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Anaerobic Digestion (AD) is a process which allows the treatment of organic waste and the production of renewable energy. In particular, dry AD allows the treatment of solid organic substrates, offering several possibilities to the enhancement of agricultural waste such as spent livestock bedding (a mixture of straw, faeces and urine). Among the available biotechnologies in AD, leach-bed reactor (LBRs) is a promising but yet poorly known process both at scientific and industrial level.

In order to develop this process, several issues have been studied: (i) the bio-physicochemical characterization of spent animal bedding and its digestion potential in LBRs; (ii) the optimization of the start-up and the operating temperature of the digesters; (iii) the co-digestion of spent animal bedding with an easily-degradable substrate and the issues connected to the management of the volatile fatty acids (VFAs) produced.

The results showed that spent animal bedding is a slowly-degradable substrate which needs a long digestion time. However, it is a substrate suitable to be treated through AD displaying high degradation and methane production rates when processed in LBRs. This substrate is, therefore, a valuable organic resource in the agricultural context.

Spent animal bedding was shown to contain an active methanogenic population able to start the process efficiently, both in thermophilic and mesophilic temperature, without requiring a specific external inoculation. An economic study at industrial scale proved that this peculiarity can be used to diminish the investment costs and then promote the development of this process. Moreover, thermophilic temperature was proved to be less advantageous over mesophilic condition.

In fact, despite the very close methane yield reached in both temperature range, the different biogas production rates in thermophilic conditions would lead to a reduction of the final electric energy production in this condition. Mesophilic temperature was then shown to be the best operating condition for this process.

Finally, the role played by the leachate recirculation in the mobilization of the VFAs accumulating in the solid bulk was highlighted in the case of a reactor co-digesting slowly-(spent livestock bedding) and easily-degradable substrates. A strategy was even proposed to efficiently face such a problem by optimizing both the VFA extraction and consumption with the objectives of increasing the overall process efficiency.

In the end, this work allowed to optimize some important parameters for the correct management of the LBRs. This technology was proved to be efficient in the treatment of spent livestock bedding, both as a sole substrate or in co-digestion with an easily-degradable substrate.

This research study demonstrates that LBRs is an adapted process for the agricultural context and this technology can easily answer to the full scale issues usually encountered. This work represents a significant advance towards the comprehension and development of LBRs to treat agricultural waste and, more generally, to the development of renewable energies based on biomass.

Sintesi

La digestione anaerobica (DA) é un processo che permette, al contempo, il trattamento di rifiuti organici e la produzione di energia rinnovabile sotto forma di biogas. La DA a secco, in particolare, permette il trattamento di substrati solidi, offrendo svariate possibilità per il trattamento di rifiuti di origine agricola come lo sterco, un rifiuto zootecnico composto da una miscela di paglia, feci e urine. Tra le biotecnologie disponibili in digestione anerobica, i "leach-bed reactors" (LBRs) costituiscono una valida opzione, tuttavia, poco conosciuta e poco sviluppata sia a livello scientifico che industriale.

Ai fini di ottimizzare questo processo, diverse problematiche sono state affrontate: (i) la caratterizzazione bio-fisico-chimica dello sterco e del suo potenziale energetico in LBRs; (ii) l'ottimizzazione dell'inoculo dei reattori e della temperatura di digestione; (iii) la codigestione dello sterco con un substrato facilmente biodegradabile e le problematiche connesse alla gestione degli acidi grassi volatili cosi prodotti.

I risultati mostrano che lo sterco é un substrato lentamente biodegradabile che necessita di tempi di digestione lunghi. Tuttavia é un residuo agricolo adatto ad essere valorizzato attraverso la DA e le rese di degradazione e produzione di metano raggiunte in LBRs sono significativamente alte. Questo substrato é di conseguenza una risorsa organica preziosa nel contesto agricolo. E stato dimostrato che lo sterco contiene una popolazione di batteri metanogeni attiva capace, sia in regime mesofilico che termofilico, di iniziare il processo di digestione anaerobica efficacemente, senza l'aggiunta di un'inoculo esterno specifico. Un'analisi economica ha dimostrato che questa caratteristica può essere sfruttata per diminuire l'investimento iniziale di un progetto a scala industriale, favorendone, quindi, lo sviluppo sul mercato. Per di più, i risultati mostrano che il regime di termofilia per la digestione dello sterco in LBR non comporta vantaggi sulla produzione finale di metano e che, al contrario, diminuisce le rese di produzione di energia elettrica. Il regime mesofilico, quindi, si è dimostrato il più adatto a questo processo.

Infine, il ruolo giocato dalla percolazione del lisciviato sulla mobilizzazione degli acidi grassi volatili, accumulatisi nella frazione solida, é stato messo in luce nello studio di un reattore di codigestione, mescolando una frazione lentamente biodegradabile (lo sterco) ed una facilmente biodegradabile. Una strategia é stata sviluppata per affrontare sia il problema dell'estrazione che quello del consumo di questi metaboliti nell'obiettivo di migliorare le rese globali del processo.

In conclusione, questo lavoro ha permesso di ottimizzare alcuni parametri fondamentali per la gestione di un LBR. Questo tecnologia si è dimostrata efficace nel trattamento dello sterco, sia in monodigestione che in codigestione con un substrato facilmente biodegradabile. Questa ricerca mostra che l'utilizzo dei LBR é appropriato al contesto agricolo e che la modificazione dei parametri di controllo permette a questo processo di rispondere efficacemente alle problematiche affrontate sul terreno. Questo lavoro rappresenta un significativo avanzamento scientifico verso la comprensione e lo sviluppo dei LBR per il trattamento di residui agricoli e più globalmente, delle energie rinnovabili a biomassa.

Résumé

La Digestion Anaérobie (DA), ou méthanisation, est un procédé qui permet le traitement de déchets organiques et la production d'énergie renouvelable sous forme de biogaz. La DA par voie sèche permet en particulier la valorisation de substrats solides, offrant plusieurs possibilités aux traitements de résidus d'origine agricole tels les fumiers, des substrats constitués d'un mélange de paille, fèces et urine accumulés dans les litières des étables. Parmi les technologies disponibles en méthanisation, les « leach-bed reactors » (LBRs), constituent une option valide mais toutefois peu connue et peu développée soit au niveau scientifique qu'industriel.

Dans le but d'optimiser ce procédé, plusieurs problématiques ont été affrontées : (i) la caractérisation bio-physico-chimique du fumier et du potentiel énergétique exprimé dans un LBR;

(ii) l'optimisation de l'inoculation des réacteurs et de la température de digestion ; (iii) la codigestion du fumier avec un substrat facilement biodégradable et la problématique reliées à la gestion des acides gras volatiles (AGVs) ainsi produits.

Les résultats montrent que le fumier est un substrat lentement biodégradable qui nécessite un long temps de digestion. Cependant, il s'agit d'un déchet agricole adapté à la valorisation par méthanisation et dont les rendements de dégradation et de production de méthane en LBRs sont intéressants industriellement. Ce substrat est par conséquent une ressource organique précieuse dans le contexte agricole.

Il a été montré que le fumier bovin contient une population méthanogène active capable de démarrer un procédé de digestion anaérobie efficacement sans l'ajout d'un inoculum externe spécifique, autant en mode mésophile que thermophile. Une analyse économique a démontré que cette propriété peut être exploitée afin de diminuer les coûts d'investissement initiaux d'un projet à l'échelle industrielle, en favorisant de cette manière le développement de la filière. De plus, les résultats montrent que pour la digestion du fumier en LBRs le mode thermophile ne comporte aucun intérêt par rapport à la production finale de méthane (qui est similaire pour les deux régimes) et que, au contraire, la valorisation par cogénération du méthane produit en thermophile diminue le rendement de production électrique surtout à cause d'une production de méthane très importante en début de digestion. Le régime mésophile parait donc être le mode de fonctionnement le plus adapté dans ce contexte.

Enfin, le rôle joué par la percolation du lixiviat sur la mobilisation des AGV accumulés dans la fraction solide a été mis en lumière dans un réacteur de co-digestion traitant une fraction de lentement biodégradable (le fumier) et une fraction facilement biodégradable. Une stratégie a été développée afin d'étudier le problème de l'extraction et de la consommation des AGV dans le but d'améliorer le rendement global du procédé.

Pour conclure, ce travail a permis d'optimiser certains paramètres fondamentaux dans la gestion d'un LBR. Cette technologie s'est révélée efficace dans le traitement du fumier, autant en mono-digestion qu'en co-digestion avec un substrat facilement biodégradable. Ces recherches montrent que l'utilisation des LBR est appropriée au contexte agricole et que la modification des paramètres de contrôle permet à ce procédé de répondre efficacement aux problématiques du terrain. Ce travail représente une avancée significative vers la compréhension et le développement des LBRs pour le traitement des résidus agricole et, plus globalement, des énergies renouvelables mobilisant des biomasses agricoles.

Samenvatting

Vergisting is een proces voor de behandeling van organisch afval en de productie van duurzame energie. Vooral, droge vergisting het behandelen van vaste organische substraten toestaat, die diverse mogelijkheden bieden om de valorisatie van landbouwafval zoals verbruikte stalstrooisel (een mengsel van stro, feces en urine). Een van de beschikbare biotechnologie in vergisting is Leach-bed reactor (LBRs), maar nog weinig bekend proces, zowel op wetenschappelijk als op industrieel niveau.

Om dit proces te ontwikkelen, zijn verschillende kwesties onderzocht: (i) de bio-fysischchemische karakterisering van verbruikte stalstrooisel en de potentieel vergisting in LBRS; (ii) het optimaliseren van het opstarten en de bedrijfstemperatuur van de kookketels; (iii) de co-vergisting van verbruikte stalstrooisel met een makkelijk afbreekbaar substraat en de kwesties in verband met de geproduceerd vluchtige vetzuren.

De resultaten toonden dat verbruikte stalstrooisel is een langzaam afbreekbaar substraat dat een lange vergisting tijd nodig heeft. Het is echter een geschikt substraat te behandelen door vergisting tonen hoge afbraak en methaan productie snelheden bij verwerking in LBRs. Dit substraat is dan een waardevolle biologische bron in landbouwcontext.

Aangetoond is dat verbruikte stalstrooisel actief methanogene populatie om het proces efficiënt te starten bevatten, zowel bij mesofiele en thermofiele temperatuur, zonder specifieke externe inoculatie. Een economische studie op industriële schaal bewezen dat deze eigenaardigheid kan worden gebruikt om de investeringskosten te verminderen en het bevorderen van de ontwikkeling van dit proces. Bovendien werd aangetoond dat thermofiele temperatuur minder gunstig dan mesofiele temperatuur. Ondanks de zeer nauwe methaanopbrengst bereikt in beide temperatuurgebied verschillende biogasproductie prijzen thermofiele omstandigheden zou leiden tot een vermindering van de uiteindelijke productie elektrische energie in deze toestand. Mesofiele temperatuur werd vervolgens naar de beste bedrijfsomstandigheden voor dit proces.

Tenslotte, de rol van het percolaat recirculatie bij het mobiliseren van de vluchtige vetzuren die is geaccumuleerd in de vaste massa werd benadrukt in de reactor voor langzaam co-vergisting (verbruikte stalstrooisel) en makkelijk afbreekbaar substraat. Een strategie werd voorgesteld om het probleem te overwinnen door het optimaliseren van zowel de vluchtige vetzuren winning en gebruik voor de efficiëntie van het totale proces te verhogen.

Uiteindelijk, in deze studie belangrijke parameters voor het juiste beheer van de LBRs werden geoptimaliseerd. Deze technologie werd bewezen efficiënt in de behandeling van verbruikt stalstrooisel te zijn, zowel als enige substraat of in co-vergisting met een gemakkelijk afbreekbaar substraat. Dit onderzoek toont aan dat LBRs is een aangepast proces voor de agrarische context en deze technologie kan eenvoudig antwoord op de volledige schaal problemen die meestal voorkomen. Dit werk is een belangrijke stap naar een beter begrip en ontwikkeling van LBRs aan agrarisch afval te behandelen en aan de ontwikkeling van hernieuwbare energie uit de biomassa. The objectives of this chapter are to describe the context and the basic knowledge developed in the frame of this PhD work. First of all, the social and economic context, mainly focusing on French case, is defined. Then, the history and basic knowledge of the anaerobic digestion process and the main process operating parameters are described. Given the extension of the scientific information on this biological process, the author focused only on the aspects playing an important role in the understanding of this PhD manuscript. Then, the focus is set on the technological features of the anaerobic digestion (AD) process studied: leach-bed reactors (LBR). Finally, based on previously reported knowledge, the cornerstones of this PhD work are presented including the structure of the thesis manuscript. It is reminded that the literature review in this chapter aims at introducing the issues of this work and, for this reason, it should be regarded as complementary to the more specific literature overview provided in the introduction section of the research chapters (2, 3, 4 and 5).

XII

List of tables

Introduction

Generalities

Population will turn from the actual 6.9 billion to 9 billion within 40 years. Population growth is then the major challenges of 21 st century and with it all problems connected to food, water, shelter and energy supply. Energy harnessing defines the lives of all organisms and superorganisms as humans. Energy is one of the aspects that will challenge the most human development in future years [START_REF] Fox | Population: one planet, too many people?[END_REF]. Human evolution, until now, was based mainly on fossil fuels (coal, charcoal, oil, etc.) exploitation. Their use increased exponentially during the industrial revolution through the invention of the steam engine and the possibility to accomplish an amount of work unexpected before, indirectly leading towards the technical development that we know today (Smil, 2004).

However, the use of fossil fuel is connected to long biogeochemical carbon cycle. The rapid liberation of huge and slowly stored amount of carbon is seriously affecting our planet leading to the well-known climate change [START_REF] Prentice | The carbon cycle and atmospheric carbon dioxide[END_REF]. This, as well as, the depletion of fossil fuel sources in future years is pushing human kind to look into new and more sustainable energy sources. Nowadays, technologies for engineering the new world exist and just need to be optimized [START_REF] Fox | Population: one planet, too many people?[END_REF] and one should contribute actively to reach sustainable development objectives. Among the existing green biotechnologies, AD allows to produce a clean fuel (based on a closed carbon cycle with respect to atmospheric carbon dioxide) but at the same time offers a valid solution to the disposal of different kind of organic waste, to the reduction odour nuisance and of pathogens [START_REF] Chynoweth | Renewable methane from anaerobic digestion of biomass[END_REF]. AD occupies then an important place among existing biotechnologies.

However, still a lot of problems are connected to its use and implementation, and research is called to actively solve them.

Methane historical discovery

We have suggestions of the use of biogas for heating bath water in Assyria already back in the 10 th century B.C. [START_REF] Bond | History and future of domestic biogas plants in the developing world[END_REF]. However, the first observations of biogas leading to our current use dates back to 1776, when father Carlo Campi observed "piccole fiammelle" (small flames) on the surface of a lake in San Colombano al Lambro (Milan) and asks Alessandro Volta (1745-1827), a young Italian physicist, to study this phenomenon. One year later, in "Lettere sull'aria infiammabile native delle paludi (Milano 1777)" A. Volta described for the first time a new gas that he supposed coming from organic matter degradation and that he addresses to with these exact words: "Quest'aria arde assai lentamente con una bella vampa azzurrina..." (this air burn quite slowly with a beautiful blue flame) [START_REF] Fondazione | Il Metano[END_REF]. Methane and its combustion properties were discovered! Many scientists were interested by that discovery and his work was translated in several languages. A. Volta corresponded with eminent scientists like A. L.

Lavoisier (1743 -1794) in France. Indeed, this latter obtained evidence that Volta's gas was "hydrogenium carbonatrum". Only later, in 1892, this gas was named officially "methane" by the international Congress on Chemical Nomenclature [START_REF] James | Methanogenesis[END_REF]. In 1920, the understanding of this process was sufficient to stimulate the development of anaerobic digesters [START_REF] Beline | La méthanisation en milieu rural et ses perspectives de développement en France[END_REF]. However, many more years were necessary to exactly identify the biological pathways and the microorganism intervening in this process. To these days new important discoveries on this process are made suggesting that we are far from the complete understanding of this complex natural process and, more in general, of the carbon cycle in such specific environment (Vanwonterghem et al., 2016).

Real applications based on the harnessing of AD gas did not wait long after methane was discovered and developed along with research. Small devices such as lamps were already tested by A. Volta himself. Nonetheless, large-scale applications were sparingly reported in New Zealand, in the United Kingdom and India to fuel street lamps in 1890s. In 1921, in China, a digester of 8 m 3 was first commercialized while, in Germany, in 1920 a sewage treatment plant was built and, in 1950, an agricultural one. Energy production through AD gained momentum during the first oil crisis in 1970s [START_REF] Bond | History and future of domestic biogas plants in the developing world[END_REF] since price of oil raised dramatically. At that period, many countries promoted the use of alternative energy source such as biogas. China, for example, promoted the installation of household anaerobic digesters which led this country to become the biggest rural biogas user in the world at that time [START_REF] Ni | New concept for the evaluation of rural biogas management in developing countries[END_REF].

Nowadays AD is considered as no way around biotechnology. It is used both for organic matter (OM) stabilization and/or energy production both in developed and developing countries for household and industrial purposes. Biogas production through AD is a green energy source, and then an important and promising part of the world energy mix, but also a very important process for managing efficiently organic waste of different kinds [START_REF] Beline | La méthanisation en milieu rural et ses perspectives de développement en France[END_REF]. However, it is important to notice a clear temporal difference in the development of AD processes: the first ones were in fact developed to treat liquid waste streams while the processes treating the solid ones developed later.

The reasons of such a delay were linked to the strong interactions between the process parameters [START_REF] Forster-Carneiro | Anaerobic digestion of municipal solid wastes: dry thermophilic performance[END_REF], the complexity of the substrate treated, the dynamic evolution of the microbial biomass and also the substrate properties (e.g. porosity, volumetric mass density, etc.) modification during digestion [START_REF] Shi | Reactor performance and microbial 194 community dynamics during solid-state anaerobic digestion of corn stover at mesophilic and thermophilic conditions[END_REF]. The need of a dynamic and global approach, mixing biological, physical and chemical knowledges affected consistently its development. Despite of that, recent studies show that a similar level of industrial development between these two process categories has been reached, in Europe at least [START_REF] Baere | State of the art of anaerobic digestion in Europe 32[END_REF]. , 2015). Nonetheless, these latter (wind and photovoltaic) are intermittent energy sources since highly dependent on meteorological conditions and daylight whereas this is not the case for AD. Thus, energy from AD, and more broadly from biomass, presents an enormous advantage in this sense since its use can be controlled and a demand-driven is possible [START_REF] Hahn | Review of concepts for a demand-driven biogas supply for flexible power generation[END_REF]. Moreover, AD is not dependent of geophysical position (impacting wind and solar power) and it is adapted to every context.

The national French interest in developing AD sector is reflected by governmental incentives which permit to promote the implementation of industrial sites on its territory. In only these last 3 years two main modifications were done to the original statute fixing the feed-in-tariffs for electrical energy produced through AD (Decree of 19 May, 2011). The first modification included specific incentives for treatment of husbandry waste (Decree of 30 July, 2013) and the second increased the incentives for electricity production but removed the ones for heat production [START_REF] Française | [END_REF]. Wilkinson, (2011), in Australia;Tranter et al. (2011), in England. Wilkinson, (2011) clearly states that decentralized energy production through AD have to face several problems related to specific context, i.e. policy, substrates availability and technology development. Each country must then face this issue specifically and no general solutions seem possible. Each country must be able to support in the best way the development of this sector within its territory by promoting the use of the technology that better suits to the kind of substrate produced. The French study from [START_REF] Buffière | Freins et développements de la filière biogaz : les besoins en recherche et développement[END_REF] already highlighted the lack of information about dry AD and the consequent difficulty in its implementation at national scale. Thus, research in this field is particularly important in order to support industrial development and increase the AD share for reaching the forecasted renewable energy targets in France.

1.1.4 Agricultural substrates: spent livestock bedding

The analysis of the resources available on a given territory is a very important step in the development of an adapted technology suitable to its treatment.

In France, agricultural waste is estimated to be 90% of the resource that can be mobilized for energy production through AD by 2030. That would mean an annual energy production of 51 TWh.

In this part, 41% is estimated coming from animal dejections which represents about 95.5 10 6 tons of raw matter (ADEME, 2013) per year. This amount is quite constant in time because of the stability of the amount of animals grown, but a slight decrease in future years could occur because of the tendency in growing animal outside the stables.

One of the main characteristic of manure is its total solids (TS) content which can vary from few percentage points, and called then slurry, to really high percentage like 30% or 40% (ADEME, 2013) which normally characterizes manure from goats, sheep and horses. Slurry is normally found in stable where animal dejections are collected by frequently scraping the soil stable, while dry manure is produced by regular addition of a bedding material in the stabling area offering higher comfort to animals. Bedding material, most of the case straw in France, mixed with faeces and urine, accumulates in the litter for several days or weeks before being cleaned out. Such a waste is referred to as spent animal bedding [START_REF] Tait | Biogasification of solid wastes by two-phase anaerobic fermentation[END_REF], and it is the main substrate investigated in this PhD work. In France, stabling practices producing spent animal bedding are very common:

75% of the animal dejections, considering all animals, are estimated to be solid (while the rest are under a liquid form) (ADEME, 2013). Cattles represent the biggest share of husbandry in France with 7.8 million of animals in 2011 [START_REF] Franceagrimer | L'observatoire national des ressources en biomasse -Évaluation des ressources disponibles en France[END_REF]. A recent study estimated that 54% of the total amount of cattle manure is solid with a minimum TS content of 18% (Degueurce et al., 2016a). Spent cow bedding is then by far the most common in France. However, even if the number of cattle overtakes those of other kinds of livestock: 5.1 million of sheep, 1.3 million of goats and 0.5 million of horses in 2011 (Agreste, 2015;[START_REF] Franceagrimer | L'observatoire national des ressources en biomasse -Évaluation des ressources disponibles en France[END_REF], the totality of the waste produced by the husbandry of these latter is dry [START_REF] Franceagrimer | L'observatoire national des ressources en biomasse -Évaluation des ressources disponibles en France[END_REF] and then interesting as a resource. In conclusion, spent animal bedding represents an important resource in the French context and its use as a second generation biomass seems important for the development of a decentralized energy production sector in this country.

Anaerobic digestion

Introduction

AD is the natural process driving the biological conversion of organic carbon into CO 2 and CH 4 , in anaerobic condition and in redox values ranging between -300 and -500 mV [START_REF] Chynoweth | Environmental impact of biomethanogenesis[END_REF]. AD is a marvellous selective natural process because it allows reducing a large varieties of different organic substrates into one very useful molecule (i.e. methane) [START_REF] Ferreira | A thermodynamic approach toward defining the limits of biogas production[END_REF]. In nature, this process has been observed in wetlands, rice fields but also in the intestines of several animals [START_REF] Chynoweth | Environmental impact of biomethanogenesis[END_REF].

Methane is a colourless and odourless hydrocarbon, combustible with an oxygen to methane ratio of 2 and with a lower heating value of 50.01 MJ kg -1 (802.32 kJ mol -1 or 35.80 MJ Nm -3 ).

Methane is also the most reduced form of carbon and the one having the highest energy potential. In fact, Gibbs free energy yield through AD is two or three times higher compared to other bioenergy process like bioethanol and biodiesel [START_REF] Kleerebezem | Anaerobic digestion without biogas?[END_REF]. Methane is a very interesting molecule since easily exploitable in engine and transported in existing infrastructures like gas pipelines. Its production is much easier than other fuels like methanol or hydrogen whose commercial development is reduced due also to their most difficult production [START_REF] Chynoweth | Renewable methane from anaerobic digestion of biomass[END_REF]. Energy balance compared to hydrogen states that even if H 2 has effectively a higher lower heating value, only a small fraction of H 2 can be produced by OM degradation compared to CH 4 .

Compared to ethanol, methane does not need pure culture or any substrate pre-treatment, resulting then in higher process efficiencies [START_REF] Chynoweth | Renewable methane from anaerobic digestion of biomass[END_REF]. Finally, stabilization of the substrate and its possible use as organic fertilizer is an additional characteristic of AD in comparison with other biotechnologies. Thus, not only the improved energy balance but also the waste treatment and the post-treatment use are real advantages of the anaerobic digestion [START_REF] Mata-Alvarez | Anaerobic digestion of organic solid wastes . An overview of research achievements and perspectives[END_REF].

Anaerobic digestion in four steps

Four distinct chronological steps are involved in the biological transformation of OM into methane and carbon dioxide: hydrolysis, acidogenesis, acetogenesis and methanogenesis (Figure 1).

These four stages are rapidly described based on these authors description mainly: Amani et al.

(2010), [START_REF] Demirel | The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: A review[END_REF] and [START_REF] Moletta | La Méthanisation[END_REF]. which fasten the breaking of the complex molecules. The rate of this step strictly depends on substrate structure and it can become the AD rate limiting one when a complex substrate, e.g. a lignocellulosic material (such as agricultural waste), is digested. In fact, the presence of recalcitrant compounds like lignin or crystallized cellulose into these substrates hampers the proper hydrolysis of the OM. This issue is often solved by undergoing through a pre-treatment step (biological, chemical or physical) of the substrate [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF].

Acidogenesis

Acidogenesis consists in the conversion of the hydrolysed compounds into volatile fatty acids (VFAs): acetic, propionic, (iso-) butyric and (iso-) valeric acids; more complex carboxylic acids (e.g. caproic, lactic acid), alcohols (ethanol, methanol) and also H 2 and CO 2 . One of the more striking properties of acidogenic bacteria is their rapid growth. Their doubling time is estimated at 30 min [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF], which is much higher than the ones of the other steps. This disequilibrium is one of the main causes of failure in anaerobic processes.

Acetogenesis

Acetogens allow converting all VFAs and alcohols into acetate, formate, H 2 , and CO 2 . Their growth rate is slightly higher than that of methanogens, and estimated ranging between 1.5 to 4 days [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. Acetogens are divided in two groups, the OHPA or Obligate hydrogen producing acetogens, which produce acetate and H 2 from acidogenesis waste, and homoacetogens producing acetate mainly through H 2 and CO 2 . OHPA reactions are endergonic (ΔG > 0) in standard condition and only the constant consumption of the H 2 (by methanogens or homoacetogens bacteria for example) permits it to continue.

Methanogenesis

The last step of the AD, methanogenesis is driven by strictly anaerobic bacteria which belong to the Archaea domain. Methanogens doubling time is estimated to range between 2 and 4 days [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. Two main processes of methane formation are known, a first one driven by hydrogenotrophic methanogens and a second one by aceticlastic methanogens. The first one, producing about 30% of the methane in a reactor, consists in the reduction of formate or CO 2 with H 2 to produce CH 4 . These reactions are thermodynamically favoured (ΔG < 0) only when H 2 partial pressure is above 10 -6 Pa. The second pathway, producing about 70% of the methane, consists in the conversion of acetate into CH 4 and CO 2 .

Parameters influencing Anaerobic Digestion

Anaerobic process is a very complex process on which chemical, physical and biological parameters have a big influence. Knowing their effects and relationships inside an anaerobic reactor is important in order to control such process. The most important parameters affecting AD process, and more in particular AD of solid substrate, are discussed hereafter.

pH and alkalinity

pH, or the concentration of hydrogen ions in the liquid phase, is a paramount parameter in AD processes. The sensitivity to this parameter is mainly due to the coexistence of four different steps in AD in a single vessel (except in specific process designs), each one having its own optimum.

Hydrolysis and acetogenesis have their optimum respectively at 5.5 and 6.5 [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF] while neutral pH is optimum for acetogenesis [START_REF] Xu | Bioconversion of H2/CO2 by acetogen enriched cultures for acetate and ethanol production: the impact of pH[END_REF] and for methanogenesis [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. Different optimum ranges for the AD analysed as a global process were reported in literature but it can be stated that they are commonly found between pH 6.5 and 8.5 [START_REF] Bernet | Caractérisation de la mise en oeuvre de la méthanisation[END_REF]. Even if pH affects directly the micro-organisms activity, it also plays a major role in VFA and ammonia inhibitions mainly by changing their speciation and making them more available to bacteria (see paragraphs 1.2.4). Given the importance of pH for correct microorganism activity and in reason of the higher sensibility of methanogens to this parameter, pH is often controlled at industrial level by addition of external compounds like sodium bicarbonate which moves pH towards neutral values [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF].

The pH stability in a given system depends on the alkalinity, or the buffering capacity of the reactive medium. The pH variation when acidic or basic compounds are added to the system is then lower when the alkalinity is significant. The main weak acid/base couples which participate to the overall buffering capacity of a digester are carbonate, ammonium, phosphate, VFAs and sulfide [START_REF] Sun | Monitoring Volatile Fatty Acids and Carbonate Alkalinity in Anaerobic Digestion: Titration Methodologies[END_REF]. However, the main contribution to the alkalinity in anaerobic digester is given by carbonates (H 2 CO 3 /HCO 3 -, pKa ~ 6.4) which are found at really high concentration in anaerobic digester. A second important source of alkalinity, especially when animal dejections which are rich in nitrogen compounds are used, is ammonia. High amounts of ammonia in the system cause an increase of the pH towards alkaline values (NH 3 /NH 4 + , pKa = 9.25) and could cause failure of the system.

Temperature

Temperature is generally known to affect each biological process by increasing its kinetics following the Vant'hoff law. Its impact is so strong that the differences in kinetics between acidogens and methanogens have often been solved by carrying out this two steps at different temperatures in order to slow down the first and speed-up the second [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF].

In nature methane is formed over a wide range of temperature form 0°C (ice fields) to 97 °C (hot springs) [START_REF] Zeeman | Psychrophilic digestion of dairy cattle and pig manure: Start-up procedures of batch, fed-batch and CSTR-type digesters[END_REF]. However, three optimal temperature ranges were identified in AD: psychrophilic (10°C to 20°C), mesophilic (20°C to 45 °C) and thermophilic (50 °C to 65 °C) [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. Indeed, each optimum corresponds to the highest activity of different specific populations.

Psychrophilic conditions are poorly studied until now and applications are often restrained to specific zones with cold climate. Because of their really slow kinetics, psychrophilic reactors often need to be inoculated massively [START_REF] Lettinga | Challenge of psychrophilic anaerobic wastewater treatment[END_REF][START_REF] Massé | Dry anaerobic digestion of high solids content dairy manure at high organic loading rates in psychrophilic sequence batch reactor[END_REF]. The most employed processes are run under mesophilic conditions [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF] since they allows to have high degradation kinetics and their energy requirements are easily filled (e.g. through the heat recovered by biogas burning in cogeneration engines). Thermophilic temperatures present, on the other side, interesting advantages in relation to kinetics and consequent reduced digestion time for several substrates [START_REF] Hegde | Comparison of thermophilic and mesophilic onestage, batch, high-solids anaerobic digestion[END_REF][START_REF] Labatut | Conventional mesophilic vs. thermophilic anaerobic digestion: a trade-off between performance and stability[END_REF][START_REF] Pohl | Thermo-and mesophilic anaerobic digestion of wheat straw by the upflow anaerobic solid-state (UASS) process[END_REF]. Moreover, this allows to reduce significantly the amount of pathogens in the digestate [START_REF] Gómez | Digestion of cattle manure under mesophilic and thermophilic conditions: Characterization of organic matter applying thermal analysis and 1H NMR[END_REF][START_REF] Jha | Comparison between Wet and Dry Anaerobic Digestions of Cow Dung under Mesophilic and Thermophilic Conditions[END_REF][START_REF] Kim | Comparative process stability and efficiency of anaerobic digestion; mesophilic vs. thermophilic[END_REF], which can be important in particular contexts where inlet substrates are biologically contaminated. However, some drawbacks are connected to thermophilic processes.

The higher kinetics can cause a rapid hydrolysis and acidogenesis leading to a VFA accumulation in the system with possible failure. Thermophilic processes are found to be more sensitive to environmental changings because of the restricted microorganisms populations involved compared to the mesophilic ones [START_REF] Moset | Mesophilic versus thermophilic anaerobic digestion of cattle manure: methane productivity and microbial ecology[END_REF]. Moreover, sensitivity to lipid-rich substrates were also recorded since thermophilic microorganism are inhibited by high concentration of long chain fatty acids (Palatsi et al., 2009). Nitrogen inhibition is also often reported in literature in relation to thermophilic conditions. Thermophilic treatment can then be problematic when treating nitrogenrich substrates like animal dejections. In fact, at this temperature the nitrogen equilibrium between NH 4 + /NH 3 is pushed toward NH 3 which is considered to be the most toxic form for microorganisms since permeable for cell (see paragraph 1.2.4). However, [START_REF] Yenigün | Ammonia inhibition in anaerobic digestion: A review[END_REF] reported also two different studies showing that the ammonia inhibition thresholds are higher in thermophilic conditions compared to mesophilic ones, reducing by far this issues.

Inoculum

Inoculation refers to the amount of active biomass brought to a system at its start-up in relation to the amount of fresh matter treated. Given the repeated start-up in a batch system, inoculation is particularly important for discontinuous process rather than for continuous ones in which inoculation is carried out only once. The inoculation is expressed in literature in very different way taking into account the VS, the TS or the raw weight of the substrate and the inoculum used [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF]. As an example: [START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF] used the substrate-toinoculum S/I on a volatile solids basis (substrate VS/ inoculum VS); [START_REF] Yap | Pilot-scale testing of a leachbed for anaerobic digestion of livestock residues on-farm[END_REF] used the inoculum-to-substrate ratio I/S on a VS basis too; [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF] and [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF] used, instead, the I/(I+S) expressed on a TS basis (inoculum TS/(inoculum + substrate) TS).

The amount of inoculum brought to a system has a big impact on the process kinetics since it accelerates the consumption of intermediate compounds and then the overall AD degradation kinetics [START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF]. Addition of high amount of inoculum permits to reduce the digestion period but inoculum occupy a significant volume in the reactor reducing in that way the volumetric productivity [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF] of the system. The look for the optimum conditions between kinetics and efficiency is then a real issue in each digester.

For optimal digestion performance, the amount of inoculum should be adapted to the kind of substrate treated. In fact, easily-degradable substrates, tending to produce rapidly VFAs would need higher amount of inoculum to counteract rapid reactor acidification. As an example, reactor loaded with easily-degradable substrates like maize silage showed risk of acidification with a S/I (VS basis) of 0.5 [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF] while a higher S/I (VS basis) between 10 and 4.4 was considered optimal to digest spent horse bedding in similar batch reactors [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF]. In this sense, [START_REF] Liew | Methane production from solid-state anaerobic digestion of lignocellulosic biomass[END_REF] found an optimum S/I (VS basis) of 2 for several lignocellulosic waste like corn stover, wheat straw, leaves and yard waste.

Inoculum can influence the digestion also through its chemical properties like C/N ratio (section 1.2.3), alkalinity, nutrients, etc. [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF]. However, a very important role is played by the adaptation of the bacteria consortium to a specific substrate composition and/or degradation conditions like temperature and nitrogen content as the following example show. The use of three different liquid inocula and three different S/I in the dry digestion of corn stover showed that to each inoculum used corresponded a different optimum S/I (VS basis), as well as a different methane yields (at the optimum of each condition) [START_REF] Xu | Comparison of different liquid anaerobic digestion effluents as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover[END_REF]. [START_REF] Guendouz | Dry anaerobic digestion in batch mode: design and operation of a laboratory-scale, completely mixed reactor[END_REF] found that a non-adapted inoculum can affect methane production kinetics when treating municipal solid waste and three consecutive batches were necessary before reaching stable methane yield. [START_REF] Zeeman | Psychrophilic digestion of dairy cattle and pig manure: Start-up procedures of batch, fed-batch and CSTR-type digesters[END_REF] found that inoculum can adapt to temperature variations but that changing from mesophilic to physchrophilic condition is slower than changing from mesophilic to thermophilic temperatures. Van Velsen (1979) reported the absence of lag phase in the methane production when varying the ammonia concentration in the digester (from 0.6 to 3 g L -1 of ammonia) where an inoculum previously adapted to high ammonia concentration (2.4 g L -1 of ammonia) was used; this was not the case when a non-adapted inoculum (adapted to 0.8 g L -1 of ammonia) was used.

To sum-up inoculation is a paramount step of the AD. The good amount and the best inoculum in respect to the substrate must be found in order to optimize a batch process.

Substrate physical properties

Physical properties like particle size, porosity and hydraulic conductivity have a significant impact on the digestion process of solid waste. Biological hydrolysis of solid substrates occurs via a surface biofilm degradation mechanism (Vandevivere et al., 2003) which makes particle size playing an important role in AD. [START_REF] Liotta | Effect of moisture on disintegration kinetics during anaerobic digestion of complex organic substrates[END_REF] showed that by increasing the particle size of carrots, an easily-degradable substrate, methane production kinetics decreased but not the final methane yield. A surface dependent disintegration kinetic was successfully used to model this process [START_REF] Esposito | Modelling the effect of the OLR and OFMSW particle size on the performances of an anaerobic co-digestion reactor[END_REF]. On the other side, [START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF] showed that by milling (and then decreasing particle size) straw, a lignocellulosic substrate, not only the degradation kinetic increased but also the methane yield, since this particle size reduction increased the accessible surface area to biological degradation by breaking recalcitrant compounds like lignin [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF].

Among physical solid properties, porosity (empty volume / total volume) and hydraulic conductivity, indirectly related to particle size (Murray, 1995) and then to mass transfer, were

shown to play an important role in percolation process as LBRs treating solid waste. In those cases, methane yield was shown to increase by enhancing the porosity of a solid substrate by addition of inert material: pistachios shell to digestate manure [START_REF] Myint | Enhancing anaerobic hydrolysis of cattle manure in leachbed reactors[END_REF] and gravel to municipal solid waste (Valencia et al., 2009). During digestion, the decrease of the porosity in a solid bulk and, as a consequence of its hydraulic conductivity, can cause problems of water channelling in percolating systems. This was shown to hamper the optimal substrate degradation [START_REF] André | Quantifying physical structure changes and non-uniform water flow in cattle manure during dry anaerobic digestion process at lab scale : Implication for biogas production[END_REF]. Physical properties of the substrates and their evolution during digestion play then a very important role in the degradation of solid substrates.

Substrate composition: C/N

Carbon-to-nitrogen ratio, C/N, is an important parameter since it reflects the substrate balance in respect to the microorganism growth, and then the efficiency of the process. Carbon quantifies the energy source in the substrate whereas nitrogen its nutritive capacity. C/N is strictly linked to the effect of nitrogen on the AD performance, indeed, limitation in nitrogen leads towards limited bacterial growth and poorer performance while too high concentration leads to inhibition (see paragraph 1.2.4). Even if the optimum C/N value varies in relation to the substrate [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF], this is commonly set between 20 and 30 [START_REF] Forster-Carneiro | Anaerobic digestion of municipal solid wastes: dry thermophilic performance[END_REF][START_REF] Hills | Effect on Carbon:Nitrogen ratio on the anaerobic digestion of dairy manure[END_REF][START_REF] Jha | Research advances in dry anaerobic digestion process of solid organic wastes[END_REF]Xu et al., 2014). Such a big range is often due to the fact that often the fractions of carbon and nitrogen considered are not only the available ones but the total amounts present in the reactor.

Optimal C/N was also shown to vary in relation to operating temperature mainly because of a variable need in both sources and a different inhibition threshold connected to nitrogen speciation:

C/N ratio of 27 and 31 were respectively found as optimum in mesophilic and thermophilic condition for reactors digesting dairy manure, chicken manure and straw [START_REF] Wang | Effects of temperature and Carbon-Nitrogen (C/N) ratio on the performance of anaerobic co-digestion of dairy manure, chicken manure and rice straw: Focusing on ammonia inhibition[END_REF].

Co-digestion is a very useful way to modify the C/N of a fed mixture. As an example, agricultural waste (e.g. straw or cereal residues) are often poor in nitrogen and have a C/N higher than 50 [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. These wastes can then be co-digested with animal manures which are known to contain high amount of nitrogen (see paragraph 1.2.4). Their co-digestion has been widely described in literature and a lot of examples has been reported by [START_REF] Mata-Alvarez | A critical review on anaerobic co-digestion achievements between 2010 and 2013[END_REF].

Total solids

The amount of water is a parameter particularly important in AD process working with high TS content. Water deficiency highly limits microorganism activity and mobility, coproducts dilution, diffusion and transfer of soluble compounds (Abbassi- [START_REF] Abbassi-Guendouz | Total solids content drives high solid anaerobic digestion via mass transfer limitation[END_REF][START_REF] Pommier | A Logistic Model for the Prediction of the Influence of Water on the Solid Waste Methanization in Landfills[END_REF][START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF]. Because of that, lower methane productions were reported by Abbassi- [START_REF] Abbassi-Guendouz | Total solids content drives high solid anaerobic digestion via mass transfer limitation[END_REF] and [START_REF] Hyaric | Influence of substrate concentration and moisture content on the specific methanogenic activity of dry mesophilic municipal solid waste digestate spiked with propionate[END_REF] when increasing TS in the digesters. In dry AD, 30% TS was found to be a critical threshold above which performance drops significantly (Abbassi- [START_REF] Abbassi-Guendouz | Total solids content drives high solid anaerobic digestion via mass transfer limitation[END_REF]. High TS content in the digester was found to cause higher accumulation of intermediate compounds, like VFAs [START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF], causing, indirectly, a process inhibition (see paragraph 1.2.4).

Finally, the low total solid content in dry AD process modifies the microorganism growth mode and, as a consequence, their morphology. The high TS content, often linked to lack of free water, hampers the development of free microorganisms and favours the development of biofilms on the substrate (Vandevivere et al., 2003). In dry system the substrate would play both the role of energy source and support for biomass which seem an advantageous strategy since the use of organic material as support material for bacterial growth increases methane production significantly [START_REF] Ward | Optimisation of the anaerobic digestion of agricultural resources[END_REF].

Inhibition

VFA

All along the AD process operation, intermediate compounds are contemporarily produced and consumed. However, when their production and consumption rate differs, intermediates accumulate and inhibitory concentrations can be reached. The intermediates accumulating the most in AD process are VFAs. The most observed VFAs are acetic, propionic and butyric acids (Karthikeyan and Visvanathan, 2012a): indeed, acetic acid is the main precursor of methane and all carbonate compounds (except CO 2 ) pass from this form before being transformed into methane;

propionic and butyric acids consumption requires specific thermodynamic conditions (ΔG > 0 in standard conditions) to be consumed, in particular propionic acid [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF], making their consumption more difficult.

VFA accumulation directly impacts the pH with a tendency to decrease its value (pKa close to 4.7). The pH drop has, as previously mentioned, a strong impact on all biochemical equilibrium in the system (see paragraph 1.2.3). At low pH, VFAs are mainly found under a unionized form which make them more toxic for cells since they can easily pass through their membrane [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF]. VFA inhibition is then strictly linked to pH (and then to alkalinity which could hamper pH drop) more then to their real amount even if some authors report inhibitory level of 0.9 g L -1 of propionic acid [START_REF] Wang | Effects of volatile fatty acid concentrations on methane yield and methanogenic bacteria[END_REF], 2 g L -1 for acetic acid and 8 g L -1 for the total acid concentration (Karthikeyan and Visvanathan, 2012a). In fact, [START_REF] Gourdon | Effects of Propionic Acid Concentration on Anaerobic Digestion of Pig Manure[END_REF] reported that no inhibition to propionic acid up to a concentration of 6 g L -1 at a pH of 7.4 digesting pig manure, and that propionic acid should simply be considered as a warning more than as a disturbance of the process. The same conclusions for butyric and isobutyric acids were found by digesting manure in continuous-stirred tank reactor (CSTR) at thermophilic temperature [START_REF] Ahring | Volatile fatty acids as indicators of process imbalance in anaerobic digestors[END_REF]. VFA toxicity should be looked with caution and analysed in every specific context in relation to the pH and the alkalinity. For this reason several indirect parameters like acetic acid-to-propionic acid ratio or VFAs-to-alkalinity ratio are often used to control possible critic states in anaerobic digesters (Karthikeyan and Visvanathan, 2012a).

Nitrogen

Proteins, urea and nucleic acids are the main source of nitrogen in anaerobic digesters [START_REF] Yenigün | Ammonia inhibition in anaerobic digestion: A review[END_REF]. More specifically, in manure the ammonia is derived mainly from the urea component contained in urines [START_REF] Whitehead | Volatilization of ammonia from urea applied to soil: influence of hippuric acid and other constituents of livestock urine[END_REF]. Cow urine, as an example, contains from 6.8 to 21.6 g N L -1 [START_REF] Bristow | Nitrogenous constituents in the urine of cattle, sheep and goats[END_REF]. During AD, the hydrolysis and solubilisation of proteins, urea and nucleic acids leads to mineralization of nitrogen and its accumulation in the systems since nitrogen in not directly ejected from the system as CH 4 and CO 2 ;

this makes the digestate a great source of nitrogen and of other fertilizer compounds. (Vaneeckhaute et al., 2013). [START_REF] Yabu | Thermophilic twostage dry anaerobic digestion of model garbage with ammonia stripping[END_REF] and [START_REF] Yenigün | Ammonia inhibition in anaerobic digestion: A review[END_REF] reported, for example, a mineralization ranging between 60% to 80% of total organic nitrogen present in substrates. This range can evidently change from process to process in relation to degradability, retention time, process design, etc.. Even if nitrogen is necessary for normal bacterial activity, high concentrations of nitrogen compounds have been reported as inhibitory of AD, especially for methanogens [START_REF] Kayhanian | Ammonia Inhibition in High-Solids Biogasification: An Overview and Practical Solutions[END_REF].

According to [START_REF] Rajagopal | A critical review on inhibition of anaerobic digestion process by excess ammonia[END_REF], concentrations between 50 and 200 mg L -1 are beneficial to AD. However inhibiting level are very disparate in literature, varying from 1.4 to 14 g L -1 (Chen et al., 2008). This is due to the difference in the system and the substrate used, the inoculum adaptation, process temperature and pH.

Inorganic nitrogen is present in anaerobic digestors in two forms: NH 3 (called free ammoniacal nitrogen, FAN) and NH 4 + . Two mechanisms are hypothesised to inhibit AD, (i) NH 4 + inhibiting the enzymes synthesing methane directly and (ii) passive diffusion of NH 3 in the cell causing proton imbalance and/or potassium deficiency [START_REF] Kayhanian | Ammonia Inhibition in High-Solids Biogasification: An Overview and Practical Solutions[END_REF]. This latter is recognized as the most important inhibitory mechanism. The FAN is related to the total ammoniacal nitrogen, TAN, by a relation varying in relation to temperature and pH (Karthikeyan and Visvanathan, 2012a). By increasing temperature, the equilibrium pushes towards NH 3 which is the reason why thermophilic conditions are often associated to ammonia inhibition. However, according to Yenigün and Demirel (2013), higher NH 3 concentration in thermophilic condition are also accompanied by higher threshold for inhibition. In addition, several authors showed also that TAN inhibition diminished in presence of other ions as K + , Ca 2+ , Mg 2+ [START_REF] Chen | Inhibition of anaerobic digestion process: a review[END_REF], but details about these interactions are still poor.

Metal ions -Potassium and Sodium

Potassium is not often directly addressed as inhibitory; however, its high concentration in spent animal bedding makes it a valuable element to consider. Indeed, macro-minerals such as potassium and sodium are essential in animal nutrition to ensure proper functioning of their metabolism, 177 g K d -1 cow -1 and 66.5 g Na d -1 cow -1 for lactating dairy cattle [START_REF] Pettygrove | Dairy Manure Nutrient Content and Forms[END_REF].

Given the animal diet it is normal to find high concentration of K + and Na + in the animal excrements. [START_REF] Fang | Anaerobic co-digestion of desugared molasses with cow manure; focusing on sodium and potassium inhibition[END_REF] and [START_REF] Pettygrove | Dairy Manure Nutrient Content and Forms[END_REF] reported a concentration of 4.4 g K + L -1 (0.57% of the total dry weight) and 1.1 g Na + L -1 in cattle manure. Cations, such as potassium and sodium, are required for the effective functioning of AD systems. However, at high concentrations, it may cause inhibition of AD process (Karthikeyan and Visvanathan, 2012b). The K + and Na + inhibitory effect is rarely mentioned in literature. Low concentrations of potassium (< 400 mg L -1 ) have demonstrated an enhancement of the AD in mesophilic and thermophilic condition [START_REF] Chen | Inhibition of anaerobic digestion process: a review[END_REF]. Concerning potassium and sodium, inhibition of AD process has been reported from 6 g K + L -1 to 28 g K + L -1 [START_REF] Chen | Effect of potassium inhibition on the thermophilic anaerobic digestion of swine waste[END_REF][START_REF] Fang | Anaerobic co-digestion of desugared molasses with cow manure; focusing on sodium and potassium inhibition[END_REF][START_REF] Jard | Batch and semi-continuous anaerobic digestion of Palmaria palmata: Comparison with Saccharina latissima and inhibition studies[END_REF][START_REF] Kugelman | Cation Toxicity and Stimulation in Anaerobic Waste Treatment[END_REF] treating sludge, swine waste, algae and desugared molasses and from 3 to 11 g Na + L -1 [START_REF] Jard | Batch and semi-continuous anaerobic digestion of Palmaria palmata: Comparison with Saccharina latissima and inhibition studies[END_REF].

Other inhibitions

A high number of elements are necessary for correct bacterial growth. At too low concentrations a deficiency can occur, while at too high concentrations an inhibition could take place. In both cases the reactor performance will be compromised [START_REF] Bernet | Caractérisation de la mise en oeuvre de la méthanisation[END_REF].

Cobalt, iron, nickel, sulfide, selenium, tungsten, molybdenum, barium, magnesium, and sodium are the micronutrients required in relatively small quantities by some microorganisms [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF] and their presence should be ensured in the digesters. Beside them, many other compounds were found to affect the AD process: light metals (e.g. Na + , K + , Ca 2+ , Mg 2+ , etc.), heavy metals (e.g. Zn 2+ , Cd 2+ , Hg 2+ , etc.), long chain fatty acids (LCFA). All these possible inhibitions

will not be discussed in this manuscript since not directly linked to the topic. The readers should refer to the work of [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF], [START_REF] Rajagopal | A critical review on inhibition of anaerobic digestion process by excess ammonia[END_REF] and [START_REF] Chen | Inhibition of anaerobic digestion process: a review[END_REF] for further details.

Anaerobic digestion technologies -Leach-Bed Reactors (LBRs)

Technology classification

In order to well understand the functioning of LBRs, a short overview on AD technologies is necessary to put LBRs in relation with all the other existing processes.

The adaptation to the context and to the variety of organic substrates led to the development of several anaerobic digestion reactor designs, and new reactor designs are still proposed. In this sense, animals represent a real source of inspiration since their digestive systems offer a very large variety of examples for designing new and more efficient AD plants [START_REF] Godon | Overview of the Oldest Existing Set of Substrate-optimized Anaerobic Processes: Digestive Tracts[END_REF]. Three main process characteristics will be highlighted here to finally define LBRs: the total solids, the operation mode and the complexity of the system.

Total solid content is a cornerstone of AD process, separating processes able to treat solid waste streams from the ones treating liquid waste streams. Two main categories are identified: wet process, working at a TS < 15% and dry process working at a TS > 15% [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. As mentioned previously, dry process evolution at industrial scale is far more recent (1980s') compared to wet technologies; this is also reflected by the scientific research in this field (see paragraph 1.1.1). However their rapid development at industrial scale in the last years [START_REF] Baere | State of the art of anaerobic digestion in Europe 32[END_REF]) is due to their interesting features in comparison to wet processes Table 1. The reduced use of water allows having a smaller working volume to build and to warm up, reducing significantly the investment costs. Moreover, this contributes to the production of a solid digestate which does not need any further post-treatment to be handled and spread onto soils.

Consequent simpler design and reduced costs for substrate transport are further positive characteristics of dry processes (Karthikeyan and Visvanathan, 2012a). Nonetheless some drawbacks are connected to them and in particular to the biological aspects. In dry processes OM in often loaded without any pre-treatment (e.g. particle size reduction) which makes of it a heterogeneous matrix more difficult to digest than aqueous streams or small particle-size substrates.

This diminishes the conversion performance and generally increases digestion time. However, biodegradability is not affected if proper conditions (like not too high TS) in the digesters are maintained [START_REF] Jha | Research advances in dry anaerobic digestion process of solid organic wastes[END_REF]. Finally, high organic load could result in reactor acidification and generally in a higher inoculation. However, the heterogeneity of the matrix can also represent a positive aspect of dry systems because of the theoretical separation of methanogenic and acidic zones inside of the reactor which make it more robust in biological terms [START_REF] Martin | The Site of Reaction in Solid-State Digestion[END_REF].

The operational mode refers to the way the reactor is fed. Two main categories are defined:

continuous and discontinuous (or batch) reactors. In the first configuration, new substrate is continuously added to the reactor and the same volume is taken out. In relation to the frequency of feeding the definition of semi-continuous reactors is sometimes used. On the other hand, in batch process the substrate is loaded in the digester which is then completely emptied after completion of the digestion (Vandevivere et al., 2003). In dry discontinuous reactors, no moving parts are present, which, besides reducing investment and maintenance costs, allows accepting undesirable like pebbles or ropes which are naturally present in the inlet OM in rural areas. However, they suffer of reduced mixing and not complete substrate degradation. Problems of clogging and percolation are often reported while discontinuous loading impact biogas production and start-ups which have to be repeated several times a year and which make the operator face risks of explosion. In spite of these drawbacks (further detailed in the next paragraph), poorer degradation efficiency are often balanced by lower investment and energy requirement which makes them suitable for farm-scale implementation [START_REF] Weiland | Biogas production: Current state and perspectives[END_REF].

System complexity refers to physical separation of the several biological steps of AD (see paragraph 1.2.2). Two main categories are defined in literature: one-stage when all the different AD steps take place in the same reactor and two-stage when hydrolysis/acidogenesis and acetogenesis/methanogenesis are separated and take place in two different reactors [START_REF] Kothari | Different aspects of dry anaerobic digestion for bio-energy: An overview[END_REF]. The choice between these two configurations is often done based on the type of substrate used and, more in particular on its aptitude to undergo acidification. Examples of single or twostage process will be given using, as an example, the LBR in the next paragraph.

1.3.2 Leach-bed reactors (LBRs)

Process description

LBRs are dry and discontinuous digesters which can be used in several configurations (complexity) because of their aptitude to be coupled. LBRs consist in the separation of a solid static phase, which is the solid dry substrate, and a liquid mobile phase called leachate. The simplest LBR configuration is the single-stage, called also one-stage or batch (Figure 2). This latter consists in the use of only one reactor where all the four steps of AD take place simultaneously (see paragraph 1.2.2). Such a configuration needs the rector to be well inoculated from the beginning in order to not fall into acidification because of the high organic load (see paragraph 1.2.3). However, even if the use of a single-stage system is common at a lab-scale, the configuration coupling several reactors is often preferred at industrial-scale: sequential LBRs configuration. This latter was first developed in the USA and known as SEBAC (Sequential Batch Anaerobic Composting) [START_REF] Chynoweth | A novel process for anaerobic composting of municipal solid waste[END_REF] (Figure 2). This consists in the use of three reactors in sequence staggered in time: new, mature and old reactor (which refers to the digestion time). In this configuration the freshly loaded reactor (new) is connected through the leachate to the old one with the aim of injecting a stabilized leachate, transporting alkalinity and inoculum, on the top of the new reactor and sending solubilised flushed compounds (mainly VFAs) from the new to the old reactor in order to consume them rapidly and do not inhibit the new reactor start-up. Once stabilized, the new reactor can be run as a single-stage and later it could help a new reactor to start (closing the cycle).

Sequential LBRs have the advantage to overtake the initial acidification because of the synergy created between new and old reactors in consuming VFAs. This configuration permits to treat very easily-degradable substrate like municipal solid waste [START_REF] Chynoweth | Sequential Batch Anaerobic Composting of the Organic Fraction of Municipal Solid Waste[END_REF] or market waste (Tubtong and Towprayoon, 2010).

Finally, LBRs were used also as simply acidification reactors aiming at producing VFAs that were extracted and sent in a methanogenic reactor, often an UASB (Upflow Anaerobic Sludge Blanket), and then called hybrid batch-UASB system (Figure 2). This configuration, completely separating acidogenesis and methanogenesis, is a classic example of a two-stage system, first proposed by [START_REF] Ghosh | Solid-Phase Methane Fermentation of Solid Wastes[END_REF] in order to optimise separately the acidogenesis and methanogenesis phase (whose optimum condition are known to be different (see paragraph 1.2.3), reaching in that way better performance. This configuration was successfully developed by [START_REF] Shin | Multi-step sequential batch two-phase anaerobic composting of food waste[END_REF] and called MUSTAC (Multi-Step Anaerobic Composting). MUSTAC consisted in several acidic

LBRs and only one UASB for VFAs conversion into methane with the double aim of increasing the total methane production and smoothing overall methane fluctuations. Acidogenic reactors were characterized by a short digestion time [START_REF] Browne | Improving hydrolysis of food waste in a leach bed reactor[END_REF][START_REF] Nizami | Role of leaching and hydrolysis in a two-phase grass digestion system[END_REF] (often coupled with a post-treatment like composting to further stabilize the substrate) and also by the recovery of certain amounts of hydrogen [START_REF] Han | Performance of an innovative two-stage process converting food waste to hydrogen and methane[END_REF]. The production of hythane, a mixture of hydrogen and methane, the first issued by the dark fermentation in acidogenic reactor and the second by the anaerobic digestion in methanogenic reactor, is then possible in such a configuration [START_REF] Liu | States and challenges for high-value biohythane production from waste biomass by dark fermentation technology[END_REF]. However, in spite of the interest of this process, hythane production is beyond the scope of this manuscript and is not described any further.

Recirculation in leach-bed reactor

A particular aspect of LBRs concerns the leachate recirculation. Indeed, this seems particularly important in dry static reactor where substrate is not mixed as LBRs. The effects of leachate recirculation on the digestion process are associated to various complementary mechanisms: humidification, transfer and inoculation [START_REF] Komilis | The effect of landfill design and operation practices on waste degradation behavior: a review[END_REF].

The first effect played by leachate recirculation is the humidification of the solid bulk phase.

It is reminded that high TS content inside the reactor decreases microorganism activity and, as a consequence the reactor performance (see paragraph 1.2.3). A not efficient aspersion of the leachate on the solid bulk top and problem of channelling, compaction and unidirectional flow are often the main cause of a delayed reach of optimal humidity inside the reactor (Uke and Stentiford, 2013). At industrial scale, the use of electrical tomography in LBRs stressed the difficulty in reaching optimal humidification of the substrate and the need in optimizing injection technologies (Degueurce et al., 2016b). To face this problem, flooding the substrate all along the digestion was tested by [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF] while [START_REF] Benbelkacem | Effect of leachate injection modes on municipal solid waste degradation in anaerobic bioreactor[END_REF] tested 12 hours flooding and drain. Both of them reported a positive impact on reactor performance compared to discontinuous recirculation: the first higher kinetics but similar methane production after 60 days and the second lower kinetics but higher methane yield after 600 days of operation (mathematical simulation between 200 and 600 days). In addition, flooding 30% of the bed was found to give better methane yield than no flooding by André et al. (2016b). However, in spite of the positive results obtained, flooding questions the design of the processes at industrial scale (e.g. garages are not designed to support such liquid pressure inside) but remains an option that needs to be further investigated to discover all its potentiality and limits.

Discontinuous recirculation is considered a good mean to improve biodegradation. In process simulating landfill degradation of municipal solid waste in bioreactors (long digestion time and small recirculated volumes) the increase of the leachate rates is found a good mean to reduce digestion time [START_REF] Benbelkacem | Effect of leachate injection modes on municipal solid waste degradation in anaerobic bioreactor[END_REF][START_REF] Chugh | Effect of recirculated leachate volume on MSW degradation[END_REF][START_REF] Šan | Impact of various leachate recirculation regimes on municipal solid waste degradation[END_REF][START_REF] Sponza | Impact of leachate recirculation and recirculation volume on stabilization of municipal solid wastes in simulated anaerobic bioreactors[END_REF]. However, compared to flooding, discontinuous recirculation resulted in lower kinetics [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF]. Continuous recirculation, on the other side, was found with no positive effect compared to discontinuous recirculation in a well inoculated reactor treating maize silage and it was found even to favour acidification in not well inoculated reactor [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF]. This

shows the importance of the inoculation and the substrate treated on the role of recirculation.

The second role played by the leachate recirculation is the transport of nutrients and intermediate compounds through the bed [START_REF] Browne | Improving hydrolysis of food waste in a leach bed reactor[END_REF][START_REF] Jha | Research advances in dry anaerobic digestion process of solid organic wastes[END_REF][START_REF] Šan | Impact of various leachate recirculation regimes on municipal solid waste degradation[END_REF]. This seems particularly important in a system as a LBR where no mixing is done. The role of transport was elucidated by testing the performance of a LBR loaded with different layer of substrate and inoculum (Veeken and Hamelers, 2000). The VFA accumulation in fresh substrate layers (leading to inhibition) when no recirculation was applied and the good performance obtained (no VFAs accumulation) when this latter was effectuated proved the importance of leachate recirculation in mobilizing intermediates in LBRs. Moreover, not only the compounds but also microorganisms were proved to be transported by leachate recirculation and to colonized the solid bed in the sense of recirculation treating food waste (Xu et al., 2014). It is easy to understand that the role of transfer acquires a greater importance in coupled systems where inoculum and nutrients can be exchanged (i.e., as in SEBAC process). The system complexity plays then a very important role on the choice of the best recirculation strategy.

If the qualitative roles of recirculation are relatively well known, their actual setting (e.g.

volume recirculated and frequency of recirculation) are difficult to generalize because of the interrelation with other parameters like substrate degradability, inoculation and, digester and process design. This means that specific assessment of these parameters in LBRs treating a slowlydegradable substrate as spent animal bedding are necessary.

Industrial LBR-based processes

Generalities

Mainly based on the process developed in France by 1937 by Ducellier and Isman to treat spent animal bedding with vegetable waste in underground silos [START_REF] Molnar | Solid-state methane generation[END_REF], several process using the principle of LBR have been industrialized. The most known in scientific literature are Biocel and SEBAC.

Based on the PhD thesis of Ten Brummeler, (1993) and his several publications (Ten Brummeler and Koster, 1990;Ten Brummeler et al., 1992, 1986), an industrial site using Biocel technology was constructed by Orgaworld in 1997 in Lelystad, The Netherlands. This site operated 14 digesters of 720 m 3 (480 m 3 working volume) working in parallel with a batch duration of 21 days at mesophilic temperature and treating 50,000 t of bio-waste (organic fraction of municipal solid waste (MSW) per year (Ten Brummeler, 2000).

On the other side, based on the work of [START_REF] Chugh | Degradation of unsorted municipal solid waste by a leach-bed process[END_REF], [START_REF] Chynoweth | A novel process for anaerobic composting of municipal solid waste[END_REF][START_REF] O'keefe | Sequential Batch anaerobic composting of municipal solid-waste (MSW) and yard waste[END_REF], an industrial SEBAC process was implemented in Florida (USA) by the company Sigarca (USA, 1995) for the treatment of different kind of bio-waste like municipal solid waste or animal waste.

However, beside these two examples, many other companies developed or are developing their own technologies in Europe: Bekon, Bioferm, Smartferm, Kompoferm, etc and finally Naskeo

Environnement. All based on the same principles, some specificity concerning the garage tightness, door opening system, building material (concrete garages or metal boxes) and specific leachate control differentiate them.

Naskeo/Methajade process : LBR configuration of this PhD thesis

This LBR-based process commercialized by Naskeo/Methajade was firstly developed by the French company Methajade for the treatment of farm waste. In 2014 Naskeo Env. acquired this company and improved this process. Nowadays, Naskeo Env. has 15 full scale plant operating on this dry discontinuous process in France [START_REF] Naskeo | Naskeo références construction[END_REF]. In the following paragraph, a general garage-system based on the Naskeo/Methajade design is described. The main problematic connected to this process will be clearly highlighted in order to better understand the issues treated in the next chapter of this manuscript.

Process description

Digesters and tank

Two main components compose a LBR process, the digesters and the tank. In the first, the solid substrate is loaded while in the second the leachate is stored (Figure 3). Digesters and tanks are connected through a leachate circuit. Leachate is sprinkled on the top of the solid substrate, let percolate through it, collected at the bottom and pumped back to the tank. The tank plays then a role of dilution and buffer volume between the digesters which are then not directly connected.

Normally four digesters work in parallel staggered in time with batch duration variable between 40 and 60 days in mesophilic conditions (e.g. with a batch duration of 60 days and four digesters, each 15 days a batch is emptied and loaded again). In this way a more stable biogas production is ensured. It is interesting noting that in this system solid and liquid fractions are separated and that, theoretically, new solid and liquid containers (digesters or tanks) can easily be added if necessary.

Loading and emptying operations

The reactors are consecutively (e.g. every 15 days in the previous example) emptied and loaded with new fresh matter which is previously mixed with digested substrate (digestate) from an old digester to ensure inoculation and a rapid start-up. The leachate is never replaced and its level is completed with fresh water if needed. The digester is closed and let run for its entire batch duration.

During substrate digestion the solid is moistened with leachate according to specific strategies (different for any process). Once the batch duration is over, the reactor is opened, emptied and reloaded and the digestion cycle continues.

Biogas conversion and energy production

Biogas produced in each digester or tank (20% of the biogas was estimated to be produced in the leachate storage by [START_REF] Kusch | Key success factors in discontinuously operated dry digestion[END_REF] in an industrial site) is collected and converted. Three conversion modes exist nowadays: direct injection into the gas grid, direct combustion into biomass boiler for heat recovery, and in a combined heat and power (CHP) unit to produce heat and electricity, better known as cogeneration. The number of French AD plants using these three different biogas conversion modalities is reported in SINOE (2003SINOE ( -2016)): 213 cogeneration, 140 biomass boiler and 9 biogas injection.

The most common practice in Naskeo sites, and thus the one considered in this manuscript, is the cogeneration: a part of the heat is used to fulfil internal process needs (like keeping digester temperature) and another is used in other external activities (like wood drying, building heating system, etc.) (no more compulsory since 2016); on the other hand, electricity is sold and the prices fixed by national decrees (see paragraph 1.1.3).

A CHP unit is a very important part of the process. A CHP unit is a combustion engine characterized by a specific combustion nominal power, or maximum combustion power, and a minimum combustion power (often close to the half of its nominal power), below which the engine shuts down, In normal conditions the CHP unit works within this power range continuously. Indeed, repeated shuts down reduce significantly its lifetime. An important parameter associated to the CHP units is the electrical power efficiency. This latter is at its highest level when the engine works at its nominal power whereas it decreases at lower powers. This makes the functioning at nominal power the most interesting since electrical production is the major source of income for an AD plant. The last limitation of a CHP is the methane content in the biogas. Variable from model to model, this threshold is often 45-50%, which means that a biogas with a lower content cannot be burned in a CHP. These specific characteristics of a cogeneration engine play a very important role in the feasibility of a process.

Process issues

The sequential LBR process described in this manuscript present several issues that affect its economic viability and development. These issues have been highlighted in Figure 3 (pink bullet points) and then discussed in the next sub-sections.

Substrate properties

In general, the biological, chemical and physical characteristics of the substrate used in a process play a key role on the process itself since they determine its range of action, i.e. limits.

Therefore, studying the characteristics of different types of spent animal bedding, their adaptability to LBRs process, the problem encountered during their digestion as well as the energy efficiency recovered from their treatment, are all key basic pieces of information. If a lot of data is available in literature about manure (animal faeces), very few studies analysed properties of spent animal bedding and even less on its digestion in LBRs. The acquisition of this knowledge was found to be an important step in order to successively improve the process efficiency.

Increasing system efficiency

The efficiency of an anaerobic digestion process is based on the amount of methane produced in a certain time slot and in relation to the energy potentially recoverable from the substrate itself.

At the same time, engineering problems connected to the reactor size of the must be considered.

Therefore, the increase of the methane production in a shorter time and a smaller digester volume is the most important objective to increase the project feasibility. The increase of the degradation rate of the substrate can be achieved through the modification of two main parameters: the inoculum quantity and quality as well as the temperature (see paragraph 1.2.3). These strategies were tested experimentally and analysed from an economic and technical angles, which often represent the real limits of the process.

VFA management and leachate recirculation

Dry AD systems and in particular LBRs are characterized by a high organic load which can lead to an acidification. Strategies like massive inoculation can be used to counteract this phenomenon but the economic impact of such approach would be significant since very little fresh matter would be loaded at every batch. Sequential LBRs based process offers the possibility to manage these VFAs through the synergic relations created by coupling several LBRs with each other (see paragraph 1.3.2). The management of VFAs through leachate recirculation is then fundamental to control the process digestion. This latter represents, in fact, the only controlling tool that the operator can use to drive the process in terms of substrate degradation and biogas production during digestion. For this reason leachate recirculation management is particularly important.

Discontinuous process and biogas exploitation

A sequential LBR system is a discontinuous process where reactors are staggered in times.

Even if this operation strategy is convenient for farm management, this represents a real drawback of the process (see paragraph 1.3.2). In this sense start-up phase needs to be perfectly managed in order to avoid serious economic loss. For each start-up phase, a reactor needs a certain time slot in order to reach a stable biogas production rate and an optimum gas quality in order to be used. The consequent discontinuous biogas production is thus a major issue in order to produce electricity in a CHP unit and the process should be operated in order to work within CHP unit technical limits.

Discontinuous biogas production is a major issue in sequential LBR site and a biogas combustion power higher or lower than the working CHP range will inevitably result in a biogas loss. The same will occur when the methane content in biogas is below the minimum acceptable threshold (often between 45% and 50%), like when a new batch is started and methane content in the head space is still low.

Thesis objectives and structure

Based on the issues highlighted above four main general objectives were identified. Each of them is treated in a separate chapter as a scientific article (published or submitted): chapters 2, 3, 4 and 5. Chapters 1 and 6 are deal with the introduction and the conclusion of the present PhD thesis, respectively.

Chapter 1 provides background information regarding anaerobic digestion, LBR process and the structure of the PhD thesis Chapter 2: focus on spent animal bedding properties and aptitude in being digested in a LBR.

Several types of spent animal bedding from different animals, all susceptible to be treated in a LBR process, were characterized and their digestion performance determined. In this chapter the evolution of the main parameters (i.e. biogas volume produced, methane content in the biogas, pH, efficiency in this kind of process and the drawbacks connected to it (Figure 4). Chapter 5 proposes a strategy to control the digestion process through a leachate management in the case of co-digestion of a slowly-and easily-degradable mixture. This chapter gives insights to the operator regarding the importance of leachate management during the acidification phase which is likely to occur seasonally in farm-scale site (Figure 7). Finally, Figure 8 allows to better visualise the PhD structure in chapters as well as the substrate and the system used in each of them. Initially spent animal bedding digestion is analysed and optimized in a single-stage process while, later, the case of co-digestion of easily and slowlydegradable matter is studied in an LBR simulating a sequential system.

Substrate

Co-digestion: easily-and slowlydegradable substrates

Process

Sequential LBRs

The objective of this chapter is to assess the influence of the physico-chemical properties of the substrate (spent livestock bedding) on the LBR process performance. Several types of spent livestock bedding were characterized in order to determine their properties (e.g. VS/TS, BMP, fibre content, etc.), variability and finally adaptability to the anaerobic digestion. In this chapter, the main issues connected to the operation of LBR and more especially the treatment of spent animal bedding Dry anaerobic digestion processes have spread widely in the past few years compared to the wet ones due to their advantages in accepting substrates with total solids (TS) higher than 20% (Karthikeyan and Visvanathan, 2012a). In countries like France, agriculture and livestock rearing are well developed and the use of primary products (e.g. cereals) as feed for anaerobic digestion (AD) plants is forbidden by national legislation (Assemblée Nationale, 2014). In such a context, manure occupies a dominant position among rural waste. Indeed, the French national audit FranceAgrimer (2012) reported an annual production of about 90 million tonnes of solid manure and 180 million tonnes of slurry.

Thanks to its particular characteristics mainly related to a high nitrogen content and alkalinity, manure have been used in AD for a long time, especially in co-digestion with other substrates. This is also linked to the very tight legislation on manure management because of problems like greenhouse gas emission, nitrogen contamination of groundwater [START_REF] Smith | Nitrogen excretion by farm livestock with respect to land spreading requirements and controlling nitrogen losses to ground and surface waters. Part 1: cattle and sheep[END_REF] and nuisance odours [START_REF] Wilkie | Anaerobic digestion : holistic bioprocessing of animal manures[END_REF]. Manure can vary greatly in relation to its TS, which mainly depends on the farm housing practices. In deep-litter housing systems, bedding is used to absorb excrements and urine, thus creating a solid waste rather than a liquid one (i.e. slurry). Such solid waste is a soiled bedding that accumulates in the stables and which is referred to as spent bedding [START_REF] Tait | Biogasification of solid wastes by two-phase anaerobic fermentation[END_REF] or spent straw if the latter is used for bedding material. In France, where deeplitter housing practices are widely used, 53.8% of the produced spent cow bedding has a TS higher than 18% (Degueurce et al., 2016a) which can in turn support the development of AD processes adapted to their treatment.

One of the AD processes gaining a foothold in the rural context is the Leach-Bed Reactor (LBR) operated in batch mode. In this dry AD process, the solid substrate is loaded into the reactor, while a liquid phase, usually stored in a separate container, is sprinkled over the solid bulk, percolates through the waste bed and finally returns to its storage tank. So far, only few authors investigated the digestion of spent animal bedding as sole substrate in LBR (Table 2), with the first example reported 30 years ago by [START_REF] Hall | Mesophilic Anaerobic Digestion of High Solids Cattle Waste in a Packed Bed Digester[END_REF]. In literature, the most common spent bedding treated in LBRs mainly originated from cow stables and only a few from horse ones. It is worth noting that very little evidence on the use of spent sheep bedding was reported [START_REF] Blanco | Batch dry anaerobic co-digestion of sheep manure and potato waste[END_REF], while nothing was ever reported regarding goat bedding.

Batch LBRs offer several complementarities to animal husbandry and in particular when deep-litter housing practices are implemented. Firstly, being a discontinuous process, it is perfectly suited to the cyclic cleaning of stables, thus allowing the reduction of storage time as well as problems arising from it, such as odour nuisances , soil contamination and the loss of volatile solids (VS) by oxidation [START_REF] Cui | Solid-state anaerobic digestion of spent wheat straw from horse stall[END_REF]. Secondly, the process accepts undesirables like pebbles or ropes, which are commonly found in farm waste [START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF] and would create operating problems in conventional continuous stirred-tank reactors (CSTRs). Thirdly, thanks to the presence of bedding material, the substrate is characterized by good porosity, which is essential for adequate percolation [START_REF] Myint | Enhancing anaerobic hydrolysis of cattle manure in leachbed reactors[END_REF]. Furthermore, the robust and simple LBR design, free of any moving parts, reduces costs related to electro-mechanical spare parts investment and maintenance, making it highly suitable for rural context (Karthikeyan and Visvanathan, 2012a). On the other hand, certain problems may originate with this process: an incomplete degradation of the substrate due to bad percolation and compaction [START_REF] André | Quantifying physical structure changes and non-uniform water flow in cattle manure during dry anaerobic digestion process at lab scale : Implication for biogas production[END_REF], an unstable biogas production due to its discontinuous loadings, as well as a difficulty to exploit all the biogas produced due to the low methane content during the first days.

Considering that, in France, spent bedding from animal stables represents the highest fraction of the feed mixture in LBRs at industrial scale, it is important to better understand its characteristics, diversity and putative differences during its digestion for industrial-scale applications. In contrast to manure and straw separately, the properties of spent bedding are scarcely described in the literature. Moreover, spent bedding has to be considered as a substrate on its own since animal mechanical action on the litter and biological degradation during litter accumulation can modify the original properties of straw and animal excrements [START_REF] Tait | Biogasification of solid wastes by two-phase anaerobic fermentation[END_REF]. 1 Spent bedding: a mixture of excrements and bedding material (straw) directly sampled from the stables 2 Synthetic mixture of excrements and bedding/bulking agent * No adequate conditions for the process Characterization of spent bedding and variability among different types is crucial to understand the properties of these substrates before loading them into an anaerobic digester. Additionally, it is worth mentioning that the few studies investigating the digestion of spent bedding in LBR operated in different conditions (e.g. inoculation, leachate recirculation, etc.), thus hampering a clear and direct comparison of the substrates and their performances in LBR.

For these reasons, the present work aims at characterizing spent animal bedding of different origins and assessing their biological degradation in a batch LBR. The main anaerobic digestion performances are described and compared, and the operating parameters that need to be carefully taken into account when treating these organic substrates in LBRs are analysed in detail.

Materials and Methods

Substrates and inoculum collection

Substrates were collected from deep-litter stables where the bedding was mainly wheat straw.

All of them were collected in winter to avoid any variability due to seasonality. Substrates were chosen to be representative of the types of spent bedding that can be found in practice, in relation to both animal species and diet. Six different types of spent bedding were collected, each from one stable: sheep (SB_sheep), goat (SB_goat), horse (SB_horse), and three different cow substrates (SB_cow). Since spent cow bedding is by far the most common in France (FranceAgrimer, 2012), a supplementary division of SB_cow into three subcategories was made, based upon the roughage used in animal diet: round bale grass silage (SB_cow_g), clamp maize silage (SB_cow_m) and hay (SB_cow_h) Table 3). This sub-division was also supported by the fact that the type of roughage is indirectly related to different breeds of livestock and therefore to the stabling objectives (i.e. milk, meat or cheese production) and it has been shown to influence faeces properties like methane potential [START_REF] Møller | Feces composition and manure derived methane yield from dairy cows: Influence of diet with focus on fat supplement and roughage type[END_REF]. All substrates were collected during stable cleaning out in order to be representative of their real properties before a possible AD treatment. Six samples of almost 20 kilos were collected from different parts of the litter and then mixed following the protocol described by [START_REF] Gy | Sampling[END_REF]. The cleaning operation and the sampling protocol ensured the collection of a sample which was representative of the entire site. The substrates were kept frozen at -20°C in plastic bags and one day before utilization they were thawed at ambient temperature.

Liquid inoculum was obtained from an UASB (Upflow Anaerobic Sludge Blanket) reactor treating sugar industry wastewater in the south of France. Before inoculation, the sludge granules were first broken up in glass reactors by continuous magnetic mixing for 3 to 5 days and were then kept in anaerobic conditions at a concentration of about 10 g VS L -1 for almost two weeks, until they reached their endogenic production level of about 1.33 NmL CH 4 g -1 VS d -1 .

Experimental apparatus

The reactors used were 7 L jacketed glass containers of 14.5 cm diameter and 43 cm high (Figure 10). The solid and the liquid fractions were separated by a mesh (1 mm holes) placed at 10 cm from the bottom of the reactor, which allowed the leachate to be stored in the volume below. A tube connecting the headspace and the volume under the mesh was added in order to equalize the pressure in the event of the bulk becoming too compact. A peristaltic pump enabled the leachate to recirculate from the bottom to the top. One small glass flask (140 mL) was fitted into the liquid circuit, in order to measure the pH using a fixed pH probe. A biogas circuit connected the top of the reactor to a volumetric biogas counter. Two ports, one on the liquid and the other on the gas circuit, facilitated the collection of samples for analysis.

Experimental set-up

All the kinds of spent bedding were tested in duplicate in two successive series of 6 reactors.

In the first, spent bedding from sheep, goat and horse was analysed separately while in the second, the three different types of cow bedding were assessed.

To better compare the results, all batches were run following the same experimental protocol.

The spent bedding load was about 410 g TS (between 1.2 and 1.6 kg of fresh substrate), whereas the total amount of water in the system was set by fixing the system TS (fresh substrate, water and sludge) between 12% and 13%. Since each type of spent bedding had a similar water-holding capacity the amount of free water for recirculation was similar in all reactors, thus ensuring a complete recirculation of the leachate each time. The systems were all inoculated at a substrate-toinoculum ratio S/X (VS substrate/VS inoculum) of 10, in order to ensure the presence of methanogens and a good start-up of the process. However, inoculum was not considered to participate to the methane production in the reactor because of its rather small concentration. Before each loading, spent bedding and the inoculum were first mixed together. Then, the inoculated substrate was manually placed inside the reactor and compacted with a weight of 8 kg, in order to simulate the compaction taking place in industrial site due to the height of the substrate pile. In a final step, the chosen amount of water was added. Once filled, the solid phase was saturated by continuous recirculation of the leachate for about 15 min. Thereafter, the liquid recirculation was run automatically 10 times a day for 1 minute at a flow rate of about 1.6 L min -1 . Such a high total amount of water recirculated per day (about 40 L kg -1 TS d -1 ) was fixed in order to provide homogenous sprinkling over the bulk and to bring the liquid phase into equilibrium with the solid phase, thus accessing the bulk conditions through leachate analysis. The digestion was stopped after 60 days which is a common digestion period for a mesophilic process digesting slowly-degradable substrate as spent animal bedding.

Sampling and analysis

Characterization of the initial substrate was carried out in triplicate and mean values were estimated with their standard deviation. The TS content was measured after drying the fresh substrate at 105 °C for 24 h, followed by 3 h of calcination at 550 °C for determination of VS. All the other analysis were carried out on substrates previously freeze-dried and milled to 1 mm. Total phosphorus was measured with the BIOTECK ELX800 microplate reader after a complete calcination in an acidic environment where sulphuric and nitro-perchloric acids were added. NTK was measured by the Kjeldahl method after 3h of calcination and successive titration with a BUCHI 370-K apparatus. Total Organic Carbon (TOC) was estimated by a carbon analyser (TOC-V CHS/CSN, Shimadzu Corporation) performing combustion of the sample at 680°C with a catalyser (cobalt/platinum) and pure oxygen. A Van Soest fractionation (Van Soest, 1963), able to separate the soluble fraction, the cellulose, the hemicellulose and the lignin, was carried out using a Fibrebag system (Gerhardt). Finally, a BMP test was performed in accordance with the protocol described by [START_REF] Motte | Morphological structures of wheat straw strongly impacts its anaerobic digestion[END_REF]. Further information about the NTK, COT and Van Soest fractionation protocols used are also given by [START_REF] Motte | Morphological structures of wheat straw strongly impacts its anaerobic digestion[END_REF]. During the digestion process, leachate was sampled after a recirculation cycle and all the analysis carried out in simplicate. At each sampling a volume between 10 and 20 mL was taken by the mean of the port present in the liquid circuit. The sampling frequency was adapted to the parameters evolution in order to precisely follow the start-up period: 4 times per week the first 3 weeks and then twice per week until the end. . Total alkalinity was measured on the raw leachate at a pH of 4.30, as recommended by [START_REF] Hill | Measuring alkalinity accurately in aqueous systems containing high organic acid concentrations[END_REF]. Soluble chemical oxygen demand (CODs) and volatile fatty acids (VFAs) were measured on a leachate previously filtered at 0.45 μm. 0-1.500 mg L -1 spectroquant kits were used to analyse COD, VARIAN I-MET-0084 gas chromatograph with helium as the gas vector was used to measure + , K + , Mg 2+ and Ca 2+ ) in the leachate were measured only at the end of the digestion using the ICS-3000 ion chromatography system after filtering the raw leachate at 0.2 μm. Analyses on biogas were done daily except during the week-end. Biogas volumetric production was measured with Ritter flow meters (Milligascounter MGC-1 V3) and its composition was analysed with a micro-GCPRO CP-4900 using helium as the gas vector.

In the graphs, the mean values between the duplicates were reported without error bars for an easier presentation of the results. The only exception concerned the biogas data of SB_cow_g. In fact, due to the malfunctioning of one of the biogas volumetric counters in the duplicate set-up, the results of only one reactor were used. On average, the error made between duplicates on each analysis (calculated as |x1 -x2| / M(x1, x2) with x1 and x2 the measures and M the mean value of the measures) was: 26% on the daily biogas volume produced, 16% on the TVFA and total alkalinity, and 2.3% and 0.4% on the CH 4 content and pH, respectively.

Statistical analysis

An analysis of variance, ANOVA, was carried out using R commander [START_REF] Fox | The R Commander: A Basic Statistics Graphical User Interface to R[END_REF] in order to evaluate the statistical significance of the results obtained from characterization (TS, VS, BMP and, carbon, nitrogen, phosphorous and fibre content) and from final methane yields. Kinetic parameters of methane production were calculated with R Grofit [START_REF] Kahm | Grofit: Fitting biological growth curves[END_REF]. The Gompertz equation was used to fit the experimental results.

A(t) =A max •exp {-exp [((μ max •exp(1))/A max ) • (λ -t) + 1)]}
Where A is the cumulative methane production (NmL CH 4 g -1 VS), t is the batch duration (day), λ is the lag phase time (day), μ max is the maximum methane production rate (NmL CH 4 g -1 VS d -1 ). Finally, linear correlations estimated with the Pearson correlation coefficients were searched among all the parameters. All these results are in reported in supplementary material (section 7)

Results and discussion

Substrate characterization

Physico-chemical characteristics

All the types of spent bedding were visually heterogeneous as a consequence of their high straw content. All of them were well compacted and in an ongoing composting process, as evidenced by the warm temperature inside the bulk (not measured). This was not the case for the SB_horse which accumulated for only a few days, which is a very short time compared to the other substrates ( Table 3). Indeed, the frequency of litter change, or the time between two stable cleanings, can vary significantly on account of sanitary requirements and farm practices. In line with this parameter, SB_horse represents an exception since horses are mostly reared for leisure and sport rather than for meat production and a much greater attention is paid to their comfort. As reported by [START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF] and [START_REF] Cui | Solid-state anaerobic digestion of spent wheat straw from horse stall[END_REF], the frequency of litter change also plays an important role in the degradation process. In fact, composting of the most degradable fraction can start easily in good biological conditions such as in a litter, even when the limited aeration and the increasing compaction diminish this process.

Another parameter that could highly affect the spent bedding properties is the amount of straw per animal (expressed in livestock unit, LU, (Eurostat, 2013)) per day. As for the frequency of litter change, the amount of straw depends on the animal and the farming practices and SP_horse represents once again an exception for its high value. By mixing the straw to the animal faeces, these latter are diluted and properties like TS, VS, C/N, fibre composition and BMP are modified.

Hence, spent bedding characteristics are often found in between the ones of raw faeces and straw.

The ANOVA, performed on TS, VS, carbon, nitrogen, phosphorous and fibre content, proved that all substrates were statistically different (p-value < 0.05).TS is a very important parameter for AD process design [START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF] because it impacts the loading charge and the methane production per volume of digester. TS of SB_cow was found, on average, lower than SB_horse and SB_sheep ( Table 3). TS values similar to the ones measured in this study were reported in literature:

26.3% [START_REF] Hall | Mesophilic Anaerobic Digestion of High Solids Cattle Waste in a Packed Bed Digester[END_REF] and 22.2% (Degueurce et al., 2016c) for spent cow bedding and 38.0% [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF] for spent horse bedding. TS of straw is generally higher than 89% [START_REF] Cui | Solid-state anaerobic digestion of spent wheat straw from horse stall[END_REF][START_REF] Wartell | Methane production from horse manure and stall waste with softwood bedding[END_REF][START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF][START_REF] Massé | Dry anaerobic digestion of high solids content dairy manure at high organic loading rates in psychrophilic sequence batch reactor[END_REF] while TS of cow faeces was estimated between 9.8 and 15.6% [START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF]. Higher values were reported for horse faeces, ranging between 20% and 42% [START_REF] Wartell | Methane production from horse manure and stall waste with softwood bedding[END_REF], as well as for sheep droppings, equal to 49% [START_REF] Blanco | Batch dry anaerobic co-digestion of sheep manure and potato waste[END_REF]. Even if goat faeces are similar to sheep ones, the effective lower value reported in Table 3 must be due to a lower amount of straw compared to SB_sheep.

A very narrow range of volatile solids (VS) contents, between 82.3% and 88.9% of TS, was found for the studied spent bedding materials. This is likely due to the similar VS content for bedding and faeces [START_REF] Cui | Solid-state anaerobic digestion of spent wheat straw from horse stall[END_REF][START_REF] Massé | Dry anaerobic digestion of high solids content dairy manure at high organic loading rates in psychrophilic sequence batch reactor[END_REF][START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF][START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF][START_REF] Wartell | Methane production from horse manure and stall waste with softwood bedding[END_REF].These values show the presence of a good fraction of degradable matter in the spent bedding and support the interest in using it for energy recovery.

Animal excrements and urine in particular are known to be rich in nitrogen compounds and a positive linear correlation (R 2 = 0.95) was found between nitrogen content and soluble fraction of the substrate. Values of 30.30 g N kg -1 TS and 27.95 g N kg -1 TS have been reported by [START_REF] Demirer | Anaerobic biogasification of undiluted dairy manure in leaching bed reactors[END_REF] and [START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF], respectively. Similar total nitrogen concentrations were measured in all the types of spent bedding, ranging between 17.6 and 23.7 g N kg -1 TS ( Table 3). Once again, SB_horse was an exception with a TS equal to 11.8 g N kg -1 TS:, probably due to the high amount of straw. The mixture of animal excrements and straw allows to decrease the total amount of nitrogen, which can be inhibitory for AD at high concentration, and to increase the C/N ratio to optimum values for AD (i.e. 20-30) (Karthikeyan and Visvanathan, 2012).

C/N value of 164 for straw is reported by [START_REF] Motte | Morphological structures of wheat straw strongly impacts its anaerobic digestion[END_REF], while a value under 10 for cow faeces was found by [START_REF] Hills | Effect on Carbon:Nitrogen ratio on the anaerobic digestion of dairy manure[END_REF]. The C/N ratios of the substrates used in this study ranged from 20 and 28, except for SB_horse whose C/N ratio was 42. A similar value (39.4) for spent horse bedding was reported by [START_REF] Cui | Solid-state anaerobic digestion of spent wheat straw from horse stall[END_REF], while Degueurce et al. (2016b) reported a value of 17 for spent cow bedding.

Phosphorous content in the spent bedding was found to vary significantly (p-value < 0.05) in relation to the animal. On average, SB_cow were found to have a lower content than the others while the highest phosphorous concentration was measured for SB_goat, 8.4 g P kg -1 TS.

Given the poor percentage of soluble and lignin fraction in straw, 10.2% and 7.4% (on a VS basis) respectively [START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF], high content of these two fractions in spent bedding is mainly due to faeces. Generally, higher values of cellulose than hemicellulose were found in all the samples ( Table 3) as reported also by [START_REF] Demirer | Anaerobic biogasification of undiluted dairy manure in leaching bed reactors[END_REF] and [START_REF] Massé | Dry anaerobic digestion of high solids content dairy manure at high organic loading rates in psychrophilic sequence batch reactor[END_REF]. This is probably related to the high percentage of straw in all the substrates [START_REF] Liew | Methane production from solid-state anaerobic digestion of lignocellulosic biomass[END_REF][START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF]. The higher fraction of cellulose in SB_horse and SB_cow_m (44.2% and 38.5% respectively) compared to other substrates can be related to the frequency of litter change, which corresponded to 3 days and 3 weeks old, respectively. Given its high content and degradation rate in lignocellulosic substrate [START_REF] Liew | Methane production from solid-state anaerobic digestion of lignocellulosic biomass[END_REF] cellulose can rapidly be degraded when good conditions of moisture and bacterial concentration are reached, as in the case of litter. This supports the view according to which a reduced time in the litter and/or in storage before AD could represent an advantageous strategy in order to increase energy production. [START_REF] Yap | Pilot-scale testing of a leachbed for anaerobic digestion of livestock residues on-farm[END_REF]. The use of straw as bedding material was proved valuable from a bioenergy perspective especially when compared with other bedding materials like softwood chips or pellets, mainly because straw contributes significantly to methane production [START_REF] Wartell | Methane production from horse manure and stall waste with softwood bedding[END_REF] Several BMPs for wheat straw were also reported in literature with values ranging from 54 to 245 NmL CH 4 g -1 VS [START_REF] Li | Comparison of methane production potential, biodegradability, and kinetics of different organic substrates[END_REF][START_REF] Liew | Methane production from solid-state anaerobic digestion of lignocellulosic biomass[END_REF][START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF][START_REF] Motte | Morphological structures of wheat straw strongly impacts its anaerobic digestion[END_REF][START_REF] Wartell | Methane production from horse manure and stall waste with softwood bedding[END_REF]. High variability in straw BMPs in relation to the crop used is also reported by [START_REF] Wu | Biogas and CH4 productivity by co-digesting swine manure with three crop residues as an external carbon source[END_REF]. The BMP of faeces has also been reported to vary significantly. Values ranging from100 to 316 NmL CH 4 g -1 VS were reported for cow [START_REF] Labatut | Biochemical methane potential and biodegradability of complex organic substrates[END_REF][START_REF] Møller | Feces composition and manure derived methane yield from dairy cows: Influence of diet with focus on fat supplement and roughage type[END_REF][START_REF] Møller | Methane productivity of manure, straw and solid fractions of manure[END_REF]) and for horse [START_REF] Wartell | Methane production from horse manure and stall waste with softwood bedding[END_REF] faeces. Finally BMP values of 204, 155 and 159 mL CH 4 g -1 VS for solid manure from dairy cows, horse and goat (no details about the bedding are given)

were recently published by [START_REF] Kafle | Comparison on batch anaerobic digestion of five different livestock manures and prediction of biochemical methane potential (BMP) using different statistical models[END_REF]. All these values show the BMP variability of faeces among different animals and within the same animal category. Thus, the addition of straw to animal faeces can either increase or decrease the final BMP in relation to the animal and the straw used. Nevertheless, the addition of straw to faeces has a great impact on the volume of methane produced per raw mass (RM) since the global TS increases. Thus, even if spent bedding has a poor biodegradability compared to other substrates like fruit and vegetable waste or kitchen waste ( 342NmL CH 4 g -1 VS and 541 NmL CH 4 g -1 VS, [START_REF] Li | Comparison of methane production potential, biodegradability, and kinetics of different organic substrates[END_REF], its high TS concentration and high VS per gram of raw matter (RM) makes it an interesting substrate for industrial purposes. In fact, 55 expressed in NmL CH 4 g -1 RM, BMP values of spent bedding (Table 4) are found between fruit and vegetable waste (11 mL CH 4 g -1 RM) and kitchen waste (123 mL CH 4 g -1 RM). Therefore, spent bedding has to be considered a valid substrate for dry AD and its physical properties make it suitable for percolation system in LBRs. For all the types of spent bedding considered, the pH ranged between 6.6 and 7.6 during the digestion process (Figure 11a). In this way, all the reactors were run in optimal conditions since AD is known to perform efficiently in a pH range of 6.8-7.6 [START_REF] Jha | Research advances in dry anaerobic digestion process of solid organic wastes[END_REF]. The decrease in pH observed during the first days of the digestion process is common in batch systems and it is due to the accumulation of VFAs (discussed later). Depending on the kind of spent bedding, the pH in this study started to increase after 3 to 10 days, reaching stable values between 7.0 and 7.5. pH is closely linked to alkalinity and therefore to several acid/base couples present in the system. Here, the most important and common ones were VFAs, with an average pKa of about 4.8, the couple CO 2 /HCO 3 -with a pKa of 6.25 and, finally, NH 3 /NH 4 + which raises pH toward alkaline values due to a pKa of 9.25. The good stability of the batch reactors studied in this work was linked to the presence of high amounts of NH 3 /NH 4 + and bicarbonates which helped to keep the pH at levels suitable for methanogenesis. During roughly the first 15 days, the alkalinity increased (Figure 11b) due to a changing balance between VFA accumulation, which tends to acidify a system, and the hydrolysis of molecules containing nitrogen and the carbonate compounds which tends to increase the pH. After this phase, given that all the VFAs were consumed and the nitrogen and carbonate compounds reached a steady concentration, the alkalinity remained constant throughout the digestion period ranging from 5.0 g CaCO 3 L -1 (SB_horse) to 9.5 g CaCO 3 L -1 (SB_cow_g).

Reactors loaded with SB_goat showed a more specific behaviour: a stable pH was reached later, most likely due to a higher VFA accumulation, and the final value of about 14 g CaCO 3 L -1 was consistently higher compared to the other substrates. Similar values for alkalinity, close to 10 g CaCO 3 L -1 , were also recorded by [START_REF] Massé | Dry anaerobic digestion of high solids content dairy manure at high organic loading rates in psychrophilic sequence batch reactor[END_REF] when treating a synthetic mixture of cow faeces and straw. The high alkalinity measured in all the systems treating spent bedding makes it, independently of its origin, a very good substrate for co-digestion as it stabilizes the pH.

VFAs

VFAs are the main metabolic intermediates of anaerobic digestion and their accumulation depends on both their rates of production and consumption. The imbalance between VFA production and consumption, processes which are carried out by different microbial populations, is one of the main challenges in batch digestion in a single stage using highly-concentred substrates.

As observed by other authors using LBRs (Ten Brummeler, 2000), an accumulation of VFAs 10 days more were needed by SB_goat, for whom a particularly high TVFA peak of 14 g COD L -1 was recorded. It is worth noting that, for SB_goat, such an accumulation of VFAs did not affect the overall reactor performance. Higher TFVA concentrations, reaching 20 g L -1 or more with no inhibition, have been observed by other authors treating more degradable substrates like vegetables and municipal solid waste (Aymerich et al., 2013;[START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF][START_REF] Shi | Reactor performance and microbial 194 community dynamics during solid-state anaerobic digestion of corn stover at mesophilic and thermophilic conditions[END_REF]Ten Brummeler, 2000) In anaerobic digesters working at high solid concentrations such as LBR, concentrations of this magnitude are common. For this reason, high levels of alkalinity, like those reported in this work with spent bedding, are essential to prevent reactor acidification.

The predominant acids produced in all the systems (Figure 12 b1-b2) were acetic and propionic acids (about 80% of the TVFA in terms of COD), which corresponds also to the data reported by [START_REF] Rico | Thermophilic anaerobic digestion of the screened solid fraction of dairy manure in a solid-phase percolating reactor system[END_REF] and [START_REF] Yap | Pilot-scale testing of a leachbed for anaerobic digestion of livestock residues on-farm[END_REF]. This seems to be specifically related to spent bedding since other authors, using the same system but treating organic fraction of municipal solid waste (Ten Brummeler, 2000), maize silage [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF] or canning plant waste (Aymerich et al., 2013), observed an additional high concentration of butyric acid.

For all the types of spent bedding treated, a delay in the degradation rate of propionic acid, compared to that of the others, was observed. Propionic acid consumption only started when all other VFAs had been degraded. In fact, the specific thermodynamic conditions necessary for the consumption of propionic acid to take place have been found difficult to reach, mainly in relation to H 2 partial pressure in the liquid phase (Aymerich et al., 2013).

Figure 12 b1-b2 clearly illustrates the profiles of acetate (C2) and propionate (C3) concentrations during batch operations. While, for SB_horse and SB_sheep, the acetic and propionic acids were consumed almost simultaneously, for the other types of spent bedding there was a gap of about 5 days. On the other hand, no clear relation was found in the magnitude of the concentration peaks of the acetic and propionic acids since the first were found to be higher with SB_sheep, SB_goat and SB_horse but lower for all the types of SB_cow.

Methane production

Variations in the amount and composition of the biogas produced give important information about system behaviour, possible inhibitions and overall kinetics. Methane is, in fact, the final product of a long sequence of reactions as well as a sought-after and valuable molecule. As observed for several lignocellulosic substrates [START_REF] Liew | Methane production from solid-state anaerobic digestion of lignocellulosic biomass[END_REF], a biphasic biogas production was found for all types of spent bedding. The biggest differences between substrates were observed during the first part of the digestion, when more easily-degradable fractions of the substrates were degraded and the highest methane production rates were measured. On the other hand, less degradable fractions played a role in maintaining a low but fairly constant production of were similar for all six types of spent bedding, the methane yield expressed on a raw mass basis is linked to the production level expressed on a VS basis by the TS content. This indicates simply that the drier the substrate, the higher the organic load in a given digester can be.

Percolation and compounds accumulation

For all kinds of spent bedding studied, percolation of the leachate through the bulk was possible thanks to the physical structure provided by the straw. No addition of extra bulking material, as performed by [START_REF] Demirer | Anaerobic biogasification of undiluted dairy manure in leaching bed reactors[END_REF] with wood chips and [START_REF] Myint | Enhancing anaerobic hydrolysis of cattle manure in leachbed reactors[END_REF] with pistachios shell, was necessary to ensure a proper leachate percolation.

By the end of the process, however, a loss of bulk volume, compaction of the waste bed and the consequent diminished potential for percolation were observed, suggesting a loss of rigidity of the straw fibres (visual observation). [START_REF] André | Quantifying physical structure changes and non-uniform water flow in cattle manure during dry anaerobic digestion process at lab scale : Implication for biogas production[END_REF] showed that during the digestion process, the permeability of the spent bedding decreases consistently and spent bedding becomes an almost impermeable substrate after 30 days of digestion. Nevertheless, at least in the first stages of the process, the amount of straw (per animal and per day), is a very important parameter insofar as straw gives spent bedding a porous structure facilitating percolation. When this latter is not optimal, problems may arise even in the early stages of the process, hampering the proper degradation of the substrate.

In the perspective of the reuse of the liquid fraction from previous batches to start up new batches, the accumulation of non-degraded compounds can become a possible cause of failure if the initial dilution is not adequate. After 60 days, Total Kjeldahl Nitrogen (TKN) and potassium were found to be the compounds of major interest. A final concentration in the liquid phase ranging from 1.7 to 4.0 g L -1 for K + and from 0.6 to 2.7 g L -1 for TKN was recorded during this experiment.

Potassium was found below the threshold considered strongly inhibitory 2.5 to 4.5 g L -1

(Karthikeyan and Visvanathan, 2012a). Acclimation to K + concentrations up to 6 g L -1 have been reported while for higher values the inhibition on methanogenic activity was persistent [START_REF] Chen | Effect of potassium inhibition on the thermophilic anaerobic digestion of swine waste[END_REF].

The nitrogen inhibition is known to depend on several factors mainly connected to the inoculum and the acclimation of bacteria. In the literature, a 50% reduction in methanogen activity has been associated with a wide range of total ammonia nitrogen (TAN), going from 1.7 to 14 g N L -1 [START_REF] Chen | Inhibition of anaerobic digestion process: a review[END_REF]. Furthermore, the importance of the speciation of nitrogen compounds has often been pointed out because any toxicity has often been ascribed to NH 3 [START_REF] Rajagopal | A critical review on inhibition of anaerobic digestion process by excess ammonia[END_REF].

Necessary for bacterial growth at concentration below 200 mg L -1 [START_REF] Chen | Inhibition of anaerobic digestion process: a review[END_REF]Karthikeyan and Visvanathan, 2012a), at high concentrations, NH 3 may inhibit growth due to its capacity to penetrate into the microbial cell. It is interesting to notice that an antagonistic effect on TAN inhibition has been related to minerals in the phosphorite ore (K + , Ca 2+ , Mg 2+ ). Hence, the presence of high amounts of K + can play a positive role in reducing the ammonia inhibition [START_REF] Chen | Inhibition of anaerobic digestion process: a review[END_REF], an important consideration for anaerobic digesters treating spent bedding which is naturally rich in both compounds. Based on that, the really high TKN concentration in the leachate of SB_goat (2.7 g L -1 coupled with the low concentration in K + measured (1.7 g L -1 ) could explain the delay in the start of methane production for this substrate. In any case, the accumulation of N in the system should be viewed with reserve and the threshold for actual inhibition needs to be evaluated in each specific case.

Finally, it is worth mentioning the presence in the leachate of a slowly-degradable COD fraction whose tendency is to accumulate in the system. Between 5 and 10 g L -1 of CODs were measured in the leachate after 60 days, however that represents a negligible fraction of the methane potential left over. High COD concentration in the leachate at the end of the process was recorded also by [START_REF] Yap | Pilot-scale testing of a leachbed for anaerobic digestion of livestock residues on-farm[END_REF].

Water balance

Spent animal bedding from deep litter stable was shown to have a high TS, ranging between 24% and 37%. However, after 60 days of digestion digestate showed a similar TS content, ranging between 13% and 15% (Table 5). A water mass balance was carried out on all the batches in order to determine the amount of water necessary to run a LBR. The error made was less than 5%

probably due to the water vapour contained in the biogas which was not taken into account.

Considering the amount of water added at the start-up and collected in the end it was possible to 65 compute the amount of water actually absorbed by the solid matrix. This latter was found between 1.2 and 1.9 kg water kg -1 TS added , or between 0.3 and 0.7 kg water kg -1 RM added . The amount of water absorbed at the end of a cycle by each substrate was found to be directly linked to its initial TS, as shown in Figure 15. This result is important since it proves that to run LBR, a considerable amount of water is needed. For every kg of spent bedding an amount of fresh water ranging from 0.3 L and 0.7 L must be added, less than in a wet process but far from being negligible. Furthermore this amount can be easily calculated by simply knowing the substrate initial TS.

Figure 15 : Amount of water absorbed after one cycle by the bulk in relation with its total solids (TS) before digestion

Digester design

In order to optimize the economic performance of batch systems such as LBRs, two specific stages, linked to the discontinuous loadings, require a particular attention: the start-up phase and the final phase. For the start-up phase, the two important parameters are the time needed to reach the minimum percentage of methane in the biogas allowing it to be burned in a CHP unit and, the percentage of methane not exploited during this period. In the final phase the most important is the batch duration, when low methane production needs to deal with economic issues.

During the start-up phase methane content in a digester headspace increases until reaching a steady level. Nowadays, CHP units with different thresholds for minimum CH 4 content in biogas, ranging from 30% to 50%, are available on the market. However, in this work, data comparison was done for biogas compositions between 20% and 40% only because, at industrial scale, the biogas injected into the CHP unit is actually a mixture of the biogas coming from several batches (operated in parallel but at different stages: multi-stage process), and hence, biogas from a new batch can be accepted even if its CH 4 content is lower than the minimum needed by the CHP unit installed on site. It is clear from Table 6a that the higher the methane content necessary for combustion in CHP TS of spent animal bedding before digestion unit, the higher the time needed to reach the desired methane content in the headspace of the digester and the higher the amount of methane not exploited. Taking the example of a biogas exploitable at a methane content of 30%, the duration needed to reach this value varied from 2.3 days (SB_sheep) to 6.0 days (SB_goat) while the methane not exploited for all the types of spent bedding varied from 1.7% to 4.2%. In this example, it is worth noting that the SB_goat needed a higher number of days to reach a usable methane content of 30% in the headspace but it was not the substrate producing the most not exploitable methane in the end (SB_horse in the present case).

Thus, the optimization of the start-up phase in relation to the substrate is an important issue for LBRs in order to improve the methanogenic kinetics, enabling the time to reach the desired CH 4 content in the biogas and the quantity of unavailable methane to be reduced to a minimum.

Furthermore, it is important to notice that the CH 4 content in the biogas at the outlet depends, among other parameters, on the headspace volume which dilutes the biogas produced during the start-up period. In the present work, the ratio between the volume of the headspace and the volume occupied by the organic substrate equalled 1, which is higher than the ratio actually found on site.

For this reason, the above-mentioned values are overestimated compared to full-scale digesters.

By drawing the attention to the final phase of the digestion process, the batch duration is the parameter to take into account in the design of a discontinuous reactor. Mesophilic LBRs are normally unloaded and reloaded every 60 days. Taking the total specific methane production on day 60 as a reference, it is possible to evaluate the methane production performance of each type of spent bedding in respect to the time and to calculate in that way the percentage of methane not exploited when reducing the batch duration. Table 6b shows, for example, that by reducing the batch digestion to 55 days or 50 days, the not exploited methane ranged, respectively, from 1.9% to 2.7% and from 3.4% to 6.4% in relation to the substrate. However, the consequent decrease in the incomes could be balanced by a smaller digester volume (if the amount of spent bedding to treat per year is fixed) for example. The search for the optimum economic balance is then necessary to set this parameter. In order to study the effect of the leachate recirculation on the digestion of the spent bedding, a further experiment was carried out. Three stainless-steel reactors of 100 L were used following the same principle than the glass reactor previously described. The details regarding this experiment are provided in section 2.1.2. Reactors were loaded with 16 kg of spent cow bedding and 7.5 kg of sludge and 3 kg of water. A very close substrate-to-inoculum ratio than the one previously used was set: S/X of 11. The same loading protocol was applied: the reactors were loaded, the liquid fraction let recirculate for 10-15 minutes and, in the end, the reactors were closed and the digestion started;

the experiment was stopped after 60 days. The amount of leachate recirculated per day was set at 10 L kg -1 TS d -1 and 1 L kg -1 TS d -1 for R1 and R2, respectively (leachate recirculation frequency was of 5 and 0.5 times per day, respectively). R3 setting was similar to R1 for the first 8 days, until the maximum concentration of VFAs was measured, and then switched to be as R2. This was done in order to study the effect of reducing the amount of leachate recirculated per day once methanogenesis was established. Figure 16 shows that methane production rates presented a similar evolution than the other substrate previously treated (Figure 13); the peak was reached after about 10 days from the start and, after that, the methane production decreased slowly until reaching 1

NmL CH 4 g -1 VS d -1 . Beside a slight difference in the peak value of methane production rates (about 9 NmL CH 4 g -1 VS d -1 for R1 and 8 NmL CH 4 g -1 VS d -1 for R2 and R3), the methane production rates were very similar all along the digestion whatever the recycling strategies. No difference was observed by reducing by 10 the amount of leachate recirculated, both by setting this parameter from the beginning of by modifying it during the digestion process, at least in the recirculation range covered. The influence of the elapsed time between two recirculations (frequency), the volume recirculated, the initial solid-to-liquid ratio (TS of the system in this manuscript) on the methane production kinetics were also studied by (Degueurce et al., 2016d). They concluded that the frequency has the highest impact while the other two factors had a minor one. They stated also that different settings of these parameters could lead to similar effects in the studied ranges: frequency from 1 d -1 to 0.16 d -1 (i.e. from once per day to once every 6 days), a recirculated volume between 0.8 and 3.5 L kg -1 TS and a TS% between 10 and 11% (as in our case study).

In contradiction with the study of Degueurce et al. (2016c), we have found here that the periodicity and the volume recirculated had no influence on the process (excluding the coincidence of having found the same performance with two different settings). This was related to different initial conditions: in the present study, the reactors were first recirculated for 10-15 minute at the start-up in order to be close to saturation whereas this procedure was not adopted by Degueurce et al. (2016c) who poured only a small initial volume of leachate on the top of the reactor. Initial TS inside the bulk were then different at the start-up and this could have affected the entire process. In this sense, leachate recirculation strategy can influence methane production kinetics by controlling the TS content in the bulk. In fact, AD is known to be affected by this parameter (see 3). This is a significant result which means that recirculation of the leachate does not affect methane production when substrate water saturation is reached at the start-up.

On the other hand, Figure 17 illustrates the VFA concentration in the leachate. Even if the values measured were quite low (i.e., less than 2 g L -1 ), some effects of the recirculation on this parameter can be observed. A higher concentration of VFAs in the leachate were found when recirculating the most, as in R1 and R3, compared to R2. This was related to the higher wash out of these compounds during the initial stage of acidification. On the other hand, a minor VFA concentration was measured in R1 compared to R3 starting from day 8, after the modification of the recirculation strategy in R3. This suggests that recirculation affects the VFAs mobility and transfer between solid and liquid phase. This phenomenon, that in this experiment was barely observable, would finally play an important role in systems accumulating a lot of VFAs like when co-digesting easily-degradable substrates (see section 5.2). Several types of spent bedding were collected from deep-litter stables, and their physicochemical properties were compared. Spent horse bedding differed from the other substrates because of a greater amount of straw added to the litter and a more frequent litter change. However, all the substrates showed similar VS/TS content, C/N (except that of horse) and BMP. In addition, LBR was shown to be an adapted process for the treatment of spent animal bedding since an average of 89 (± 11)% of the BMP was reached after 60 days of operation. Spent goat bedding and spent sheep bedding behaved similarly to the other substrates even if this first showed a difficult start-up leading to an acidification phase. Therefore, spent animal bedding is a promising substrate for farm-scale LBR plant and its digestion as sole substrate is feasible. Moreover, the overall process stability suggests that a co-digestion with more easily-degradable substrates could be done. Long term accumulation of nitrogen and potassium in the leachate was identified as the main concern with this kind of substrate. Batch duration and start-up strategies appeared as two possible levers for enhancing performance at industrial scale. Finally, the recirculation appeared to affect the VFA concentration in the leachate; this phenomenon is supposed to play a significant role when digesting an easily-degradable mixture.

This chapter focused on one specific phase of the digestion process in a LBR: the start-up.

The role played by the leachate and the digestate on the digestion performance (i.e. methane production and process stability) were studied in detail. In addition, given the big impact of the start-up phase at industrial scale an economic analysis aiming at the comparison of these to possible inoculation was effectuated. In the end, the best strategy for inoculating a LBR treating spent animal bedding was proposed based on experimental and economic results. Anaerobic Digestion (AD) is a widespread biotechnology converting different kinds of organic residue into biogas, a mixture of carbon dioxide and methane. Among the existing dry AD processes, leach-bed reactors (LBRs) were proved to efficiently treat spent animal bedding from cow (André et al., 2016b;Degueurce et al., 2016d;Riggio et al., 2017a), horse [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF]Riggio et al., 2017a), pig [START_REF] Yap | Pilot-scale testing of a leachbed for anaerobic digestion of livestock residues on-farm[END_REF], goat and sheep (Riggio et al., 2017a) stable. At farm scale, several reactors are operated in batch mode and run in parallel in order to obtain a fairly stable biogas production. The digesters are regularly emptied and reloaded at different periods with the help of a wheel loader [START_REF] Kusch | Dry digestion of organic residues[END_REF]. Two separated phases, the liquid and the solid, coexist throughout the process, and both are usually involved in the start-up phase of a LBR.

A batch anaerobic digestion process needs the implementation of an appropriate start-up phase in order to create the best conditions for the establishment of the biological activity and thus generate as much biogas as possible within a limited time slot. Microbial inoculation is particularly important for discontinuous processes since it has to be repeated at every digester loading event. For example, at an industrial site using four LBRs in parallel with a batch duration of 50 days, about 30 reactor loadings per year are carried out. Hence, the assessment and optimization of the start-up strategy is critical.

With this aim in mind, the liquid fraction and/or the solid fraction obtained from a previous LBR can be incorporated into the new substrate in order to facilitate the start-up of the biological reactions. The liquid fraction, named leachate, is generally used to humidify the solid waste in order to start the AD process which could be hampered if the total solids concentration is too high (Abbassi- [START_REF] Abbassi-Guendouz | Total solids content drives high solid anaerobic digestion via mass transfer limitation[END_REF][START_REF] Bollon | Development of a kinetic model for anaerobic dry digestion processes: Focus on acetate degradation and moisture content[END_REF]. In LBRs, the addition of the leachate alone has been shown to improve the start-up phase of the anaerobic digestion process thanks to the microbial inoculation as well as the nutrients and alkalinity addition as previously described by [START_REF] Pognani | Optimization of solid state anaerobic digestion of the OFMSW by digestate recirculation: A new approach[END_REF] treating organic fraction of municipal solid waste (OFMSW) and, Kusch The solid fraction, called digestate (digested solid material from a previous treatment cycle), is generally mixed with the fresh substrate in order to enhance the overall process kinetics as well as to avoid process failures due to the accumulation of inhibitory intermediate compounds [START_REF] Di Maria | Optimization of Solid State Anaerobic Digestion by inoculum recirculation: The case of an existing Mechanical Biological Treatment plant[END_REF][START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF][START_REF] Motte | Dynamic effect of total solid content, low substrate/inoculum ratio and particle size on solid-state anaerobic digestion[END_REF]Riggio et al., 2017b;[START_REF] Saady | A start-up of psychrophilic anaerobic sequence batch reactor digesting a 35 % total solids feed of dairy manure and wheat straw[END_REF].

However, the volumetric methane productivity (methane production per digester volume) may be affected since the solid digestate occupies a significant space in the digester working volume [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF]. Increasing the volumetric methane productivity is indeed one of the major challenges in the anaerobic digestion of solid substrates [START_REF] Yang | Challenges and strategies for solid-state anaerobic digestion of lignocellulosic biomass[END_REF]. Furthermore, in this context, the addition of an external solid source of inoculum raises questions because manure is known to contain methanogens insofar as AD is a process taking place in the digestive system of all ruminants [START_REF] Solli | A metagenomic study of the microbial communities in four parallel biogas reactors[END_REF][START_REF] Sun | Characterization of microbial community structure during continuous anaerobic digestion of straw and cow manure[END_REF].

Even though the role of leachate and digestate as sources of microbial inoculum in LBRs has been studied and reported separately in the literature, a direct and parallel comparison of their influence on the start-up phase of a LBR treating spent cow bedding has not yet been described, nor has their respective economic impact. In the scientific literature, only an economic analysis using a LBR process was found for the treatment of the OFMSW in a landfill context [START_REF] Chynoweth | Sequential Batch Anaerobic Composting of the Organic Fraction of Municipal Solid Waste[END_REF]. On the other side, the use of dry animal manure such as spent cow bedding, was shown to be an interesting organic waste material for significantly increasing the economic performance of biogas plants [START_REF] Asam | How can we improve biomethane production per unit of feedstock in biogas plants?[END_REF] because of its dry content and subsequent high methane potential.

The objectives of this study are then: 1) to identify the impact of adding leachate or solid digestate for starting up LBR on the degradation kinetics of spent cow bedding and methane production yield; 2) to carry out an economic simulation based on these experimental data in order to highlight the economic issues related to the two start-up strategies; and 3) to propose the best inoculation strategy when treating spent cow bedding in LBRs. Both experimental investigation and economic analysis of a LBR process for the treatment of spent cow bedding represent a novelty in the scientific literature and an important support for industrial development.

Materials and Methods

Substrates and inoculum

Spent cow bedding, corresponding to a mixture of wheat straw and faeces, was collected in October 2014 in a cow dairy farm located in South-West France. This farm used a deep-litter housing system and grew 30 Blonde d'Aquitaine cows whose roughage feeding was composed by 50 % hay and 50 % grass silage. During the cleaning-out of the litter, after accumulation for about 1 month in the stable, six samples of about 10 kg each were sampled from different parts of the litter and were then mixed together manually following the protocol described by [START_REF] Gy | Sampling[END_REF] to ensure homogeneity. This overall sample was stored in plastic bags of 8 kg each and kept at ambient temperature for one week before use in order to maintain its natural biological activity. Spent bedding from only one bag was used to load all the reactors.

The solid digestate and leachate used for inoculation were collected from a previous experiment using the same experimental system and treating spent cow bedding similar to the one used in the present study. Table 7 reports the main physico-chemical characteristics of the spent cow bedding, the digestate and the leachate used. The methane production rate of this digester, run for more than 60 days, was about 0.06 NL CH 4 kg -1 VS h -1 (VS, volatile solids). This value was low enough to consider negligible the methane production originating from digestate in the new batch.

The leachate contained 13.0 g L -1 of total chemical oxygen demand (CODt) and 4.0 g L -1 of soluble chemical oxygen demand (CODs), no trace of volatile fatty acids (VFAs) and, 1.0 g L -1 and 2.0 g L - 1 of NH 4 + and K + , respectively.

Experimental apparatus

The 6 reactors used were identical: 7 L jacketed glass containers and 14.5 cm in diameter (Figure 19). In each reactor, the solid and liquid fractions were kept apart by a sieve (1 mm holes) placed at 10 cm up from the bottom of the reactor, which enabled the leachate to be retained in the lower volume at the bottom. A tube connecting the headspace and the volume under the mesh was added in order to equalize the pressure in the event of the bulk becoming too compact. A peristaltic pump enabled the leachate to recirculate from the bottom to the top. One small glass flask of 140 mL was fitted into the liquid circuit in order to measure pH using a fixed pH probe. Two ports, one in the liquid and one in the gas circuits, facilitated the collection of samples for analysis. The reactors were maintained in mesophilic conditions (37°C) using a thermo-regulated water bath.

Figure 19: Experimental leach-bed reactor (LBR)

Experimental setting

Three conditions were tested in duplicate to evaluate the different start-up strategies for a LBR packed with spent bedding (Table 8): the addition of a liquid fraction (leachate, L), a solid fraction (digestate, D), and tap water only (blank, B).

For condition D, the substrate-to-inoculum ratio on a volatile solids (VS) basis was 6.8 while on a total solids (TS) basis, expressed as TS digestate / (TS digestate + TS substrate), it reached 13.5 %. [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF] reported an optimum between 10 % and 20 % (on a TS basis) when treating spent horse bedding. This inoculation ratio is also representative of the one implemented at farm-scale. The leachate used for the condition L, on the other hand, was set using a leachate completed with tap water in order to reach the total amount of liquid desired: the total solid concentration (liquid and solid phase) of all the reactors was set between 10 % and 11 %. In this case, the substrate-to-inoculum ratio was 45.3 on a VS basis and 4.2 % on a TS basis. This indicated that the VS brought via the inoculation were less when using the leachate compared to the digestate.

For the reactors B, only tap water was used to moisten the spent bedding and no further inoculation was added to that already present in the spent cow bedding. The mixture (spent bedding and digestate, when applicable) was compacted with a weight of 8 kg in order to ensure the same compaction in all the reactors. Finally, the liquid fraction (composed of leachate and/or water) was poured over the solid bed and left to percolate down. A continuous recirculation for 15 min at a flow-rate of about 1.6 L min -1 allowed to humidify homogeneously the bulk phase (Abbassi- [START_REF] Abbassi-Guendouz | Total solids content drives high solid anaerobic digestion via mass transfer limitation[END_REF]. Thereafter, the recirculation was ensured automatically 10 times per day for 1 minute at a flow-rate of about 1.6 L min -1 . This high total amount of water recirculated daily, set at 40 L kg -1 TS d -1 as done by Riggio et al. (2017a) in a similar system, served to ensure the liquid phase remained in equilibrium with the solid phase, thus permitting the assessment of the bulk conditions through analysis of the leachate.

details used cannot be disclosed and the results obtained have been arbitrarily normalized with respect to the values obtained at 11.5 kt y -1 (kilometric ton per year).

The biogas plant considered in the simulations was a farm-scale (100 kW el to 300 kW el ) AD Along with the capital expenditure (CAPEX), two additional economic parameters were selected based on [START_REF] Karellas | Development of an investment decision tool for biogas production from agricultural waste[END_REF] to evaluate the performance of the two start-up strategies:

the Payback Period (PP) and the Internal Rate of Return (IRR). The PP, expressed in years, is the duration required to recover the cost of an investment. Its equation is:

𝑃𝑃 = 𝐶/𝐼 (Eq. 1)
with C, cost of project (investment -grants) and I, annual cash inflows (annual revenueoperating costs). The IRR is defined as the value of the Discount Rate necessary to the Net Present

Value function (NPV) (the sum of the present values of all the benefits and cost cash flows) to equal zero, which means that the present value of the investment funds equals the present net revenues from an operation. Its equation is:

NPV = ∑ 𝐶 𝑛 (1+𝑟) 𝑛 -𝐶 𝑜 = 0 𝑁 𝑛=0 (Eq. 2)
with Cn: net cash inflow during the period n; Co: total initial investment costs; n: number of time periods (N= 15 years); r: discount rate. The higher the IRR, the more profitable the project.

To sum up, PP has the advantage of being explicit but it does not take into account important factors like the value of money over time (money at the present time is worth more than the same amount in the future due to its potential earning capacity) and the risk of an investment; IRR is a more precise but complicated parameter that takes these latter into account, permitting a comparison of projects of different size. More information about the hypotheses and the economic inputs are reported in the supplementary material (section 7.2).

Results and discussion

Process performance

Biogas production

The analysis of the cumulative volume of methane produced and the methane yield provides a comprehensive evaluation of process performance for the three different operating conditions (Figure 20 a -b). The error between duplicate was really low indicating the precision of measures in the three conditions and the reproducibility of the reactor set-up. The blank, where only tap water was added, showed a cumulative volume of methane produced that was 9.0 % lower compared to the batches where leachate was added (condition L). However, after 60 days, the blank reached a methane yield of 169.0 NL CH 4 kg -1 VS on average, which represents 82 % of the BMP of spent cow bedding (205.7 NL CH 4 kg -1 VS). Very similar methane yields were reported by Riggio et al. (2017a) when treating spent cow bedding for the same batch duration. These results suggest that the AD process in batch mode treating spent cow bedding with only the addition of water can start up reasonnably well and also the microbial inoculation provided by the solid manure itself is sufficient.

Similar results were found by [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF] with spent horse bedding and water.

The cumulative volume of methane produced in conditions L and D (addition of leachate and digestate, respectively) were linked to the difference in the amount of fresh matter loaded (Table 8).

In fact, after 60 days of operation, condition D produced 13.5 % less volume of methane than condition L (Table 9). Nevertheless, the methane yield, expressed as the ratio of the total volume of methane produced during a batch to the quantity of VS added as spent bedding at the beginning of the batch, was 15.8 % higher for condition D. The contribution of digestate amendment to the methane produced was estimated to be lower than 6.0 % of the total methane produced (by considering a constant methane production rate of 0.06 NL CH 4 kg -1 VS h -1 ). Hence, the part of methane produced from digestate degradation cannot fully explain the higher methane yield which was certainly related to faster overall degradation kinetics.

Higher kinetics of substrate degradation after inoculation with digestate were also recorded by Di [START_REF] Di Maria | Optimization of Solid State Anaerobic Digestion by inoculum recirculation: The case of an existing Mechanical Biological Treatment plant[END_REF] and [START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF]. These results can be explained by the possible greater amount of specific microorganisms brought into the system through the addition of solid digestate and/or by the microbial consortia present in the digestate which seem better adapted to solid substrate degradation. The observation that the microbial consortia in the leachate and solid phase differ, with little exchange between them (Degueurce et al., 2016c), could support this explanation.

The biogas production rates in Figure 20c indicate that faster kinetics were obtained with condition D, followed by condition L and then B. Different production rates were observed up to about 40 days; thereafter, for all the conditions, very close values were recorded until the end. The methane concentration in biogas displayed in Figure 20d was found slightly better when adding digestate, as also reported by Di [START_REF] Di Maria | Optimization of Solid State Anaerobic Digestion by inoculum recirculation: The case of an existing Mechanical Biological Treatment plant[END_REF]. However, the very tiny difference observed between the use of leachate or water during the first 10 days proved the very limited inoculation capability provided by the leachate in comparison to water. A more significant delay was observed for condition B after 10 days, attesting of the positive effect played by the leachate or the digestate.

In conclusion, the addition of digestate led to increased methane production kinetics and permitted a higher methane yield at 60 days while the addition of leachate led to higher volumetric methane production.

pH, VFAs and alkalinity

In the three conditions tested, the process was stable overall and no significant inhibition was detected. The lowest pH values were observed for the blank condition (Figure 21a). However, the pH remained above 6.5, even during the first days during which the highest VFA peak was monitored (Figure 21b). After the acidification phase, the pH stabilised at values ranging between 7.0 and 7.4 for all three conditions. A constant difference between condition L and the other two is explained by the higher alkalinity of the leachate (Figure 21c) mainly because of the presence of NH 4 + in the solution. Indeed, alkalinity in L was about 2 g CaCO 3 L -1 and 3 g CaCO 3 L -1 higher than in B and in D, respectively. Hence, leachate can contribute significantly to a greater stability of the process.

The concentration of total volatile fatty acids (TVFA) in conditions D and L (Figure 21b) followed a similar trend: a peak was reached after 7 days of operation (8 and 5 g COD L -1 , respectively), and, after 15 days, no more VFAs were recorded. The lower TVFA concentration in condition D in comparison to L was mainly related to a lower load of spent bedding (Table 8). The difference between conditions L and B was due to slower propionic acid consumption in the latter (Figure 21d), meaning that the acetogenesis kinetics were slower in the blank. A faster degradation of propionic acid could be linked to a higher alkalinity and/or pH rather than to the inoculation, since this leachate inoculation was poorer compared to that of the digestate. A faster consumption of propionic acid observed in condition L when compared to condition D, indicates that the leachate played a more important role than the digestate in propionic acid consumption.

Economic analysis

A qualitative economic analysis was carried out in order to compare the two inoculation strategies: conditions D and L. Real values could not be provided for confidentiality reasons.

The evolution of the Payback Period (PP), the Internal Rate of Return (IRR) and the Capital Expenditure (CAPEX) were analysed in relation to the annual tonnage processed (capacity) and the batch duration. Further results about the sensitivity analysis of four important external process parameters (grants accorded to the project, the fraction of heat sold, the preparation time for loading and emptying the digesters and the haulage distance for substrate collection) were provided in the supplementary materials (section 7.3).

Capacity (annual tonnage processed)

The CHP power chosen (100 kW el to 300 kW el ) and the experimental methane yields determined for each strategy (condition D or L) set the lower and upper capacity limits for the simulation: 5.0 kt y -1 and 15.0 kt y -1 . Figure 22 a -b shows that the PP decreased and the IRR increased with the capacity of the plant. These trends are common in AD processes [START_REF] Walla | The optimal size for biogas plants[END_REF] and show an improved economic margin for bigger plants due to economies of scale. [START_REF] Lantz | The economic performance of combined heat and power from biogas produced from manure in Sweden -A comparison of different CHP technologies[END_REF] agreed on the importance of the scale of biogas production at farm-scale plants.

In our conditions, a comparison was considered economically interesting only for capacities higher than 8.0 kt y -1 because for lower quantities a rapid increase of the PP was observed along with a decrease in the IRR (Figure 22 a -b). To further support this choice, it is interesting to note that below a capacity of 8.0 kt y -1 the IRR was too low, turning negative at about 6.0 kt y -1 (an IRR of approximatively 10 % is generally required in farm-scale projects [START_REF] Wresta | Economic Analysis of Cow Manure Biogas as Energy Source for Electricity Power Generation in Small Scale Ranch[END_REF]).

When increasing the annual capacity, the discontinuous variation of the feed-in tariffs, the number of digesters and the CHP power installed (i.e. five consecutive drops in the feed-in tariffs, a gradual increase from 4 to 9 digesters, 25 kW el step-by-step increase in the CHP power from 100 kW el to 300 kW el ) led to irregularities in the evolution of the IRR and PP (e.g. for 8.0 kt y -1 and 9.5 kt y -1 ). However, these latter did not affect the overall economic analysis.

In the selected capacity range, condition L performed, overall, slightly better than condition D: condition L's IRR and PP values better than, or similar to, those of D. That means that the 15.8 % higher methane yield in condition D counterbalanced the 34 % higher digester working volume needed in this condition but it did not allow overtaking condition L from an economic point of view. In spite of these results, the difference found in the PP and IRR were not judged significant to prefer one strategy over the other, and this for the entire range of capacity analysed.

Batch duration

Given the more rapid kinetics observed when using digestate instead of leachate as inoculum, batch duration represents an important parameter requiring close analysis. Figure 22 c-d represent the variation over time of the PP and IRR, respectively, for an average capacity of 11.5 kt y -1 .

Similarly as for the batch duration, it is important to note that below 30 days the process is not economically viable because of the low IRR, and that a more stable evolution (especially of the IRR) is obtained after 40 days. That supports the choice of a batch duration not below this threshold at industrial scale. Moreover, batch durations below 40 days would mean lower degradation and treatment of the organic substrate (environmentally not advisable) and an under-conversion of its energy potential (about 15 % of methane lost at day 40 compared to day 60). Therefore, the impact of batch duration on the economic assessment was studied between 40 days and 60 days.

Both the PP and the IRR in the selected range of analysis were found very close regarding the conditions tested (Figure 22 c-d) and, as for the capacity, batch duration was not found a discriminant parameter allowing the choice between the two strategies.

For capacities different (varying from 8.0 and 15.0 kt y -1 ) from the one studied (11.5 kt y -1 ) similar results were observed (not shown). However, it is interesting to note that for smaller capacities, the batch duration was found to impact significantly the economic performance whereas for bigger no particular effect resulted from its modification. As a consequence, batch duration of 60 days should be preferred for small capacities while the choice of batch duration would depend on a specific objective for bigger sites (i.e. to treat the maximum amount of substrate or to increase its degradation).

Capital expenditure

CAPEX is a very important parameter to consider from an economic point of view because it quantifies the capital needed to practically undertake a new project. Figure 23 shows the CAPEX evolution (normalized) in condition D and L, and the additional investment of condition D over condition L. CAPEX normally increases in relation to the capacity (Figure 23a) and the batch duration (Figure 23b) in both conditions. However, these evolutions follow significantly different trends. When considering these growths in the range of interest (8.0 -11.5 kt y -1 for the capacity and 40 -60 days for the batch duration) the additional investment in condition D is estimated to be 18% higher, on average, compared to L. This represents a significant percentage, especially for small project whose feasibility is often difficult to reach. This result underlines that the addition of digestate affects significantly the initial investment by the mean of the increase of the digesters volume. In fact, engineering construction represents a big share of the CAPEX. In view of the above, condition D has a significant disadvantage compared to condition L and this latter is then the best choice from an economic point of view.

Conclusion

Two start-up strategies for LBR treating spent cow bedding were tested: the addition of leachate or solid digestate. Both were found to impact positively the overall performance of the process, the first improving the process stability (pH and higher alkalinity) and the second enhancing the substrate degradation kinetics. The economic analysis showed no significant differences in the payback period and internal rate of return connected to these two start-up strategies. However, despite these results, the bigger working volume (34 % in this case study) required when adding solid digestate affected considerably the initial investment: 18 % higher compared to the use of leachate. Therefore, leachate addition, associated to a smaller working volume and lower initial costs, seems acceptable from the economic point of view when treating a substrate already rich in methanogens like spent cow bedding. These economic results promote further development of farm-scale AD process and the increase of decentralized energy production. leachate stored at the bottom of the reactor to the top of the bulk. A valve in the leachate circuit allowed taking leachate samples while a port on the biogas circuit allowed gas sampling for analysis. The operation principle of the present LBRs is further detailed by Riggio et al. (2017a).

Substrate collection and experimental conditions

Three conditions were tested: two reactors were inoculated with a mixture of digestate and leachate and were operated under mesophilic (37°C) or thermophilic (55°C) conditions (reactors named M_inoc and T_inoc, respectively); a third reactor was started up in thermophilic conditions but without specific inoculation (T_no inoc). Two successive runs were carried out in order to establish the process's stability and repeatability. The spent cow bedding used in each run was collected at the same farm during two different stable cleanings. The substrates sampled were stored for 2-3 days in plastic bags at ambient temperature before being used. The solid digestates and leachates used in run 1 were sampled from two previous batches adapted to thermophilic and mesophilic conditions for over three months (with two consecutive loadings). For run 2, the digestates and leachates collected at the end of run 1 were used instead. Total solids (TS), volatile solids (VS) and biomethane potential (BMP) tests (on raw matter) of digestate and spent cow bedding used in both run are reported in Table 10a. The protocols used have been described by Riggio et al. (2017a).

The operating conditions for each run are described in Table 10b. In each reactor, about 1.5 kg of total solids (TS) of spent bedding was added. For the reactors inoculated, solid digestate was mixed to the spent bedding to reach a digestate TS/(substrate TS + digestate TS) of 13%. The leachate was diluted before being added to the reactor in order to keep a N-NH 4 + concentration in the leachate below 0.9 g L -1 at the start-up and to avoid any risk of nitrogen inhibition [START_REF] Angelidaki | Thermophilic anaerobic digestion of livestock waste : the effect of ammonia[END_REF]. The total amount of leachate to be added was chosen to keep the initial TS of the mix (manure + digestate + leachate) at 11.5%, close to the one reported by Riggio et al. (2017a) in similar systems. Before starting the digestion process, leachate was recirculated continuously for 98 10 min in order to achieve water saturation of the waste bed. Finally, the reactors were closed and the internal recirculation of the leachate was scheduled twice a day for a total volume of 1 L kg -1 TS d -1 . During digestion, volatile fatty acids (VFAs), pH, alkalinity and biogas volume and its composition were monitored. The frequency of analysis and the protocols used have been described in section 2.1.2.

Hypothesis for electrical production

Electrical production in a CHP unit from biogas produced in mesophilic and thermophilic conditions was compared using data from run 1. The simulation considered the following hypothesis: treatment of 9,400 tons/year of spent cow bedding and 4 LBRs working in parallel, staggered over time with a batch duration of 44 days: every 11 days a LBR was emptied and reloaded. 4 LBRs were chosen as a representative number of digesters in this kind of farm plant. In fact, more digesters would affect the economic feasibility of the project. 44 days were chosen not only because it is a realistic digestion time for this kind of substrate but also because the methane yield of the two conditions tested were the same after this time slot, cancelling the influence due to this factor on the comparison. Finally, a staggering time of 11 days between digesters start-ups was indirectly set after the previous choices: 4 digesters and 44 days of batch duration.

Biogas storage was not considered. A CHP unit with an electrical nominal power (P nom ) of 250 kW el [START_REF] Schnell | Food waste decomposition in leachbed reactor: Role of neutralizing solutions on the leachate quality[END_REF] was chosen, based on the average annual energy production of the site (considering the annual amount of substrate and the duration time evoked above). A minimum methane content of 45% was set for injection into the CHP unit and an electrical efficiency (η el ) of 45.5% was considered at P nom on manufacturer recommendation. The electrical efficiency was considered to vary linearly [START_REF] Bianchi | Performance prediction of micro-CHP systems using simple virtual operating cycles[END_REF] between electrical nominal power (max_η el = 45.5%) and the electrical minimal power P min (min_η el = 41.0%) with the equation min_η el = max_η el × 0.9. It is important to note that the electrical minimal power P min (50% of P nom ) corresponds to the power under which the CHP shuts down.

Effect of inoculation in thermophilic conditions

Figure 25a permits a comparison to be made between the specific methane production (SMP) rates in thermophilic conditions with and without the addition of solid digestate as inoculum. SMP rates between the two conditions were different: for the first 12 days in run 1 and 24 days in run 2.

Moreover, in both runs a lower SMP rate was measured during the first 5 days when the LBR was not inoculated with solid digestate. This result indicates that an inoculation with solid digestate mostly influenced the start-up phase of the process leading to higher SMP peaks. Figure 25b shows that the VFAs which accumulated during this period were rapidly consumed after 5 days in both conditions and runs.

Small differences were observed when adding solid inoculum, while different VFA concentration peaks between runs were due to the use of different samples of spent cow bedding. All along the digestion process, the pH remained in a suitable range for an optimized anaerobic digestion (i.e., 7.6 -8.2).

In addition to the results of [START_REF] Chachkhiani | 16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure[END_REF] showing that thermophilic microbial community in cattle manure is present even if at a subdominant level compared to mesophilic communities, these results show that these microorganisms were sufficiently active to permit spent cow bedding to be digested without a specific addition of solid inoculum. The slightly faster startup was not found significant enough to justify the use of solid inoculum when using spent cow bedding, a substrate which, in any case, needs a long time to be degraded. Figure 26b presents the methane yield (MY) for both temperature conditions and runs. Run 2 was carried out to confirm SMP rates and MYs, but a technical problem required the interruption of the experiment after 37 days. However, MYs measured in runs 1 and 2 were very similar, thus indicating that the inoculum had already adapted in run 1 and that the results were repeatable. More precisely, after 37 days the errors on the MYs between the two runs (calculated as |x1 -x2| / M(x1,x2) with x1 and x2 the measures and M the mean value of the measures) were 3.5% an 2.1%

for thermophilic and mesophilic reactors, respectively. Because of the higher initial SMP rates, the MY in thermophilic conditions were higher and only after about 42 days of operation the same MY was reached in thermophilic and mesophilic reactors. The additional amount of methane produced in thermophilic, in comparison to mesophilic conditions, is depicted in Figure 26c.

Initially, the surplus was significant in both runs but it rapidly decreased to reach an average value of 27% at 13 days and 0% at 42 days. Methane yield at 42 days corresponded to 80% of the BMP (run 1). This means that if 80% of the potential energy were recovered, then thermophilic temperatures would not offer any advantage over mesophilic temperatures. However, for lower BMP value, the interest in using thermophilic conditions by reducing batch duration should be assessed economically.

In batch digesters fed with corn stover and operated at 20% TS, [START_REF] Shi | Reactor performance and microbial 194 community dynamics during solid-state anaerobic digestion of corn stover at mesophilic and thermophilic conditions[END_REF] also reported similar MY in thermophilic and mesophilic conditions after 38-45 days of operation. However, when treating easily-degradable substrates such as vegetable waste, [START_REF] Hegde | Comparison of thermophilic and mesophilic onestage, batch, high-solids anaerobic digestion[END_REF] reported better performance under thermophilic conditions with a significant reduction of the time (10 days) to reach the 95% of the BMP. The particular behaviour observed when treating spent cow bedding suggests that a thermophilic temperature had an impact mainly on its easilydegradable fraction (higher methane production rates over the first 7-8 days) and not on the slowlydegradable one (similar SMP rates after 12 days). Since the easily-degradable compounds represent only a small fraction of spent cow bedding which is known to be rich in lignocellulosic material (Riggio et al., 2017a), the advantage of operating under thermophilic conditions was thus extremely 104 limited. It is interesting to note that the degradation kinetics recorded were not influenced by nitrogen inhibition (N-NH 4 + kept below 0.9 g L -1 ) and only the effect of temperature on kinetics was observed. Nitrogen inhibition should be considered as a further problem requiring a solution in thermophilic conditions.

Higher VFA concentrations were observed in thermophilic condition as a consequence of a faster hydrolysis (Figure 27a). However, after 5 days very low concentrations were measured in thermophilic condition while 12 days were needed to degrade completely the accumulated VFAs in mesophilic condition. The delay observed was due to an accumulation of propionic acid in mesophilic condition (Figure 27b). In fact, thermophilic temperature favours the consumption of propionic acid because it lowers the Gibbs free energy of the reaction [START_REF] Amani | Anaerobic digestion from the viewpoint of microbiological, chemical, and operational aspects -a review[END_REF]. The high alkalinity in the system (higher than 5 g CaCO 3 L -1 at start-up), hampered a too important drop of the pH which remained between 7.3 and 8.2 considering both runs and temperature conditions.

Electricity production

Methane production rates have a significant impact on methane conversion into electricity through CHP units, mainly on account of their technical constraints (i.e. minimum methane content and maximum combustion power). Figure 28a depicts the combustion power (P biogas ) associated to the cumulated biogas of the four digesters at the inlet of the CHP. First, to prevent dropping below the minimum methane content (i.e. 45%), the use of biogas produced from a new batch can start only when the methane content is high enough and, hence, a part of the initial biogas produced is not exploited. For both temperatures, the amount of methane lost is quite similar: the difference is a mere 1.4%. In addition, in thermophilic conditions, high SMP rates during the first days of each batch cause important fluctuations of the total methane flow at the entrance of the CHP unit (Figure 28a). When P biogas overtakes the maximum power accepted by the CHP (i.e. 550 kW comb or 250 kW el ), the surplus biogas is burned in a torch and then lost if no storage is provided. As a consequence, about 7.9% of the methane is not converted into electrical power in thermophilic conditions compared to the 2.3% in a mesophilic environment. Another interesting aspect is showed by Figure 28b which illustrates the electrical power (P el ) produced by the CHP from biogas collected in mesophilic and thermophilic conditions. In mesophilic conditions, the CHP is operated at its P nom for a longer than in thermophilic conditions (i.e. 54% and 36% of the time, respectively). As a consequence, a better exploitation of the biogas energy can be achieved in mesophilic conditions, since η el is the highest at P nom whereas it decreases for lower powers.

To sum up, higher methane production rates in thermophilic conditions during the first days of the batch process are proved to induce, on one hand, the loss of a part of the biogas produced because the CHP power limits are overtaken, and, on the other hand, a poorer exploitation of the biogas's combustion power due to variable electrical efficiency. Based on this simulation, 5.9% less electrical energy can be produced for an annual period in thermophilic conditions (1.84 ×10 6 kW•h el ) as opposed to mesophilic conditions (1.95 × 10 6 kW•h el ). This result represents a further drawback when using thermophilic temperatures in a discontinuous process, in addition to the higher energy consumption, higher investment cost because of the use of thermo-resistant materials, as well as further biological issues as nitrogen inhibition.

Conclusions

The digestion of spent cow bedding, a slowly-degradable substrate, was compared at mesophilic and thermophilic conditions. Thermophilic temperature increased methane production but this effect was restricted to the start-up period (degradation of the easily-degradable fraction), with a reduced advantage for the cumulated methane recovered over the long term. Furthermore, higher kinetics during the first days caused higher fluctuation of the methane flow at the inlet of a CHP unit, with consequent lower electrical energy production. These issues should be considered when assessing advisability of implementing thermophilic conditions for the digestion of spent cow bedding in LBRs. 10.1016/j.biortech.2017.01.060. Supplementary data were added in section 5. 1 and 5.2.3.2 110 In the previous chapter the treatment of spent animal bedding as a sole substrate was studied and some optimization strategies were established. In this chapter, the co-digestion of spent animal bedding with an easily-degradable substrate is discussed since such configuration can occur seasonally at industrial scale. The system setting used in this section was modified and a new design, simulating the presence of other rectors as in a sequential system at industrial scale, was considered. In this chapter, the influence of the addition of different fractions of an easilydegradable substrate in such a system is tested and compared (section 5.1). Afterwards the effect of the reactor flushing with different amount of leachate is studied and a strategy to manage VFAs in sequential process proposed (section 5.2).

Figure 29 : Graphical abstract of the fourth issue faced in this PhD thesis: the extent of inhibition when adding a significant fraction of an easily-degradable substrate and the use of the leachate flush to manage volatile fatty acid accumulation 5.1 Co-digestion of spent cow bedding with an easily-degradable substrate

Introduction

Spent animal bedding was shown to be easily digested in a LBR (see chapter 2). However, at industrial scale, spent animal bedding is often mixed with other substrates. The substrate composition is often seasonal and easily-degradable waste or acidic waste such as ensiled maize, generating a high production of VFAs at the beginning of the batch operation, can be added. It is then important to manage properly this acidification phase and assess properly the amount of easilydegradable substrate that can be mixed without affecting too much the reactor performance.

Single-stage systems, where leachate is recirculated within the same reactor, were shown to suffer of longer methanogenesis phase leading even to complete failure if the initial inoculation is not adapted to the substrate. Massive inoculations are then made to treat easily-degradable substrate. For example, [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF] digested maize silage at a S/I of 0.5 compared to spent horse bedding easily digested at a S/I of 10.

In addition, [START_REF] Dearman | Methane production and microbial community structure in single-stage batch and sequential batch systems anaerobically codigesting food waste and biosolids[END_REF] showed that single-stage system suffered of VFAs accumulation and lower methane yield when compared to sequential system exchanging leachate and loaded with the same substrate mixture: 65% food waste, 25% digested biosolids and 10% seeds on a weight basis. Sequential systems are then more adapted to treat easily-degradable waste avoiding massive inoculation. In addition, this configuration represents the one often used on Naskeo sites.

The objective of this test was then to compare the influence of the addition of an increasing amount of easily-degradable substrate on the digestion process of a LBR operated as in a sequential system.

Materials and Methods

Three different co-digestion mixtures were tested. In each reactor 360 g of dry matter was loaded and the mixture fractions were calculated on a TS basis. In addition to a fixed amount of cereal residues (15% TS), different proportions of rapidly and slowly-degradable substrates (carrots and spent cow bedding, respectively) were added in the following proportions on a TS basis: 10% -75% in condition L, 25% -60% in condition M and 40% -45% in condition H (Table 11). These mixtures were inoculated with solid digestate using an inoculation ratio of 13% TS (digestate TS / total TS) as reported for previous experiments. Reactors were loaded and bulk phase brought close to water saturation through continuous recirculation of leachate for about 10-15 minutes. This latter was then extracted, the reactors were closed and the experiment started. The liquid phase used for extraction was the mixture of a leachate and water buffered with NaHCO 3 in order to keep total alkalinity at 11.4 g CaCO 3 L -1 (partial alkalinity at 9.4 g CaCO 3 L -1 and initial pH of 8.6). It was kept at room temperature and the reactors were flushed every day (excepted during the week-end) with 360 mL of the liquid phase: the leachate flush ratio was of 1 L kg -1 TS d -1 . The leachate was sprinkled on the bulk, let percolating and then collected after three hours for analysis.

The substrate and leachate properties are reported in Table 12. Further details about the system used, its set-up, the analysis carried out and the protocol used are provided in section 5.2.

Table 12 : Characterization of the substrates loaded: total solids (TS), volatile solids (VS) of spent cow bedding, cereal residues, carrots and digestate

Spent cow bedding

Carrots

Cereal residues Digestate TS 24.3% +/-0.6% 12.4% +/-0.1% 89.6% +/-0.4% 15.9% +/-0.5% VS 20.6% +/-0.8% 11.6% +/-0.1% 71.9% +/-0.5% 10.4% +/-0.3% VS/TS 84.7% +/-3.8% 93.8% +/-1.0% 80.2% +/-0.7% 65.1% +/-2.7%

Results and discussion

Figure 30 a-b shows the methane production rate and the methane content in the biogas.

Methane production rate is modified by the increasing addition of easily-degradable fraction: the higher this fraction the longer the lag phase and the lower the methane peak reached. The methane content clearly shows a methanogenesis inhibition: no inhibition is observed with 10%TS of carrots, the lag phase lasts 9 days with 25%TS of carrots and 18 days with 40%TS of carrots. The methane content stabilized between 55% and 60% after 10 days from the end of the lag phase in all the conditions.

Figure 31 shows respectively the VFA concentrations and the pH in the leachate after percolation through the bulk and extraction from the reactors. The VFA peak were higher when a bigger amount of easily-degradable fraction was loaded in the reactor: 14 g COD L -1 in condition L, 20 g COD L -1 in condition M and 24 g COD L -1 in condition H. In addition, significant VFA concentrations (> 1g COD L -1 ) were extracted for a longer time when adding easily-degradable substrate: 21 days, 34 days and 44 days respectively. Since the same amount of leachate was used to flush the reactors all along the process, higher VFA concentrations mean that the total amount of VFAs extracted were higher when increasing the amount of carrots added: 60.9 ± 1.1 g COD in condition H, 41.8 ± 2.1 g COD in condition M and 19.7 ± 1.8 g COD in condition L. The high concentrations in VFAs were reflected also by the pH level which reached very low values especially in conditions M and H, respectively 5.9 and 5.3, while the minimum was 6.5 in condition L. Low pH values were related to the low methane content and to the duration of the methanogenesis lag-phase.

Conclusions

The increase of the fraction of easily-degradable substrate in total mixture leads to increasing inhibition due to the accumulation of VFAs and low pH. 10% TS of easily-degradable fraction did especially if easily-degradable substrates (e.g. fruits or vegetable waste), produced seasonally in large quantities, are mixed to slowly-degradable substrates such as spent animal bedding. The management of VFAs will be different in relation to the process configuration adopted. In a LBR with internal recirculation of the leachate, the VFAs accumulated must be consumed in the same reactor and, often, considerable amounts of digestate are used to increase the reactor inoculation with methanogens in order to prevent acidification [START_REF] Kusch | Effect of various leachate recirculation strategies on batch anaerobic digestion of solid substrates[END_REF]. VFAs produced in a LBR can also be extracted and eliminated separately by coupling the LBR to an external AD reactor wherein VFAs are consumed (Viétez and [START_REF] Tait | Biogasification of solid wastes by two-phase anaerobic fermentation[END_REF], making this configuration particularly advantageous. In this last case, leachate management plays a very important role because it can enhance the transport and degradation of VFAs.

In the literature, several coupling strategies have been tested. The use of an external liquid AD reactor such as a UASB (up-flow anaerobic sludge-blanket) reactor (LBR-UASB coupling) has been proposed for the treatment of easily-degradable compounds (i.e. food waste, municipal solid waste and grass silage) [START_REF] Browne | Improving hydrolysis of food waste in a leach bed reactor[END_REF][START_REF] Han | Performance of an innovative two-stage process converting food waste to hydrogen and methane[END_REF][START_REF] Nizami | Role of leaching and hydrolysis in a two-phase grass digestion system[END_REF][START_REF] Shin | Multi-step sequential batch two-phase anaerobic composting of food waste[END_REF][START_REF] Xu | Optimization of food waste hydrolysis in leach bed coupled with methanogenic reactor: effect of pH and bulking agent[END_REF]. In such a configuration, one or more LBRs were operated as acidogenic reactors with small batch durations, while the UASB was used as a methanogenic reactor, as in a two-stage process. However, the use of a specific methanogenic reactor is not the only option for managing VFAs when treating easily-degradable substrates. The coupling of two LBRs (LBR-LBR coupling) was proposed in a sequential process where the VFAs produced in a freshly-loaded LBR (new) were consumed in a more mature LBR (old) at the end of the digestion. First used in its simplest configuration by [START_REF] Hall | Mesophilic Anaerobic Digestion of High Solids Cattle Waste in a Packed Bed Digester[END_REF] to treat manure, a more complex configuration known as SEBAC (sequential batch anaerobic composting) was then developed in the USA by [START_REF] Chugh | Degradation of unsorted municipal solid waste by a leach-bed process[END_REF] and [START_REF] Chynoweth | A novel process for anaerobic composting of municipal solid waste[END_REF] to treat municipal solid waste (MSW). They suggested to use three LBRs, the new and the old one coupled until stabilization of the new reactor, and a third one already stabilized (with an incubation time between new and old) with internal leachate recirculation. With no particular modification in the coupling strategy (new-old) yet paying more attention to the coupling period, further work was carried out in Australia [START_REF] Dearman | Anaerobic digestion of food waste: Comparing leachate exchange rates in sequential batch systems digesting food waste and biosolids[END_REF][START_REF] Nopharatana | Evaluation of methanogenic activities during anaerobic digestion of municipal solid waste[END_REF]in Thailand (Tubtong et al., 2004) in the treatment of MSW, food waste, the organic fraction of MSW (OFMSW) and market waste.

Although the LBR-LBR coupling was proven to be less efficient than the LBR-UASB coupling in terms of VFA conversion, the use of a continuous external methanogenic reactor increases costs, demands more accurate process control and results in a more complex design [START_REF] Poggi-Varaldo | A side-by-side comparison of two systems of sequencing coupled reactors for anaerobic digestion of the organic fraction of municipal solid waste[END_REF]. The latter constitutes a real drawback for LBR systems whose simplicity is one of their main advantages, in particular in a farm-scale context. Moreover, the co-digestion of rapidly-and slowly-degradable substrates, which can occur seasonally, requires a long digestion time to recover the energy potential of the slowly-degradable organic compounds. This makes the LBR-LBR coupling a very beneficial option for treating this kind of mixture.

Very few data are available in the literature to help understanding and optimizing leachate and VFA management in a LBR-LBR system. Only [START_REF] Dearman | Anaerobic digestion of food waste: Comparing leachate exchange rates in sequential batch systems digesting food waste and biosolids[END_REF] and Tubtong et al.

(2004) analysed in details the effect of leachate recirculation rate in a LBR-LBR system. They both pointed out the importance of increasing the volume of the leachate exchanged between a new and a mature reactor, observing an increase in the methane production rate.

In the literature, the extraction and the consumption of VFAs (from and in LBRs respectively)

have always been studied simultaneously as a consequence of the chosen experimental design which consists in the direct coupling of two (or more) LBRs. Additionally, this configuration does not permit the clear assessment of the impact of VFA accumulation on the process performance.

However, since extraction and consumption of VFAs are crucial aspects for understanding and managing the leachate flush in a LBR, a novel approach is proposed in the present study by always injecting a new leachate with known properties (i.e. with or without VFAs). This strategy permitted to provide separate data on both VFA extraction and consumption in LBRs.

Consequently, the objectives of this paper are the following: to test the influence of the leachate flushing rate on the VFA extraction and the overall substrate degradation performance in a new reactor, and to study the consumption of VFAs in mature reactors. Based on the above, the final goal is to propose practical guidelines on the leachate flush strategy in order to improve the VFA management for the treatment of a mixture of rapidly-and slowly-degradable substrates in a LBR-LBR system.

Materials and Methods

Experimental apparatus

Six LBRs were operated in parallel. Each reactor consisted in a 7 L jacketed glass cylindrical reactor of 14.5 cm diameter and 43 cm internal height. The LBRs were kept in mesophilic condition (37°C) by water recirculation from a thermo-regulated bath (Figure 32). A mesh with 1 mm holes was placed at 10 cm from the bottom of the reactors to hold the solid substrate. This allowed creating a 1.7 L volume at the bottom of the reactors for temporary leachate storage. Solid substrate occupied 3 L and the head space was of 2.3 L. A tube connecting the headspace and the volume under the mesh was added in order to facilitate percolation by equalizing the pressures in case of compaction of the bulk solid. A peristaltic pump was used for injection of fresh leachate on the top of the bulk and a sprinkler was added for a homogeneous distribution on the entire surface. The same pump was used to extract the leachate which accumulating at the bottom of the reactor. A Tedlar Biogas bag and a valve were added to the biogas circuit in order to regulate the pressure during the injection and the extraction process. A port in the biogas circuit allowed the collection of gas samples for analysis. The substrate loaded in the reactors was a mixture of spent cow bedding and cereal waste (collected from a dairy farm in the South of France and kept at -20°C before use), both of which are commonly available in a rural context and made-up the slowly-degradable substrates. Additionally, carrots from a supermarket were added as a model for an easily-degradable co-substrate. The mixture contained, on a TS basis, 45% of raw spent cow bedding, 15% of raw cereal residues and 40% of carrots. These latter were freshly grated to roughly 3-cm long bits with a Moulinex ME415 vegetable mincer before being mixed in with the rest. A total mass of 360 g TS or 2,100 g RM (raw matter) was loaded into each reactor and compacted. The bulk density was about 630 kg m -3 . The spent cow bedding, thanks to its porous structure, permitted good leachate percolation [START_REF] Demirer | Anaerobic biogasification of undiluted dairy manure in leaching bed reactors[END_REF][START_REF] Myint | Enhancing anaerobic hydrolysis of cattle manure in leachbed reactors[END_REF]Riggio et al., 2017a;[START_REF] Xu | Optimization of food waste hydrolysis in leach bed coupled with methanogenic reactor: effect of pH and bulking agent[END_REF]. In order to ensure digester inoculation, solid digestate from a previous batch was mixed to the solid bulk before loading and represented 13% of the mixture (digestate TS/total TS), as previously done by Riggio et , (2016). The leachate used for flushing the solid bulk was collected at an industrial agricultural site using sequential LBRs to treat mainly spent cow bedding and green waste. This was diluted 1:1.7 (vol/vol) and stored at 37°C throughout the whole experiment in order to maintain microorganism activity as high as in a real system. The properties of the substrates and leachate before loading are summarized in Table 13.

Table 13 : Characterization of the substrates loaded: (a) total solids (TS), volatile solids (VS), and BioMethane potential (BMP) of spent cow bedding, cereal residues, carrots and digestate; (b) total and soluble chemical oxygen demand (CODt and CODs), volatile fatty acid (VFA), total and partial alkalinity and pH of the leachate

Experimental procedure

Every day except for the week-ends the reactors were flushed once: a given volume of fresh leachate was injected into the reactor and drawn off 3 hours later (i.e. after percolation) from the bottom of the reactor in order to be analysed. The injection of three different volumes of fresh leachate was tested in duplicate (Figure 33): high (H1-H2) with 720 mL flush - with 360 mL flush -1 and low (L1-L2) with 180 mL flush -1 . Only one flush per day was effectuated in each reactor. These volumes corresponded to a volume of leachate injected per day and per amount of total solids loaded of, respectively, 2.0, 1.0 and 0.5 L kg -1 TS d -1 , for one flush per day (or, to facilitate comparison with the literature: 0.34, 0.17 and 0.09 L kg -1 RM d -1 or 0.22, 0.11 and 0.05 L L -1 subst d -1 ). These leachate flush-rates were chosen to be representative of those applicable at industrial scale.

Figure 33 : Experimental planning and configuration. Three conditions are tested in duplicate: in L1-L2 a low leachate flush rate is applied (0.5 L kg -1 TS d -1 ), in M1-M2 a medium leachate flush rate is applied (1.0 L kg -1 TS d -1 ) and in H1-H2 a high leachate flush rate is applied (2.0 L kg -1 TS d -1 ).

During the "reactor flush" period, a fresh leachate is pumped in and out every day while, during the "internal recirculation" period, the same leachate is recirculated through the bed. Additionally, M2 and H2 are used to carry out an activity test.

The reactors were flushed until no more VFAs were observed in the extracted leachates: 31 days in condition H, 34 days in condition M and 45 days in condition L. Starting from this moment and until day 90, an internal recirculation mode was adopted: the leachate was recycled within the same reactor and never renewed. Throughout the internal recirculation mode, the amount of leachate recirculated was the same as during the reactor flush.

123

Given the good repeatability of the pairs H and M, one of each pair (H2 and M2) was used to carry out two activity tests. These tests were done by adding acetic acid to the fresh leachate and then flushing the reactor with 360 mL of the prepared solution (as in condition M). Two different acetic acid concentrations were tested: 5 g L -1 in H2 and 10 g L -1 in M2. The first test was done at 31 days and 34 days for H2 and M2, respectively, whereas the second test at the end of the digestion process for both conditions.

Sampling and analysis

Characterization of the initial substrates was carried out in triplicate and the average values with standard deviations were reported. The TS content was measured after drying at 105 °C (378 K) for 24 h, followed by 3 h of calcination at 550 °C (823 K) for determination of VS. BioMethane Potential (BMP) tests were carried out on the raw samples in batches of 500 mL inoculated with anaerobic sludge from a sugar industry anaerobic treatment plant of, in accordance with the procedure described by Riggio et al. (2017a).

The industrial leachate used for flushing was characterized in terms of partial and total alkalinity, VFA concentration, pH, and total and soluble chemical oxygen demand (CODt and CODs, respectively) (Table 14).During the digestion process, after every reactor flush, pH, VFAs and CODs were measured. Specifically, pH, alkalinity and CODt were measured in the raw leachate, while VFAs and CODs were measured in a leachate previously filtered at 0.45 μm. The pH was measured with Mettler Toledo InPro 4260i probes connected to a Mettler Toledo pH M300 operational manual transmitter. Partial alkalinity was measured at a pH of 5.75 and total alkalinity at a pH of 4.30, as advised by [START_REF] Hill | Measuring alkalinity accurately in aqueous systems containing high organic acid concentrations[END_REF]. VFAs were analysed in a VARIAN I-MET-0084 gas chromatograph with helium as the gas vector. COD was measured using 0-1.500 mg L -1

Aqualytic tube tests after 2 h of oxidation at 160 °C in a HACH COD reactor with readings via Aqualytic Multi Direct spectrometer. During digestion, biogas volume was recorded manually once a day using the Ritter flow meter Milligascounter MGC-1 V3. Biogas flow was recorded every 5 min only during the activity tests, with the help of a home-made software connected to the Ritter flow meter. Biogas composition was analysed with a micro-GCPRO CP-4900 gas chromatograph using helium as the gas vector. Finally, biogas volume was expressed in standard conditions: 273.15 K and 101,325 Pa.

Table 14 : Characterization of the substrates loaded: total solids (TS), volatile solids (VS), and BioMethane potential (BMP) of spent cow bedding, cereal residues, carrots and digestate; total and soluble chemical oxygen demand (CODt and CODs), volatile fatty acid (VFA), total and partial alkalinity and pH of the leachate

Data processing

Conditions H and M represent the average values of the pairs (H1-H2 and M1-M2, respectively) with their variation range (maximum and minimum values). Since H2 and M2 were used to carry out activity tests (on days 31 and 34, respectively), H and M only correspond to H1 and M1 after these days (without error bars). On the other hand, since L1 and L2 showed significant differences, both reactors are described for the whole operation.

Spent cow bedding

Results and discussion

Reactor performances

Operating periods

The following parameters were monitored along timeline: pH (Figure 34a), methane production rate (Figure 34b) and total VFA (TVFA) concentration in the extracted leachate (Figure 34c). As a representative example, the TVFA composition of condition H is also reported (Figure 34d). As an aid, it should be noted that the amount of VFAs extracted through a flush represents only a fraction of the total VFAs present in the bulk phase. As a consequence of that, TVFA concentration and pH in the solution are only indicative of the actual amounts in the bulk. Overall, the experiment can be divided into three main periods, which can be identified by analysing the biogas directly produced by the solid phase along with the characteristics of the leachate after percolation through the bulk.

Period I (0-15 days): Acidification Period I was characterized by a strong acidification, with pH dropping below 6 (Figure 34a), an absence of methane production indicating a complete methanogenesis inhibition due to low pH (Figure 34b), and high TVFA concentrations in the leachate (Figure 34c). During the first days of the process, the CODs of the extracted leachate was slightly higher than the COD of VFAs, meaning that other metabolites (e.g. alcohol or more complex acids) were also produced in acidic TVFA concentrations dropped significantly to values lower than 12 g COD L -1 and methane production was lower than 0.2 NL CH 4 d -1 .

Period II (15-35 days): Methanogenesis establishment

This period was characterized by the establishment of the methanogenic activity until the maximum methane production rates were reached (Figure 34b). In the first days of this period, pH in the leachate switched towards values higher than 7 and proved that methanogenesis inhibition in phase I was effectively linked to a low pH. In fact, pH higher than 7 are known to be favourable for anaerobic digestion [START_REF] Jha | Research advances in dry anaerobic digestion process of solid organic wastes[END_REF]. However, it is reminded that the pH measured in the extracted leachate only partially represents the effective pH in the reactor bulk, which might be little lower inside the bulk. In period II, TVFA concentrations were lower than in period I and decreased all along. TVFA decrease was related not only to their extraction but also to their conversion into methane (Figure 34b). A slightly higher concentration of propionic acid was observed in this period compared to period I. In fact, propionic acid is known to be more difficult to degrade than the other VFAs and its complete degradation occurred only after depletion of both butyric and acetic acid (Öztürk, 1991).

Figure 34b shows that the methane production kinetics were similar for all the conditions.

Similarly, the methanogenesis lag-phase were also comparable across all the conditions but L1

which means that leachate flush rate had a very little influence on this parameter: the methanogenesis lag-phases were about 15 days on average with only a difference of 2 to 3 days between conditions H and L2 (L1 will be discussed later). Literature reports a stronger influence of the leachate flush rate on the methanogenesis lag-phase: Tubtong and Towprayoon (2010) observed a difference of 10 days between two LBRs treating market waste and flushed once every two days with 0.8 and 2.3 L kg -1 TS flush -1 (15 and 25 days respectively), while [START_REF] Dearman | Anaerobic digestion of food waste: Comparing leachate exchange rates in sequential batch systems digesting food waste and biosolids[END_REF] observed a difference of 20 days between two LBRs treating organic fraction of municipal solid waste and flushed once per week with 0.8 and 1.6 L kg -1 TS flush -1 (30 and 50 days respectively). Although the real reasons of these differences are difficult to define as many factors could play a role, the minor flush frequency (0.5 flush day -1 or 1 flush week -1 compared to 1 flush day -1 in this study) causing a delayed mobilization of the VFAs and accumulation in the bulk phase could be the cause.

Even if high leachate flush rates showed little effect on methanogenesis lag-phase some differences were observed when decreasing it to value close 0.5 L kg -1 TS d -1 , as for reactors L1 and Activity tests were performed on H2 and M2 by injecting acetic acid (through the addition of fresh leachate) at two periods, after the peak of the methane production rate (34 and 31 days for M2 and H2, respectively) and right before stopping the batch (90 days) (Figure 34). Three injections were realized in each condition. A mass balance between the VFAs injected with the fresh leachate and the VFAs extracted allowed determining that 36% ± 3% of VFAs were hold in the bulk after each injection (Table 15). Figure 35 presents the biogas flow rate due to acetic acid consumption (average of the 3 injections). The results show that the biogas production rate was about four-fold higher in the first period than at the end of the batch treatment, indicating that methanogenic activity was higher when the injections were carried out right after the biogas peak rather than at the end of a batch. Indeed, by sprinkling a leachate with an acetic acid concentration of 10 g L -1 , the amount of acetic acid held in the bulk was consumed faster, namely one day for the 1 st injection and two days for the 2 nd injection. The reason of a reduced methanogenic activity over time is linked to the reduction of available substrates for methanogens during the digestion process.

Figure 35 : Activity test: biogas production rate after injection of leachate containing acetic acid. 1 st injection is effectuated after the methane peak (after about 30 days) and 2 nd injection at the end of digestion (after about 80 days). Two acetic acid concentrations in the leachate were tested: 5 g L -1 and 10 g L -1 . VFAs produced through acidification of the substrate, since it is not possible to assess the real quantity of VFAs accumulated within the solid bulk phase. However, since no VFAs were extracted, there were no VFAs retained in the bulk during period III, and the accumulated amounts of H 2 , VFAs and CH 4 presented in Figure 36b corresponded to the quantity of substrate degraded (while, before this period, they corresponded only to the COD accessed and available to the operator, i.e. inferior to the real substrate degradation).

Subtrate degradation

. At 35 days, 34%, 29%, 21% and 20% of the BMP were reached in conditions H, M, L1 and L2, respectively (Table 16). A significant difference was still measurable at 60 days: 81%, 77%, 72% and 69% in conditions H, M, L1 and L2, respectively. Since mesophilic processes with slowlydegradable compounds are usually not operated for more than 60 days at industrial scale, condition H proves to be a better condition to apply in order to increase substrate degradation within this limited time frame. In a coupled system (LBR-LBR coupling), the increase of the leachate flushrate would mean a better overall methane production rate, as effectively observed by Tubtong et al.

( 2004) and [START_REF] Dearman | Anaerobic digestion of food waste: Comparing leachate exchange rates in sequential batch systems digesting food waste and biosolids[END_REF]. After 90 days of operation, the degradation curves converged towards similar values due to the similar loading, thus further confirming the accuracy of the measurements. By this time, between 81% and 87% of the BMP was reached. despite the differences observed in the methane produced and the VFAs extracted (Figure 36a). It means that the VFA extraction and the delayed methane production were equivalent (in COD terms) and that VFA extraction was essential to make the COD entrapped in the bulk available during period II. the higher the leachate flush-rate, the higher the amount of VFAs extracted and the lower the amount of CH 4 produced in the digester.

However, the percentage of VFAs extracted and the leachate flush-rate were not directly proportional (e.g. by doubling the amount of leachate injected, the mass of VFAs extracted increased by only 23% when passing from condition M to H). This suggests that an optimum should be found between the amount of leachate used to flush, the amount of VFAs extracted and the VFA concentration in the extracted leachate. These parameters should then be assessed when conceiving a process using LBRs, but they will not be considered in this context. 37 a-b show the daily and the accumulated mass of VFAs that were extracted from the bulk thanks to the flush. These figures show that most of the VFAs were extracted during period I, only a small fraction in period II, while almost no VFAs were extracted in period III. VFAs are known to inhibit hydrolysis at very high concentrations, while their extraction permits to reduce it [START_REF] Cadavid-Rodriguez | Production of volatile fatty acids from wastewater screenings using a leach-bed reactor[END_REF]. Such reduced VFA inhibition in the first stage of the process is therefore essential to decrease the overall batch duration and achieve higher performance.

Hence, a high leachate flush-rate is important during period I. Furthermore, a high leachate flush-rate in period I will reduce the methanogenesis lag-phase problem observed in L1 and L2. Considering that the problems of homogenous percolation and channeling are bigger at industrial scale than at lab scale, the reduction of the leachate volume injected is not recommended during this phase.

The very small amount of VFAs extracted in period II and the very similar methane production kinetics obtained (Figure 34b) in all three conditions indicate that the leachate flush-rate does not have a great impact in this period and that, consequently, the lowest and least expensive leachate flush-rate could be chosen. It would also be of interest to lower the amount of VFAs extracted as well as the problem connected to their consumption. To this end, several authors have even advised to switch to an internal recirculation mode (zero extraction) in this period, once specific conditions confirming a stabilized methanogenesis have been reached, such as: a pH above 6.5 [START_REF] Chugh | Degradation of unsorted municipal solid waste by a leach-bed process[END_REF], a methane production rate higher than 0.5 L CH 4 kg -1 VS d -1 [START_REF] Dearman | Anaerobic digestion of food waste: Comparing leachate exchange rates in sequential batch systems digesting food waste and biosolids[END_REF] and a methane content in biogas exceeding 30% (Tubtong and Towprayoon, 2010).

Consumption of external VFAs in LBRs

After extraction, VFAs have to be converted into methane and the treatment of the large amounts of VFAs extracted (e.g. 41% of the degraded COD in condition H) has to be properly managed. Nonetheless, the availability of readily-degradable substrates like VFAs can be considered an advantage, since their conversion and subsequent methane production can be easily controlled. This would permit a smoothing-out of the methane flow-rate sent to the cogeneration engine (an important issue at plant scale (Degueurce et al., 2016d)) but also, in a more global context, adapting the biogas production to a demand-driven biogas supply [START_REF] Hahn | Review of concepts for a demand-driven biogas supply for flexible power generation[END_REF].

Figure 37a permits estimating the maximum quantity of COD to be sent to a methanogenic reactor (if no storage volumes are considered) for conversion into methane. The maximum values are reached during period I when the most VFAs were extracted: 27 mg COD g -1 VS d -1 in condition H, 19 mg COD g -1 VS d -1 in condition M and 15 mg COD g -1 VS d -1 in condition L. These organic compounds could be easily treated via a very common process, such as an UASB [START_REF] Browne | Improving hydrolysis of food waste in a leach bed reactor[END_REF][START_REF] Han | Performance of an innovative two-stage process converting food waste to hydrogen and methane[END_REF][START_REF] Nizami | Role of leaching and hydrolysis in a two-phase grass digestion system[END_REF][START_REF] Shin | Multi-step sequential batch two-phase anaerobic composting of food waste[END_REF][START_REF] Xu | Optimization of food waste hydrolysis in leach bed coupled with methanogenic reactor: effect of pH and bulking agent[END_REF]. However, a LBR-LBR coupling (VFAs are consumed in a LBR) will be considered since this would allow to keep the process simple, a real advantage for a process intended for rural context. In this case study, lower TVFA concentrations than those found in period I were chosen for activity tests (Figure 35 and Table 15) as if leachate storage tanks had been added to the system, thus leading to a dilution of the organic load sent to the LBR. Indeed, as shown in Figure 35, the methanogenic activity of H2 and M2 was almost two-fold higher right after the biogas peak as compared to the value reached at the end of digestion. It is interesting to note that during injection 1 the maximum biogas production rate doubled, rising from 80 NmL h -1 to 160 NmL h -1 (the small volume produced in comparison to the head space did not reveal any difference in the methane content), whereas during injection 2 the maximum biogas production rate increased from 10 NmL h -1 to 35 NmL h -1 , much lower than the previous rate (160 NmL h -1 ). Therefore, for more efficient VFA degradation in LBRs, VFAs should be injected a few days after the biogas production peak, when no more VFAs are extracted and when hydrolysis becomes the limiting step in the LBR (beginning of period III). Clear details about the methanogenic activity of the mature LBR used in coupled systems are not given in the literature since the attention was mainly focused on the stabilization of the new reactor [START_REF] Chynoweth | A novel process for anaerobic composting of municipal solid waste[END_REF][START_REF] Dearman | Anaerobic digestion of food waste: Comparing leachate exchange rates in sequential batch systems digesting food waste and biosolids[END_REF]Tubtong and Towprayoon, 2010). However, reactor maturity and methanogenic activity must surely have impacted their results via the VFA consumption rate.

Figure 35 also shows that by injecting a leachate with acetic acid at a concentration of 5 g L -1 , the maximum biogas rate was already reached. In fact, by using a leachate with an acetic acid concentration of 10 g L -1 , similar biogas rate were obtained up to 6-7 h after injection and only the total time to consume the entire amount of acetic acid varied between the two conditions (5 and 10 g L -1 ) (Table 15). Although injecting a leachate with a higher VFA concentration clearly increased the amount of VFAs held in the bulk phase, a concentration of 5 g L -1 was found to be enough to maintain a high biogas production rate and not to drop below the half the height of the biogas peaks 138 (at 6-7 hours). Thus, an injection of a leachate with an acetic acid concentration varying between 5 and 10 g L -1 at a frequency of every 6-7 hours seems to be the optimal strategy to allow the maximum consumption of VFAs and maintain a high biogas production rate. Finally, along with this frequency, small volumes per injection seem reasonable. In this study, sprinkling 360 mL of leachate over the bulk substrate (18.5 cm high and 14.5 cm in diameter) resulted in a retention of only 36% ± 3% of the mass of the acetic acid injected into the reactor (Table 15). Even if this percentage was dependent on the geometry of the reactor, meaning that this result cannot be generalized, higher volumes of leachate would probably have increased only the channeling with no significant impact on the mass retained in the bulk. This view is further corroborated by the increasing compaction of the bulk over time and the consequent reduction of percolation efficiency [START_REF] André | Quantifying physical structure changes and non-uniform water flow in cattle manure during dry anaerobic digestion process at lab scale : Implication for biogas production[END_REF].

Overall strategy for VFA management in LBRs

A LBR treating a mixture of easily and slowly-degradable substrates passes through three periods. Table 18 summarizes the recommendations to properly manage VFAs based on the results obtained in each of them. During initial acidification (period I), a large amount of VFAs are produced which must be extracted rapidly. Hence, a high leachate flush-rate, ≥ 2 L kg -1 TS d -1 , has to be applied in order to reduce the risk of inhibiting hydrolysis while increasing the overall substrate degradation rate. During methanogenesis establishment (period II), less amounts of VFAs are present in the substrate bulk and their extraction can be reduced (≤ 1 L kg -1 TS d -1 ) or even stopped (internal recirculation), making sure to avoid negative impact on the methanogens growth activity. Once the biogas peak is reached, a reactor could be left to complete its degradation. As such, the leachate flush would no longer play any role in VFA transfer and could be stopped.

However, a LBR could also be used to consume external VFAs (coming from other reactors). In this case, leachate injection should be regulated in order to keep a high biogas production rate in order to convert the majority of external VFAs. To exploit the highest methanogenic activity, external VFAs should be injected right after the methane peak, when a clear lack of easilydegradable substrate is observed. Moreover, by using a leachate containing only acetic acid, an injection every 6-7 hours with a concentration between 5 and 10 g L -1 was found to be the best strategy. Small volumes are also advised (here 360 mL flush -1 was tested).However, since different combinations of volume per flush and frequency can give similar results (Degueurce et al., 2016d), the most suitable combination must be found in relation to the technical possibilities linked to the digester and substrate properties (e.g. digester geometry and percolation capacity). Similarly, more complex mixtures of organic acids than straightforward acetic acid should be tested.

Effect of frequency

In the previous experiment the frequency of flushing was kept constant (i.e. once per day). However, its influence could play an important role and its impact on the VFA extraction capacity was studied more in detail in a separate context. 1,200 g of spent cow bedding (28.1% TS and 22.9% VS) and 400 g ensiled maize (12.3% TS and 11.7% VS) were loaded in two different reactors. 1500 mL of water were added in each reactor and digestion started as in a single-stage process (see section 2.1.2 for system design). After 5 days, a high concentration of VFAs was measured in the leachate, the reactors were stopped and initial liquid extracted. Afterward, the reactors were flushed following different strategies: the first was flushed 12 times by injecting 200 mL (corresponding to 0.5 L kg -1 TS flush -1 ) and the second 10 times with 800 mL (corresponding to 2 L kg -1 TS flush -1 ). After that phase, 1 L of water was injected in each reactor and let recirculate for over 1 hour continuously in order to homogenise the remaining VFAs and to identify its amount. The cumulated amounts of VFAs extracted, respectively in the reactor flushed with 200 ml and in the one flushed with 800 mL, were 11.1 g and 12.8 g. A difference of only 13% allowed us to compare the two extraction strategies.

Figure 38 shows the cumulated mass of VFAs extracted over the cumulated amount of water used for extraction. The results highlight that using a bigger volume of water for extraction allowed extracting more VFAs. However, for a given amount of water used, bigger amount of VFAs were extracted flushing with a smaller volume. This means that an optimum should be found between VFAs extracted and leachate pumped (in an industrial system), and that the frequency is a very important parameter affecting the extraction efficiency (since a same amount of VFAs can be extracted using a smaller amount of water but split in several flushes). This test allowed also to quantify the amount of VFAs extracted compared to the 142 total amount of VFAs accumulated: by using a leachate flush rate of 0.5 L kg -1 TS flush -1 about 9% of the total VFAs in the bulk were extracted with one flush while 14% were extracted with 2.0 L kg -1 TS flush -1 . That means that, after a flush, most of the VFAs are still in the bulk and only a very small fraction is washed out. In this last chapter the major findings of the previous chapters are reported and discussed. This will allow having an overall view of the entire study, to draw the main conclusions and to open new perspectives.

Context

Anaerobic digestion is known worldwide to be an efficient bioprocess allowing the simultaneous stabilization of organic waste and energy recovery. Because of its advantages, AD is a no way around biotechnology and its role in the future energetic mix is undiscussed.

As many other European countries, France is investing on its development to reach the objective fixed by the "Loi sur la transition énergétique" asking for 32% of the total energy production coming from renewable sources by 2030. Decentralized energy production is a very important development axis because it allows the spreading of energy sources on all the territory, which will directly reduce the loss connected to the energy transport and favour the local conversion of organic resources. The development of farm-scale anaerobic digestion plants is a very promising sector in France whose 90% of the available bioresources are estimated to originate from agricultural sector by 2030. 41% of those are animal dejections and 75% are estimated being under a dry state. Spent animal bedding, a mixture of animal faeces, urines and bedding material (most of the case straw in France) is then a very interesting waste to be considered for France decentralized energy production network.

Moreover, the uncontrolled management of this waste can lead to environmental issues like soil and water pollution as well as odour nuisances and uncontrolled emission of methane.

Anaerobic digestion is able to efficiently solve these issues and leach-bed reactors, a dry discontinuous AD process, is adapted to this context and kind of organic substrates. Several small French companies recently decided to invest on its development to treat organic farm waste. However, the treatment of spent animal bedding in LBRs is not well known since this technology was initially designed for the treatment of easily-degradable waste. As a consequence, many problems connected to the use of LBRs for the treatment of slowlydegradable waste have not been faced, and solved, yet. In addition, even if some industrial plants treating spent animal bedding exist, a lot of operational parameters should be still assessed to optimize this process and make it more profitable and competitive with respect to other technologies. In this context, research is required to answer industrial questions and help the implementation of farm-scale AD plants based on LBRs.

Digestion of spent livestock bedding in a single-stage LBR

In chapters 2, 3 and 4, the digestion of spent animal bedding in single-stage LBR was studied and some optimization levers were proposed to improve the methane production.

These objectives were reached through: (i) the characterization of spent animal bedding and the identification of possible variability of its properties; (ii) the study of the LBRs advantages and limits as a process, as well as in relation to spent animal bedding digestion; (iii) the study of specific strategies to optimize methane production.

The substrate properties

The characterization of six different types of spent animal bedding (one from sheep, one from goat, one from horse and three from cows) allowed to sketch common properties of this kind of waste (Figure 39). Two very poorly studied types of spent animal bedding and two more common ones were chosen: the ones from sheep and goat belonged to the first group, and the ones form horse and in particular from cows to the second group. An overall analysis show that spent animal bedding has a high total solids content which enables them to be easy to handle, a VS/TS ratio close to 85% on average, which indicates a high organic matter content, and a balanced C/N ratio ranging between 20-28 (except for the spent horse bedding which displays a C/N ratio of 42), which means that spent bedding is a substrate whose digestion does not lack of nitrogen. Fibre analysis reveals that spent animal bedding is a lignocellulosic material, with presence of significant amount of cellulose, hemicellulose and lignin, but also a significant fraction of soluble compounds (between 20% and 37%). The coexistence of an easily-degradable fraction and a slowly-degradable one has a great impact on the dynamic of spent bedding degradation: a long digestion time and an initial high methane peak (further discussed later). Finally, the biomethane potential (BMP) measured for the substrates studied are very close, in a range between 192 and 239 NmL CH 4 g -1 VS, which indicates that their biodegradability is quite low compared to other substrates. However, expressed on a raw matter basis, spent animal bedding shows values ranging between 40 -71

NmL CH 4 g -1 RM, which suggests that these substrates are interesting for energy recovery from an industrial point of view.

In spite of the common properties shared by different kinds of spent bedding, mulching, or the amount of straw used as bedding in relation to the number of animals (related to the husbandry practices), is found to be a parameter potentially affecting significantly the properties of spent animal bedding

In spite of the common properties shared by different kinds of spent animal bedding, some differences can appear within this waste category. A very important one is the mulching or the amount of straw used as bedding in relation to the number of animals, which a parameter strictly related to the husbandry practices. A low amount of straw can decrease the TS of the substrate, modify its physical structure, and then reduce the percolation efficiency (and degradation) in LBRs, while a high straw content can significantly increase the C/N which could lead to a non-optimal substrate for AD. Among the tested substrates spent horse bedding is found to be the one with the highest amount of straw. Even if its digestion did not Concerning the inoculation, it is also proved that spent cow bedding, the most common type of spent animal bedding, easily starts AD both in mesophilic and thermophilic temperature conditions without solid inoculum addition. This is a very interesting result since this proves that S/X of this substrate (considering it as a mixture of straw and faeces) is already sufficient and no further addition of methanogenic microorganism's inoculum is necessary. That can be of significant help when starting a new process when solid digestate is not available or, more generally, to reduce costs of a plant site.

Despite the different methane kinetics observed, the final degradation efficiencies of all the spent animal bedding tested are similar. The methane yield (MY)-to-BMP ratio is on average 89% ± 11% after 60 days of operation in LBRs. This proves that LBR is a suitable process to efficiently treat spent animal bedding. Some drawbacks and optimization levers have been also identified in the digestion of spent animal bedding in a LBR. The long digestion time for these slowly-degradable substrates is recognized as the main weakness of this substrate when treated through AD. Shortening the digestion time could be an interesting option since, during the last 20 days, the methane production is really low and represents only 9% to 15% of the total methane production when a digestion time of 60 days is considered.

Further optimizations concern the start-up strategy. Indeed, this is a crucial phase during which methanogens grow in the digester and methane starts to be produced. However, the low initial methane content in the biogas during the start-up phase can hamper its combustion in a CHP unit. As an example, up to 8.1% of the total methane produced can be lost if a threshold of 40% of methane content in the digester head-space is considered as prerequisite prior injection in the CHP unit. Increasing methanogenesis rate can represent an interesting option since it allows a rapid increase of methane concentration in the biogas, but this can also induce a high methane production peak that could not be converted by the CHP because of the sudden flux of fuel to deal with. An optimum between these two parameters should then be found.

Another important aspect about LBRs is the water management and the reuse of the liquid fraction after an AD cycle. It has been highlighted that in LBRs a significant amount of water is needed and the required quantity is directly proportional to the initial substrate TS.

As an example, considering an initial TS between 24% and 37% from 0.3 to 0.7 kg of water per kg of fresh matter is needed to run the process. This factor should not be underestimated at industrial scale, even if the advantage of this dry system remains relevant compared to a wet process.

Moreover, the reuse of the liquid fraction for the next AD cycle can cause accumulations of non-degraded compounds, and some of them can reach inhibitory

Inoculation

Inoculation in LBRs is particularly important since it has to be repeated at each start-up phase, that is to say several times per year and per reactor at industrial scale. Its assessment is then critical. Inoculation can be done through the liquid leachate or the solid digestate.

Leachate is shown to play mainly a role of process stability inside a reactor by providing the system with the needed alkalinity whereas digestate is found to act mainly on the biogas production. It is shown that 15.8% higher methane yield in 60 days could be reached by adding digestate (digestate TS/ total TS = 13%) compared to a simple leachate addition. This better depletion of the organic matter is an interesting result for a slowly-degradable substrate such spent cow bedding. However, the addition of digestate has to deal with the digester size since its addition leads to an increase of the digester working volume (if the same amount of fresh organic matter is treated). In chapter 3, it was shown that from an economical point of view, the use of the digestate or of the leachate gave the same economic results for two economical parameters: the internal rate of return and the payback period. This means that the economic impact due to the digester volume increase could be counterbalanced by the higher

In other words, the advantages of thermophilic over mesophilic temperatures concerning the methane yield are negligible after 42 days of digestion.

Furthermore, the use of thermophilic temperature and a consequent increase in the biogas production rate during the first days of digestion displays a significant impact on the biogas conversion in CHP unit. Because of the power operating range (i.e. between maximum and minimum powers) of the cogeneration engine, a too high fluctuation of the biogas production causes the loss of a part of the biogas, which is then sent to a torch. In the end, in thermophilic conditions, the loss of electric energy is estimated up to 5.9% (compared to the one produced in mesophilic condition). Furthermore, additional energy costs for maintaining a higher temperature in the digesters as well as the use of more expensive material for the construction of the structure in the case of thermophilic conditions would decrease even more its economic attractiveness. For this reason, thermophilic operating temperature to treat spent animal bedding is shown to provide no real advantage if spent animal bedding is considered as a resource. The search of an economic optimum between operation time and methane yield is then considered beyond the scope of the PhD thesis and not studied.

Leachate recirculation

Previous results (section 2. 1.3.2) showed that the accumulation of VFAs is not particularly significant when digesting spent animal bedding as a sole substrate (maximum VFA concentrations generally lower than 5 g L -1 except for spent goat bedding which reached 11 g L -1 ). Therefore, leachate recirculation does not influence significantly the digestion process and the modification of the leachate recirculation from 10 L kg -1 TS d -1 to 1 L kg -1 TS d -1 gave the same results in term of methane yield and methane production rate if the bulk water saturation is reached at the beginning. As a consequence, in order to reduce costs related to pump use and maintenance the lowest leachate recirculation rate should be chosen and a value of 1 L kg -1 TS d -1 is considered an upper limit for this parameter.

6.3

The co-digestion of spent livestock bedding with an easily-degradable substrate in a sequential LBR system Spent bedding is the substrate which is mainly found in countryside and for this reason the most used in farm-scale AD process. Usually, spent livestock bedding is showed to be easily digested in LBRs and does not involve acidification problems because of its high alkalinity. Co-digesting spent bedding with other more easily-degradable substrates seems then a possible and effective option to increase the methane production of the site and, as a consequence, its economic feasibility. Moreover, the addition of a second substrate is a common practice. Most of the time, this latter is an easily-degradable substrate that is added seasonally and sometimes can lead to acidification problems that should be further well controlled.

In chapter 5 the co-digestion of spent bedding with an easily-degradable substrate was studied. The system used was no more a single-stage digester but one simulating a sequential system as on a real site (the leachate was never recirculated within the same reactor and an external leachate always used). It is observed that methanogenesis inhibition is proportional to the fraction of easily-degradable substrate added (Figure 41): 10% (on a TS basis) of easilydegradable can be added to spent animal bedding without any particular risk of acidification, with 25% of TS, a 10 days lag-phase is observed while with 40% of easily-degradable the lagphase reached 20 days. An increased accumulation of VFAs and the consequent decrease of the pH in the bulk in relation to the fraction of easily-degradable substrate added were found the main cause of inhibition. However, in any case the leachate flush allowed the system to recover and establish proper methanogenic conditions.

Figure 41 : Effect of the addition of a fraction of easily-degradable substrate to spent animal bedding

Coupling two reactors and flushing VFAs out of the inhibited reactor in order to consume them in a methanogenic one is shown in literature to be an effective strategy to treat easily-degradable mixture and/or recover inhibited reactors. In chapter 5, the influence of the leachate flush rate on the recovery of a reactor with high risk of acidification (40% TS of easily-degradable substrate) was investigated (Figure 42). Three leachate flush rate conditions were tested as representative of the ones applicable at industrial scale: 2, 1 and 0.5 L kg -1 TS d -1 . This allowed to highlight that during the acidification phase, when no methane is produced, the leachate flush rate should be kept at its highest level (≥ 2 L kg -1 TS d -1 ) since the objective is to remove the VFAs produced in the bulk. This is shown to have a significant impact on the degradation rate of the substrate which at 35 days presented a degradation of 65% (COD H2+CH4+VFA -to-BMP) when flushing with 2 L kg -1 TS d -1 and 45%-48% when flushing with 0.5 L kg -1 TS d -1 . During the establishment of the methanogenesis, when methane production starts, the amount of VFAs extracted decreases considerably. For this reason a lower leachate flush rate is advised in this phase: ≤ 1 L kg -1 TS d -1 .

It should also be noted that at the end of this step, a large amount of VFAs is extracted from the bulk and that this could represent up to 41% of the total degraded COD. Good management of this great amount of VFAs is then particularly important in real systems since they could accumulate in the leachate storage and cause inhibition of the process. For this purpose, VFA consumption is at least as much important than VFA extraction. In order to better understand the consumption of external VFAs in LBRs, a leachate rich in acetic acid was used. The results showed that for an optimal acetic acid consumption in LBRs, this should be sent when the methanogenic activity in the batch is still high, i.e. right after the methane peak is reached. In this condition, their consumption time was reduced by half compared to an addition at the end of a batch. Furthermore, in order to keep a high methane production rate in methanogenic stable reactors, a leachate rich in easily-degradable compounds (like acetic acid) should be injected frequently (every 6-7 h in our conditions). In addition, because of the bulk compaction and the problem of channelling, small volumes per flush are advised.

In the above mentioned tests, the frequency of recirculation was not considered and only the total volume recirculated was considered (frequency fixed at once per day).

However, this parameter is very important and its role during extraction was highlighted in another test. Results in section 5.

Perspectives

One of the major concerns in the LBR process optimization is the scale-up. Since LBR constitutes a dry and unmixed process, parameters linked to the physical properties of the bulk (e.g. particle size, medium porosity, etc.) highly impact the real performance at industrial site, much more than at lab scale. The difficulty in directly applying to the industrial scale the results obtained at lab one is the major reason of delay in the development of dry AD processes in spite of the undiscussed simplicity of operation on field.

Phenomena related to the modification of the physical substrate properties during digestion, like compaction of the bulk, intimately affect the biological activity and the percolation in LBRs. As an example, leachate flush strategy developed for lab-scale process could not be applied directly at industrial scale and would probably need further adjustment making the search for the optimum leachate management a complicated quest at industrialscale. Related to that, the treatment of easily-degradable compounds, whose performance has been shown to strictly depend on flush management can be difficult to optimize at this scale and empirical rules are often used.

The first action to undertake should be to consider physical substrate properties during experiments. Few authors already studied these aspects while working on spent cow bedding digestion in LBRs: [START_REF] André | Quantifying physical structure changes and non-uniform water flow in cattle manure during dry anaerobic digestion process at lab scale : Implication for biogas production[END_REF] determined the relation between percolation and compaction all along the digestion and [START_REF] Shewani | Assessment of percolation through a solid leach bed in dry batch anaerobic digestion processes[END_REF] worked on the static and dynamic absorption of water in a LBR. However, a lot of questions still need to be answered: what is the relation between degradation and compaction? What is the optimum substrate column height in a LBR? What is the minimal amount of bulking agent required to ensure percolation even at later stage? All these are very important parameters to couple to biological tests like the ones carried out in this thesis.

The second important aspect is modelling. Modelling of AD in LBRs is considered one of the most complex models to develop in this field [START_REF] Batstone | Mathematical modelling of anaerobic digestion processes: applications and future needs[END_REF] because of the higher number of parameters to consider compared to classic wet digestion, already efficiently described by ADM1 [START_REF] Batstone | [END_REF]. Beside the high number of parameters already considered in the ADM1, some others should be considered: physical substrate properties and their modification with TS content and digestion time, and data about water flow properties inside the bulk. Even if such a complex model was not yet developed, a first attempt to model the water flow through a bed composed of spent cow bedding and a coupling with a simplified AD biological model was tested by Shewani (2012). This work focused on the transfer of soluble compounds between solid and liquid phase during percolation. This interesting study, if well calibrated, could allow to finally answering problematic dealing with the optimum leachate management in percolation reactor and easily-degradable substrate digestion. However, calibration is a difficult step because of the need of including parameters coming from a full scale process.

Another important aspect of percolation systems such as LBRs is the role of immersion.

Few authors already worked on that aspect (see paragraph 1.3.2) but a lot of questions remain unanswered. Immersion could be a very useful mean to solve problems connected to reach optimal initial bulk TS, but also to solve problems related to compaction and bad percolation which hamper degradation of the substrate. Also, the increase of the consumption of external VFAs, which is nowadays a very limiting aspect in sequential LBR process treating very easily-degradable mixture, could be improved by applying this strategy. Immersion is thought to be a very interesting approach that should be further investigated and considered seriously as a solution to all percolation problems observed and be an active part of a control strategy in LBR systems.

Finally, three PhD thesis [START_REF] André | Étude de verrous scientifiques et technologiques pour la compréhension et l ' optimisation du procédé de méthanisation voie sèche discontinu de sous -produits d ' origine agricole[END_REF][START_REF] Degueurce | La méthanisation par voie sèche agricole appliquée aux fumiers de bovins : optimisation de la recirculation des lixiviats[END_REF][START_REF] Shewani | Méthanisation discontinue en voie solide statique à percolation : caractérisation[END_REF] recently completed in addition to this one were carried out simultaneously in France between 2013 and 2016 in order to solve the drawbacks connected to the implementation of LBR process and adaptation to spent animal bedding. Researches focused most of the time on different aspects of the process: Shewani (2016) on modelling water flows and mass transfer in LBRs, system, André (2016) mainly on microbial community development and interaction in solid and liquid phase. Some research teams also focused on the use of the electrical tomography techniques to study the water flow inside real reactors and find correlation with substrate TS and degradation (André et al., 2016a;Degueurce et al., 2016b). Complementary results, most of them confirming each other, were produced in these past years and LBR technology surely made a big step towards a better understanding and practical implementation. The next step to validate this process, not only at lab scale but especially at industrial scale, should focus on the collection of all the results established in these last years and the design of new tests considering all the LBR aspects studied in these PhD works. -A coefficient of -7.5 % was applied to the experimental values of methane yield in order to simulate a worse degradation at farm-scale. On average, 86 % of the BMP is reached at labscale with spent bedding after 60 days of digestion while, at farm-scale, installations are close to 80 %, on average, (ADEME, 2014).

-The spent bedding and the digestate were considered to have a volumetric mass of 600 kg m -3 , as observed on site.

-The minimum number of digesters was set at four in order to ensure a constant biogas production over time. No maximum number was fixed.

-The only adjustable dimension in the digester volume was the length, with a maximum of 30 m.

The working volume was fixed at 64 % of total volume.

-The total volume distribution was based on always preferring the minimum number of reactors.

The addition of one more reactor took place when the maximum length was reached for all the reactors.

-Fixed electrical powers (for the Combined Heat and Power, CHP), ranging from 100 kW el to 300 kW el with 25 kW el steps, were used. Real CHP characteristics were reported for levels of (100, 150, 200, 250, and 300) kW el in accordance to manufacturer's technical specifications. The other intermediate power values used were simulated by limiting the nominal power of the next higher CHP unit. To simulate such cases, the efficiency of the CHP unit directly below the case studied and the cost of the directly above were used (e.g. for 125 kW el , the efficiency of a 100-kW el CHP unit and the cost of a 150-kW el CHP unit were used).

-Electrical CHP power efficiency was considered to range between 39 % and 41 % in relation to the CHP size. The heat CHP power efficiency was fixed at 45 %.

-The number of hours operating at full-load was set at 8,000 hours per years as done by [START_REF] Caputo | Economics of biomass energy utilization in combustion and gasification plants: Effects of logistic variables[END_REF] and [START_REF] Lantz | The economic performance of combined heat and power from biogas produced from manure in Sweden -A comparison of different CHP technologies[END_REF].

-The feed-in tariffs used are those described by French legislation in government orders (Decree of 19 May, 2011) and (Decree of 30 July, 2013). To a base tariff set in relation to the CHP electrical power installed, two bonuses are added: one for energy efficiency and the other for the amount of animal manure used. The base tariff (for the electrical power considered in this work)

was set between 0.1337 € kWh -1 el for power ≤ 150 kW el and 0.1267 € kWh -1 el for power between 150 kW el and 300 kW el ; the energy efficiency bonus, independent of the electrical power, was set between 0 to 0.04 € kW -1 el for energy efficiency values comprised between 35 % and 70 %;

for electrical power ≤ 300 kW el ; the animal manure bonus was set between 0 and 0.026 € kW -1 el for 20 % and 60 % of animal manure used, respectively. Linear interpolation was applied for intermediate values. It is important to note that the animal manure bonus was always at its maximum value (since 100 % animal manure).

-The selling and purchase of spent bedding and digestate was not taken into account since in small agricultural projects the digestate produced is returned back to the farmers who brought the spent bedding initially. More details about the hypotheses made are reported in the supplementary material.

-The total time (N) used in Eq. 2 is 15 years, which corresponds to the number of years the project can benefit from advantageous electricity prices fixed by French legislation.

-The external process parameters chosen were selected for analysis since: the fraction of heat sold is the second source of revenue in co-generation AD plants (if the digestate is not sold, as in this case) and the preparation time for emptying and loading substrate from the digester is the most time-consuming activity in an LBR process (ADEME, 2010); the haulage distance has been described as a very important parameter in a lot of economic analysis and grants are always identified as the most important factor influencing a project's viability (ADEME, 2010;[START_REF] Massaro | The sustainability of anaerobic digestion plants: a win-win strategy for public and private bodies[END_REF][START_REF] Pantaleo | Assessment of optimal size of anaerobic codigestion plants: An application to cattle farms in the province of Bari (Italy)[END_REF][START_REF] Walla | The optimal size for biogas plants[END_REF].

Sensitivity analysis

A sensitivity analysis was performed in order to identify which external process parameters (i.e. investment grants, fraction of heat sold, preparation time for loading and emptying the digesters and haulage distance) can affect the economic evaluation and whether differences exist in this respect between conditions D and L. The bar values presented in Figure 43 correspond to a variation of the IRR, or PP, in respect to the nominal value (e.g. (IRR-IRR nom )/ IRR nom ). As an example, the impact of the variations in the investment grants on the IRR for 11.5 kt y -1 in condition L was studied (second bar from left side in Figure 43c). At its nominal value of 30 % (Table 22), the impact was zero since it corresponds to the reference case (IRR = IRR 11.5 k t -L ). By increasing its value to 50 % (> nominal), the upper range value (Table 22), the IRR increased by 71 % compared to the IRR 11.5 kt -L . Similarly, a value of -43 % was found for the lower range limit (< nominal).

The sensitivity analysis showed that all four external process parameters selected had a great impact on economic performance, particularly on the IRR. Their variations in respect to the nominal values were often found symmetrical for nearly all capacities, which means that the evolutions of IRR and PP in relation to the external process parameters are quite linear in the ranges studied, without any optimum to be found. The heat sold and the substrate preparation had a minor impact overall whereas the haulage distance and the investment grants had a considerable effect. In particular, as also highlighted by [START_REF] Walla | The optimal size for biogas plants[END_REF] and [START_REF] Massaro | The sustainability of anaerobic digestion plants: a win-win strategy for public and private bodies[END_REF], the investment grants were found to be by far the most influential parameter since they directly affect the investment and, consequently, the PP .

VFigure 1 :Figure 2 :Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 10 :Figure 11 :Figure 12 :Figure 15 :Figure 17 :Figure 19 :Figure 22 :Figure 24 :Figure 28 :Figure 31 :Figure 33 :Figure 37 :

 123456789101112151719222428313337 Figure 1: Biological phases of the anaerobic digestion process (adapted from Demirel and Scherer (2008)) ................................................................................................................................. 9

Figure 38 :

 38 Figure 38 : Cumulated mass of VFAs extracted from a LBR after several flushes .........................

Figure 39 :

 39 Figure 39 : Spent animal bedding (SaB) average properties and digestion performance in a leachbed reactor (LBR) ...........................................................................................................

Figure 40 :

 40 Figure 40 : Results of the parameters optimization and optimum process conditions to digest spent animal bedding in a LBR ................................................................................................

Figure 41 :

 41 Figure 41 : Effect of the addition of a fraction of easily-degradable substrate to spent animal bedding ............................................................................................................................

Figure 42 :

 42 Figure 42 : Parameters evolution in a LBR treating an acidic mixture of spent cow bedding and an easily-degradable substrate; leachate flush and VFA management in relation to the phase of degradation; schematic functioning of a coupled LBR-LBR system: the new reactor from where VFAs are extracted and the mature reactor where VFAs are consumed .....

Figure 43 :

 43 Figure 43 : Sensitivity analysis results: variability of the IRR and PP for three capacities: (8.0, 11.5, 15.0) kt y -1 . (a-b) Payback Period for condition D and L; (c-d) Internal Rate of Return for conditions D and L ..........................................................................................................

Figure 1 :

 1 Figure 1: Biological phases of the anaerobic digestion process (adapted from Demirel and Scherer (2008))

Table 1 :

 1 Strengths and weaknesses of dry processes based on[START_REF] Jha | Research advances in dry anaerobic digestion process of solid organic wastes[END_REF],Karthikeyan and Visvanathan (2012a) andKothari et al.

Figure 2 :

 2 Figure 2: Existing LBR configurations(Vandevivere et al., 2003)

Figure 3 :

 3 Figure 3: Operating principle of the Naskeo/Methajade sequential LBRs process

Figure 4 :

 4 Figure 4 : Graphical abstract of the first issue faced in this PhD thesis: physico-chemical characterization of several types of spent animal bedding, description of the problematics connected to a LBR process and biogas performance reached

Figure 5 :

 5 Figure 5 : Graphical abstract of the second issue faced in this PhD thesis : the role played by both leachate and digestate on the digestion process and the economic comparison of these two strategies

Figure 6 :

 6 Figure 6 : Graphical abstract of the third issue faced in this PhD thesis: the advantages and drawback of treating spent animal bedding in thermophilic conditions

Figure 7 :

 7 Figure 7 : Graphical abstract of the fourth issue faced in this PhD thesis: the extent of inhibition when adding a significant fraction of an easily-degradable substrate and the use of the leachate flush to manage volatile fatty acid accumulation

Figure 8 : 1 • 2 •

 812 Figure 8: Structure of the PhD manuscript

  are discussed: biogas loss at the start-up, batch duration, overall methane production and accumulation of intermediate metabolites. This chapter developed the knowledge necessary to propose some new operating strategies to optimize the treatment of spent animal bedding in leachbed reactors.

Figure 9 :

 9 Figure 9 : Graphical abstract of the first issue faced in this PhD thesis: physico-chemical characterization of several types of spent animal bedding, description of the problematics connected to a LBR process and biogas performance reached

Figure 10 :

 10 Figure 10 : Experimental leach-bed reactor

-

  VFAs and Mettler Toledo InPro 4260i probes connected to a Mettler Toledo pH M300 operational manual transmitter were used to measure pH. Ion concentrations (i.e. Cl -, NO 2

Figure 11 :

 11 Figure 11 : Parameters monitored in the leachate during the digestion process in a LBR of six different spent animal bedding: (a1 -a2) pH and (b1 -b2) total alkalinity in g CaCO 3 L -1

(

  Figure 12 a1-a2) was observed during the first days, indicating an effective imbalance between hydrolysis/acidogenesis and acetogenesis/methanogenesis. All the various types of spent bedding behaved similarly and the total VFA (TVFA) peaks corresponded to the minimal pH values. TVFA peaks were reached between day 3 and day 10 and then VFA concentrations decreased at different rates. VFA concentrations below 1 g COD L -1 were reached at different times starting from day 12.

Figure 12 :

 12 Figure 12 : Volatile fatty acids measured in the leachate during the digestion process in a LBR of six different spent animal bedding: (a1 -a2) total volatile fatty acids concentration in g COD L -1 , (b1 -b2) acetic (C2) and propionic (C3) acid concentrations in g COD L -1

  Figure13a1-a2 shows the biogas production rate during the experiments. Three main groups were identified based on the lag phase, which was estimated by fitting the experimental data with the Gompertz equation (data not shown). SB_sheep and SB_horse had an average lag-phase of 1.1 days, SB_cow of 4.2 days and SB_goat of 6.4 days. However, the methane production rate peaks among these groups were not found statistically different. The reasons of such a different initial methanogenic activity were not clear. No significant correlations were found between the lag-phase and the substrate properties highlighted through characterization. Problems related to the original 60 methanogenic population present in the substrate, specific inhibition or nutrients deficiency could be one of the causes.

Figure 13 :

 13 Figure 13 : (a1 -a2) methane production rate in NmL CH 4 g -1 VS d -1 and (b1 -b2) methane content in biogas during the digestion process of six different spent animal bedding in a LBR

Figure 14 :

 14 Figure 14 : (a1 -a2) methane yield in NmL CH 4 g -1 VS, (b1 -b2) methane yield in NmL CH 4 g -1 RM (raw mass) during the digestion process of six different spent animal bedding in LBRs

Figure 16 :

 16 Figure 16 : Methane production rates of three LBRs with different internal recirculation conditions

Figure 17 :

 17 Figure 17 : Volatile fatty acids concentration in three LBRs with different internal leachate recirculation conditions

Figure 18 :

 18 Figure 18 : Graphical abstract of the second issue faced in this PhD thesis : the role played by both leachate and digestate on the digestion process and the economic comparison of these two strategies

(

  2008) andDegueurce et al., (2016c) using spent cow and horse bedding, respectively. The importance of the adaptability of the inoculum to the substrate and to the process in terms of nutrients (e.g. nitrogen) and trace elements concentration was studied by[START_REF] Xu | Comparison of different liquid anaerobic digestion effluents as inocula and nitrogen sources for solid-state batch anaerobic digestion of corn stover[END_REF] on corn stover digestion and[START_REF] Facchin | Effect of trace element supplementation on the mesophilic anaerobic digestion of foodwaste in batch trials: The influence of inoculum origin[END_REF] on food waste treatment; those results higlighted the complexity of the inoculation step and the importance to well assess this parameter in order to reach optimal digestion performance. In addition,Degueurce et al., (2016c) supported the idea that leachate has poor inoculation properties by demonstrating that the bacterial consortia present in the solid and liquid phases are different, with no significant microbial population intermixing. Then the role of leachate still remains an open question.

  plant using several LBRs to treat spent cow bedding. The site design comprised a minimum of four digesters operated in parallel with a certain interval between each reactor start-up (e.g. 4 digesters, batch duration of 60 days and 15 days of interval) and a tank collecting the leachate. The considered incomes were those from the selling of heat and electricity produced through a Combined Heat and Power (CHP) unit. The feed-in tariffs used were those in accordance with actual French legislation (Decree of 19 May, 2011, Decree of 30 July, 2013). The selling and purchase of spent bedding and digestate were not considered.

Figure 20 :

 20 Figure 20 : Comparison of the biogas parameters analysed all along the digestion process of three different experimental loadings: spent cow bedding and water (B), spent cow bedding and leachate (L) and spent cow bedding and digestate (D). (a) cumulative methane produced, (b) methane yield, (c) biogas production rate and (d) methane content in biogas produced; (b) methane yield; (c) biogas production rate; (d) methane concentration in biogas

Figure 21 :

 21 Figure 21 : Comparison of the leachate parameters analysed all along the digestion process of three different experimental loadings: spent cow bedding and water (B), spent cow bedding and leachate (L) and spent cow bedding and digestate (D). (a) pH; (b) total volatile fatty acid concentration; (c) total alkalinity; (d) acetic (C2) and propionic (C3) acid concentrations

Figure 22 :

 22 Figure 22 : Results of the economic simulation performed on an industrial site using a sequential LBR process considering a digester number varying from 4 to 9 and an electric cogeneration power ranging from 100 to 300 kW el . Evolution of the (a) Payback Period and Internal (b) Rate of Return in relation to the annual capacity. Evolution of the (c) Payback Period and (d) Internal Rate of Return in relation to the batch duration

Figure 23 :

 23 Figure 23 : Results of the economic simulation performed on an industrial site using a sequential LBR process considering a digester number varying from 4 to 9 and an electric cogeneration power ranging from 100 to 300 kW el . Evolution of the CAPEX in relation to (a) the annual capacity and (b) the batch duration

Figure 25 :

 25 Figure 25 : Comparison of thermophilic conditions with (T_inoc) and without (T_no inoc) solid digestate, in run 1 and run 2: (a) specific methane production rate; (b) VFA concentration

Figure 27 :

 27 Figure 27 : Comparison of mesophilic (M_inoc) and thermophilic (T_inoc) conditions: (a) VFA concentration in run 1 and run 2; (b) acetate and propionate concentrations in run 1

Figure 28 :

 28 Figure 28 : Comparison of a simulated industrial plant operated in mesophilic and thermophilic conditions. The simulation, based on the data from run1, consider the treatment of 9,400 tons/year of spent cow bedding, the installation of a combined heat and power (CHP) unit of 250 kW el and the use of 4 digesters staggered in time with a batch duration of 44 days. (a) heat power contained in the cumulated biogas at the inlet of the CHP unit; (b) electrical power produced considering the CHP power working range: the maximum (P_max_CHP) and the minimum (P_min_CHP)

  this chapter has been published to as:Riggio, S., Torrijos, M., Vives, G., Esposito, G., van Hullebusch, E.D., Steyer, J.P., Escudié, R. -Leachate flush strategies to manage volatile fatty acids accumulation in leach-bed reactors -Bioresource Technology -2017 -DOI :

Figure 30 :

 30 Figure 30 : Biogas parameters monitored during the digestion process considering three different fractions of easily-degradable substrate loaded in the LBRs: (H), medium (M) and low (L). (a) Methane production rates and (b) methane content in the biogas.

Figure 31 :

 31 Figure 31 : Parameters monitored in the leachate during the digestion process considering three different fractions of easily-degradable substrate loaded in the LBRs: high (H), medium (M) and low (L). (a) Volatile fatty acids concentration and (b) pH of the leachate in LBRs with different fractions of easily-degradable substrate

Figure 32 :

 32 Figure 32 : Experimental design. Fresh leachate without volatile fatty acids (VFAs) is stored in a 50 L volume. A fixed amount of leachate is pumped into the leach-bed reactor (LBR) through a peristaltic pump. After percolation through the bed, the leachate enriched in VFAs is pumped out and analysed.

  , the biogas associated with the degradation of acetic acid was determined by subtracting the volume of biogas produced by the reactor receiving acetic acid from the blank reactor run without it (i.e. H2 minus H1 and M2 minus M1). Three successive injections of acetic acid were made to evaluate the methanogenic activity. The average values of the three tests are presented with their standard deviation.

L2.

  In fact, a reduced volume of leachate injected could induce differences in the VFA extraction capacity because of the heterogeneity of the matrix and the channeling. Altogether, this suggests that reducing too much leachate flush rate degrades the extraction capacity and leads to variable results.

Figure 34 :

 34 Figure 34 : Parameters measured during the digestion process: (a) pH; (b) methane production rate; (c) total volatile fatty acids (TVFA) concentration; (d) volatile fatty acids (VFAs) concentration (condition H). H and M refers to the mean value of H1-H2 and M1 -M2, respectively, during period I and period II while, they correspond to only H1 and M1, respectively, during period II

Figure

  Figure36apresents the daily production of H 2 and CH 4 collected in the biogas and VFAs

Figure 36 :

 36 Figure 36 : Dynamic presentation of all the metabolites extracted or produced during the digestion process: (a) extraction rate of volatile fatty acids (VFAs), and production rate of methane (CH 4 ) and hydrogen (H 2 ) in conditions L1 and L2; (b) accumulated amounts of VFAs, CH 4 and H 2 over time. H and M refers to the mean value of H1-H2 and M1 -M2, respectively, during period I and period II while, they correspond to only H1 and M1, respectively, during period III

Figure 37 :

 37 Figure 37 : Dynamic presentation of total volatile fatty acids (TVFA) extracted: (a) extraction rate of TVFA; (b) accumulated TVFA extracted over time. H and M refers to the mean value of H1-H2 and M1-M2, respectively, during period I and period II while, they correspond to only H1 and M1, respectively, during period III

Figure 38 :

 38 Figure 38 : Cumulated mass of VFAs extracted from a LBR after several flushes

Figure 39 :

 39 Figure 39 : Spent animal bedding (SaB) average properties and digestion performance in a leach-bed reactor (LBR)

Figure 40 :

 40 Figure 40 : Results of the parameters optimization and optimum process conditions to digest spent animal bedding in a LBR

  2.3.2 show that a big volume per flush could be replaced by smaller volumes recirculated more frequently. As an example, a frequency of 2 times per day with a leachate flush ratio of 0.5 L kg -1 TS flush -1 (i.e. 1 L kg -1 TS d -1 ) are proved to extract the same amount of VFAs than one flush of 2 L kg -1 TS flush -1 . The extraction efficiency should then be considered when fixing the recirculation strategies in the site and smaller volume sent more frequently could be preferred. However, a uniform percolation should be also ensured, otherwise the extraction efficiency could rapidly decrease as proved by the variability of the results obtained with a low leachate flushing rate 0.5 L kg -1 TS d -1 .Frequency and volume are then operating parameters that should be fixed together and an optimum looked in relation to the physical properties of the solid matrix and digester geometry.

Figure 42 :

 42 Figure 42 : Parameters evolution in a LBR treating an acidic mixture of spent cow bedding and an easily-degradable substrate; leachate flush and VFA management in relation to the phase of degradation; schematic functioning of a coupled LBR-LBR system: the new reactor from where VFAs are extracted and the mature reactor where VFAs are consumed

7. 2

 2 Hypothesis for the economic simulation -The average daily experimental values of methane yield presented in Figure 20b (conditions L and D) were used -The biogas produced was considered exploitable when the concentration in methane reached 30 %.

  Figure 43 : Sensitivity analysis results: variability of the IRR and PP for three capacities: (8.0, 11.5, 15.0) kt y -1 . (a-b) Payback Period for condition D and L; (c-d) Internal Rate of Return for conditions D and L

Table 1 :

 1 Strengths and weaknesses of dry processes based on Jha et al. (2011), Karthikeyan and Visvanathan (2012a) and Kothari et al. (2014) ................................................................. 22

Table 2 :

 2 Comparison of literature results: performance of leach-bed reactor (LBR) treating spent livestock bedding or a synthetic mixture of livestock faeces and an external bulking agent ........................................................................................................................................... 43

Table 3 :

 3 Characterization of six types of spent animal bedding ....................................................... 53

Table 4 :

 4 BMP test results and methane yields measured in leach-bed reactors ............................... 55

Table 5 :

 5 Analysis of the water mass balance on the LBRs. The results of both duplicates for each

substrate studied are reported ............................................................................................ 65 Table 6 : Optimization of start-up and batch duration ....................................................................... 68 Table 7 : Characterization of the solid and liquid substrates: total solids (TS), volatile solids (VS), VS to TS ratio and biomethane potential (BMP) .............................................................. 78 Table 8 : Experimental loading of the three tested conditions: blank (B), condition D and condition L ........................................................................................................................................ 80 Table 9 : Average methane performance in the three tested conditions after 60 days of digestion .. 85 Table 10 : (a) Total solids (TS), volatile solids (VS), ratio of VS to TS (VS/TS) and the BioMethane potential (BMP) of the substrates loaded in the reactors in run 1 and run 2; (b) loading set-up of run1 and run 2 .................................................................................................... 98 Table 11: Composition of the co-digestion mixture used in each condition. For each substrate used, the amount of fresh matter added and the share that it represents in term of TS are provided .......................................................................................................................... 112 Table 12 : Characterization of the substrates loaded: total solids (TS), volatile solids (VS) of spent cow bedding, cereal residues, carrots and digestate ........................................................ 113 Table 13 : Characterization of the substrates loaded: (a) total solids (TS), volatile solids (VS), and BioMethane potential (BMP) of spent cow bedding, cereal residues, carrots and XIII digestate; (b) total and soluble chemical oxygen demand (CODt and CODs), volatile fatty acid (VFA), total and partial alkalinity and pH of the leachate ...................................... 121

Table 14 :

 14 Characterization of the substrates loaded: total solids (TS), volatile solids (VS), and BioMethane potential (BMP) of spent cow bedding, cereal residues, carrots and digestate; total and soluble chemical oxygen demand (CODt and CODs), volatile fatty acid (VFA), total and partial alkalinity and pH of the leachate ...................................... 124 Table : Activity test. Reactor H2 corresponds to a duplicate operated with a high flush-rate and M2 to a duplicate operated at a medium flush-rate. ........................................................ 130

Table 16 :

 16 COD H2+CH4+VFA -to-BMP ratio: evolution over time of the ratio of the metabolites collected as hydrogen (H 2 ), methane (CH 4 ) and volatile fatty acids (VFAs) to the

	Literature review

BioMethane Potential (BMP) of the loaded substrates (expressed in COD). ................ 133 Table 17 : Overall amount of volatile fatty acids (VFAs), methane (CH 4 ) and hydrogen (H 2 ) collected after 90 days, expressed in gram of chemical oxygen demand (COD) per gram of volatile solids (VS) initially loaded in the reactor. The percentage values are calculated based on the final amount of COD collected in each reactor .......................................... 134 Table : Recommendations about volatile fatty acids (VFAs) management in a sequential leachbed reactor (LBR) process .............................................................................................. 140 Table : Results of the ANOVA comparing the results of characterization and methane production of the substrates studied in leach-bed reactor ................................................................. 164 Table 20 : Results of the modelling of the methane production in LBRs using the Gompertz equation ........................................................................................................................... 165 Table : Pearson correlation coefficients found among parameters from characterization, methane production in LBR and modelling .................................................................................. 166 Table : Selected ranges for sensitivity analysis .......................................................................... 172 XIV 1 CHAPTER 1

  The development of decentralized AD facilities like farm-scale processes, consisting in the creation of local and numerous sources of energy in the territory, is one important axis supported by the French government. The objective of constructing 1000 anaerobic digesters by the end of 2020 and 1500 by the end of 2030 proves this commitment. However, nowadays, France still has relatively few farm-scale AD plants: less than 210 in 2014. This number is very low when compared to Germany which is the European leader with about 8,000 agricultural plants in2015

("IEA Bioenergy," 2015)

. As a consequence, a real need in developing this sector is highlighted. But France is not the only country interested in the development of decentralized energy production as in farm-scale anaerobic digester. Indeed, this interest is shared by several other countries as numerous recent scientific publications reported:

[START_REF] Karellas | Development of an investment decision tool for biogas production from agricultural waste[END_REF]

, in Greece;

[START_REF] Klavon | Economic analysis of small-scale agricultural digesters in the United States[END_REF]

, in United States;

Table 2 :

 2 Comparison of literature results: performance of leach-bed reactor (LBR) treating spent livestock bedding or a synthetic mixture of livestock faeces and an external bulking agent

	Substrate	Reactor Temperature	Batch	Methane	References
				duration	yield
	1 spent cow straw	LBR	mesophilic	40 days	166 L kg -1	(Hall and
	bedding		(30°C)	70 days	VS	Hawkes, 1985)
					215 L kg -1
					VS
	1 spent cow straw	LBR	mesophilic	30 days	114.5 NL kg -	(Degueurce et al.,
	bedding		(35°C)		1 VS	2016c)
	1 spent cow straw	LBR	mesophilic	32 days	-	(André et al.,
	bedding		(37°C)			2015)
	1 spent cow straw	LBR	-	-	-	(Shewani et al.,
	bedding					2015)
	1 spent cow straw	LBR	mesophilic	30 days	114.5 NL kg -	(Degueurce et al.,
	bedding		(35°C)		1 VS	2016c)
	1 spent pig/swine	LBR	mesophilic	50 days	not provided	(Yap et al., 2016)
	straw bedding		(37°C)		clearly
	1 spent horse straw	LBR	mesophilic	45 days	170 NL kg -1	(Kusch et al.,
	bedding		(35°C)		VS	2008)
	2 cow manure	LBR	psychrophilic	84 days	147 NL kg -1	(Massé and
	(faeces only) +	sequence	(20°C)	(21 days	VS	Saady, 2015)
	straw			per batch)
	2 sheep manure +	LBR	mesophilic	94 days	184 NL kg -1	(Blanco et al.,
	straw		(35°C)		VS	2010)
	2 raw manure slurry	LBR	psychrophilic	-	-	(Myint and
	+ pistachios half-		(22 °C)			Nirmalakhanda,
	shell					2009)
	1 spent horse	LBR	mesophilic	57 days	44.8 L kg -1	(Wartell et al.,
	softwood-pellet		(34-36°C)		VS *	2012)
	bedding
	solid phase of raw	LBR	thermophilic	60 days	214 -227 L	(Rico et al., 2015)
	dairy manure slurry		(50°C)		kg -1 VS
	2 cow manure +	LBR	psychrophilic	-	not provided	(Demirer and
	wood powder/chips		(20-24°C)		clearly	Chen, 2008)

Table 3 :

 3 Characterization of six types of spent animal bedding This information has not been measured directly but provided by the farmers as average value on their farm b LU: livestock unit. Milk cows = 1LU, goat and sheep = 0.100 LU and horse =0.800 LU up to the European legislation c Mean value ± standard deviation of the triplicates d On a volatile solid basis * The exact value is not known by the farmer. However it was visually higher than the one of the other substratesMethane potentialBMP represents the amount of CH 4 that can be produced by anaerobic digestion per mass of substrate usually expressed on a VS basis. Even if the BMPs measured were significantly different (p-value < 0.05), their range of variation was quite narrow: 192-239 NmL CH 4 g -1 VS with the lowest values belonging to SP_cow_g and highest to SB_horse (Table4). Few BMPs of spent livestock bedding are reported in literature: 277 NmL CH 4 g -1 VS for horses[START_REF] Kusch | Biogas production with horse dung in solidphase digestion systems[END_REF], 173.5 NmL CH 4 g -1 VS for cows(Degueurce et al., 2016c) and 195-218 NmL CH 4 g -1 VS for pigs/swine

	Substrate		SB_sheep	SB_goat	SB_horse	SB_cow_h	SB_cow_g	SB_cow_m
	Breed		-	-	-	Simmenthal	Blonde d'Aquitaine	Prim'Holstein
	Roughage feed		-	-	-	Hay	Baled grass silage	Clamp maize silage
	a Amount of straw 1 b kg LU -1 d -1 9.8	4.5	11.3	6.5	>3	8-10
					(Wartell et al., 2012) *
	Freq. litter change		2-3 months 2-3 months 2-3 days	2 months	2-3 months	3 weeks
	c TS	%	37.0  1.2	29.8  0.8	34.3  0.4	23.7  0.1	25.6  0.8	28.8  0.3
	c VS/TS	%	88.9  4.5	84.4  2.7	86.8  1.2	83.7  0.3	82.3  3.8	84.0  1.7
	c C	g C kg -1 TS	427.4  2.7 438.1  2.0 498.5  5.5	505.6  5.9	525.1  2.4	498.6  2.3
	c N	g N kg -1 TS	19.1  0.2	21.7  1.2	11.8  0.7	17.6  0.8	23.7  0.7	17.7  0.5
	c P	g P kg -1 TS	6.8  0.6	8.4  0.0	5.6  0.4	4.1  0.3	3.7  0.4	4.6  0.8
	C/N/P	-	62 / 2 / 1	51 / 2 / 1	88 / 2 / 1	121 / 4 / 1	141 / 6 / 1	109 / 3 / 1
	C/N	-	22 / 1	20 / 1	42 / 1	28 / 1	22 / 1	28 / 1
	c Soluble	d% VS	31.8  1.2	36.7  1.3	19.0  0.3	25.9  0.3	38.1  1.5	25.9  2.2
	c Hemicellulose	d% VS	23.8  0.1	20.6  0.8	28.5  0.4	28.4  1.5	21.2  1.0	21.2  1.0
	c Cellulose	d% VS	30.7  1.0	34.7  1.7	44.2  1.1	33.0  2.5	28.4  0.8	38.5  2.0
	c Lignin	d% VS	13.7  2.0	8.0  0.6	8.3  1.0	12.8  1.0	12.3  1.0	12.0  1.5

a

Table 4 :

 4 BMP test results and methane yields measured in leach-bed reactors

			SB_sheep	SB_goat	SB_horse	SB_cow_h	SB_cow_g	SB_cow_m
	a BMP	NmL CH 4 g -1 VS	204  8	227  8	239  7	216  6	192  3	228  2
		NmL CH 4 g -1 RM	65  3	57  2	71 3	43  2	40  1	55  1
	b Methane yield in	NmL CH 4 g -1 VS	201  31	196  33	206  32	185  4	168 *	204  20
	LBR (60 days)	NmL CH 4 g -1 RM	64  10	49  8	61  10	37  2	35 *	49  5
	LBR/BMP		(99  16)% (86  15)% (87  14)% (85  3)%	88% *	(89  9)%
	ratio

a Mean value ± standard deviation b Mean value ± range (maximum/minimum) * Value from only one reactor of the pair 2.1.3.2 Process analysis pH and alkalinity

Table 5 :

 5 Analysis of the water mass balance on the LBRs. The results of both duplicates for each substrate studied are reported

		TS spent	TS	Mass Balance	Water absorbed by the substrate
		bedding	digestate	(water loss)	in a cycle
		%	%	%	kg water kg -1 TS added kg water kg -1 RM added
	SB_sheep1	37%	15%	3%	1.9	0.7
	SB_sheep2	37%	15%	3%	1.9	0.7
	SB_goat1	30%	14%	4%	1.4	0.4
	SB_goat2	30%	15%	3%	1.2	0.4
	SB_horse1	34%	14%	3%	1.6	0.6
	SB_horse2	34%	14%	4%	1.7	0.6
	SB_cow_h1	24%	14%	4%	1.4	0.3
	SB_cow_h2	24%	14%	4%	1.3	0.3
	SB_cow_g1	26%	14%	5%	1.4	0.4
	SB_cow_g2	26%	14%	2%	1.2	0.3
	SB_cow_m1	29%	14%	5%	1.8	0.5
	SB_cow_m2	29%	13%	4%	1.8	0.5

Table 6 :

 6 Optimization of start-up and batch duration a -the time (days) needed to reach the specific CH 4 content in the headspace of the digester and the percentage of methane not exploited (compared to the total production after 60 days)

	b -the percentage of CH 4 not exploited (compared to the total production after 60 days) when
	diminishing the batch duration
	a	Analysis of the start-up
		Time
	CH 4 content	SB_sheep SB_goat SB_horse SB_cow_h SB_cow_g SB_cow_m
	20%	1.5 d	3.4 d	1.9 d	3.1 d	3.4 d	3.1 d
	25%	1.8 d	4.4 d	2.5 d	3.3 d	3.7 d	3.6 d
	30%	2.3 d	6.0 d	3.2 d	3.6 d	4.1 d	4.2 d
	35%	2.9 d	7.0 d	3.9 d	3.9 d	4.9 d	5.3 d
	40%	3.5 d	8.3 d	4.6 d	4.4 d	5.7 d	6.1 d
		Percentage of CH 4 not exploited
	CH 4 content	SB_sheep SB_goat SB_horse SB_cow_h SB_cow_g SB_cow_m
	20%	0.5%	1.3%	0.8%	1.2%	1.0%	1.3%
	25%	0.7%	2.0%	2.3%	1.8%	1.4%	2.1%
	30%	1.7%	2.9%	4.2%	2.4%	1.9%	3.3%
	35%	3.4%	4.1%	6.2%	3.0%	3.0%	6.0%
	40%	5.2%	6.0%	8.2%	4.4%	4.1%	8.1%
	b	Analysis of the batch duration
		Percentage of CH 4 not exploited
	Days	SB_sheep SB_goat SB_horse SB_cow_h SB_cow_g SB_cow_m
	40	8.9%	12.6%	11.3%	12.9%	15.2%	13.6%
	45	5.7%	8.0%	7.0%	8.4%	10.9%	9.2%
	50	3.4%	4.4%	3.8%	4.7%	6.4%	5.7%
	55	2.0%	2.5%	1.9%	1.5%	2.7%	2.5%
	60	0.0%	0.0%	0.0%	0.0%	0.0%	0.0%

Table 7 :

 7 Characterization of the solid and liquid substrates: total solids (TS), volatile solids (VS), VS to TS ratio and biomethane potential (BMP)

		TS (wet basis)	VS (wet basis)	VS/TS	BMP
		%	%	%	NL CH 4 kg -1 VS
	Spent cow bedding	29.8  1.6	23.7  0.2	79.5	205.7  15.4
	Digestate	13.7  0.3	10.4  0.3	75.6	-
	Leachate	1.03  0.01	0.38  0.02	36.8	-
		The results are mean values of the triplicates  standard deviation

Table 8 :

 8 Experimental loading of the three tested conditions: blank (B), condition D and condition L

			Spent cow bedding	Digestate	Leachate	Water
			kg	kg	L	L
	Blank (B)	B1 B2	1.30 1.32	--	--	2.28 2.28
	Condition D	D1 D2	0.97 0.97	0.33 0.33	--	2.10 2.10
	Condition L	L1 L2	1.30 1.30	--	1.69 1.70	0.59 0.58

Table 9 :

 9 Average methane performance in the three tested conditions after 60 days of digestion

		Cumulative
		volume of	Methane yield
		methane
		NL CH 4	NL CH 4 kg -1 VS
	Blank (B)	52.5 ± 0.8	169.0 ± 1.5
	Condition D	49.9 ± 0.6	216.5 ± 2.4
	Condition L	57.7 ± 1.0	187.0 ± 3.1
	Comparison of conditions D and L	-13.5%*	15.8%*
	The results are mean values of the duplicate  range of
	variation (minimum and maximum value)

*(D-L)/L

Table 10

 10

	: (a) Total solids (TS), volatile solids (VS), ratio of VS to TS (VS/TS) and the BioMethane
	potential (BMP) of the substrates loaded in the reactors in run 1 and run 2; (b) loading set-up of
	run1 and run 2

Table 11 :

 11 Composition of the co-digestion mixture used in each condition. For each substrate used, the amount of fresh matter added and the share that it represents in term of TS are provided

			Low (L)	Medium (M)	High (H)
	Spent cow	g	1011	808	607
	bedding	% (TS)	75%	60%	45%
	Carrots	g	244	611	976
		% (TS)	10%	25%	40%
	Cereal	g	244	244	245
		% (TS)	15%	15%	15%

Table 15 :

 15 Activity test. Reactor H2 corresponds to a duplicate operated with a high flush-rate and M2 to a duplicate operated at a medium flush-rate.

		Reactor	Acetic acid	Acetic acid	Peak
		used	concentration	mass hold	duration
			g L -1	% (w/w)	hour
	1° test	H2	5	37% ± 1%	12
		M2	10	39% ± 1%	22
	2° test	H2 M2	5 10	35% ± 0% 32% ± 1%	22 35-50
	Total			36% ± 3%

Table 16 :

 16 COD H2+CH4+VFA -to-BMP ratio: evolution over time of the ratio of the metabolites collected as hydrogen (H 2 ), methane (CH 4 ) and volatile fatty acids (VFAs) to the BioMethane Potential (BMP) of the loaded substrates (expressed in COD).Table17reports the amount of total COD extracted as VFAs, CH 4 and H 2 during the 90 days of the experiments. Hydrogen production constituted only 3% of the overall COD, a small fraction of total substrate degradation. CH 4 represented the highest fraction of the total COD, with values ranging between 56% and 70%. High amount of VFAs (up to 41% in condition H) were extracted:

		COD H2+CH4+VFA -to-BMP ratio
	Day	H	M	L1	L2
	15	34%	29%	21%	20%
	35	65%	59%	45%	48%
	45	74%	70%	60%	60%
	60	81%	77%	72%	69%
	90	87%	84%	85%	81%
	It is worth noticing that the overall degradation kinetics in L1 and L2 were very similar

Table 17

 17

		: Overall amount of volatile fatty acids (VFAs), methane (CH 4 ) and hydrogen (H 2 )
	collected after 90 days, expressed in gram of chemical oxygen demand (COD) per gram of volatile
	solids (VS) initially loaded in the reactor. The percentage values are calculated based on the final
	amount of COD collected in each reactor
		H1 (1)		M1 (1)		L1		L2
		g COD g -1 VS	%	g COD g -1 VS	%	g COD g -1 VS	%	g COD g -1 VS	%
	H 2	18.0	3%	18.0	3%	17.3	3%	17.1	3%
	VFA	258.2	41%	209.7	34%	191.8	31%	160.7	27%
	CH 4	355.4	56%	383.6	63%	410.6	66%	411.6	70%
	Total	631.7	100% 611.3	100% 619.7	100% 589.4	100%
		(1) because of the activity test, only the values of H1 and M1 are provided

Table 21 :

 21 Pearson correlation coefficients found among parameters from characterization, methane production in LBR and modelling

		A max	BMP	C	cellulose	hemicellulose	lignin	MY (RM)
	A max	100.0%
	BMP	33.2%	100.0%
	C	-8.6%	-17.9%	100.0%
	cellulose	42.4%	87.5%	-4.2%	100.0%
	hemicellulose	8.9%	25.0%	21.2%	26.9%	100.0%
	lignin	-2.8%	-62.9%	47.5%	-47.5%	9.2%	100.0%
	MY (RM)	79.5%	22.7%	-51.2%	36.3%	5.6%	-15.9%	100.0%
	MY (VS)	99.7%	31.2%	-5.8%	40.1%	6.5%	1.7%	76.3%
	N	-35.2%	-70.6%	4.1%	-81.2%	-74.7%	23.0%	-38.4%
	P	24.0%	27.8%	-72.4%	0.3%	-47.0%	-58.8%	41.6%
	soluble	-36.8%	-59.7%	-23.6%	-75.8%	-76.6%	-2.0%	-26.1%
	TS	38.4%	9.7%	-73.4%	20.3%	-5.0%	-25.5%	86.1%
	VS	40.0%	11.0%	-72.4%	22.1%	0.3%	-24.7%	87.0%
	λ	-34.1%	-4.2%	17.7%	-27.8%	-64.1%	-20.2%	-62.9%
	μ max	74.8%	18.8%	-35.0%	8.4%	-29.7%	0.2%	62.2%

m : maximum methane production; λ: lag phase; A max : maximum cumulative methane yield

Acknowledgements

First of all I thank the Erasmus Mundus Joint Doctoral Program Environmental Technologies for Contaminated Solids, Soils and Sediments (ETeCoS 3 ), the National institute of Agronomy Research (INRA) and the French company Naskeo Environnement for having built the frame of my work and having given me the opportunity to carry on this research activity.

Among the people collaborating to this PhD study I thank in the first place Renaud Escudié and Michel Torrijos. I am grateful to them for their daily support to my research work, their

Abbreviations

This chapter has been published as: Riggio, S., Torrijos, M., Debord, R., Esposito, G., van Hullebusch, E.D., Steyer, J.P., Escudié, R. -Mesophilic anaerobic digestion of several types of spent livestock bedding in a batch leach-bed reactor: substrate characterization and process performance -Waste Management journal -2017-DOI: 10.1016/j.wasman.2016.10.027 -Supplementary data were added in section 2. 1.3.2 and 0. methane which was very similar for all the substrates, as showed by the methane production rates after 25 days of operation.

Finally, the presence of one or two peaks in the methane production rate was related to the rate of VFAs consumption. Therefore, the bigger the gap in degradation between the acids produced the more likely would there be two biogas peaks (as for SB_cow_m).

The methane content in the biogas showed a similar trend for all substrates except SB_goat.

Methane content increased rapidly during the first 10 days, reaching stable values comprised between 55% and 60% (Figure 13), whereas the increase in methane content was slower for SB_goat, indicating some difficulty in the establishment of methanogens in this last system, as the high lag phase showed.

For all the substrates, methane yield expressed in terms of VS ranged between 168 and 206

NmL CH 4 g -1 VS (Figure 14 a1-a2). Since the inoculum used was estimated to produce less than 4% of the total methane in one batch reactor, all the methane measured was considered to be produced only by spent bedding. The errors in the measured biogas volume did not allow differentiating the studied substrates based on this parameter (p-value = 0.82). However, similar methane yields in LBR were reported by treating spent bedding from cow, horse and sheep stables in literature (Table 2). Further comparison in batch system for spent straw was reported by [START_REF] Cui | Solid-state anaerobic digestion of spent wheat straw from horse stall[END_REF], 150 NmL CH 4 g -1 VS after 30 days. Other comparisons are difficult to make because few authors have used spent bedding as a sole substrate. It is important noting that, after 60 days of digestion in LBR, methane yield was 89 (± 11)% (error calculated as a standard error of laboratory, SEL) of the of the BMP (Table 4) and VS degradation of the substrate (48 ± 3)%.This proved that an LBR process is efficient in treating spent livestock bedding.

CHAPTER 3

Study of the start-up phase

This chapter has been submitted to Biochemical Engineering Journal as: Riggio, S., Torrijos, M., Mingam, A., Esposito, G., van Hullebusch, E.D., Steyer, J.P.,Comas, J., Escudié, R. -Start-up strategies for leach-bed reactors treating spent cow bedding: impact on process and economic performance

Analysis

Characterization of the initial raw spent cow bedding was carried out. The TS concentration was measured after drying at 105 °C (378 K) for 24 h, followed by 3 h of calcination at 550 °C (823 K) for determination of VS. Finally, the spent bedding was freeze-dried and crushed to 1 mm particle size prior to BioMethane Potential (BMP) tests. These tests were carried out in batch of 500 mL inoculated with anaerobic sludge from a sugar treatment plant in accordance with the procedure described by Riggio et al. (2017a). The analyses were performed in triplicate and the average values with standard deviation were reported.

During the digestion process only the liquid phase and gas were analysed. The analysis frequency was adapted to the parameters evolution in order to precisely follow the start-up period:

3-4 times per week at the beginning until once per week in the end. The analyses of the liquid phase were done after a recirculation cycle. A volume between 10 and 20 mL was sampled by the mean of the port present in the liquid circuit. Total alkalinity and pH were measured on the raw leachate.

Alkalinity was measured at a pH of 4.30, as advised by [START_REF] Hill | Measuring alkalinity accurately in aqueous systems containing high organic acid concentrations[END_REF] and, the pH with Mettler Toledo InPro 4260i probes connected to a Mettler Toledo pH M300 operational manual transmitter. Volatile fatty acids (VFAs) were measured on a leachate previously filtered at 0.45 μm using the VARIAN I-MET-0084 gas chromatograph with a 15 m × 0.53 mm column (Alltech FFAP EC TM 1000) and a flame ionization detector (FID) using helium as the gas vector. Biogas volume

was measured with a Ritter flow meter (Milligascounter MGC-1 V3) and its composition was analysed with a micro-GCPRO CP-4900 using helium as the gas vector. The biogas volumes were expressed in standard condition: 273.15 K and 101,325 Pa. The average values of the pairs and their variation ranges (minimum and maximum values) are presented.

Economic assessment

The experimental data were processed together with the economic information delivered by the French company Naskeo Environnement. For industrial confidentiality reasons, all the exact CHAPTER 4

Comparison of the mesophilic and thermophilic anaerobic digestion of spent cow bedding in leach-bed reactors

This chapter has been published as: Riggio, S., Torrijos, M., Vives, G., Esposito, G., van Hullebusch, E.D., Steyer, J.P., Escudié, R. -Comparison of the mesophilic and thermophilic anaerobic digestion of spent cow bedding in leach-bed reactors -Bioresource Technology -2017-DOI:10.1016/j.biortech.2017.02.056 94 In this chapter, the effect of operating the LBR in thermophilic condition as a tool to diminish batch duration and consequently improve the overall performance of the process was investigated.

In fact, in chapter 2, the long incubation time necessary to reach a significant BMP when treating spent animal bedding had already been identified as one of the levers for possible process optimization. Furthermore, the problematic connected to the exploitation of the biogas in a

Combined Heat and Power (CHP) unit were analysed as well as their impact on the plant performance. Anaerobic digestion (AD) is a very widespread biological process aiming at the treatment of organic waste and the production of green energy. Spent animal bedding, a mixture of animal faeces and a bedding material both accumulating in the stall, has been proved to be efficiently treated in discontinuous dry anaerobic digesters such as leach-bed reactors (LBRs) (Riggio et al., 2017a).

Spent animal bedding constitutes a slowly-degradable substrate mainly because of its high content in lignocellulosic material [START_REF] Buffiere | Towards new indicators for the prediction of solid waste anaerobic digestion properties[END_REF]; for this reason digestion times ranging from 40 to 60 days are generally used in discontinuous LBRs at industrial scale. In order to improve the economic performance of the entire process, there is a real interest in increasing the substrate degradation kinetics and then reduce the overall digestion time. The use of thermophilic conditions in LBRs treating spent cow bedding could represent an effective solution to enhance the process performance. Indeed, by comparing the influence of mesophilic and thermophilic temperatures, several authors agreed on the faster degradation kinetics when using thermophilic conditions to treat different substrates: spent horse bedding [START_REF] Böske | Upflow anaerobic solid-state (UASS) digestion of horse manure: Thermophilic vs. mesophilic performance[END_REF], the organic fraction of municipal solid waste [START_REF] Fernández-Rodríguez | Comparison of mesophilic and thermophilic dry anaerobic digestion of OFMSW: Kinetic analysis[END_REF], spent cow bedding [START_REF] Gómez | Digestion of cattle manure under mesophilic and thermophilic conditions: Characterization of organic matter applying thermal analysis and 1H NMR[END_REF], wood chips [START_REF] Hegde | Comparison of thermophilic and mesophilic onestage, batch, high-solids anaerobic digestion[END_REF] and cow dung [START_REF] Jha | Comparison between Wet and Dry Anaerobic Digestions of Cow Dung under Mesophilic and Thermophilic Conditions[END_REF].

In the literature, few data comparing mesophilic and thermophilic treatment are available on spent animal bedding consisting of faeces and straw. [START_REF] Böske | Upflow anaerobic solid-state (UASS) digestion of horse manure: Thermophilic vs. mesophilic performance[END_REF] used a continuous upflow anaerobic solid-state (UASS) reactor to treat spent horse bedding whereas [START_REF] Gómez | Digestion of cattle manure under mesophilic and thermophilic conditions: Characterization of organic matter applying thermal analysis and 1H NMR[END_REF] used a dry unmixed batch system to digeste spent cow bedding. In thermophilic conditions, the first authors observed higher kinetics and methane yield than at a mesophilic temperature while the second reported higher kinetics and a lower methane yield. In addition to the discrepancy in their results, these latter hardly seem applicable to a different system such as a LBR.

The use of LBRs in thermophilic condition is not very common in scientific literature: [START_REF] Koppar | Anaerobic digestion of peel waste and wastewater for on site energy generation in a citrus processing facility[END_REF] used it to treat citrus peel waste, [START_REF] Liang | Effect of pretreatment and total solid content on thermophilic dry anaerobic digestion of Spartina alterniflora[END_REF] to treat smooth cordgrass and [START_REF] Rico | Thermophilic anaerobic digestion of the screened solid fraction of dairy manure in a solid-phase percolating reactor system[END_REF] to treat raw dairy manure (among other types). Moreover, no direct comparison has been made between LBRs run in mesophilic and thermophilic temperatures and nor has research involved two specific challenges connected with this discontinuous process: the startup conditions (i.e. the inoculation) and the discontinuous biogas production. The first, repeated at every digester loading, affects the methane production rates if not well managed, while the second causes problems for combustion in a Combined Heat and Power (CHP) unit. More particularly, this latter issue is very important when dealing with LBR plants in rural areas since a limited number of reactors (the main cause of a fluctuating biogas production) is often chosen to make this process economically feasible.

The efficiency of thermophilic treatment of spent cow bedding in LBRs remains an open question. Therefore, the aim of this work was to investigate if the operation of thermophilic mode when treating spent cow bedding in LBRs could be an effective measure for reducing the digestion time and increasing methane yield. In order to reach this objective, specific problems related to inoculation and the challenge connected to the combustion of biogas in a CHP unit were analyzed in detail.

Material and Methods

Experimental set-up

The three leach-bed reactors used in the present study were made of stainless-steel and the internal diameter and total height were 40 cm and 80 cm, respectively, for a total volume of about 100 L. A mesh (3 mm holes) placed at 20 cm from the bottom separated the solid and the leachate volumes:

75 L and 25 L, respectively. Each reactor was connected to a dedicated thermo-regulated water bath to maintain the temperature. A centrifugal pump (Rover Pompe BE-M 20) was used to sprinkle the not cause any particular inhibition and methane was rapidly produced. From 25%TS of easilydegradable matter, the inhibition was significant and the time to recover longer, meaning that the addition of such an amount should be done with caution at industrial-scale. However, independently of the percentage added, the system recovered its methanogenic capacity thanks to the flushing.

Flushing is then important in order to overcome reactor acidification.

5.2 Leachate flush strategies to manage volatile fatty acids accumulation in leach-bed reactors

Introduction

Anaerobic digestion (AD) is a very efficient biological process facilitating the treatment of organic solid waste and the production of biogas which can be used for energy purposes. In relation to total solids (TS), the reactor operating modes are divided in three main groups: wet (TS < 15%), semi-dry (10% < TS < 20%) and dry (TS > 20%) (Karthikeyan and Visvanathan, 2012a). Dry AD processes are the best-suited to an agricultural context where solid waste such as cereal residues and spent animal bedding have high TS content and fairly low biodegradability. Among dry processes, leach-bed reactors (LBRs) present economic and technological advantages thanks to their simple design and easy operation. A LBR usually consists of a batch digester in which the solid substrate is loaded with a wheel loader, and a liquid tank storing a leachate which is discontinuously sprinkled over the substrate top during the whole process. Therefore, leachate plays important roles by helping increasing the moisture content, improving mass transfer and diluting inhibitory compounds (Degueurce et al., 2016d). In comparison to other technologies, LBRs are recognized for their many advantages: high loadings of solid waste, reduced water consumption, unnecessary digestate posttreatment, reduced investment costs and greater biological stability compared to classic wet processes (Karthikeyan and Visvanathan, 2012a). However, this batch system entails certain disadvantages: i) discontinuous biogas production caused by frequent loading and emptying of the digester ; ii) incomplete degradation of the substrate, mainly due to its heterogeneity, leachate channeling and progressive bulk compaction [START_REF] André | Quantifying physical structure changes and non-uniform water flow in cattle manure during dry anaerobic digestion process at lab scale : Implication for biogas production[END_REF]; iii) accumulation of intermediate compounds formed during the first days of a batch operation due to the initial high loading of fresh substrate. In this sense, volatile fatty acids (VFAs), one of the main intermediates of the anaerobic process, represent a major problem for the proper management of LBRs. In fact, they may be produced in quite high concentrations which can cause inhibition and decrease methane production.

In LBRs treating agricultural waste, VFA accumulation can represent an important problem, conditions, as suggested also numerous authors [START_REF] Cadavid-Rodriguez | Production of volatile fatty acids from wastewater screenings using a leach-bed reactor[END_REF][START_REF] Cysneiros | The effect of pH control and "hydraulic flush" on hydrolysis and Volatile Fatty Acids (VFA) production and profile in anaerobic leach bed reactors digesting a high solids content substrate[END_REF][START_REF] Dogan | Performance of leaching bed reactor converting the organic fraction of municipal solid waste to organic acids and alcohols[END_REF]Selvam et al., 2010). These metabolites represented a maximum (condition H) of 4% of the total COD degraded. CODs was exclusively composed by VFAs after the first 3 days. As a consequence, for a clearer presentation of the results, only VFAs will be considered in this work. During the first 3 days, a significant production of hydrogen (average of all reactors: 27.6 ± 1.1 NmL H 2 g -1 VS of fresh matter), was measured. This is typical during an acidification phase as reported also by [START_REF] Chugh | Degradation of unsorted municipal solid waste by a leach-bed process[END_REF] and [START_REF] Han | Performance of an innovative two-stage process converting food waste to hydrogen and methane[END_REF] during the initial days of digestion in LBR treating unsorted municipal solid waste and food waste, respectively.

The highest TVFA concentration (24 g COD L -1 ) was reached with the lowest leachate flushrate applied (digesters L1 and L2) and corresponded to roughly twice the concentration observed for the highest leachate flush-rate (digester H). This indicates that the dilution factor played an important role in TVFA concentrations in the extracted leachate and, as a consequence, on its pH.

Condition H is used as an example to illustrate the composition of the extracted VFAs (Figure 34d) during this period, since their relative composition was similar for the three conditions. The main VFAs produced were butyric, acetic and, to a lesser extent, propionic acids in period I, while all the other VFAs (iso-butyric, valeric and iso-valeric) were recorded at very low concentrations. Bearing in mind that no VFA conversion into methane took place, Figure 34d shows that butyric acid was produced in great amount only during the earliest days; acetic acid in less quantity yet for a longer time (as suggested by a less sharp peak); whereas propionic acid was produced almost constantly throughout. The predominance of butyric and acetic acids in an acidification phase has already been reported in acidogenic LBRs (Cadavid-Rodriguez and Horan, 2014; [START_REF] Cysneiros | The effect of pH control and "hydraulic flush" on hydrolysis and Volatile Fatty Acids (VFA) production and profile in anaerobic leach bed reactors digesting a high solids content substrate[END_REF]. The high percentage of butyric acid extracted and the production of H 2 in the first days are a clear indication of a dark fermentation pathway, which was then interrupted because of an increase in the pH due to the daily flush. At the end of period I, the pH was higher than 6.8 for all the conditions, Attention to the reduced homogeneity in percolation (2) (Switching to a self-recirculation mode when methanogenesis is established)

Acetic acid concentrations between 5 and 10 g L - 1 , injection every 6-7 h and small volumes per recirculation (3) (Last days stop leachate addition to complete VFA consumption in the bulk) *these ranges are average for the three conditions tested (2) Tested by other authors (3) Volume conditions were not tested. However, they seem the best solutions CHAPTER 6

Discussion and perspectives

seem to be affected by the consequent high C/N ratio, mulching is a parameter that should be kept under control since it indirectly defines the faeces-to-bedding ratio.

Digestion of spent animal bedding in LBRs

The advantages and limits of the LBRs were studied together with the digestion of spent animal bedding and its degradation performance (Figure 39).

Digestion of spent animal bedding in LBRs is found to proceed efficiently without inhibition: the pH never drops below 6.5 and the methane content in biogas reaches values ranging between 55% -60%. The initial accumulation of VFAs in the system, which characterizes a sensitive phase in the LBR process, is rapidly replaced by methane production whose peak appears straight after VFA consumption. Initial high biogas production (as well as the acids accumulation) is related mainly to the degradation of the easily-degradable fraction contained in the spent animal bedding while the significantly lower biogas production after 30 days is connected mainly to the degradation of its slowly-degradable fractions.

Among all the types of spent animal bedding studied only spent goat bedding is characterized by a particularly high VFA accumulation (peak of ~11 g L -1 ) and a significant lag phase while all the other types have a much more similar degradation process with low VFA peak of (~5 g L -1 ) and almost no lag-phase. A reduced amount of active methanogens naturally present in the substrate could be the cause of this different behaviour. In this sense, a higher inoculation could be necessary when treating spent goat bedding in order to increase its methanogenesis kinetics and reduce its lag-phase.

Therefore, spent animal bedding of different types have been found to behave similarly when treated in LBRs which make it possible to consider them as similar substrates.

However, despite of this generalization few exceptions as the one of spent goat bedding can significantly affect the process and should be taken into consideration concentration levels. With spent animal bedding the main concern are nitrogen and potassium accumulation. A final concentration in the liquid phase ranging from 1.7 to 4.0 g L -1 for K + and from 0.6 to 2.7 g L -1 for TKN with respect to the substrate treated was recorded at labscale (up to 5 g L -1 of N-NH 4 and 9 g L -1 of K + are measured in industrial leachate after several cycles). Their possible inhibitory effect should be better assessed and leachate dilution after several AD cycles should be considered as a possible option. However, the choice of a more appropriate system TS (i.e. the ratio of the volume of the tank containing the leachate and the total volume of the solid digesters) could delay the occurrence of inhibitory concentrations and the need of frequently diluting the leachate to bring these concentrations back to acceptable levels.

The optimization strategies

Once set the basic knowledge on the LBR process, the substrate properties and its digestion, the next step dealt with the optimization strategies. The main objective of those was to increase methane yield and methane rate: i.e. produce the maximum amount of methane in the shortest operation time and working volume. Two main operating parameters were selected, the inoculation (discussed in chapter 3) and the temperature (discussed in chapter 4); the first acting through an increase of the number of adapted microorganism able to degrade the substrate and the second by enhancing the activity of the already present micro-organisms.

The optimization of the process certainly needed to deal with some technical and economic aspects related to LBR technical features. Those were then taken into account in order to propose the best option to be implemented at industrial-scale (Figure 40). methane production. However, the investment cost significantly changed and for the addition of digestate (30% of the volume considering the same density for spent animal bedding and digestate), the initial investment was found 18% higher. These results suggest that the use of leachate is a better economic strategy than the addition of digestate since a higher investment could represent a real barrier to the feasibility of an industrial project. It is important to stress that such a strategy is possible because spent cow bedding already contains a significant amount of active methanogens. The addition of solid inoculum is then not necessary as it could be for other type of substrates (e.g. straw or maize silage). This is a rather great advantage connected to the use of spent animal bedding.

Thermophilic temperature

The temperature increase from mesophilic to thermophilic conditions was also evaluated on the digestion of spent cow bedding. The use of thermophilic conditions instead of mesophilic ones was listed as a possible method to increase methane production rate and reduce batch duration. The results showed that temperature effectively enhanced the methane production rate but this effect was mainly important on the easily-degradable fraction of the substrate and then on the first 10 -15 days (only 14% higher methane yield in thermophilic condition after 15 days). Since spent animal bedding is mainly a lignocellulosic substrate (i.e.

slowly-degradable substrate), the effect of temperature on the overall digestion was poor.

Indeed, one of the main problems while treating spent animal bedding is to recover most of its potential energy because of its relatively low methane potential (measured through the BMP).

Nonetheless, thermophilic and mesophilic temperature are showed equivalent in the attempt to reach the 80% of the BMP which is reached after 42 days of operation in both conditions.

SUPPLEMENTARY MATERIAL 164 43 a-b). The higher variations and its sensitivity to the capacity, make the IRR a crucial parameter.

Statistical and modelling data on spent animal bedding characterization and digestion in LBR

In conditions D and L, while the relative difference in sensitivity to each parameter was similar, the parameters that most distinguished the two conditions were found to be substrate preparation and haulage distance. In fact, by adding digestate, the total substrate volume to be loaded and then emptied is higher; on the other hand, by using a leachate, less energy, is recovered from the same amount of matter transported. Within the chosen ranges, these differences were not enough to justify choosing one start-up strategy over another. However, these differences could increase significantly for values out of the range limits. For example, for haulage distances above 10 km (up to 70 km which is still an environmentally interesting solution [START_REF] Massaro | The sustainability of anaerobic digestion plants: a win-win strategy for public and private bodies[END_REF], the digestate option could turn out to be a better strategy than using a leachate. However, attention should be paid to the overall performance which will inevitably decline.