Merci Également

Olivier Chapleur

Andrea Schievano

Claire Dumas

Michel Gautier

Dominique

Romain G Et Clément

Loïc, Perrine, Yann, Coline, Kévin Merci À Lucas

Je souhaite tout d'abord remercier José Martinez et Eric Dufour pour leur

RÉSUMÉ

Le phosphore est un élément clé des mesures prises en faveur de l'économie circulaire en Europe car c'est un élément irremplaçable, indispensable à tous les organismes vivants et déterminant pour l'autonomie alimentaire du continent. Or, il est issu de ressources minières limitées fortement concentrées dans quelques pays et dont l'Europe est presque totalement dépourvue.

L'approvisionnement et son coût sont dépendants du contexte géo-politique, soumis aux fluctuations de l'offre et de la demande mondiale. Paradoxalement, dans les régions d'élevage intensif, le phosphore importé massivement avec les aliments du bétail mais mal retenu par les animaux se retrouve épandu en excès avec les effluents d'élevage conduisant à l'eutrophisation des eaux de surface avec des impacts économiques importants sur la potabilisation de l'eau ou son utilisation pour la pisciculture ou les loisirs. C'est également le cas autour de certaines zones urbaines où le phosphore immobilisé dans les boues de station d'épuration est épandu plus que valorisé sur les terres agricoles.

Au contraire, l'approvisionnement des régions de grandes cultures repose presque exclusivement sur les importations d'engrais chimiques. L'enjeu est donc de pouvoir recycler le phosphore des effluents humains et animaux, en excès par rapport au besoin des cultures dans certaines régions, sous une forme utilisable dans les zones de grandes cultures. Dans ces travaux, nous nous sommes intéressés spécifiquement au P des lisiers de porcs qui représente une source importante facilement mobilisable du fait de la concentration des élevages et de l'existence de procédés de traitement de l'azote et/ou de valorisation énergétique.

Des procédés de recyclage du P ont été développés produisant majoritairement de la struvite qui est un engrais à diffusion lente dont les performances agronomiques sont au moins équivalentes à celles des engrais minéraux fabriqués à partir de ressources minières. La plupart de ces procédés ont été développés pour recycler le P issu des boues de stations d'épuration urbaines de taille importante. Le plus souvent, ils ne concernent que le P dissous, ce qui limite fortement le taux de recyclage. Le prétraitement des effluents pour augmenter la fraction de P dissous est souvent coûteux particulièrement dans le cas des effluents d'élevage qui sont des milieux fortement tamponnés dans lesquels la dissolution du P majoritairement sous forme minérale particulaire exige d'importantes quantités d'acide. Cette étape de dissolution est le verrou au développement de la plupart des procédés.

Certains procédés biologiques comme l'ensilage ou la production d'hydrogène reposent sur le potentiel acidifiant de micro-organismes qui peuvent se développer dans certaines conditions dans les milieux fortement chargés en matière organique. La baisse du pH significative engendrée par ces métabolismes pourrait être substituée au prétraitement par acidification chimique pour dissoudre le P des lisiers.

Cette thèse accompagne donc le développement et l'optimisation d'un procédé innovant basé sur la l'acidogénèse, production d'acide in situ par les bactéries endogènes, permettant la dissolution d'une large fraction du P total initial qui constitue la première étape du recyclage du P des lisiers de porcs.

Après séparation, le liquide enrichi en P est ensuite soumis à l'étape de cristallisation pour produire de la struvite tandis que les boues appauvries en azote et en phosphore peuvent être utilisées comme apport de matière organique pour les sols déjà très fortement pourvus en P des régions d'élevage intensif. Du fait de l'hydrolyse acide de déchets organiques générée par ce procédé, il peut s'inscrire en amont ou en parallèle des procédés de valorisation énergétique par production de méthane dont le développement est fortement encouragé.

Objectifs

Les objectifs de cette thèse peuvent se résumer ainsi :

• Déterminer à l'aide d'un co-substrat modèle (saccharose) l'effet de l'acidogénèse sur le pH, la dissolution du P mais aussi des autres ions impliqués ou interférant dans la cristallisation de la struvite • Etudier l'effet de l'utilisation de divers déchets organiques comme co-substrats sur le développement de la flore acidogène et leurs métabolismes • Evaluer la possibilité de disposer d'un outil simple de prédiction de la baisse du pH en fonction du type de co-substrat et des caractéristiques du lisier.

• Etudier la possibilité de développer une flore acidogène adaptée en travaillant sur un réacteur semi-continu.

• Vérifier la faisabilité de cristalliser de la struvite à partir du liquide enrichi en P et la qualité du produit obtenu.

• Etablir un bilan chiffré du recyclage et proposer des pistes d'améliorations.

Résultats

La présentation des résultats est organisée autour de trois publications dont l'une est publiée, l'une est soumise et l'autre prête à soumettre.

Les objectifs des travaux décrits dans le premier article sont de tester la faisabilité de l'acidification biologique et d'évaluer la quantité de sucre nécessaire en fonction des caractéristiques des lisiers (pH initial, pouvoir tampon …). L'effet de l'acidification biologique sur la dissolution des autres composés de la struvite (ammonium et magnésium) et du calcium qui peut interférer avec la cristallisation de la struvite a aussi été étudié. Pour cette première approche du saccharose (sucre blanc du commerce) a été utilisé. Les résultats obtenus avec le sucre permettront d'évaluer le potentiel de dissolution du P par voie biologique sur différents lisiers.

Quatre lisiers différents ont été utilisés. Les essais réalisés avec sept concentrations de sucre entre 0 et 60 g.L -1 montrent que la baisse de pH obtenue est directement liée à la concentration en sucre ajoutée.

Un pH de 3.99 a été atteint avec la concentration la plus forte. Le dosage des acides organiques a montré que la production d'acide lactique est le principal responsable de la baisse du pH. Le faible pH atteint avec les concentrations en sucre les plus fortes permettrait d'inhiber les métabolismes de dégradation de l'acide lactique et de maintenir le pH stable. Les concentrations en phosphore, calcium et magnésium dissous sont liées à l'évolution du pH. Jusqu'à 95% du P total initial du lisier ont été dissous avec les trois concentrations en sucre les plus élevées.

La dissolution du P et du magnésium en fonction du pH n'est pas linéaire. Elle intervient principalement pour des pH compris entre 5.5 et 6.5. En revanche la dissolution du calcium en fonction du pH est quasi-linéaire entre pH 7 et pH 4.5. Cette évolution différente permet de définir une zone de pH comprise entre 5.5 et 6 pour laquelle la dissolution du Ca comparée au P et au Mg est moindre ce qui favoriserait la cristallisation du P sous forme de struvite lors des étapes ultérieures. Le fait d'opérer la séparation avant cristallisation à ce pH réduirait également la quantité de réactif nécessaire à la remontée du pH lors de la cristallisation. En revanche la faible stabilité du pH dans cette zone demande une gestion précise du procédé d'acidification.

Finalement la régression linéaire mise en oeuvre sur les données obtenues avec les quatre lisiers a permis d'établir une relation bien ajustée entre le pH minimal, la quantité de sucre, le pH initial du lisier et son pouvoir tampon pour une zone de pH entre 4 et 8. Cette relation pourra être utilisée pour calculer la quantité de co-substrat à ajouter. Ces travaux ont montré le lien étroit entre la production d'acide lactique et le pH. Or l'acide lactique est le produit majoritaire de dégradation des sucres simples très facilement dégradables.

Les travaux décrits dans le second article ont pour objectif de s'assurer que l'acidification peut être obtenue avec les co-substrats riches en glucides complexes disponibles sur les unités de méthanisation et de tester la faisabilité d'un recyclage en parallèle de la méthanisation pour exploiter la présence d'une flore déjà accoutumée à la dégradation anaérobie de co-substrats complexes.

Un lisier brut et deux digestats issus du même digesteur mais prélevés à deux périodes différentes ont été utilisés. Avec le digestat, le pH décroît presque linéairement jusqu'à des concentrations de sucre de 30 g.L -1 . La baisse est moins importante à quantité de sucre équivalente avec le lisier brut et 50 g.L -1 sont nécessaires pour obtenir la valeur minimale et ce malgré un pH initial plus faible. La présence d'AGV dans le lisier avant le test peut expliquer à la fois le pH plus faible et le pouvoir tampon plus élevé. Le lactate est l'acide organique majoritaire pour les fortes concentrations en sucre dans les deux matrices utilisées. Pour des concentrations inférieures, le lactate a été produit pendant les premières 48 heures mais consommé ensuite lors de fermentations secondaires qui ont pu avoir lieu grâce à un pH insuffisamment acide pour les inhiber. Un pH inférieur à 5 est obtenu avec les pommes, les carottes à 50g.L -1 , les biscuits et la farine de blé noir stockés. Cette baisse de pH peut être attribuée à la production d'acide lactique. Aucune baisse de pH ni production d'AGV n'est observée pour la paille.

Pour les autres co-substrats, le pH atteint des valeurs comprises entre 5 et 5.7. Les valeurs atteintes sont corrélées à la teneur en glucides. La conversion de la matière organique en acides organiques est comprise entre 25 et 56% inférieures aux valeurs obtenues avec le sucre (44-78%).

Les communautés microbiennes impliquées dans l'acidification ont été caractérisées par séquençage haut débit des gènes des ARNr 16S. L'analyse des indices de diversité suggère une large diversité initiale avec quelques espèces dominantes. La diversité se réduit pour les essais pour lesquels les pH les plus bas sont atteints. Le faible pH et la concentration en lactate exerceraient une pression de sélection ne permettant de conserver que les micro-organismes tolérants aux conditions acides que sont les Lactobacillus. Ces essais montrent que la fermentation lactique avec des déchets organiques est possible et que la production d'acide lactique dépendra de la disponibilité de sucres facilement dégradables, qui favorisent le développement rapide des Lactobacillus, avec un effet inhibiteur empêchant les fermentations secondaires.

L'objectif des travaux décrits dans le troisième article est de définir un « équivalent sucre » pour quelques déchets issus de la production agricole régionale puis de vérifier la relation établie précédemment pour calculer la quantité de co-substrats à ajouter pour atteindre le pH désiré en fonction des caractéristiques du lisier. Enfin l'influence de l'acidification biologique sur la cristallisation du P sous forme de struvite et la qualité du solide final a été testée.

Quatre lisiers issus d'élevages industriels ont été utilisés et 5 co-substrats, dont le sucre, y ont été ajoutés à la concentration de 50 g de matière organique par litre pour déterminer l'équivalent sucre.

Les 4 substrats sont des légumes ou des fruits habituellement produits en Bretagne et dont les invendus où les déchets de transformation alimentent les méthaniseurs. Ce sont des co-substrats riches en sucre mais avec des teneurs ou une accessibilité différente. Il s'agit des carottes, pommes petits pois et haricots verts. L'équivalent sucre est le rapport entre la quantité de sucre et la quantité de co-substrat permettant d'obtenir un pH équivalent. Des valeurs respectives de 0,74, 0,69, 0,42 et 0,42 ont été calculées pour les pommes, les carottes, les haricots et les petits pois. Les quantités pour atteindre des valeurs de pH de 4, 5 et 6 ont été calculées d'après l'équation citée plus haut et ont été ajoutées aux 4 lisiers. L'adéquation entre le pH obtenu et le pH calculé a été estimée par la moyenne des valeurs absolues des différences ou par la moyenne des différences positives ou négatives. L'adéquation est satisfaisante pour le sucre comme pour la plupart des co-substrats sauf les petits pois. Dans ce dernier cas, les acides majoritairement produits ne sont pas le lactate mais l'acétate, le propionate, le butyrate et le valérate. Ces acides ayant un pKa supérieur à 4.5, il n'est pas possible d'atteindre un pH de 4. La suppression des valeurs obtenues pour les essais dont l'objectif était d'atteindre ce pH permet d'améliorer l'adéquation entre les valeurs de pH attendue et mesurée. La différence est alors au maximum de 0,45 point de pH. Pour des valeurs de pH proches de la valeur optimale de 5.5-6 une fermentation secondaire intervient systématiquement ce qui confirme l'hypothèse émise précédemment selon laquelle le procédé devra être piloté avec un suivi rapproché du pH pour réaliser la séparation lorsque le ratio Ca/P est optimal. Comme lors des essais précédents, l'évolution de la concentration en P dissous lors de l'acidification avec les co-substrats est étroitement liée à l'évolution du pH pour 3 des quatre lisiers. Un comportement différent a cependant été observé pour un des lisiers qui avait une concentration initiale en P total très faible. Dans ce cas et surtout lorsque les quantités de co-substrat ajoutées sont importantes l'assimilation du P pour la croissance de la biomasse n'est plus négligeable et réduit la concentration en P dissous observée. Si le co-substrat est riche en P comme c'est le cas des petits pois, la concentration en P dissous sera liée à l'hydrolyse progressive des formes organiques contenant le P sous l'effet de l'acidification.

Afin d'évaluer l'intérêt de conserver un inoculum acidogène adapté au co-substrat des essais en semicontinu ont également été réalisés. Le pH est descendu rapidement à 5.7 pendant les 4 premiers jours puis le pH minimum a baissé progressivement jusqu'à 5.4. La concentration en P dissous est passée de 150 à 680 mg.L -1 en quatre jours puis a augmenté progressivement pour atteindre 747 mg.L -1 soit 57% du P total du mélange au bout des 16 jours. L'effluent recueilli pendant la durée du test a été centrifugé puis analysé avant d'être utilisé pour des essais de cristallisation. La centrifugation a permis de récupérer 83% du volume sous forme liquide soit 47% du P total initial. La cristallisation a été réalisée en bécher agité. Environ 3.5g d'hydroxyde de magnésium ont été nécessaire pour atteindre pH 8. Le mélange a été laissé sous agitation pendant 2 heures. Plus de 99% du P dissous a été précipité.

Le solide produit est composé à 69% de struvite, le reste étant probablement composé d'autres composés minéraux phosphatés, de l'hydroxyde de magnésium non dissous et de la matière organique.

Le faible temps de contact et le mode d'agitation ont conduit à l'obtention de cristaux de petite taille et hétérogènes. Aussi, malgré une précipitation presque totale du P dissous, le solide issu de la filtration à 100 µm n'a permis de recueillir que 26% du P total du mélange initial. L'obtention d'un pH légèrement inférieur lors de l'acidification aurait probablement permis d'améliorer le taux de dissolution du P et la réalisation de la cristallisation dans un équipement industriel optimisé d'obtenir des cristaux plus gros. Ces deux étapes sont déterminantes pour améliorer le rendement global du procédé. Si des équipements industriels développés spécifiquement pour la cristallisation de la struvite sont disponibles sur le marché, une bonne connaissance des co-substrats et notamment de leur équivalent sucre est déterminante pour choisir les cocktails qui favoriseront la production d'acide lactique et limiteront les fermentations secondaires. Des études complémentaires sur l'influence des paramètres de l'étape d'acidification biologique (temps de séjour, température …) devraient également permettre d'orienter les métabolismes.

Conclusion et perspectives

L'hypothèse de départ suivant laquelle l'utilisation du métabolisme acidogène de la flore endogène pourrait provoquer in situ la baisse de pH nécessaire à la dissolution du P a été vérifiée. Entre 40 et 90% du P ont été dissous suivant les co-substrats. Pour obtenir ce résultat, des co-substrats riches en sucres facilement dégradables favorisant le développement des bactéries lactiques devront être privilégiés. La quantité à apporter pourra être calculée en fonction des caractéristiques du lisier avant ou après digestion grâce au modèle simple proposé dans ces travaux.

Les facteurs clés déterminant le rendement du procédé sont la concentration en P dissous au moment de la séparation et la taille des cristaux obtenus tandis que le coût du produit sera plutôt lié à la quantité de réactif nécessaire à l'augmentation du pH lors de la cristallisation. . Determination of the acidifying power for each co-substrate compared to sucrose ......................... 4. Biological acidification of pig slurry in batch tests using complex organic 1. Model fitness ...................................................................................................................................... 2. Dissolution of P,Mg,Ca and N ...................................................................................................... Phosphorus is also one of the limiting nutrients in agriculture with nitrogen (N) and potassium (K) (Sattari et al., 2014). As a result, those nutrients need to be added to the soil using mineral fertilizers and animal manure [START_REF] Vitousek | Nutrient imbalances in agricultural development[END_REF]. Historically, crop production relied on natural levels of soil-P and addition of locally available organic matter like manure and human excreta [START_REF] Mårald | I mötet mellan jordbruk och kemi: agrikulturkemins framväxt på Lantbruksakademiens experimentalfält 1850-1907[END_REF].

Molar ratios of interest for struvite

LIST OF TABLES

LIST OF ABBREVIATIONS AND ACRONYMS

In the twentieth century, increased food demand due to rapid population growth has led to the massive application of phosphate rock based mineral fertilizers to crop soils (Smil, 2000). This "green revolution", which combines technical and agronomic progress with large application of mineral fertilizer enabled major improvements in crop yields and associated food production but also led to the accumulation of P in soils and a significant amount can be lost due to runoff to surface waters [START_REF] Carpenter | Eutrophication of aquatic ecosystems: Bistability and soil phosphorus[END_REF]. Nutrient over enrichment in fresh water bodies can lead to excessive biomass production, a harmful phenomenon known as eutrophication. In this situation, phytoplankton community may shift to toxic algal bloom [START_REF] Smith | Eutrophication science: where do we go from here?[END_REF]. Its decomposition leads to foul odors and oxygen depletion, killing the fish population [START_REF] Carpenter | Eutrophication of aquatic ecosystems: Bistability and soil phosphorus[END_REF]. Other problems associated with eutrophication include the presence of toxins [START_REF] Lawton | Cyanobacterial (blue-green algal) toxins and their significance in UK and European waters[END_REF], extirpation of native plants [START_REF] Gleick | Water in crisis: paths to sustainable water use[END_REF], and loss of biodiversity [START_REF] Maitland | Restoration of aquatic ecosystems: Science, technology, and public policy[END_REF]. In fresh water undisturbed by anthropogenic activity, P is usually the limiting nutrient. As a result, increase in P concentration is the main cause of eutrophication [START_REF] Foy | The contribution of agricultural phosphorus to eutrophication[END_REF]Schindler et al., 2008). State regulations have been enacted during the last 20 years in the developed countries to limit P concentration in municipal and industrial wastewater effluents in order to fight against eutrophication. Indeed, P concentration is relatively high in municipal wastewaters, coming mostly from human feces and detergents. Municipal and industrial wastewaters are now fully treated under stringent discharge limits, reducing drastically the "point sources" of P in fresh waters. As a result, it is now necessary to redirect the efforts towards the management of "diffuse sources" of P, i.e. agriculture (Dupas et al., 2015;[START_REF] Grizzetti | Changes of nitrogen and phosphorus loads to European seas[END_REF]Némery et al., 2005;[START_REF] Van Drecht | Global nitrogen and phosphate in urban wastewater for the period 1970 to 2050[END_REF]. Among agricultural activities, intensive livestock breeding can be a main cause of diffuse P emissions as a result of high application rates of animal waste and the resulting enrichment of soils in phosphorus [START_REF] Heckrath | Phosphorus Leaching from Soils Containing Different Phosphorus Concentrations in the Broadbalk Experiment[END_REF][START_REF] Ringeval | Contribution of anthropogenic phosphorus to agricultural soil fertility and food production[END_REF]. The recent focus on modern agriculture's role in diffuse P emissions and associated eutrophication of freshwater bodies has led to modest but significant improvements with lower P fertilizer application, reduced transport to aquatic ecosystems thanks to retention basins [START_REF] Bennett | Human Impact on Erodable Phosphorus and Eutrophication: A Global Perspective: Increasing accumulation of phosphorus in soil threatens rivers, lakes, and coastal oceans with eutrophication[END_REF] and individual or collective treatment systems. These efforts have recently been heightened by the realization that the only resource for P fertilizer manufacturing, phosphate rock, is a fossil material under threat of exhaustion in a near future. A still growing world population as well as a phosphate rock resource of lower quality, less accessible, with higher processing and shipping cost has led to a steady increase in fertilizer price and consequently higher food price, while depleting the resource at an increasingly faster pace (Smil, 2000). The threat to worldwide food security makes phosphorus a new global sustainability challenge intertwined with other major threats such as climate change, water scarcity and energy production [START_REF] Cordell | Phosphorus vulnerability: A qualitative framework for assessing the vulnerability of national and regional food systems to the multi-dimensional stressors of phosphorus scarcity[END_REF]. Therefore, reducing the diffusion of P to freshwater ecosystems to limit eutrophication is now part of a larger goal that targets a sustainable management of the P resource to guaranty long term food security. One of the main impediments to optimal phosphorus usage is the disparities between geographical areas in term of agricultural needs for P on one hand and production of P waste flows on the other hand. Crop-oriented agricultural regions rely heavily on mineral fertilizers while areas with intensive livestock farms struggle to find outputs for their P-rich manure. At a larger geographical scale, economic and development disparities also affect the utilization of phosphorus: while Western Europe continues to over apply phosphorus in agricultural soils with harmful impacts on the environment, countries in Eastern Europe have steadily relied on an undersupply of P to the crops due to their economic downfall following the collapse of the Soviet Union, diminishing progressively the crop yields [START_REF] Csatho | Two worlds within EU27: Sharp contrasts in organic and mineral nitrogen-phosphorus use, nitrogen-phosphorus balances, and soil phosphorus status: Widening and deepening gap between Western and Central Europe[END_REF]. A possible solution to these disparities could be to recycle phosphorus from livestock waste streams to produce concentrated, easily transportable P-based mineral fertilizers to be used in agricultural areas in need.

Brittany, France is a good example of an agricultural area with excess P that has to be exported away.

With a local climate enabling intensive livestock breeding and a soil poorly suited for crops, Brittany became the leading region in France for pig breeding during the mid-twentieth century and maintained its position until now, with 60% of the swine production on only 6% of the arable land in the country.

The discrepancy between the amount of P-rich waste streams produced and the crop needs as well as the progress in domestic wastewater treatment make agriculture the main cause of P emissions to fresh water in the area, with 60% of dissolved P and 80% of particulate P [START_REF] Colmar | Synthèse du projet TRANS-P[END_REF], despite an 80% decrease in P fertilizer in the last 20 years. Numerous treatment units have been implemented, mainly activated sludge systems initial solid liquid separation and treatment of the liquid phase with nitrification denitrification to convert ammonia into nitrogen gas. Since the beginning of the 21 st century, 51 digestion units have also been erected, co-digesting animal manure with crop residues and industrial organic waste. Because phosphorus in pig slurry is mostly under inorganic, solid forms, it necessarily ends up in the solid phase at the end of either treatment pathways. In Brittany, this P rich solid phase is usually composted with the intent to be exported as an organic fertilizer in cereal producing regions, such as "la Beauce" in the south west of Paris. However, the product obtained is far less concentrated in phosphorus than mineral fertilizers and is usually sold at a very low price. Pig slurry is a suitable effluent to precipitate struvite (Kataki et al., 2016), a P rich, easily transportable mineral fertilizer. Recovering P from pig slurry as struvite would lower the transport costs compared to compost and maintain the organic matter locally. In order to maximize the amount of P recovered, it is necessary to dissolve it first by lowering the pH before a crystallization step at alkaline pH. Several organic acids have been tested for acidification, such as formate and acetate, leading to the dissolution of 75-80 % of TP (Daumer et al., 2010). However, the cost of the acids was too high to compete with conventional mineral fertilizer and the environmental assessment was very poor, with chemical acidification as the main influence in the Life Cycle Analysis. Such acids can be avoided by taking advantage of a biological process known as acidogenesis: under anaerobic conditions, acidogenic bacteria in the slurry are able to produce organic acids from organic matter, leading to a pH decrease.

In the context of anaerobic digestion development in Brittany, it could be interesting to place an acidogenic process prior to (or in parallel of) the digester in order to recover phosphorus and produce organic acids that would subsequently be converted to methane. The objective of this thesis was to study the biological acidification of pig slurry with various organic co-substrates and investigate the precipitation of phosphorus as struvite.

The first part of this manuscript is dedicated to the context of the study. The problematic of phosphorus regarding eutrophication, resource use and waste is investigated in a first section. In a second section, the agronomic effect of struvite as fertilizer is reviewed, as well as the existing processes for P recovery from domestic and livestock wastewater. In a third and last section, the biological process of acidogenesis is investigated, describing the acidogenic bacteria involved and their metabolic pathways, with a focus on lactic acid fermentation, a favored reaction regarding efficient phosphorus dissolution and recovery as struvite.

In chapter 1, biological acidification of pig slurry was performed in 96 h batch tests using sucrose as a model organic co-substrate and four different slurries. The evolution of pH, the organic acids produced and the dissolution of P, N, Mg and Ca were studied. A mass balance was calculated and expressed as carbon recovery. A simple model was set up to predict the lowest pH based on the slurry's characteristics (initial pH and buffer capacity) and initial sucrose concentration.

In chapter 2, the same biological acidification protocol in batch reactors was tested on raw and digested swine slurry using sucrose and various organic wastes (potato, biscuit, buckwheat flour, apple, carrot, bean, pea and wheat straw). A microbiological study was conducted prior to fermentation and at the end of the batch tests in order to evaluate the changes in term of bacterial diversity and dominating groups. The co-substrates were characterized in term of acidifying capacity in order to expand the model predicting the lowest pH reachable.

In chapter 3, biological acidification of raw pig slurry was conducted in batch tests using carrots, green beans, peas and apples to verify the pH model validity. These co-substrates were chosen because they can easily be found as agricultural, agro-industrial or municipal waste in Brittany. Dissolution of P, Mg, N and Ca was investigated again. A semi-continuous reactor was set up to investigate the potential adaption of the biomass towards higher acidification and phosphate dissolution, using a mix of carrots and peas as organic co-substrates. The effluent was used to precipitate phosphorus and the dried solid obtained was dissolved in an acidic solution to study its ionic composition.

Finally, in the last part of the manuscript, the main results obtained are discussed and additional considerations are provided in order to improve upon this work and make the process fully implementable.

CONTEXT

II. PHOSPHORUS, A NUTRIENT AT A CROSSROAD

ROLE OF PHOSPHORUS IN NATURE AND ANTHROPOGENIC ACTIVITIES

Phosphorus is a fascinating nutrient at the nexus of several local, national and global problematics. It is at once critical to life on earth, severely limiting primary production in most ecosystems, paradoxically the main cause of eutrophication in fresh waters and, as recently realized, a rather scarce resource that could impact global food security in the mid-term [START_REF] Elser | Phosphorus: a limiting nutrient for humanity?[END_REF].

Phosphorus is involved in the cell membrane through the bilayer of phospholipids, which make up 5-10% of the cellular mass [START_REF] Sterner | Ecological stoichiometry: the biology of elements from molecules to the biosphere[END_REF]. Even more importantly, adenosine monophosphate (AMP), diphosphate (ADP) and triphosphate (ATP) are the universal molecules acting as energy carrier in any living organism. Finally, phosphorus is involved in the genetic material and its expression through DNA and RNA. In humans, phosphorus constitutes a major part of bones and teeth under various calcium phosphate salts, or 0.7 Kg of total body mass (Ashley et al., 2011).

On Earth, two natural phenomena are responsible for phosphates (PO 4

3-

) fluxes: weathering and leaching. Weathering corresponds to the chemical and physical processes responsible for exposed rock to decompose, while leaching is the phenomenon by which molecules are carried away from the soil by water flowing through. Weathering and leaching are relatively slow processes that represent the main constraint on terrestrial productivity [START_REF] Lambers | Plant nutrient-acquisition strategies change with soil age[END_REF]. Indeed, P is the least mobile and available nutrient in most soil conditions due to its high reactivity with other molecules causing a strong retention [START_REF] Gull | Phosphorus uptake and growth promotion of chickpea by co-inoculation of mineral phosphate solubilising bacteria and a mixed rhizobial culture[END_REF]. As a result, P constitutes a limiting nutrient in agriculture, along with nitrogen and potassium [START_REF] Cleveland | Patterns of new versus recycled primary production in the terrestrial biosphere[END_REF]. Some natural processes occurring locally have enabled sufficient replenishment of soil P to allow agriculture to flourish early in human history, such as the flooding of Nile and Indus basins [START_REF] Nixon | The Artificial Nile The Aswan High Dam blocked and diverted nutrients and destroyed a Mediterranean fishery, but human activities may have revived it[END_REF][START_REF] Weil | The rivers come: Colonial flood control and knowledge systems in the Indus basin, 1840s-1930s[END_REF]. However, under regular conditions, the phosphorus removed from soil through the harvest of crops needs to be replaced artificially [START_REF] Ngoze | Nutrient constraints to tropical agroecosystem productivity in long-term degrading soils[END_REF]. Humans understood that as long as 5000 years ago when they started to apply their excreta as well as animal manure to the fields [START_REF] Mårald | I mötet mellan jordbruk och kemi: agrikulturkemins framväxt på Lantbruksakademiens experimentalfält 1850-1907[END_REF]. Other secondary sources of phosphorus such as ashes and harvest residues were also applied (Cordell et al., 2009). Erosion, a major source of P loss, was controlled through terracing as early as 2000 BC in Arabia and later in all inhabited areas, including remote pacific islands [START_REF] Mcneill | Breaking the Sod: Humankind, History, and Soil[END_REF]. Complex sewer systems were implemented during Roman and Greek era, enabling both high hygiene levels in the cities and organic matter collection points outside the urban areas for reuse in surrounding agricultural fields [START_REF] Dersin | What life was like when Rome ruled the world: The Roman Empire 100 BC-AD 200[END_REF] This very efficient use and reuse of P made for a closed cycle that started to "leak" during the Middle Age with often a dysfunctional sewer system and a partial diversion of sewage towards nitrate production for gun powder instead of field application [START_REF] Kirchmann | Recycling municipal wastes in the future: from organic to inorganic forms?[END_REF]. As a result of soil deterioration and deadly famines in Europe during the 17 th and 18 th century, new sources of P were actively sought outside of the traditional and local resources [START_REF] Emsley | 13th element[END_REF].

Thus crushed bones were used as P-fertilizer in England and US in the early 19 th century (Ashley et al., 2011). Shortly after, other concentrated sources of P were discovered such as guano and phosphate rock. Guano consists in a dense mix of sea bird excrement, feathers and bones, first found in Peru and south pacific islands [START_REF] Hutchinson | Survey of contemporary knowledge of biogeochemistry 3. The biogeochemistry of vertebrate excretion[END_REF] [START_REF] Mårald | Everything circulates: agricultural chemistry and recycling theories in the second half of the nineteenth century[END_REF]. Industrial, economic and scientific development, as well as the discovery of phosphate rock deposits and the Haber Bosch process for artificial nitrogenous fertilizer production have all enabled the green revolution to take place. Through massive irrigation, chemical fertilizer and pesticides application and new varieties of crops, agricultural yields doubled and sustained a 50% world population growth during the 20 th century (Tilman, 1998). However, this new agricultural paradigm also led to a massive waste at every step of the food production chain. While population on earth increased by half, P fertilizer consumption was multiplied by 5 compared to pre-industrial times, which is far more than carbon (+13%) and nitrogen (+100%) [START_REF] Falkowski | The Global Carbon Cycle: A Test of Our Knowledge of Earth as a System[END_REF]. Put another way, only 20% of the P extracted to produce food actually ends up in our plates (Cordell et al., 2009). Two major leaks in the P cycle were originating from excessive application of chemical fertilizers on one hand and poor treatment of human excreta on the other hand. Widespread availability of cheap chemical fertilizer led to overuse in the developed countries while wastewater treatment initially focused only on reducing the oxygen demand without regard for nutrients. As a result, the P content of freshwater systems worldwide is now at least 75% greater than preindustrial levels, and oceans face an inflow of P from 8 MT per year to 22 MT per year [START_REF] Bennett | Human Impact on Erodable Phosphorus and Eutrophication: A Global Perspective: Increasing accumulation of phosphorus in soil threatens rivers, lakes, and coastal oceans with eutrophication[END_REF]. As described in the introduction, the shift of water bodies from oligotrophic to eutrophic with regard to phosphorus and nitrogen has major and costly consequences. Beyond the physical, chemical and biological changes induced by eutrophication, there are also significant socioeconomic impacts on health, commercial fisheries, tourism, recreational activities and mitigation measures undertaken [START_REF] Anderson | Estimated annual economic impacts from harmful algal blooms (HABs) in the United States[END_REF]. The economic impact in the US alone was estimated very conservatively at 2.2 billion US$ annually [START_REF] Dodds | Eutrophication of U.S. Freshwaters: Analysis of Potential Economic Damages[END_REF]. It is through the prism of environmental damage and economic loss that new regulations regarding wastewater treatment were taken in the late 20 th century in developed countries, focusing mostly on domestic wastewater (point source pollution) and much less on nutrients runoff from agricultural fields (diffuse source pollution). Ashley et al. (2011) described the livestock manure treatment processes as having a medieval level of sophistication. In any way, reducing P input to freshwaters from either agriculture or domestic waste through conventional treatment processes without return to arable soil does not disrupt the linear flow of phosphorus and associated waste. The notion that phosphorus was a finite and irreplaceable resource threatening food security only came to the forefront after the major price hike (+700%) in [START_REF] Levlin | Phosphorus recovery with acid and base from inorganic sewage sludge residues[END_REF][START_REF] Franz | Phosphate fertilizer from sewage sludge ash (SSA)[END_REF](Cordell et al., 2009)). In the last ten years, rigorous research have eventually been conducted to assess the resource stock, quantify P flux, forecast future needs, identify causes of waste and suggest various initiatives to close again the phosphorus cycle.

PHOSPHORUS RESERVE, CURRENT AND FORECASTED USES

Large uncertainties remain regarding the amount and quality of phosphorus deposits in the world [START_REF] Elser | Phosphorus cycle: a broken biogeochemical cycle[END_REF]. Beyond the inherent uncertainties regarding yet to be discovered underground resources, a major cause for this lack of knowledge stems from the low transparency of producing countries [START_REF] Gilbert | Environment: the disappearing nutrient[END_REF]. Now that P has become a strategic commodity, producing countries are reluctant to disclose their reserves.

Current estimations have been re-evaluated upward, varying from 5.2 to 38.8 GT-P (Table 1)

depending on the methodology and the type of resource considered (e.g. including or not additional

resource not yet discovered, currently inaccessible with current technics or too costly for now). The reference in the field, the United State Geological Survey (USGS) gave its last estimate at 9 GT-P, considering only the P that can be extracted/produced economically under the current situation [START_REF] Ober | Mineral commodity summaries[END_REF]. The initial prediction made by Cordell et al. (2009), forecasting a P production peak in the short term (2035) followed by a sharp decrease due to a depleting resource has eventually been judged

unlikely [START_REF] Heckenmüller | Global availability of phosphorus and its implications for global food supply: an economic overview[END_REF][START_REF] Van Vuuren | Phosphorus demand for the 1970-2100 period: A scenario analysis of resource depletion[END_REF]. Indeed, at the current extraction rate of 34 MT-P/year, the reserve could last for 265 years. Currently China is the main producer of phosphate rock, exceeding US production since 2006, with major changes in the repartition of producing countries in the last 20 years (Figure 1). There is still a large diversity of countries producing significant amounts of P rock. However, the extreme concentration of underground P resources in very few countries (even more than oil) or one could say, in one country alone (Morocco, Figure 2) could cause huge tensions for access to the commodity and major price hikes due to monopoly/oligopoly pricing [START_REF] Elser | Phosphorus cycle: a broken biogeochemical cycle[END_REF][START_REF] Scholz | Sustainable use of phosphorus: A finite resource[END_REF][START_REF] Taylor | The fertilizer oligopoly: The case for global antitrust enforcement[END_REF]. Economic growth in a country would not depend anymore on access to USGS [START_REF] Ober | Mineral commodity summaries[END_REF] affordable oil, but affordable P, making the world a P-based economy instead of oil-based [START_REF] Gilbert | Environment: the disappearing nutrient[END_REF]. As described earlier, phosphate rock production has increased steadily since the beginning of the green revolution, and is now more than 5 times what it was in 1960 (Figure 3). Indeed, 80 to 90% of the phosphate rock extracted is used to produce P fertilizers [START_REF] Heifer | The 82nd IFA Annual Conference[END_REF][START_REF] Koppelaar | Assessing phosphate rock depletion and phosphorus recycling options[END_REF][START_REF] Prud'homme | challenges and opportunities for the fertilizer industry: fertilizers and agricultural production[END_REF][START_REF] Smil | Phosphorus: global transfers[END_REF], making fertilizer demand the main component for the increase in P rock extraction. Minor uses for P-rock are feed additives (7%), food additives (2%), detergents (5%) and industrial applications such as lightning and electronics [START_REF] Scholz | Sustainable phosphorus management: a transdisciplinary challenge[END_REF]. In all these minors applications, P could actually be substituted with other compounds [START_REF] Scholz | Sustainable phosphorus management: a transdisciplinary challenge[END_REF]. Indeed, the use of zeolite in laundry detergent has already decreased P consumption by half in that domain [START_REF] Shinh | The outlook for industrial & food phosphates[END_REF]. The slight recess in P rock production observed in 1990 corresponds to the fall of the USSR. Since then, most of the P-fertilizer demand comes from China and the developing countries in Latin America (Figure 4), while the western world has reduced its use after 50 years of over application. Future demand for P-fertilizer depends on several factors: population growth, changes in food diet, evolution of agri-systems (intensive/extensive, specialized or mixed crop-livestock agriculture), trade fluxes (globalization vs. regionalization of the economy), residual-P content in soils, availability of fertilizers and whether or not active measures are taken to reduce the waste and recycle P. Since the beginning of the millennium, available phosphate rock (after beneficiation) has increased from 19.8 MT-P in 2000to 20.5 MT-P in 2005, 22.8 in 2009and 25.2 in 2016[START_REF] Heffer | Fertilizer Outlook 2016-2020[END_REF].

Until 2050, population is expected to grow by 30% (medium scenario) to reach 9.2 billion (FAO, 2013), requiring agricultural production to grow by 60-110% with crop demand increasing by 100-110% [START_REF] Alexandratos | World agriculture towards 2030/2050: the 2012 revision[END_REF][START_REF] Tilman | Global food demand and the sustainable intensification of agriculture[END_REF]. Such demand might not be met due to underachieving gains in crop yields, well below what would be necessary [START_REF] Ray | Yield Trends Are Insufficient to Double Global Crop Production by 2050[END_REF]. In any case, in order to sustain this population growth and associated need for larger food production, Pfertilizer consumption is expected to grow steadily until 2050. [START_REF] Koppelaar | Assessing phosphate rock depletion and phosphorus recycling options[END_REF]

MITIGATING MEASURES ENVISIONED TO CLOSE THE PHOSPHORUS CYCLE

As described above, P loss to the environment is both a threat to ecological systems (and humans when toxins are produced) through eutrophication of freshwaters and to food safety via a depleting stock. Closing the P cycle would mitigate both adverse consequences of P wasted to the environment.

In order to do so, it is necessary to identify the mains flows of P and where they could be either reduced or re-directed toward safe crop fertilization. Replacing P with equally efficient compounds in detergents and other industrial use would have a significant impact on overall P consumption and this process is already underway in the European Union and the US [START_REF] Scholz | Sustainable phosphorus management: a transdisciplinary challenge[END_REF]. However, as discussed above, the overwhelming majority of P is used in agriculture through P fertilizer and spreading of manure. As a result the main levers to improve P efficiency and reduce the amount of P extracted from the earth are to be found in the domain of agriculture. Similarly, within the P cycle, human excreta and landfilled matter represent respectively 2-5.7 and 2.5-3.8 MT-P/year [START_REF] Reijnders | Phosphorus resources, their depletion and conservation, a review[END_REF][START_REF] Scholz | Sustainable phosphorus management: a transdisciplinary challenge[END_REF]. In comparison P fertilizer and manure represent 14.2-20.4 and 5-28.2 MT-P/year respectively [START_REF] Liu | Global Phosphorus Flows and Environmental Impacts from a Consumption Perspective[END_REF]MacDonald et al., 2011;[START_REF] Sheldrick | Contribution of livestock excreta to nutrient balances[END_REF]. These are both the main flows of phosphorus within the system and the less efficient. Annually, 10-13.7 MT-P/year are lost from arable land into freshwaters while only 12-13 MT-P/year are taken up by crops [START_REF] Koppelaar | Assessing phosphate rock depletion and phosphorus recycling options[END_REF][START_REF] Scholz | Sustainable phosphorus management: a transdisciplinary challenge[END_REF]. Several explanations exist for this inefficient use of P. Firstly, the global amount of P mineral fertilizer used worldwide does not reflect the major disparities in application rates among countries and even within a country. Indeed, 30% of worldwide cropland is facing a P-deficit (Africa and South America) while 12.8% of arable land (located in western Europe, USA and East China) receives more than 50% of P fertilization (MacDonald et al., 2011;[START_REF] Potter | Characterizing the Spatial Patterns of Global Fertilizer Application and Manure Production[END_REF]. Excess fertilization results from the increase of livestock production, specialization and intensification of animal breeding and spatial disconnection between livestock and crops [START_REF] Reijnders | Phosphorus resources, their depletion and conservation, a review[END_REF]. The disparities at global scale are also found at domestic scale. Indeed, areas specialized in intensive livestock production often cannot find arable land within an (economically)

reasonable radius to apply their manure due to high transport costs for a low fertilizing content [START_REF] Kellogg | Manure Nutrients Relative to The Capacity Of Cropland And Pastureland To Assimilate Nutrients: Spatial and Temporal Trends for the United States[END_REF]Schipanski and Bennett, 2012). [START_REF] Bateman | Closing the phosphorus loop in England: The spatio-temporal balance of phosphorus capture from manure versus crop demand for fertiliser[END_REF] described the case of England, in which despite an overall soil P deficit and excess manure, full reuse is not achieved due to physical distance and time disconnection between crop need and manure production. A similar situation occurs in France too, with regions specialized in crops such as cereals and other focused on intensive animal breeding. The result is a high heterogeneity in soil P balance, which generates the need for imported mineral fertilizer and reduces the recycling of P from manure to crops (Senthilkumar et al., 2012). A local example would be Brittany, where swine producers struggle to find outputs for their manure, giving it for free or converting it to economically non-competitive organic fertilizers such as compost.

Part of the solution would be to come back to integrated crop-livestock systems with sufficient cropland available, where manure reuse enables high P efficiency [START_REF] Russelle | Reconsidering Integrated Crop-Livestock Systems in North America[END_REF]. This would require agricultural policy changes at national and continental level. At the technological level, a promising possibility would be to recover the P contained in animal manure under an easily transportable form, adapted to the needs of crop producers, and cheap enough to compete with mineral P fertilizers. This would reduce the over application of manure in intensive breeding areas, limit runoff and leaching to freshwater and diminish the need for imported mineral fertilizers, thus slowing down the depletion of P reserves.

Scientific research on P treatment from industrial, domestic and livestock wastewaters has long been focused on its removal in order to discharge clean water and limit eutrophication. However a major shift toward P recovery instead of mere removal occurred in the last 20 years. In the next sections, those technologies are investigated.

III. PROCESSES FOR PHOSPHORUS RECOVERY

As discussed in the previous section, recent concerns about P resource depletion have led the scientific community institutional and industrial stakeholders to shift their aim from phosphorus removal for clean water discharge to P-recovery under reusable forms for agriculture. A wide range of feedstock is suitable for P-recovery, with domestic wastewater and animal manure as the most abundant effluents and most adapted for P-recovery. Several industrial wastewater types with high P content also make for worthy sources to recover P from. Struvite (magnesium ammonium phosphate hexahydrate) is overwhelmingly the main product targeted, followed by calcium phosphate and K-struvite (in which magnesium is replaced by potassium). In this literature review, the agronomic value of struvite is examined, before describing the existing P-recovery processes for struvite as well as other P-based mineral fertilizers. This review highlights the need for phosphorus dissolution in pig slurry in order to achieve a significant recovery rate. Because the strategic option for P dissolution developed in this PhD is a biological acidification under anaerobic conditions using sucrose and organic waste as cosubstrate, the final section of this review focuses on the metabolic processes involved in acidogenesis and particularly lactic acid fermentation.

AGRONOMIC VALUE OF STRUVITE

Struvite precipitation from waste streams requires very specific conditions that can be both difficult to attain and financially expensive [START_REF] Hao | Looking beyond struvite for P-recovery[END_REF]. As a result, the added value of struvite has to be high enough in term of practical use, safety for the environment and agronomic value in order to justify its production.

So far, some advantages have been observed when struvite was applied compared to conventional fertilizers. The main quality of struvite is its low solubility leading to slow assimilation in arable soil.

As a result, nitrogen leaching is severely reduced compared to conventional fertilizers (Rahman et al., 2011). Struvite is particularly suited for plants grown in pots since a lot of water is drained in this situation and a slow release fertilizer prevents nutrients runoffs [START_REF] Antonini | Greenhouse evaluation and environmental impact assessment of different urine-derived struvite fertilizers as phosphorus sources for plants[END_REF]. Another advantage of the slow nutrient release provided by struvite is to prevent the chemical burning of crop when used in a single, large application [START_REF] Fattah | Development of control strategies for the operation of a struvite crystallization process[END_REF]. Magnesium present in struvite also improves P uptake compared to conventional P fertilizer thanks to a synergistic effect on P absorption by the plant [START_REF] Li | Recovery of ammonium-nitrogen from landfill leachate as a multi-nutrient fertilizer[END_REF]. Struvite effect on crop yield depends on the type of soil and nutrient content.

Thus, better fertilizing effects were observed compared to chemical fertilizers in slightly acid soils [START_REF] Barak | Struvite: A recovered and recycled phosphorus fertilizer[END_REF][START_REF] Ghosh | Characterization of soil-fertilizer P reaction products and their evaluation as sources of P for gram (Cicer arietinum L.)[END_REF]Kataki et al., 2016), as well as P depleted soils [START_REF] Vogel | Phosphorus application with recycled products from municipal waste water to different crop species[END_REF].

Many studies found at least similar performances compared to conventional P fertilizers (Achat et al., 2014a;Achat et al., 2014b;[START_REF] Gell | Safety and effectiveness of struvite from black water and urine as a phosphorus fertilizer[END_REF][START_REF] Li | Recovery of ammonium-nitrogen from landfill leachate as a multi-nutrient fertilizer[END_REF]Liu et al., 2011;[START_REF] Pérez | Plant availability of Pfertilizers recycled from sewage sludge and meat-and-bone meal in field and pot experiments[END_REF][START_REF] Plaza | Greenhouse evaluation of struvite and sludges from municipal wastewater treatment works as phosphorus sources for plants[END_REF][START_REF] Thompson | Field evaluation of the availability for corn and soybean of phosphorus recovered as struvite from corn fiber processing for bioenergy[END_REF] yet sometimes observed slightly lower yields [START_REF] Ackerman | Evaluation of manure-derived struvite as a phosphorus source for canola[END_REF][START_REF] Ganrot | Plant availability of nutrients recovered as solids from human urine tested in climate chamber on Triticum aestivum L[END_REF]. These lower performances seemed to be related to the alkaline pH of the soil, a condition that reduces the availability of struvite's nutrients compared to conventional fertilizers [START_REF] Ackerman | Evaluation of manure-derived struvite as a phosphorus source for canola[END_REF]. A potential long term issue with struvite application is the potential accumulation of magnesium, noted by [START_REF] Gell | Safety and effectiveness of struvite from black water and urine as a phosphorus fertilizer[END_REF]. Indeed, if natural variations of the Mg:Ca ratio don't affect crop yields [START_REF] Schulte | Soil Calcium to Magnesium Ratios--should You be Concerned?[END_REF], an artificially large ratio induced by struvite application would change soil conductivity and aggregates stability, negatively impacting crop yields [START_REF] Zhang | Effect of exchangeable Mg on saturated hydraulic conductivity, disaggregation and clay dispersion of disturbed soils[END_REF]. Besides, excess magnesium also reduce Ca uptake by certain plants, which could lead to calcium deficiency [START_REF] Stevens | Soil calcium: magnesium ratios and lime recommendations for cotton[END_REF]. Finally, the low N:P ratio of struvite (1:1) is not perfectly suited for plant growth, which requires usually an N:P:K ratio of 4.5:1:0.94.

Consequently, struvite should be used in P depleted soil or in combination with other fertilizers to adjust the NPK ratio applied to the crop.

Beyond its intrinsic characteristics, struvite recovered from waste needs to meet certain physical and chemical criteria in order to find a market. Thus, high crushing strength is absolutely required in order to ensure durability and prevent the loss of fine material during manufacturing, transport and field application. Highest strength was found in crystals of 2-2.5 mm [START_REF] Fattah | Development of control strategies for the operation of a struvite crystallization process[END_REF], while [START_REF] Forrest | Optimizing struvite production for phosphate recovery in WWTP[END_REF] advised for larger crystals (3-5mm) with sufficient strength as demonstrated by [START_REF] Adnan | Pilot-scale study of phosphorus recovery through struvite crystallization examining the process feasibility[END_REF]. When the crystals are too small and too brittle, only low economic returns can be expected [START_REF] Forrest | Optimizing struvite production for phosphate recovery in WWTP[END_REF]. Another critical aspect factoring into marketability of struvite recycled from waste is its content in heavy metals (HM) and pathogens. There is still a lack of information regarding the transfer of HM to the precipitate based on the waste stream used, yet most some studies indicate that the HM content of struvite was well within the European norm for fertilizers [START_REF] Gell | Safety and effectiveness of struvite from black water and urine as a phosphorus fertilizer[END_REF] and below the HM values found in phosphate rock [START_REF] Wollmann | Assessment of Alternative Phosphorus Fertilizers for Organic Farming: Sewage Precipitation Products[END_REF]. Pathogens have been found in struvite recovered from human urine, but improvement in the drying process (temperature, air moisture content) could mitigate this transfer [START_REF] Decrey | Fate of the pathogen indicators phage ΦX174 and Ascaris suum eggs during the production of struvite fertilizer from source-separated urine[END_REF].

From this review it appears that a strong emphasis should be put on improving struvite crystallization process to meet the physical characteristics required by the fertilizer/agricultural sector. The low HM content compared to traditional chemical fertilizer should help in commercializing struvite recovered from organic waste. Further studies are needed to determine the best climate and edaphic conditions to maximize struvite efficiency (Kataki et al., 2016). Even though recovered struvite can be sold directly to large fertilizer manufacturers and incorporated in their process [START_REF] El Rafie | Evaluation of struvite precipitated from chemical fertilizer industrial effluents[END_REF], the buying price would be low if the final product is not marketed as environment-friendly. Due to its relatively high cost of production compared to conventional P fertilizer from P rock (at least in the short term) , the most likely outputs for struvite will remain high value added crops such as golf turf, tree seedlings, ornamentals, vegetables and flower boards [START_REF] Munch | Controlled struvite crystallisation for removing phosphorus from anaerobic digester sidestreams[END_REF].

EXISTING PROCESSES FOR PHOSPHORUS RECOVERY

Phosphorus recovery technologies for municipal effluents and to a lesser degree industrial wastewaters have been largely developed since the 90's, mostly as a mean to save money on maintenance, dewaterability and disposal costs of sludge. Meanwhile, recycling of P from livestock waste streams remains at laboratory and pilot scale. Before delving into the variety of stream sources and technologies used to recover P, a common aspect among all those processes is the precipitation of phosphate ions into low soluble salts that can be recovered. The most common precipitate targeted is struvite, formed according to Equation 1. In the case of calcium phosphate, different forms are possible: hydroxyapatite (HAP), hydroxydicalciumphoshate (HDP) (Equation 2), brushite or amorphous calcium phosphate. Usually Mg 2+ and Ca 2+ are the limiting ions for full P precipitation and are added as magnesium chloride and calcium chloride. Because precipitation only takes place at pH above 8 for struvite and above 9 for calcium phosphate, caustic soda (NaOH) or alkaline salts can be used to increase pH. Magnesium oxide (MgO), magnesium hydroxide (Mg(OH) 2 ) or their calcium equivalents enable both pH and stoichiometric adjustment. Carbonate stripping through air bubbling is also a common technic used to elevate the pH. -Sludge ashes [START_REF]Pyruvate formation from glucose[END_REF] These processes are able to recover phosphorus initially present in the liquid phase, in the sludge or in sludge ashes.

Recovery from treated effluents

Only one technology exists to recover P from treated effluents of a municipal WWTP. The REM-NUT process uses two ion exchange units to remove phosphate, ammonium and potassium from low concentration effluents (PO 4 -P = 5-10 mg/L) [START_REF] Liberti | REM NUT Ion Exchange Plus Struvite Precipitation Process[END_REF]. Sodium chloride (NaCl) as brine from seawater is used to regenerate the ionic exchangers. pH is raised to 9.5 caustic soda and MgCl 2 is added to form struvite and K-struvite. Up to 90% of influent P can be recovered using this process, yet low selectivity from the ion exchange unit for P leads to the adsorption of undesirable compounds, frequent regeneration of the exchanger (as often as every 3 hours [START_REF] Brett | Phosphorus removal and recovery technologies[END_REF]) and lower recovery (50-70%). This selectivity problem and its consequences severely impact the economic sustainability of the process. Logically this technology has been evaluated as the most expensive compared to the amount of P recovered (Egle et al., 2016).

Recovery from the liquid phase of anaerobic digestion

Most technologies for P recovery are designed to precipitate the dissolved P from the supernatant of anaerobic digestion (Desmidt et al., 2015) (stream 4 in Figure 5). These are the simplest and cheapest processes since they focus only on the dissolved phosphate of the liquid phase, but the recovery rate is usually very low, in the 10-25% range compared to P in WWTP influent (Egle et al., 2015;[START_REF] Melia | Trends in the recovery of phosphorus in bioavailable forms from wastewater[END_REF]. Indeed, most of the phosphorus is located in the sludge after anaerobic digestion [START_REF] Pastor | Sewage sludge management for phosphorus recovery as struvite in EBPR wastewater treatment plants[END_REF]. Beside the low cost of these technologies, the main interest is to reduce the amount of P returned to the head of the treatment plant and thus reduce incrustation and scaling in pipes. Perhaps because the finality of these processes is not to reach a high recovery rate of incoming phosphorus to the WWTP, the scientific literature offers very little data on this parameter while describing extensively the proportion of phosphate (not total-P) recovered (Abma et al., 2010;Cullen et al., 2013;[START_REF] Moerman | Phosphate removal in agro-industry: Pilot-and full-scale operational considerations of struvite crystallization[END_REF]. Several versions of this type of process have been commercialized, some of which possess relevant improvements. Four examples are given below. The first describes the very basic technology for P recovery from digester supernatant. In the second one, a similarly simple process is applied to industrial wastewater. In the third example, the crystallization step is placed before centrifugation of the digestate, improving massively the dewaterability of the sludge. The fourth and final example presents a dual process recovering P from both digester supernatant and undigested EBPR sludge, improving the recovery rate significantly and reducing incrustation/scaling.

• Basic recovery from anaerobic supernatant

The oldest phosphorus recovery process on full scale is the Phosnix technology developed by Unitika in Japan in 1987 [START_REF] Munch | Controlled struvite crystallisation for removing phosphorus from anaerobic digester sidestreams[END_REF]. The supernatant from anaerobic digestion containing 110mg of dissolved P per liter is fed at the bottom of a fluidized bed reactor. The bed contains fine particles of struvite acting as seed material on which newly formed crystals can grow. Mg(OH) 2 and NaOH are added to reach a stoichiometric ratio of Mg:P and increase the pH to 8.2-8.8 respectively. Air stripping provides the necessary mixing to maintain the particles in suspension. When the crystal size reaches 0.5-1mm, the pellets are removed from the reactor while the liquid containing too fine crystals is returned to the reactor. The pellets are stored until the water content is less than 10% before being transported to a processing plant and used as a raw material to produce a fertilizer equilibrated in N, P and K [START_REF] Ueno | Three Years Experience of Operating and Selling Recovered Struvite from Full-Scale Plant[END_REF]. During three year study, Ueno could demonstrate that 90% of dissolved P entering the fluidized bed reactor was effectively recovered, leading to a 50% P-recovery at the scale of the WWTP. This high recovery value is unusual for such a process. Indeed, on the schematic displayed by Ueno, 55% of TP is dissolved in the digester supernatant, a surprisingly high value, compared to other digesters, e.g. <10% [START_REF] Wild | Prediction of recycle phosphorus loads from anaerobic digestion[END_REF], 15% [START_REF] Marti | Struvite precipitation assessment in anaerobic digestion processes[END_REF], 19% [START_REF] Pastor | Sewage sludge management for phosphorus recovery as struvite in EBPR wastewater treatment plants[END_REF], 20% [START_REF] Martí | Sludge management modeling to enhance P-recovery as struvite in wastewater treatment plants[END_REF], 23% (Cullen et al., 2013).

• Struvite recovery from potato processing and dairy effluents

The NuReSys® process is a completely stirred tank reactor (CSTR) with prior CO 2 stripping treating anaerobic effluent from an upflow anaerobic sludge reactor (UASB) [START_REF] Moerman | Phosphate removal in agro-industry: Pilot-and full-scale operational considerations of struvite crystallization[END_REF]. Sodium hydroxide and magnesium chloride are used to adjust pH to 8.5-8.7 and Mg/P ratio to 1-1,2. At a full scale dairy plant, no struvite precipitation occurred initially despite a 70% phosphate removal. The high calcium concentration compared to phosphate (140 vs 42 mg/L) caused the concurrent precipitation of an amorphous solid containing Ca, P and Mg. When the P content of the UASB effluent increased, calcium phosphate precipitation ceased, replaced by 100% struvite crystals of 2-6mm, with 78% of TP removed. The same process was tested at pilot scale on a UASB effluent of a potato processing facility, reaching up to 88% TP (117 mg-TP/L and 15 mg/L in the influent and effluent). Very similar results (76% TP removal) were obtained on potato processing anaerobic effluents with the PHOSPAQ technology, a similar process using a CSTR reactor and integrated air bubbling for CO 2 stripping (Remy et al., 2013). Therefore, struvite recovery from anaerobic effluent appears particularly suited to industrial effluents with high dissolved P and low particulate P.

However, effluents with interfering ions, such as calcium in the case of dairy effluents might not the most suited effluent for P recovery as struvite.

• Precipitation of struvite prior to sludge dewatering: the AirPrex™ process At the WWTP of Waßmannsdorf, Berlin (Germany), severe scaling was noticed in the centrifuge dewatering digested sludge as well as in the pipes leading to it and exiting it [START_REF] Heinzmann | Induced Magnesium Ammonia Phosphate Precipitation to Prevent Incrustations and Measures for Phosphorus Recovery[END_REF]. This resulted in operational disturbances and frequent interruptions (mechanical cleaning every two weeks). Uncontrolled precipitation of struvite occurred due natural CO 2 degassing once the anaerobic sludge had exited the digester. Indeed, CO 2 release increases the pH, lowering the solubility product of struvite. In order to reduce the incrustation problem, a controlled precipitation reactor was installed prior to sludge dewatering (Figure 6). MgCl 2 provides the necessary Mg 2+ ions for full precipitation of phosphorus. CO 2 is stripped on purpose through fine bubble aeration, raising the pH to 8 and creating an internal recirculating flow, allowing the struvite crystals to grow. The crystals are removed at the bottom of the air lift reactor while sludge flocs, lighter, are washed out toward the dewatering unit. 90-95% of phosphates are precipitated (Kataki et al., 2016). Struvite solids go through a sand washer for cleaning and purification, leading to low organic contamination. The product is marketed as a mineral fertilizer called "Berliner Pflanze", sold since 2008 within the Berlin and Brandenburg region in retail stores or shipped to farmers/traders. The fertilizer contain a N:P 2 O 5 :MgO ratio of 7%:21%:8%. The P recovery rate corresponds to 18% of total P entering the WWTP, while saving 474 000 € per year. Commercialization of the fertilizer represents only 10% of the savings, reduced maintenance 15% (less incrustation) and better dewaterability / lower disposal cost 75%. Five Airprex™ units have been built so far, in Germany and in the Netherlands. • Enhanced recovery from anaerobic supernatant of digested EBPR sludge When Bio-P is used on the mainstream line of the WWTP, phosphates are released by the PAO during anaerobic digestion but re-precipitate immediately [START_REF] Doyle | Struvite formation, control and recovery[END_REF][START_REF] Marti | Struvite precipitation assessment in anaerobic digestion processes[END_REF]. As a result, this uncontrolled precipitation leads to higher maintenance costs to remove the scaling, decreased dewaterability and lower P available for recovery (Cullen et al., 2013). [START_REF] Martí | Sludge management modeling to enhance P-recovery as struvite in wastewater treatment plants[END_REF] showed that dissolved P concentration decreased during anaerobic digestion due to the release of poly-P by PAO and re-precipitation, leading to only 9% of TP entering the WWTP available for recovery.

The Wasstrip® process (Figure 7) was developed to solve this problem by diverting a large fraction of the P flow from the digester to the crystallization stage (Cullen 2013). The struvite reactor (Pearl®) is a fluidized bed treating anaerobic supernatant of digested domestic EBPR sludge. Developed by the company Ostara, it was first installed in Durham, Oregon (USA) in 2009. In 2011 the aerated holding tank for EBPR sludge prior to digestion had its aeration turned off and volatile fatty acids (VFA) were added from the anaerobic primary sludge thickener. Under anaerobic conditions and in presence of VFAs, PAO release their stored phosphates and magnesium while taking up VFAs. Because ammonium concentration is still low compared to the digester where mineralization of organic matter leads to high ammonium concentration, no struvite precipitation takes place in the holding tank. The liquid phase, rich in P and Mg is sent to the struvite reactor while the impoverished sludge is sent to digestion. The anaerobic centrate from digestion, rich in ammonium, is then sent to the struvite reactor. The fluidized bed for struvite crystallization has several compartments of decreasing sizes from top to bottom, where MgCl 2 is added. This allows a good segregation of crystals, with the largest at the bottom and smaller particles higher the reactor where they have longer contact time with the liquid and more surface area. Besides, the high flow velocity at the bottoms leads to the wash out of remaining sludge solids that were not retained during prior centrifugation. Consequently, the struvite obtained as a low contamination in terms of organic content and pathogens. The product obtained is marketed as a mineral fertilizer "Crystal Green®" with an NPK content of 5:28:0 and 10% Mg [START_REF] Britton | P-Recovery in North America-Ostaras Pearl Process[END_REF]. The P flow from the holding tanks represented 70% of all incoming P to the struvite crystallizer during a three year study, leading to a 60% increase in struvite production (Cullen et al., 2013) and an improved dewaterability of digested sludge [START_REF] Britton | Industry Update for WASSTRIP® impacts on nutrient flows, digestion and dewatering of EBPR solids[END_REF]. However, postulating a yet it also contains significant quantities of heavy metals [START_REF] Ignatowicz | The impact of sewage sludge treatment on the content of selected heavy metals and their fractions[END_REF][START_REF] Lake | Fractionation, characterization, and speciation of heavy metals in sewage sludge and sludge-amended soils: a review[END_REF], persistent organic pollutants (POP) [START_REF] Jelic | Occurrence, partition and removal of pharmaceuticals in sewage water and sludge during wastewater treatment[END_REF] such as pharmaceutical compounds and endocrine disruptors pesticides [START_REF] Stamatis | Occurrence and removal of fungicides in municipal sewage treatment plant[END_REF] and pathogens [START_REF] Sahlström | Bacterial pathogen incidences in sludge from Swedish sewage treatment plants[END_REF]. Presence of these harmful compounds as well as uncertainties regarding plant availability of P [START_REF] Kahiluoto | Phosphorus in Manure and Sewage Sludge More Recyclable than in Soluble Inorganic Fertilizer[END_REF][START_REF] Kidd | Bioavailability and plant accumulation of heavy metals and phosphorus in agricultural soils amended by long-term application of sewage sludge[END_REF][START_REF] Krogstad | Influence of chemically and biologically stabilized sewage sludge on plant-available phosphorous in soil[END_REF] make municipal digested sludge application in agricultural fields heavily regulated and suspicious by citizens of European countries [START_REF] Ott | The European phosphorus balance[END_REF]. As described above, phosphorus in municipal digested sludge is mostly in the solid phase, making its extraction a necessary step if high recovery rate is desired. The existing processes for P recovery from municipal sludge rely on energy and/or chemical intensive technologies associated with challenging post treatments for residual by products.

Pre-treatments of municipal sludge

• Thermal pre-treatments of municipal sludge Wasted activated sludge (WAS) from WWTP using chemical precipitation for P is not impacted by thermal treatment. Indeed, Phosphorus is mostly present as an insoluble salt, e.g. FePO 4 and cannot be solubilized when treated at high temperature [START_REF] Appels | Influence of low temperature thermal pre-treatment on sludge solubilisation, heavy metal release and anaerobic digestion[END_REF]. However, most of the P is released during thermal treatment of EBPR sludge, at temperature and duration as low as 70°C and one hour [START_REF] Takiguchi | Pilot plant tests on the novel process for phosphorus recovery from municipal wastewater[END_REF][START_REF] Xue | Releasing characteristics of phosphorus and other substances during thermal treatment of excess sludge[END_REF]. Thermal hydrolysis is always performed on thickened WAS and primary sludge, primarily to enhance sludge dewaterability and methane production during subsequent anaerobic digestion [START_REF] Graja | Reduction of sludge production from WWTP using thermal pretreatment and enhanced anaerobic methanisation[END_REF][START_REF] Hii | A review of wet air oxidation and Thermal Hydrolysis technologies in sludge treatment[END_REF][START_REF] Kepp | Enhanced stabilisation of sewage sludge through thermal hydrolysis -three years of experience with full scale plant[END_REF][START_REF] Pickworth | Maximising biogas in anaerobic digestion by using engine waste heat for thermal hydrolysis pre-treatment of sludge[END_REF]. While moderate temperatures (70°C) can be used as pasteurization/hydrolysis treatment of WAS, most commercial technologies implemented on full scale are 30 minutes high-pressure thermal hydrolysis processes (HPTH) with temperature between 130 to 180 C and pressure in the 6-12 bar range [START_REF] Morgan-Sagastume | Production of volatile fatty acids by fermentation of waste activated sludge pre-treated in full-scale thermal hydrolysis plants[END_REF]. HPTH is an efficient pre-treatment that has been enabled a near doubling in destruction of volatile content of waste activated sludge by anaerobic digestion [START_REF] Norli | The cambi thermal hydrolysis process (THP)-dramatically improving sludge digestion and dewatering-11 years experience of full scale project[END_REF]. The increase in methane production could potentially compensate the energy necessary to produce the steam [START_REF] Kepp | Enhanced stabilisation of sewage sludge through thermal hydrolysis -three years of experience with full scale plant[END_REF]. HPTH treatment of EBPR sludge combined with the Airprex process is currently under development in Germany (Egle et al., 2015). Laboratory studies have shown that thermal pre-treatments, by hydrolyzing organic matter into VFAs, can lower the pH and prevent re-precipitation of P. [START_REF] Yu | Simultaneous phosphorus and nitrogen recovery from anaerobically digested sludge using a hybrid system coupling hydrothermal pretreatment with MAP precipitation[END_REF] obtained an increase in dissolved P from 370 to 580 mg/L, or a 25% increase relatively to TP, at 220°C during 60 minutes.

• Wet chemical treatments Contrary to thermal pre-treatment, wet chemical treatment is used on digested sludge where complex organic matter has already been reduced. One full scale wet chemical treatment technology for P recovery and two pilot scale units using different processes have been implemented so far.

The Seaborne process has been built on full scale in Gifhorn, Germany. The digested sludge is acidified with sulfuric acid, down to an unknown pH, leading to the dissolution of phosphorus and part of the HMs (Figure 8). After centrifugation the solids are incinerated. Dissolved HM are removed from the supernatant thanks to the hydrogen sulfide contained in the biogas from the digester, thereby purifying it to be converted into heat. The HM-free liquid can then undergo P recovery using magnesium hydroxide and sodium hydroxide in two consecutive steps with pH adjusted to 9. Severe problems were encountered regarding the removal of precipitated HM, due to colloid formation passing through the filter and interfering with the subsequent P recovery step [START_REF] Müller | Nutrient recycling from sewage sludge using the Seaborne process[END_REF]. The process was modified: now, immediately after acidification, iron and other interfering ions are precipitated by adding sodium hydroxide and sodium sulfide to reach a pH of 5.7, and removed with the solids during centrifugation (Egle et al., 2015). Depending on the pH targeted during acidification with sulfuric acid, 30 to 90% of TP can be dissolved. P recovery rate is not described but Egle et al.

(2015) estimated it at 50% of incoming TP to the WWTP. In the Stuttgart process pilot installed at a WWTP in southwest Germany, the digested sludge is acidified using sulfuric acid down to pH 4 and centrifuged (Figure 9). Contrary to the Seaborne process, the dissolved metals are removed using citric acid as chelating agent [START_REF] Antakyali | Large-scale application of nutrient recovery from digested sludge as struvite[END_REF].

Magnesium oxide is added first to the supernatant in a batch reactor to increase magnesium concentration and sodium hydroxide is used to adjust the pH to 8.5. When the targeted pH is reached, the liquid is transferred to a conic reactor to allow sufficient retention time for crystal growth. The liquid is recirculated from top to bottom for mixing. The final effluent after struvite precipitation is returned to the mainstream treatment line of the WWTP. 700-800 mg-P/L were obtained from the acid dissolution of digested sludge, corresponding to 75% of TP in the sludge. 98% of the dissolved P was subsequently precipitated, leaving only 5-10 mg-P/L in the effluent. Even though the pilot did not treat all the digested sludge produced at the WWTP, it could recover approximately 35-45% of incoming P to the plant, two to three times as more than processes focusing on dissolved P in digested supernatant.

However, the effluent returned to the mainline lead to accumulation of heavy metals in the sewage sludge and the P precipitate obtained had high contamination with Al, Fe and Ca. The chemical costs of the Stuttgart pilot process (construction and energy costs not taken into account) amounted to 2-3 € per Kg of struvite produced, or 15.8-23.7 € per Kg P. These costs are far above any potential selling price for struvite fertilizer but once again the main interest of P recovery in municipal WWTP is to reduce the costs of maintenance and sludge disposal.

Interestingly, large variations of dissolved P occurred during acidification even at identical pH (e.g.

72% and 52% TP at pH 4.2). [START_REF] Antakyali | Large-scale application of nutrient recovery from digested sludge as struvite[END_REF] hypothesized that it could be due to differing quantity and type of phosphorus compound. Such phenomenon also occurred during the work undertaken in this PhD thesis, and similar hypothesis were made. The final wet chemical technology for P recovery, currently at pilot scale, is the Budenheim process (Figure 10). Contrary to the Seaborne and Stuttgart process, no sulfuric acid is used. Instead, CO 2 gas is applied at a flow rate of 400 L/h at a pressure of 10 bar during 30 minutes, lowering the pH down to 4-5. The pH range represents a compromise between P recovery rate and dissolution of interfering ions and HMs. At pH 5, dissolution of P is not maximal but the HM remain in the sludge phase. The process enables 50% dissolution of TP without any need for chemical addition. 90% of dissolved P is then recovered as di-calcium phosphate by adding lime (CaOH).

As it could be expected, P recovery from municipal sludge using wet chemical treatment leads to significantly higher recovery rate. However, a balance has to be found between maximum P recovery and minimization of issues arising from the byproducts. Acidification below pH 4 led to foaming problems, the acidified sludge needs additional treatment (corrosion induced by high sulfur content), return of HMs to the mainstream treatment line and contamination of the product with iron. 3) destruction of pathogens and sterilization of material. One of the advantages of wet air oxidation is that dewatering of sludge is no longer necessary [START_REF] Baroutian | Formation and degradation of valuable intermediate products during wet oxidation of municipal sludge[END_REF][START_REF] Bernardi | Performances of soluble metallic salts in the catalytic wet air oxidation of sewage sludge[END_REF].

The level of solubilization and oxidation of organic matter increase with the amount of oxygen added, temperature and pressure [START_REF] Chung | Effects of operational conditions on sludge degradation and organic acids formation in low-critical wet air oxidation[END_REF]. Under moderate conditions, wet air oxidation leads mostly to the formation of dissolved volatile fatty acids [START_REF] Suárez-Iglesias | Valuable compounds from sewage sludge by thermal hydrolysis and wet oxidation. A review[END_REF]. No dissolution of P occurs when chemical P sludge is used since iron ions re-precipitate concomitantly with phosphates to form FePO 4 [START_REF] Niewersch | Nanofiltration for Phosphorus Recycling from Sewage Sludge[END_REF].

The Phoxnan technology avoids the precipitation of FePO SCWO functions at pressure and temperature above 220 bar and 375°C, conditions in which water is above its critical point. The properties of water change regarding density, viscosity, diffusivity and solvating ability (water becomes a non-polar solvent). In this state where gas and liquid cannot be distinguished, mass transfer resistance disappears, favoring faster reactions [START_REF] Goto | Supercritical water oxidation for the destruction of municipal excess sludge and alcohol distillery wastewater of molasses[END_REF][START_REF] Svanström | Environmental assessment of supercritical water oxidation of sewage sludge[END_REF]. When dioxygen is added, the organic matter is completely oxidized within a few minutes leaving only inorganic ashes and a clean liquid phase. Phosphorus needs to be extracted from the ashes using acid or alkaline treatment. 80-100% of TP was dissolved using acid leaching but iron was also leached, making FePO 4 the only recoverable product [START_REF] Levlin | Phosphorus recovery from sludge incineration ash and Supercritical Water Oxidation residues with use of acids and bases[END_REF]. Alkaline leaching enables the selective precipitation of phosphorus but initial dissolution is significantly lower.

Consecutive acid and alkaline leaching was tested, leading to an increase in P dissolution compared to alkaline only treatment (+23%, reaching 53% of TP). The cost of chemicals remained acceptable (1.2 €/Kg-P) but did not include the costs for subsequent precipitation [START_REF] Levlin | Phosphorus recovery with acid and base from inorganic sewage sludge residues[END_REF]. Two processes have already been tested at pilot scale, yet no upscaling has apparently been planned (Egle et al., 2015).

• P-recovery from sewage sludge ash (SSA)

Due to concerns about pollutants in sewage sludge, a large fraction of it is now incinerated (100% in the Netherlands).The sludge is first dewatered to >25% dry solids content and introduced in a fluidized sand bed at 800-900 °C in presence of oxygen (Figure 11). Organic matter, including organic pollutants is oxidized to CO 2 , NOx and SOx gases. The ashes generated contain all the P initially present. Fe and Al, as well as significant quantities of HM similarly end up in the inorganic ashes [START_REF] Adam | Thermochemical treatment of sewage sludge ashes for phosphorus recovery[END_REF]. P concentration is usually around 60 g-P/Kg, within a range going from 50 to 110 g/Kg [START_REF] Donatello | Production of technical grade phosphoric acid from incinerator sewage sludge ash (ISSA)[END_REF]. The mineral form and proportion of P in SSA is actually similar to that of low grade phosphate rock (average phosphate rock contains 130 g-P/Kg). This phosphorus is mostly present as whitelockite [START_REF] Sturm | Electrokinetic phosphorus recovery from packed beds of sewage sludge ash: yield and energy demand[END_REF] a mineral insoluble in water and therefore not available to plants. As a result, further processing is necessary to remove the HMs and convert P to a water soluble form. A process that can be used is actually one of the technologies developed for fertilizer production from phosphate rock: sulfuric or phosphoric acid is added to SSA, producing single super phosphates (Ca(H 2 PO 4 ) 2 ) [START_REF] Donatello | Recycling and recovery routes for incinerated sewage sludge ash (ISSA): A review[END_REF]. The RecoPhos® process produces single super phosphates by adding phosphoric acid to SSA. The final product can be sold as a mineral fertilizer with a P content of 16.6%, demonstrating similar effect on crops compared to conventional fertilizer, a logical result since the product is very similar. The contamination with HM is low compared to phosphate rock but mostly because phosphoric addition in the process reduces the relative concentration of HM compared to P. The fate of Fe and Al is not described, but they are likely still present in the fertilizer product. This type of P extraction from SSA enables a 99% P recovery, corresponding to 85% of P entering the WWTP (Egle et al., 2015).

In order to remove the HMs from the final product, a stepwise increase in pH after dissolution enables the segregated precipitation of P and HMs [START_REF] Petzet | Towards a complete recycling of phosphorus in wastewater treatmentoptions in Germany[END_REF]. In the Sephos process, sodium hydroxide is added to reach pH 3.5, precipitating aluminum phosphate while trace metals remain in solution [START_REF] Schaum | Phosphorus recovery from sewage sludge ash-a wet chemical approach[END_REF]. Al-P can be used as a raw material by the fertilizer industry. After further alkaline treatment on the aluminum phosphate product, a re-precipitation of P with calcium is possible [START_REF] Schaum | Management of Residues Emanating from Water and Wastewater Treatment[END_REF].

Another strategy to separate trace metals from P is to use ion exchangers. [START_REF] Xu | Recovery of phosphorus as struvite from sewage sludge ash[END_REF] used hydrochloric acid to dissolve P from SSA rather than sulfuric acid alleging its poor solubilizing capacity [START_REF] Cohen | Phosphorus dissolution from ash of incinerated sewage sludge and animal carcasses using sulphuric acid[END_REF], though others found similar capacity between HCl and H 2 SO 4 [START_REF] Biswas | Leaching of phosphorus from incinerated sewage sludge ash by means of acid extraction followed by adsorption on orange waste gel[END_REF]. By applying 25mmol HCl per gram SSA during 2 hours, 95% of TP was dissolved, along with significant amounts of Cu, Pb, Zn, Cr and Ni. Then a cation exchange resin was added to the liquid phase to remove the trace metal elements. Struvite was then precipitated with the addition of MgCl 2 , NH 4 Cl and NaOH. The final product exhibited 97% struvite purity.

Finally, the AshDec® process, operated at pilot scale in Austria, is able to remove part of the HM by feeding the SSA into a rotary kiln heated up around 1000°C with the addition of calcium, potassium or magnesium chloride. The HMs combines with chloride transfer to the gaseous phase, leaving the system with the exhaust before being removed during flue gas cleaning [START_REF] Petzet | Towards a complete recycling of phosphorus in wastewater treatmentoptions in Germany[END_REF]. A removal rate of 70-90% was reached for Cd, Cu, Pb, Zn, Mo and Sn, while As, Cr and Ni concentrations remained unchanged [START_REF] Adam | Thermochemical treatment of sewage sludge ashes for phosphorus recovery[END_REF]. The product obtained depended on the chloride source: Chlorapatite (Ca 5 (PO 4 )3Cl) when CaCl 2 was used and stanfieldite (Ca 4 Mg 5 (PO 4 ) 6 )

when MgCl 2 was used [START_REF] Peplinski | Reaction sequences in the thermochemical treatment of sewage sludge ashes revealed by X-ray powder diffraction-a contribution to the European project SUSAN[END_REF]. A recent modification using sodium sulfide led to the precipitation of rhenanite (CaNaPO 4 ). technologies from digester supernatant will likely see a large development in the near future. The processes recovering P from the sludge itself or its ashes could theoretically reach much higher recovery rate compared to dissolved P technologies, yet much optimization is needed. The primary issue in this type of process is the high contamination in HM, requiring complex technology and high costs for removal. Beyond the technical and economical barrier, the environmental impact of these processes remains largely unknown, or if such assessments have been conducted, they are rarely in the public domain. One life cycle analysis was conducted on struvite recovery from human urine provided by source-separated sanitation system [START_REF] Remy | Ecological assessment of alternative sanitation concepts with Life Cycle Assessment[END_REF]. A positive balance was found compared to conventional treatment of phosphorus, yet such study should be conducted in every case using a uniformed method in order to be able to compare the processes together. This task was very recently undertaken by (Egle et al., 2016) leading to the following conclusions:

• Recovery processes for dissolved P in digested sludge obtain relatively clean products (HMs, POP, pathogens), significantly less contaminated than the original source, and with a pollution potential inferior to that of a commercial fertilizer. They are plant available and perform as well as chemical P fertilizer, depending on the soil properties and climate. Nevertheless, the recovery rate of these processes is low relatively to the P flow to the WWTP (25% maximum). • Recovery from sewage sludge requires significantly more complex technology, enabling nonetheless high removal of HMs and pathogens, with medium recovery rate (40-50%).

• Sewage sludge ash appears as the most promising source of P in municipal WWTP, with processes reaching 60-90% recovery rate. Because sludge incineration is centralized, regrouping several WWTP, economy of scale is possible. Pathogens and organic pollutants are obviously removed during the process, yet the costs associated with this type of process strongly depend on the SSA quality, i.e. HM contamination. Synergy with other industrial processes nearby is strongly advocated as a mean of cost saving.

These recommendations are quite reasonable in the context of large, semi-public WWTP situated in heavily populated areas. Beyond the lack of environmental assessments for advanced, energy and technology intensive processes, their cost and the practical implications of implementing such large units in agricultural areas make them irrelevant for the treatment of livestock effluents. Dissolution of solid P is indeed a necessity to achieve a good recovery rate but chemicals and energy intensive processes are not adapted. Existing lab scale and full scale unit for P recovery from animal waste streams rely on the same type processes, either simple technology applied to dissolved-P only or chemical/energy intensive technology to access solid P. These processes are described in the next section.

b. PHOSPHORUS RECOVERY FROM LIVESTOCK EFFLUENTS

In Europe, P flow from animal manure represents 1.5 times the amount of chemical P fertilizer applied (1.6 and 1.1 MT-P/year) [START_REF] Withers | Stewardship to tackle global phosphorus inefficiency: The case of Europe[END_REF]. In France, both flows are approximately equal (0.31 and 0.29 MT-P/year) ( (Senthilkumar et al., 2012). The P balance in soils of Europe and France is still largely in excess despite a significant decrease in the last 30 years. Indeed, 42% of P currently applied is not exported and accumulated every year (21% in France). As described previously, these mean values hide the massive spatial variations in P applied and soil P accumulation/deficit across Europe and between regions of a same country [START_REF] Csatho | Two worlds within EU27: Sharp contrasts in organic and mineral nitrogen-phosphorus use, nitrogen-phosphorus balances, and soil phosphorus status: Widening and deepening gap between Western and Central Europe[END_REF][START_REF] Gissol | L'état des sols en France[END_REF], mostly due to livestock density and associated manure application in the fields nearby. Therefore, the challenge is to recover manure P from intensive livestock regions under a form that can be transported easily and at low cost to regions with soil P deficit. Intensive pig breeding is the primary livestock production in need of manure processing [START_REF] Schoumans | Phosphorus management in Europe in a changing world[END_REF] due to its enclosed nature, without enough arable land for spreading. Currently, excess pig manure is exported mostly as compost for the solid phase, while the liquid phase undergoes nitrification/denitrification for nitrogen removal. Very few technologies for P recovery from pig manure or any livestock effluent currently exist on full scale.

Therefore, this section will describe both these full scale processes and the one only tested at lab scale.

The types of P recovery techniques for animal manure are similar to the ones in municipal WWTP.

Thus P can be recovered from the liquid phase of digested or undigested manure, P can be recovered after pre-treatment of manure to extract P from, or concentrate in, the solid phase, and finally P can be converted into ashes through incineration.

Phosphorus recovery from the liquid phase

Two full scale units currently exist to recover phosphorus from animal manure. The first one treats 115,000 m 3 of denitrified calf manure in Putten (Netherlands) since 1998, producing K-struvite. The process consists in 3 consecutive CSTRs, a clarifier and a struvite buffer tank (Schuiling and Andrade, 1999). pH is increased to 9 using MgO. Few details are known regarding the influent or effluent characteristics. Since the manure has already been denitrified, ammonium concentration is very low.

As a result, the rise in pH leads to the precipitation of K-struvite, a process that would not occur at high ammonia concentration due to the lower solubility product of N-struvite compared to K-struvite.

No information is given concerning the proportion of dissolved P vs. TP or the recovery rate. The solid recovered had low calcium or ammonium contamination (<2%) and a relatively low amount of organic matter (5.5%). Suspended solid concentration had a negative impact on the process when above 1 g/L, a phenomenon also noticed by [START_REF] Shen | Abating the effects of calcium on struvite precipitation in liquid dairy manure[END_REF] and [START_REF] Zeng | Nutrient removal from anaerobically digested cattle manure by struvite precipitation[END_REF]. Cattle slurry has a very high suspended solid content, and even after centrifugation the supernatant can still contain 10 g-TSS/L or more [START_REF] Zeng | Nutrient removal from anaerobically digested cattle manure by struvite precipitation[END_REF]. The negative impact of suspended solids on struvite precipitation has also been observed in the case of pig slurry, with a decrease in the kinetics of precipitation, contamination of the solid obtained and smaller crystals (Capdevielle et al., 2016;Cerrillo et al., 2015;Zhang et al., 2012) The second full scale unit is operating in North Carolina, recovering phosphorus as calcium phosphate from the supernatant of centrifuged raw swine manure since 2003 (Vanotti et al., 2007) (Figure 12).

The liquid phase undergoes nitrification denitrification in order to remove nitrogen as N 2 gas and reduce alkalinity. Then, lime (Ca(OH) 2 ) is added to adjust the pH to 10.5-11 and promote calcium phosphate precipitation. The solid obtained is withdrawn from the settling tank afterward and filtered with the help of an anionic polymer. N removal and associated alkalinity consumption helped reduce the amount of lime needed to 567mg/L, for 118 mg-P/L were removed. The crystallization step enabled a removal rate of 95% of soluble P entering the reactor. However, most of the phosphorus has been removed during the initial solid liquid separation step. As a result, only 20% of TP contained in the raw slurry is recovered as calcium phosphate. P contained in the solid phase is composted at a centralized solid processing plant, and therefore can be applied on nearby arable soil. However, compost is not as concentrated in P as a mineral fertilizer and cannot be transported very far away. The solid obtained contained 10.7%-P and a moisture content of 77%. No details were given regarding the use or commercialization of the product. • Lab studies on supernatant of digested manure No full scale process currently exists to recover P from the liquid phase of digested manure. Most lab studies on struvite precipitation from these effluents focus on ammonium removal to meet discharge standards. Because nitrogen is mineralized during anaerobic digestion while phosphorus remains solid, ammonia is in large excess compared to phosphate in the supernatant. Consequently, when ammonium removal is the goal, a source of phosphorus has to be added, on top of magnesium a chemicals from pH adjustment. The P recovery rate is therefore irrelevant in these studies and rarely mentioned. Karakashev et al. (2008) tested a multi steps process in which swine raw manure was first digested, the supernatant used for P recovery before a second chemical oxygen demand (COD) removal stage in UASB reactor and finally N removal using partial nitrification denitrification. The raw manure had 1.6 g-TP/L, the supernatant after digestion 600 mg-TP/L including 160 mgPO Inhibiting effect of cations on anaerobic digestion has been noticed elsewhere in the scientific literature [START_REF] Chen | Inhibition of anaerobic digestion process: A review[END_REF][START_REF] Soto | Methanogenic and non-methanogenic activity tests. Theoretical basis and experimental set up[END_REF]. In this PhD, magnesium hydroxide is used to adjust the pH and steer the precipitation toward struvite rather than calcium phosphate. As a result, magnesium concentration after crystallization can be in large excess (up to 100 mg/L) and its effect on subsequent anaerobic digestion should be investigated.

Because magnesium is always the limiting nutrient regarding struvite precipitation, MgO and MgCl 2

were often added as a magnesium source (Cerrillo et al., 2015;Perera et al., 2007;Perera et al., 2009).

Bittern, a residual product of seawater evaporation after halite removal was also used as a cheap source of magnesium removal (Ye et al., 2011). NaOH was systematically added for pH control, sometimes in combination with CO 2 stripping through air bubbling (Cerrillo et al., 2015;Song et al., 2011). Air agitated reactors can reduce the cost of chemicals, it also results in massive ammonia stripping, as high as 90% of ammonia removed (Song et al., 2011). Sequencing batch reactors and continuous feed reactors have been tested for phosphorus precipitation. Retention times in SBR were lower than in continuous reactors, with 0.6-4 h (+ 30 minutes settling) and 6-20 h respectively.

Interestingly no fluidized bed reactors have been tested for P recovery from supernatant of digested animal manure. When mixing is brought only by a rotating propeller, there is a high risk that precipitation will occur onto it [START_REF] Le Corre | Phosphorus Recovery from Wastewater by Struvite Crystallization: A Review[END_REF].

Metals such as iron and aluminum are in low concentration in pig manure, even more so in the digester supernatant. Zinc and copper content can be relatively high in the remaining suspended solids of the supernatant, especially when compared to phosphorus, but the solids recovered after P recovery had a contamination below legal limits in all the studies that measured it. Thus (Cerrillo et al., 2015) found that Zn and Cu accounted only for 0.3% and 0.006% of the recovered solid, while struvite represented approximately 50%, due to organic contamination. To improve the purity of the crystals, solid accumulations devices can be added, usually made of stainless steel wire mesh. Perera et al. (2009) found that such device did not increase the P recovery rate, but the 25% of recovered P accumulated on it were far less contaminated in organic matter and free of trace metals (Cu, Zn) compared to the settled solid recovered at the bottom of the reactor. (Song et al., 2011) obtained a higher proportion of recovered P on the accumulation device compared to Perera et al. (2009), with 40% of all recovered P on the stainless steel mesh, during both SBR and continuous operation.

Overall, clean struvite from the collector represented 30% of phosphate initially contained in the supernatant. He also found that the struvite had less impurity on the accumulating device, with struvite representing 90% of the solid recovered on the accumulating device versus 67% in settled solids.

Therefore, accumulating devices situated above the settling zone offer the possibility to recover a purer product. It should be noted that no such device exists in full scale processes for digester supernatant such as Pearl, Phospaq or Nuresys. It would have to be mechanically extracted from the reactor and scrapped, which does not seem suitable for a full scale process.

All the studies on pig slurries found an optimal pH at 8.5-9.5. Concurrent precipitation of calcium phosphate as amorphous calcium phosphate (Ye et al., 2011) and calcium carbonate (Song et al., 2011) was observed in several studies, especially when high pH (10 or above) was tested or an insufficient amount of excess Mg was introduced, regardless of the initial P:Ca ratio.

In the case of digested dairy manure supernatant without pretreatments, several studies have been conducted so far for P recovery. Contrary to the full scale unit in the Netherlands treating calf manure, they investigated struvite precipitation. [START_REF] Uludag-Demirer | Ammonia removal from anaerobically digested dairy manure by struvite precipitation[END_REF] exclusively focused on ammonia removal and added 4.3 g-P/L as Na 2 HPO 4 (300mg-TP/L initially present, unknown proportion of phosphates), for only 500 mg NH 4 -N/L. With the addition of MgCl 2 , logically more than 95% of ammonium precipitated thanks to the large excess of Mg and P. Calcium is not mentioned but it is likely that the solid obtained contained some calcium phosphate. [START_REF] Zeng | Nutrient removal from anaerobically digested cattle manure by struvite precipitation[END_REF] noticed that simply by raising the pH to 9, 30% of the phosphates precipitated, without any Mg addition, concluding that a mix of struvite and calcium phosphate precipitated. An enormous excess of Mg compared to P was necessary to recover a high percentage of P (73% of P recovered at a molar Mg:P ratio of 22:1). This severe inhibition of P precipitation likely resulted from a very high ionic strength, suspended solid concentration and COD content. Similarly only 50% of ammonium precipitated when P:N ratio was adjusted to 1:1. The absence of K struvite precipitation confirms that N-struvite has a far higher formation reactivity preventing potassium removal as struvite when ammonium is in excess.

Most studies on P recovery from the supernatant of digested swine manure had a phosphate concentration between 22 and 160 mg/L and a TP concentration (though often not given) below 160 mg/L, except for Karakashev et al. (2008) cited above. Phosphorus content in digested dairy manure supernatant was slightly higher with 100-450 mg-P0 4 3--P/L. Phosphorus concentration in raw manure can vary widely, depending on the type of livestock, handling process at the farm and resulting suspended solid content. However, it can be safely assumed that phosphates available for precipitation in the supernatant of digested manure do not represent more than 20% of TP (Barnett, 1994;Daumer et al., 2008;Toor et al., 2006). It appears that the supernatant of digested swine manure is more suited to struvite recovery than cattle/dairy manure due to the high ionic strength in the latter, as well as the difficult solid/liquid separation and associated high suspended solid content.

• P recovery from raw swine manure (recovery of dissolved P only)

Multiplies studies have been conducted on raw swine manure, with differing objectives. P-recovery as struvite was often seen as a mean of decreasing the dissolved phosphate or ammonium concentration. All the studies on P recovery from raw swine manure never used truly "raw" swine manure and systematically screened it through a sieve with 0.5-1.5 mm mesh opening. The studies conducted on actual raw slurry spiked large amounts of phosphorus to match ammonium concentration. As a result, despite high P removal rate, the actual amount of P recovered was either artificially inflated or representing a relatively low amount compared to the true initial TP of the slurry. Compared to the supernatant of digester, the overall recovery of phosphorus is largely higher with raw slurry when the settled solid are taken into account (50-70% of TP). However without a dedicated struvite collector the settled solids are similar to the solid phase obtained from a centrifuge or a screw press, with high organic matter and suited only to composting. With struvite collector, the maximum P recovery obtained was 18% TP. In order to achieve higher recovery rate, it is necessary to dissolve the inorganic P first.

Pre-treatments prior to phosphorus recovery

As discussed above, dissolving P is a necessity to achieve decent P recovery rates. Several pretreatments of dairy and swine manure have been tested at lab scale with the objective to evaluate the extraction of P from the solid phase. They involve wet chemical treatments (chemical acidification), and thermochemical processes.

• Acidification of animal slurry

Pig slurry Daumer et al. (2010) studied the recovery of P as struvite from pig manure using organic acids (formate and acetate). Because the P recovery step would occur prior to anaerobic digestion, sulfuric acid was not used due to its inhibitory effect while organic acids could be converted to methane. The pig manure had already undergone nitrification denitrification in order to decrease the buffer capacity.

7 and 20 g of formic and acetic acid were necessary to decrease the pH to 4.5-5, leading to an increase in ortho-P from 267 mg/L to 920-930 mg-P/L, or 77% of TP. Because simple decantation was used to recover the supernatant, the mass balance indicated that "only" 50% of initial phosphorus was available in the liquid phase despite 77% dissolution. The ratio of acid to dissolved P was approximately 7.5 g-formate per gram P. Supernatant of settled untreated pig manure was then added to the acidified slurry to provide the necessary ammonium for struvite precipitation. Szögi et al. (2015) developed a process called "quick wash" consisting in applying citric or hydrochloric acid to the solid fraction of swine manure (213-300g-TS/Kg) to lower the pH down to 3-5 and dissolve P. After centrifugation, Ca(OH) 2 is then added to the liquid fraction rich in P and poorer in N (partly undissolved during acidification) to increase the pH to 8-10 and precipitate P as calcium

phosphate. An organic anionic polymer is then added to enhance the precipitation. Dissolution of P was a function of the amount of acid added, and citric acid was slightly more efficient than HCl. Two g of manure and 50 mL of acid were mixed together, leading to dissolution of 87% TP (or 3.5 g-TP per Kg of manure) with 10 mmol/L citric acid and 40 mmol/L HCl. Lime at different concentrations was added to the supernatant obtained after centrifugation of the acidified manure, at a ratio of 2% w/v (Ca(OH) 2 ). This ratio seems to indicate that 20 g of supernatant were added into 1L of lime solution.

After lime and polymer addition, 87% of TP initially contained in the manure was recovered in the precipitate at pH 8. The precipitate contained 5.6% P. Overall the process enables the recovery of 80% TP or 5.7 Kg-P per ton of "solid" manure with, according to the author, 4.8Kg of citric acid, 40 Kg of lime and 0.35 Kg of anionic polymer. However, the amounts of citric acid and lime seem to be ten times lower than what was used in the experiments. Indeed, 50 mL of citric acid at 10mmol/L mixed with 2 g of manure corresponds to 0.25 mol/Kg manure, and with a molar mass of 192 g/mol, one obtains 48 g-citrate/Kg manure or 48 Kg per ton of manure, not 4.8 Kg per ton. As a result, the economic analysis seems flawed. It claims that to process one ton of manure, the cost of chemicals would be 11.93 $, while the product has a fertilizer value of 22.43 $ on a P 2 O 5 basis. That would be a profit of 50% considering a selling price equal to that of regular fertilizers, a very conservative hypothesis. Considering the more likely amount of citric acid necessary, the selling price would cover 60% of the chemical cost. No analysis of heavy metals, organic pollutants or pathogens has been conducted a prerequisite for commercialization.

Dairy manure

Zhang et al. ( 2012) postulated that most of the P in dairy manure is under an inorganic, solid form, bound to calcium. As a result, to recover P as struvite it is necessary to separate it from calcium by acidic dissolution. HCl was used to lower the pH to 4 in the supernatant of digested dairy manure, dissolving 90% of TP (dissolved P increased from 20 to 250 mg/L). In order to re-precipitate P as struvite, a chelating agent was added to bind the dissolved calcium and MgCl 2 was introduced in large excess to promote struvite formation. The tests confirmed that P is initially bound to calcium and that adding EDTA and Mg enable struvite precipitation. However, the amount of EDTA and Mg necessary is too large to make the process economically viable.

• Thermochemical pretreatments

Pyrolysis of swine manure

Pyrolysis is the thermal degradation at ambient pressure of inorganic material under anoxic conditions and at temperatures between 400 and 800°C [START_REF] Furness | Thermochemical Treatment of Sewage Sludge[END_REF]. Before pyrolysis the material needs to be dried (dry matter > 95%). [START_REF] Azuara | Recycling phosphorus by fast pyrolysis of pig manure: Concentration and extraction of phosphorus combined with formation of value-added pyrolysis products[END_REF] studied the extraction of phosphorus by chemical acidification after pyrolysis of dried pig manure. The manure was first pressed and then dried. The dried fraction contained 94% of TP initially contained in the manure. The solids were introduced in a fluidized bed reactor at a rate of 0.2 Kg.h -1 . A N 2 flux was used to maintain anoxic conditions. The retention time was set at 90 minutes, and three temperatures were tested: 400-500-600°C. 92-96% of P contained in the dried manure remained in the solid phase, called "char", at the end of pyrolysis. In order to extract phosphorus from the char, the solids were dissolved in a solution of sulfuric acid at 200 mmol.L -1 . 90% of the P from the char was leached, 60 to 75% as orthophosphates. The author also used incineration on the dried slurry and found that all of the P could be dissolved as orthophosphate with 5 Kg-H 2 SO 4 per Kg-P. Consequently, he advised that pyrolysis should be used to obtain a liquid fraction rich in dissolved organic (called "oil", with the properties of diesel oil), while the char would be incinerated and P leached from the ashes. Indeed, leaching from the char after pyrolysis required 100 Kg of acid per Kg of P. Another solution is to use directly the char as a fertilizer.

Hydrothermal treatment and acid leaching of P from manure

These pretreatment can be conducted on the raw manure itself and it does not require concentration/drying processes. [START_REF] Ekpo | Influence of pH on hydrothermal treatment of swine manure: Impact on extraction of nitrogen and phosphorus in process water[END_REF] Hydrothermal treatment of cow manure was evaluated in two consecutive studies by [START_REF] Dai | Immobilization of phosphorus in cow manure during hydrothermal carbonization[END_REF][START_REF] Dai | A synergistic combination of nutrient reclamation from manure and resultant hydrochar upgradation by acidsupported hydrothermal carbonization[END_REF] with the objective to immobilize P in the char in the first experiment, and extract it with hydrochloric acid in the second experiment. Hydrothermal treatment alone (200°C, 4-24 h) enabled a large increase in phosphorus content in the solid phase and resulted in a lower extractability. This resulted from a conversion of water-extractable P and Ca into apatite. Depending on the objective for manure processing, hydrothermal treatment could be used as a mean to concentrate and immobilize phosphorus into the char. In the second study, Dai obtained the full extraction of TP with a 2% HCl solution. More than 90% of dissolved P was present as ortho-P. The acid to P ratio was strikingly low, with 2.2 Kg-HCl per Kg-P. There might be some ambiguity regarding the volume in which dissolved P is measured. No explicit value for TP in the initial manure was given. If the acid to P ratio is exact, the manure would have a P content of 4.2% P (% DM), a surprisingly high value, since most cow manure have a P content around 1-2% of dry matter.

Microwave heating (MW), acidification and wet oxidation of dairy manure with hydrogene peroxide per Kg of P. The pre-treatment was used for both struvite precipitation and anaerobic digestion. MW alone did not increase the methane production, and H 2 SO 4 logically inhibited anaerobic digestion due to sulfur inhibition.

Cow manure is extremely challenging regarding solid liquid separation [START_REF] Hotaling | Struvite recovery from digested dairy manure and regional manure anaerobic digestion study New York State Energy Research and Development Authority[END_REF] to remove 90% of Ca without binding with Mg. In the final stage, struvite was precipitated without addition of Mg since P was the limiting nutrient. 95% of P was removed from the liquid. No details were given regarding pH adjustment. The product was very pure, between 91 and 97%, with very small amounts of Ca, Na, and K.

MW, hydrothermal treatment and pyrolysis have positive effects on transforming the organic matter into a useful liquid product. However, on top of the relatively advanced technological level involved in these pre-treatments, their effect on P dissolution in very limited or absent. Only chemical acidification has a significant and positive effect on P dissolution with as much as 95% TP solubilized into ortho-P. H 2 SO 4 was slightly more efficient than HCl in certain studies and both were equivalent in others. The exact amount of acid was not described in most studies on MW pre-treatment but it seems to have been used in very large excess.

A recapitulative table (Table 2) sums up the amount and type of acid used, as well as the ratio of acid to P dissolved during acidification of swine manure and sewage sludge ashes. It appears that ashes from swine manure and sewage sludge give similar results in term of acid to P ratio. Acidification from char or raw manure requires significantly more acid. While no hypothesis was made by [START_REF] Azuara | Recycling phosphorus by fast pyrolysis of pig manure: Concentration and extraction of phosphorus combined with formation of value-added pyrolysis products[END_REF] regarding the higher need for acid during P extraction from char compared to ashes, the reason for higher acid need in raw slurry is quite straightforward: many compounds in raw slurry can act as pH buffer and some of them have been removed from the ashes are transformed into other compounds without buffering capacity. In raw slurry, the ratio of acid to dissolved P is minimized when TP concentration is high and on the contrary the ratio is larger at very low TP concentration, since inorganic P is not the only compound reacting during acidification.

Chemical acidification, contrary to more advanced processes, does not require large investment costs, massive energy consumption, and skilled operators. Microwave, hydrothermal carbonization, pyrolysis and incineration are processes suited for very large treatment plants. In rural areas with low population densities, P recovery processes should be kept as simple as possible to be installed on site or regrouping other livestock breeders living nearby. As a result, chemical acidification is the most low tech process, relatively easily applied locally. However, it still constitutes the main post for expenses for struvite recovery from swine manure. In Szögi et al. (2015), after correcting the amount citric acid necessary, acidification represented 76% of chemicals price. (Daumer et al., 2010) found that acidification represented 85% of chemical costs and 65% of the overall process counting investment, energy and maintenance. Biological acidification of pig slurry using organic co-substrate could reduce significantly the cost of P recovery from pig manure by replacing the chemicals by free (or cheap) organic waste. Biological acidification of pig slurry has never been studied with the objective of P recovery, however it has been used in lab study for other purposes such as two-stage hydrogen and methane production or ammonia emission control.

Strong acidification down to pH 1-2 is not necessary since a large fraction of TP in swine manure can be dissolved at pH 4-6 (Christensen et al., 2009;Daumer et al., 2010;Sharpley and Moyer, 2000).

Such pH can be reach during biological acidification. Hjorth et al. (2015) obtained a pH of 4.3 with 30 g-glucose per liter of swine manure, as a mean of reducing ammonia loss. Schievano et al. (2012) obtained a pH between 5-6 using swine manure and market biowaste as organic co-substrate. He similarly reached a pH below 5 with vegetable market waste at a ratio of 35% (weight waste / weight manure). As described by Tenca et al. (2011), this fermentation process facilitates the subsequent methane production step. Therefore, a struvite recovery step could be put prior to anaerobic digestion, with dissolution of P and hydrolysis or organic matter in the first stage, solid liquid separation to recover P as struvite, and a return of the solid fraction and liquid phase after P recovery to an anaerobic digester.

IV. ACIDOGENESIS

As discussed above, biological acidification (or anaerobic acidogenesis, or fermentation) of pig slurry using organic co-substrates appears as a cheap, low-tech process yet efficient enough to bring the pH down to 4-6, a range in which P dissolution is relatively high.

Acidogenesis is a microbial process in which organic matter is hydrolyzed and converted into organic acids, alcohols, hydrogen gas and CO 2 under anaerobic conditions. This process takes place naturally in soil, human/animal gut, silage and fermenters. It is usually presented as the second step of anaerobic digestion, after the hydrolysis step of macromolecules, and producing the VFA and hydrogen and CO 2 gas that are used thereafter in the acetogenic and methanogenic steps to produce methane (Figure 13).

The amount and type of product obtained depends on the organic substrate (carbohydrate, lipid, and protein content), the microorganisms present and the environmental conditions (pH, substrate concentration, temperature). process similar to what would be needed to dissolve P in swine manure. Because the P recovery process investigated in this PhD is conceptualized as a pre-treatment for anaerobic digestion, the existing two-stage processes are of interest, by providing hindsight into the optimal conditions for acidification and its consequences in the subsequent methanisation stage. Fermentation of waste with or without anaerobic inoculum has also been the subject of many scientific investigations in order to produce biofuels or molecules with high added value such as lactate, succinate, propionate and butyrate. These scientific studies can provide information regarding the types of metabolite produced for each kind of waste and the conditions applied.

One process uses acidogenesis with the same purpose as this PhD research: ensiling. This process consists in the storage or crops under anaerobic conditions in order to preserve it all year long and use it as an energy rich feed for ruminant livestock (Dunière et al., 2013). Silage relies on lactic acid fermentation of water soluble carbohydrates leading to a quick drop in pH, inhibiting global bacterial activity thus preventing further degradation of organic matter. This fast acidification is exactly what is sought for P dissolution in pig slurry. The fact that lactic acid bacteria (LAB) are found in large amounts in pig slurry (Snell-Castro et al., 2005), food waste and municipal organic waste (Abdel- [START_REF] Abdel-Rahman | Recent advances in lactic acid production by microbial fermentation processes[END_REF][START_REF] Bonk | Exploring the selective lactic acid production from food waste in uncontrolled pH mixed culture fermentations using different reactor configurations[END_REF]Probst et al., 2015;[START_REF] Tang | Lactic acid fermentation from food waste with indigenous microbiota: Effects of pH, temperature and high OLR[END_REF][START_REF] Tang | Dynamic membrane-assisted fermentation of food wastes for enhancing lactic acid production[END_REF][START_REF] Yin | Metabolic pathways for glycerol degradation during acidogenesis[END_REF] tends to indicate that lactic acid fermentation could be possible in swine manure. Hjorth et al. (2015) effectively obtained a high lactate concentration and low pH using glucose as organic cosubstrate in swine slurry. Lactate as an (almost) exclusive end product of acidogenesis has two advantages: high acidification efficiency and low buffer capacity during pH adjustment for struvite crystallization. The efficient acidification is due to the low pKa of lactate compared to other organic acids (Table 3). Malate, succinate and formate are often transient and used as intermediaries, not end products contrary to lactate. From this low pKa results a low buffer capacity, i.e. a low proportion of protonated acid in the targeted acidification range (4)[START_REF] Yin | Metabolic pathways for glycerol degradation during acidogenesis[END_REF][START_REF]Pyruvate formation from glucose[END_REF]. Only 5% of lactate is protonated at pH 5, versus 36-43% for acetate, propionate, butyrate and valerate, the other major products of acidogenesis (Figure 14). During pH adjustment to promote struvite precipitation these protonated acids would act as acidic buffers, releasing their proton into solution and increasing the need for alkali to increase the pH. Consequently, this section on acidogenesis is oriented toward lactic acid fermentation.

CATABOLISM OF ORGANIC MATTER UNDER ANAEROBIC CONDITIONS

Carbohydrates, lipids and amino acids can be used by bacteria under anaerobic conditions to produce simpler organic compounds and energy (as ATP) for maintenance and growth (Figure 15). In absence of oxygen acting as final electron acceptor, the redox reactions mediated by co-enzymes such as nicotinamide adenine dinucleotide (NAD) require a neutral electron balance. Pyruvic acid appears a major hub in the acidogenesis process from which most final compounds originate. Interestingly, lipids, polysaccharides and amino-acids can all be converted to pyruvate and subsequently organic acids or ethanol through a various number of redox reactions. It should be noted that several major product of acidogenesis such as lactate, acetate, propionate and ethanol can be reused, transformed and combined to generate other compounds. Some of those reactions will be described in a dedicated section below. During acidogenesis, two processes are used by bacteria to degrade amino acids. The Stickland reaction consists in a paired deamination redox reaction between two amino acids. As shown in Figure 16, an amino acid, e.g. alanine is oxidized to form pyruvate while the electrons are used to reduce two glycines. Ammonia is released and energy is recovered through formation of three acetate molecules [START_REF] Thauer | Energy conservation in chemotrophic anaerobic bacteria[END_REF]. The second process is the decarboxylation of the amino acid, releasing CO 2 and an amine (Equation 3). The balance between deamination and decarboxylation is effectuated as a mean to adjust the pH toward neutrality. Production of organic acids by deamination takes place at alkaline pH and tends to lower it. Decarboxylation occurs at low pH, the amines released having an alkaline effect on pH [START_REF] Stancik | pH-dependent expression of periplasmic proteins and amino acid catabolism in Escherichia coli[END_REF]. Stickland reactions can represent up to 90% of protein degradation [START_REF] Nagase | Interactions between amino-acid-degrading bacteria and methanogenic bacteria in anaerobic digestion[END_REF] leading to a majority of acetate as end product. Thus [START_REF] Liu | Acidogenic fermentation of proteinaceous sewage sludge: Effect of pH[END_REF] found that 80% of the organic acids produced during fermentation of protein rich sewage sludge were acetate (Equation 4). Similarly Yin et al. (2016) found that 70% of the COD from the proteinaceous substrate (peptone) converted to organic acid was acetate, using sludge from a UASB reactor as seed.

The pH was adjusted to 6 throughout the batch test study.

Protein conversion to organic acids seems to be partial most of the time, contrary to carbohydrates which are often completely converted to organic acids. Thus, Yin et al. (2016) 5), with COD loss corresponding well with the theoretical biohydrogen production. Once again it should be noted that pH was maintained at 6, which necessarily impacts the metabolic pathways occurring in the mixed culture. 6). Formate is converted further to molecular hydrogen and carbon dioxide by the Formate Hydrogen Lyase (FHL) complex. This pathway is present in Enterobacter for example [START_REF] Converti | Use of carbon and energy balances in the study of the anaerobic metabolism of Enterobacter aerogenes at variable starting glucose concentrations[END_REF]. In the absence of an exogenous final electron acceptor, the different pathways used for the degradation of pyruvate and the relative proportion of each final compound are driven by the need for the bacteria to regenerate NAD + for glycolysis and maintain the redox balance. Thus, this can be achieved by producing two moles of lactate per mole of glucose (homolactic fermentation, see dedicated section below) or a 50/50 mix of acetate and ethanol [START_REF] Alam | Anaerobic fermentation balance of escherichia-coli as observed by invivo nuclear magnetic-resonance spectroscopy[END_REF][START_REF] Redwood | Dissecting the roles of Escherichia coli hydrogenases in biohydrogen production[END_REF]. pH also affects the relative proportion of end-products. The conversion of formate to H 2 and CO 2 depends on pH and formate concentration [START_REF] Hallenbeck | Fermentative hydrogen production: Principles, progress, and prognosis[END_REF][START_REF] Rossmann | Mechanism of regulation of the formate-hydrogenlyase pathway by oxygen, nitrate, and ph -definition of the formate regulon[END_REF][START_REF] Zhang | The influence of pH on hydrolysis and acidogenesis of kitchen wastes in two-phase anaerobic digestion[END_REF]. Below pH 7, the FHL is progressively activated to reach full activity at pH 6, countering the acidifying effect of formate.

Clostridia, a bacterial group present in large proportion in pig slurry can degrade pyruvate to a mix of acetate and butyrate during acidogenesis (Equation 7 and8). Pyruvate is converted to acetyl-CoA and CO 2 thanks to the pyruvate ferredoxin oxidoreductase enzyme (PFOR), which reduces ferredoxin. A hydrogenase can then convert two protons to hydrogen by re-oxidizing the ferredoxin. This assures the production of two moles of hydrogen per mole of glucose consumed. Under low H 2 partial pressure, the acetyl-CoA is converted to acetate, producing one ATP and allowing the NADH produced during glycolysis to be re-oxidised to NAD + while reducing ferredoxin via an NADH:ferredoxin oxidoreductase enzyme [START_REF] Hallenbeck | Fundamentals of the fermentative production of hydrogen[END_REF]. The reduced ferredoxin is used subsequently to produce two extra H 2 per mole of glucose [START_REF] Jungermann | Function of reduced pyridine nucleotide-ferredoxin oxidoreductases in saccharolytic Clostridia[END_REF]. Under higher H 2 pressure, acetyl-CoA is converted to butyrate [START_REF] Chatellard | Trends and Challenges in Biohydrogen Production from Agricultural Waste[END_REF][START_REF] Hallenbeck | Fermentative hydrogen production: Principles, progress, and prognosis[END_REF], in which case NADH is used in the process and no extra H 2 is produced. The maximum VFA and hydrogen production occurs during the initial exponential growth phase [START_REF] Lay | Modeling and optimization of anaerobic digested sludge converting starch to hydrogen[END_REF]. pH once again affects the distribution of end product by modifying the enzyme activity in the different catabolic pathways [START_REF] Zhu | Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum[END_REF]. Similarly to Enterobacter, the redox balance through the relative proportion of the reduced and oxidized form of NAD is also a crucial parameter to explain the changes in the distribution of end products (Wang et al., 2012).

Propionibacterium [START_REF] Papoutsakis | Fermentation equations for propionic-acid bacteria and production of assorted oxychemicals from various sugars[END_REF], Bacteroidetes [START_REF] Macy | Pathway of succinate and propionate formation in Bacteroides fragilis[END_REF] and several

Firmicutes pertaining to the class of Negativicutes [START_REF] Reichardt | Phylogenetic distribution of three pathways for propionate production within the human gut microbiota[END_REF] are able to produce propionate and acetate (Equation 9) from glucose using the succinate pathway (Figure 15). nLDH does not exist solely in LAB, as seen above in the case of Enterobacter. Two types of nLDH lead to two isomers, L and D lactic acid [START_REF] Garvie | Bacterial lactate dehydrogenases[END_REF]. LAB can form one or the other, or a racemic (1:1) mixture. Conversely, certain homolactic fermentation LAB have the capacity to synthetize other enzymes and adapt their catabolism routes to environmental changes. Thus in presence of excess pyruvate from outside redox reactions (e.g. from citrate), Lactococcus lactis is able to produce diacetyl and acetoin using the enzymes diacetyl synthase and acetolactate synthase, when pH is low and carbohydrates are in limited supply [START_REF] Salminen | Lactic acid bacteria: microbiological and functional aspects[END_REF]. Other LAB such as Lactobacillus casei possesses the PFL system evoked earlier in the case of Enterobacter. This pathway is used under substrate limitation and long retention time, leading to a change from homolactic fermentation to mixed acid fermentation with the production of lactate, acetate, formate and ethanol.

The assertion that LAB can only grow on carbohydrates is not considered absolute any longer. Thus ATP can be obtained from glutamate, arginine and histidine [START_REF] Christensen | Peptidases and amino acid catabolism in lactic acid bacteria[END_REF][START_REF] Fernández | Amino Acid Catabolic Pathways of Lactic Acid Bacteria[END_REF]. Pyruvate can be formed by LAB from Aspartate, cysteine, methionine, serine and alanine, meaning that lactate could be produced from the catabolism of these amino acids [START_REF] Fernández | Amino Acid Catabolic Pathways of Lactic Acid Bacteria[END_REF]. This could be an explanation for the high lactate concentration obtained from egg white by Shen et al. (2017). Initially (Figure 18), the remaining intact plant cells and aerobic microorganisms consume the oxygen left by oxidizing easily available carbohydrates, such as glucose and fructose. Once oxygen is depleted, facultative heterolactic LAB naturally present on the crops (such as Lactobacillus plantarum) are the quickest to develop on water soluble carbohydrates compared to the other bacteria or even other LAB, thanks to their capability to use hexose and pentose for lactic fermentation [START_REF] Beck | The quantitative and qualitative composition of the lactic acid population of silage[END_REF]Dunière et al., 2013;[START_REF] Grazia | A survey of lactic acid bacteria in Italian silage[END_REF][START_REF] Holzer | The role of Lactobacillus buchneri in forage preservation[END_REF]. Once pH has dropped acid tolerant and acetate tolerant LAB can colonize the silage, usually strict heterofermenters such as Lactobacillus Brevis, L. buchneri and Leuconostoc [START_REF] Mcdonald | Acid Tolerance Of Leuconostoc-Mesenteroides And Lactobacillus-Plantarum[END_REF]McDonald, 1982). pH is stabilized around 4 and no hydrolysis of organic matter occurs, thanks to the inhibiting effect of pH, undissociated lactic acid, and synergetic antimicrobial effect of lactic and acetic acid produced by heterofermenters such as L. buchneri [START_REF] Adams | Growth inhibition of food-borne pathogens by lactic and acetic acids and their mixtures[END_REF][START_REF] Holzer | The role of Lactobacillus buchneri in forage preservation[END_REF].

When the toxic effect of lactic acid fermentation is not enough (too much residual oxygen initially, too high water content, not enough epiphytic LAB to start with), several bacteria can grow and use lactate, leading to the degradation of silage protein content and production of toxic compounds for animal and human health. pH re-increases due to the conversion of lactate into other organic acids with an higher pKa (Dunière et al., 2013). This secondary fermentation in which lactate is converted to other VFAs when the source of carbohydrate has been exhausted is not unique to silage. It happens in human/animal gut and plays an important role in digestion. The metabolic routes by which lactate is consumed are explored in the next sections. contaminating silage are able to consume lactate to produce butyrate, CO 2 and hydrogen gas while recovering energy (Equation 10) (McDonald, 1982;[START_REF] Mcdonald | Energy changes during ensilage[END_REF]Oude Elferink et al., 1999). Other microorganisms such as Megasphaera elsdenii, found in the rumen and swine feces [START_REF] Hino | Presence of lactate dehydrogenase and lactate racemase in Megasphaera elsdenii grown on glucose or lactate[END_REF]Tsukahara et al., 2002) perform the same reaction [START_REF] Prabhu | Lactate and acrylate metabolism by Megasphaera elsdenii under batch and steady-state conditions[END_REF].

Bacteria isolated from human feces, Eubacterium halii and Anaerostipes caccae were able to produce butyrate from lactate and acetate [START_REF] Duncan | Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product[END_REF]) (Equation 11and Figure 19).

d. OTHER ORGANIC ACIDS PRODUCED FROM LACTATE

Lactate can be converted to propionate via the acrylate pathway (Equation 12and Figure 20) by Clostridium propionicum [START_REF] Cardon | Two New Amino-Acid-Fermenting Bacteria, Clostridium propionicum and Diplococcus glycinophilus[END_REF][START_REF] Leaver | The fermentation of three carbon substrates by Clostridium propionicum and Propionibacterium[END_REF] and Megasphaera elsdenii [START_REF] Counotte | Role of Megasphaera elsdenii in the fermentation of DL-[2-13C] lactate in the rumen of dairy cattle[END_REF][START_REF] Ladd | FERMENTATION OF LACTIC ACID BY THE RUMEN MICROORGANISM, PEPTOSTREPTOCOCCUS ELSDENII[END_REF]. This last bacterium can also convert lactate back to pyruvate to produce acetate, CO 2 and H 2 [START_REF] Grause | Lactic acid as a substrate for fermentative hydrogen production[END_REF][START_REF] Prabhu | Lactate and acrylate metabolism by Megasphaera elsdenii under batch and steady-state conditions[END_REF]) (Equation 13).

Valerate and hydrogen can be produced from lactate via the condensation of acetate and propionate themselves derived from lactate fermentation. Megasphaera elsdenii is capable of this reaction [START_REF] Counotte | Role of Megasphaera elsdenii in the fermentation of DL-[2-13C] lactate in the rumen of dairy cattle[END_REF][START_REF] Ladd | The fermentation of lactic acid by a gram-negative coccus[END_REF], which was also noticed in human faecal microflora enriched in f. LACTIC ACID FERMENTATION FOLLOWED BY METHANE PRODUCTION [START_REF] Bo | Influence of lactic acid on the two-phase anaerobic digestion of kitchen wastes[END_REF] found that fermentation of kitchen waste led to a conversion of 50%-COD to lactate.

The acidified mixture was sent to anaerobic digestion and compared with glucose fed anaerobic digestion. The reactor receiving fermented kitchen waste had a lower methane production due to the accumulation of propionic acid, a known product of secondary fermentation of lactate. These inhibitory effects of lactate on anaerobic digestion were not observed by Daumer (unpublished data).

This review of acidogenesis provides useful hindsight regarding biological acidification of pig slurry to dissolve inorganic solid phosphorus. The low pH obtained by Tenca et al. (2011), perfectly sufficient for high P dissolution, indicates that the high alkalinity of swine slurry can be overcome at co-substrate concentration similar to what is commonly used in anaerobic digestion processes. The capacity of LAB to outcompete other bacteria for easily accessible carbohydrates suggests the need to use sugary co-substrates for an optimal acidification process.

V. CONCLUSION

The double problematic associated with phosphorus has been assessed. Depletion of this fossil resource is threatening global food security and runoffs from agricultural fields are severely polluting freshwater ecosystems. The role of agriculture in the inefficient use of P fertilizer and animal manure has been documented as well as its consequences in term of eutrophication. A major cause of inefficient use of phosphorus resources is the specialization of agriculture toward intensive livestock breeding in certain regions and high yield crops in others. As a result the latter relies exclusively on imported chemical P fertilizer since secondary sources of P such as animal manure are too far away to be transported there. Conversely, the regions specialized in intensive livestock breeding do not possess sufficient crop fields to spread their manure, leading to over-application, P build-up in soils, leaching and runoffs, polluting local water ecosystems. P-recovery processes have been developed to produce struvite, a slow release fertilizer with similar performances compared to conventional fertilizers. These processes have been implemented in a few large municipal wastewater treatment plants but most of them only recover already dissolved-P, leading to limited P-recovery rates. Scientific research for Precovery from animal manure similarly focuses on dissolved-P or involves costly, high technology and chemical intensive pre-treatments. In this PhD, an innovative process has been developed to recover P as struvite from swine manure, using acidogenesis as a biological process to acidify the manure and dissolve a large proportion of total phosphorus. Because local organic waste is hydrolyzed and organic acids are produced during acidogenesis, the process could be implemented in parallel of, or prior to, anaerobic digestion (Figure 21). The solid fraction obtained after digestion would have a dramatically decreased P content, allowing it to be used as organic amendment to local arable soils. Conversely, struvite could be sold as P fertilizer and be exported at limited transport costs to regions specialized in crop production, thanks to the high P content of struvite compared to raw manure.

OBJECTIVES OF THESIS

• Determine the effect of acidogenesis on pH, production of organic acids and dissolution of P, Mg, NH 4 + and Ca when sucrose is used as a model organic co-substrate for biological acidification of pig slurry.

• Verify that lactic acid bacteria are responsible for lactate production during biological acidification of raw and digested swine manure using sucrose as co-substrate.

• Determine the effects of various organic wastes on acidification, the bacterial communities involved and the metabolic pathways they used.

• Evaluate the possibility to predict the lowest pH reached during acidogenesis based on cosubstrate type and characteristics of swine slurry (initial pH, buffer capacity).

• Investigate the possible adaptation of acidogenic biomass to acidic conditions using semicontinuous feeding.

• Produce struvite from dissolved P, Mg, NH 4 + in the supernatant after acidification and centrifugation.

• Determine the purity of struvite in term of P content and organic, calcium and heavy metal contamination.

• Evaluate the mass balance for phosphorus throughout the process to calculate the associated P recovery.

L'étude bibliographique a montré que pour optimiser la valorisation du phosphore il est nécessaire d'obtenir un produit à l'efficacité agronomique au moins comparable à celle des engrais phosphatés les plus efficaces. La struvite répond à ces conditions. Or, la production de struvite se fait à partir de P dissous. Le phosphore des lisiers étant majoritairement sous une forme minérale particulaire mélangée à la matière organique, une étape de dissolution suivie d'une étape permettant de séparer le liquide enrichi en P de la matière organique est nécessaire pour valoriser une fraction significative du P des lisiers de porcs. L'acidification chimique testée lors de travaux précédents s'est révélée efficace mais coûteuse sur le plan économique. De plus, elle impacte fortement le bilan environnemental du procédé.

Des procédés d'acidification biologique comme l'ensilage pour la conservation des fourrages permettent de faire produire aux bactéries endogènes de l'acide in-situ. Ce type de procédé a également été utilisé pour acidifier le lisier avec l'objectif de réduire les émissions d'ammoniac. Dans ce cas l'apport d'un co-substrat facilement dégradable est nécessaire. La production d'acide in situ en quantité suffisante pour obtenir une baisse significative du pH à partir de déchets organiques utilisés habituellement comme co-substrat de la digestion anaérobie est une alternative qui permettrait de s'affranchir du coût de l'acide et de l'impact environnemental lié à sa production. L'étude bibliographique a également montré que les co-substrats sucrés sont les plus efficaces car ils permettent de produire de l'acide lactique qui est l'un des plus forts des acides organiques.

Les objectifs des travaux décrits dans ce premier article sont de tester la faisabilité de l'acidification

III. INTRODUCTION

The anthropogenic use of phosphorus presents a double problematic: on one hand it is contributing to water bodies pollution due to a combination of fertilization excess and leakage/runoff (MacDonald et al., 2011), and on the other hand it is a fossil resource critical for agriculture and worldwide food production (Cordell et al., 2009). As a result, recovering phosphorus from pig slurry as a mineral fertilizer could be a useful tool to mitigate water pollution from pig breeding and participate in a more sustainable food production (Ashley et al., 2011). Brittany (France) possesses only 6% of the national arable land but represents 60% of French the pig production (Comité régional porcin de Bretagne, 2013). Stringent regulations now prevent the unlimited spreading of the slurry into the fields and various handling strategies are currently used in the region [START_REF] Landrain | Le phosphore: comprendre, maîtriser, valoriser, Chambres d'agriculture de Bretagne[END_REF]. Among them is anaerobic digestion [START_REF] Levasseur | Etat des lieux du traitement des lisiers de porc en France[END_REF], in which various organic wastes are co-digested with the slurry in order to produce energy and heat. Exportation of manure out of Brittany is another strategy to reduce the environmental impact on soil [START_REF] Martinez | Livestock waste treatment systems for environmental quality, food safety, and sustainability[END_REF]. Part of the digested and undigested manure is separated using screw press or centrifuge decanter to collect a liquid phase rich in nitrogen that is treated via biological processes, while most of the phosphorus remains in the solid phase under a mineral form (Christensen et al., 2009;[START_REF] Daumer | Traitement biologique des lisiers de porcs en boues activées. Guide technique à l'usage des concepteurs, exploitants et organismes de contrôle des stations[END_REF]. A large part of this solid phase has to be exported out of Brittany (usually as organic fertilizer) but this product has trouble finding its market. It cannot compete with compost because of its high P-content when organic matter is the customer's driving choice, and cannot compete either with mineral fertilizers because of the transport cost of organic matter when P is the component demanded.

Phosphorus recovery as struvite (NH 4 MgPO 4 , 6 H 2 O), a slow-release fertilizer, would make the export of P more cost effective, meet the market demand for a concentrated, P-based mineral fertilizer and allow the organic matter in the digestate/compost to be maintained locally thanks to its low P content.

Phosphorus recovery as struvite has been studied in a large variety of N and P concentrated waste streams: WWTP centrate liquor from anaerobic digestion [START_REF] Battistoni | Phosphorus removal from a real anaerobic supernatant by struvite crystallization[END_REF][START_REF] Jaffer | Potential phosphorus recovery by struvite formation[END_REF], industrial wastewater (Abma et al., 2010), municipal landfill leachate [START_REF] Di Iaconi | Nitrogen recovery from a stabilized municipal landfill leachate[END_REF], dairy wastewater (Zhang et al., 2010) as well as swine lagoon effluent [START_REF] Nelson | Struvite precipitation in anaerobic swine lagoon liquid: effect of pH and Mg:P ratio and determination of rate constant[END_REF], digested swine wastewater (Song et al., 2011;[START_REF] Vanotti | Recovery of ammonia and phosphate minerals from swine wastewater using gas-permeable membranes[END_REF]Ye et al., 2011) and raw swine manure [START_REF] Burns | Laboratory and in-situ reductions of soluble phosphorus in swine waste slurries[END_REF][START_REF] Çelen | Using a chemical equilibrium model to predict amendments required to precipitate phosphorus as struvite in liquid swine manure[END_REF]. Several full scale reactors have been built for struvite crystallization, such as the OSTARA, WASSTRIP (Cullen et al., 2013) and PHOSPAQ processes (Remy et al., 2013).

However these technologies are only used so far to treat municipal and industrial wastewater. In the case of animal manure, precipitation of phosphorus as struvite is still investigated at laboratory and pilot scales [START_REF] Rahman | Production of slow release crystal fertilizer from wastewaters through struvite crystallization -A review[END_REF], focusing mainly on reducing the dissolved phosphate concentration to meet the discharge standard for the liquid effluent. However, if the goal is to recover phosphorus, dissolving the inorganic solids that contain most of the phosphorus is necessary. It has been demonstrated that up to 70% of TP could be dissolved at pH 5.5 (Christensen et al., 2009). P recovery as struvite can therefore be achieved through acidic dissolution, solid-liquid separation and pH increase in the liquid phase using magnesium oxide (MgO) or magnesium hydroxide (Mg(OH) 2 )

leading to phosphorus precipitation (Capdevielle et al., 2016). The use of chemicals for acidic dissolution was demonstrated as too expensive and having a negative relative impact on the environment (Daumer et al., 2010). For the process to be efficient and sustainable, changing this acidification step is required. A biological process known as acidogenesis enables the natural acidification of a mixture under anaerobic conditions. During acidogenesis, organic matter is hydrolyzed and converted by fermenting bacteria into VFAs, lactic acid and alcohols. This acidifying reaction process is a crucial step in silage production (Dunière et al., 2013), a technic used to preserve the nutritive value of forage for cattle feeding [START_REF] Buxton | Silage science and technology[END_REF]. Acidogenesis also occurs during anaerobic digestion of organic waste. The syntrophic cooperation of acidogenic, acetogenic and methanogenic organisms enables the conversion of organic waste into methane and carbon dioxide.

Because acidogens have different optimal conditions compared to acetogens and methanogens [START_REF] Liu | Hydrogen and methane production from household solid waste in the two-stage fermentation process[END_REF], and especially a higher maximum growth rate, an overloading of a digester with easily fermentable substrates often lead to acidosis and inhibition of methanogens [START_REF] Mccarty | Volatile acid toxicity in anaerobic digestion[END_REF]. As a result, the process can be physically separated into two consecutive reactors one acidic and one methanogenic, each of them functioning under their respective optimum [START_REF] Cohen | Anaerobic digestion of glucose with separated acid production and methane formation[END_REF].

This two stages process has been applied to crop residues [START_REF] Kalia | Biomethanation of plant materials[END_REF][START_REF] Parawira | Energy production from agricultural residues: High methane yields in pilot-scale two-stage anaerobic digestion[END_REF], agro-industrial waste [START_REF] Dareioti | Effect of hydraulic retention time (HRT) on the anaerobic codigestion of agro-industrial wastes in a two-stage CSTR system[END_REF], and food waste (Uçkun [START_REF] Uçkun Kiran | Bioconversion of food waste to energy: A review[END_REF].

Biological treatment of swine slurry in a two stage anaerobic process has recently been studied [START_REF] Ren | Performance and microbial community dynamics in a two-phase anaerobic co-digestion system using cassava dregs and pig manure[END_REF]Schievano et al., 2012), often with the purpose of biohydrogen and methane production [START_REF] Choi | A low-energy, cost-effective approach to fruit and citrus peel waste processing for bioethanol production[END_REF]Wu et al., 2010). It has been shown that a two-stage anaerobic digestion of fruit and vegetable waste mixed with swine manure at a ratio of 70/30 (w/w) lead to a pH of 4.2 in the acidogenic stage (Tenca et al., 2011), a pH at which phosphorus is known to be mostly dissolved in pig slurry (Sharpley and Moyer, 2000). Therefore, biological acidification of swine slurry with organic co-substrates could be proposed as the first stage in a bioprocess aimed at nutrient recovery (N & P)

and green energy production (methane). Acidogenesis would dissolve P from pig slurry at lower cost than chemical acidification while providing VFAs and potentially increasing the biodegradability of the waste for the subsequent anaerobic digestion.

This batch test study focuses on the biological acidification of pig slurry using various concentrations of sucrose as a model organic co-substrate, adapting the methods developed by [START_REF] Braak | Phosphorus recycling potential assessment by a biological test applied to wastewater sludge[END_REF] and [START_REF] Guilayn | Sequencing biological acidification of waste-activated sludge aiming to optimize phosphorus dissolution and recovery[END_REF] as a first step for phosphorus recycling. Biological acidification of pig slurry using glucose, cellulose or lactic acid has been tested to minimize ammonia emission by lowering the pH (Hjorth et al., 2015). The results in terms of pH decrease and organic acids produced were very similar to this study. The objective here was to determine the relationship between on one hand initial amount of sucrose and slurry's characteristics (initial pH, buffer capacity) and on the other hand pH change, fermentation products and dissolution of phosphorus, magnesium, ammonia (the three molecules forming struvite) and calcium (which lead to the undesired precipitation of calcium phosphate). This endeavor could then translate into a targeted acidification step using real organic waste that would maximize P-dissolution, favor struvite formation over calcium phosphate and minimize the amount of reactant necessary to re-increase the pH.

IV. MATERIALS AND METHODS

PIG SLURRY

The raw swine slurry ("slurry 1") was collected at a commercial pig fattening farm in Melesse (Brittany, France). Its composition can be found in Table 4. It was used to study the metabolism of acidogenesis in details. Three additional pig slurries (Table 4) were also tested in order to correlate the level of sucrose added with the initial and lowest pH reached, as well as the buffer capacity of the slurry. The buffer capacity was measured in a beaker containing 100 mL of raw pig slurry with a magnetic stirrer, a pH probe and a graduated burette containing sulfuric acid (1 mol/L). The buffer capacity was calculated has the concentration of sulfuric acid necessary to reach pH 4, the lowest pH obtained during the biological acidification of the slurries (pH 3.99 was reached in slurry 2 with 60 g/L of sucrose). One liter bottles were inoculated with 640 g of raw pig slurry 1 and six different sucrose concentrations [START_REF]Butyrate formation from lactate[END_REF]20,30,40,50 and 60 g/kg-slurry, in reactors called R10, R20, R30, R40, R50, R60 respectively) in triplicate, with an additional bottle inoculated with slurry only and used as a control.

The bottles were sealed once inoculated. Temperature was set at 38°C on heating plates and magnetic stirrers provided continuous mixing at 300 rpm. Samples were collected simply by removing the lid and pouring the liquid from the bottles. Contrary to previous batch tests with wastewater sludge, purging the gas in the head-space of the bottles at the beginning and after sample collection had no effect on the system when applied to pig slurry, therefore the bottles were never flushed with inert gas.

Sample collection occurred after 12,24,36,48,72 and 96 hours. The same protocol was applied to slurry 2, 3 and 4 using only 5 different sucrose concentrations for each, from 0 to 60 g/L, in order to investigate the variations between slurries.

ANALYSIS

pH was measured using a WTW probe right after each sample collection. Total solids (TS), volatile solids (VS), total suspended solids (TSS), and volatile suspended solids (VSS) were measured in triplicates on the raw slurry with standard methods (APHA, 1998). An acidic dissolution of the ashes was realized to measure TP, T-Ca and T-Mg. 200mg of ashes were added to 0.5g of K 2 SO 8 and 5mL of a mix of H 2 SO 4 / HNO 3 (75:25) in triplicate. The samples were autoclaved at 110°C during one hour at 1 bar.

The concentrations in TP, T-Ca and T-Mg (expressed in gram per liter of raw manure in Table 4) were measured using automated colorimetric methods on a spectrophotometer (Gallery, Thermoscientific).

For TP measurement, two reactants (containing sulfuric acid, potassium antimonyl tartrate ammonium molybdate and ascorbic acids, Thermoscientific products) were added automatically to the sample.

Phosphates react with ammonium molybdate under acid conditions to form a 12-molybdo-phosphoric acid complex. The complex is reduced by the ascorbic acid to form a blue compound. The absorbance was measured by spectrophotometry at the wavelength of 880 nm. For T-Ca measurement, a reactant containing Arsenazo III and an imidazole buffer was added to the sample. Calcium ions form a highly colored complex at neutral pH. The absorbance of the complex was measured at 660nm. For T-Mg measurement, a reactant containing Xylidyl I blue, EGTA, K2CO3 and a Tris buffer was added to the sample. At alkaline pH magnesium ions react with Xylidyl I blue to form a red-colored complex. The absorbance was measured at 520 nm. . Supernatants were obtained after 20 min of centrifugation (4°C, 20,000 g) and filtration on a 0.45 µm polypropylene membrane. Phosphate concentration was measured with automated colorimetric methods on the Gallery. Cationic composition of the supernatant was measured by ionic chromatography Metrohm 940 Professional Vario IC with a Metrosep C4 -250/4,0 column. Sucrose, glucose, fructose and VFA composition of the supernatant was measured by HPLC (Ultimate 3000, Dionex) with a Hiplex-H column (Agilent).

STATISTICS

In order to evaluate the effect of sucrose concentration on pH and ionic concentrations, an ANOVA was performed at each sampling time (12, 24, 36, 48, 72 and 96 hours). The conditions of application for ANOVA (normal distribution of the residuals and equal variance of the residuals for each modality) were verified with a Shapiro-Wilk test and a Bartlett test. The different modalities (i.e. level of initial sucrose concentration) were then compared two by two with a Tukey's HSD test.

V. RESULTS AND DISCUSSION

EFFECT OF SUCROSE CONCENTRATION ON PH AND FERMENTATION PRODUCT IN SLURRY 1 a. PH CHANGE

Figure 22 shows the effect of sucrose addition on pH in slurry 1. Considering that sucrose would likely be fermented mostly into VFAs and other organic acids, the pH drop was logically absent in R0, moderate at R10, R20, R30 and severe in R40, R50, R60 (Figure 22). Indeed, the pH initially dropped in all the reactors except in the control where it remained stable throughout the experiment. The pH drop was significantly more pronounced and longer at higher initial sucrose concentration. After 24 to 36 h, pH re-increased in the reactors with an initial sucrose concentration of 10, 20 and 30 g/L whereas at higher initial sucrose content (40, 50, 60 g/L), pH dropped below 5 at 36 hours and remained stable or kept decreasing afterwards. The pH re-increase in R10, R20, R30 could have resulted from either methanogenesis (consumption of VFAs to form methane and CO 2 ) or secondary fermentation (conversion of VFAs initially produced into other, less acidic compounds). Since no significant amount of methane could be detected (measurement at 24 and 48 h, data not shown), a secondary fermentation seemed the most likely and was later confirmed (see below).

b. EVOLUTION OF LACTIC ACID, VFAS AND SUCROSE ACROSS TIME

As shown in Figure 23A and 23B (R30 and R50 representing moderate vs. high initial sucrose content), a lactic acid fermentation occurred in all the reactors with lactate as the main product as long as sucrose was still present. The preponderance of lactic acid in this first phase can be seen through both its proportion among the acids produced and its strong linear correlation with pH (r² = 0.97).

Once sucrose had been removed, lactate disappeared in the next 12 to 24 h in reactors R10, R20, R30 while acetate and propionate concentrations increased and butyrate and valerate appeared. The replacement of lactate by less acidic VFAs (acetate, propionate, butyrate and valerate have a pKa of 4.75, 4.87, 4.82 and 4.86 respectively versus a pKa of 3.89 for lactate) could explain the pH reincrease. On the contrary, lactate concentration remained stable in R40, R50 and R60 once sucrose had been consumed, without any increase in other VFAs concentration or any apparition of butyrate and valerate, while pH stayed low. Carbon recovery, calculated as the ratio between the net production of organic acids (VFAs + lactic acid) and the removed sucrose, can be found in Table 5 At 12 hours, the carbon recovery was relatively poor (67-89% and high standard deviations) possibly due to variations in the lag phase between triplicates and the transitory storing or initial catabolic steps of glucose and fructose (glycolysis) in the bacterial cells. At 36 h, the C-recovery was around 95% in all the reactors, with most of the sucrose converted to lactate. The C-recovery in R40 to R60 remained similar until the end since the metabolic activity was severely reduced due to the low pH. In R10 to R30, the Crecovery decreased down to 80-85% at 48 hours due to the conversion of lactate to acetate, propionate, butyrate and valerate, metabolic processes involving CO 2 production, which was not measured in this study. The C-recovery in those reactors increased slightly at 72 and 96 hours to 87-93%, likely due to the fermentation of organic matter naturally present in the slurry. The differing behaviors between low and high initial sucrose concentration in terms of pH change and whether or not lactate was converted to other VFAs could be explained by the physiology of the bacterial community during the batch tests and the scientific literature on ensiling process. Pig slurry is known to contain relatively high amounts of LAB and Clostridia (Snell-Castro et al., 2005), two bacteria critical in the silage process. During ensiling, in presence of a sufficient amount of carbohydrates (sucrose in this case), amino acids and vitamins (both present in large amounts in pig slurry), the LAB can outcompete the other microorganisms and dominate the fermentation thanks to their higher growth rate (McDonald, 1982). If the pH reached at the end of the lactic acid fermentation is around 5 or lower, the activity of other anaerobic organisms, such as Enterobacteria and Clostridia is completely inhibited (Pahlow et al., 2003), preventing the conversion of lactate to other fermentation products and further degradation of the organic matter [START_REF] Kempton | Chemistry and Microbiology of Forage-Crop Silage[END_REF].

Therefore the fermentation process in R40, R50 and R60 could reasonably be compared to a successful ensiling (yet at a much quicker pace and with a lower dry matter content), since lactate was the main product throughout the test and pH remained below 5 after 36 hours.

In a failed ensiling process, the initial carbohydrate content is too low and the pH reached after the lactic acid fermentation is too high to prevent a secondary fermentation of lactate and protein degradation into biogenic amines, ammonia and organic acids [START_REF] Rooke | Biochemistry of ensiling[END_REF]. That seems to be what took place in R10, R20 and R30. After 36 hours, pH was still at 5.5 or higher (Figure 22), sucrose could not be detected anymore and the only substrates available were lactate and organic matter from the slurry. Lactic acid fermenting Clostridia are known to have the ability to convert lactate to acetate, propionate [START_REF] Johns | The Mechanism of Propionic Acid Formation by Clostridium propionicum[END_REF][START_REF] Kuchta | Lactate reduction in Clostridium propionicum. Purification and properties of lactyl-CoA dehydratase[END_REF] and butyrate (McDonald, 1982;[START_REF] Woolford | The silage fermentation[END_REF]. Some Clostridia are highly proteolytic and can use amino acids in their catabolism, producing acetate, propionate [START_REF] Wood | Microbiology of fermented foods[END_REF], butyrate [START_REF] Elsden | Amino acid utilization patterns in clostridial taxonomy[END_REF] and valerate [START_REF] Dehority | Studies on the metabolism of valine, proline, leucine and isoleucine by rumen microorganisms in vitro[END_REF]. Pig slurry contains also large amounts of Selonomonas Ruminantium and to a lesser extent Megasphaera elsdenii (Tsukahara et al., 2002), two bacterial species able to grow on lactate. Megasphaera elsdenii can use lactate to form acetate, propionate, butyrate and valerate from lactate in absence of glucose [START_REF] Rogosa | Transfer of Peptostreptococcus elsdenii Gutierrez et al. to a New Genus[END_REF]. Several strains of Selonomonas Ruminantium produce acetate, propionate and CO 2 from lactate [START_REF] Wallace | Control of Lactate Production by Selenomonas ruminantium: Homotropic Activation of Lactate Dehydrogenase by Pyruvate[END_REF]. Therefore, the disappearance of lactate and higher production of acetate, propionate, butyrate and valerate were likely the result of increased activity from lactic acid fermenters.

Coming back to the goal of building a process allowing biological acidification of pig slurry as a first step of phosphorus recycling, these results indicate that the most suitable co-substrates for pig slurry acidification would contain high amounts of soluble carbohydrates to favor LAB, and low quantities of proteins, whose degradation produces amines and NH 3 with an alkaline effect on pH [START_REF] Rooke | Biochemistry of ensiling[END_REF]. Fruits and vegetable waste (apples, carrots, potatoes) as well as corn waste could be the most promising co-substrates and will be investigated in the near future. The patterns of dissolution of phosphorus, magnesium and calcium were similar. Initially these compounds were mostly under solid form (>94%). At low initial sucrose concentration (R0 and R10), no additional dissolution occurred. A moderate dissolution followed by partial or total re-precipitation took place at moderate sucrose concentrations (R20 and R30), and at high sucrose concentration the dissolution was almost total (R40 to R60).

As seen in Figure 24A, less than 80 mg-P/L (<5% TP) was dissolved at any time in R0 and R10, an amount significantly lower than the other reactors at 24 and 36 hours. In R20 and R30, respectively 400 and 1300 mg-P/L were dissolved at 24 and 36 hours, but re-precipitated afterwards, down to levels statistically similar to R0 and R10. In R40, R50 and R60, dissolved P was always statistically similar among them, and became significantly higher than the other reactors from 36h to the end, with up to 1600 mg-P/L (95% TP).

The same pattern occurred for Mg 2+ (Figure 24B). In R0 and R10, less than 160 mg/L (or 15% T-Mg)

were dissolved throughout the experiment. In R20, 400 mg/L or 36% of T-mg were dissolved at 24 and 36 hours but the precipitation that followed was only partial, with approximately 220 mg/L or 20% remaining dissolved until the end of the test, a number significantly higher than in R0 and R10.

The same but amplified phenomenon occurred in R30, with around 1100 mg/L (or close to 100% T-Mg) at 24 and 36 hours, followed by a partial re-precipitation with still 600 mg/L or 50% T-Mg. In R40, R50 and R60, dissolved magnesium concentrations were always statistically similar and became significantly higher than the other reactors from 48h to the end, with up to 1100 mg/L (100% of T-Mg).

Similarly in the case of calcium (Figure 24C), no more than 200 mg/L or 6% of T-Ca were dissolved in R0 and R10 during the whole duration of the test. Respectively 500 and 1000 mg/L (or 14 and 28%

of T-Ca) were dissolved in R20 and R30 at 24 and 26 hours but only approximately 300 and 400 mg/L remained afterwards, concentrations still significantly higher than in R0 and R10. R40, R50 and R60 had statistically similar calcium concentrations until 36 hours but R40 then stagnated around 2200 mg/L or 60% of T-Ca while R50 and R60 had a further increase in Ca concentration with around 2700mg/L or 75% of T-Ca at 48 hours before remaining stable until the end of the test.

Interestingly, the evolution of P, Ca and Mg in all the reactors followed closely the changes in pH (Figure 25A). The dissolved P concentration did not increase linearly with the decreasing pH. A clear and relatively narrow threshold appeared between 5.9 and 6.3, through which dissolved P went from less than 10% to 80% of TP. Below pH 5.9, only 10 additional percent of TP could be gained. Mg 2+ followed a similar pattern, with less than 20% of T-Mg above pH 6.5 and more than 95% below pH 5.9. Calcium dissolution was more gradual; with less than 20% of T-Ca above pH 6.2, 40% at pH 5.5

and more than 95% below pH 4.5.

The change in ammonium concentration did not follow the same pattern (Figure 24D) and was apparently less linked to pH (Figure 25B). In Figure 24D, NH 4 + initially dropped in all the reactors except R0. The drop was more pronounced in the case of high initial sucrose concentrations. After this initial drop, NH 4 + concentration in R10 to R60 re-increased and three statistically different groups appeared after 48h. R40, R50, and R60 had a stable NH 4 + concentration around 2800 mg/L or 60%-TN until the end of the test. R30 had around 2500 mg/L or 55%-TN. R10 and R20 had still a slightly increasing NH 4 + concentration from 2100 mg/L at 48h to 2300 mg/L or 50%-TN at the end. In R0 ammonium concentration increased linearly for 48 hours and stabilized around 2500 mg/L, similarly to R30. The increase in NH 4 + in absence of sucrose and despite the high pH in R0 contrasted with the absence of any dissolution of P, Ca and Mg in the same reactor. The overall increase in ammonium concentration over time in all the reactors with and without pH decrease could be explained by the enzymatic degradation of organic nitrogenous compounds (e.g. amino acids). Initial sucrose concentration has an impact on two phenomena with opposite effects on ammonia concentration: dissolution of inorganic solids through acidification on one hand and bacterial growth on the other hand. The low pH reached at high sucrose concentration enabled a higher ammonia release through the dissolution of inorganic N-containing compounds (e.g. struvite initially present in the raw slurry). In the case of phosphorus, the inflexion point noticed for slurry 1 can be seen again around a pH of 6-6.2. However, the maximum proportion of dissolved P varied significantly in one slurry, with only 58 % of TP in slurry 2 compared to 95, 89, and 78% in slurry 1, 3 and 4 respectively. No analysis was performed to determine the inorganic compounds that contained phosphorus in the slurries. However, based on the limited calcium dissolution in slurry 2 (60% of T-Ca maximum), it could be hypothesized that this slurry contained an important amount of calcium phosphate under a low solubility form (e.g. hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 ). Maximum P dissolution was obtained when the minimum pH was reached in slurry 1 and 3 but not in slurry 2 and 4 where maximum P dissolution occurred at a moderate pH around 5 while dissolved P decreased at lower pH. A possible explanation could come from a lower TP content in those slurries compared to slurries 1 and 3. This low TP content means that at high sucrose concentration and consequently at low pH, the P incorporated into the biomass would not be negligible and impact negatively the ratio dissolved-P/TP. Considering a biomass chemical formula as C 5 H 7 NO 2 P 0.074 (Droste, 1997), and a yield on sucrose of 0.14 (biomass COD.sucrose COD - 1 ) (Gujer and Zehnder, 1983), the consumption of 60g/L of sucrose corresponds to the incorporation of 126 mg-P/L, i.e. 33 and 23% of TP in slurries 2 and 4, but only 7 and 9% in slurries 1 and 3. Another solids. Addition of MgO or Mg(OH) 2 to the liquid phase can provide the pH increase necessary and shift the precipitation process toward struvite rather than calcium phosphate thanks to their magnesium content. The optimal parameters to get a maximum P-recovery and favor struvite versus calcium phosphate are a pH close to 7, a molar N:P ratio above 3:1, a Mg:P ratio above 1:1, a Mg:Ca ratio around 2.25:1 or higher, and an addition of MgO as low as possible to favor the formation of large crystals (Capdevielle et al., 2013). In the pH range where most of the P was dissolved (pH 4 to 6), the N:P ratio never went below 3.9 in any of the four slurries, indicating that the concentration of ammonium and phosphorus in acidified pig slurry are very suitable for struvite crystallization. The Mg:P ratio was between 1 and 2.7 in all the slurries. Since MgO would be added to increase the pH, this ratio would increase even further, leaving large amounts of Mg in excess. Impact of this excess on subsequent anaerobic digestion should be investigated. The Mg:Ca ratio however was always below 2.25 in all the slurries, with a value of 2 at pH 6 and a decrease to 0.5 at pH 4. Magnesium dissolution stopped at pH 6 while calcium concentration continued to increase until pH 4, which explains why the Mg:Ca ratio decreased with the decreasing pH. The acidic buffer created by the production of VFAs during the acidifying step could require a large amount of MgO or Mg(OH) 2 to bring the pH up. This would reduce the crystals size and represent an additional cost. As a result, an optimal acidification would lead to a pH low enough for maximum P dissolution but also as close as possible to 7 to minimize Ca dissolution and the need for MgO. As seen in Figure 26A, the gain in dissolved P below a pH of 5.5 was minimal, null, or negative. On the other hand, above pH 6, dissolved P was always lower than 50%. As a result, targeting a pH between 5.5 and 6 should provide a relatively high dissolved P, allow for a minimum addition of MgO while dissolved magnesium is already high and

calcium not yet completely dissolved. These recommendations only apply to biological acidification of pig slurry using sucrose. When organic waste is used, hydrolysis and mineralization takes place, releasing into the liquid phase part of the nitrogen, phosphorus, magnesium and calcium contained in the waste [START_REF] Mehta | Nutrient solubilization and its availability following anaerobic digestion[END_REF], changing the ionic equilibriums. As a result, the effect of organic waste on acidification and ionic equilibrium in pig slurry should be investigated.

CORRELATING LOWEST PH WITH INITIAL SUCROSE CONCENTRATION AND BUFFER

CAPACITY

Considering that the ideal pH range to maximize P recovery and minimize MgO is 5.5-6, it would be practical to be able to predict the amount of co-substrate necessary to reach it in any given pig slurry.

Indeed, an excess of co-substrate would guarantee a maximum dissolution of P, but require an important amount of MgO to precipitate it. Conversely an insufficient addition of co-substrate would lead to a low P dissolution. Applying a linear regression on the data obtained from the batch tests on four pig slurries, it was possible to correlate the initial sucrose concentration, initial pH, buffer capacity (as the explanatory variables) with the lowest pH reached (as the variable to be explained).

Residuals' normality was verified with a Shapiro-Wilk test. Other characteristics of the pig slurries were also tested (such as total solids, inorganic solids) but none had a statistically significant effect.

The distribution of the observed pH plotted against their predicted value can be seen in Figure 27. The relatively good fitting of the model was possibly due to the dominance of lactic acid during the initial acidifying phase, i.e. the lowest pH was always reached at maximum lactic acid concentration, which corresponded to 70-100% of the VFAs produced at the moment. The equation was rewritten to predict the amount of sucrose necessary to reach a given pH [START_REF]A Net production of VFAs and lactate, sucrose removed and pH across time in R10. Standard deviation shown for the sucrose removed and the sum of VFAs and lactate[END_REF]. This model cannot be used as such for real co-substrates. As a result, waste commonly used in Brittany in centralized biogas plants will be tested in batch tests in order to rate their acidifying capacity versus sucrose. The equivalency to be found should allow the substitution of sucrose in the equation (Equation 14) with its equivalent in cosubstrate, expressed as volatile solids. Les travaux précédents réalisés avec du saccharose ont montré que la production d'acide lactique, métabolite presque exclusif issu de la dégradation des sucres simples était déterminante pour acidifier le lisier biologiquement. L'étude bibliographique laisse à penser que cette voie n'est pas toujours majoritaire lors de la dégradation de substrats complexes comme les déchets utilisés en méthanisation.

Le choix des substrats et la flore initialement présente peuvent également déterminer les voies métaboliques actives lors du procédé. Les travaux présentés dans ce chapitre ont pour objectif de vérifier que l'acidification peut être obtenue avec des sucres complexes et de tester l'influence de la nature de la flore présente au début du procédé en comparant les résultats obtenus avec du sucre ajouté au lisier brut et à du digestat de lisier.

Un lisier brut et deux digestats issus du même digesteur mais prélevés à deux périodes différentes ont été utilisés (Table 6). Une première série d'expérimentation a été conduite avec le lisier brut et le digestat 1 auxquels ont été ajoutés 5 concentrations de sucre différentes de 10 à 50 g.L -1 . Trois cosubstrats complexes (pommes de terre, déchets de biscuits et déchets de farine de blé noir) ont également été testés à une concentration de 50 g de MV.L -1 avec le digestat 1.

Dans la deuxième série d'expériences 8 nouveaux co-substrats ont été testés à la même concentration avec le digestat 2, les déchets de biscuits et de farine de blé noir stockés à 4 °C ainsi que des carottes, des haricots verts, des petits pois de la macédoine de la paille de blé et des pommes. Un dernier essai avec une concentration moindre de carottes (30 g.L-1) a également été effectué. La composition en glucides, lipides et protéines et en sucres sont présentées dans la Table 7.

Avec le digestat, le pH décroît presque linéairement jusqu'à des concentrations de sucre de 30 g.L-1.

La baisse est moins importante à quantité de sucre équivalente avec le lisier brut et 50 g.L -1 sont nécessaires pour obtenir la valeur minimale et ce malgré un pH initial plus faible (Figure 28). La

III. INTRODUCTION

The green revolution during the 20 th century enabled massive increase in crop yield and food production, thus sustaining a large population growth during the same period (Tilman, 1998). This achievement relied heavily on generous application of chemical fertilizer providing N, P and K to the crops. Because P is the main limiting nutrient in freshwater ecosystems [START_REF] Mainstone | Phosphorus in rivers -ecology and management[END_REF]Sattari et al., 2014), a small increase in concentration due to runoff and leaching from agricultural fields can lead major ecological perturbations known as eutrophication (Schindler et al., 2008;[START_REF] Sharpley | Strategies for the Sustainable Management of Phosphorus[END_REF]. While the treatment of domestic wastewater vastly improved in the last 50 years, diffuse pollution by agriculture becomes more prevalent (Dupas et al., 2015;Némery et al., 2005). More recently, the realization that the source of P for fertilizer manufacture was limited, more contaminated in cadmium and other heavy metals (Smil, 2000) and concentrated in very few countries, led to a major shift in evaluating the question of phosphorus. Indeed, P chemical fertilizer is the main industry using phosphate rock (80%), and tits use efficiency is very limited, since only 20% of extracted P is actually consumed as food (Cordell et al., 2009). A major cause of inefficient use of P is the localized excess of livestock manure (Senthilkumar et al., 2012). In highly specialized agricultural areas, livestock farmers struggle to find an output for their manure (Schipanski and Bennett, 2012).

The amount of manure produced is exceeding by far the needs of local crops, and transportation of manure to long distance is both economically unprofitable and damaging to the environment. Actually, phosphorus could be recovered from animal manure as struvite, a highly concentrated and slow released P fertilizer that could be exported and sold to agricultural regions specialized in crop production, reducing their need for chemical P fertilizer (Daumer et al., 2010). Pig slurry contains a significant amount of P, 200-1500mg/L (Karakashev et al., 2008;Suzuki et al., 2005), but mostly as inorganic solids (Christensen et al., 2009). Several processes for P recovery from animal manure have been tested on lab scale (Cerrillo et al., 2015;Perera et al., 2007;Perera et al., 2009) and full scale (Schuiling and Andrade, 1999;Vanotti et al., 2007), however most of them focus on already dissolved phosphorus. As a result, the recovery rate cannot excess 10 to 30% (Egle et al., 2015). Chemicals such as hydrochloric acid (Zhang et al., 2012), formate (Daumer et al., 2010) and citrate (Szögi et al., 2015) have proven efficient to dissolve P. However, these chemicals represent an important cost and make the process economically uncompetitive (Egle et al., 2016;[START_REF] Molinos-Senante | Economic feasibility study for phosphorus recovery processes[END_REF]. They are also dangerous when used for farmers and the environment. Biological acidification of pig slurry, i.e.

the natural production of organic acids by endogenous acidogenic bacteria under anaerobic conditions, has shown promising results when sucrose was used as organic co-substrate. Up to 90% of TP was dissolved at pH 4-5.5 with 30 to 60g-sucrose per liter of raw slurry (Piveteau et al., 2017). The main organic acid produced was lactate, when pH dropped low and fast enough. Lactic acid fermentation presents several advantages regarding pig slurry acidification for P recovery: lactate has a low pKa compared to other organic acid, meaning that a limited amount of co-substrate could be sufficient to reach the desired pH, and the proportion of undissociated acid after acidification is lowered compared to usual VFA. As a result the amount of alkaline compound needed thereafter in the process to increase the pH and favor struvite precipitation would be reduced. In this paper, several organic waste as well as sucrose were tested as organic co-substrate for biological acidification of raw swine slurry and digested swine slurry. pH, amount and type of organic acids produced were investigated and correlated to the waste composition. Microbial community analysis was performed in order to identify the bacterial populations responsible for organic acid production and understand how to favor lactic acid bacteria during biological acidification of pig slurry.

IV. MATERIALS AND METHODS

CHARACTERIZATION OF PIG SLURRY AND DIGESTED PIG SLURRY

Raw swine slurry was collected from a pig fattening farm in Brittany (France). Digested swine slurry (digestate) was withdrawn at two different dates from a 87 L laboratory digester treating liquid swine slurry supplemented with horse feed (Table 6).

BATCH EXPERIMENTS

Batch acidification experiments using sucrose as organic co-substrate were conducted on raw swine slurry and digestate 1 using 1 L bottles inoculated with 400 mL of raw slurry or digestate 1 and five different sucrose concentrations [START_REF]Butyrate formation from lactate[END_REF]20,30,40,50 g/ L-slurry). The batch bottle for raw slurry and 10g-sucrose/L was mishandled and broke, the partial results obtained were not included. Batch acidification test were also conducted on digestate 1 with the addition of potato, grounded biscuit and buckwheat waste at 50g-MV per liter of digestate. The bottles were sealed once inoculated, and placed in a thermostatic shaker, at 120 rpm and 38°C. Purging oxygen from the head space was not done since it had been shown to have no impact on acidification (Piveteau et al., 2017). The batch test ran for four days with daily sample collection for pH and organic acid analysis.

A second set of batch tests were conducted using digestate 2 and different organic waste (Table 7) at 50g-MV per liter of digestate. In addition of carrot, bean, pea, macédoine (diced mixed vegetables), wheat straw and apple, the same buckwheat flour and biscuit waste were tested again (hereby referred as "buckwheat flour 2" and "biscuit 2") , after 3 months of storage at 4°C in order to evaluate the effect of aging and degradation of waste. An additional batch test was conducted using a lower concentration of carrot, 30 g-MV/L instead of 50. All batch tests were done in duplicate and gave very similar results (not shown).

A sample of liquid was taken at the end of the batch test, centrifuged at 20,000g for 20 minutes and the pellet was frozen immediately at -20°C for microbiological analysis. pH was measured using a WTW probe right after each sample collection. TS, VS, TSS and VSS were measured in triplicates on the raw slurry with standard methods (APHA, 1998). Supernatants were obtained after 20 min of centrifugation (4°C, 20,000 g) and filtration on a 0.45 mm polypropylene membrane. Sucrose, glucose, fructose, lactic acid and VFA composition of the supernatant was measured by HPLC (Ultimate 3000, Dionex) with a Hiplex-H column (Agilent).

MICROBIOLOGICAL ANALYSIS

Total DNA was extracted from about 200 mg of pellet. Extraction was performed using the Macherey Nagel Genomic DNA for Soil kit according to the manufacturer's instructions. The extracted DNA was eluted in 100 µL of sterile water and stored at -20°C until further analyses. Concentration and purity of extracted DNA were checked by spectrophotometry (ND-1000, NanoDrop Tech.).

Bacterial and archaeal diversity were studied by amplifying the hypervariable region V4-V5 of the 16S rRNA genes using primers 515F (5'-GTGYCAGCMGCCGCGGTA-3') [START_REF] Wang | Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[END_REF] and 928R [START_REF] Wang | Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies[END_REF] as described in detail in Poirier et al. (2016a); Poirier et al. (2016b). The amplified DNA fragments were then sequenced using high throughput sequencing Ion Torrent™ technology and methods (Life Technologies, USA) at the NGS facility of the BIOMIC Team of IRSTEA (Antony, France). Quality filtered sequences were exported as FastQ files (47751 to 382063 reads for each sample). These reads were then quality checked by two amplicon read processing pipelines: QIIME 1.8.0 [START_REF] Caporaso | QIIME allows analysis of high-throughput community sequencing data[END_REF]USEARCH v5.2.136 (Edgar, 2013). Sequences with low quality score (<20) and chimeric sequences were removed while the remaining sequences were clustered into Operational Taxonomic Units (OTUs) at 97% sequence similarity using Usearch quality filter reference set (http://www.drive5.com/usearch/) [START_REF] Edgar | Search and clustering orders of magnitude faster than BLAST[END_REF]. The longest sequence of each OTU was chosen as representative and used for taxonomic identification using the Ribosomal Database Project (RDP II).

Phylogenetic alignment of sequences was done with the PyNAST program with a minimum length of 150 bp and a minimum percent identity of 75.0. Alpha diversity was used to describe the microbial richness, diversity and evenness of each sample using Shannon, Simpson and equitability metrics respectively.

In each sample, OTUs whose number of reads was greater than 1% of the total number of reads were considered as predominant OTUs. Predominant OTUs who had a low affiliation score were further identified by phylogenetic analysis using the ARB software [START_REF] Ludwig | ARB: a software environment for sequence data[END_REF] andSSURef_Nr99 123.1 SILVA 03032016 database. OTUs were aligned and edited against the 59847 sequences of the database using the ARB fast aligner software and fitted in the global phylogenetic tree using the ARB parsimony interactive tool with E. coli and termini filters.

STATISTICAL ANALYSIS

The microbial community structure of samples was compared by statistical methods using the open source software R (V3.2.3) [START_REF] Venables | An introduction to R. Notes on R: a programming environment for data analysis and graphics[END_REF] including the vegan package (v2.3-2) [START_REF] Oksanen | The vegan package[END_REF]. Non-metric multidimensional scaling (nMDS) based on Bray-Curtis distances was conducted using the metaMDS function of vegan. Correlations between pH, lactate concentration and microbial community structures were searched by fitting data of each factor to the nMDS ordination of the microbial community using the envfit function of vegan. The 20 tests run, with 999 permutations each, gave an excellent representativeness value of stress at 0.366994 (Table 8). Final pH logically decreased with increasing sucrose concentration, almost linearly until 30 g/L but not afterward (Figure 28). pH did not decrease below 4 despite higher initial sucrose concentration.

Those results are in line with those of Piveteau et al. (2017). The effect of sucrose on pH was stronger in the digestate, indicating a lower buffer capacity. This lower buffer capacity could be due to the little suspended solid concentration compared to the pig slurry, and the absence of VFA initially while the slurry had 10 g-VFA/L (which could itself explain the low initial pH in the raw slurry, 6.73 vs 7.86)

Lactate was the main organic acid produced after 96 hours in batch tests with 30 g-sucrose/L and higher, while very little to none was measured at 10 and 20 g/L. Homolactic fermentation of sucrose in pig slurry was observed before by Hjorth et al. (2015) and (Piveteau et al., 2017). Homolactic fermentation of glucose also occurred in digested cattle manure used for biohydrogen production [START_REF] Chatellard | The type of carbohydrates specifically selects microbial community structures and fermentation patterns[END_REF]. Lactate was produced at 10 and 20 g-sucrose/L but only during the first 48 hours before being converted to other organic acids, leading to a pH re-increase. This secondary fermentation is a common phenomenon in spoiled silage (Oude Elferink et al., 1999), and also occurred in lactic acid fermenters when pH was above 5, leading to the conversion of lactate to acetate, propionate and/or butyrate [START_REF] Kim | Hydrolysis and acidogenesis of particulate organic material in mesophilic and thermophilic anaerobic digestion[END_REF][START_REF] Komemoto | Effect of temperature on VFA's and biogas production in anaerobic solubilization of food waste[END_REF][START_REF] Li | Lactic acid accumulation from sludge and food waste to improve the yield of propionic acid-enriched VFA[END_REF][START_REF] Wu | Lactic acid production from acidogenic fermentation of fruit and vegetable wastes[END_REF].

The carbon recovery was only partial in the two batch tests series with 44 to 78% for digestate and 67-97% for raw slurry (Table 9). All sucrose was removed in all the batch reactors except for the one with digestate and 50 g-sucrose/L, where 5.6 g/L remained. The slower pH decrease in the raw slurry and the relatively low initial pH could explain why sucrose was entirely consumed in the batch with raw slurry and not with digestate. Based on the stoichiometric equation for acetate, succinate and butyrate production, a theoretical CO 2 production was calculated. The carbon recovery improved for the raw pig slurry with 90-110%, yet not for the digestate. Because the HPLC analysis was not conducted at the same time, lactate concentration in digestate batch test could have been underestimated by the machine due to a calibration issue. No production of organic acid and nor pH change occurred in the batch with wheat straw, indicating the inability of the biomass to degrade it. Similar low degradation of wheat straw was noticed by [START_REF] Chatellard | The type of carbohydrates specifically selects microbial community structures and fermentation patterns[END_REF] during co fermentation of digested cattle manure and wheat straw for biohydrogen production. In the other batch tests, pH decreased below 5 only with apple, carrots (at normal 50 g/L VS concentration) biscuit 2 and flour 2, corresponding to the production of lactate, an organic acid with a lower pKa compared to VFA (Figure 29). In the other batch tests, acetic acid was the dominant VFA followed by butyrate and propionate. The conversion of organic matter to organic acids was between 25 and 56% for macédoine and buckwheat flour 2 respectively (Table 10). A positive correlation was found between acidification and the amount of carbohydrates added to each batch test (Figure 30). Interestingly, while the repetition of the test for macédoine gave almost identical result (not shown), the same co-substrates at the same initial concentration in two different batch tests gave very different results in the case of biscuit and buckwheat flour. The two co-substrates had certainly started to degrade when used in the second batch experiment, with more accessible carbohydrates this time, leading to lactic acid fermentation rather than mixed acid. This indicates that predicting the acidifying capacity of a given organic waste is very uncertain depending on the age of the waste and how it has been stored.

In the case of carrots, the two concentrations tested also gave very different results with a lower acidification and the absence of lactic acid in the batch with 30 g-VS/L. Lactate had been produced during the first 48 hours but disappeared afterward, a secondary fermentation similar to that of acidification with sucrose. The diversity indexes calculated for the raw slurry and digestate before acidification were above 7.0 and 0.97 for Shannon and Simpson indexes respectively (Figure 31). Their equitability index was 0.7.

These values are high but in the range of usual values for these types of microbial communities [START_REF] Chen | Linkages of Firmicutes and Bacteroidetes populations to methanogenic process performance[END_REF][START_REF] Leite | Lessons learned from the microbial ecology resulting from different inoculation strategies for biogas production from waste products of the bioethanol/sugar industry[END_REF][START_REF] Lu | Bacterial communities in manures of piglets and adult pigs bred with different feeds revealed by 16S rDNA 454 pyrosequencing[END_REF]. They suggest a high microbial diversity with a few dominant species. As shown in Figure 31, the addition of co-substrate induced a drop of the three diversity indexes when accompanied by a large pH decrease. pH values far from neutrality are known to reduce bacterial diversity [START_REF] Guo | Hydrogen production from agricultural waste by dark fermentation: A review[END_REF]. Thus, the co-substrates with easily accessible carbohydrates present in large amounts (sucrose, apple, carrot 50 g-VS/L, buckwheat, flour 2) had a high lactate production, leading to a strong pH drop and decreasing bacterial diversity. This was not the case for co-substrates with fewer carbohydrates or less accessible ones (wheat straw, macédoine, bean, pea). The pH decrease was lower with those co-substrates and bacterial diversity remained stable. It appears likely that low pH and high lactate concentration acted as a selection pressure on bacterial populations, with only acid tolerant microorganisms growing in these conditions. their corresponding values of pH and lactate was performed using the nMDS method (R vegan package v2. . The nMDS representation showed several correlations (Figure 32). First, the raw slurry samples clustered all away from the digestate samples in the lower right square of the Figure 32, suggesting different global communities between these two types of matrices. Second, 9 out of the 11 samples that possessed a pH below 5 clustered in the higher right square of the Figure 32 and faraway from similar samples with higher pH, suggesting that samples with strong acidification undertook similar changes in their microbial communities. This hypothesis is comforted by the clear evolution of the slurry and digestate communities under increasing sucrose addition. While the communities belonged to diametrically opposed location initially, they gradually joined the "lactic acid area".

Finally, the distribution of the samples shows clear gradients of distribution along 2 axes influenced by the pH and lactate concentration. Thus the majority of the community was made of Bacteria that grouped within 628 OTUs identified up to the family or genus rank.

Figure 33A represents the microbial community structure, at the phylum level, of the 153 OTUs that contained at least 1% of the total sequences collected and that were assumed to represent the dominant species of the communities. In all samples, the dominant phyla were Firmicutes and Bacteroidetes, commonly found in anaerobic/acidogenic reactors [START_REF] Chen | Linkages of Firmicutes and Bacteroidetes populations to methanogenic process performance[END_REF][START_REF] Fitamo | Microbial population dynamics in urban organic waste anaerobic co-digestion with mixed sludge during a change in feedstock composition and different hydraulic retention times[END_REF][START_REF] Leite | Lessons learned from the microbial ecology resulting from different inoculation strategies for biogas production from waste products of the bioethanol/sugar industry[END_REF][START_REF] Regueiro | Microbiome response to controlled shifts in ammonium and LCFA levels in co-digestion systems[END_REF]Shen et al., 2017). In raw slurry these phyla represented 57% and 23% of the total sequences respectively and were dominated by members of Clostridiaceae (10% of sequences) and Prevotella (7% of sequences) respectively, both microbial groups widely present in swine slurry (Snell-Castro et al., 2005). In the initial digestate, Bacteroidetes was the main phylum with about 37% of the total sequences. It contained several genera with 2 representing 7 and 10% of the sequences that were related to uncultured species retrieved from anaerobic digesters (Accession numbers: AB494402 and CU922250 respectively). The Firmicutes represented 30% of the initial digestate sequences with Clostridium as the most represented genus and sequences close to sequences retrieved from anaerobic digesters (Accession numbers: MF769126, KY224082, GQ135833).

The comparison of all the samples highlights the decisive role of a few dominant species during acidification (Figure 33B). In all reactors, acidification was associated with an increase in the proportion of Firmicutes and a reduction of Bacteroidetes. In the case of slurry with sucrose,

Clostridiales increased massively at 20 and 30 g-sucrose/L but were dominated by Lactobacillus at higher sucrose concentrations. The dominant species of Lactobacillus were L. amylophylus and L.

amylovorus, both strict homofermentative lactic acid bacteria (LAB) [START_REF] Mercier | Kinetics of lactic acid fermentation on glucose and corn by Lactobacillus amylophilus[END_REF][START_REF] Weinberg | The effect of temperature and Lactobacillus amylovorus and Lact. plantarum, applied at ensiling, on wheat silage[END_REF], as reflected by the high proportion of lactate in the batch test with high initial sucrose concentration. These LAB species were discovered in the fermentation broth of swine wastewater and corn [START_REF] Nakamura | Lactobacillus amylophilus, a new starchhydrolyzing species from swine waste-corn fermentation[END_REF] and in the fermentation broth of cattle waste and corn respectively [START_REF] Nakamura | Lactobacillus amylovorus, a New Starch-Hydrolyzing Species from Cattle Waste-Corn Fermentations[END_REF]. In the batch test with digestate and sucrose, Clostridiales remained stable at any sucrose concentration while both Lactobacillus and Streptococcus increased massively with sucrose concentration, Streptococcus at 10 and 20 g/L, Lactobacillus at 30 and 40 g/L. Thus, it appears that Clostridiales and Lactobacilliales were able to compete successively for sucrose compared to Bacteroidetes and other bacteria, but also competed with each other, Lactobacillus dominating at higher initial sucrose concentration and associated low pH. The success of Clostridiales at moderate sucrose concentration was likely due to their capacity to grow on lactate.

In the batch tests with digestate and organic waste, Lactobacillus systematically dominated when the co-substrate had a high content in easily accessible carbohydrates, e.g. apple, carrot (at high initial concentration, biscuit 2 and buckwheat flour 2). In contrast, Clostridiales performed better than Lactobacillus in biscuit 1 and buckwheat flour 1, indicating a better capacity for hydrolysis.

Streptococcus was significantly present in most samples, indicating a capacity to function and grow with more diverse or more limiting substrate than Lactobacillus. Indeed, Lactobacillus was absent in the batch with low concentration of carrot, while Streptococcus represented 20% of the population. It has been documented that Streptococcus is able to grow on a large variety of carbohydrates (Holzapfel and Wood, 2012) and shift its metabolic pathway from homolactic fermentation to mixed acid under limiting substrate concentration [START_REF] Thomas | Change from homo-to heterolactic fermentation by Streptococcus lactis resulting from glucose limitation in anaerobic chemostat cultures[END_REF]. However, Streptococcus has a very low tolerance to acidic condition [START_REF] Axelsson | Lactic acid bacteria[END_REF], which would explain its inability to dominate at high initial sucrose concentration where pH fell below 5.

Among the Bacteroidetes, Porphyromonadaceae were able to increase their share of the population from 6% initially to 30-40% in macédoine and biscuit 1, and to at least 10% in all the batch tests at pH above 5. This family of bacteria is strictly anaerobic and originates from the digestive tract of mammal, hence its presence in raw and digested swine slurry. Moreover, they have been observed in several food waste anaerobic digesters [START_REF] Fitamo | Microbial population dynamics in urban organic waste anaerobic co-digestion with mixed sludge during a change in feedstock composition and different hydraulic retention times[END_REF][START_REF] Dai | A synergistic combination of nutrient reclamation from manure and resultant hydrochar upgradation by acidsupported hydrothermal carbonization[END_REF]Jang et al., 2016;[START_REF] Leite | Lessons learned from the microbial ecology resulting from different inoculation strategies for biogas production from waste products of the bioethanol/sugar industry[END_REF][START_REF] Chen | Linkages of Firmicutes and Bacteroidetes populations to methanogenic process performance[END_REF]. They are able to grow on various carbohydrates and produce mainly butyric acid [START_REF] Sakamoto | Butyricimonas synergistica gen. nov., sp. nov. and Butyricimonas virosa sp. nov., butyric acid-producing bacteria in the family 'Porphyromonadaceae' isolated from rat faeces[END_REF]. These bacteria were found in significant amounts during the fermentation of swine slurry at 35°C with initial pH adjustment to 10 (Lin et al., 2015).

This microbiological analysis demonstrated that even though Clostridiaceae were a major cluster of bacterial population in all the batch tests, they did not systematically dominate as it is often the case during acidogenic fermentation [START_REF] Feng | Enhancement of Waste Activated Sludge Protein Conversion and Volatile Fatty Acids Accumulation during Waste Activated Sludge Anaerobic Fermentation by Carbohydrate Substrate Addition: The Effect of pH[END_REF][START_REF] Ren | Assessing optimal fermentation type for bio-hydrogen production in continuous-flow acidogenic reactors[END_REF]. High initial sucrose concentration and easily degradable carbohydrate rich co-substrates enabled LAB and especially Lactobacillus to outcompete other bacteria for substrate and remain dominant thanks to its tolerance for acidic pH (Holzapfel and Wood, 2012) and possibly the production of inhibiting substances such as undissociated lactic acid and bacteriocins [START_REF] Monlau | Specific inhibition of biohydrogen-producing Clostridium sp. after dilute-acid pretreatment of sunflower stalks[END_REF][START_REF] Noike | Inhibition of hydrogen fermentation of organic wastes by lactic acid bacteria[END_REF]. Pour des valeurs de pH proches de la valeur optimale de 5.5-6 une fermentation secondaire intervient systématiquement ce qui confirme l'hypothèse émise précédemment selon laquelle le procédé devra être piloté avec un suivi rapproché du pH pour réaliser la séparation lorsque le ratio Ca/P est optimal.

Comme lors des essais précédents, l'évolution de la concentration en P dissous lors de l'acidification avec les co-substrats est étroitement liée à l'évolution du pH pour 3 des quatre lisiers. Un comportement différent a cependant été observé pour un des lisiers qui avait une concentration initiale en P total très faible. Dans ce cas et surtout lorsque les quantités de co-substrat ajoutées sont importantes l'assimilation du P pour la croissance de la biomasse n'est plus négligeable et réduit la concentration en P dissous observée. Si le co-substrat est riche en P comme c'est le cas des petits pois, la concentration en P dissous sera liée à l'hydrolyse progressive des formes organiques contenant le P sous l'effet de l'acidification.

La dissolution du Ca est plus variable suivant les lisiers. Elle est comparable à celle observée précédemment avec 2 des 4 lisiers. Dans le lisier pauvre en P et en Ca, une part importante du Ca vient du co-substrat et comme pour le P sa dissolution est liée à l'hydrolyse du co-substrat. Dans le lisier le plus riche en Ca, on observe une augmentation brutale de la concentration dissoute lorsque le pH est proche de 5 ce qui laisserait supposer que la forme sous laquelle se trouve le Ca dans ce lisier est différente de celle des autres lisiers. La concentration en ammonium évolue peu avec l'acidification sauf dans les essais avec les petits pois dans lesquels l'évolution constante semble être liée à l'hydrolyse du co-substrat. Afin d'évaluer l'intérêt de conserver un inoculum acidogène adapté au co-substrat des essais en semicontinu ont également été réalisés. Une alimentation tous les jours ou tous les quatre jours a été mise en oeuvre. Les résultats étant identiques, seul l'essai avec une alimentation quotidienne sont présentés dans l'article. Après avoir rempli un réacteur avec 4 litres du lisier 4, un mélange petits pois carottes a été ajouté et le réacteur placé sous agitation pendant 4 jours. Ensuite, un quart du réacteur était remplacé quotidiennement par un mélange lisier petits pois et carottes dans les mêmes proportions (temps de séjour de 4 jours) pendant 12 jours. Le pH est descendu rapidement à 5.7 pendant les 4 premiers jours puis le pH minimum a baissé progressivement jusqu'à 5.4. La concentration en P dissous est passée de 150 à 680 mg.L-1 en quatre jours puis a augmenté progressivement pour atteindre 747 mg.L-1 soit 57% du P total du mélange au bout des 16 jours. L'effluent recueilli pendant la durée du test a été centrifugé puis analysé avant d'être utilisé pour des essais de cristallisation. La centrifugation a permis de récupérer 83% du volume sous forme liquide soit 47% du P total initial. Sa composition est proche de celle observée lors des tests batchs avec un ratio N :P :Mg :Ca de 6.5 :1 :0.9 :0.4 soit une quantité d'ammonium favorable à la cristallisation de struvite, un léger déficit en magnésium et la présence de calcium en proportion assez importante.

La cristallisation a été réalisée en bécher agité. Environ 3.5g d'hydroxyde de magnésium ont été nécessaire pour atteindre pH 8. Le mélange a été laissé sous agitation pendant 2 heures. Plus de 99% du P dissous a été précipité. Après cristallisation le solide a un ratio N :P :Mg :Ca de 1 :1.19 :1.32 :0.05 proche de celui de la struvite avec un léger excès de magnésium et de phosphore et une faible contamination par le calcium. La struvite étant l'un des seuls minéraux contenant de l'ammonium susceptible de se former dans nos conditions la quantité de struvite a été calculée d'après la concentration en ammonium. Le solide produit est composé à 69% de struvite, le reste étant probablement d'autres composés minéraux phosphatés, de l'hydroxyde de magnésium non dissous et de la matière organique.

Le faible temps de contact et le mode d'agitation ont conduit à l'obtention de cristaux de petite taille et hétérogènes. Aussi, malgré une précipitation presque totale du P dissous, le solide issu de la filtration à 100 µm n'a permis de recueillir que 26% du P total du mélange initial. Population growth increases the need for higher crop yield and therefore P-based fertilizers. Despite varying estimations regarding the amount, quality and accessibility of P reserves, it is commonly accepted that a sustainable agricultural system assuring long term food security requires a more efficient use of fertilizer and the large scale implementation of P recovery processes for industrial and municipal wastewaters as well as livestock waste streams (Cordell et al., 2009). Agronomical progress to optimize fertilizer use and development of P recycling technologies would also severely limit soil leaching and associated eutrophication of water bodies. The agricultural specialization of large areas into crop farming on one hand and livestock breeding on the other hand has led to major imbalances in term of localized phosphorus need/excess. While crop-oriented regions rely heavily on fertilizer to compensate their P exports, regions focused on intensive livestock production struggle to find outputs for their manure. Recovering phosphorus from livestock farms under a concentrated and easily transportable form would limit the over application of manure in P-saturated soils and provide an alternative P-based fertilizer for other areas in need.

Many full-scale P-recovery processes have been successfully implemented for a wide range of domestic and industrial wastewaters, e.g. production of calcium phosphate from the supernatant of anaerobic EBPR sludge through the Crystalactor® process, [START_REF] Giesen | Crystallisation Process Enables Environmental Friendly Phosphate Removal at Low Costs[END_REF], production of a N-P-K fertilizer from sewage sludge ashes using the Ash Dec® process [START_REF] Mattenberger | Sewage sludge ash to phosphorus fertiliser (II): Influences of ash and granulate type on heavy metal removal[END_REF], production of struvite (NH 4 MgPO 4 ,6H 2 O) from reject water of a municipal digester and anaerobic effluent of a potato processing plant with PHOSPAQ TM technology (Abma et al., 2010), from municipal EBPR sludge dewatering liquid using the WASSTRIP® system [START_REF] Schauer | Full-scale implementation of the WASSTRIP™ process: plant-wide impact of struvite recovery[END_REF] and from acidified digested sludge through the Seaborne process [START_REF] Günther | Technical and scientific monitoring of the large-scale seaborne technology at the WWTP Gifhorn[END_REF]. However, very few P-recovery technologies have been implemented on full scale to treat livestock waste (Desmidt et al., 2015). A full scale demonstration unit for P-recovery as calcium phosphate from liquid swine slurry was tested during one year in Mount Olive, North Carolina using nitrification to reduce the buffering capacity and calcium hydroxide to bring the pH up into precipitation range and promote calcium phosphate precipitation (Vanotti et al., 2007). A full-scale plant producing K-struvite from the liquid phase of calf manure has been operating in Putten (Netherlands) since 1999, using magnesium oxide to increase the pH (Schuiling and Andrade, 1999). Several P-recovery technologies applied to swine wastewater are still tested on lab-scale. Because magnesium is usually the limiting ion for struvite precipitation, MgCl 2 is added, while aeration and/or NaOH are used for pH adjustment [START_REF] Burns | Optimization of phosphorus precipitation from swine manure slurries to enhance recovery[END_REF]Liu et al., 2011;Rahman et al., 2011;Ye et al., 2011). Each livestock wastewater treatment described above only recovered precipitated phosphorus from the liquid phase, leaving P in the solid phase untouched. Because phosphorus in pig slurry is mostly present in inorganic solids (Barnett, 1994;Daumer et al., 2008;Toor et al., 2006), precipitation of orthophosphate in the liquid phase enables a low concentration in the effluent that meets discharge standards, but also a very limited P-recovery as mineral fertilizer overall. For example, in the Mount Olive treatment unit described earlier, the solid phase containing 70% of total phosphorus (TP) was not directed to the calcium phosphate recovery process but sent instead to a centralized solid processing unit nearby for aerobic composting.

Therefore, acidification of pig slurry to dissolve inorganic phosphorus is a necessary step if high Precovery as mineral fertilizer is the goal. It has been shown that 70% of TP could be dissolved when pH was decreased to 5.5 (Christensen et al., 2009). Chemical acidification of swine slurry has been investigated on lab-scale using organic acids such as acetate and formate allowing a dissolution 89% of TP at pH 4.9 and 95% at pH 4.75 respectively (Daumer et al., 2010). Following this dissolution experiment, phosphorus was precipitated as struvite using magnesium oxide (MgO), but the process was considered economically noncompetitive due to the cost of formic acid. A possible solution to avoid the cost of chemical acidification is to take advantage of the biological process known as acidogenesis. Under anaerobic conditions, organic co-substrates added to pig slurry are fermented by endogenous acidogenic bacteria into organic acids, which decreases pH. Besides, the organic acids produced during acidogenesis could be used for methane production in a subsequent digestion process.

Biological acidification of swine slurry using glucose as co-substrate (30g/L) led to the production of lactic acid and a pH of 4.3 (Hjorth et al., 2015). Another experiment with sucrose as co-substrate showed that 60 to 90% of TP could be dissolved when pH reached 4-5.5, with lactic acid as the main fermentation product (Piveteau et al., 2017).

In Brittany, France, intensive pig breeding and over application of slurry in the fields has led to the contamination of fresh waters and coastal algal blooms. Due to the regulatory limits imposed on spreading of slurry during the 90's, around 500 treatment units have been built in the region, mostly with nitrification-denitrification and activated sludge processes. Since the beginning of the new millennium, 50 anaerobic digesters have also been installed to produce energy by co-digesting pig slurry with organic waste from agricultural, industrial and municipal activities nearby. In both treatment pathways, the solid phase that contains most of the phosphorus is partially spread while complying with the stringent regulatory guidelines or composted, exported and sold as an organic fertilizer in other regions. Struvite, a P-based, slow-release mineral fertilizer would be more easily exported due to its higher P content and the organic, solid phase could be kept in Brittany. The hydrolysis taking place during biological acidification of organic waste and pig slurry could also enhance the energy production through higher methane yield.

In this study, biological acidification of pig slurry was investigated in anaerobic batch tests using apples, carrots, green beans, peas and sucrose as organic co-substrates. Those fruits and vegetables were chosen because there are common products grown in Brittany and available locally as agricultural or industrial waste. They possess different levels of sugar content, apple having the highest content followed by carrot, green bean and pea. Sugars are the most effective organic compounds to produce organic acids and lower the pH (Yin et al., 2016). The model linking buffer capacity of the slurry, initial pH and sucrose concentration with lowest pH reached during acidification (Piveteau et al., 2017) was extended to those co-substrates and its reliability was tested in batch tests with four slurries and three different concentrations of co-substrates. pH, organic acids concentration and dissolution of P, Mg, N and Ca were studied throughout the batch tests. A semi continuous reactor fed with swine slurry, carrots and peas was set up to produce a stable acidified effluent (pH = 5.42).

After centrifugation, magnesium hydroxide was added to the supernatant to bring the pH to 8 and precipitate phosphorus. The dried solid recovered was dissolved at acidic pH and ionic concentrations were measured to verify that struvite was effectively produced.

IV. MATERIALS AND METHODS

1. PIG SLURRIES Four raw swine slurries were collected from four different commercial pig fattening farms in Brittany.

The composition of the slurries can be found in Table 11. Their buffer capacity was measured in a beaker containing 100 mL of slurry with a magnetic stirrer, a pH probe and a graduated burette containing sulfuric acid (1 mol/L). The buffer capacity was expressed as milliequivalent per liter per pH point and calculated according to the equation (Equation 15) below:

ORGANIC CO-SUBSTRATES

Frozen carrots, beans and peas as well as fresh apples from the local supermarket were used as complex organic co-substrates for pig slurry biological acidification. Their composition can be found in Table 12. A biological acidification with each organic co-substrate at 50g-VS per liter of digested slurry was performed in simplicate. Based on the lowest pH obtained with the co substrate, Eq.1 could be solved for the corresponding sucrose concentration. The sucrose equivalent (g-sucrose/g-VS) was calculated as the corresponding sucrose concentration (g-sucrose/L) divided by 50 (g-VS/L). The test was repeated with a lower concentration of co-substrate in the case of carrots and apples (30 and 36g-VS/L for carrot and apple respectively) due to an acidification that went beyond the linear pH range. The sucrose equivalent of each co substrate can be found in Table 13. The tests were conducted in 1L bottles inoculated with 400 mL of raw pig slurry. Three different concentrations for each organic co-substrate and sucrose were added to the bottles before sealing them with a lid. The three concentrations of co-substrate ("low", "medium", "high") were calculated using a simple empirical model adapted from Piveteau et al. (2017) oxygen removal was performed in the headspace since it has no effect acidification (Piveteau et. al. 2017). The bottles were placed in a thermostatic shaker, at 120 rpm and 38 °C. Samples were taken every 24 hours during 4 days for analysis. The same protocol was applied to the four slurries. Co-substrate (g-VS/L) = Sucrose 0 (g/L) / sucrose eq (g-sucrose/g-VS)

Table 14. Co substrates and sucrose concentrations in each batch test (g/L)

SEMI CONTINUOUS REACTOR

A semi-continuous reactor for biological acidification was set up with the objective of studying the effect of semi-continuous feeding on acidogenic biomass and potential improvements in term of pH decrease, organic acids production, and P dissolution. Slurry 4 (3 L), carrots (560 g) and peas (440 g) were introduced in a 4 L reactor under anaerobic conditions with a helix for mixing. After 4 days, semi continuous operation of the reactor was started with one fourth of the volume withdrawn and replaced once a day with the same proportion of pig slurry, carrots and peas (HRT = solid retention time = 4 days). The reactor was operated during 16 days, 4 days as batch and 12 days (3 HRT) in semicontinuous mode. pH, organic acids and ionic concentrations were measured daily in the effluent.

CRYSTALLIZATION PROCESS

The effluent collected during the last five days of semi continuous operation in the semi continuous reactor was used for struvite precipitation. again the main acid when minimum pH was reached but in lower proportion than sucrose and apples (30-75%).Secondary fermentation of lactate occurred systematically at medium and low initial concentration Batch tests with green beans had 41 to 48% of the added VS converted to organic acids, none of it lactate. Finally, batch tests with pea had the lowest conversion to organic acids with only 18 to 34% of the added VS converted to organic acids. The hierarchy in acidifying capacity of each cosubstrate, expressed via their "sucrose equivalent" (Table 13) was confirmed by the similar ranking of their conversion to organic acid and their proportion of lactic acid.

As described in the case of pea and bean, the use of a fixed sucrose equivalent to define the acidifying capacity of an organic co-substrate relatively to sucrose was inaccurate when a low pH (4-5) was targeted, due to the higher pKa of the organic compounds produced compared to lactate. However, when biological acidification of pig slurry is used for maximum P recovery as struvite, a pH of 5.5-6

was considered as optimal by Piveteau et.al. (2017). As a result, this sucrose model expanded to organic co-substrate could still be accurate enough for limited biological acidification. very loosely linked to pH in slurry 1 (Figure 39A). This difference could be explained by the relative proportion of P originating from the slurry compared to the co-substrates themselves. Slurry 1 had only 230 mg-TP/L. As a result, P from co-substrates represented up to 72% of TP (Table 16). It could be postulated that orthophosphate concentration was determined by both the kinetics of hydrolysis/degradation of the co-substrates and pH (Figure 40). Indeed, as seen in Figure 40, phosphate concentration for P-rich co-substrates (in decreasing order: pea, bean, carrot) evolved similarly to a first order hydrolysis reaction. In the case of sucrose, P concentration and pH remained stable from 24h until the end, indicating logically that only inorganic P was dissolved, driven by pH.

Low phosphate concentration measured in the batch tests with apple, carrots and sucrose despite the low pH value (4)[START_REF] Yin | Metabolic pathways for glycerol degradation during acidogenesis[END_REF] was likely due to P incorporation during biomass growth. Indeed, considering a biomass chemical formula as C 5 H 7 NO 2 P 0.074 (Droste, 1997), and a yield on sucrose of 0.14 (biomass COD.sucrose COD -1 ) (Gujer and Zehnder, 1983), 121, 93 and 65 mg-P/L were necessary at high, medium and low sucrose concentration for biomass growth, representing 52, 40 and 28% of TP in slurry 1.

In slurry 2, 3 and 4, dissolved P concentration increased with decreasing pH down to pH 5.5 and then increased slower until pH 4 in slurry 2 and 4, and decreased in slurry 3. The lower phosphate concentration in slurry 3 at pH 4-5 was associated with the high initial concentration of carrots, apple and sucrose. Consequently, biomass growth and associated need for P was high in those batch tests while carrot, apple and sucrose provided little or no P. The maximum proportion of dissolved P/TP was 85, 80, 94 and 73% in slurry 1, 2, 3 and 4 respectively (Figure 41A, slurry 1 was not included since P was not directly correlated with pH). Those values are relatively homogenous however it should be noted that the high maximum of dissolved-P / TP in slurry 1 was mostly due to P originating from peas and beans, not the slurry. This indicates that in the case of pig slurries with low P content, the choice of co-substrate should not only be guided by its acidifying capacity but also its P content. In slurry 1 the concentration of Ca 2+ expressed as percentage of T-Ca appeared to be both pH and time dependent. Indeed, in the case of carrots batchs, pH dropped in the three reactors during the first 24 h and remained stable afterwards, while Ca 2+ concentration kept on increasing. At low initial concentration, Ca 2+ increased from 60% at 24h to 80% of T-Ca at 96h (pH stable at 5.5), and at high initial concentration, Ca 2+ increased from 80 to 100% of T-Ca (pH stable at 4.3). While pH dependency tends to indicate dissolution of inorganic solids and time dependency usually results from hydrolysis and degradation of organic matter, it is also possible that inorganic solids dissolution was simply not instantaneous. However, 48, 41 and 33% of T-Ca came from the carrots added to the slurry (at high, medium and low initial concentration respectively), and 103, 90, 84% of T-Ca was dissolved at the end of the batch test, indicating that a large part of the dissolved calcium came from the carrots.

As a result, it seems likely that decomposition of organic matter participated in Ca 2+ increase, not solely pH-driven dissolution of inorganic solids. In the other slurries, calcium dissolution was driven by pH change (Figure 41C). The trends were similar in slurry 2 and 3 with a linear dissolution from approximately 10% at initial pH, 30-50% at pH 5.5 and 50-80% at pH 4.5. The dissolution of calcium in slurry 4 was slightly different, with no dissolution from initial pH (6.5) to pH 5.5 (less than 10% of even though carrot has been shown to lead to lactic acid fermentation in batch tests. It appears likely that the 4 days retention time and limited acidification has led to secondary fermentation of lactate in the semi-continuous reactor. This reactor had been operated for 16 days only. Possibly shorter retention time or longer operation of the reactor could have led to the selection of LAB versus other acidogenic bacteria. The supernatant obtained after centrifugation was used to recover P as struvite.

83% of the volume centrifuged was recovered as supernatant thanks to an efficient coagulant. More than 99% of phosphates precipitated, corresponding to 41.5% of initial TP. The actual recovery rate was not 41.5% but 26% because some of the struvite crystals were too small to be retained by the 100 µm mesh sieve. However, a simple system with longer retention time and recirculation of the effluent should lead to near complete recovery of precipitated P. The organic content of the solid recovered was relatively high 10-20%. A washing process of the crystals with clean water might help remove some of the organic matter contamination. Even though no heavy metal analysis had been performed when the work in chapter 2 was submitted for publication, certain heavy metal contaminants have been measured since. Zinc and copper represented 152 and 157 mg/Kg-DM, below the 200 mg/Kg-DM recommended for organo-mineral fertilizer by the European Sustainable Phosphorus Platform.

Legislative efforts should be pursued in order to obtain a certification for struvite fertilizer derived from animal manure. Certified struvite with lower contamination rates compared to conventional fertilizers would increase the marketability of the product and its selling price.

Current estimations put the selling price of struvite between 188 and 763 $/T [START_REF] Molinos-Senante | Economic feasibility study for phosphorus recovery processes[END_REF]. The process described in this PhD would reduce the price of struvite recovery by up to 65% compared to chemical acidification. Taking into account the cost of energy, generic flocculant/coagulant and magnesium hydroxide, the total cost would be between 500 and 1500 $ per ton of struvite produced, depending on quality of chemicals used and the rebate obtained on bulk purchase. As a result, the process would be profitable if the selling price of struvite is in the upper range and if coagulant and magnesium hydroxide can be bought at minimum price.

As described in section II.1. of the context (agronomic value of struvite), the physical aspect of the product obtained is critical for its re-use as fertilizer. The precipitation pilot developed by IRSTEA should be used to optimize the parameters applied to obtain a product with sufficient length and crushing strength to be directly usable as a mineral P fertilizer.

A potential source of improvement to lower the cost of struvite recovery would be to minimize the need for magnesium hydroxide. This chemical was used to bring the pH from 5.4 to 8 to enable struvite crystallization. The supernatant contained approximately 24g of organic acids per liter of acidified slurry. Because this was not a lactic acid fermentation, acetate propionate butyrate and valerate were the main organic acids, representing respectively 42, 21, 25 and 10% of all acids produced (g/g). As a result, 63 mmol or 20% of these acids were under an undissociated form at pH 5.4. Deprotonation of VFAs accounted for 70% of the acid buffer capacity contained in the

  Des travaux complémentaires sur les paramètres du procédé pour orienter la microflore et le métabolisme vers la production d'acide lactique permettraient de s'assurer d'un pH suffisamment bas et stable pour optimiser la dissolution du P. De plus la production majoritaire d'acide lactique réduirait le pouvoir tampon du lisier lié à la présence d'acides gras non dissociés ( acides organiques dont le pKa est plus faible) et ainsi la quantité de réactif nécessaire à ajuster le pH lors de la cristallisation de III. INTRODUCTION ....................................................................................................................................... IV. MATERIALS AND METHODS ....................................................................................................................

  .. V. RESULTS AND DISCUSSION ......................................................................................................................

  ... a. Phosphorus ..................................................................................................................................................... b. Magnesium calcium and nitrogen dissolution ................................................................................................

  .. VI. CONCLUSIONS ......................................................................................................................................... VII. FUNDING SOURCE ............................................................................................................................... VIII. REFERENCES ......................................................................................................................................

Figure 13 .Figure 15 :Figure 16 :Figure 18 :Figure 28 .Figure

 1315161828 Figure 13. metabolic pathways for the degradation of organic matter under anaerobic conditions. Adapted from[START_REF] Moletta | La digestion anaérobie: du plus petit au plus grand[END_REF] ................................................................................................................ 39 Figure 14: proportion of protonated acid as a function of pH ............................................................... 40 Figure 15: metabolic pathways during acidogenesis. Dotted lines correspond to secondary fermentation of primary molecules ....................................................................................................... 41 Figure 16: Stickland reaction between alanine and glycine .................................................................. 42 Figure 17: catabolic pathways in LAB for carbohydrates (adapted from Wang, 2015). ....................... 46 Figure 18: Shifts in bacterial population during the different steps of silage processing (Dunière et al., 2013) ...................................................................................................................................................... 47 Figure 19: Conversion of lactate and acetate to butyrate hydrogen and CO 2 (Duncan, 2004) .............. 48 Figure 20: Acrylate pathway for lactate fermentation to propionate by Clostridium propionicum (http://microbiochem.weebly.com/propionate.html) ............................................................................. 49 Figure 21. Envisioned implementation of struvite recovery within an anaerobic digestion process: parallel biological acidification. Implementation of biological acidification prior to digestion is also possible .................................................................................................................................................. 51 Figure 22. Evolution of pH across time for each initial sucrose concentration (standard deviation displayed) .............................................................................................................................................. 77 Figure 23. Net production of VFAs and lactate, sucrose removed and pH across time. Standard deviation shown for the sucrose removed and the sum of VFAs and lactate. A: Low initial sucrose concentration (30 g/L). B: High initial sucrose concentration (50 g/L) ................................................ 80 Figure 24. A: Dissolved phosphorus across time for each sucrose concentration. B: Dissolved magnesium. C: Dissolved calcium. D: Ammonium .............................................................................. 82

Figure 1 :Figure 2 :

 12 Figure 1: Production of phosphate rock in 1997 and 2016

Figure 3 :Figure 4 :

 34 Figure 3: World phosphate rock production from 1960 to 2017 (USGS data)

Equation 1 : 2 :

 12 Formation of struvite Equation Formation of HAP and HDP a. TECHNOLOGIES FOR PHOSPHORUS RECOVERY FROM MUNICIPAL / INDUSTRIAL WASTEWATERSIn municipal wastewater treatment plants (WWTP), a significant fraction of dissolved P (30-40%) is taken up during biomass growth thus ending up in the sludge. In order to meet the discharge requirements, iron and/or aluminum addition leads to the co-precipitation of P, trapping it in the sludge phase too. A biological process known as Enhanced Biological Phosphorus Removal (EBPR) or Bio-P uses Polyphosphate Accumulating Organisms (PAO) to naturally store P in the sludge through the alternation of anoxic and aerated phases. Because the biomass can be inhibited by toxic compounds and variations of load and temperature, Bio-P is often used in combination with chemical precipitation to reach discharge limits. Use of EBPR is increasing in order to reduce the cost associated with Fe and Al addition. Besides, P recovery is much more difficult once P is bound to metals. Technologies have been developed to recover phosphorus at different location within a WWTP (Figure5):-Final effluent[START_REF]A Net production of VFAs and lactate, sucrose removed and pH across time in R10. Standard deviation shown for the sucrose removed and the sum of VFAs and lactate[END_REF] -Liquid phase of anaerobic thickener[START_REF]APPENDIX B Net production of VFAs and lactate, sucrose removed and pH across time in R20[END_REF]

Figure 5 .

 5 Figure 5. Access points for P recovery in municipal WWTP (adapted from Egle et al., (2015))

Figure 6 .

 6 Figure 6. Aiprex process for struvite recovery (adapted from P-rex)

7

  mg-P/L in the influent to the WWTP (the actual value was not given), the recovered P represented only 16% of the total P flow.Egle et al. (2015) estimated the recovery rate at 20%. The Ostara Company has now 14 Pearl® reactors, 10 of which equipped or planned to be equipped with the Wasstrip® process.

Figure 7 .

 7 Figure 7. Combination process of Pearl and Wasstrip

Figure 8 .

 8 Figure 8. Seaborne process (adapted from Müller et al. (2007))

Figure 9 .

 9 Figure 9. Stuttgart process

Figure 10 .

 10 Figure 10. Budenheim process

  4 by using a prior wet chemical treatment identical to the Seaborne and Stuttgart technology: acidification with sulfuric acid, this time at a pH as low as 1.5-2. The acidified sludge then undergoes a low pressure wet air oxidation treatment (160-200°C, 20 bar) using the LOPROX® technology. Residual solids naturally settle while the liquid is filtered by ultrafiltration and nanofiltration in order to remove HMs, Fe and Al. Despite the use of advanced technology, only 55% of TP is obtained in the filtrate due to poor selectivity during filtration.

Figure 11 .

 11 Figure 11. Ash Dec process

Figure 12 .

 12 Figure 12. P-recovery process for pig manure. Adapted from Vanotti et al. (2007)

  Thus,[START_REF] Ryu | Application of struvite precipitation as a pretreatment in treating swine wastewater[END_REF] focused on ammonium removal: they used a CSTR reactor with mechanical mixing and MgCl 2 , K 2 HPO 4 , NaOH to adjust the N:P:Mg ratio to 1:1:1 and increase the pH to 9 with an hydraulic retention time (HRT) of 16-48 h. A nitrification denitrification reactor received the effluent from the struvite reactor. Overall, complete ammonium removal was achieved, with 80-90% during struvite precipitation. Because the raw slurry had 2-5 g-TSS/L and no struvite accumulation device was used, the solid obtained had a high contamination by organic matter (35%). TP content of the slurry was relatively low, 100-200 mg-TP/L, with only 30% as phosphate. 95% of TP was effectively removed from the effluent, but the solid obtained potassium, sodium, calcium and silicon on top of the organic matter making it unsuitable for processing as a mineral fertilizer without further treatment. Similarly,Rahman et al. (2011) achieved a 92% PO 4 -P recovery rate (no TP given) in an aerated reactor with MgCl 2 addition and an HRT of 18 hours. However, due to the settling of half of the suspended solids, struvite represented only 6.6% of the solid recovered. Air agitation also led to the removal of 30% or 1.2 g/L of ammonia. With the same reactor configurationLiu et al. (2011) observed the co-precipitation of half of dissolved Na, K, Zn, Cu, and Ca in the influent.[START_REF] Taddeo | Sustainable nutrients recovery and recycling by optimizing the chemical addition sequence for struvite precipitation from raw swine slurries[END_REF] obtained relatively pure struvite without a dedicated accumulation device in an aerated batch reactor with Mg(OH) 2 addition, an aeration time of 1.5 hour and settling of 30 minutes. However, the phosphate concentration had been multiplied by 75 (from 64 to 4776 mg/L) to reach a molar ratio N:P of 1:1. As a result, the amount of struvite is artificially inflated compared to the suspended solids initially present and compared to the co-precipitation of phosphorus with calcium.Suzuki et al. (2005) used an aerated reactor with a settling zone and a stainless steel mesh wire recovery device, with no addition of magnesium. The effluent contained 50% less phosphate 65% less TP. The struvite recovered on the accumulated device was very pure (98%) without Zn and Cu, but represented only 4.5% of TP introduced in the reactor. In a follow-up study[START_REF] Suzuki | Removal and recovery of phosphorous from swine wastewater by demonstration crystallization reactor and struvite accumulation device[END_REF] added bittern to reach a Mg:P ratio of 1.6:1 and improved the recovery device. During a 70 days period, the process achieved a 66% phosphate removal, a similar TP removal, with 50% of TP in the settled solids and 18%-TP in very pure struvite (95%, no Zn or Cu).

  , as described earlier. The resulting high suspended solid concentration severely impedes struvite crystallization. In order to obtain a liquid with low SS content and high ortho-P/TP ratio,[START_REF] Zhang | Recovery of phosphorus from dairy manure: a pilot-scale study[END_REF] tested several pre-treatments on liquid cow manure. An unknown amount of sulfuric acid was added initially to dissolve P by lowering the pH to 3-4. The acidified manure underwent a microwave (MW) process with the addition of H 2 O 2 . Solid and liquid were separated through a simple clarification stage.Microwave and oxidation massively lowered the SS content by solubilization/destruction and by improving the settleability of the remaining suspended solids. By clarification, 70% of the volume was recovered as SS-free supernatant. Initially, 40% of the 220 mg-TP/L were ortho-P. With acidification to pH 4, the ratio of ortho-P / TP increased to 60%. With acidification plus MW-H 2 O 2 (0.1-0.3% H 2 O 2 ), the ratio increased to 90-95%, indicating that the increase in dissolved P was due in equal part to acidification and MW-H 2 O 2 . Oxalic acid was added to the supernatant to bind to calcium and prevent re-precipitation of phosphorus with it. A ratio of 2 moles oxalic acid per mole Ca was enough

Figure 13 .

 13 Figure13. metabolic pathways for the degradation of organic matter under anaerobic conditions. Adapted from[START_REF] Moletta | La digestion anaérobie: du plus petit au plus grand[END_REF]

Figure 14 :

 14 Figure 14: proportion of protonated acid as a function of pH

Figure 15 :

 15 Figure 15: metabolic pathways during acidogenesis. Dotted lines correspond to secondary fermentation of primary molecules

  had protein conversion to VFA of 40-70% while glucose conversion was 88%. Similarly,[START_REF] Liu | Acidogenic fermentation of proteinaceous sewage sludge: Effect of pH[END_REF] found that 40-50% of sludge proteins were converted to VFA despite a thermochemical pre-treatment of the domestic sludge. Interestingly, despite an incomplete protein conversion to organic acids (65%) a high lactate concentration was obtained byShen et al. (2017) during the fermentation of egg white with UASB sludge as inoculum, pH adjustment at 6 and a temperature of 30°C. 22% of the proteins (in COD) were converted to lactate by Leuconostoc and Lactobaccillus. The incomplete conversion of proteins to organic acids could simply be due to ammonia toxicity, since protein degradation releases ammonium. NH 4 + -N concentration was as high as 6 g/L in Shen's batch experiment.

Figure 16 :

 16 Figure 16: Stickland reaction between alanine and glycine

Equation 5 .

 5 Metabolic pathways for glycerol degradation during acidogenesis (Yin, 2016) c. CATABOLISM OF GLUCOSE Under anaerobic conditions, most bacteria are using the Embden-Meyerhof-Parnas pathway (EMP, or glycolysis) to oxidize glucose to pyruvate. In the process two moles of ATP and two moles of reduced Nicotidamide Adenine Dinucleotide (NADH) are produced (Equation

Figure

  Figure 17: catabolic pathways in LAB for carbohydrates (adapted from Wang, 2015). Enzymes 1, galactokinase; 2, arabinose isomerase; 3, xylose isomerase; 4, mannose phosphotransferase system; 5, hexokinase; 6, glucose-6-phosphate dehydrogenase; 7, 6-phosphogluconate dehydrogenase; 8, ribulose-5-phosphate 3epimerase; 9, transketolase; 10, transaldolase; 11, 6-phosphofructokinase; 12, fructose-bisphosphate aldolase; 13, triosephosphate isomerase; 14, lactate dehydrogenase; 15, phosphomannose isomerase; 16, phosphoglucose isomerase; 17, phosphoglucomutase; 18, galactose-1-phosphate uridyl transferase; 19, glucosyltransferase; 20, ribulokinase; 21, xylulokinase; 22, phosphoketolase; 23, acetate kinase; 24, phosphotransacetylase; 25, aldehyde dehydrogenase; 26, alcohol dehydrogenase

Figure 18 :

 18 Figure 18: Shifts in bacterial population during the different steps of silage processing (Dunière et al., 2013)

Equation 10 .

 10 Figure 19: Conversion of lactate and acetate to butyrate hydrogen and CO 2[START_REF] Duncan | Lactate-utilizing bacteria, isolated from human feces, that produce butyrate as a major fermentation product[END_REF]

Figure 21 .

 21 Figure 21. Envisioned implementation of struvite recovery within an anaerobic digestion process: parallel biological acidification. Implementation of biological acidification prior to digestion is also possible

  Les résultats obtenus avec les autres lisiers ont des tendances similaires. Ils ont permis de montrer que la dissolution du P et du magnésium en fonction du pH n'est pas linéaire. Elle intervient principalement pour des pH compris entre 5.5 et 6.5. En revanche la dissolution du calcium en fonction du pH est quasi-linéaire entre pH 7 et pH 4.5. Cette évolution différente permet de définir une zone de pH comprise entre 5.5 et 6 pour laquelle la dissolution du Ca comparée au P et au Mg est moindre ce qui favoriserait la cristallisation du P sous forme de struvite lors des étapes ultérieures. Le fait d'opérer la séparation avant cristallisation à ce pH réduirait également la quantité de réactif nécessaire à la remontée du pH lors de la cristallisation. En revanche la faible stabilité du pH dans cette zone demande une gestion précise du procédé d'acidification. Finalement la régression linéaire mise en oeuvre sur les données obtenues avec les quatre lisiers a permis d'établir une relation bien ajustée entre le pH minimal, la quantité de sucre, le pH initial du lisier et son pouvoir tampon pour une zone de pH entre 4 et 8. Cette relation pourra être utilisée pour calculer la quantité de co-substrat à ajouter. Ces travaux ont montré le lien étroit entre la production d'acide lactique et le pH. Or l'acide lactique est le produit majoritaire de dégradation des sucres simples très facilement dégradables. Les travaux suivants ont pour objectif de s'assurer que l'acidification peut être obtenue avec les co-substrats riches en glucides complexes disponibles sur les unités de méthanisation et de tester la faisabilité d'un recyclage en parallèle de la méthanisation pour exploiter la présence d'une flore déjà accoutumée à la dégradation anaérobie de co-substrats complexes comme cela a été envisagé à la fin de l'étude bibliographique.

Figure 22 .

 22 Figure 22. Evolution of pH across time for each initial sucrose concentration (standard deviation displayed)

Figure 23 .

 23 Figure 23. Net production of VFAs and lactate, sucrose removed and pH across time. Standard deviation shown for the sucrose removed and the sum of VFAs and lactate. A: Low initial sucrose concentration (30 g/L). B: High initial sucrose concentration (50 g/L)

Figure 25 .

 25 Figure 25. A: Dissolved phosphorus expressed as percentage of the total amount, plotted against pH. B: Dissolved magnesium. C: Dissolved calcium. D: Ammonium

Equation 14 .Figure 27 .

 1427 Figure 27. Measured pH plotted against model-predicted pH

  présence d'AGV dans le lisier avant le test peut expliquer à la fois le pH plus faible et le pouvoir tampon plus élevé. Le lactate est l'acide organique majoritaire pour les fortes concentrations en sucre dans les deux matrices utilisées. Pour des concentrations inférieures, le lactate a été produit pendant les premières 48 heures mais consommé ensuite lors de fermentations secondaires qui ont pu avoir lieu grâce à un pH insuffisamment acide pour les inhiber.Un pH inférieur à 5 est obtenu avec les pommes, les carottes à 50g.L -1 , les biscuits et la farine de blé noir stockés. Cette baisse de pH peut être attribuée à la production d'acide lactique (Figure29). Aucune baisse de pH ni production d'AGV n'est observée pour la paille. Pour les autres co-substrats, le pH atteint des valeurs comprises entre 5 et 5.7. Les valeurs atteintes sont corrélées à la teneur en glucides. La conversion de la matière organique en acides organiques est comprise entre 25 et 56% inférieures aux valeurs obtenues avec le sucre (44-78%). Les communautés microbiennes impliquées dans l'acidification ont été caractérisées par séquençage haut débit des gènes des ARNr 16S. L'analyse des indices de diversité suggère une large diversité initiale avec quelques espèces dominantes. La diversité se réduit pour les essais pour lesquels les pH les plus bas sont atteints. Le faible pH et la concentration en lactate exerceraient une pression de sélection ne permettant de conserver que les micro-organismes tolérants aux conditions acides. L'analyse statistique de la structure des communautés microbiennes montre une différence importante entre le lisier brut et le digestat mais cette structure évolue pendant l'acidification et se rapproche d'autant plus à la fin des essais que la concentration en sucre est importante. Dans tous les échantillons les phyla majoritaires sont les Firmicutes et Bacteroidetes qui sont les phyla classiquement retrouvés dans les réacteurs anaérobies acidogènes. Dans tous les réacteurs l'acidification est associée à une baisse de l'abondance relative des Bacteroidetes et une augmentation des Firmicutes. Lactobacillus domine pour les concentrations en sucre les plus élevées tandis que l'adaptation des Clostridiales aux plus faibles concentrations serait due à leur capacité à croître sur le lactate. Dans les essais digestats et déchets organiques, Lactobacillus domine systématiquement lorsque le contenu en sucre facilement dégradable est important comme dans les carottes, les pommes ou les biscuits et sarrasin dégradés. Les Clostridiales dominent dans les échantillons moins dégradés indiquant une meilleure capacité à l'hydrolyse. Streptococcus est présent de façon significative dans la plupart des échantillons indiquant une capacité à agir et se développer sur des substrats plus divers ou en conditions limitantes. Il est capable de modifier son métabolisme passant de la fermentation homolactique à une fermentation produisant divers acides mais il est peu tolérant au milieu acide ce qui explique qu'il ne soit pas dominant dans les échantillons avec les plus fortes concentrations initiales en sucre dans lesquels le pH est inférieur à 5. Ces essais montrent que la fermentation lactique avec des déchets organiques est possible et que la production d'acide lactique dépendra de la disponibilité de sucres facilement dégradables, qui favorisent le développement rapide des Lactobacillus, avec un effet inhibiteur empêchant les fermentations secondaires.Afin de prédire la quantité de substrats à ajouter d'après l'équation décrite dans le chapitre précédent pour obtenir le pH optimal en vue du recyclage du P sous forme de struvite, un équivalent sucre sera calculé pour quelques-uns des co-substrats testés. Un test sur un réacteur semi-continu permettra de voir s'il est possible d'adapter la flore pour augmenter l'efficacité de la dégradation des glucides complexes et de la fermentation lactique.

Figure 28 .

 28 Figure 28. Organic acids produced and pH after 4 days. A: batch tests with digestate 1. B: batch tests with raw slurry. *:not applicable

Figure 29 .

 29 Figure 29. Final pH and organic acids produced after 4 days

Figure 31 .

 31 Figure 31. Comparison of the microbial community diversity indexes (Shannon, Simpson and equitability) and pH of the different samples

Figure 32 .

 32 Figure 32. nMDS statistical analysis of samples microbial communities

Figure 33 .

 33 Figure 33. A: community structures and corresponding value of pH the swine slurry and digestate samples at the phylum taxonomic rank. B: inclusion of dominant Orders (Porphyromonadaceae), Families (Clostridiales, Lactobacilliales) and Genera (Lactobacillus, Streptococcus)

  L'obtention d'un pH légèrement inférieur lors de l'acidification aurait probablement permis d'améliorer le taux de dissolution du P et la réalisation de la cristallisation dans un équipement industriel optimisé d'obtenir des cristaux plus gros. Ces deux étapes sont déterminantes pour améliorer le rendement global du procédé. Si des équipements industriels développés spécifiquement pour la cristallisation de la struvite sont disponibles sur le marché, une bonne connaissance des co-substrats et notamment de leur équivalent sucre est déterminante pour choisir les cocktails qui favoriseront la production d'acide lactique et limiteront les fermentations secondaires. Des études complémentaires sur l'influence des paramètres de l'étape d'acidification biologique (temps de séjour, température …) devraient également permettre d'orienter les métabolismes. Biological acidification of pig slurry using organic co-substrates: An efficient process for phosphorus dissolution prior to struvite crystallization Piveteau Simon a,b, *, Dabert Patrick a,b , Daumer Marie-Line a,b a Irstea, UR OPAALE, 17 Avenue de Cucillé-CS 64427, F-35044 Rennes, France b Univ de Bretagne Loire, France. * Corresponding author. Simon Piveteau. UR OPAALE, Irstea. E-mail address: simon.piveteau@irstea.fr III. INTRODUCTION Phosphorus, a necessary nutrient for life (DNA, RNA, ATP) is a fossil resource in limited supply.

Equation 15 .

 15 Calculation of buffer capacity of the slurryBuffer capacity (mEq/L) = A / 0.1 / (pH 0 -4) A: amount of sulfuric acid in milliequivalent added to 0.1L of slurry to reach pH 4. pH 0 : initial pH of the slurry

Equation 17 .

 17 Calculation of initial sucrose concentration necessary to reach a targeted pH Sucrose 0 (g/L) = -105.404 -16.879 pH targeted + 27.271*pH 0 + 0.283*Buffer (mEq/L) Equation 18. Calculation of initial co-substrate concentration based on its sucrose equivalent

Figure 36 .

 36 Figure 36. Organic acids produced during biological acidification of slurry 2 with pea (A) and sucrose (B) as cosubstrates

Figure

  Figure 39. Phosphate concentration plotted against pH in slurry 1 (A), slurry 2 (B), slurry 3 (C) and slurry 4 (D)

Table 1 .

 1 Phosphate rock reserves estimations ......................................................................................... 7

Table 2 .

 2 Amount of acid needed to dissolve P under various forms ..................................................... 37

Table 3 :

 3 pKa of organic acids produced during acidogenesis ............................................................... 40

Table 4 .

 4 Composition of the pig slurries (concentrations expressed per liter of raw manure) .............. 75

Table 5 .

 5 Carbon recovery in R10 to R60 at each sampling time. Mean of three incubations and standard deviation are displayed. Carbon recovery at maximum lactic concentration shown in bold .. 78

Table 6 .

 6 Characteristics of pig slurry and digested slurries (standard deviation displayed between brackets) .............................................................................................................................................. 100

Table 7 .

 7 Composition of the organic waste used during biological acidification of pig slurry. Standard deviation of triplicate measures for TS and VS displayed between brackets. ..................................... 101

Table 8 .

 8 Statistical results for nMDS analysis ..................................................................................... 102

Table 9 .

 9 Carbon recovery: C-mol organic acids produced / C-mol sucrose consumed (%)................ 104

Table 10 .

 10 Conversion ratio of organic matter to organic acids ........................................................... 105

Table 11

 11 Figure 1: Production of phosphate rock in 1997 and 2016...................................................................... 8 Figure 2: Phosphate rock reserves in the world in 1999 and 2017 (USGS data) .................................... 8 Figure 3: World phosphate rock production from 1960 to 2017 (USGS data) ....................................... 9 Figure 4: P fertilizer demand and expected growth until 2020 ............................................................... 9 Figure 5. Access points for P recovery in municipal WWTP (adapted from Egle et al., (2015)) ......... 15 Figure 6. Aiprex process for struvite recovery (adapted from P-rex).................................................... 18 Figure 7. Combination process of Pearl and Wasstrip .......................................................................... 19 Figure 8. Seaborne process (adapted from Müller et al. (2007)) ........................................................... 21 Figure 9. Stuttgart process ..................................................................................................................... 22

. Composition of the pig slurries ........................................................................................... 124 Table 12. Composition of the organic co-substrates ........................................................................... 125 Table 13. Sucrose equivalents of the co-substrates ............................................................................. 126 Table 14. Co substrates and sucrose concentrations in each batch test (g/L) ...................................... 127 Table 15. Average relative and absolute error of the model for sucrose and each co-substrate .......... 130 Table 16. Proportion of total phosphorus in each batch test originating from the co-substrate expressed as percentage of TP ............................................................................................................................. 132 Table 17. Composition of acidified slurry and filtrate after crystallization, including mean value and standard deviation................................................................................................................................ 136 LIST OF FIGURES Figure 10. Budenheim process .............................................................................................................. 23 Figure 11. Ash Dec process ................................................................................................................... 25 Figure 12. P-recovery process for pig manure. Adapted from Vanotti et al. (2007) ............................. 29

Table 1 . Phosphate rock reserves estimations

 1

rock production (MT-P/year)

	P-1997 2016
	US	Morocco	China	rest of the
				world
	P-
	Morocco	China	rest of the	total
			world

rock reserves (GT-P) 1999 2017

world annual growth rate 2015-2020 MT-P/year demand growth rate

	Latin america Central/East	Africa	Asia	Oceania North Am. +
	Eur.+Russia			West. Eur.

  Conclusion on phosphorus recovery from WWTPPhosphorus flow to WWTP in Europe (0.4 MT-P/year) corresponds to 20% of imported P or 37% of chemical P fertilizer[START_REF] Withers | Stewardship to tackle global phosphorus inefficiency: The case of Europe[END_REF]. Recovering P from this stream or "secondary deposit" would have a major impact in term of resource efficiency and advance toward closing the P cycle. However a little less than 30 full scale plants for P-recovery currently exist in Europe (50 in the world). All of them are located in Western Europe (and the US). Most only recover phosphorus from the supernatant of digested sludge. The main incentive for implementing such processes is related to the cost associated with maintenance (less incrustation/scaling), chemical demand (flocculants and Al/Fe for chemical P removal) and disposal of residual sludge. Indeed, the market for mineral P fertilizer derived from municipal sludge does not really exist yet, due to a heterogeneous legislation regarding this type of product and the uncompetitive price compared to conventional P fertilizer. However, the large benefits regarding cost saving enabled by such technology should already appeal to many treatment plants. Because the product obtained has very good fertilizing value, less metal contamination than conventional fertilizer, and limited chemical and energy costs, the P-recovery

  studied the effect of temperature and type of acid or base on P dissolution in batch experiments lasting one hour, at 120, 170, 200 and 250°C. Without acid, temperature had a negative on P dissolution, with 500 and 10 mg-P/L after the 120°C and 250°C treatment respectively. The best results were obtained at 170°C with 0.1M H 2 SO 4 .79% of TP initially present in the manure was dissolved, 92% of it as ortho-P. The ratio of sulfuric acid to dissolved P was 5.6 Kg-H 2 SO 4 /Kg-P, a value similar to what is obtained by direct acidification on raw manure. It should be noted that in this experiment, the swine manure was dried at 60% and re-dissolved afterward to undergo thermal treatment. Consequently, it was not direct use of raw manure.In a similar experiment, Heilmann et al. (2014) tested hydrothermal treatment on pig slurry during 40 minutes runs at 200-260°C. Hydrochloric acid was not added before treatment, but used instead on the char obtained. 90% of the P contained in the char was extracted jointly with calcium, indicating that most of the calcium in the pig manure was initially present as calcium phosphate. The acid to P ratio to reach this level of extraction was 108 Kg-HCl / Kg-P. Struvite was tentatively precipitated from the acid extract by adding stoichiometric amounts of Mg and NH 4+ , but only calcium and phosphate precipitated. It appears that extraction of P from biochar is too expensive and the product obtained does not possess sufficient added value.

  [START_REF] Pan | Microwave pretreatment for enhancement of phosphorus release from dairy manure[END_REF] applied H 2 SO 4 + MW + H 2 O 2 pre-treatment to dairy manure based on its success on domestic sludge. The results indicated that MW in itself does not affect P dissolution, contrary to what occurs with EBPR sludge. Cell lysis induced by MW is efficient to release P from PAO in EBPR sludge but has no effect on inorganic P as calcium phosphate in dairy manure. Only temperature at 170°C, H 2 SO 4 , and H 2 O 2 treatment led to the release of phosphates. MW only seemed to slightly accelerate the effect of H 2 O 2 on P dissolution. No details were given regarding the exact amount of sulfuric acid used, making it impossible to calculate the ratio of acid to dissolve phosphorus.[START_REF] Qureshi | Microwave treatment and struvite recovery potential of dairy manure[END_REF] similarly found a positive effect of temperature but no effect of hydrogen peroxide on P dissolution during MW treatment in combination with H 2 SO 4 . Unfortunately, the effect of H 2 SO 4 alone is never presented in any of the publications on MW treatment of cow manure. Again, Less efficient results were obtained by[START_REF] Jin | Enhancing anaerobic digestibility and phosphorus recovery of dairy manure through microwave-based thermochemical pretreatment[END_REF]. MW treatment alone did not lead to any dissolution of P. With 2% H 2 SO 4 + MW, dissolved P increased from 20%-TP to 39%. A similar result was obtained with a lower amount of H 2 SO 4 (0.5%) and the addition of H 2 O 2 (1%), indicating that both acidification and wet oxidation increase P dissolution. It should be noted that if the H 2 SO 4 added at 2% v/v is pure acid, then the acid to dissolved P ratios are extremely high, with 80-320 Kg-H 2 SO 4

the exact concentration of H 2 SO 4 was not given. However, pH was decreased to 1.4, indicating a rather large amount of acid and making acidification the main reason for P dissolution. Phosphate concentration increased from 40 mg/L (or 20% TP) to 160 or 86% at 170°C, with and without H 2 O 2 .

Table 2 . Amount of acid needed to dissolve P under various forms

 2

	Source of P	Type of substrate	acid	mmol H+/g-P g-acid/g-P
	Sewage sludge	SSA a	HCl	455	16.6	(Schaum et al., 2007)
		SSA	HCl	145	5.3
		SSA	HCl	200	7.3	(Montag and Pinnekamp, 2009)
		SSA	HCl	142	5.2
		SSA	HCl	222	8.1	(Xu et al., 2012)
		SSA	H 2 SO 4	271	13.3	(Franz, 2008)
		SSA	H 2 SO 4	171	8.4
		SSA	H 2 SO 4	194	9.5
		SSA	H 2 SO 4	87	4.3	(Takahashi et al., 2001)
		SSA	H 2 SO 4	232	11.4	(Biswas et al., 2009)
	Swine manure	Ashes	H 2 SO 4	102	5.0	(Azuara et al., 2013)
		Char	H 2 SO 4	367	18.0
		Raw	formic	152	7.0	(Daumer et al., 2010)
			acetic	333	20.0
		Solids (30% DW) citric	35	6.7	(Szögi et al., 2015)
		Raw	H 2 SO 4	180	8.8	This author
			H 2 SO 4	352	17.2
			H 2 SO 4	268	13.2

a SSA: sewage sludge ash

Table 3 : pKa of organic acids produced during acidogenesis

 3

		pKa
	lactate	3.86
	succinate	4.2-5.6
	malate	3.4-5.2
	formate	3.77
	acetate	4.76
	propionate	4.88
	butyrate	4.82
	valerate	4.82

  This reaction is thermodynamically unfavorable and thus requires ATP. Butyrate and acetate can then be produced. Glycerol enters glycolysis as dihydroxyacetone

phosphate, which leads to phosphoenolpyruvate, itself converted to a molecule of pyruvate. Then, as described in Figure

15

pyruvate can theoretically be converted into a wide variety of products such as acetate, propionate, butyrate lactate, ethanol and valerate. However,

Yin et al. (2016)

obtained mostly propionate, acetate and butyrate (Equation

  Pourles fortes concentrations le pH minimum est atteint entre 36 et 48 h puis reste stable. Un pH de 3.99 a été atteint avec la concentration la plus forte (Figure22). Le dosage des acides organiques a montré que la production d'acide lactique est le principal responsable de la baisse du pH. Le bilan carbone montre que la presque totalité du sucre a été convertie en acide lactique et en acide gras volatils. Le faible pH atteint avec les concentrations en sucre les plus fortes permettrait d'inhiber les métabolismes de dégradation de l'acide lactique et de maintenir le pH stable. Les concentrations en phosphore, calcium et magnésium dissous sont liées à l'évolution du pH. Jusqu'à 95% du P total initial du lisier ont été dissous avec les trois concentrations en sucre les plus élevées. Une re-précipitation est observée lorsque le pH remonte pour les concentrations intermédiaires de sucre. Le même phénomène est observé avec le magnésium dont la dissolution atteint 100% avec les trois concentrations en sucre les plus fortes. Cependant la re-précipitation observée pour les valeurs moyennes est plus faible que pour le P. La fraction de calcium dissous est inférieure. Les deux plus fortes concentrations en sucre permettent de dissoudre 75% tandis que la concentration de 40 g.L-1 permet de dissoudre seulement 60% du calcium. Les courbes de dissolution de l'ammonium montrent que de l'azote est consommé pour la croissance de la biomasse acidogène dans les essais avec sucre. Une minéralisation des formes

	Quatre lisiers différents ont été utilisés. Leurs caractéristiques ont été analysées. Le pouvoir tampon a
	été estimé à partir de la quantité d'acide sulfurique 1mol/L nécessaire pour atteindre un pH de 4 qui
	correspond au pH le plus faible observé par acidification biologique sur du lisier (Table 4). Les essais
	ont été réalisés dans des conditions définies lors de travaux précédents sur les boues. Cinq ou sept
	concentrations de sucre comprises entre 0 et 60 g.L-1 ont été testées. Des prélèvements ont été réalisés
	toutes les 12 heures en début d'incubation puis toutes les 24 heures jusqu'à 96 heures. Les essais
	réalisés avec le lisier n°1 et sept concentrations de sucre différentes montrent que la baisse de pH

biologique et d'évaluer la quantité de sucre nécessaire en fonction des caractéristiques des lisiers (pH initial, pouvoir tampon …). L'effet de l'acidification biologique sur la dissolution des autres composés de la struvite (ammonium et magnésium) et du calcium qui peut interférer avec la cristallisation de la struvite a aussi été étudié. Pour cette première approche du saccharose (sucre blanc du commerce) a été utilisé. Les résultats obtenus avec le sucre permettront d'évaluer le potentiel de dissolution du P par voie biologique sur différents lisiers. obtenue est directement liée à la concentration en sucre ajoutée. Très peu de variations sont observées pour les faibles concentrations (0 et 10 g.L-1). Pour les valeurs intermédiaires (20 et 30 g.L-1) le pH minium, 6.2 et 5.5 est atteint en 24 h mais la baisse est suivie d'une remontée rapide 12 h après. organiques sous l'effet du pH serait observée ensuite. La concentration en ammonium dissous dépend de l'équilibre qui s'établit entre ces deux mécanismes (Figure

24

).

Table 4 . Composition of the pig slurries (concentrations expressed per liter of raw manure)

 4

	TSS VSS TS VS TP	TN T-Ca T-Mg pH	Buffer	PO 4 -P NH 4 -N Ca 2+ Mg 2+
	g/L		mEq/L	g/L
	slurry 1 75,8 61.0 83.4 65.1 1.68 4.60 2.65 1.10 7.15	443	0.005 2.03 0.21 0.07
	slurry 2 15.3 10.4 22.0 13.8 0.38 2.346 1.22 0.29 7.70	99	0.006 1.62 0.11 0.03
	slurry 3 64.3 42.9 77.5 49.2 1.37 5.742 3.59 1.34 7.92	283	0.018 3.42 0.07 0.03
	slurry 4 41.9 33.8 58.0 41.4 0.53 4.774 1.59 0.50 6.70	380	0.055 2.51 0.59 0.33
	2. BIOLOGICAL ACIDIFICATION OF PIG SLURRY IN BATCH TESTS USING SUCROSE AS CO-
	SUBSTRATE

Table 5 . Carbon recovery in R10 to R60 at each sampling time. Mean of three incubations and standard deviation are displayed. Carbon recovery at maximum lactic concentration shown in bold

 5

	time (hours)	R10	R20	R30	R40	R50	R60
	12	82 ± 15	84 ± 41	67 ± 40	89 ± 59	78 ± 60	67± 9
	24	95 ± 11	83 ± 11	91 ± 8	90 ± 4	103 ± 1	99 ± 4
	36	94 ± 5	96 ± 1	97 ± 5	93 ± 11	93 ± 3	96 ± 2
	48	85 ± 18	83 ± 9	81 ± 7	97 ± 5	94 ± 9	103 ± 4
	72	89 ± 16	89 ± 5	87 ± 3	97 ± 9	97 ± 6	97 ± 2
	96	93 ± 10	92 ± 12	88 ± 12	100 ± 6	97 ± 9	101 ± 3

Table 6 . Characteristics of pig slurry and digested slurries (standard deviation displayed between brackets)

 6

		TS	VS	TSS	VSS	NH4-N	pH
				g/L
	digestate 1	19.9 (0.0)	10.1 (0.1)	10.4 (0.1)	6.5 (0.0)	1.3 (0.0)	7.86
	digestate 2	22.1 (0.3)	10.2 (0.1)	10.9 (0.2	7.1 (0.1)	1.4 (0.0)	7.99
	raw slurry	58.0 (2.2)	41.4 (2.0)	41.9 (3.9)	33.9 (3.0)	2.5 (3.0)	6.73

Table 7 . Composition of the organic waste used during biological acidification of pig slurry. Standard deviation of triplicate measures for TS and VS displayed between brackets.

 7

			TS	VS	Carbohydrates* Sugars* Proteins* Lipids*
				g/L			% dry weight
	potato		192.3 (0.2) 175.1 (0.2)	76	2	10	1
	apple		145.4 (0.3) 139.8 (0.1)	75	75	2	1
	macédoine		161.8 (2.7) 114.8 (3.0)	50	18	14	6
	wheat straw	893.4 (4.3) 833.4 (3.3)	87	2	3	7
	carrot		83.9 (3.0) 79.1 (2.8)	66	49	8	3
	bean		109.5 (4.6) 101.8 (4.4)	51	23	14	2
	pea		205.1 (1.9) 198.7 (2.0)	40	16	25	3
	buckwheat flour 852.1 (2.2) 762.2 (1.7)	71	2.6	13	3
	biscuits		857.3 (2.1) 769.5 (1.4)	59	32	7	2
	*average	values	from	literature	http://www.aprifel.com/profils-des-fruits-et-legumes.php,
	https://ndb.nal.usda.gov/ndb/foods/show/6482
	3. PHYSIC AND CHEMICAL ANALYSES

Table 8 . Statistical results for nMDS analysis

 8

	Parameters	nMDS 1	nMDS 2	R²	Pr (>r)
	pH	-0.55605	0.83115	0.9126	0.006	** (signification 0.001)
	Lactacte	0.96560	-0.26004	0.7754	0.010	** (signification 0.001)
	V. RESULTS AND DISCUSSION
	1. EVOLUTION OF PH AND ORGANIC ACIDS CONCENTRATION IN BATCH TESTS WITH
	SUCROSE

Table 9 . Carbon recovery: C-mol organic acids produced / C-mol sucrose consumed (%)

 9

	Carbon recovery (%)

Table 10 . Conversion ratio of organic matter to organic acids

 10

	Co-substrate	organic acids/VS (% g/g)
	potatoe	41
	apple	45
	macédoine	25
	carrot 50g-VS/L	32
	carrot 30g-VS/L	39
	bean	32
	pea	31
	biscuit 1	28
	biscuit 2	47
	flour 1	31
	flour 2	56

Figure 30. Correlation between carbohydrate content and final pH

	6 3. MICROBIAL DIVERSITY DETECTION USING 16S RDNA HIGH THROUGHPUT SEQUENCING 350
	4 5 The microbial community of all samples was analyzed by 16S rRNA gene high throughput sequencing 50 100 150 200 250 300 final pH valerate butyrate propionate acetate lactate giving between 47 751 and 382 063 reads for each sample that grouped within a mean of 861 +/-166 OTUs per sample (minimum number at 583 and maximum at 1135) at 97% similarity. The estimated coverage of the samples diversity was above 98% showing a good representativeness of the data (not organic acids (mmol/L) formate shown).
	0	3	succinate
			malate
			pH

Table 11 . Composition of the pig slurries

 11

Figure 34. Lowest pH reached at different initial sucrose concentrations. Example of sucrose-equivalent determination for apple

		TS	VS	TP	TN	T-Ca	T-Mg	pH	Buffer	PO 4	3--P	NH 4	+ -N	Ca 2+	Mg 2+
			g/L						mEq/L			g/L
	slurry 1	28.45 17.16 0.23 4.06 0.44	0.38	7.63	71.6	0.019	3.237	0.191 0.306
	slurry 2	50.50 36.79 0.99 3.50 1.93	0.65	7.05	87.4	0.073	2.259	0.217 0.055
	slurry 3	15.58 9.47	0.48 2.50 1.35	0.48	7.74	45.5	0.032	1.702	0.176 0.071
	slurry 4	68.69 50.81 1.56 4.25 3.23	0.90	6.53	110.7	0.150	1.836	0.264 0.030

Table 13 . Sucrose equivalents of the co-substrates 4

 13 . BIOLOGICAL ACIDIFICATION OF PIG SLURRY IN BATCH TESTS USING COMPLEX ORGANIC CO-SUBSTRATES

  linking initial concentration of sucrose, buffer capacity, initial and lowest pH (Equation17, Equation 18 and Table14), with the objective to target three minimum pH between 4 and 6, a range in which phosphorus is largely dissolved. No

		carrot	green bean	pea	apple
	VS added (g/L)	30	50	50	36
	lowest pH reached	5.49	5.44	5.42	4.66
	corresponding sucrose concentration (g/L)	20.52	20.95	21.08	26.73
	sucrose eq. (g-sucrose/g-VS)	0.69	0.42	0.42	0.74

  The effluent was centrifuged immediately at 10000 RPM

		Concentration	carrot fresh VS	green bean fresh VS	pea fresh VS	apple fresh VS	sucrose
	slurry 1	high medium low	1058 815 571	88 68 48	1348 136 1038 104 728 73	700 539 378	144 111 78	600 462 324	76 58 41	58 44 31
	slurry 2	high medium low	616 386 156	51 32 13	511 320 129	51 32 13	410 257 103	84 53 21	379 237 96	48 30 12	36 22 9
	slurry 3	high medium low	789 554 324	68 48 28	649 458 269	63 45 26	520 367 215	111 78 46	481 340 198	65 46 27	45 32 19
	slurry 4	high medium low	382 237 91	36 22 9	589 365 139	68 42 16	291 180 69	59 37 14	265 164 63	28 17 7	25 15 6

Table 15 . Average relative and absolute error of the model for sucrose and each co-substrate Figure 35. Measured minimum pH plotted against model-predicted pH for sucrose, apple, carrot, pea and green bean batch tests

 15

		relative error	absolute error	absolute error (pH>5)
	sucrose	0.03	0.37
	carrot	0.23	0.34
	green bean	0.23	0.38	0.23
	pea	0.72	0.79	0.45
	apple	-0.15	0.43

Table 16 . Proportion of total phosphorus in each batch test originating from the co-substrate expressed as percentage of TP

 16

	concentration	carrot	bean	pea	apple
	slurry 1

lactate [START_REF] Bourriaud | Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident[END_REF]. Finally, Clostridium kluyveri can produce valerate from ethanol and propionate [START_REF] Stadtman | Tracer experiments on the mechanism of synthesis of valeric and caproic acids by Clostridium kluyveri[END_REF] Equation 12. Consumption of lactate via the acrylate pathway Equation 13. Conversion of lactate into acetate, CO 2 and biohydrogen

e. FERMENTATION OF SWINE MANURE

Fermentation of swine manure and other agricultural waste streams have been studied in the context of hydrogen production [START_REF] Chatellard | Trends and Challenges in Biohydrogen Production from Agricultural Waste[END_REF], but using swine manure mostly as a medium where an hydrogen producing inoculum is added [START_REF] Wu | Optimization of continuous hydrogen production from co-fermenting molasses with liquid swine manure in an anaerobic sequencing batch reactor[END_REF]2010;2009;2017), with additional dilution of the manure making its bacterial content very low [START_REF] Zhu | Swine manure fermentation for hydrogen production[END_REF]. Tenca et al. (2011) however used swine manure at 8 g-VS/L and added fruit and vegetable waste, as well as an inoculum for hydrogen production. The organic co-substrate represented between 15 and 55% of the total weight, an amount that would be suitable for biological acidification of pig slurry and P recovery. Operated in semi continuous mode with 1 to 3 days HRT, the pH obtained was 4. 3-5.6, values perfectly suitable for P dissolution in pig slurry. The details of the organic acids produced were not given unfortunately.

CHAPTER 1 DISSOLUTION OF PARTICULATE PHOSPHORUS IN PIG SLURRY THROUGH BIOLOGICAL ACIDIFICATION

Piveteau S., Picard S., Dabert P., Daumer M-L. (2017). Dissolution of particulate phosphorus in pig slurry through biological acidification: a critical step for maximum phosphorus recovery as struvite. Water Research. DOI: 10.1016/j.watres.2017.08.017 E-mail address: simon.piveteau@irstea.fr However, initial sucrose concentration is also proportional to biomass growth and associated Nassimilation. The assimilation of NH 4 + for bacterial growth could explain for example the drop at 12h in all the reactors except R0. The balance between these contradicting effects could explain why after 96 hours R0 and R30 have a similar ammonium concentration, higher than R10 and R20 but lower than R40, R50 and R60. phenomenon potentially disconnecting dissolved P from pH is the hydrolysis of solid organic P and the mineralization of dissolved organic P. Organic phosphorus can represent up to 22% of TP (Daumer et al., 2008). Since hydrolysis and mineralization are partially time-dependent it could explain why in the case of slurry 4 the maximum dissolved P concentration was reached despite a pH re-increase (from 68% of TP at pH 4.9 to 78% at pH 5.2)

Calcium dissolution was not necessarily as linear as in slurry 1. Slurries 2 and 4 had Ca dissolution as high as 50% of T-Ca at a pH of 6.5, while the 50% value was reached only at pH 5.5 in slurries 1 and 3. The mineral form of calcium should be investigated to explain this difference.

Magnesium dissolution varied somewhat among the slurries, with the 50% threshold reached between pH 6 and 7. The maximum dissolution was reached around pH 6, and varied between 70 and 100%.

Ammonia was not linked with pH in any slurry, for the same reasons described above.

POTENTIAL FOR STRUVITE CRYSTALLIZATION

In order to precipitate the dissolved phosphorus into struvite, pH needs to be increased to 7 or above.

This could be done through acetogenesis and methanogenesis with the conversion of lactate and VFAs into acetate, H 2 and subsequently methane and carbon dioxide. However, phosphorus solids would be mixed with anaerobic sludge and inorganic solids, making the segregation of struvite crystals difficult.

Therefore, the acidified slurry should be centrifuged to separate the dissolved P from the suspended

VI. CONCLUSIONS

This work demonstrated that most of the phosphorus (60-90% of TP) present in pig slurry can be dissolved using a simple biological process known as acidogenesis. A limited acidification down to a pH of 5.5-6 using sucrose allowed maximum P dissolution while providing optimal conditions for P recovery as struvite. The model correlating the amount of co-substrate with buffer capacity, initial and lowest pH can be used to adjust the amount of co-substrate to reach the desired pH range. It should be improved and expanded using real organic waste. The full conversion of sucrose into lactic acid and VFA indicates that organic waste with high soluble carbohydrates content should be used as cosubstrate for biological acidification. The synergy/antagonism between P recovery and renewable energy production in the subsequent methanisation stage needs to be investigated.

VII. ACKNOWLEDGEMENT

IRSTEA would like to thank Naïs Le Quenellec for her dedication and skills in HPLC analysis.

II. ABSTRACT

Swine manure contains large amounts of phosphorus, ammonium and magnesium, making this waste stream suitable for P-recovery as struvite, a slow release P-fertilizer. Phosphorus has to be dissolved through acidification to become available for struvite precipitation. Biological acidification of swine slurry under anaerobic conditions (i.e. acidogenesis) using sucrose as organic co-substrate led to a dissolution of as much as 90% of Total-P thanks to a high production of lactic acid. In this study, several organic waste and sucrose were tested as organic co-substrate for the biological acidification of raw and digested swine slurry in batch tests. Lactic acid fermentation occurred when the co-substrate had a high content in easily accessible carbohydrates and when a sufficient amount of waste was added to the slurry (50g-VS/L). A microbiological analysis indicated that acidogenesis led a significant reduction in bacterial diversity. The dominating clusters at the end of acidification were

Clostridiales and Streptococcus when moderate acidification occurred and Lactobacillus when pH dropped below 5. The successful acidification of digested and raw pig manure when using easily biodegradable, carbohydrate rich organic waste is promising for developing an inexpensive, low-tech recovery process for struvite recovery.

Streptococcus at medium concentration and Lactobacillus at high initial concentration. Despite quite different initial microbial community compositions between raw and digested slurry, the presence of increasing amounts of easily biodegradable carbohydrates systematically shifted the bacterial populations toward a homogenous, Lactic Acid Bacteria rich community, confirming other lactic acid fermentation studies using undefined mixed culture as inoculum [START_REF] Dreschke | Lactic acid and methane: Improved exploitation of biowaste potential[END_REF][START_REF] Liang | Comparative analysis of microbial community of novel lactic acid fermentation inoculated with different undefined mixed cultures[END_REF]Probst et al., 2015). These batch tests and microbiological results indicate that lactic acid fermentation of raw or digested swine slurry with cheap/free organic waste is easily achievable if the waste is rich enough in easily accessible carbohydrates. The organic acid rich supernatant, after P recovery, could be used as feed for anaerobic digestion and energy production from methane.

CHAPTER 3 BIOLOGICAL ACIDIFICATION OF PIG SLURRY USING ORGANIC CO-SUBSTRATES

II. ABSTRACT

Pig slurry is a suitable waste stream for phosphorus recovery as struvite but requires a dissolution step through acidification prior to crystallization. Biological acidification of pig slurry was studied in batch tests by adding several co-substrates (carrots, beans, peas, apples and sucrose). Dissolved phosphorus increased from 7-10% of total-P at initial pH (6.5-7.7) to 60-90% when low pH (4-5.2) was reached. A simple crystallization step using magnesium hydroxide to increase pH to 8 led to complete phosphate removal. The dried solid obtained contained 69% of struvite and less than 0.5% of calcium based on ionic concentrations obtained after acidic dissolution. The loss of small crystals through the sieve (100 µm pore size) limited the recovery to 58% of dissolved-P and 26% of Total-P, but simple optimization steps such as longer retention time and recirculation of the filtrate should enable the formation of larger crystals and substantially improve the P-recovery rate.

DETERMINATION OF THE ACIDIFYING POWER FOR EACH CO-SUBSTRATE COMPARED TO

SUCROSE

During biological acidification of pig slurry with sucrose, the lowest pH reached decreases linearly with the initial amount of sucrose added down to pH 4. Biological acidification of digested swine slurry was conducted with 7 different sucrose concentrations (in simplicate): 0, 10, 20, 30, 40, 50, and 60 g/L (Figure 34). The digested pig slurry was obtained from an 87L in-house digester that provides a stable effluent all year long. Samples were taken at 24, 48, 72 and 96 h for pH measurement. A linear equation (Equation 16) was obtained, linking lowest pH reached during the 96 h batch with the initial sucrose concentration. For solid analysis from the crystallization process, 100 mg of dried solid recovered after crystallization and filtration was introduced in in a beaker with 150 mL of distilled water and pH was adjusted at 1.8

with sulfuric acid (1 mol/L). Volume was adjusted to 200 mL with distilled water. P0 4 3-concentration was measured on the Gallery while Mg 2+ , NH 4 + and Ca 2+ were measured by ionic chromatography.

V. RESULTS AND DISCUSSION

MODEL FITNESS

The deviation of the model was calculated for each co-substrate as the average absolute difference between the measured lowest pH and the targeted lowest pH (Table 15 and Figure 35). The average relative difference between measured lowest pH and targeted lowest pH, also displayed in Table 15, reflects the overall overestimation (positive average pH difference) and underestimation (negative average pH difference) of the acidifying capacity of the co-substrate. The model demonstrated a moderate fitness (absolute deviation) in the case of sucrose and for most of the co-substrates but showed a relatively poor fitness in the case of pea. The deviation could be due to an overestimation of the sucrose equivalent for peas. Indeed, the lowest pH obtained was almost always above the targeted pH. This overestimation could itself be explained by the fact that the fermentation products of pea differs from those of sucrose. Lactate was the main molecule produced during biological acidification of pig slurry using sucrose (Figure 36B) and this organic acid has a low pKa compared to usual volatile fatty acids (VFA). On the contrary, fermentation of pig slurry with pea as organic co-substrate produced mainly acetate, propionate, butyrate and valerate (Figure 36A). As a result, while sucrose had a linear effect on minimum pH down to pH 4.5-4 (Figure 37A), that was not the case for pea, with an inflexion around 6-5.5 (Figure 37B). This makes the calculated sucrose equivalent for pea a function of pH, not a fixed value as initially postulated. The sucrose equivalent of pea was determined during a batch test in which the lowest pH reached was 5.4 (Table 13). Consequently, when lower pH values were targeted, the amount of co-substrate necessary was underestimated. To a lesser extent, the same conclusion applied to green beans, which also did not produce any lactic acid. However, the batch tests with carrots and apples did have lactic acid production and were able to reach lower pH.

The absolute error of the model for pea and green bean was significantly lower when only the targeted pH above 5 were taken into account (Table 15).

The batch test with slurry 4 led systematically to a minimum pH higher than the targeted value. Based on the proportion of added VS converted to organic acids, it could be postulated (no gas analysis was performed) that methanisation occurred, converting some of the organic acids into acetate and then methane, limiting the acidification. Indeed, as seen in Figure 38A, the proportion of co-substrate converted into organic acids was significantly lower for every co-substrate at high initial concentration during batch 4 compared to the others. Besides, remaining sucrose (plus glucose and fructose) concentration was null after 48 hours, indicating that sucrose had been consumed but not converted into organic acids, confirming further the hypothesis of methanisation.

Sucrose was entirely converted into organic acids during the first three batchs (98-116% of initial sucrose added, Figure 38B), mostly as lactate, which confirms why sucrose is the most acidifying cosubstrate. Apple had the second highest conversion into organic acid (58-73% of VS) with lactic acid as the main product (65-85% of organic acids produced in mol/mol) when minimum pH was reached.

A secondary fermentation occurred when pH did not decrease below 5, with lactic acid being converted into VFAs. The phenomenon of secondary fermentation leads to a pH re-increase (McDonald, 1982), which implies that a targeted acidification meant to reach a given pH requires frequent pH monitoring in order to stop the process when the desired pH has been reach. Batch tests with carrots had the third highest conversion into organic acids (41-58% of VS). Lactate was once T-Ca) and a sudden increase to 60% at pH 5. The inorganic, solid forms of Ca in slurry 4 may have been different than those in slurry 2 and 3, with different dissolutions constants.

Dissolution of nitrogen was not influenced by pH in any of the slurries (Figure 41D). Some mineralization of organic matter from the complex co-substrates occurred but not from the slurries themselves. Indeed, in the batches with sucrose as co-substrates, ammonia concentration never increased, while in the case of N-rich co-substrates ammonium concentration increased over time gaining progressively 10 to 30 additional percent of TN during the four days of experiment in the batch tests with peas in each slurry.

MOLAR RATIOS OF INTEREST FOR STRUVITE PRECIPITATION

An equimolar amount of P, N and Mg is necessary to precipitate all the phosphorus as struvite. To favor the precipitation of phosphorus as struvite and prevent calcium phosphate formation, a high molar ratio N:P (>3) as well as an excess of Mg compared to Ca are required (Capdevielle et al., 2013). Ammonium was always largely in excess, with an N:P ratio above 4.5 in all the slurries at any sampling time. Mg 2+ was often the limiting ions (i.e. a molar concentration lower than phosphates),

with Mg:P ratio sometimes as low as 0.5. Yet the amount of Mg necessary to match the phosphate concentration was never higher than 185 mg/L. When Mg(OH) 2 was used to precipitate struvite, 3.4g/L on average were necessary to reach pH 8, i.e. 1.4 g-Mg 2+ /L, more than 8 times the amount of Mg required for equimolarity (see section V.4).

SEMI-CONTINUOUS REACTOR OPERATION AND PHOSPHORUS RECOVERY

As seen in Figure 42, pH decreased rapidly during the first 4 days (reactor operated in batch mode) to reach 5.7, and progressively decreased to 5.4 at the end of the experiment of semi continuous operation. Phosphate concentration increased quickly from 150 to 680 mg/L during the four days of batch operation and kept on increasing during semi-continuous operation, albeit at a slow pace, to reach 747 mg/L at the end. This experiment indicates a semi-continuous process for biological acidification of swine slurry with organic waste is at least as efficient as a batch process in terms of pH reached and dissolved phosphorus.

The effluent withdrawn daily during the last five days of operation of the semi-continuous reactor was used for five identical struvite crystallization experiments. Centrifugation was efficient thanks to the coagulant, with 83% (± 2.1) of the initial volume recovered in the supernatant. The detailed composition of the biologically acidified slurry can be found in Table 17. At pH 5.4, the concentration of P, Ca, Mg and N were similar to those found during the batch experiment with slurry 4, with approximately 50% of TP (including the phosphorus contained in the co-substrates). The N:P:Mg:Ca molar ratio was 6.5:1:0.9:0.4, indicating both a favorably large excess of ammonium yet also a slight deficit in magnesium and a non-negligible amount of calcium. The dried solid obtained after crystallization had an Mg:N:P:Ca ratio of 1.32:1:1.19:0.05 (standard deviation of 0.1:0:0.04:0.02). The relative proportion of each compound is relatively close to struvite, with a small excess of P and Mg (the excess of magnesium could have resulted from undissolved magnesium hydroxide in the solid), and very low contamination by calcium. The proportion of struvite in the solid could be extrapolated based on ammonia concentration measured from the dissolved solid. Indeed, struvite is the only inorganic precipitate containing N that could have been formed during the pH increase from 5.4 to 8.

Based on ammonia, 69.1% (± 6.9) of the solid recovered consisted in struvite crystals. The rest of the solid was most likely composed of other phosphorus precipitates, undissolved magnesium hydroxide and organic matter.

In order to precipitate phosphorus, 3.4 g-Mg(OH) 2 (± 0.3 g) per liter of supernatant were necessary to reach pH 8. As a result, a large amount of Mg 2+ remained in the liquid after crystallization (Table 17).

Nevertheless, addition of magnesium hydroxide enabled the precipitation of 99.2% (± 0.3) of the phosphates from the supernatant (calculation based on the difference between phosphate concentration in the acidified slurry and in the liquid after precipitation and filtration).Not all precipitated P was retained by the 100 µm sieve, with 4.5g of dried solid recovered per liter of supernatant (± 1g/L), most likely due to heterogeneous crystal sizes (Figure 43). As a result, despite the complete precipitation of P, "only" 58% (± 9) of dissolved-P or 26% (± 5) of TP was effectively recovered as solids. A simple optimization of the crystallization process (e.g. mixing adjustment, rinsing of the solids to remove adsorbed organic acids, longer time allowed for crystal growth and dissolution of magnesium hydroxide) should enable a larger and more homogeneous crystal size, leading to near complete recovery of the 750 mg P0 4 3--P/L or 50% of TP with low contamination of inorganic and organic matter, only limited by the loss of the liquid trapped in the solid phase during centrifugation.

VI. CONCLUSIONS

The batch experiments demonstrated that the organic co-substrates used were effective to acidify pig slurry and dissolve a large proportion of TP. The release of P, Ca and N due to hydrolysis of organic co-substrate and P assimilation for bacterial growth were non negligible phenomena in the slurries with lower solid content.

The semi-continuous process for biological acidification proved to be as efficient or slightly more compared to batch operation. The crystallization tests demonstrated complete phosphorus removal, resulted in a solid with low calcium contamination and an N:P:Mg ratio very close to the one of struvite. The process should be optimized to obtain larger crystals and thus a higher recovery rate. The effects of biological acidification and P-recovery on the subsequent anaerobic digestion in terms of methane potential should be investigated.

DISCUSSION AND PERSPECTIVES

The main objective of this PhD was to use acidogenesis, a biological process instead of chemical acidification to dissolve phosphorus in raw and digested pig slurry for struvite recovery.

Using sucrose as a model organic co-substrate, lactic acid fermentation took place in swine slurry, leading to a pH decrease proportional to the initial amount of sucrose added down to pH 4. A simple model predicting the lowest pH reachable was developed based on initial pH and sucrose concentration as well as buffer capacity of the slurry. 60 to 90% of TP was dissolved when pH decreased to the range 5-6. Because calcium dissolution was more progressive and increased down to pH 4, it could be determined that an optimal acidification for struvite recovery should not go further than 5.5, balancing the need for high phosphorus dissolution and low calcium concentration.

Secondary fermentation of lactic acid toward acetate, propionate, butyrate and valerate occurred when pH did not drop below 5 during lactic acid fermentation. A pH re-increase followed, re-precipitating most of the phosphorus together with calcium.

Since the process could potentially be implemented in parallel of anaerobic digestion, biological acidification was also tested on digested swine manure and similar lactic acid fermentation took place.

Various organic wastes were tested but lactic acid fermentation did not occur systematically. Only the wastes with high, easily degradable carbohydrates content led to lactic acid and very low pH, while the other co-substrates led to mild acidification with mostly acetate, butyrate, propionate and valerate as final products. The microbiological analysis using 16S rRNA gene high throughput sequencing confirmed that lactic acid bacteria (Lactobacillus) were responsible for the lactic acid production while

Clostridiales dominated during more moderate acidification.

Several co-substrates were ranked in term of acidification capacity compared to sucrose in order to expand the initial model predicting lowest pH to these co-substrates. Experimental results indicated that such predictions were accurate when lactic acid production occurred (e.g. apple or carrots) or for mild acidification only (pH 5-6). Interestingly, part of the dissolved-P obtained came from the cosubstrates themselves as a result of organic matter hydrolysis. Consequently, during biological acidification of pig slurry with low P content, P dissolution was only partly a function of pH, with time dependent increase or decrease of P concentration as a result of hydrolysis and biomass assimilation respectively. Therefore the P content of waste should be taken into account in low P content slurry, with longer acidification time to allow its release.

A semi continuous reactor fed with raw swine slurry, carrots and peas (HRT = 4 days) led to a progressive decrease in pH from 5.7 to 5.4 and a slight improvement in phosphate concentration from 45 to 50% of TP. While this dissolution rate is slightly lower than previous results of biological acidification (60-90% TP), this corresponds to 750 mg PO 4 3--P/L, i.e. a very concentrated stream compared to most processes reviewed in the Context section. However, no lactic acid was present, supernatant, with ammonium representing 9% and phosphates 21%. At such initial pH, only 3% of lactic acid would have been protonated. By optimizing HRT and cultivating progressively a biomass rich in lactic acid bacteria, the amount of magnesium hydroxide could be significantly reduced.

Further investigation should also be conducted in order to assess the impact of this P-recovery process on subsequent / parallel digestion process. It has been shown in chapter 2 and 3 that consequent hydrolysis of the co-substrate occurred. This could potentially increase the methane potential during anaerobic digestion or reduce the hydraulic retention time in the digester.

To conclude, this work has demonstrated that a large fraction of total phosphorus in pig slurry (40-90%) could be dissolved by adding organic waste collected locally and with relatively short retention time (48 hours for sucrose, apples and carrots, 96 hours for beans and peas). Lactic acid fermentation provides the quickest and most efficient dissolution process with little acidic buffer to overcome when precipitating struvite. Implementing this P recovery process in some of the many digestion units existing in Brittany for the treatment of livestock, industrial and municipal organic waste would significantly reduce the impact of animal breeding on the local environment and provide a clean Pfertilizer for crop producers, thus participating in progressively closing the phosphorus cycle.

ABSTRACT

Phosphorus is a crucial nutrient for life, implicated in cellular bioenergetics as well as storage and processing of genetic information. It is also one of the limiting nutrients in agriculture with nitrogen and potassium. Since the green revolution in the middle of the 20 th century, agriculture has relied on increasing amounts of cheap mineral P-fertilizers produced from a fossil resource to improve crop yields and sustain population growth. However, the resource is depleting and its use efficiency is poor: less than 20% of extracted P is actually consumed in food. One of the reasons for this is the specialization of entire regions into on type of agricultural production or another. Thus, regions focusing on high-yield crops require large applications of fossil mineral fertilizers while intensive livestock breeding areas cannot find an output for their P-rich manure due to the distance with crop fields in need of P fertilization. Over application of animal manure in Brittany is the main cause of eutrophication in the area. Phosphorus could be recovered from pig manure as struvite, a concentrated, slow-release mineral fertilizer easily transported to croporiented regions in need of P fertilization. P in pig slurry is mostly under a solid inorganic form, requiring dissolution prior to precipitation as struvite. Because chemical acidification is too expensive and harmful to the environment, the process developed in this PhD relied on acidogenesis, a biological process in which organic matter is converted to organic acids under anaerobic conditions, thus naturally acidifying the swine slurry. Various organic wastes were tested as organic co-substrates on raw and digested pig slurry, leading to lactic acid fermentation when the co-substrate had a high content in easily biodegradable carbohydrates and a fermentation with diverse organic acids produced at low content in easily biodegradable carbohydrates. Lactobacillus was the genus responsible for lactic acid fermentation and various Clostridiales dominated otherwise, producing acetate, propionate, butyrate and valerate. A reactor was operated with semi-continuous feeding of raw swine slurry and carrot/pea, leading to the dissolution of 50% total-phosphorus or 750 mg-P/L. After centrifugation, struvite was precipitated in the supernatant by adding magnesium hydroxide to increase the pH to 8. 99% of dissolved P precipitated. The solid recovered contained 70% of struvite, a slight excess of P and Mg as well as organic matter. Because hydrolysis of organic matter and production of organic acids occur during acidogenesis, the process could be implemented in the many anaerobic digestion units installed in Brittany treating animal manure and agricultural, industrial and municipal organic waste. The struvite recovered could be sold to regions in need while the digestate impoverished in P and rich in organic matter could be kept locally. Such process would reduce eutrophication due to over application of pig manure and reduce as well the reliance on fossil P-fertilizer by offering an alternative source with equivalent fertilizing performances.

Keywords: phosphorus recovery, struvite, swine manure, pig slurry, lactic acid fermentation