composition and Enzymatic Digestibility of Napier grass Ensiled with Additives[END_REF]. This may influence digestibility of lignocellulosic compounds during ensiling, as particle size reduction is frequently linked to the increase of the available specific surface area and to the reduction of both degree of polymerization and cellulose crystallinity [START_REF] Agbor | Biomass pretreatment: fundamentals toward application[END_REF]. Furthermore, unlike part of these works [START_REF] Pakarinen | Evaluation of preservation methods for improving biogas production and enzymatic conversion yields of annual crops[END_REF][START_REF] Chen | Ensiling agricultural residues for bioethanol production[END_REF], the lignification degree of the feedstock used in this study was very low, as initial lignin values ranged from 3.8-4.6%VS. Finally, the results of the aforementioned works were mostly presented on a TS/VSadded basis. This may carry an underestimation of cell wall breakdown if substantial organic matter losses occurred during ensiling.

Evolution of BMP during storage

Discrepancies between results of open-air and ensiling conservation were even more evident if we compare the evolution of the methane potential (BMP) along storage, Figure 4. Methane yields for the aerobic treated sample ranged between 336 and 153 L/kgVSadded. While taking into account storage losses (L/kgVSoriginal), this represented more than 80% of energy losses from 7 to 98 days of storage. On the opposite, the 96hwilted silage recorded slightly increasing values of BMP during storage, even considering organic matter losses. At the end of the 98 days, the BMP based on VSoriginal of 96h-wilted biomass open-air stored was only 18% of the one for the ensiling of the same feedstock. Nevertheless, aerobic treatment does not appear to be harmful for the methane yield of 96h-wilted biomass in the first week of storage, despite important VS losses. Indeed, similar BMP values based on VSoriginal were obtained for open-air and ensiling of 96h-wilted biomass at 7 days. This was a result of an enhanced biochemical accessibility of aerobic storage in early days, as reflected by the higher BMP referring to VSadded for this condition after the first week. Today aerobic pretreatments for biomass are based on the delignification functions of microorganisms such as white rot fungi [START_REF] Kumar | Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production[END_REF][START_REF] Monlau | Lignocellulosic Materials Into Biohydrogen and Biomethane: Impact of Structural Features and Pretreatment[END_REF]. In this work, besides the use of a feedstock with low lignin content, no biological agent was added. Yet, fungi development that was observed during open-air storage could have the above-mentioned properties. Nevertheless, characterization of microbial biomass produced was not further investigated, since the main goal was to follow BMP evolution along storage.

Regarding ensiling, long-term storage had little effect on BMP based on VSoriginal.

Methane potential varied from 317 to 304 L/kgVSoriginal for non-wilted biomass, 307-275 L/kgVSoriginal for 72h-wilted silage and 261-280 L/kgVSoriginal for 96h-wilted biomass between 7 and 98 days of ensiling. Nevertheless, it is possible to identify distinct impacts on methane potential for different periods of storage. First of all, it is interesting to notice that, despite higher VS losses, low TS crops had higher BMP values after 7 days of ensiling. This should be a consequence of heat damage during wilting process of TS39% silage. Indeed, as previously mentioned, extremely warm and ventilated conditions used at the end of the 96h-wilting led to important WSC losses. In addition, higher rates of hydrolysis and fermentation of lignocellulosic fractions during the first 7 days of ensiling for low TS crops should have enhanced their biochemical accessibility and therefore their methane potential. Between 7 and 14 days of storage, BMP increased for all silages. This corresponds to a period of organic acid production from structural carbohydrates, which rendered biomass more available for methanogens.

The increase was more expressive for TS10% and TS39% silages, for which higher VFA accumulation occurred during this period. After 14 days and until at least the end of first month of storage, BMP based on VSadded continued to increase for all silage conditions. However at this time, stability of silage began to play an important role on methane potential conservation. Indeed, while taking into account VS losses, BMP decreased for low TS crops. This evidenced a harmful effect of secondary clostridial fermentation for energy conservation. Between 28 and 90 days of storage, instability of non-wilted and 72h-wilted silage led to losses of BMP based on VSoriginal of around 10%.

Results of BMP based on VSoriginal after 98 days of storage suggest that wilting was incapable of improve energy conservation before and during ensiling. Despite its more constant and easily predictable fermentation pathways, 96h-wilted biomass probably lost part of its methane potential by heat damage (or volatilization) during wilting.

Concerning low TS crops, especially fresh ensiled biomass, the gains in biochemical accessibility overcame their instability and VS losses. Moreover, the results suggest that the higher fermentation rates for low TS crops are not inevitably damaging for BMP conservation, especially for shorter storage durations.

In short, the catch crop used in these tests should be promptly ensiled after harvest if unsuitable weather conditions are present for wilting. However, one should take into attention the possibility of leachate production during ensiling of high moisture crops.

Concerning these laboratorial assays, liquid effluent production was around 23% of total biomass weight (result not shown) after 98 days of ensiling for fresh biomass (TS10%E). In a field scale silo, packing density should be at least equal to the one used in this work. Therefore, important leachate production should occur while using the same feedstock. Indeed, the early correlations done by Bastiman [START_REF] Bastiman | Factors affecting silage effluent production[END_REF], Sutter [START_REF] Sutter | Problem of waste effluent from silage making and feeding of silage[END_REF] and Zimmer [START_REF] Zimmer | The influence of pre-wilting on nutrient losses, particularly on the formation of gas, Tagungsberichte Der[END_REF] predict an effluent production that range from 34 to 45% of the total weight for a silage with 10% of TS. Moreover, results presented in Figure 3 show that VS water-soluble fraction will increase along storage duration, resulting in more than 65% of total VS content after 98 days of storage for TS10%E condition. In this case, important losses of methane potential will occur if effluent produced is not collected and promptly added to the anaerobic digester downstream.

Conclusions

Ensiling is a far more appropriated method than open-air storage for conservation of the catch crop studied before anaerobic digestion. Wilting was inefficient while witnessing unfavorable weather conditions at harvesting. Even if prolonged wilting periods under warmer/dryer conditions led to a decrease of the moisture content, it did not provide improvements on long-term conservation of the methane potential. For high moisture products, instability of fermentation is offset by gains in biochemical accessibility. In such conditions, biomass should be promptly ensiled without wilting. Liquid effluent that will likely be produced, should be collected and used for biogas production.

Figure 4 -

 4 Figure 4 -BMP evolution over storage duration. (A): methane potential based on VSadded; (B): methane potential based on VSoriginal, therefore considering storage losses.

Table 1 -

 1 Chemical characteristics of feedstock and impact of wilting TS10%E represents the ensiling of non-wilted biomass; TS14%E concerns the ensiling of the 72hwilted biomass; TS39%E and TS39%A symbolize the ensiling and open-air storage of the 96-wilted biomass, respectively. Results presented as b % of total sample weight; c %VSoriginal, d %TKN

	Biomass	Non-wilted	72h-wilted	96h-wilted
	Condition a	TS10%E	TS14%E	TS39%E / TS39%A
	pH	6.4	6.2	5.8
	TS b	10.1	14.1	38.7
	VS b	8.4	11.6	32.3
	VFA c	0.3	0.4	0.1
	WSC c	12.0	12.7	5.7
	HEM c	11.3	10.1	10.5
	CEL c	20.4	18.3	16.8
	LIG c	3.8	3.4	3.0
	TKN c	4.7	5.1	5.1
	NH3-N d	2.6	7.0	4.5

a

Table 2 -

 2 Fermentation profiles of biomass over storage time (%VSadded/original unless otherwise specified) LA stands for Lactic Acid, AA for Acetic Acid and BA for Butyric Acid; b results based on %VSadded;

	Condition	Storage duration (days)	VS losses	pH	LA b	Chemical characteristics a AA b BA b VFA b WSC b NH3-N (%TKN)	TKN c
		0	-	6.4	0.0	0.0	0.2	0.3	12.0	2.6	4.7
		7	5.3	4.0	18.9	2.1	0.0	22.1	2.3	8.2	4.7
	TS10%E	14	8.1	4.2	23.1	3.3	0.0	28.2	0.3	12.3	4.7
		28	20.9	6.7	0.5	5.0	6.2	17.6	0.4	23.7	5.0
		98	25.8	5.6	0.0	7.2	8.3	19.5	0.1	40.2	5.6
		0	-	6.2	0.0	0.0	0.3	0.4	12.7	7.0	5.1
		7	3.9	4.2	18.9	2.5	0.0	22.4	0.3	12.7	5.3
	TS14%E	14	5.2	4.4	17.9	3.6	0.0	22.6	0.4	15.3	5.3
		28	10.4	5.9	8.1	7.9	0.9	18.6	0.8	24.0	6.0
		98	16.8	7.0	0.0	6.4	5.5	13.6	0.2	37.0	6.2
		0	-	5.8	0.0	0.0	0.1	0.1	5.7	4.5	5.1
		7	2.1	4.9	7.7	1.2	0.0	9.4	2.9	11.4	5.7
	TS39%E	14	2.6	4.6	10.8	1.3	0.0	12.5	1.6	13.1	5.4
		28	3.0	4.4	11.0	1.8	0.2	13.6	0.6	12.9	5.5
		98	3.4	4.4	11.6	2.6	1.0	16.2	0.6	12.9	5.5
		0	-	5.8	0.0	0.0	0.1	0.1	5.7	4.5	5.1
		7	21.9	8.4	1.5	1.5	0.0	3.3	0.1	34.7	4.7
	TS39%A	14	38.6	9.0	0.6	0.4	0.0	1.1	0.2	40.9	3.8
		28	48.3	9.3	0.4	0.0	0.2	0.7	0.2	46.8	3.8
		98	67.8	9.6	0.0	0.0	0.0	0.0	0.0	14.1	2.0
	c results based on %VSoriginal

a

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

Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?

Keywords: Cattle manure, Storage, Ensiling, Anaerobic digestion, Methane potential , biochemical methane potential, CEL, cellulose, 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

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

Cattle manure for biogas production. Does ensiling and wheat straw addition enhance preservation of biomass and methane potential?

Introduction

Several European countries are currently mobilizing for the energy recovery from cattle manure through anaerobic digestion (AD). In France, the annual production of cattle manure is estimated at nearly 87 Mt, which represents around 28.3 MWh of potential primary energy [START_REF] Degueurce | Fumiers de bovins, une ressource à fort potentiel pour la filière de méthanisation en France ?[END_REF]. Nevertheless, due to its production fluctuations and in order to enable a continuous supply of biogas plants throughout the year, cattle manure needs to be stored before use, in some cases for extended periods. For this feedstock, a common practice is the open-air storage before AD. The reason for this is mainly related to the simplicity and low cost of the operation. However, this practice has several drawbacks. Generally speaking, air presence is perceived as a spoiling agent for biomass conservation, leading to important energy losses during long-term storage with aerobic spoilage of easily biodegradable organic fraction. Recent works with different catch crops showed that more than 80% of biochemical methane potential (BMP) was lost after 3 months of storage in the presence of air [START_REF] Franco | Optimizing storage of a catch crop before biogas production: impact of ensiling and wilting under unsuitable weather conditions[END_REF][START_REF] Franco | Optimizing agricultural wastes storage before anaerobic digestion : impact of ensiling on methane potential of lignocellulosic biomass[END_REF]. In the specific case of cattle manure, this reality is further compounded by the fact that open-air storage facilities are important sources of ammonia and odor emissions [START_REF] Berg | Mitigation of methane emissions during manure storage[END_REF].

Regarding conservation methods before AD, ensiling is usually pointed out as the logical choice to store wet biomass. This process is typically divided into four phases, based on different biochemical transformations [START_REF] Egg | Feedstock storage, handling and processing[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 available carbohydrates 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 feed-out. After unloading the silo for AD feeding, biomass enters once again into aerobic environment, which may spoil the silage. While well succeeded, ensiling enhances biomass conservation and minimize energy losses. For instance, Herrmann et al. [START_REF] Herrmann | Effects of ensiling, silage additives and storage period on methane formation of biogas crops[END_REF] observed an average of 2% difference between BMP of fresh and ensiled material after 1-year ensiling of maize, sorghum hybrid, forage rye and triticale.

Given the high efficiency of ensiling for other biomass crops, it should be expectable to obtain good preservation of cattle manure using the same method. However, ensiling success is extremely dependent on several biochemical characteristics of feedstock, such as moisture content, water-soluble carbohydrates (WSC) content, buffering capacity and endogenous microflora [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF][START_REF] Franco | Ensiling for biogas production: Critical parameters. A review[END_REF]. Indeed, cattle manure does not fulfill the chemical requirements for an efficient ensiling, especially due its lack of WSC. Without those, organic acids cannot be produced in a significant extent and then, the desired level of acidity for silage stabilization would not be achieved.

Even if cattle manure does not naturally lead to an optimal ensiling conservation, it is possible that an anaerobic environment reduces organic matter and energy losses during storage. Furthermore, efficiency of cattle manure ensiling may be improved by costorage with wheat straw. In fact, this co-substrate is considered to have an impact on organic losses of traditional crops ensiling [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. This may be related to the fact that straw addition decreases the moisture content and microbial concentration of the feedstock.

Therefore, straw may act as a delaying agent of bacterial growth, improving energy conservation during storage. Likewise, wheat straw addition to cattle manure should decrease soluble organic matter concentration on the feedstock, which may facilitate silo compaction and prevent energy losses through leachate formation during storage. However, to the best of our knowledge, no study addressing these subjects exists today.

This work established a comparison between open-air storage and ensiling of cattle

manure during 120 days at laboratory scale. The effects of different degrees of wheat straw addition on silage conservation were also addressed. Storage performance was evaluated, above all, by its impact on BMP conservation of raw material.

Notwithstanding, other parameters, such as organic matter losses or eventual gas emissions, were examined during storage as well. This study contributes to the optimization of practices for cattle manure management before biogas production, which will definitively have an impact on the energy recovered downstream.

Material and methods

Substrates

Fresh cattle manure was collected on 10 March 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. Fresh raw material had 13% of total solids (TS) content and it was tested in both aerobic and ensiling conditions. In addition, two mixtures of fresh cattle manure and wheat straw were prepared at the laboratory and tested for ensiling. Wheat straw had 10 cm maximum length and it was mixed with cattle manure in order to achieve final TS contents of 19% and 22%, respectively.

Storage conditions and description of respective raw materials are summarized in Table 1.TS13%E and TS13%A represent the ensiling and open-air storage of fresh cattle manure, respectively. TS19%E corresponds to the co-ensiling of cattle manure and straw with final TS content of 19%. TS22%E is related to the co-ensiling of cattle manure and straw with final TS content of 22%.

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 air-open. 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) conservation assays were done.

Chemical analysis

For each sample time, one silo (per tested condition) was sacrificed. It was opened and weighed, biomass was homogenized and two samples were taken. One 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 use.

Crude material was analyzed for its TS content, volatile solids (VS) content and BMP.

For the water-soluble phase, besides TS/VS content and BMP, pH, WSC, volatile fatty acids (VFA), total Kjeldahl nitrogen (TKN) and ammonia nitrogen (NH3-N) fractions were determined. Particulate solid was analyzed for its TS/VS, TKN and cell wall constituents 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 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 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 using 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 XP U44-162 French standard. Hemicellulose content (HEM) was calculated as NDF minus ADF; cellulose (CEL) as ADF minus ADL and; lignin (LIG) as approximatively equal to ADL. TKN and NH3-N were determined through the procedure described in the NF EN 25663 French standard.

The interest of our experimental procedure is to enable the assessment of the composition and BMP based on the initial mass of product used, since the weight loss was 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 allows the study of the results based on the VS of the initial material.

Biochemical methane potential tests

Batch anaerobic digestion tests were performed for crude material and water-soluble phase samples. Tests were conducted in a temperate room at 35 °C using glass vessels of 2 L for crude material and 0.1 L for water-soluble phase. 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 3.0-3.2%wt; VS 2.0-2.2%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].

In order to assess the kinetics of methane production, the experimental data of BMP assays was fitted to the following equation:

𝑉 𝐶𝐻 4 (𝑡) = 𝑉 𝑚𝑎𝑥 (1 -𝑒 -𝑘𝑡 )
Where, 𝑉 𝐶𝐻 4 corresponds to the volume of methane produced; 𝑡 is the time of the assay; 𝑉 𝑚𝑎𝑥 is the maximum volume of methane produced, which was equivalent to the experimental BMP value and; 𝑘 is the rate coefficient of CH4 production. Since gas production started almost immediately after reactors were sealed, lag time was not considered in the calculations.

Results and discussion

Feedstock characterization

Chemical characteristics of raw materials and stored biomass are presented in Table 2.

Besides low TS content (12.8%), fresh cattle manure was mainly characterized by a negligible content of WSC and pH value of 7.9. These were unsuitable features for energy conservation through an acidification-based process as ensiling. First of all, as lower TS crops are more unstable and susceptible to deterioration, lower silage pH is required for an efficient ensiling [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. By analogy with the data published by Kalač [START_REF] Kalač | The required characteristics of ensiled crops used as a feedstock for biogas production: a review[END_REF] for ensiled forage, a critical pH value of around 4.0 is required to preserve fresh cattle manure. In order to achieve such degree of acidification, extensive production of lactic acid should occur in the first days of ensiling [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. However this would require an adequate level of readily available carbohydrates, i.e. high WSC content. In fresh cattle manure, energy sources were mainly concentrated in the form of lignocellulosic compounds. Indeed, cellulose and hemicellulose contents of fresh cattle manure were estimated as 26.8 and 32.5% of organic matter, respectively. Nevertheless, it should not be expected a prompt and wide VFA production from these polymers. Their biochemical accessibility is rather limited by lignin protection (6.1%VS) [START_REF] Liu | Evaluation of the correlations between biodegradability of lignocellulosic feedstocks in anaerobic digestion process and their biochemical characteristics[END_REF] and depolymerization/hydrolysis reactions should take several weeks to occur in substantial extent.

Structure of cell wall constituents changed with wheat straw addition to cattle manure. This was, in fact, the only remarkable effect of wheat straw beyond the increase of TS/VS content. Cellulose content increased to 36.0-37.2%VS after wheat straw addition. In turn, hemicellulose content decreased to 27.5-28.7%VS. No noticeable effect on lignin content was recorded with wheat straw addition.

Effects of storage method on biomass preservation

Chemical characteristics

Aerobic storage was mainly characterized by a pH increase and modifications on organic matter structure, Table 2. Structural carbohydrates were uninterruptedly degraded along the 4 months, culminating in both cellulose and hemicellulose losses of around 60% of their initial content. Furthermore, no accumulation of WSC nor VFA was recorded for this condition. This suggests that the degradation of structural carbohydrates led to severe VS and energy losses. Lignin content did not vary significantly, meaning that no (or limited) delignification occurred during aerobic storage. In addition to these chemical modifications, fungi development was noticeable throughout open-air storage. Therefore, microbial degradation must have occurred through an aerobic metabolism producing carbon dioxide and microbial biomass.

Storage in anaerobic conditions of fresh cattle manure led to VFA production and pH decrease until 30 days. Nevertheless, the acidification was slow since only structural carbohydrates were available for fermentation. This should explain the significant reduction of (hemi-) cellulose content during the first month of ensiling. The major components of VFA were acetic and butyric acid, with other small fractions of propionic, valeric and caproic acid. No formation of lactic acid was detected during ensiling. Since the fermentation products of TS13%E are weaker acids than lactic acid, pH drop for stored cattle manure was not as marked as in lactate silages. In addition, between 14 days and 30 days of ensiling, VFA content increased from 6.0% to 12.9%VSadded but pH value remained stable at 6.9. This evidenced that fresh cattle manure had a strong buffer capacity at neutral pH conditions. Altogether, efficient pH values around 4.0 were not achieved for ensiling of fresh cattle manure and further biomass degradation occurred for prolonged storage periods. Indeed, between 30 and 120 days, fermentation profile reversed and more than 90% of VFA were consumed until the end of storage. This caused an increase of pH, which reached a value of 8.4 at 120 days. The fact that this pH value were superior to the one of the raw material can be explained by the ammonia production and accumulation during ensiling. Moreover, structural carbohydrates continued to be degraded until the end of the storage. After 4 months, 48% and 37% of original hemicellulose and cellulose were biodegraded, respectively. Thus, non-negligible organic matter and energy losses must have occurred for long-term ensiling of fresh cattle manure.

Equivalent fermentation profiles were found for TS13%E and co-ensiling conditions in the first month of storage. However, the rate of acidogenesis decreased with wheat straw addition. This may be explained by the fact that wheat straw mostly contains hardly accessible carbohydrates. Moreover, wheat straw addition has decreased the initial microbial concentration and moisture content, which delayed bacterial growth.

For instance, it is well known that at least part of silage bacteria have lower activity for higher TS contents [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]. Nevertheless, wheat straw addition enhanced stability of cattle manure silages. For both TS19%E and TS22%E, pH value decreased from 8.2-8.3 before storage to around 5.5-5.6 after 30 days and remained constant until 4 months of ensiling. Additionally, even if VFA production was more restricted for co-ensiling, fermentation had a much higher impact on silage acidification than in TS13%E. This suggests that wheat straw addition reduced the buffer capacity of the raw material at neutral pH value. Despite the apparent stability of wheat straw silages, degradation of structural carbohydrates occurred until the end of the storage. After 4 months, 28% and 29% of the sum of original (hemi-) cellulosic compounds were missing for TS19%E and TS22%E, respectively. However, this should not have led to substantial energy losses, since VFA accumulation occurred until the end of storage for both co-silages.

The data presented here evidenced that the recommended pH value of around 4. 10-4.35 for TS content between 15-25% [START_REF] Kalač | The required characteristics of ensiled crops used as a feedstock for biogas production: a review[END_REF] was not applicable for the conservation of cattle manure, or at least for its co-ensiling with wheat straw. It must be remembered that this value of pH is the required acidity to prevent lactate degradation. In our experiments, no lactate was produced during ensiling. This means that either lactic acid was quickly consumed as an intermediate for acidogenesis, or that monosaccharides were degraded by other pathways than LAB fermentation. In any case, the stability of cattle manure did not relied on lactic acid formation but on the accumulation of VFA during storage.

Therefore, the critical pH in cattle manure silages should be considered as the required acidity to prevent methanogenic activity and further VFA damage for long-term storage, which was achieved for both TS19%E and TS22%E conditions.

TKN balance

Effects of storage method on total Kjeldahl nitrogen balance are presented in Figure 1.

This evolution is related to the TKN content of feedstocks, which was 4.0%VS for fresh cattle manure, 2.7%VS for TS19%E and 2.3%VS for TS22%E.

Regarding open-air storage, there was a continuous elimination of TKN, resulting in losses of 58.6% of its original content after 120 days. Mechanisms of degradation can be suggested through analysis of TKN composition along storage. First, there was a considerable decrease of particulate TKN content, which ranged from 52% to 39% of original TKN during the 4 months of aerobic treatment. Major part of particulate loss occurred during the first 7 days of storage, during which the content of the watersoluble phase of organic nitrogen increased. This indicates that some proteins were subject to hydrolysis under aerobic conditions, therefore increasing the amount of organic nitrogen compounds that can be processed by microorganisms capable of nitrogen mineralization and nitrate/nitrite production. However this may only partially explain the original source of TKN losses, due to the limited degradation extent of organic nitrogen in the particulate phase during storage. In fact, the majority of structural modifications of TKN during aerobic treatment occurred in the water-soluble phase. From 7 days of storage and until the end of conservation, the content of organic nitrogen in the liquid fraction decreased, which suggests that the rate of amino acid degradation was significant during aerobic treatment. Even if the previous mechanisms may lead to inorganic nitrogen production, this was not observed during storage. Indeed, ammonia concentration decreased all along storage duration, from 1.30%VSoriginal before storage to 0.02%VSoriginal after 120 days of aerobic treatment. This may be explained by the constant perturbations in the NH4 + /NH3 equilibrium (pKa = 9.25) during conservation. In open-air storage, NH3 emissions did not have any impact on its concentration in the gas phase, since ammonia produced was instantly diluted in the atmosphere. Thus, in such conditions, nitrogen was considered to be lost through NH3 production, even at relatively low pH. The rate of ammonia loss improved with time, as pH increased for long periods of aerobic treatment. Furthermore, nitrification reactions may have contributed to the elimination of organic nitrogen in aerobic conditions. This would lead to nitrate and nitrite emissions, which are not measured in the TKN procedure. Considering that all TKN losses led to ammonia emissions, around 40.1 LNH3/kgVSoriginal were expected to be released into the atmosphere during the 4 months of open-air storage of fresh cattle manure. Ammonia emissions were reduced through ensiling of fresh cattle manure. Indeed, in the first 30 days of ensiling there was a complete preservation of TKN content and at the end of storage only 12% of its original content was lost. This latter value corresponded to a theoretical value of 9.0 LNH3/kgVSoriginal for gas emissions during the 4 months of ensiling. Nevertheless, there was a larger transformation of TKN structure for ensiling than for open-air storage. On the one hand, the organic nitrogen content in both particulate and water-soluble phase decreased almost linearly with time, from a total 68%TKNoriginal before storage to 35%TKNoriginal after 4 months. On the other hand, ammonia content clearly increased along ensiling. In such conditions, TKN degradation must have occurred through two successive reaction mechanisms: hydrolysis of proteins into amino acids and subsequent fermentation in ammonia. These are typical reactional mechanisms that arise during anaerobic fermentation [START_REF] Batstone | The IWA Anaerobic Digestion Model No 1 (ADM1)[END_REF] and poor silages (high pH values) [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF]. Besides NH4 + , these reactions generally lead to other side-products, such as a pool of organic acids, CO2 and H2, generating energy losses, regardless of how small they may be. Even if there is a high production of ammonia during ensiling, gas emissions should be avoided as long as the silo is airtight and a low pH medium is present. This explain why degraded nitrogen was stored under the form of NH4 + in the first month of ensiling. However, during the last 3 months of storage, pH increased to values near the pKa of NH4 + /NH3, which led to TKN losses through ammonia production.

Co-ensiling of cattle manure with wheat straw did not have a significant impact on the reactional mechanisms related to TKN. However, lower degradation rates of TKN were obtained for TS19%E and TS22%E. In fact, organic nitrogen content (from both particulate and water-soluble phases) was around 70%TKN for all three feedstocks. This amount decreased to 34.8%TKNoriginal for TS13%E, 41.4%TKNoriginal for TS19%E and 51.9%TKNoriginal for TS22%E at the end of storage. These results may be in part linked to the effect of lower growth of proteolytic clostridia for higher TS contents, which has been reported in the literature for other types of silages [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[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]. Nitrogen losses were even lower than in TS13%E condition, reaching at the most 2.3%TKNoriginal during the co-ensiling. This corresponded to theoretical ammonia emissions of 1.2 LNH3/kgVSoriginal at the end of the storage. Enhanced conservation of nitrogen was related to the stable low pH value obtained during the 4 months for silages with wheat straw.

Organic matter losses

Higher damage of organic matter was found for the aerobic treatment, as shown in Figure 2. After one month, around 25% of organic matter of open-air stored cattle manure was lost, which ascended to more than 50% after 4 months. This was linked to the constant loss of structural polymers during storage.

Silage quality had an important effect on VS conservation during storage. Anaerobic storage of fresh cattle manure limited the organic losses to less than 10% in the first month, the period in which there was an accumulation of VFA. Nevertheless, silage instability of low TS crop aforementioned led to damage of almost 40% of original VS content at the end of the 4 months.

Co-ensiling with wheat straw had a positive effect on organic matter conservation. For TS19%E and TS22%E, most part of the VS losses occurred in the first month and corresponded to 5% of original VS. During this period, pH kept decreasing. Therefore, other bacteria populations capable of VS degradation, such as methanogens, must have proliferated under such conditions. Furthermore, fermentation of monosaccharides may lead to several side-products. For instance, reactional mechanisms of acetate or butyrate production include CO2 and H2 formation. Besides organic matter damaging, this would cause slight energy losses, as hydrogen would be lost into the environment during silo opening. In addition, nitrogen mineralization contributed to VS losses, although to a minor extent. During the last 3 months of storage, organic matter suffered minimal damage, due to silage stabilization. After 4 months, 92% of original VS was conserved for both co-storage conditions. These are remarkable results regarding silage conservation, since they are equivalent [START_REF] Franco | Optimizing agricultural wastes storage before anaerobic digestion : impact of ensiling on methane potential of lignocellulosic biomass[END_REF][START_REF] Herrmann | Effects of ensiling, silage additives and storage period on methane formation of biogas crops[END_REF] or even better [START_REF] Franco | Optimizing storage of a catch crop before biogas production: impact of ensiling and wilting under unsuitable weather conditions[END_REF][START_REF] Lehtomäki | Biogas Production from Energy Crops and Crop Residues[END_REF][START_REF] Pakarinen | Storing energy crops for methane production: Effects of solids content and biological additive[END_REF] than data found in the literature concerning good quality of crop silage. All things considered, there was a positive effect on the conservation of the biomass inherent to the wheat straw addition. This is in agreement with McDonald et al. [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF], who considered straw as an additive for ensiling, due in part to its effect on the reduction of organic matter losses.

Effects of storage method on methane potential

BMP evolution

Evolution of methane potential along storage is presented in Figure 3. The results exposed are related to both VSoriginal and VSadded, which allows the monitoring of original BMP conservation and eventual modifications on the chemical accessibility of the remaining biomass. BMP of aerobic treatment (TS13%A) ranged from 288 L/kgVS before storage to 157 L/kgVSadded and 74 L/kgVSoriginal after 4 months. BMP losses were divided in two distinct chronologic phases. Around half of energetic damage occurred during the first month of storage. This was expected since in first moments of long-term storage, besides the particulate phase, the major part of soluble organic matter should be within reach of aerobic microorganism. In this period, losses followed a linear tendency, equivalent to 1.2% original BMP loss per day. Methane potential losses slowed down somewhere between the second and fourth month and led to 74% of BMP lost at the end of aerobic storage.

Energy conservation was improved with ensiling of fresh cattle manure (TS%13E), since constantly higher BMP values during storage were obtained rather than when aerobic assays were carried out. As in the preceding condition, two chronologic phases of BMP evolution were observed for TS13%E. A slight BMP increase was found during the first weeks of storage for low TS silage. Indeed, after 1 month of storage original methane potential was improved by 11%, which is substantially higher than the limit of accuracy of BMP tests. Enhanced biochemical accessibility of feedstock must have been the source of this energetic increment, as BMP related to VSadded varied from 288 to 348 L/kgVSadded during the first month. As previously discussed, during this period, production and accumulation of VFA took place through degradation of (hemi-) cellulosic compounds. These latter polymers are not fully biodegradable in such soft condition as that experienced within the mesophilic AD process [START_REF] Ghosh | Hemicellulose conversion by anaerobic digestion[END_REF]. Therefore, their hydrolysis/acidogenesis during ensiling is a reasonable explanation for the BMP increase. This trend did not last until the end of storage, since in the last 3 months BMP of fresh ensiled cattle manure abruptly decreased, either considering organic matter losses or not. Consequently, TS13%E lost 46% of its original methane potential after the end of the 4 months of storage. One can assume that this energetic loss was linked to the consumption of VFA and structural carbohydrates during this period of storage.

In order to evaluate the tradeoff between higher energy conservation rates during ensiling and cost increase by silo construction, a financial estimation was performed.

For silo construction, a medium practiced cost in France of 40 €/m 3 (57 €/ton for a packing density of 0.7 kg/m 3 ) was used. Likewise, a reference value in France of 80 €/MWh was applied to the purchase price of additional bio-methane produced. Taking into account the energy conservation rates of TS13%E and TS13%A, methane potential gain through ensiling should be of 172 L/kgVS after a 4-month storage batch. This corresponds to around 6.3 €/ton of additional revenue by ensiling batch. Therefore, the investment made in silo construction should be recovered after 3 years (considering 3 storage batches per year). Is important to notice that the real recovery period should be slightly higher, since neither depreciation, nor interest nor other operational costs (compaction, silo covering, biogas treatment, etc.) were considered in this financial estimation.

Wheat straw addition had a positive effect on BMP conservation of cattle manure during ensiling in the long term. In fact, minor variations were recorded on the methane potential along the 4 months of storage for both conditions using straw: TS19%E showed BMP values of 277-304 L/kgVSadded and 271-287 L/kgVSoriginal; while for TS22%E it were observed 285-321 L/kgVSadded and 265-305 L/kgVSoriginal. Although less clear than in previous storage conditions, BMP evolution during storage of both TS19%E and TS22%E can be separated in two periods of time. After one month of storage, BMP related to VSadded increased by about 10% and 4% for TS19%E and TS22%E conditions, respectively. This reflects a slight enhancement of biochemical accessibility of raw materials and it should be interrelated with the production and accumulation of VFA in silage from hardly biodegradable carbohydrates during this period. Nevertheless, this effect was counteracted by organic matter losses.

Consequently, the improvement of BMP related to original VS was not relevant (within the precision limits). Between the end of the first and fourth month, BMP related to VSadded decreased by 3% for TS19%E and 11% for TS22%E. This evidenced that biodegradability of stabilized silages may decrease in long-term storage, which may arise since the so-called biomass stabilization occurred at relatively high pH values. At the end of the 4 months of ensiling, 98% and 86% of feedstock's methane potential was conserved for TS19%E and TS22%E, respectively. This proves that wheat straw addition was in both cases an asset to BMP preservation for long-term ensiling of cattle manure and that use of 19% TS should lead to optimal energy recovery. Furthermore, using the same parameters of the preceding financial estimation, higher energy conservation rates of TS19%E and TS22%E (compared to open-air storage) should lead to 25.7 €/ton and 23.9 €/ton of additional income per storage batch. This indicates that the investment for silo construction should be covered in less than 1 year for efficient ensiling conditions. The price of wheat straw was not included in these calculations, since its energy potential should be recovered in methane production.

Finally, our results suggest that there is a strong correlation between fermentation profiles, organic matter conservation and BMP for prolonged storage durations. At the same time, one may take into account that BMP assays may last for about 2 to 3 months to be completed. Therefore, whenever either a quick analysis of several storage conditions is required or only simple approaches are available, organic matter losses determination (e.g., or even total weight loss) may be a resourceful method for qualitative analysis on optimization of storage conditions for a specific feedstock before biogas production.

BMP distribution in particulate and water-soluble phase

The contribution of the soluble and particulate fraction to the total BMP is presented on Minor changes on BMP contributions (between particulate and soluble part) were detected for ensiling of fresh cattle manure (TS13%E) during the first month of storage, with water-soluble phase representing 25-32% of total methane potential. Significant degradation of water-soluble phase only occurred during the last 3 months, decreasing its fraction to 15% of total BMP at the end of storage. These results partially agree with the data afore discussed. On the one hand, a clear increase tendency of water-soluble phase of BMP was expected over the first month of ensiling. This would be the result of the increase of VS soluble fraction along storage duration, which was indeed recorded either by direct measure of VS fractions (result not shown) or by degradation of structural carbohydrates and VFA accumulation. On the other hand, the decrease of water-soluble fraction of BMP in the last 3 months of storage is in line with the loss of almost the totality of VFA that was observed during this period.

Analogous evolutions of BMP fractions during ensiling were observed for both degrees of wheat straw addition. For instance, regarding TS19%E condition, water-soluble fraction of BMP increased from 19% prior to storage to 34% after 4 months. This is in agreement with data from fermentation profiles, and confirms that complex energy sources have not only been degraded but also solubilized and conserved in that form for prolonged periods of ensiling. However, this mechanism of energy conservation may not be applicable to all types of cattle manure. 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 may depend on the type of cattle, animal's diet, as well as, on the time of the year [START_REF] Marañón | Anaerobic mesophilic treatment of cattle manure in an upflow anaerobic sludge blanket reactor with prior pasteurization[END_REF]. Therefore, for several types of manures, the methanogens concentration, e.g., may be far superior to the one of the feedstock used in these tests. In such cases, solubilization of particulate would still occur but VFA produced would be degraded due to a higher methanogenic activity. This will weaken ensiling efficiency, as it will lead to substantial energy losses before AD.

Moreover, wheat straw addition had an impact on BMP distribution before storage: water-soluble phase of BMP decreased from 25% for TS13%E (and TS13%A) to 20% and 19% for TS19%E and TS22%E, respectively. This was expected since organic matter of wheat straw is mostly under particulate form. Nevertheless, these are promising results for ensiling low TS feedstocks. In field scale silage of standard crops, low TS content should be avoided in order to prevent leachate formation during storage [START_REF] Bastiman | Factors affecting silage effluent production[END_REF][START_REF] Sutter | Problem of waste effluent from silage making and feeding of silage[END_REF][START_REF] Zimmer | Efficient silage systems[END_REF]. Since part of the BMP of silages are present in water-soluble form, important energy losses may occur due to seepage of eventual liquid effluent production. The use of cattle manure will increase TS content and reduce leachable methane content, therefore decreasing the risk either of effluent production [START_REF] Mcdonald | The Biochemistry of Silage, Second Edi[END_REF] and eventual energy losses that it may entail. Indeed, no production of liquid effluent was recorded during ensiling of cattle manure with wheat straw (result not shown). These outcomes were considered to be representative of a field scale, as cattle manure was ensiled with a commonly used packing density in agricultural silage.

Kinetics

Besides the maximum value of methane produced, kinetics of BMP assays were characterized by the 1 st -order rate coefficient (k) of biomass degradation, Figure 5. This parameter is closely linked to the biochemical accessibility of biomass, hence the interest for biomass conservation purposes. Nevertheless, while testing prolonged storage, different inoculum should be used for part of BMP series. Contrary to methane potential value, modifications on inoculum properties have an impact on the rate of biomass degradation during BMP assays. Therefore, an indirect approach was used to analyze evolution of k values during storage, which consisted in only comparing results between different conditions for the same storage period (BMP tests were performed with the same inoculum).

Concerning the feedstocks, higher rate coefficient was found for fresh cattle manure, being the decrease of biochemical accessibility proportional to the amount of wheat straw added. This confirms that the majority of wheat straw VS was hardly reachable, which was previously suggested by the increase of cellulose content and particulate phase of BMP related to straw addition.

Storage method had a significant effect on biochemical accessibility of fresh cattle manure. In the first 30 days of storage, the difference between the rate coefficients of the two methods progressively increased, benefiting ensiling. This must have been an outcome of: in first place, the degradation of structural carbohydrates into VFA and their accumulation during this period for TS13%E and; secondly, the important loss of water-soluble compounds in the first month of open-air storage. This scenario was reversed in the last 3 months and the degradation rate of ensiled cattle manure became weaker than the one for the aerobic treatment. On the one hand, this difference cannot be attributed to an increase of the biochemical accessibility for the open-air stored feedstock, since its BMP declined significantly in this period and its water-soluble fraction did not increase. This means that the shift should have been a consequence of VFA degradation for TS13%E, which led, in addition to the energy damaging, to a great loss of accessibility of the organic matter during the 3 last months of storage.

Despite different initial values, similar rate coefficients were obtained for TS19%E and TS22%E from 7 days of storage. Furthermore, both conditions with wheat straw presented lower k than TS13%E until 30 days of ensiling. This was explained by the fact that wheat straw led to a more controlled fermentation, and as formerly discussed to scarcer gains related to the BMP kinetics. At the end of storage, TS19%E and TS22%E had far higher degradation rates during the BMP assays than ensiling of fresh cattle manure. This evidenced that wheat straw is an asset on the conservation of great rates of biochemical accessibility for extended periods of ensiling. Restricted fermentation and VFA accumulation in water-soluble phase appear to be the key factors for the success of co-silages with cattle manure and wheat straw.

Conclusions

Ensiling led to lower energetic losses and NH3 emissions than open-air storage during conservation of cattle manure. Yet, chemical properties of fresh cattle manure hindered silage stabilization for extended periods. Co-ensiling of cattle manure with wheat straw enhanced biomass acidification, inducing a preservation of more than 86% of methane potential after 4 months. In addition, wheat straw suppressed ammonia production and limited the risks of energy losses through liquid effluent. In summary, this work demonstrated that long-term conservation of cattle manure can be achieved if correct management practices are used. This will impact the performance of agricultural biogas plants.

Figure 4 .

 4 Figure 4. During aerobic storage (TS13%A) the water-soluble contribution of BMP

Figure 1 -Figure 2 -Figure 3 -Figure 4 -Figure 5 -

 12345 Figure 1 -TKN balance for: (A) TS13%A; (B) TS13%E; (C) TS19%E; (D) TS22%E

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.