Elisabeth Neuhauser

Thomas Le Floch

Gaëlle Bulteau

Marie -Cécile

Julie Gwénaëlle

Coline Pierre Mathias

Siyu Ainsi

Chen Wang

Abdel Lakel

Isabelle Bourven

Michel Baudu

Christophe Dagot

VDW: volatile dry weight (X)DLVO: (extended) Derjaguin

Keywords: packed bioreactor, filtration, purification, river sand, crushed aggregate, biomass

Je tiens à adresse un merci particulier aux personnes qui ont

ma gratitude sincère à Julie, Anthony, Vincent, Thomas et Julia pour leur aide énorme sur l'accessibilité et la maintenance de la plate-forme (i.e. fosses toutes eaux), ainsi de leur aide à tout moment des travaux lourds. Je remercie également Bruno, Stéphane, et Yvan pour le transport de l'effluent d'entrée du pilote tous les semaines durant un an et demi! J'exprime également ma gratitude à tous mes collègues du laboratoire GRESE à Limoges et je garderai un excellent souvenir de mes visites à GRESE où j'ai toujours été accueille avec la plus grande sympathie. Je tiens à remercier particulièrement Karine, Alexandra, Gosia, et Asmaa pour leur aide et leurs conseils inspirants. Je remercie sincèrement à mes compatriotes: Lulu et Feishu, pour leur accompagnement et leur aide durant la période de soutenance.

Enfin, je remercie ma famille, surtout mon grand-père qui était toujours fier de moi et nous avait quittés cet été, qui me manque et manquera toujours. Je remercie et dédie ce manuscrit à mes parents, qui m'avoir toujours donné énormément d'amour et de soutien. Je ne saurai trouver les mots pour exprimer toute ma gratitude pour eux... height and hydraulic loading ................................................ 102 L'absence de mise à jour des connaissances limite le développement des technologies de l'assainissement autonome et laisse de nombreuses questions rencontrées dans la pratique des SPANC sans réponse.

(Article 1) Evolution of purification efficiencies of different filter materials and impact of filtration packing

List of Figures

List of Tables

L'exploitation des sables alluviaux est aujourd'hui limitée dans un contexte réglementaire européen à travers des notions de continuité morphologique des rivières. Son utilisation dans certains domaines tend à être limitée, et des matériaux de substitution comme les granulats concassés sont envisagés pour le garnissage de filtres. Cependant l'impact de l'application de ces nouveaux matériaux à la filtration d'effluents septiques est très peu documenté.

Éléments scientifiques de l'étude

Les systèmes d'assainissement autonomes

Les travaux de recherche sur les systèmes d'assainissement autonome ont débuté au milieu du 20e siècle et ont concerné essentiellement les mécanismes de rétention et de dégradation dans les sols ou dans les lits filtrants constitués de matériaux granulaires et surtout de sables. Ils ont également permis de déterminer les conditions opératoires et d'exploitation des filtres afin de prolonger leur durée de vie et notamment dans le cas du traitement secondaire par infiltration-percolation dans des parcelles de sol ou sur les lits de filtration. Les fosses toutes eaux sont en mesure de réduire très largement les matières en suspension par décantation, réduire la quantité d'azote par digestion anaérobie et ammonification. Cependant, les effluents septiques sont toujours considérés comme l'une des raisons la plus fréquente de l'eutrophisation des milieux aquatiques et de la contamination eaux souterraines (Withers et al., 2011;[START_REF] Stanford | The impact of co-contaminants and septic system effluent quality on the transport of estrogens and nonylphenols through soil[END_REF]. L'existence d'un traitement secondaire par infiltration dans le sol ou sur des tertres garnis de sable permet, avec des conditions d'écoulement insaturés, l'amélioration de l'épuration de l'effluent en engageant différents mécanismes dans le milieu poreux: filtration mécanique des matières en suspension, dégradation biologique de la matière organique par la biomasse hétérotrophe développée au sein du matériau filtrant, conversion de l'azote organique ou l'ammoniac en ions nitrates par la biomasse autotrophe également présente dans le réacteur de filtration (Van Cuyk et al., 2001;. Rodgers et al., 2005;Gill et al., 2009). Les lits de filtration en écoulement insaturé permettent également la réduction des microorganismes par rétention mécanique, adsorption aux interfaces solide/eau ou gaz/eau ou par prédation par des protozoaires [START_REF] Wan | Influence of the Gas-Water Interface on Transport of Microorganisms through Unsaturated Porous Media[END_REF][START_REF] Stevik | Retention and removal of pathogenic bacteria in wastewater percolating through porous media: a review[END_REF]Chabaud et al, 2006). Les tertres d'infiltration, notamment les filtres à sable sont les plus recommandés et couramment installés en raison de leurs bonnes performances, leur facilité d'installation, et pour les aspects économique et esthétique.

Même si les mécanismes de dégradation de la matière carbonée de l'élimination de l'azote et du phosphore ont fait l'objet d'études, les informations concernant la nature de la biomasse et du biofilm développé sur les supports utilisés en assainissement autonome sont incomplètes. Les principales approches sur le biofilm ont été motivées par un élément de dysfonctionnement des filtres fréquemment rapporté, le colmatage. Les travaux menés dans les années 2000 ont concerné le développement de la biomasse totale et parfois des éléments qualitatifs du biofilm : protéines, polysaccharides. Ces travaux ont montré des modifications du biofilm en fonction de la température, du temps de fonctionnement et suivant la profondeur du réacteur filtrant [START_REF] Le Bihan | Monitoring biofilter clogging: biochemical characteristics of the biomass[END_REF]Ragusa et al., 2004;Zhao et al., 2009). La matrice des EPS contient diverses macromolécules biologiques (protéines, polysaccharides, les lipoprotéines, glycoprotéines ... synthétisées par la biomasse cellulaire ou issue de la lyse des cellules) ainsi que les composés organiques adsorbés de l'environnement (substances humiques-like, substrats, minéraux) [START_REF] Frølund | Enzymatic activity in the activated-sludge floc matrix[END_REF]Higgins & Novak, 1997). Des auteurs ont affirmé que la présence d'EPS induit le colmatage du filtre ou une conductivité hydraulique réduite (Vandevivere & Baveye, 1992). Cependant, la compréhension du rôle et de l'implication négative ou positive des matériaux dans le processus de développement du biofilm et dans la production des EPS reste très limitée.

Facteurs modifiant le biofilm

Le rôle des matériaux filtrants sur le développement d'un biofilm ou sur la structuration de celui-ci dont les constituants de la matrice extracellulaire, a également été documenté (Rodgers et al., 2004a;[START_REF] Kim | Biofilm morphology as related to the porous media clogging[END_REF]. Ces travaux montrent une influence de la taille des matériaux sur la quantité totale de biofilm présente et une modification par la vitesse du fluide de la structure du biofilm.

Le temps de rétention des solides (SRT) ou l'âge des boues, a également un effet considérable sur la production d'EPS, mais les résultats rapportés dans la littérature sont quelque peu contradictoires. Des chercheurs ont constaté que la quantité d'EPS dans divers agrégats microbiens augmente avec une augmentation du SRT, ce qui implique que les bactéries produisent plus d'EPS dans des conditions endogènes. [START_REF] Sesay | Enzymatic extraction of activated sludge extracellular polymers and implication on bioflocculation[END_REF] ont constaté que l'augmentation du SRT était positivement corrélée avec la quantité totale d'EPS dans des boues activées ainsi qu'avec la quantité de protéines et de glucides contenue dans les EPS. Le rapport entre les protéines et les sucres augmente également de 1,5 à 2,5 avec une augmentation de la SRT de 4 à 20 jours. Cependant, certains chercheurs ont suggéré que la production d'EPS est indépendante du SRT. [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF] ont constaté que la teneur totale en EPS n'a pas changée significativement avec une augmentation du SRT alors que le ratio protéine/glucide augmente lorsque le SRT est porté de 4 à 12 jours et reste inchangé alors que le SRT a été augmentée de 12 à 16 jours. Li & Yang (2007) ont rapporté que les TB-EPS (tight bound EPS) de boues activées n'ont aucun rapport avec le SRT, mais que la quantité de LB-EPS (loosely bound EPS) diminue avec une augmentation de SRT.

L'hydrodynamique du réacteur a également été considérée via le régime d'écoulement (cisaillement). La force des cisaillements dans un réacteur peut également influencer la composition en EPS. L'augmentation du taux de cisaillement (dF/dS) ou l'augmentation de l'intensité de l'aération dans un réacteur SBR (réacteur batch séquentiel) semble augmenter la quantité d'EPS par unité de masse de boues (Adav et al., 2007). Des travaux réalisés sur ce même type de réacteur, montrent que la quantité de polysaccharides extraite des boues activées augmente avec le taux d'aération alors que le celui de protéines reste constant (Shin et al., 2001). Ces résultats montrent donc un comportement différent des bactéries soumis au cisaillement. [START_REF] Ramasamy | Effects of shear stress on the secretion of extracellular polymeric substances in biofilms[END_REF] ont montré qu'une augmentation brutale des forces de cisaillement entraine une augmentation de la quantité de sucres contenue dans les EPS, mais après un certain temps la composition des EPS retrouve son état initial. La libération des EPS des boues dans le milieu peut être accentuée dans certaines conditions hydrodynamiques par un effet mécanique de cisaillement [START_REF] Aquino | Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds[END_REF]Sheng et al., 2006a).

Les conditions d'aérobie ou d'anaérobie peuvent également modifier la production d'EPS avec l'observation d'une diminution du taux d'EPS dans des boues activées dans des conditions d'anaérobie (Nielsen et al., 1996). La désintégration de boues activées dans des conditions de carence ou de limitation en oxygène a été observée [START_REF] Wilén | Short term effects of dissolbed oxygen concectration on the turbidity of the supernatant of activated sludge[END_REF][START_REF] Afnor | [END_REF][START_REF] Gaval | Impact of the repetition of oxygen deficiencies on the filamentous bacteria proliferation in activated sludge[END_REF]. Ce phénomène pourrait être attribué à l'arrêt de la production d'EPS ou à l'hydrolyse de celles-ci. Shin et al. (2001) a comparé la production des EPS par des boues activées dans trois bioréacteurs et ont observent qu'un taux d'oxygénation augmente la production de polysaccharides avec le temps alors que le taux de protéines reste inchangé. Pour de faibles taux d'oxygénation, les concentrations de polysaccharides comme de protéines sont inchangées.

Problématique et objectifs de l'étude

Comme nous venons de le voir, l'hydrodynamique, l'oxygénation ou l'âge des boues sont des paramètres de la quantité et de la qualité du biofilm dont les EPS. Ces éléments sont susceptibles de modifier deux critères essentiels de l'évaluation d'un système d'assainissement autonome, i.e. les rendements épuratoires et le colmatage du filtre. La nature du garnissage est susceptible d'impacter l'hydrodynamique d'un réacteur qui, par son fonctionnement insaturé, modifiera les conditions d'oxygénation du milieu et par un cisaillement plus ou moins important du biofilm, probablement l'âge des boues. La substitution du sable de rivière par des granulats peut donc entrainer des modifications majeures dans le fonctionnement et donc dans l'efficacité des massifs filtrants. Peu d'études ont été réalisées avec des matériaux concassés issus de carrières et aucune n'a fait l'objet d'une approche du fonctionnement biologique d'un filtre comparativement au sable. Liénard et al. (2001) a comparé les caractéristiques de matériaux de substitution et de sable comme la taille des grains, la porosité et la présence de particules fines. Une étude de l'échelle du laboratoire à court terme a été axée sur l'efficacité du traitement et de la biomasse sur agrégat écrasé (Wanko et al., 2005).

Ainsi, même si le rôle de la structure des matériaux sur le comportement de la biomasse fixée est méconnu, la modification des matériaux va modifier des éléments de l'hydrodynamique et donc les conditions de développement du biofilm et des EPS liées à ce biofilm.

Afin de mettre en évidence un comportement différent des filtres en fonction de la nature du garnissage, nous avons donc choisi de concentrer nos expériences et nos observations sur des paramètres globaux de fonctionnement (rendements épuratoires) et sur des éléments apriori plus sensibles comme le biofilm et son organisation. Ces derniers éléments seront interprétés en tenant compte des caractéristiques des matériaux et de l'hydrodynamique des réacteurs.

Méthodologie et organisation de la thèse

La méthodologie retenue pour ce travail est une approche comparative d'installations pilotes sur une longue période afin d'observer des différences de comportement dans un régime pouvant être interprété comme stationnaire. Cette expérimentation longue permet d'observer d'éventuelles différences de comportement de réacteurs soumis aux mêmes conditions extérieures (T°, nature de l'alimentation) et donc à leur même variabilité. Cette expérimentation longue, sera validée par des duplicatas de colonnes garnies par les mêmes matériaux mais de hauteurs différentes, la hauteur ne devant pas interférer sur la colonisation du réacteur (pour une profondeur identique dans le lit). Des effets de charges volumiques (expérimentation à deux charges volumiques) doit permettre d'accentuer l'expression de certaines différences dans le comportement des matériaux (accumulation/rétention/croissance de biofilm).

Quatre matériaux ont été choisis : deux sables et deux granulats concassés avec 1 sable et 1 concassé dont les diamètres moyens sont proches.

L'approche méthodologique retenue est donc :

• Etude comparative des caractéristiques des matériaux entre les sables des rivières et des granulats concassés et impact sur l'hydrodynamique; • Comparaison des capacités et de l'efficacité épuratoire des 4 matériaux choisis.

• Comparaison de la production et de la qualité du biofilm et des EPS et facteurs d'influence apportés par les différents matériaux.

Afin de répondre aux objectifs, ce travail de thèse est présenté en quatre parties :

-La première partie présente un état de l'art sur les réacteurs filtrants dans le cadre de l'assainissement non collectif. Les données concernant ce procédé et les caractéristiques des matériaux de garnissage sont discutées dans cette partie. Les mécanismes et les principaux facteurs contrôlant l'épuration de l'effluent septique dans un système de filtration sont évoqués en détails. Les mécanismes du colmatage biologique, le développement du biofilm, la matrice extracellulaires et ses composants structurel majeurs sont ensuite présentés. La fin de cette partie est consacrée aux moyens de caractérisation du biofilm (populations et composants structurels) afin de nous orienter sur des choix méthodologiques.

-La seconde partie est consacrée à la caractérisation des matériaux filtrants et à leur mise en oeuvre en réacteurs. Le comportement des différents réacteurs matériaux a également été intégré à cette partie. Quatre matériaux, deux sables roulés et deux agrégats concassés, sont caractérisés et comparés en fonction de paramètres de forme et de compositions minérales. Les réacteurs sont étudiés notamment sur leur comportement hydrodynamique qui est représenté par le temps de séjour en écoulement discontinu.

-La partie suivante est consacrée à l'étude comparative des efficacités épuratoires des In this chapter, a literature review offers an overview of relevant and important studies in the research area of on-site wastewater treatment systems. It revisits the current knowledge on the treatment plants of domestic sewage in decentralized areas, including backgrounds, technique choices and treatment efficiencies. It also reviews the challenges encountered in the practice of vertical drained sand filters such as the clogging problem. It allows summarizing the various mechanisms involved in the process under unsaturated conditions of sand filter, and finally brings out the objectives and focus of this study. This chapter also includes currents studies on biological aspects of biofilm and its compositions and properties resulted from wastewater purification process.

I.1. On-site wastewater treatment systems (OWTS)

OWTS concerns 12 to 15 million people in France. According to French Standard of 07 September 2009, OWTS are defined as the technologies insuring the collection, the transport, the treatment and the evacuation of domestic wastewater from the buildings that cannot be connected to municipal wastewater network. This order also gives the prescriptions of applicable techniques for on-site treatment plants receiving a raw organic pollutant charge inferior or equal to 1.2 kg/j of BOD 5 (Arreté 07/09/2009, mortified by Arreté 07/03/2012).

The technique choices and solutions are summarized by French standard NF DTU 64.1 [START_REF] Afnor | [END_REF]. The OWTS are generally categorized as soil (or reconstructed media) infiltration systems and other installations with other treatment dispositive (micro-station, rotating biological disk...). The qualities of treated effluent request to be in accordance to receipting environment requirements: BOD 5 <35mgO/L and TSS <30mg/L.

I.1.1. Domestic wastewater (Raw water)

The domestic wastewater is produced by inhabitation. The flow rate of effluent collected from the outlet of an individual house in France varies from 80 to 180 L/habitation/day. The domestic wastewater is the combination of 2 major waste sources: about 25% of the effluent is toilet waste (blackwater) from WC flushes concentrated in human excreta such as faeces, Part Ι: Literature review urine and companied by toilet paper disposal; the rest (about 75%) combines wastewater (greywater) from other sources: bath, shower, wash basin, kitchen sink and washing machine (Almeidaet al., 1999). Blackwater and greywater have different characteristics, but both contain pollutants and disease-causing agents that require treatments. The black water contributes the majority of organic pollutants and pathogenic agents. The greywater brings in about 70% of the total volume that contains detergents, washing powder, and greases (Chocat, 1997). [START_REF] Eriksson | Characteristics of grey wastewater[END_REF] indicated that there were about 900 different xenobiotic organic compounds in greywater.

The domestic wastewater has similar compositions to municipal wastewater. The wastewater components that most wastewater facilities are designed to remove, are suspended solids, organic matters, nutrients and pathogenic organisms: Suspended solids (SS) consist of inorganic and organic materials. The suspended solids must be significantly reduced by treatment or they can increase the chemical oxygen demand (COD) when discharged to receiving water and provide sorption sites for microorganisms and other pollutants. They can also clog the soil adsorption fields in onsite systems.

Organic matters (OM) in wastewater are traditionally characterized by COD, total organic carbon (TOC) and biological oxygen demands (BOD) and are often composed of particulate and dissolved fractions (size). However, the chemical compositions of OM are highly heterogeneous and are ranged from simple compounds like acetic acids to more complex polymers. The major chemical fractions in organics of wastewater are proteins, lipids and sugars, and their fractions can be changeable according to various conditions (Raunkjaer et al., 1994). [START_REF] Huang | Chemical composition of organic matters in domestic wastewater[END_REF] indicated that the largest organic group in domestic wastewater was fibers (major components characterized as cellulose, hemicelluloses and lignin) which represented 20.64% of TOC, followed by proteins (12.38% of TOC) and sugars (10.65% of TOC), and the sum of volatile fatty acids, soluble proteins and soluble sugars represented about 30% of total COD. Part Ι: Literature review Typical nutrients in domestic wastewater comprise of inorganic and organic compounds of nitrogen and phosphorus, and also salts of certain metals (Na, Ca, K, Mg, Co, Mo, Cu, Mn). Nitrogen is reported as the sum of organic nitrogen, nitrate salts and ammonium. In a household, over 85% of the nitrogen load originates from the blackwater, ranging from 130 to 180mg/L (Palmquist & Hanaeus, 2005). Concentrations of Total Kjeldahl Nitrogen (TKN) in greywater range from 1 to 50mg/L [START_REF] Eriksson | Characteristics of grey wastewater[END_REF]. The phosphorus presents in forms as orthophosphates, polyphosphate, and organic phosphorus (Crites & Tchobanoglous, 1998). The human body wastes, especially the urine are the major source of nitrogen and phosphorus. Food residue, laundry detergents, personal care products and cleaning products also contribute to the concentration of nitrogen and phosphorus and the presence of certain metals.

The main source of microorganisms in domestic wastewater is human feces.

Various types of microorganisms present in the domestic wastewater: bacteria, virus, and protozoa [START_REF] John | Review of Factors Affecting Microbial Survival in Groundwater[END_REF], including: Bacteria: Escherichia coli, (E. coli), Salmonella spp., Shigella spp., Vibrio cholera, Yersinia, Pseudomonas (Matthess & Pekdeger, 1985); Virus: Poliovirus, Rotavirus, Hepatitis, Echovirus… [START_REF] John | Review of Factors Affecting Microbial Survival in Groundwater[END_REF]; Protozoa: Giardia lamblia, Entameobahistolytica, Cryptosporidium parvum [START_REF] Macler | Current knowledge on groundwater microbial pathogens and their control[END_REF].

The main contaminants contents vary with the countries and their own living habits. The data of the concentrations of the critical characteristics of domestic wastewater must be summarized from three studies in France (Table 1

I.1.2. Primary treatment and Septic effluent

The fate of main pollutants in septic tank

The raw wastewater is generally collected, transported and pre-treated by septic tank. A septic tank is a buried watertight container that served as the combination of settling tank and unheated unmixed anaerobic digester. The pre-treated effluent is called septic effluent. The septic tank insures the separation of floated materials and most of settable solids from liquid, and certain solids and non-settable organics undergo the decomposition by anaerobic digestion. The anaerobic digestion can be separated into two steps: fermentation (results in organic acids) and methanization (results in biogas). During the fermentation, the hydrolysis of complex organic matters firstly takes place and leads to an incomplete degradation of these organics into simple monomers; secondly these monomers are degraded directly into acetones or biogas (CO 2 , H 2 ); or undergo the acidogenesis and results in organic acids (propionic acids, Part Ι: Literature review butyric acids, acetic acids); During the methanization, the organic acids or monomers undergo an acetogenesis and a methanogenesis, then finally degraded into biogas (CO 2 , CH 4 ).

However, in septic tanks, methanogenesis bacteria hardly survive, and fermentation process predominates. This fact results in an important accumulation of partly degraded organics and volatile grass acids [START_REF] Philip | La fosse septique en assainissement individuel: CAractéristiques et mécanismes de l'épuration, effets de bioactivateurs Université de Montpellier[END_REF].

Before entered in septic tank, 73% of nitrogen presented in domestic water is organic nitrogen and 24% exists as ammonium. The nitrogen in raw wastewater is partially treated in conventional septic tanks due to the anaerobic conditions. During this process, much of organic nitrogen is converted to ammonium known as ammonification; as a result, the nitrogen in septic effluent is 70-90% of ammonium and 10-30% of organic nitrogen [START_REF] Lowe | Influent constituent characteristics of the modern waste stream from single sources: Literature review[END_REF]. Some removal (20-30%) of phosphorus in raw wastewater is attributed by the accumulation in residual sludge that settled at the bottom. As a result, the septic effluent still contains 80-100% of phosphorus concentration found in the raw wastewater [START_REF] Lowe | Controlled field experiment for performance evaluation of septic tank effluent treatment during soil infiltration[END_REF]. The removal of microorganisms is also relatively low.

Environmental impact of septic effluent

The widespread use of septic tank in suburban and rural areas contributes the disposal of septic effluent in receiving environments, and becomes one of concerns of groundwater, surface water and soil contaminations that are susceptible provoking waterborne diseases. In United States, septic tanks are one of the most frequently reported sources of groundwater contaminations (Yates, 1985). The septic tank effluents which contain phosphorus and ammonia are also potential source of eutrophication risk in rural aquatic environments, and the ammonium and the phosphorus absorbed by the sediments may become a source of nutrients for submerged macrophytes (Withers et al., 2011). The organic matters partially removed by anaerobic digestion remain in septic tank and septic effluent. The steroid estrogens and nonylphenols remained in septic effluent have the possibility of penetrating in soil adsorption systems and being transported into groundwater [START_REF] Stanford | The impact of co-contaminants and septic system effluent quality on the transport of estrogens and nonylphenols through soil[END_REF]. Septic effluent or pre-treated domestic wastewater, being highly risky to natural environment contaminations, should not be discharged directly and further treatments are necessary. The secondary treatment of domestic wastewater should respond to the conceptions of on-site sanitation: the treatment should be built in situ and technically simple to be carried out, controlled and monitored. The treatment should not also consume much energy. The design should not affect the landscape, etc.

I.1.3. Secondary treatment of OWTS -Soil infiltration systems

The secondary treatments of OWTS or the treatments of septic effluent are generally based on soil adsorption fields by using the infiltration and purification capacities of the natural soil. The field is sometimes reconstructed by other packing materials having similar structure but with characteristics more suitable for the treatment.

The natural soil adsorption are generally referred to soil infiltration trenches (underground), spreading bed of small depth (underground) and infiltration mounds (aboveground). The principle of these different configurations is similar and it is based on the infiltrationpercolation theory: the effluent percolates through several meters in the unsaturated zone and at the same time combining the purification phenomena. The purified effluent will be recharged in the water table, or will be drained outside.

The structure and the composition of soil decide the soil permeability. Natural soil is a mixture of various kinds of grains: clay, silt, sand, gravel, etc. The soil texture impacts physically and chemically the functioning of infiltration system: a clayish soil indicates a better chemical capacity of pollutants, but worse physical behaviors (poor porosity, low permeability, terrible air exchange ability…); a sandy soil presents a higher permeability and a better air exchange, but a weak adsorption capacity [START_REF] Hillel | The effect of nutrients on extracellular polymeric substance production and its influence on sludge properties[END_REF]. The permeability of soil K is determined by the following equation:

K = Quantityofwaterinjected (m ) Infiltrationsurface (m ) × Testperiode (h) (mm/h)
The permeability K varies with the texture of the soil, thus the permeability. The following table (Table1.3) summarizes the values of K and the permeability for some textures of soils: According to NF DTU 64.1, for a clayish soil with K<10, the infiltration-percolation systems by natural soil are not recommended. In such case, infiltration systems can be designed with other packing materials, such as constructed wetlands, horizontal or vertical drained sand filters with or without plants. Among these systems, single pass vertical drained sand filter with discontinuous dosing is preferred, due to its good performances against minor energy consumption.

I.1.4. Current problems of vertical unsaturated sand filter

Filter sizing is empiric. The lack of scientific base of filter conception often leads to oversized filtration beds in order to meet the potential needs of long-term functioning. Efforts and criteria have been made with the purpose of finding the correlation between filter sizing and long term functioning (clogging) since 1970s: Authors tented to propose the operation conditions and sizing criteria that provide a balance between necessary nutrients loads and Part Ι: Literature review clogging provoked by organic and particulate matters accumulation [START_REF] Healy | Site Evaluation and Design of Seepage Fields[END_REF];

Later, design criteria have been improved since more full scale soil infiltration beds and sand filters have been investigated [START_REF] Jenssen | Technology Assessment of Wastewater Treatment by Soil Infiltration Systems[END_REF].

Packing materials: not all the granular materials can be used in sand filter. Proper, durable, inert and granulometric well defined materials are required. In general, the alluvial sands are recommended. However, due to the excessive exploration, the alluvial sands are less available that drives more attention to other suitable, potential materials, for example, the crushed aggregates resulted from massive rocks reducing is nowadays considered as appropriate substantial choice, however their characteristics still need analysis and comparisons to the common materials. The characteristics of materials are discussed in the next chapter.

Clogging: filtration system failures reported are mainly caused by clogging.

Investigations of clogging lead to three major mechanisms: pore blocking caused by suspended solids (DeVries, 1972); over developed biomass and accumulation of extracellular polymers substances, such as proteins and polysaccharides (Pell & Nyberg, 1989b); and precipitation of certain metals such as FeS and CaCO 3 (Cooke et al., 1999). First of all, the extraction of alluvial sands can be effectuated directly, however, for compact deposits like massive rock, the explosive have to be employed. One explosion can result in cutting down of a large quantity of rocks (more than 10 000 tons). Then exploded rocks are transported to the treatment site for following operations: crushing, cleaning, and screening in order to get the desired granulometry.

The crushing is performed by crushers in order to reduce successively the various sizes of raw materials. There are several types of crushers: jaw crushers, gyratory crushers, cone crushers, impact crushers (horizontal shaft impactor and vertical shaft impactor). The principles of these crushers are similar: the force is given mechanically by rock breakers (jawstock, eccentrically gyrating spindle, or hammers), in order to break the rocks into smaller pieces before going into the screening machines. Screening (or sieving) allows selecting the grains.

The sieve permits only to let pass the elements inferior of certain size. Granulates of desirable size can be chosen and obtained by several time of crushing and screening. The washing has to be carried out in order to remove dusts during production process and keep the final products clean which is necessary to industrial utilizations. The operations of screening and washing are often combined together under water ramps during the sieving. Once granulates are reduced, treated, and classified, the products are transported to storage areas.

I.2.2. Physical characteristics of materials

Granulometry and effective diameter

Granulometric analysis (EN 933-1, AFNOR, 1997) permits the determination of material grain sizes and the distribution of grains in a granular material. This analysis consists of fractioning the material by screens with different opening which are defined by European standard EN 933-2. With granulometric analysis, some parameters can be determined, such as effective diameter, average diameter, uniformity coefficient, and fines particles contents. The effective diameter is defined as the screen opening letting pass 10% of sand sample which is referred to D 10 , similarly D 50 and D 60 are also defined. For the infiltration, most studies have agreed that the d10 should be at least 0.2mm [START_REF] Gold | Wastewater Renovation in Buried and Recirculating Sand Filters[END_REF][START_REF] Guilloteau | Epuration des eaux usées urbaines par infiltration-percolation Techniques sciences méthodes[END_REF]. The ideal d10 is recommended in the range of 0.2-2mm by DTU 64.1.

Part Ι: Literature review

Uniformity and porosity

Both the river sand and crushed aggregates presents the variety of grain size, with less or more fine particles filling in the space between larger grains. The Uniformity coefficient (UC)

is defined as the ratio between D 60 (the screen diameter passing 60% of sand sample) and D 10 , estimated from the grain size distribution. The sand is less uniform with larger UC. The recommended UC value is between 3 and 6 in France. Sands with higher UC value are more heterogeneous and more unpredictable. They may contain tortuous path for water to move through, thus different hydraulic and hydrodynamic behaviors may be observed. A good filter media should have large porosity to amplify air exchange (Ball, 1994). Studies have showed that the degree of heterogeneity has less impact on infiltration rate than D 10 and fines content (Liénard et al., 2001). The UC also has less impact on the treatment performance than the grain sizes [START_REF] Darby | Shallow intermittent sand filtration performance evaluation[END_REF].

Fine particles content

The recommended fine particles content does not exist. But the fine particles content in sand could interfere the infiltration process: [START_REF] Revil | Permeability of shaly sands[END_REF] indicated that the permeability of a clayish sand decreases with the increasing of fine clay particle percentage.

Then authors revealed by studying different types of sands, that the infiltration rate decrease in the sands with higher fine particles content thus the permeability also decreases (Liénard et al., 2001). This study recommended also the utilization of sand with fines content inferior of 2.5%.

Particle shape

The shapes of material grains are determined by mechanical and chemical forces.

Chemical action and abrasion increase with age and older sands tend to be rounder regardless of particle size. The larger the particle (typically in grain size>0.4mm) is, the higher imperfections and brittle fracturing. Conversely, smaller particles are stronger because of the lack of imperfections [START_REF] Margolis | Processes of formation and environmental occurrence of microfeatures on detrital quartz grains[END_REF].

I.2.3. Impact of physical characteristics

According to French standard DTU 64.1, the grain size distribution is the key to sand filter design and functioning. The grain size manages the percolation behaviors and Part Ι: Literature review purification behaviors: the larger the grains result rapid percolation, however, hydraulic residence time and pollutant treatment efficiency reduce (Boller & Kavanaugh, 1995). In the other hand, a filter of finer material is more fragile to the clogging: a contrast study showed that compared to coarse sand (D 10 =0.8mm), the oxygen content decreased over time in finer sand (D 10 =0.35mm) which led to the unequilibrium between oxygen diffusion and consumption. On contrary, for the coarse sand, oxygen content is always stable even during continuous loading (Rolland et al., 2009). Authors proposed a narrower range of D 10 (0.2<D 10 <0.4) in order to insure an effective treatment (Liénard et al., 2001).

I.2.4. Comparison between river sands and crushed aggregates

The river sands has been applied for a long time for filtration process of municipal wastewater treatments and infiltration discharge of household wastewater treatments due to its good structural stability, chemical inert and infiltration behavior. But alluvial sands have limited source. The possibility of crushed aggregates application has been studied in several parameters which control the infiltration and purification behaviors as sand filter media.

Comparing to the alluvial sand, the crushed aggregate as filter medium is rarely carried out.

Crushed aggregate has been analogized with alluvial sand in terms of geometry characteristics and of biological treatment performance (Liénard et al., 2001;Wanko et al., 2005;Rolland et al., 2009).

The crushed aggregate is well graded and less uniform (higher UC) [START_REF] Gold | Wastewater Renovation in Buried and Recirculating Sand Filters[END_REF]. The mineralogical composition shows that the crushed aggregate contains higher limestone content (Wankoet al., 2004). Trails in France, 21 alluvial sands and 5 crushed aggregates have been compared in terms of their granulometric parameters and hydraulic behavior. Later, similar tests have been carried (Liénard et al., 2001;Wanko et al., 2004). The particles shapes of the alluvial sands and crushed aggregates are also different: the grains of crushed aggregates exhibit higher angularity and higher roughness (Tsomokos & Georgiannou, 2010).

The differences in the physic and composition of the two types of materials can induce the differences in biological treatment efficiencies. By comparing the treatment efficiencies of synthetic wastewater with the alluvial sand and crushed aggregates of similar effective diameter, Wanko et al., (2005) showed that no significant difference in organic removals using the crushed aggregates as filter media if the granulometry carefully chosen. On the contrary, another study showed that crushed aggregates exhibited low efficiencies than river sand due to the differences in particle shapes [START_REF] Schäfer | Transport of bacteria in unsaturated porous media[END_REF]. Thus the influences of sand type on the filter performance are still not quite clear.

I.2.5. Mineralogical characteristics of river sands and crushed aggregates

The filter materials are composed by various minerals. The compositions of most minerals are dominated by silica (SiO 2 ), lime (CaO) and alumina (Al 2 O 3 ), and other major oxides of sodium (Na 2 O), potassium (K 2 O), iron (Fe 2 O 3 ), magnesium (MgO) and titanium (TiO 2 ). The silica content varies widely from less than 50% to more than 99%. Even pure quartz sand may be bound with chemical cement, such as calcite. Comparing to alluvial sands, the crushed aggregates contain more calcites [START_REF] Collis | Aggregates: sand, gravel and crushed rock aggregates for construction purposes[END_REF]. The composition is as function of the weather, the grain size and the source of the sand. For example, the silica content decreases with the grain sizes (Pettijohn et al., 1973).

I.3. Mechanisms involved in vertical filtration systems in unsaturated conditions

The complex environment in the unsaturated filtration systems involves physical, chemical and biological phenomena, not only the filtration process but also a biological contact process. During the passage of feeding water, dissolved matters transported by liquid phase are uptaken by fixed biomass or free bacteria, or carried along with dynamic phase, or sorbed and settled in immobile phase; particulate matters are brought into contact with the surface of sand grains. Inert matters are retained on the surface or blocked in the pore throat, undergo the biological degradation and then converted into simpler forms for subsequent removal.

I.3.1. Solute transport mechanisms in porous media

The septic effluent discontinuously dosed on the filter surface, percolates slowly through the filter medium and the unsaturated conditions provide the presence of three phases: air, liquid and solid phase. The unsaturated conditions result in complex interaction, increase retention time, and facilitate aerobic bacteria growth (Kristiansen, 1981a;Boller & Kavanaugh, 1995).

I.3.1.1. Water saturation

The water flow can be described by Darcy's law as function of the gradient of the matric pressure and the hydraulic conductivity:

Equation 1: q !" = -K(θ)∇H = - '(() )* ( +, +- + ρg)
The hydraulic conductivity is filter media attitude to transmit the water, depends on the pore space, which is severely impacted by the grain sizes, shapes and connected channels of the filter media. For an unsaturated media, the conductivity depends strongly on water content θ:

as water content decreases, fewer pores are filled to conduct water and the paths of water flowing through the medium become discontinuous and tortuous, so the medium is less conductive. Thus, under the unsaturated conditions, the hydraulic conductivity K is a function of water content θ. According to van Genuchten (1980), the effective water saturation S e varies from 0 to 1 and can also be expressed by the capillarity:

Equation 2: S 2 = ( (3( 4 )
(( 5 3( 4 ) = 6 (67(8,) 9 ) :

With: θ r and θ s represent respectively the residual and saturated water content; ψ represents the capillary pressure; α, n and m are shape parameters.

I.3.1.2. Theoretical approach of solute transport in porous media

The classical theories of solute transport in saturated or unsaturated porous media are based on the hypothesis: the solute transport takes place only in the liquid phase. The transport of non-reactive solute into a porous medium is quantified by two terms: 1)

Convection (average velocity of flow) and 2) Dispersion. When a solution passes through a porous media at high velocities, molecular diffusion can be overlooked. In the cases of slow filtration, both the two mechanisms can be significant to solute transport behavior.

1) Convection: the inert solutes (concentration: C [ML -3 ]) are transported generally by water flow which is described by Darcy velocity q as mentioned as Darcy's law;

2) Dispersion: the dispersion includes molecular diffusion and mechanical dispersion. In the unsaturated flow environment, it is accepted that an immobile liquid phase exists, and this immobile phase does involve in the process of solute transport [START_REF] Van Genuchten | Mass Transfer Studies in Sorbing Porous Media: II. Experimental Evaluation with Tritium[END_REF]. The ratio between the mobile and immobile liquid phase depends on the flow velocity. The two domains convection-dispersion equation is expressed by the following two equations with solute exchange process between the two regions: The retardation coefficient caused by sorption process R is added to mobile and immobile phase. The water content θ is the sum of mobile and immobile water: θ = θ m + θ im . Later, authors have claimed that the liquid domain in unsaturated porous media consists of the mobile zone, the stationary zone comprising the irreducible water content and an immobile liquid which moves very slowly [START_REF] Kartha | Effect of immobile water content on contaminant transport in unsaturated zone[END_REF].

I.3.2. Particulate matters transport in porous media

Besides the desolved minerals and organic matters, particulate and colloidal matters are also transported with the feeding water flow. As regards to the retention by filter media are colloids and suspended small solids. The particles are brought into contact with sand grains (collectors) could be intercepted by 1) straining (or screening); 2) sedimentation; 3) physicochemical factors and 4) diffusion [START_REF] Huisman | Slow sand filtration[END_REF].

1) Straining: the particulate matters larger than the pore throats are either intercepted at the surface of the filter or blocked in the pore throat (McDowell-Boyer et al., 1986);

2) Sedimentation: this process is important only for particles larger than 10µm. The settling action within the pores is comparable to conventional settling tank where the process of sedimentation is generally described by Stocks regime (Elimelech, 1995);

3) Multi-particle bridging: the bridging takes place when multiple particles interact and form a particulate arch against a pore throat that is larger than single particle.

The likelihood of bridging depends on particle shape and the number of available particles in the pore throat [START_REF] Santos | Multiple Particle Retention Mechanisms during Filtration in Porous Media[END_REF]; 4) Mass attraction, electrostatic interactions and diffusion: these mechanisms act on very small particles. The electrostatic forces play an important role to colloidal particles and their attachment process due to charged surface for both colloids and grains. The diffusion process resulted from Brownian movement brings particles into contact with containing surface and acts independently of filtration rate throughout the whole depth of filter medium, even when water is not flowing.

I.3.2.1. Macroscopic equations

The deposition of particulate matters causes the flow head loss and permeability damage.

The macroscopic convection dispersion equations and kinetic equation have been proposed in order to integrate different mechanisms impacting the transport process of particles or colloids. These studies have been performed mostly in saturated porous media, but the unsaturated porous media has become a highlight since the gas-water interface has been noticed.

This macroscopic transport model accounts Brownian diffusivity into convection dispersion equation (Tien, 1989):

Equation 6:

;

< ;= + v !"∇C = ∇ D∇C + mC∇∅ψ
The term φ describes the colloid interaction energy and m describes the particle mobility which is related to Brownian movement by the following expression: The term ∂σ/∂t is usually called filtration rate and σ is the concentration of retained particles.

The filtration rate depends on the amount of particle available for deposition (C p : concentration of particles in the fluid) and the deposit concentration (σ: particles that have already deposited). σ varies during the course of operation and with spatial coordinate. Thus the filtration rate is a time dependent local function.

I.3.2.2. Presence of air-liquid interfaces in unsaturated environment

The unsaturated porous media contains three phases: air, water and solid, and the interactions mostly occur at the solid-water interfaces (SWI) and the air (or gas) -water interfaces (AWI or GWI) [START_REF] Schijven | Removal of viruses by soil passage: Overview of modeling, processes, and parameters[END_REF]. The particles are initially transported by liquid flow. When they move close to the AWI, they slow down due to the restricted flow around the air bubble. Once the colloids are near the AWI, they are attracted by the attractive interactions or through direct collision and finally attached to the AWI [START_REF] Sirivithayapakorn | Transport of colloids in unsaturated porous media: A pore-scale observation of processes during the dissolution of air-water interface[END_REF]. [START_REF] Torkzaban | Transport and fate of bacteria in porous media: Coupled effects of chemical conditions and pore space geometry[END_REF] showed, in their study of quartz sands, that the colloids retention increases with lower water flow velocity, lower water contents and higher ionic strength. These authors also indicated that neither the SWI nor the AWI dominates the colloids retention, but the straining remains the primary mechanism.

Part Ι: Literature review

I.3.3. Hydrodynamic in unsaturated porous media: residence time distribution

Definition

A flow of matters enters in a reactor, is composed by different fractions. The residence time of one fraction equals to the necessary time to run through the distance between the entrance and the exit. The matters can be simple molecular or aggregates of matters of a size more or less important, and all the particles of same fraction have identical residence time in the reactor. However, when a flow of matters crosses the entrance of reactor, different fractions are not crossing at the same instant. Because of this, the time passed of different fractions inside the reactor is variable. This phenomenon is represented by residence time distribution (RTD) [START_REF] Danckwerts | Continuous flow systems: Distribution of residence times[END_REF]Villermaux & Van Swaaij, 1969).

Considering a system with a constant volume V, the permanent regime is established with a flow rate q. The average concentration of concerned matter cross the outlet section is expressed as C y (t), and the mass has stayed inside during the time interval dt(from t to t+dt) can be expressed as qC y (t)dt. The pollutant hydraulic residence time distribution can be described by the following function:

Equation 9: E t = C< ^ = _ C< ^ = à and_ E t dt b c = 1
The RTD can be considered as the responds of instant injection of an ideal tracer. This function is often transformed in Laplace field:

Equation 10: G s = C S C f s andG s = _ E t e 3g= dt b c
Considering the moments of a residence time distribution are a number series of order n, the any moment of order k is represented by the product of convolution of the function E(t) and the function t k :

Equation 11: μ O = _ t O E t dt b c
The mean residence time is defined as the moment of the first order:

Equation 12: t ̅ = _ tE t dt b c
The dispersion of time distribution around the mean residence time is defined as the variance:

Equation 13: σ = _ t -t ̅ E t dt b c

Hydrodynamic models

The hydraulic residence time (HRT) in the filter medium affects numerous treatment efficiencies of sand filtration system. A different HRT means a different interaction time between pollutants and biomass. Residence time distribution varies with the hydraulic load, feeding-drainage frequencies, amount of the biomass present and the intrinsic characteristics of the packing materials.

The measurements of hydrodynamics are directed at two features of ideal flow conditions:

plug flow and continuously stirred tank reactors (CSTR). Under plug flow conditions, the concentration-time distribution is simply a spike with a very small standard deviation about the nominal mean residence time; and for continuously stirred flow conditions, the distribution takes the form of an exponential function where the effect of flow dilution in steady flow conditions progressively reduce the concentration at the outlet. The circulations take place in the real reactors can be more complex with the presence of exchanges, retard or dead zones. Many attempts used a series of CSTR with dead zone, but the model did not always fit the observed RTD [START_REF] Wen | Models for flow systems and chemical reactors[END_REF][START_REF] Riemer | Multi-component diffusion in denitrifying biofilms[END_REF].

In certain media, especially under unsaturated flow conditions, an immobile and a mobile zone exist at the same time and exchanges take place between the two phases, and this exchange is the main cause of the single dragging. By introducing volume parameters θ im and θ m representing the two zones [START_REF] Van Swaaij | Residence time distribution in the liquid phase of trickle flow in packed columns[END_REF] , the tracer mass assessments on the two phases are established: The k m [T -1 ] is transfer coefficient. Considering the system is closed at two extremities, the RTD function is given in Laplace transform [START_REF] Sardin | Modeling the nonequilibrium transport of linearly interacting solutes in porous media: A review[END_REF]: In wastewater reactors with fixed biomass, the RTDs in such systems usually present an asymmetric behavior called "tailing" due to the tracer exchange within the biofilm by diffusion [START_REF] Riemer | Residence time distribution in submerged biofilters[END_REF][START_REF] Stevens | The effect of tracer diffusion in biofilm on residence time distributions[END_REF]. Under this circumstance, the biodiffusion models were developed by [START_REF] Riemer | Residence time distribution in submerged biofilters[END_REF] considering that the transport phenomena exist in the phase of liquid flow are: convection, axial dispersion and molecular diffusion into the biomass and only the diffusion exists in the biomass section, summarized in the following With k e : the exchange coefficient, and S m , S im : the section of mobile and immobile zone.

Equation 16: G s =

I.4. Treatment efficiencies and functioning of vertical drained packed filter

The filtration reactor functions as an aerobic, fixed biomass reactor. The mechanisms of pollutants removal involve physical, chemical and biological aspects. The eliminations of pollutants and pathogenic germs depends on the characteristics of materials and the states of filter functioning. Infiltration and purification capacities evolve with the development of clogging which has two actions on the filter: favorable to biological treatment by biofilm and unfavorable due to overly accumulated matters (SS from feed water + biofilm developed in the filter). This is a complex process with various interactions among the feed water input, the secretion of biofilm and the surface of sand grains. In Figure 1.2, some current facts of sand filter functioning is presented in an overview schema.

I.4.1. Mechanisms of retention and/or degradation and purification

Suspended solids

The elimination of suspended solids depends on the particle sizes and the pore space of the sand bed. The mechanical retention takes place when suspended particles are larger than the pore throats; smaller particles could be removed by the interception and the adsorption. [START_REF] Gougoussis | Assainissement individuel et aptitude des sols à l'élimination et à l'épuration des effluents domestiques[END_REF]McDowell-Boyer et al., 1986).

Organic matters

The organic pollutants represented by organic carbon can be particulate or soluble, inert or readily biodegradable. The particulate organic matters retained on the surface of the sand massive undergo the hydrolysis and become less complex or soluble which can be readily assimilated. The soluble organic matters are retained at various depths. The complete mineralization is achieved by the aerobic biodegradation under the actions of heterotrophic bacteria which are fixed in biofilm. Under the anaerobic conditions, organic matters are only incompletely, slowly degraded into organic substances easily degradable such as proteins and also some polymers difficult to degrade such as celluloses. The degradation sometimes leads to the formation of methane, hydrogen, sulfide… [START_REF] Lefevre | Epuration des eaux usées urbaines par infiltration-percolation: étude expérimentale et définition de procédés Academie de Montpellier Université des sciences et techiniques du Languedoc[END_REF]Rodgers et al., 2005). Nitrosomonas (1) and Nitrobacter (2):

(1) Nitritation:

NH m 7 + 3 2 oe O = NO 3 + H O + 2H 7
(2) Nitration:

NO 3 + 1 2 oe O = NO 3
The nitrification can be quite performing for the cases of filtration systems because the oxygen is not limited. The authors indicated that for a hydraulic loading around 5 cm/day, a delay of latency about 19 days was necessary for establishing the fauna of Nitrobacteriaceae and after this latency, the explosion of Nitrobacteriaceae has been found between the surface and the depth of 12cm (Ardakani et al., 1974). For a strong loading, the oxidation from nitrites to nitrates becomes incomplete since the oxygen is not enough. The aeration of the filtration milieu is the necessary condition for the nitrification.

The process of denitrification exists in two ways: the NO ; the phosphorus can shortly stabilized by biological immobilization [START_REF] Gougoussis | Assainissement individuel et aptitude des sols à l'élimination et à l'épuration des effluents domestiques[END_REF]Zanini et al., 1998;Robertson, 2003).

Microorganisms

The microorganisms behave like other particles in the filtration massive where several mechanisms can involve: sedimentation, filtration, adsorption, predation… The major processes of bacterial elimination in the sand filter are the filtration, adsorption and microbial degradations which include the predation, the nutritive competition, and the parasitism [START_REF] Lefevre | Epuration des eaux usées urbaines par infiltration-percolation: étude expérimentale et définition de procédés Academie de Montpellier Université des sciences et techiniques du Languedoc[END_REF]Gammack et al., 1992;Chabaud et al., 2006). The viruses are so small that the elimination by filtration cannot be effective enough, but the adsorption and inactivation can be involved. The attenuation of bacteria and virus varies from case to case, and depends on various factors such as the pH, the temperature, the gas-water interface which is quite noted in unsaturated porous media [START_REF] Wan | Influence of the Gas-Water Interface on Transport of Microorganisms through Unsaturated Porous Media[END_REF].

I.4.2. Influencing factors of treatment efficiencies

Both environmental conditions and operating parameters impact the pollutant removals by sand filtration. Numerous studies were conducted by the alternation of the filter design in order to optimize the filter performance, such as daily hydraulic loads, dosing frequencies, the presence of plants and types of materials. The performance is an interactive, dynamic process between the hydraulic and purification behaviors. Some studies are summarized in Table 1.5:

Part Ι: Literature review [START_REF] Kauppinen | Sand filters for removal of microbes and nutrients from wastewater during a one-year pilot study in a cold temperate climate[END_REF] By reviewing previous studies, the purification efficiencies of a packed filter often undergo the influences of several factors at once, but the hydraulic retention time seems to be the governing factor. The traditional single pass filters packed with grained materials (soil, gravel, sand…) show less effective capacities in total nitrogen and phosphorus removals (about 30% of Total N and unstable removal of P) which are predictable under the aerobic filtration conditions. The filtration system will also clog, and their performances are considered being affected.

Part Ι: Literature review

I.4.3. Causes of clogging (total suspended solids + biofilm) and impacts of clogging on the filter performance

The filter media clogging has been recognized as common and significant problem that affects the hydraulic and treatment efficiencies of the system, especially the long-term performance (Kristiansen, 1981a;Siegrist & Boyle, 1987). The clogging consists of the obstruction of the pore space, and it tends to appear in the upper layer of the sand filter where the substrates and microorganisms are most abundant [START_REF] Rodgers | Surface Clogging in an Intermittent Stratified Sand Filter[END_REF]. The causes of clogging can be related to various factors, the environmental and operational conditions both influence the process. By the nature of these mechanisms, the clogging is generally categorized as physical, chemical and biological process (biofilm) (Baveye et al., 1998).

I.4.3.1. Causes of clogging

The occurrence of clogging is related to physical, chemical and biological process. The clogging development is complex because these processes take place simultaneously, continuously and interact with the substratum and also among the aggregates themselves.

Physical clogging (origin of feed water)

The deposition of inorganic and organic solids at the surface develops into a physical clogging. Feeding waters with high concentration of suspended solids have been frequently

observed to lead to a severe clogging through the process of filtration [START_REF] Rice | Soil clogging during infiltration of secondary effluent[END_REF]. The organic particulate matters difficultly or non-biodegradable can also accumulate inside the pore throat causing pore blockage (Siegrist & Boyle, 1987;Nguyen, 2000). Under high loading conditions, this accumulation of partly hydrolyzed organic particle matters reduces more rapidly the pore space than the development of biofilm (Zhao et al., 2009).

Chemical clogging (origin of feed water + filter media)

The chemical clogging is mainly caused by mineral precipitation in filter media due to the chemical properties of media grains, mineral components of wastewater, biochemical activities of biomass, and environmental factors like temperature and pH that are able to modify the solubility of these minerals (Baveye et al., 1998). The most commonly observed precipitates are CaCO 3 ; Fe, Al or Mn hydrous oxides; phosphate precipitates; and sulfate, sulfide precipitates. The carbonate and phosphate precipitates highly depend on the calcareous Part Ι: Literature review content and pH value of filter. In alkaline and calcareous environment, the free calcium in the material is responsible for the high correlation between calcium and phosphorus in phosphate minerals, and the high pH value results in an increase in CO 3 2-concentration, allowing or accelerating CaCO 3 precipitation (Cooke et al., 1999). Organic matters in septic effluent cause irons in non-calcareous sites to become soluble and subsequently results in Al-P precipitation coating on the surface of sand grains below in the filtration beds (Robertson, 2003). The formation of sulfide is generally microbial catalyzed. The sulfate reducing bacteria "breathing" sulfate rather than oxygen are known to oxidize incompletely a number of organic acids and alcohols into acetate where sulfates act as electron accepters with sulfides as byproducts. The ferrous iron (Fe 2+ ) existing already or resulting from bacteria activities readily associates sulfides and forms into FeS which are precipitates as black colloids and has a very low solubility. The FeS colloids bond strongly with organic matters, humus and do not seem to accumulate on mineral soil particles, but they form a black layer in clogged filter media (Kristiansen, 1981a;[START_REF] Gottschalk | Bacterial metabolism[END_REF]. The chemical clogging is less pronounced than physical clogging.

Biofilm

The biological clogging mainly consists of biofilm which leads to the decreasing of pores space results in clogging. Net-work surrounding the microcolonies with extracellular polymers substances (EPS) [START_REF] Kim | Biofilm morphology as related to the porous media clogging[END_REF] by bacteria adhered to material grains is the main cause of the biological clogging (Rodgers et al., 2004a). The biofilm plays an important role on the colonization ability of bacteria to solid surface to protect themselves from environment in order to survive [START_REF] Costerton | The Bacterial Glycocalyx in Nature and Disease[END_REF][START_REF] Ronner | Characterization of microbial clogging in wastewater infiltration systems[END_REF]Van Cuyk et al., 2001;Mauclaire et al., 2002).

I.4.3.2. Influencing factors on clogging process

The clogging process is impacted by various factors which can be operational, environmental and of bacteria themselves: such as materials characteristics, feeding water characteristics, and surface properties of bacterial stains. The interactions are especially noticed between the material surface and the particulate or colloids matters and the microorganisms. Some factors are summarized in the From the above table, the filter media has great impact on the development of clogging, not only on the physical process (size of particulates vs pore space), but also on the bacteria adhesion and further biofilm formation (cell surface vs sand grain surface). The process of clogging is rather complex, and the impacts of material properties are not quite clear.

I.4.3.3. Impacts of clogging on the filtration and treatment performance

As mentioned in the overview schema of sand filter functioning (Figure 1.1), the actions brought by clogging to the filtration and purification process can be favorable and unfavorable. The transition between the two actions is a dynamic process involving mainly the overly accumulation of particulate matters and biofilm.

Favorable impacts

The presence of slight clogging generated by the accumulation of biomass and particulate matters favorites the filter performance at the early stage of operation [START_REF] Okubo | Effect of infiltration rate on biological clogging and water quality changes during artificial recharge[END_REF]. The pore size in the upper layer is partially reduced by the particulate matters straining and the surface coating on the grains, and the network among these retained matters and newly generated biomass benefit to achieve more uniform infiltration (Kristiansen, 1981c).

As mentioned, the hydraulic retention time plays an important role in purification process. A slight clogging reduces the initial hydraulic conductivity thus hydraulic residence time (HRT)

creases comparing to the "clean" bed which increases the chance of pollutant adsorption, exchange, biodegradation of organic carbon by heterotrophic bacteria and bacterial adhesion (Healy et al., 2007). Furthermore, the microbial communities (nitrifying and denitrifying bacteria) developed in sand filter are also responsible for the biotransformation of the nutrient pollutants (Pell et al., 1990). The local denitrification conditions sometimes can be satisfied beneath the surface biomat zone where the environment is enriched in organic carbon with less content in dissolved oxygen (Gill et al., 2009). When an excessive surface crust has formed, the terminal head loss can occur. In the study of [START_REF] Campos | Biomass development in slow sand filters[END_REF], the sand filter with higher developed biomass did not show the improvement in TOC removals in the effluent.

Unfavorable impacts

The clogging is often recognized as problematic functioning deficiencies and observed in laboratory sand columns or onsite sand (or soil) filters (Siegrist & Boyle, 1987). The functioning deficiencies include purification difficulties that usually companied by hydraulic difficulties over time, such as: the influent ponding, the decreased infiltration rate (field study), the hydraulic head loss and the decreased hydraulic conductivity (Rice,1974;Kristiansen, 1981a;[START_REF] Mays | Hydrodynamic Aspects of Particle Clogging in Porous Media[END_REF]Beach et al., 2005). The loss of initial infiltration rate is inevitable. According to [START_REF] Okubo | Biological clogging of sand and changes of organic constituents during artificial recharge[END_REF], under saturated conditions, the evolution of infiltration rate over time consists of three successive stages: (i) the aerobic period with a rapid decrease of infiltration rate companied with the reduction of dissolved oxygen, (ii) the transitional period (constant or slight increase of infiltration rate with low dissolved oxygen (DO) concentration) and (iii) an anaerobic period in which infiltration rate decreased rapidly until the clogging developed under anaerobic conditions. The clogging may Part Ι: Literature review be delayed under unsaturated flow conditions. This decreased capacity is the result of the abatement of the pore size and of their interconnections or even of a complete filling of pores.

The influent ponding represents the serious threat to the use of the sand filter. When the ponding occurs, even not at the entire filter surface, some regions (especially near the inlet) can be under saturated conditions with reduced air exchange and limited dissolved oxygen contents. During the decomposition process of organic pollutants under the action of heterotrophic bacteria, the dissolved oxygen in the liquid phase is consumed, when the air phase in the medium fails to renew itself, the mineralization in aerobic way may be interrupted and even stopped. With the presence of severe clogging, the microflora becomes specific and less diverse in an environment poor in oxygen and rich in organic matter. The degradation mechanism alters to anoxic anaerobic, enriched in the carbon dioxide (CO 2 ), and the methane (CH 4 ). The lack of dissolved oxygen also blocks the nitrification. The accumulation in the pores of partially degraded organics and particulate matters enhances the clogging even more and the filter reaches the terminal use lifetime. The following scheme describes the development of clogging and the interaction with the hydraulic and purification behavior (Figure1.3):

The current knowledges of packed filtration beds suggest that the development of clogging is inevitable. More and more studies showed that the evolution of biofilm tends to be an important process since the suspended particulates are brought by the feed water and the precipitation has little impacts, but the biofilm develops around the material grains, and occupies more and more the pore throats until reaches the equilibrum. However, informations are lacking, espeicially on the evolution in time and depthes of biofilm compositions, their characteritics and quantities, which also the focus of this study. In the two following sections, an bibliographic overview revisits some key information of biofilm.

. Biofilm definition and fixed bed systems

Biofilm is microbial derived sessile community characterized by cells that are irreversible attached to a substratum or an interface or to each other are embedded in a matrix of extracellular polymeric substances (EPS) that they have produced and exhibit an altered phenotype with respect to growth rate and gene transcription. These microorganisms may undergo physiological and genetic modifications during their transition from planktonic mode of growth (Donlan & Costerton, 2002).

Compared to suspended microbial growth systems, wastewater treatment with fixed biomass (fixed bed or moving bed systems) has several advantages: increase of biomass residence time which results in lower space requirement and reduced hydraulic residence time, high active biomass concentration, as well as lower sludge production [START_REF] Verma | Aerobic Biofiltration Processes-Advances in Wastewater Treatment[END_REF]. Fixed bed systems often use porous mediums that contain pores (or voids) and provide large specific surface for the development of biofilm: it can be natural materials (soils, sand, river sediments, aquifer zone…) or artificial mediums (crushed aggregates, glass or plastic solid materials…).

In porous media, microbial cells in suspension may adhere firmly to solid surface that comprises the effective pore space. Under favorable conditions, adhered cells reproduce at the surface and increase the amount of attached biomass. The Figure 1.4 shows a biofilm coating on quartz sand grains:

.2. Introduction of extracellular matrix of biofilm

Biofilm offers their member cells the benefits of the protection from environmental stress.

The spatial distribution of biofilms presents various structures and sizes: monolayer biofilm of thickness of 5-25µm resulted from laboratory pure culture with single species [START_REF] Andersson | Biofilm formation and interactions of bacterial strains found in wastewater treatment systems[END_REF]; and biofilm of 40µm thickness in anaerobic wastewater treatment plant [START_REF] De Beer | Effects of biofilm structures on oxygen distribution and mass transport[END_REF]. In fixed biomass systems, biofilm is heterogeneous and organized by microcolonies structures including void space and water channels. These water channels or pore spaces within biofilms associate with bulk liquid and allow the transport of substrates including oxygen, and free cells, and the convey of metabolic wastes [START_REF] Lawrence | Determination of Diffusion Coefficients in Biofilms by Confocal Laser Microscopy[END_REF][START_REF] Massol-Deyá | Channel structures in aerobic biofilms of fixed-film reactors treating contaminated groundwater[END_REF].

This "gel like" structure of biofilm is mainly composed by the extracellular matrix of biofilm: EPS represented 50 to 90% of total organic matters of biofilm (Wingender et al., 1999). The matrix of EPS is highly hydrated (90% water). The majority of EPS is composed by mostly macromolecular complex polysaccharides, proteins, small quantities of lipids, humic substance-like and nucleic acids (Flemming & Wingender, 2001a). The fraction of these macromolecules varies according to the type of microorganisms, cell physiological state and

Quartz sand grains

Biofilm

Part Ι: Literature review environmental conditions: the quantity of polysaccharides and proteins represents 75-89% of the composition of EPS (Tsuneda et al., 2003a). Minerals are also found trapped in the matrix and associated with the EPS [START_REF] Landa | Particle size distribution in an efluent from an advanced primary treatment and its removal during filtration[END_REF] and minerals can represented 10-48% of biofilm dry weight depending on the extraction methods (Bourven et al., 2011;D'Abzac et al., 2012).

I.5.2. Biofilm development steps

Before the biofilm formation, the bacterial cells attach themselves on the substratum surface, which start with the very first possible step: the formation of conditioning film.

Having been observed in some medical biofilm cases, the conditioning film is realized by adsorbing macromolecules originated from bacterial metabolism and neutralizing the excessive free energy at the proximity between the cells and the solid surface [START_REF] Marsh | Dental plaque as a biofilm[END_REF].

The conditioning film has not been reported in cases of biofilm in filter media. The development of biofilm begins under favorable conditions, followed by several steps: cell proliferation, production of EPS, maturation and detachment. As shown in Figure 1.5, the development of biofilm is generally described as following stages (1-conditioning of the surface and reversible adhesion, 2-irreversible adhesion, 3, 4-proliferation and accumulation,

5-maturation and detachment):

Proliferation and accumulation

Resulted from the step of irreversible adhesion, the initial coating of cells and EPS are established which favorites the growth of cells inside the initial film. The cells obtained from cells divisions form clusters or communities of bacteria. There is also the recruiting of free cells in liquid milieu. The heterogeneity of the biofilm increases during the accumulation and there is not necessarily only uniform layer but the colonization and architecture of different bacterial communities [START_REF] Stoodley | Biofilm as complex differented communities[END_REF].

Maturation and detachment

Equilibrium state achieves between the production and accumulation of new cells and EPS and the detachment of the aged biofilm, and the quantity the biofilm reaches a limit value and maintains constant (Liu et al., 2003). Besides the cells mortality, the cells derives inside the biofilm and certain biofilm constituents including the cells can be transferred and released back to the bulk liquid. The process of detachment is governed by biotic and abiotic factors:

I.5.3. Bacterial adhesion

As to initial the solid surface colonization, bacterial cells have to approach and adhere to the surface. The process of bacterial attachment can be divided into two steps: reversible adhesion and irreversible adhesion. The process is governed by various interactions.

I.5.3.1. Initial stage: physical transport and retention

1) Hydraulic transport: convection-diffusion

The microorganism migration occurs in the bulk liquid and at the liquid-solid interfaces, as well as the air-liquid interfaces under the unsaturated conditions. Similar to the colloids, the bacteria are transported with liquid flow towards the solid phase. This step includes convection and diffusion transport. Higher flow velocity induced higher bacterial cell penetration or earlier breakthrough [START_REF] Huysman | Water-facilitated transport of bacteria in unsaturated soil columns: Influence of cell surface hydrophobicity and soil properties[END_REF][START_REF] Sarkar | Transport of bacteria in porous media: I. An experimental investigation[END_REF].

Hydraulic loading rate and effective grain size impact directly the convection transport of bacteria by governing flow velocity and capillary force [START_REF] Stevik | The influence of physical and chemical factors on the transport of E. coli through biological filters for wastewater purification[END_REF]. Diffusion transport intervenes when Brownian movement cannot be neglected, especially when the porous media is under rest conditions (no loading) [START_REF] Corapcioglu | Microbial transport in soils and groundwater: A numerical model[END_REF].

2) Physical retention (Straining)

The straining is physical mechanism of movement blocking through of the bacteria larger than pores. The straining mechanism highly depends on filter media characteristics, degree of water saturation, bacterial cells and the development of clogging zone [START_REF] Ausland | Intermittent filtration of wastewater-removal of fecal coliforms and fecal streptococci[END_REF].

I.5.3.2. Reversible adhesion: physico-chemical stage

The reversible adhesion takes place when bacteria are close enough to the solid surface.

This process involves the interactions between the natures of the two surfaces (solid surface vs. cell surface), under the chemical environment of the liquid phase. The reversible adhesion is based on the extended DLVO (XDLVO) (extended Derjaguin-Landau-Verwey-Overbeek) theory which includes three groups of interactions: generally attractive Lifshitz-Van der Waal interactions (LW), electrostatic interactions (EL) and Lewis Acid-base interactions (AB) (electron donating and receiving) [START_REF] Van Oss | Energetics of cell-cell and cell-biopolymer interactions[END_REF]. The attraction or repulsion of bacteria cells to the solid surface is described by total Gibbs energy which is the sum of these three interactions:

∆G = ∆G ™R + ∆G ¡ + ∆G ¢
When ∆G is negative, the attraction takes place and ∆G is positive, the repulsion occurs. The hydrophobicity of cell surface is another interaction playing important role in bacteria adhesion to solid surfaces.

1) Nature of material surface

Compared to the cell surface, the sand surfaces are more stable and uniform. The study of Jacobs et al., (2007) suggested that natural quartz sand particles have slightly hydrophilic negatively charged surface and the surface roughness may reduce the repulsion. A study of surface chemistry showed that natural quartz sand surface contains mainly the oxide of silica and iron, also presents weak contents of Ca and Al [START_REF] Shani | Colloid transport through saturated sand columns: Influence of physical and chemical surface properties on deposition[END_REF].

Presence of multi-or divalent cations

Divalent ions have shown to enhance the bacterial adhesion to surface by the compression of electrical double layer (EDL) [START_REF] De Kerchove | Calcium and Magnesium Cations Enhance the Adhesion of Motile and Nonmotile Pseudomonas aeruginosa on Alginate Films[END_REF]. Ca 2+ ions in particular have shown to play an important role by involving in non-specific such as a neutralization of surface charge and reduce the repulsive energy barrier [START_REF] Kuznar | Adhesion kinetics of viable Cryptosporidium parvum oocysts to quartz surfaces[END_REF]. Ca 2+ ions can also act as bridging ions by forming the cationic bridges between the cell surface and solid surface [START_REF] Rose | A quantitative study of calcium binding and aggregation in selected oral bacteria[END_REF]. The divalent ions can be brought by feed water and may be released by the sand medium.

2) Nature of bacterial cell surface (DLVO interactions + steric interactions)

The bacterial cell surface is a highly dynamic surface responding to environmental changes. Charged group may associate or dissociate upon pH, ionic strength of suspending fluid, and also upon the approach of a charged surface. Under most physiological conditions, bacterial cell surface carries a net negatively charge with a few exceptions.

DLVO interactions

The LW interactions are always attractive and contribute the adhesion. In contrary, the electrostatic interactions are dominated and repulsive in a low ionic solution of pH 7 (I<0.001M) [START_REF] Redman | Bacterial adhesion and transport in porous media: Role of the secondary energy minimum[END_REF]. Today it is well known that increasing ionic strength reduces the EDL thickness and the cell may be brought close enough to the solid surface so that attractive LW interaction dominates [START_REF] Rijnaarts | DLVO and steric contributions to bacterial deposition in media of different ionic strengths[END_REF].

Steric interactions (non -DLVO interactions)

Macromolecular-substratum interactions are generally called steric interactions which mediate both reversible and irreversible adhesion. The steric interaction can be repulsive or attractive, which depends on the hydrophobicity of cell surface and substratum [START_REF] Rijnaarts | Reversibility and mechanism of bacterial adhesion[END_REF]. Steric repulsion occurs when cell surface macromolecules are hydrophilic and have no affinity for the substratum (ex: glass is highly hydrophilic surface); on the other hand, if the macromolecules or parts of them have an affinity for the substratum (ex: Teflon is hydrophobic surface) and exceeding certain critical value, attractive bridging may take place.

While at higher ionic strength, the shift from DLVO controlled adhesion to steric controlled adhesion is observed and the adhesion efficiency depends on the type of cell surface coating [START_REF] Rijnaarts | DLVO and steric contributions to bacterial deposition in media of different ionic strengths[END_REF]. The nature of cell surface coating determines the complexity of steric interactions between bacteria and surface which may reduce the rate of deposition, but at the same time, may cause the irreversible adhesion [START_REF] Tsuneda | a) Extracellular polymeric substances responsible for bacterial adhesion onto solid surface[END_REF]. The Figure 1.6 shows the main interactions involved in the reversible adhesion.

Studies showed that the bacteria movements also depend on their electrophoretic mobilities. Following the definition given by [START_REF] Ohshima | Electrophoresis of soft particles[END_REF], the bioparticles such as bacteria possessing the hard core and an adsorbed permeable gel-type layer are considered as soft particles, and exhibit the electrophoretic behaviors. The electrophoretic mobility of the soft particles is influenced by the interface properties and described by Ohshima formalism where the interphase between the soft particle and the outer medium is considered as homogeneous. Under the high ionic strength condition, this model suggests a finite of nonzero plateau that the electrophoretic mobility depends on the space charge density of the soft layer and the soft layer parameter and its dynamic viscosity. However the Ohshima's prediction deviates when the partial dissociation of microbial interphase charge becomes significant under low ionic strength condition [START_REF] Duval | Humic substances are soft and permeable: evidence from their electrophoretic mibilities[END_REF]. Face with the limitations of Ohshima's model, the diffuse soft particle electrokinetic formalism was developed by Duval and Ohshima (2006): under decreasing ionic strength condition, the inhomogeneous of the polymer segments affects significantly the electrophoretic mobility and the interphasial diffuseness may evolve with the physical-chemical composition of the medium. The literature review of [START_REF] Duval | Progress in electrohydodynamics of soft microbial particle interphases[END_REF] combined both numerical and experimental data and allowed going beyond the classic approaches of impermeable particles and revaluating the Part Ι: Literature review impacts of the chemical heterogeneities and physical diffuseness of soft particle interphase on the electrophoretic motions.

Irreversible adhesion: biochemical stage

Comparing to reversible adhesion, this stage is permanent between bacterial cells and solid surface involving biochemical phenomenon. The steric attraction promotes the irreversible adhesion. The cells presented on the surface attach themselves by intermediary of their metabolism activities, EPS and multivalent cations in EPS. The irreversible adhesion occurs through the implication of cellular appendices, such as adhesine (protein and polysaccharides) and/or the production of EPS that connect bacteria and adsorbent. This process less or more rapid depends on different types of microorganisms, environment and the contact time (Characklis & Marshall, 1990).

Sand grain

I.5.4. Matrix of extracellular polymeric substances

The biofilm well developed also forms a "compact" and "gelatinous" structure where the bacteria are "stuck" in the "cement" of extracellular matrix. The extracellular matrix plays two major roles: i) in the cells adhesion to sand grain surface; ii) in the cells cohesion among themselves. The next section discusses this main structure of biofilm: extracellular matrix and more detailed: the organic fractions of extracellular matrix: extracellular polymeric substances (EPS).

I.5.4.1. Spatial organizations of extracellular matrix

The EPS can bond tightly around the outer space of bacterial cells, as capsules-like closely associated. The bacteria also produce slime-like EPS loosely bond with cells and soluble EPS which do not associate directly to the cells (Wingender et al., 1999). Besides the organic fractions, the minerals are also bound in the extracellular matrix. The Figure 1.8

demonstrates the organizations bound EPS and soluble EPS: (Wingender et al., 1999) The way to separate two fractions is usually by centrifugation, where the polymers in the supernatant are soluble EPS and the polymers bound with the pellet are bound polymers:

Bound EPS

The bound EPS can also be grouped into two subcategories: tight bound (TB) EPS and loosely bound (LB) EPS. The TB-EPS are closely associated with cellular envelops, and the LB-EPS are gel-like substances surrounding the cells, and can be considered as slime.

Soluble EPS

The soluble EPS regroups the substances that are weakly associated and free molecule:

including some slimes and soluble or colloidal macromolecules which however are not dissolved [START_REF] Sardin | Modeling the nonequilibrium transport of linearly interacting solutes in porous media: A review[END_REF]. Wingender et al., (1999) indicated that the separation of soluble EPS from the matrix demands an extraction of weak energy (centrifugation…) while the bound EPS needs treatments allowing the dispersion of biomass (heat, reactive chemicals…).

I.5.4.2. Biochemical composition of EPS and mineral fractions

As mentioned, the matrix of EPS contains proteins, polysaccharides, nucleic acids, lipids as major biochemical components and also minerals…The biochemical fractions are changeable depends the nature of biomass, the type of substrates and other environmental factors.

Mineral fractions

The mineral composition (cations, anions) plays an important role in the metabolism of bacterial cells. Mineral fractions present in the matrix in the different forms: ionized or particulates. The particulate mineral solids are formed by precipitation, such as Ca salts (CaCO 3 , Ca5(PO4)3(OH)) and Fe salts (FeO(OH), FePO4 2H2O) [START_REF] Juang | Stable aerobic granules for continuous-flow reactors: Precipitating calcium and iron salts in granular interiors[END_REF]. The multivalent cations (Mg, Ca, Fe…) create the ionic interactions with the ionized functional groups of EPS. The cations in the matrix are mobile and interchangeable (Higgins & Novak, 1997).

Proteins

Proteins have an amazing range of structural and catabolic properties as a result of their various amino acid compositions. The proteins are amino acids chains linked by peptide bonds of various lengths. The side chains of α carbon atom of amino acids possess various functional groups (electrically charged, polar, or nonpolar) bring in various interactions like ionic, hydrogenous, or hydrophobic with other molecules [START_REF] Riemer | Multi-component diffusion in denitrifying biofilms[END_REF]. The properties and functions of proteins are related to their spatial configurations which depends on the amino acids compositions (water solubility, colloids solution, denaturation, and spectrofluorimetric properties…). The mechanisms of protein secretion are the synthesis takes plan in the cytoplasm and the molecules are then excreted into the environment [START_REF] Rodgers | Surface Clogging in an Intermittent Stratified Sand Filter[END_REF]. Proteins can also associate with polysaccharides (glycoprotein) and lipids (lipoprotein) (Bourven et al., 2012). [START_REF] Okubo | Effect of infiltration rate on biological clogging and water quality changes during artificial recharge[END_REF] utilizing the combination of size exclusion and infrared microscopy on EPS from activated sludge, found that except high molecular weight proteins (from 45 to 670kDa), it existed also a group of small size proteins (<1kDa) in smaller amount that were associated with polysaccharides. Similarly, the presence of lipoproteins as one subfamily of proteins was also proven by the same technique (Kristiansen, 1981c). The proteins represent a group of molecules possessing various functions: i) metabolism with the enzymes [START_REF] Frølund | Enzymatic activity in the activated-sludge floc matrix[END_REF]; ii) structural: The structural function is first due to the diversity of functional group of amino acid which can interact with other molecules/mineral element and second with specific structure (in gram negative bacteria), the lectins-like proteins which link sugar group (Higgins & Novak, 1997).

Polysaccharides

The polysaccharides are the biopolymers the most studies since they represent the majority of EPS matrix components in pure bacteria cultures and it is also the reason that some studies referred the exopolysaccharides as the EPS [START_REF] Costerton | Microbial biofilms[END_REF]. Most microorganisms secrete polysaccharides. The majority of these compounds are neutral sugars (glucose, galactose, and mannose) [START_REF] Dignac | Chemical description of extracellular polymers: Implication on activated sludge floc structure[END_REF]. Polysaccharides are ubiquitous biopolymers build up by monosaccharide of variable molecular weight. Most of them are hydrophilic with the presence of hydrogen bond. The synthesis of the polysaccharides can be intracellular or extracellular (Kumar et al. 2007). The polysaccharides in EPS matrix are either in forms of capsule covalently associated with cellular membrane (lipopolysaccharides)

or in forms of slime weakly joined with the cells (Kumar et al.,2007). Certain polysaccharides also contain variable groups such as: hydroxyl, acetyl, phosphate. Some of them are capable to bond with Ca 2+ ions, for example, bacterial alginates which represent a few exopolysaccharides. The alginates have certain capacity to form a gel and this capacity depends on the presence of mannuronic and guluronic acids. Many alginates possess the sequence of poly-L -guluronic acid which can bind Ca 2+ very effectively within the "egg-box" structure where the distribution of Ca 2+ in the gel were most observed on the surface [START_REF] Geddie | The effect of acetylation on cation binding by algal and bacterial alginates[END_REF]Lin et al, 2010).

Humic substances

The reference of humic substances as the EPS compounds is abusive. By definition, the humic substances come from the soil, where they can be resulted from long evolution process.

Thus researchers refer these compounds as humic-like substances and this group of molecules is very heterogeneous. They come from the biodegradation or chemical degradation of environment organic residues (Franciesco et al, 2002). Their main components are phenol, amino acids, and sugars of small sizes [START_REF] Wuertz | A new method for extraction of extracellular polymeric substances from biofilms and activated sludge suitable for direct quantification of sorbed metals[END_REF]. They contain functional groups such as: alcohol, phenol, carboxylic, lactones…

Nucleic acids

DNA and RNA are two groups of polymers of nucleotide chains. One nucleotide contains a sugar of five carbon atoms (deoxyribose for DNA and ribose for RNA), a phosphate group, and an amino group. The nucleic acids represent only a small amount in EPS matrix extracted from wastewater biomass (Frølund et al, 1996). At first, the extracellular nucleic acids were considered as a product and an indicator of cellular lyses. Later, researchers found that many microorganisms secrete large amount of extracellular nucleic acids (Steinberger & Holden 2005). In multi-species biofilm, high content nucleic acids were detected at the outer layer of micro-colonies [START_REF] Allesen-Holm | A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms[END_REF] which is in accordance with their roles in the communication among the cells.

I.5.4.3. Influencing factors of EPS secretion

The EPS play a role of tampon as protection and nutrients for the microorganisms in the matrix, for example, the soluble EPS are substances easy to metabolize to be the carbon and energy source (Laspidou & Rittmann, 2002). Some environmental factors affect the secretion of EPS:

Stress

The presence of the stress factors simulates the EPS synthesis: low substrate content (Hoa et al., 2003); salinity [START_REF] Mishra | Isolation and characterization of extracellular polymeric substances from micro-algae Dunaliellasalina under salt stress[END_REF]; presence of metal ions (Mikes et al., 2005). An increase in soluble EPS was observed at low substrate condition [START_REF] Aquino | Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds[END_REF]; Avella et al, (2010a) have observed an increase of soluble EPS and that the protein fraction increased during the presence of antibiotic in activated sludge biofilm; A higher amount of polysaccharides has been observed in some resistant bacterial strains pure culture with the presence of metal elements [START_REF] Kazy | Extracellular polysaccharides of a copper-sensitive and a copper-resistant Pseudomonas aeruginosa strain: synthesis, chemical nature and copper binding. World Journal of Microbiology and Kristiansen, R[END_REF].

Physical conditions

The shear forces influent the composition of EPS matrix. The EPS quantity of sludge increased under higher shears and higher aeration conditions (Adav et al., 2008a). The soluble EPS increase by the release from the aggregates took place under enhanced flow hydrodynamics and shears [START_REF] Aquino | Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds[END_REF]. The increase in sugar fraction in EPS Part Ι: Literature review 56 were shown under higher aeration condition (Shin et al, 2001), and the loss in EPS were observed under anaerobic sludge (Nielson et al, 1996).

Characteristics of substrates

The ratio of nitrogen/carbon in substrates seems affects the productions of EPS, however this effect of the nitrogen ration is not quite clear and accordant in studies. Some authors claimed higher productions both in soluble and bound EPS while the increase of the N/C ratio (caseins/starch from 2 to 8) [START_REF] Arabi | Impact of protein/carbohydrate ratio in the feed wastewater on the membrane fouling in membrane bioreactors[END_REF], and some authors found that the increase in N/C ratio only induced the increase in weakly bound EPS production [START_REF] Ye | Effect of C/N ratio on extracellular polymeric substances (EPS) and physicochemical properties of activated sludge flocs[END_REF].

I.5.4.4. Functions of EPS

As mentioned in the "bacterial adhesion", one of the functions of EPS is to facilitate the irreversible adhesion and to initiate the colonization. Besides these fundamental functions, the EPS also benefit the stabilization of biofilm by involving in different interactions in the biofilm, especially in the biofilms of wastewater treatment. These particular functions will be developed after a summary of EPS functions.

General functions of EPS as biofilm

Some other general functions have been attributed to EPS which are summarized in Table 1.8:

Part Ι: Literature review 57 Table 1.8 : Summary of functions of EPS (Wingender et al., 1999) Function

Roles of EPS in biofilm structural stability

The stability of biofilm refers to the capacity of the aggregates to resist to the hydrodynamic constrains and to the shear forces (Sheng et al., 2006b).The EPS play an important role in the stabilization by interacting between cells or with multivalent cations and also by the hydrophobicity… -The proteins and sugars contribute to the stability of biofilm which are confirmed by the deflocculation after the hydrolysis of proteins and polysaccharides by adding the enzymes [START_REF] Cammarota | Metabolic blocking of exopolysaccharides synthesis: effects on microbial adhesion and biofilm accumulation[END_REF][START_REF] Yang | Inhibition of free ammonia to the formation of aerobic granules[END_REF].

-The production of EPS also neutralizes the negative charge of cell surface and reduces the repulsive electrostatic force and the cell adhesion to the aggregates can be benefited by the polymeric interactions (Tsuneda et al., 2003a).

-The hydrophobicity helps the cells attach to aggregates and allows them to stick and keep the stability of the aggregates [START_REF] Zita | Effects of bacterial cell surface structures and hydrophobicity on attachment to activated sludge flocs[END_REF]. The hydrophobicity is associated with the presence of fibrillar structures and specific proteins, but not with the presence of polysaccharides (hydrophilic) [START_REF] Mcnab | Cell wall-anchored CshA polypeptide (259 kilodaltons) in Streptococcus gordonii forms surface fibrils that confer hydrophobic and adhesive properties[END_REF][START_REF] Singleton | Cloning and analysis of a Candida albicans gene that affects cell surface hydrophobicity[END_REF]Arabi & Nakhla, 2008b).

-The lectin-like proteins located in the appendices of cells interact with the bacterial polysaccharides by the hydrogen link [START_REF] Mirelman | Microbial lectins and agglutinins: properties and biological activity[END_REF][START_REF] Bush | Structure and conformation of complex carbohydrates of glycoproteins, glycolipids, and bacterial polysaccharides[END_REF].

-The presence of divalent cations also reduce the repulsive force between cells and favorites the aggregation. The non-monovalent cations also intervene the stabilization of EPS molecules by ionic interactions (Higgins & Novak, 1997).

Role of EPS in the sand filtration process

The EPS are considered as the main components of biofilm which constitute the biological clogging. Studies showed that the presence of EPS at mature state reduces the infiltration rate (Rodgers et al., 2004a;[START_REF] Schijven | Removal of viruses by soil passage: Overview of modeling, processes, and parameters[END_REF]. But the spatial distribution of EPS around sand grains also ammeliorates the repartition of substrates and increases the residence time which benefits the purification process of sand filtration.

However the EPS evolution during operating time and as function of filter depth is rarely documented and few studies have been carried out on the their more detailled characteristics such as their compositions or molecular sizes.

I.6. Characterization-monitoring of biofilm (cells + EPS)

The complex nature of biofilm makes the investigations difficult. The biofilm is investigated by various techniques from different aspects. The most of these techniques are destructive to the biofilm. General information can be provided by simple measurements, such as: totality of biofilm (amount as organic matters, or as Carbon or Nitrogen), thickness and structures of biofilm; more detailed information on the cells and EPS may need several steps of treatments and followed by specific analytical techniques. An overview of several categories of biofilm investigation techniques is presented in Table 1.9:

Table 1. 9 : Biofilm investigation techniques overview Microscopic techniques have high potential in analysis of biofilms, but natural biofilm can develop into thick package that may limit light penetration into the biofilm matrix hindering the use of optical techniques, such as the light microscopy, which is advantaged with its simplicity and rapidity; however also limited by its low resolution [START_REF] Lazarova | Biofilm characterization and activity analysis in water and wastewater treatment[END_REF]. The development of Confocal Laser Scanning Microscopy (CLSM) and epifluorescence microscopy has extended the possibilities of in-depth visual 3-dimensional (3-D) observation of biofilm structure and provided a bridge between light microscopy and electron microscopy [START_REF] Zhang | Quantification of extracellular polymeric substances in biofilms by confocal laser scanning microscopy[END_REF]. The carbohydrate of Pseudomonas aeruginosa biofilm has been visualized and characterized by using fluorescently labeled lectins in combination with a epifluorescence microscope and a CLSM [START_REF] Strathmann | Application of fluorescently labelled lectins for the visualization and biochemical characterization of polysaccharides in biofilms of Pseudomonas aeruginosa[END_REF]. The Scanning Electron Microscopy (SEM) provides high resolution images that allows the visualization of the biofilm microstructure including the visualization of EPS and cells (Eighmy et al., 1983). The Scanning Transmission X-ray Microscopy (STXM) shares similar principal with SEM. A multi -microscopy combination (CLSM-STXM-SEM) has demonstrated the distribution of macromolecules (protein, polysaccharides, lipid, and nucleic acids) of the biofilm in river sediments [START_REF] Lawrence | Microscale Analyses of the Formation and Nature of Microbial Biofilm Communities in River Systems[END_REF]. STXM is also capable of detecting the absorbed metals in biofilm.

I.6.2. Characterization of the population of biofilm -Cells

The cells are considered as the biomass which is responsible of the wastewater purification process for both suspended and fixed biomass reactors. It has always been the subjects in water treatment research the total content, the biodiversity and the physiology of the cells, otherwise the biomass in the biofilm.

I.6.2.1. Total content assessment of biomass

Volatile Dry Weight (VDW): Total organic matters

The VDW (weight loss between 105 and 550°C) is often used to estimate the total amount of biomass and this method is also generally used to assess the total organic matters of soil samples. The biomass may be overestimated by heating weight loss, since the structural water of clay minerals also loss weight during heating along with the organic materials [START_REF] Rice | Soil clogging during infiltration of secondary effluent[END_REF].

Biomass carbon and nitrogen determination

The biomass carbon and nitrogen assessments are generally applied to fixed biofilm samples, especially often carried out to quantify the living microbial biomass in soil or in sediments. The techniques are referred to fumigation -extraction (incubation) methods or substrate -induced respiration method [START_REF] Wardle | A comparative assessment of factors which influence microbial biomass carbon and nitrogen levels in soil[END_REF].

Revivifiable cells count-Extraction and enumeration of cultivable microbes

Quantitative and representative recovery of microorganisms from environmental samples is essential in understanding the ecosystem function. The microbial enumeration is a screening-level tool which can be used to evaluate the response of soil microorganisms or attached bacteria in artificial porous media, and the geometric distribution of microbial populations and communities. The enumeration of microbes in soil or other filter media requires the sample preparations which generally involves the dispersion of substratum sample and the extraction of attached cells from substratum which involves a variety of binding mechanisms [START_REF] Trevors | Nucleic acids in the environment[END_REF]. Because of the strong binding between cells and substratum, the severe cell damage may be a result of breaking these binding. The ultrasonic treatment and low speed centrifuge as dispersion and extraction can only recover a fraction of dislodged cells [START_REF] Lindahl | Evaluation of methods for extraction of bacteria from soil[END_REF]. The enumeration of cells is based on the revivifiables aerobic flora which includes aerobic and facultative anaerobic bacteria. In soil, population densities of TRHs within background soils usually range between 10 4 and 10 7

CFU/g soil [START_REF] Maila | Evaluation of microbial diversity of different soil layers at a contaminated diesel site[END_REF]. The study of [START_REF] Chabaud | Influence du biofilm sur les performances des systèmes de traitement par infiltration dans le sol: application à l'assainissement non collectif[END_REF] showed that the number of cultivable cells recovered from a sand filter were relatively stable during the operating period.

I.6.2.2. Biodiversity -Molecular techniques

Genetic information is often required in microbial biodiversity and taxonomy investigations to explore the molecular basis of biofilm formation. A reconstruction of near compete genomes or genome sequences of microorganisms is possible using genomic datasets in combination with molecular techniques. These techniques include polymerase chain reaction (PCR) for DNA amplification, followed by pulse field gel electrophoresis (PFGE), denaturing-gradient gel electrophoresis (DGGE) and more detailed information can be obtained by coupling with other analytical methods like mass spectrometry and microscopic techniques [START_REF] Aoi | In situ identification of microorganisms in biofilm communities[END_REF][START_REF] Meays | Source tracking fecal bacteria in water: a critical review of current methods[END_REF]. There are also molecular fingerprint techniques that do not involve PCR amplification, such as fluorescence in situ hybridization (FISH), by applying ribosomal RNA-targeted oligonucleotide probes could be visualized with the help of fluorescence quenching, or technique such as ribosomal intragenic spacer analysis (RISA) based on PCR amplification products. RISA has been employed to evaluate the evolution of bacterial communities' complexity in sand filtration columns and demonstrated an increase of bacterial communities throughout the operation time (0-138 days) [START_REF] Chabaud | Influence du biofilm sur les performances des systèmes de traitement par infiltration dans le sol: application à l'assainissement non collectif[END_REF]. FISH has been successfully employed to explore bacterial communities in activated sludge, marine, freshwater environments, marine sediments, soil [START_REF] Wagner | Probing activated sludge with oligonucleotides specific for proteobacteria: inadequacy of culture-dependent methods for describing microbial community structure[END_REF][START_REF] Amann | The identification of microorganisms by fluorescence in situ hybridisation[END_REF].

FISH conjunction with CLSM has allowed defining the 3-dimentional distribution of microbial populations in mixed species biofilm of different environments [START_REF] Moller | Activity and threedimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy[END_REF][START_REF] Manz | Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy[END_REF].

I.6.2.3. Physiology of cells -Enzymatic activities

The enzymatic activities can be associated with active cells, cell debris as well as being complexed with minerals or colloids [START_REF] Burns | Enzyme activity in soil: location and a possible role in microbial ecology[END_REF]. Various techniques of quantifying microbial activities by enzymatic measurements exist in the literature. While the activity of many extracellular hydrolases is probably a result of enzymes associated with some or all these compounds, dehydrogenase assays measures intracellular catalysis and are more likely to be correlated with the activity of extant cells [START_REF] Dick | Soil enzyme activities as integrative indicators of soil health[END_REF]. The fluorescein diacetate (FDA)

hydrolysis is used as a general indicator of soil hydrolytic activity. The determination of FDA hydrolysis is simple, rapid and sensitive, except when activities are low, it requires long time of incubation [START_REF] Okubo | Biological clogging of sand and changes of organic constituents during artificial recharge[END_REF]. The adenosine triphosphate ATP content can be associated with the soil biomass, however the ATP content represents only a small portion which is active (Beach et al., 2005).

I.6.3. Characterization of the EPS (separation + quantification + analysis (monitoring))

The matrix of EPS forms a shelter to protecting the cells from environmental stress and providing suitable conditions for proliferation. The choice of extraction method applied in each case remains a compromise between the high yields of biochemical components and the absence of contamination by chemical reagents or by intercellular materials for further investigations. The involving of chemical reagents and thermal treatments often interfere the chromatograms of size exclusion chromatography due to the denaturation and disruption of macromolecules, such as protein-like compounds [START_REF] Bourven | Influence of extraction method on size exclusion chromatography fingerprints of EPS from wastewater sludges[END_REF]. Physical methods such as ultrasound appear having modest recovery efficiencies but no interferences to any other analysis followed. In this case, the ultrasound was chosen over CER in cases of EPS extraction since the ultrasound has fairly equal extraction efficiencies for the major groups of EPS [START_REF] Comte | Effect of extraction method on EPS from activated sludge: An HPSEC investigation[END_REF]. The ultrasound treatment: probe (Probe Bandelin GM 70, 60W during 90s, [START_REF] Labanowski | Matière organique naturelle et anthropique : vers une meilleurecompréhension de sa réactivité et de sa caractérisation[END_REF] might also lead to cell lysis (Yu et al., 2009).

I.6.3.2. Physic-chemical properties of EPS and Characterization methods of EPS

Quantification of organic components with classical colorimetric methods

The contents of the main EPS components are often assessed via the colorimetric methods. The chemicals interact with the certain functional groups which are specific for target components, and the intensity of generated color is measured by UV/Visible spectrophotometer. The result relies on a known reference which establishes the calibration curve with a series of known concentrations. Several methods exist for each component in order to improve the reliability. Some common used methods are summarized in the Table 1.11.

The EPS resulted from the biomass of wastewater purification is highly heterogeneous and this great diversity reduces the efficiencies of colorimetric assays. For example, the different colorimetric assays for proteins present their own advantages and inconveniences: the protein contents determined by Lowry method are interfered with humic-like substance and thus overestimated. Lowry method has been modified in order to take account the humic-like compounds (Frølund et al., 1996). On contrary, this modification tends to underestimate the protein contents by taking account humic-like compounds even when they are absent [START_REF] Ras | Protein extraction from activated sludge: An analytical approach[END_REF][START_REF] Avella | The pitfalls of protein quantification in wastewater treatment studies[END_REF]. The Bradford method does not interfere with other organic compounds but also underestimates the protein contents due to different colorimetric reactions according to the type of proteins (Raunkjaer et al., 1994). According to [START_REF] Ras | Protein extraction from activated sludge: An analytical approach[END_REF] and Raunkjaer et al., (1994), the BCA method also has different reactions with the types of proteins and overestimates the proteins by taking account some polysaccharides. (Frølund et al., 1996). The infrared spectroscopy is sometimes coupled with SEC to identify the chemical nature. It has been successfully applied to characterize wastewater sludge exopolymers (Frølund et al., 1996;Görner et al., 2003;[START_REF] Garnier | Characterization of activated sludge exopolymers from various origins: A combined size-exclusion chromatography and infrared microscopy study[END_REF][START_REF] Comte | Effect of extraction method on EPS from activated sludge: An HPSEC investigation[END_REF][START_REF] Simon | Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges[END_REF], and the HPSEC fingerprints were generally recorded at the wavelength of 210nm and 280nm, detected by UV absorbance: these authors suggested that proteins have relatively large apparent MW ranged from 10 to 670kDa, and that polysaccharides have low aMW smaller than 1kDa. Frølund et al. (1996) found similar profiles of HPSEC fingerprints from the EPS of different origins. [START_REF] Comte | Effect of extraction method on EPS from activated sludge: An HPSEC investigation[END_REF] demonstrated that HPSEC fingerprints differed with the chemical extraction whereas the control and physical extractions exhibited similar fingerprints. Higher aMW (up to 1200kDa) compounds presented in strongly bond EPS or capsular EPS and the majority was proteins and nucleic acids. Whereas in weakly bond EPS or slime EPS, the aMW ranged from 0.3 to 20kDa [START_REF] Andersson | Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans[END_REF]Yu et al., 2009). With the combination of SEC-fluorescence detection, the protein-like fingerprints have been specified by the chosen Ex/Em wavelength and exhibited heterogeneous MW ranged broadly from large sizes of >600kDa to small sizes of <9kDa (Bourven et al., 2012). Later, humic-like compounds fingerprints were also specified by fluorescence detector, the main aMW of humic-like substances of both activated sludge and anaerobic granular sludge ranged in low MW region: from 1.4 to 6kDa (Bhatia et al., 2013).

Surface properties: Hydrophobicity and surface charge

Both hydrophobicity and surface charge belongs to the surface properties of EPS and these two characteristics are often associated in the studies. The hydrophobicity is one of the fundamental interactions in bacterial aggregates and the hydrophobic interactions have great influence on the settleablity, dewaterability and bioflocculation of sludge. The hydrophobicity of EPS is characterized by XAD-8 and XAD-4 resin which divide soluble EPS (at pH=2) into three fractions filtering through XAD resins: 1) fraction sorbed onto the XAD-8 resin: hydrophobic substances; 2) fraction sorbed onto the XAD-4 resin: hydrophilic substances and 3) unsorbed fraction: non-sorbed hydrophilic substances [START_REF] Martin-Mousset | Distribution et caractérisation de la matière organique dissoute d'eaux naturelles de surface[END_REF].

Several techniques can estimate the cell surface charge: the determination of zeta potential, the acid-base titration and the colloids titration [START_REF] Jorand | Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances[END_REF]. The soluble EPS at pH=2 showed a significant protein fraction and that no carbohydrates have been found in the Part Ι: Literature review hydrophobic fraction which indicated that the carbohydrates do not involve in the hydrophobic interactions [START_REF] Jorand | Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances[END_REF]Arabi & Nakhla, 2008a). The surface charge is related to the ionizable groups presented on the sludge surface, and also strongly depends on the composition of EPS matrix. Studies indicated an inverse correlation between the surface charge and hydrophobicity, both are strongly influenced the proportion of EPS components, especially increased with the increment of the ratio proteins/carbohydrates [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF][START_REF] Mikkelsen | Physico-chemical characteristics of full scale sewage sludges with implications to dewatering[END_REF][START_REF] Sponza | Extracellular polymer substances and physicochemical properties of flocs in steady and unsteady-state activated sludge systems[END_REF][START_REF] Wang | Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors[END_REF]. Extracellular proteins contribute the hydrophobic properties of EPS due to their high proportions of hydrophobic amino acids alanine, leucine and glycine (Higgins & Novak, 1997). The study of [START_REF] Wang | Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors[END_REF] showed that the negative surface charge exhibited a positive correlation with the increase of total EPS content; however the EPS presented hydrophobic property due to the increased ration PN/PS, which indicated with abundant EPS, the attractive force was dominated by polymeric interactions in cell adhesion.

Identification of EPS components

There are analytic tools permitting the identification of certain molecules of classic EPS components after an appropriate separation and purification. amino acids have been separated with the help of high performance liquid chromatography (HPLC) and detected by spectrofluorimetric detector and the results also showed that several possibilities of proteins source in EPS matrix including bacterial excretion and influent residues [START_REF] Dignac | Chemical description of extracellular polymers: Implication on activated sludge floc structure[END_REF]. Gas chromatography (GC) coupled with mass spectroscopy or flame ionization detector has been often applied to identify the monosaccharaides and lipids presented in the extracts according to their different retention time [START_REF] Dignac | Chemical description of extracellular polymers: Implication on activated sludge floc structure[END_REF][START_REF] Gloaguen | The extracellular polysaccharide of Porphyridium sp.: an NMR study of lithium-resistant oligosaccharidic fragments[END_REF][START_REF] Andersson | Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans[END_REF].

Part Ι: Literature review

General view of protein-like and HS-like with Excitation/Emission Matrix

The [START_REF] Domínguez | Effect of different extraction methods on bound EPS from MBR sludges. Part I: Influence of extraction methods over threedimensional EEM fluorescence spectroscopy fingerprint[END_REF]Bhatia et al., 2013). This technique has been considered as a rapid tool of qualitative analysis of EPS extracts in sludge.

I.7. Conclusion of literature review

From the information given by the previous studies, the essential characteristics of the technique, the conception and the functioning of OWTS systems, especially the sand filters have been explained. The majority of these studies allow identifying the operating parameter (loading charge, dosing frequency, material natures…) which influence greatly the purification performance and the duration of the installations. The milieu of sand filter receiving septic effluent is an environment very complex and heterogeneous, and the complexity and the nature of the interactions among physical, chemical and biological process taking place in this unsaturated environment makes it difficult to renew the knowledge. It appears that the biofilm develops mainly at the surface layer of the filter and participates importantly the reduction of different pollutant. At the same time, it provokes the phenomena of clogging which may interfere the functioning of the filter. Furthermore, the influence of the filter material is still quite unclear and the lack of natural deposit of alluvial sands in certain areas simulates the researches of similar materials, such as crushed sands. In this study, the interests were brought to the characteristics of crushed sands (materials, hydrology, purification and biomass distribution evolution and microbial activities) and the influence of these characteristics on the possibility of using them as filter media for domestic wastewater treatment (purification efficiencies; biological clogging caused by biofilm).

The strategy of experimental study:

With these interests, this study is based on the comparative characterization between selected alluvial and crushed sands. The experimental study was carried out around following points (Table 1.12): The monitoring of the abatements of main pollutants was referred to the classic physicochemical analysis of inlet and outlet effluents. The analyzed parameters represented the organic pollutants, nitrogen and phosphorous pollutants.

The system allowed the sampling in depths inside the sand columns. The monitoring of biomass (totality) and their components (cells and extracellular matrix respectively) was based on the combination of simple rapid methods and more pushed forward analysis: the choice of methods was based on the purpose, quantity and quality of samples. Due to the nature of comparative study, identical analysis was applied to all the sands.

Part ΙΙ: Experimental study and result discussions

Le sable de rivière est couramment utilisé comme matériaux de remplissage pour les biofiltres utilisés en système d'assainissement autonome selon les recommandations de la Norme Française (DTU 64.1). Les réacteurs de filtration biologique garnis de sable ont été largement étudiés en termes de procédé et de comportement hydrodynamique. Ce système avec comme matériau un sable de rivière, est considéré comme stable et son efficacité épuratoire parfaitement décrite et maitrisée (Wanko et al., 2005). Le changement de matériaux de garnissage peut modifier les qualités attribuées au procédé en considérant que peu d'études ont été réalisées, en particulier sur les agrégats concassés. L'objectif de ce travail est de regarder l'impact de la nature du garnissage des filtres biologiques sur:

le fonctionnement épuratoire du procédé

-

Granulometric characteristics

The granulometric analysis provides important information for grained materials as the The effective diameter is the opening indicated by the distribution curve at 10% passing.

Average diameter (mm) D m

D D = ∑ m F D F ∑ m F
With m i : the refused mass retained on the sieve i [M]; and D i : the average opening of two consecutive sieves [L].

Uniformity coefficient(-) UC UC=D 60 /D 10 With D 60 is defined similarly as the opening indicated by the curve of 60% passing sample Fine particle content (%) Fine%

The fine particle percentage is the percentage of passed mass though 0.08mm sieve to total sample mass.

Physical characteristics

Some physical characteristics were determined by laboratory methods, such as volumic mass density, relative density, porosity, and specific area is based on estimated value. These parameters were determined according to Liénard et al., (2001).

Volumic mass density (real density) (ρ)

The real density is estimated by placing the sample of known mass slowly into a graduated test tube which has known volume of clear water. This method is rapid and simple however less precise, so the test should be carried out at least 3 times to have an average value. About 300 g of dried material is weighed precisely, and then carefully and slowly poured into the known volume water. The volume moved from V 1 to V 2 , and the difference of volume represents the real volume of dried grains without pores, and the real density ρ (kg/m 3 ) is calculated by the following equation:

Equation 1: ρ = S ¤ ¥ H 3¥ '
With M g : mass of dried sample (kg); V g =V 2 -V 1 is the volume difference of after and before placing the sand in water, representing the dried grain volume.

Porosity (φ)

The porosity can be estimated at the same time with the real density. The total volume (V t ) is measured by graduated test tube for the same dried sample. The void volume is represented the difference between the total dry volume of sample and the volume of the grains:

Equation 2: V ‡ = V = -V * Equation 3: φ = ¥ ¥ ©

Bulk density (apparent density: ρ app )

The bulk density is estimated by the mass and the total volume of sand sample:

Equation 4: ρ -ZZ = S ¤ ¥ ©

Specific surface

The specific surface is the total surface of grains per volume or mass unity. It depends on the size and the shape of testing material. The specific surface can only be estimated since the measurement of grain shapes is complicated. The specific surface of spherical grains of uniform size is estimated by the average size:

Equation 5: A g = U« ¬ H - ® « ¬ ¯= °«¬
For the materials of rounded grains of the various sizes, such as the alluvial sands, the specific surface is estimated by the sum of mass fraction (%) of each size (average opening of two consecutive sieves):

Part ΙΙ-Chapter 1: Study of filter materials and filtration reactor Equation 6:

A g = 6 ∑ ± G L G
The above equation can be only served to estimate the specific surface of rounded grains.

However, even the river sands sometime can hardly considered being rounded, and for crushed sands, the shape is much more irregular. Lakel (1995) has proposed a modification by introducing a shape factor φ into the equation:

Equation 7: A g = °² ∑ ± G L G
With φ=1 for sphere and φ=0.7-0.95 for sands. In this study, the factor is 0.95 for river sands and 0.7 for crushed aggregates, and the specific surface is expressed on m 2 /kg, by dividing real density: A s (m 2 /kg) = A s (m -2 )/ρ (kg/m 3 ).

Grains shapes observation and image analysis

Attempt has been made to access the grain shapes of two different types of sands in this study.

Observations by camera and by microscope showed a poor representation of the entire sand sample. The images of the bulk sample (dried, fine particle eliminated) were taken by camera, and the some grains of certain sizes (0.25-0.4mm; 0.6-0.8mm) have been sampled after the sieving tests. The microscopic approach is based on the studies (Cho et al., 2006). Pictures of aggregates were processed by ImagJ software. After an 8 bit and a binary conversion the picture scale was set and the analysis of particles were run with a specific plug in. Filter were used with a limitation of size (0.1 to 4 mm) and circularity (>0.75) for a good isolation of particles. The isolation process is presented in Figure 2.1: However, the surface can contain exchangeable cations such as: K + , Na + , Ca 2+ , Mg 2+ , and Al 3+ that may alter the ionic strength. The mineralogical analysis was effectuated by local laboratory IDAC (Inovalys, Nantes). The nominal method of released elements does not exist.

In this study, a procedure is proposed in order to study the contents of certain released elements, and four elements have been analyzed by Atomic Emission Spectroscopy.

Released cations

The materials are dried and sieved through 0.08mm. Three different masses were sampled, placed into 50mL plastic tubes, and washed with 20mL ultrapure water for 3 times. The experimental conditions are summarized in

Mineralogical analysis

The mineralogical analysis was conducted by local laboratory IDAC (Inovalys, Nantes). The materials were attacked by nitric acid under high heat or microwave and dissolved into the mix solution of LiBO 2 and Li 2 B 4 O 7 . The major elements were quantified by Atomic Absorption Spectroscopy.

Calcareous (Limestone) contents

The calcareous (limestone) content measurements were also carried out by IDAC (Inovalys, Nantes).

Process design: filtration reactors

The process of filtration reactors is composed of three parts: mixing and feeding, main pilot and evacuation. An overall view of the pilot is simplified in Figure 2.2:

Feeding tank

Due to the distance of septic effluent sampling site, the feeding tank was also served as a mixed storage tank. This tank had a capacity of 1m 3 which insures one week of feeding. The septic effluent was renewed once a week and the tank was continuously mixed. The feeding water was pumped by a peristaltic pump.

Filtration pilot and cylinder reactors

Four selected packing materials, including two types of river sands (RS1 and RS2), and two types of crushed aggregates (CA1 and CA2) have been tested, and their position arrangements in the reactors are presented in Table 13. The pilot is composed by 12 cylinders of three different heights: 15cm (Reactors 1-4), 30cm (Reactors 5-8) and 70cm (Reactors 9-12). The diameter for all the columns was identical: 30cm. For the reactors of 70cm (9-12), sampling ports were installed at 5, 10, 15, 30, 45cm. The inclined ports with half tube were supposed to serve effluent receptions which were not achieved during the operation, so these ports were also served for sand sampling. The Figure 2.3 shows the positions of the sampling ports:

Estimation of saturate hydraulic conductivity at laboratory scale

The method for saturated hydraulic conductivity determination is normalized for on-site soil tests (NF X30-441, AFNOR, 2008). A simplified method for rapid determination at laboratory scale is proposed by Grant (Liénard et al., 2001). This method allows the determination of saturated hydraulic conductivity (k s, m/s) through the measurements of the infiltration time (Grant time: t g ). The calculation is based on the Darcy's law: The experimental device proposed in this study is according to Liénard et al., (2001). The sand was fed with clear water several times until meet saturated condition (thin constant film of water on the surface). Then 500mL clear water was poured slowly on the surface and the passing time of this 500mL water was noted. This measurement should be at least repeated for 5 times to obtain an average value. According to the authors, Grant infiltration time should be fitted the threshold between 50s and 150s, corresponding to k s between 3.7×10 -4 and 1.1×10 -3 .

The Figure 2.4 shows the experimental conditions of this test:

. Water retention capacity

Beside the capacity of conducting liquid, grained materials also have the capacity of retaining certain portion of liquid under the effect of capillary pressure after the drainage. This capacity is characterized by two methods proposed by [START_REF] Mohammedi | Etude de matériaux de garnissage en vue d'une réduction géométrique des filtres biologiques dans le cadre de l'assainissement autonome[END_REF]: method of permanent flow rate, and drainage method. The two methods provide respectively the access to the total volume retained in porous media and the volume retained after the drainage (stagnant water).

Method of permanent flow rate

Several columns of 70cm height (material layer height), 8cm diameter are used in this study.

The column is packed with dried material sample, and the column is fed with a hydraulic load of 2cm/min which corresponding to a flow rate of 10mL/min. The flow rate at the outlet of the column is noted as function of the time. When the flow rate is stable and constant, the feeding is stopped.

Part ΙΙ-Chapter 1: Study of filter materials and filtration reactor

Drainage method

The flow rate decreases after stopping the feeding. The recording of flow rate is continued until no water comes out from the column. The Figure 2.5 shows the principle of the two methods: The capacity is expressed either by the ratio between the V total and the volume of material (C v %) or between the M total and the mass of material (C m %). The massive capacity is characterized by the measurements of humidity (mass loss at 105°C). The total water involved in the sand column includes the drainable water and retained water. The volume of drained water is represented by the area under the curve after stopping the feeding (V drained ).

Reactor characterization

In an unsaturated filtration rector, except the solid phase provided by the filter media, characterized by the packing materials, the liquid phase and the air phase also present and both involve in the treatment process. In this study, the hydrodynamic characteristics and oxygen gas contents in reactors (or in certain reactors) have been examined.

Part ΙΙ-Chapter 1: Study of filter materials and filtration reactor

Hydrodynamic characteristic

The flow characteristics are often represented by the hydrodynamic behavior. The hydrodynamic behavior impacts the functioning of filtration reactor. The initial hydrodynamics depends on the intrinsic characteristics of packing materials, operating conditions and also the reactor itself. This parameter is often characterized by the distribution of residence time which gives access to the average hydraulic residence time (HRT)of filtration reactors.

Tracer choice

The choice of the tracer should respond to the following requirements: 1) detectable of small quantities; 2) neutrality by bioreactors; 3) no nuance for the biomass; 4) no retention in the biomass.In this study, lithium chloride (LiCl) was used as the tracer.

Injection

The quantity of tracer injected into the systems varies from case to case. For packing material systems, the tracer quantity injected is supposed to be 0.5-1g of Li + per m 3 material which his suggested by [START_REF] Maillard | Les filtres à sable verticaux en assainissement autonome regroupé: rôle des conditions opératoires, des écoulements et des transferts gazeux sur les performances épuratoires et le vieillissement Université de Renne[END_REF]. In this study, 1g Li/L solution was prepared. 20mL and 30mL solution were injected respectively for 12cm/day and 20cm/day loading rate. The reactors were dosed by 10 batches per day for both loading rates. The tracer was injected with certain batch.

Sampling

The effluents were sampled right after the injection. Before the peak detected, more samplings were effectuated. After the passing of the peak, the sampling frequency was decreased.

Detection

The detection of Li concentration in effluents was realized by atomic absorption spectroscopy (SpectrAA 220, VARIAN). The detection limit was between 0.5 and 8mg Li/L, so the dilutions were necessary for certain samples.

E t = < G ∑ < G ∆= G With C i (mg/L)
is the tracer concentration of sampling moment t i ; and ∆t i is the delay between two consecutive samplings.

The average hydraulic residence time (HRT)t s is calculated as:

Equation 10: t g = ∑ < G = G ∆= G ∑ < G ∆= G 1.3.2.

Estimation of oxygen gas variation

During each batch of feeding water pumping, the oxygen gas level fluctuates with the passage of the liquid flow. The estimation of oxygen gas variations was effectuated with 70cm reactors at 10cm layers by optical oxygen sensor (OXROB10, Pyroscience sensor technology). The excitation signal was sent at the wavelength of 620nm and the emission signal was 760nm processed at by optical oxygen meter (Fire Sting O2, Pyroscience sensor technology). The data was recorded every 10 seconds during 24h. The detection limit of oxygen sensor is 0.02% O 2 .

Results and discussions

Material characteristics

In this section, the main characteristics of selected filter materials are discussed, including granulometric, physic and chemical characteristics. These characteristics are intrinsic properties of materials, invariable during the operating.

Granulometric characteristics

The size distribution curves of selected materials are presented in Figure 2.6 (river sands)

and Figure 2.7 (crushed aggregates). These curves are compared to the minima and maxima of French standard DTU 64.1.

The selected materials including two river sands and two crushed aggregates are all in the proposed range of filtration use according to DTU.64.1. From the distribution curves, it appears that RS1 is relatively fine sand and RS2 is very coarse. On contrary, the crushed aggregates are well graded and heterogeneous in size, presenting both fine and coarse particles. With the help of size distribution curves, several parameters can be defined for each material. The detailed information of these parameters is provided in Table 2.1.4: Diameter and uniformity:

The effective diameter of each sample was determined from the granulometric curves (Figure 2.1; 2.2). The results showed that the river sand: RS2 were coarse sand. Even though the D 10 of RS2 was in accordance with DTU 64.1, it exceeded the limits of filtration sand choice recommended by Liénard et al., (2001) who indicated that the sand of effective diameter between 0.2 and 0.4mm conducted practically better biological treatments. In this case, RS1, CA1 and CA2 are in accordance of this requirement. The CA1 presents fine effective diameter (D 10 =0.17mm) but relatively large average diameter (D m =1.36).

The uniformity coefficients show that the grain size distributions of crushed aggregates (CA1 and CA2) are more heterogeneous than river sands, especially CA1. Liénard et al., (2001) suggested in their study that an UC inferior of 4 would suit better for filtration treatments and also indicated that crushed aggregates tend to be well graded in size (9~27). The river sands in this study show accordingly satisfied uniformity. Even though RS2 has a better uniformity, the grains have relatively larger sizes.

Fine particles content:

Crushed aggregates show higher content of fine particles (size inferior of 0.08mm), especially CA1. Liénard et al., (2001) indicated that the permeability decreased with the increase of fine particle content and the percentage should not be superior of 2.5% for filtration process.

During the packing, materials are washed and compacted by water in order to eliminate the dust or very fine particles, even though they are not totally removed.

From the effective diameters and uniformity coefficients, the RS1 and CA2 are more in accordance to the material recommendations for filtration bed packing and CA1 can be considered as the extreme situation of granulate materials that their consequences in filtration process should be fully examined. The results show that the RS1 is more appropriate for filtration use. In this study, this material is considered as the reference material. The crushed aggregates show great differences in the size distribution, and their consequences of applying these materials may lead to various functioning. The materials have similar physical properties, and the real density and bulk density are in the range of sandy soil: 2600 and 1600kg/m 3 [START_REF] Hillel | The effect of nutrients on extracellular polymeric substance production and its influence on sludge properties[END_REF]. The porosity is an indicator of relative volume of pore space in the minerals. Except for the fine river sand (RS1), the coarse sand (RS2) and crushed aggregates (CA1 and CA2) have slightly higher porosity, but the porosities of four materials are all in the range of sandy soil: 30-60% [START_REF] Hillel | The effect of nutrients on extracellular polymeric substance production and its influence on sludge properties[END_REF]. The physical characterization can only provide basic and loose information that indicate that river sands and crushed aggregates physically showed no significant differences except the roundness. The image analysis shows the crushed aggregates are more angular, less spherical (less roundness and circularity) and is in agreement with previous studies (Wanko et al., 2005;Cho et al, 2009).

Chemical and mineralogical characteristics

The river sands are considered as inert materials and their chemical characteristics do not impact the filter functioning. The crushed aggregates, as exploited from rocks are also chemically stable. Authors have indicated that crushed aggregates show slightly difference in mineral composition (Wanko et al., 2005).

The mineral compositions can affect the surface properties of the materials, such as the cations associated onto the grain surface. The chemical properties are particularly concerned in the study of the materials for wastewater treatments because the interactions taken place at liquid/solid interfaces may influence the adhesion of bacteria and the abatements of certain pollutants. The mineral compositions of filter materials are presented in The results show that both river sands and crushed aggregates are silicon based materials but crushed aggregates are more heterogeneous in mineral compositions due to the origins of materials. The two river sands present similar compositions since both materials are exploited from Loire River. The crushed aggregates are sandstone origins (CA1: Feldspathic sandstone and CA2: Precambrian sandstone). According to [START_REF] Mcbride | A classification of common sandstones[END_REF] the feldspars are often pink to red in color, calcite or quartz based and angular in forms and tend to weather to clay minerals under humid conditions but depend on the depositional environment; Pettijohn (1987) has also pointed out that the feldspars are more alkaline and present higher contents of Na, K and Ca. The material CA1 in this study can be recognized in several characteristics of feldspars. The release cations which were in accordance with grain compositions showed higher released Ca and Na with crushed aggregates. These elements are susceptible contributing some anions precipitation (ex: phosphate). The presence of metal elements at the surface of feldspars is also associated with their oxides (-O) or hydroxides (-OH) which contributes as well as the precipitation of some anions.

Reactors characteristics 1.4.2.1. Hydraulic characteristics

The water movement characteristics in a filter medium are one of the important properties in filtration beds, which basically depend on the filter materials. The filter material has the capacity of conducting the liquid phase flowing through the media known as the hydraulic conductivity, which can be estimated trough the infiltration test by measuring the time of infiltration. The filter material also has the capacity of retaining the liquid in the media and the liquid phase attached to the grain surface loosely or firmly bounded. This capacity is estimated by the water retention test. The results of and infiltration time (t s ) and estimated saturated hydraulic conductivity (K s ) are presented in Table 3.6. The results of capacity of water retention: volumic capacity (C v ) and massive capacity (C m ) are summarized in Table 2.1.9. The coarse river sand (RS2) shows the highest hydraulic conductivity and also the lowest water retention capacity. The crushed aggregate especially CA1 shows relatively low hydraulic conductivity which suggests that the flow is relayed in this material due to the differences in size and shape of particles. Authors have indicated that the estimated hydraulic conductivity should be ranged between 1.1×10 -3 and 3.7×10 -4 for filtration use. The water retention capacity also shows that CA1 holds more water and retains within the medium.

From the above, the differences in materials intrinsic characteristics are not only observed in size distributions, but also in forms and mineral compositions which indicate the problematic of altering filter materials from sands to crushed aggregates. The consequences of applying filtration reactors based on these materials can be dramatic.

Hydrodynamic behaviors

The hydrodynamic behaviors in filtration reactors are one of the most important parameters that govern the purification process the development of biomass. The reactors of 70cm and 30cm are the main reactors in this study and the flow characteristics are studied as hydrodynamic behavior by the distribution of hydraulic residence time. The average HRT of each reactor are summarized and the oxygen gas variation in Table 2.1.10. The hydrodynamic behavior of a filtration reactor is not only affected by the packing materials, but also the operational parameters and the reactor size. The results of 30cm reactors at 12cm/day show in the accordance with the hydraulic conductivities and water retention capacities of the filter materials. The coarse river sand (RS2) which is very permeable material shows low water residence time, poor water retention capacity and suggests that the solutes stay relatively shorter time in this material. As the contrary, the CA1 presents higher average HRT value, which suggests the existence of the solute exchange or interaction between the liquid phase and the material. As observed with granulometric analysis, this material contains more fine particles, even though the washing took place during the packing, some smaller or fine particles still stay in the system. Authors also suggested that the crushed aggregates with very angular, irregular particle shapes tend to form more tortuous flow paths and make the paths longer than in round, smooth materials (Wanko et al., 2005).

The 70cm reactors show much higher HRT volume in CA1. The HRT values decrease in 70cm reactors with higher hydraulic loading (20cm/day) for both RS1 and CA1. The oxygen gas sensor were applied one time for the two materials and the oxygen gas variations showed that in RS1, there was saturated/unsaturated phases during the batch inlet, but in CA1, no alternations of phase have been found and always in unsaturated conditions.

Conclusion:

A L'efficacité épuratoire donnera une vision globale de l'impact des matériaux puisque plusieurs acteurs interviennent dans la dépollution : le matériau et la biomasse.

-Les caractéristiques morphologiques des matériaux sont au moment de la mise en place du procédé :

i. via leur arrangement dans le réacteur, directement responsables de la rétention des particules solides de l'effluent ii. via leur surface spécifique, susceptibles de permettre une colonisation différente du réacteur -L'hydrodynamique influencera en particulier la mise en place du biofilm par rapport à son accès au substrat alors que la nature chimique du matériau est susceptible d'influencer l'adhésion et le développement du biofilm, mais aussi une précipitation éventuelle des phosphates (Zanini et al., 1998;Robertson, 2003).

Par ailleurs, l'impact sur la biomasse sera abordé par une estimation de la flore autotrophe nitrifiante et hétérotrophe à différentes hauteurs du réacteur.

Experimental procedures of purification efficiencies analysis

The feeding water and treated effluent characterization was carried out every 2 weeks in order to follow the filters purification performance. Both feeding water and treated effluents were sampled and stored at 4°C.

Feeding water distribution, treated effluent sampling and dosing schedule

Feeding water distribution and treated effluent evacuation

The feeding water was spread by the distribution disk, which was punched of 15 holes. The homogeneous distribution is difficult to achieve. The evacuation of treated effluents was achieved by the 3 way valves which provided the access to the effluent sampling tanks and to the drainage.

Dosing planning

The program of pilot feeding: openness and close of peristaltic pump and electric valves were controlled by computer, so these parameters could be altered or deposited. Filtration reactors were supplied discontinuously by batches with septic effluent in order to keep the unsaturated conditions. The hydraulic charge described by the thickness of effluent above material layer per day (charge: 12cm/day or 20cm/day corresponding to 8.5L/day or 14L/day) were fractionated by 10 batches at each column during 24 hours. The Table 2

Physico-chemical parameters analysis

The purification by sand filtration mainly concerns the treatments of particulate pollutants, organic carbon pollutants, nitrogen pollutants and phosphorous pollutants. The treatment efficiency is calculated as:

∆% = C c -C F C c × 100%
The tested parameters and their methods are presented in Table 2

Feeding water origin and characteristics

The packed reactors were fed with septic effluent during the long-term monitoring. The wastewater collected from La Chapelle sur Erdre (3km from Nantes, 33.42km 2 , 17 709 habitants) was pretreated in three septic tanks for 5 days in order to separate the liquid/solid phase. The settled sludge is rested over 2 years. The pretreated effluent from 3 septic tanks is called septic effluent and collected from a decanter before pumped to the reactors. The main characteristics of septic effluent are presented in Table 2.2.3.

Cells extraction and assessment

Sampling

The cells have been extracted from material samples and revivifiable aerobic heterotrophic flora and autotrophic flora (nitrifying bacteria) counts were carried out based on the culture of harvested cells. Due to the coarseness of river sand 2 (RS2), this test was only carried out with three other materials from 3 layers of reactors: 0~5cm, 10~15cm and 30cm. The fresh materials were sampled and processed as soon as possible.

Cell extraction 10g of sample was washed with 100mL of 9% NaCl solution and the washed and the cells were extracted by sonication (sonication bath, 60Watts, 15 min at 4°C). The samples received same ultrasound energy by putting in concentric circle with same separated space from the bath wall (Bigois, 1985;[START_REF] Lakel | Analyse déterministe de systèmes de biofiltration en écoulement saturé et insaturé dans le cadre d'un procédé d'assainissement autonome avec recyclage permettant l'élimination des composés azotés[END_REF].

Assessment

The extracted cell suspend was diluted with 9% NaCl solution and inoculated to corresponding culture mediums.

-The revivifiable aerobic flora (heterotrophic) was cultured on solid medium of plate count agar (PCA). 1mL of cell suspension was seeded. The incubation was effectuated at 30°C during 3 days.

-The procedure of nitrifying autotrophic flora assessment was adapted from the work of Aragno (1974). The liquid mineral medium enriched with ammonium ions was used and the composition is summarized in Table 2.2.4. 0.2mL of cell suspension was seeded in 1.8mL microplate. After 3 days of incubation at 30°C, the presence of nitrites was recovered by adding sulfuric acid and alpha-naphtylamine (reagent Nit 1 and Nit 2 of analytical profile index: API20E) in each cupule. In case of the absence of nitrites, zinc powder was added in order to recover the nitrates. Five copies were effectuated for each dilution.

Introduction

Domestic wastewater is produced on a per-dwelling basis, in combining sewage from toilet waste with washing machine and kitchen wastewater (Reneau et al., 1989;Almeida et al., 1999); the characteristics of this untreated effluent are similar to those of municipal wastewater (Raunkjaer et al., 1994;Crites and Tchobanoglous, 1998;Palmquist and Hanaeus, 2005). In rural France, this flow rate can be as low as 180 liters / inhabitant/day. On-Site

Wastewater Treatment (OWTS) plants typically comprise a septic tank for primary wastewater treatment and infiltration/filtration systems (featuring soil infiltration or sand filtration) for secondary treatment. In France, 15 million residents rely on such plants.

The septic tank enables solid/grease/liquid separation and settling, in addition to the liquefaction of solids, digestion of certain organic matter and transformation of organic nitrogen into ammonium (Reneau et al., 1989). The digestion and transformation of pollutants are often incomplete, leaving high concentrations still detectable in treated effluent, known as septic effluent, which is considered to be a source of groundwater contamination and water resources eutrophication in the presence of phosphorus and nitrogenous pollutants and microorganisms (Yates, 1985;Withers et al., 2011). This septic effluent is spread over soil absorption fields or sand filtration systems flowing under unsaturated conditions. This process takes place in the presence of air and water and is thus capable of efficiently removing carbon, nitrogen and suspended solids.

Secondary treatments are often applied with on-site soil absorption fields. Soil conditions however are sometimes unsuitable for wastewater treatment, hence the necessity of soil reconstruction with other packing materials, with the most widely used filter medium being natural sands from alluvial deposits, such as river sands. These packing materials must be carefully chosen in order to meet long-term operating requirements and modify purification behavior. Grain size distribution is the most important characteristic for a porous medium.

French and American authorities have imposed that the particle size distribution of a filter medium meets the following requirements: an effective diameter (D 10 ) of between 0.2and 2mm, and a uniformity coefficient (D 60 /D 10 ) of between 3 and 6 (French Standard DTU 64.1;USEPA, 2002).Some authors have also suggested a narrower range of D 10 (i.e. 0.2mm<D 10 <0.4mm) so as to ensure an effective purification process (Liénard et al., 2001).

The drained single-pass sand filters with discontinuous feeding are often encountered due to the simplicity of the process involved. The discontinuous feed keeps the filter medium unsaturated and aerated in addition to facilitating aerobic bacterial growth; consequently, sand filters can be considered as aerobic fixed-biomass reactors (Kristiansen, 1981a). The biomass developed inside the filter medium contributes to purification performance through the degradation of organic matter consumed by heterotrophic bacteria. The particulate organic matter is retained by the filter medium and then undergoes hydrolysis (Rodgers et al., 2005).

Larger particulate suspended solids are mechanically removed by straining, while the removal of smaller particles involves interception and adsorption (McDowell-Boyer et al., 1986). The transformation of ammonium nitrogen into nitrates is achieved by autotrophic bacteria from the Nitrobacteriaceae family (Rodgers et al., 2005). A number of authors have indicated that nitrification can be easily obtained in an unsaturated environment within the granular filter medium; moreover, Nitrobacteriaceae has been found between the surface and a depth of 12cm (Ardakani et al., 1974). Nitrification often results in the high nitrate contents of treated effluent and a low total nitrogen removal rate due to a slight denitrification under aerobic conditions (Van Cuyk et al., 2001). Low removal efficiencies of phosphorus pollutants (85% orthophosphates and other compounds in organic form) are prevalent with adsorption and precipitation. The adsorption of PO 4 3-is mainly correlated with the type of filter materials and enhanced by a greater content of clay, lime and iron and aluminum hydroxides in the materials along with a basic pH environment. Precipitation is correlated with the ionic composition of the effluent being treated: the presence of Fe 3+ , Al 3+ , Ca 2+ promote the precipitation of PO 4 3-(Zanini et al., 1998; Robertson, 2003). The elimination of microorganisms involves various mechanisms related to the cell size and activities, including straining, adsorption and predation, plus the presence of an air-water interface under unsaturated conditions [START_REF] Stevik | Retention and removal of pathogenic bacteria in wastewater percolating through porous media: a review[END_REF]Chabaud et al., 2006). The purification process is influenced by both environmental and operational conditions, such as: temperature, packing material characteristics, hydraulic loading, organic loading, and dosing frequencies (Ellis and Aydin, 1995;Rodgers et al., 2005;Gill et al., 2009) Due to overexploitation, river sands are less available in some areas. Other materials are substituted for use as packing materials, e.g. crushed aggregate resulting from the quarrying of massive rocks is considered as a potentially suitable material. Some studies have shown that crushed aggregate offers a very broad size distribution with angular, irregular particle shapes (Liénard et al., 2001;Cho et al., 2006). In introducing a synthetic septic effluent, some

Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials authors have indicated that removal efficiencies for organic and suspended solids appear to be similar for two different kinds of materials. This study however focused on short-term colonization between river sands and crushed aggregate through drawing experimental comparisons, while more long-term filter operating data were excluded (Wanko et al., 2005).

It was conducted using actual septic effluent in order to examine the impact of the various packing materials on filter operations, and especially on purification efficiency.

Materials and methods

Pilot batch experiment

The batch experiment was carried out on a laboratory-scale pilot equipped with 12 columns 30cm in diameter and 3 different heights: 15, 30, and 70 cm. The feed water was stored in a mixed storage tank and replenished about every 7 to 10 days. The columns were supplied discontinuously by batches of septic effluent in order to maintain the unsaturated conditions. The hydraulic loading was set at 12cm/day for all reactors, except for columns 10 and 12, which were set at 20 cm/day.

Two natural materials (river sands RS1 and RS2) and two crushed aggregates (CA1 and CA2) were tested. The filter materials were analyzed before column packing since the river sands and two crushed aggregates were of a different type and underwent a different treatment process in the quarries. All four aggregates were studied and compared in terms of particle size distribution, mineralogical, physical and hydrodynamic characteristics. Hydraulic conductivities were estimated by infiltration tests using the columns with an 8-cm diameter and 20-cm height (Liénard et al., 2001). The hydraulic residence time (HRT) was characterized in the 30-and 70-cmreactors by means of instantly injecting inert tracer lithium chloride (LiCl, 1g Li/l) and monitoring Li concentration in the treated effluents. Li was analyzed using Atomic Absorption Spectroscopy (SpectrAA 220, VARIAN).

Pictures of the sand samples were processed by ImagJ Software. After an 8-bit and binary conversion, the picture scale was set and the particle analyses performed with a dedicated plugin. The filter was introduced with limitations of both size (0.1-4 mm) and circularity (>0.75) to ensure effective particle isolation. Two parameters were determined: Circularity = 4π× (Area) / (Perimeter) 2 ; Roundness = 4× (Area)/π× (Major axis) 2 .

Packing material characteristics are shown in Table 1, while reactor configurations and feeding conditions are described in Table 2.

Characterization of feed water and treated effluent

The feed water was collected from the septic effluent settling tank. The main characteristics were monitored throughout the operating period. The average values of each characteristic are listed in Table 2. The treated effluents from 12 columns were characterized by physico-chemical analysis along with Merck ® photometric tests for chemical oxygen demand (COD), total nitrogen (Tot-N), ammonium (NH 4 + ), nitrates (NO 3 -) and orthophosphates (PO 4 3 ). Suspended solids were measured by weight loss after heating at 105°C for 24h. All feed water characteristics are summarized in Table 3. The total organic matter attached to the various materials was quantified as volatile dry weight (VDW), with the weight loss of sampled materials being measured by incineration between 105° and 550°C (Rolland et al., 2009). The total organic distribution across the depths of the four materials was evaluated at the end of the second study period.

The revivifiable aerobic heterotrophic and autotrophic flora were extracted from the three different layers in river sand 1 and crushed aggregates 1 and 2 at the end of the first period. A 20-g mass of colonized materials was sampled from 0~5cm, 10~15cm and 30cm. These materials were then washed with 100ml of a 9% NaCl solution, and 10 g of washed sample

were extracted using a 20-ml phosphate tampon (0.1M at pH=8)by sonication for 15min. The suspended slurry was diluted and inoculated with nutrient agar for aerobic heterotrophic flora.

The nitrifying bacteria were also assessed by a mineral liquid medium containing ammonium ions. After 3 days of incubation at 30°C, the presence of nitrites was showed by adding sulfuric acid and alpha-naphtylamine (reagent Nit 1 and Nit 2 of analytical profile index: API20E) in each cupule. In case of the absence of nitrites, zinc powder was added in order to recover the nitrates. Five copies were effectuated for each dilution.

Results and discussion

While practicing on-site sanitation, the packed filtration bed must contain appropriate material with a bed height of at least 70cm in order to ensure the long-term performance of the installation. As an initial step, the purification performance was compared by running the filtration reactors with a 30-cm diameter and 30-cm thickness on four different packing materials (river sands RS1 and RS2, and crushed aggregates CA1 and CA2). The upper 30-cm

Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials 109 filter medium is the more active part in a vertical filtration bed; also, the impact of various materials on treatment efficiencies is more significant over the upper part of the filter.

Operational parameters were modified on two selected materials for subsequent comparisons; these parameters were: bed thickness and hydraulic loading. The purification efficiencies with four different filter materials were monitored in terms of particulate matter (TSS), organic matter (COD), nitrogenous pollutants (ammonium, nitrates and total nitrogen) and phosphate pollutants.

Treatment efficiencies of the filtration process with various filter materials and under different operating conditions

The filtration reactors with a 30-cm active layer were packed with four different materials (river sands RS1 and RS2, crushed aggregates CA1 and CA2) and fed with feeding water at a rate of 12 cm/day. Results obtained during 360 days of operations are presented in Figure 2.

The TSS removals (Fig. 2a) showed similar and satisfactory efficiency rates with the fine river sand (RS1) and both crushed aggregates (CA1 and CA2) after 60 days of operations.

RS1 demonstrated quick and effective removals at the beginning of the process. On the other hand, the coarse river sand (RS2) yielded lower and less stable TSS removals throughout operations, typically below 80%. COD removals behaved similarly to TSS. The coarse river sand (RS2) was rated at the lowest efficiency (<70%, >125mgO/l) throughout the operating period, while the fine river sand (RS1) and crushed aggregate 1 (CA1) exhibited a similar removal rate (85%, 60mgO/l) after running for 120 days (Fig. 2b). Compared to RS1, the two crushed aggregates took longer (100 days for CA1, 180 days for CA2) to reach a similar efficiency level. While an increase in removals can be noticed in RS2, this material still produced the lowest and least stable organic removals.

Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials The nitrogenous pollutants in feeding water were dominated by organic and ammonium nitrogen; nitrate traces could be neglected. As indicated in the TSS and organic removals, RS1 and CA1 also showed a similar rate of satisfactory efficiency recordings in ammonium removals after 100 days, with RS1 displaying early stabilization (Fig. 2c). The removals in CA2 indicated slightly less stability. Meanwhile, the removals with coarse sand RS2 tended to be less than 80%. The increases in nitrate concentration were observed in all treated effluents (Fig. 2d). The conversion of nitrogen forms indicate that the activities of nitrifying bacteria and nitrification taking place had been gradually optimized in the given environment (Pell and Nyberg, 1989c;Van Cuyk et al., 2001;Rodgers et al., 2005). The ammonium and nitrate results suggest significant nitrification in RS1 and CA1 and relatively less nitrification in CA2 (after 180 days), as well as an incomplete nitrification with RS2. The increases in nitrate concentration led to poor or even decreased total nitrogen removals (Fig. 2e) due to N 2 stripping after denitrification; however, denitrification remained limited due to the lack of an available carbon source and conditions favoring reduction following nitrification (Van Cuyk et al., 2001). The initially higher Tot-N removal rates and the non-simultaneity between ammonium removal and the appearance of nitrates suggests the possible existence of sorption or assimilation by fast-growing heterotrophic bacteria in the unstable systems packed with fine river sand and crushed aggregate (De Vries, 1972;[START_REF] Hinkle | Mass balance and isotope effects during nitrogen transport through septic tank systems with packed-bed (sand) filters[END_REF].

Phosphorus pollutants in the feeding water were dominated by phosphates; their removal rates were typically below 60% (2~9 mgP/l). Figure 2f shows that phosphate removals by the filtration reactors were variable and less effective. The two crushed aggregates were responsible for somewhat greater removal at the beginning of the process. Removal rates decreased after about 120 days of operations, falling in general to below 60%. The low or decreasing P-removal efficiencies have been observed by other authors as the result of an aerobic or anoxic environment; moreover, phosphorus elimination requires an anaerobic condition, which is limited in filtration beds (Gill et al., 2009;[START_REF] Torrens | Impact of design and operation variables on the performance of vertical-flow constructed wetlands and intermittent sand filters treating pond effluent[END_REF], and might be explained by the depletion of adsorption sites or release by the biomass [START_REF] Arias | Phosphorus removal by sands for use as media in subsurface flow constructed reed beds[END_REF][START_REF] Vohla | Filter materials for phosphorus removal from wastewater in treatment wetlands -A review[END_REF].

From the above discussion, RS2 presents an exceptional case due to the coarseness of this material. As the reference material, the fine river sand shows predictable and satisfactory removals in the TSS, organic matter and ammonium. The crushed aggregates, especially CA1, yield similar purification efficiencies. A further comparison of treatment efficiencies is Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials needed in order to differentiate the purification capacities of RS1 and CA1. The next step therefore consists of distinguishing between the reference material (RS1) and the typical crushed aggregate (CA1) during 360 days with operational alternations in bed thickness and hydraulic loading rate (HLR).

Effect of operating conditions on the crushed aggregate filter

The treatment efficiency results for river sand and crushed aggregate according to the various heights and the two hydraulic loads are summarized in Table 4.

Generally speaking, these results demonstrate, for both RS1 and CA1-packed reactors, that reducing bed thickness (from 30 to 15cm) leads to a lower purification efficiency, especially with respect to organic and ammonium removals. In contrast, the thicker beds improve efficiency and provide for both satisfactory removals and better stability due to the longer HRT (Table 2) [START_REF] Torrens | Impact of design and operation variables on the performance of vertical-flow constructed wetlands and intermittent sand filters treating pond effluent[END_REF]. With 70-cm filtration reactors, RS1 led to slightly better efficiencies in organic and ammonium removals, but also to significantly higher total nitrogen removal rates (39% for RS1 and 22% for CA1). P-removals, on the other hand, were higher in crushed aggregate (44% for RS1 and 65% for CA1).

The other comparison drawn was between HLR of 12cm/day vs. 20 cm/day with two selected filter media. Put briefly, increasing HLR led to a decrease in the HRT value, especially for RS1 (Table 2), and a slight decrease in the efficiency of several parameters (Table 4): TSS, organic matter, ammonium and total nitrogen, specifically at the beginning of operations. Removal rates gradually rose with operating time, and the bed thickness also compensated the resistance to hydraulic loads, given that some authors have indicated that a higher hydraulic loading significantly reduces organic removal efficiency in shallower beds (38cm) (Rodgers et al., 2005).

From the above discussion, even though RS1 and CA1 yield similar purification efficiencies, differences could still be detected during the first 120 days, particularly in the total nitrogen and phosphate removals, which presumably should be correlated with the differences in material characteristics (Fig. 3). Ammonium removal efficiencies presented fluctuations under higher hydraulic loading, yet both materials seemed to be resistant enough and removals generally remained above 90%. The early stabilizations were established under the lower hydraulic loading, notably in the fine river sand (RS1), whereas a longer operating time was needed for the two materials at a higher loading. At the beginning of operations, both adsorption and incomplete nitrification were present in the systems (Wanko et al., 2005). The higher water flow reduced ammonium adsorption and limited the nitrification process (Van Cuyk et al., 2001;Hedström and Rastas Amofah, 2008). This impact of hydraulic loading has been confirmed by nitrate contents as well as total nitrogen removals in the higher loading reactors, where denitrification has considerably limited the reduction of HRT values. Phosphate removals were not improved under the environment of unsaturated filter media and were based mainly on particulate retention and adsorption. As mentioned in the previous section, efficiencies declined for both the two hydraulic loadings and the two types of materials. 48 [START_REF] Cooke | Modeling biochemically driven mineral precipitation in anaerobic biofilms[END_REF]65] 17 [START_REF] Van Cuyk | Hydraulic and purification behaviors and their interactions during wastewater treatment in soil infiltration systems[END_REF][START_REF] Bhatia | Fluorescence detection to determine proteins and humic-like substances fingerprints of exopolymeric substances (EPS) from biological sludges performed by size exclusion chromatography (SEC)[END_REF] 66 [START_REF] Appels | Principles and potential of the anaerobic digestion of waste-activated sludge[END_REF]91] Fig

Consequences of different filter materials on filter operations: Biomass distribution and purification efficiencies

Impact of material characteristics on biomass distributions in filtration reactors

With the observations recorded for the various filter media at the end of the study, total organic contents (expressed as volatile dry weight, i.e. mgVDW/g material) and the bacterial flora of extracted cell cultures could be assessed by core sampling from 3 layers in the filtration reactors (0~5cm, 10~15cm and 30cm). Due to the poor colonization onto coarse sand (RS2), cell extractions were not carried out with this material. The results of VDW in four media are presented in Table 5, while the results of total heterotrophic flora and autotrophic (nitrifying) flora are listed in Table 6. Total organic contents decreased from the top layer throughout the bed in all materials studied.

The fine river sand (RS1) showed higher accumulations at the surface and less organic matter in the deeper part. The total VDW is highest for RS1, in accordance with grain size (see Table 1, average grain diameter of 0.82 mm for RS1, vs. 2.66 mm for RS2). This fineness results in the surface retention of particulate matter and the straining of bacterial cells input by the feed water. In contrast, both crushed aggregate specimens revealed a slightly higher penetration of particulates and cells into the beds due to the size heterogeneity of these materials (Table 1:

uniformity coefficients of 10 and 5 for CA1 and CA2, respectively, vs. 3 and 2 for RS1 and RS2, respectively).

The distribution of heterotrophic bacteria in the fine river sand appeared to be homogeneous in the top 15 cm of medium and then decreased significantly in the deeper layer, which is in agreement with the VDW values. These results indicate that in fine filter media, filtration is a surface phenomenon active over the top part of the filter, hence leading to colonization where Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials substrates, bacterial cells and oxygen are relatively concentrated compared to the deeper part (Pell et al., 1990;Ellis and Aydin, 1995). This surface filtration phenomenon is less pronounced with crushed aggregate (CA1 and CA2), despite a more heterogeneous grain size even though the top 0~5 cm still show a greater colonization of heterotrophic bacteria, although without any significant decrease from 15cm to 30cm, unlike the fine uniform river sand. The distribution of nitrifying bacteria is less prevalent at the surface of the three filter media because the presence of higher substrate concentrations inhibits the activities of autotrophic flora. For the fine river sand (RS1), the nitrifying bacteria are mainly concentrated in the 10~15cmlayer but not in the deeper part (30cm), which suggests that the distribution of nitrifying bacteria is also influenced by another factor, namely oxygen content. The lower oxygen gas contents (Table 2) in RS1 during batch operations indicates a relatively saturated environment and the lack of access to oxygen in the deeper layer. On the other hand, with a lower substrate, the nitrifying flora is embedded relatively deeper in crushed aggregate, due to the size heterogeneity and angularity of crushed aggregate; moreover, the porosities are relatively higher and the media less saturated (e.g. CA1, Table 2). Some authors have pointed out that nitrifying bacteria are most active in the 10-12 cm below the infiltrative surface (Ardakani et al., 1974;[START_REF] Bahgat | Sand-filters used for wastewater treatment: buildup and distribution of microorganisms[END_REF], but with more permeable material nitrification also occurs between depths of 0.3 and 1m in the medium (Pell et al., 1990).

Bacteria influence the treatment efficiencies (through the final removal value and stabilization time, as described above) of soluble pollutants, especially given that heterotrophic metabolisms remove organic carbon and may cause denitrification in an anoxic environment while the nitrification metabolism removes ammonium ions.

Impact of material characteristics on the treatment performance of filtration processes:

Given that packing material characteristics govern the mechanical retention phenomena and biomass implementation, they ultimately alter the treatment performance of filtration reactors.

The removal of particulates and organic pollutants depends to a large extent on the material grain size. As observed in Figure 2 with RS1 compared to two crushed aggregates (CA1 and CA2), a medium composed of finer material provides for earlier stabilization in both TSS (particulates) and organic (particulates and dissolved forms) removals under the action of mechanical retention by the reactor surface layer (McDowell-Boyer et al., 1986;Rolland et al., 2009). Efficiency can also be improved and enhanced within relatively coarser media (CA1 and CA2), with an accumulation of retained particulates by reducing pore space and increasing surface area; the hydrolysis and assimilation by heterotrophic biomass also play an important role in organic pollutant reduction (Siegrist and Boyle, 1987;[START_REF] Schwager | Transport phenomena in intermittent filters[END_REF]Rodgers et al., 2005). Results from this study indicate that for a filter medium with an average grain size smaller than 1.6mm (i.e. D 10 <0.44mm, CA2), 30-cm media are sufficiently effective for TSS removals (Ellis and Aydin, 1995;[START_REF] Elbana | Effectiveness of sand media filters for removing turbidity and recovering dissolved oxygen from a reclaimed effluent used for micro-irrigation[END_REF]. Crushed aggregate exerts no significant impact on TSS and organic removals provided an adequate stabilization time has been allocated (i.e. more than 60 days of operations).

The highly permeable material (RS2) with a coarse size does not improve ammonium elimination (< 75%) due to its poor HRT (Table 2), which leads to an incomplete nitrification.

Aside from the exceptionally coarse sand, RS1 and the two crushed aggregates reveal no significant difference in ammonium removal after 100 days of operations. An earlier and faster ammonium removal by RS1 (over the first 60 days) was due to the assimilation by bacteria growth, which takes place higher in the top layer of river sand.

By drawing another comparison between RS1 and CA1 with 70-cm media, the material characteristics indicate a significant influence on total nitrogen removal efficiency. Using the 70-cm reactor packed with RS1, the improved removal (39% for RS1, 22% for CA1) is due to the more extensive denitrification within the fine medium and under a low hydraulic loading.

Relatively saturated conditions (oxygen gas variation, see Table 2) when the effluent inlet arrives and stagnates at the surface of the fine uniform medium (Table 1) are temporarily created. Moreover, the pore seal caused by an accumulation of particulates and biomass further improves the appearance of local anoxic microsites and favors denitrification (Gill et al., 2009). The denitrification condition is fragile to a point that increasing HLR serves to limit denitrification by reducing the corresponding HRT value (23% for RS1 at 20cm/day and 39% for RS1 at 12cm/day).

Unlike with organic and nitrogenous pollutants, phosphate elimination is dictated by the physical retention of phosphate particulate sand the physico-chemical mechanisms of soluble phosphates [START_REF] Clark | Phosphorus removal by chemical precipitation in a biological aerated filter[END_REF]Molle et al., 2005). This difference is mainly based on the type of materials and not on the bed thickness. Crushed aggregate yields a higher phosphate removal efficiency, even though its efficiencies also decrease. The presence of a greater fine particle content and the mineralogical heterogeneity in crushed aggregate may improve the adsorption onto material surfaces and lead to temporarily immobilization inasmuch as desorption may be taking place.

Conclusion

This study has been based on comparisons of pollution reduction in the bed filters of On-Site Wastewater Treatment between two types of packing material, river sand and crushed aggregate, with different physical and mineralogical characteristics.

The elimination of suspended solids and organic matter is mainly governed by the size of granular materials. Effective removals (>90%) of TSS and COD were achieved by both types of materials once the material fineness was deemed appropriate. Stable removal rates were observed by increasing the filtration bed height with selected materials that possessed very distinct characteristics. An increase in hydraulic loading slightly decreased he removal rate in a crushed aggregate (CA1), yet also resulted in a longer time before stabilization in both media.

Ammonium removals, accompanied by the enrichment of nitrate content in treated effluent, led to low total nitrogen removals. The excessive size of filter medium RS2 did not suggest a complete ammonium removal nor a complete nitrification. Longer HRT with the relatively fine media significantly improved ammonium elimination but still increased nitrate concentrations. With the deeper fine river sand medium, the nitrate level decreased after a certain operating time, which indicates denitrification is potentially more readily favored than with crushed aggregate. The exceptional loading results with a shorter HRT and the Tot-N removals decreased in both materials.

The P-removal efficiencies were relatively unstable across the tested filtration systems.

The elimination rates were mainly governed by physicochemical mechanisms (adsorption onto media or organic matter and precipitation) rather than biological ones (assimilation) and moreover were not significantly affected by a hydraulic loading increase. With greater Ca contents and mineral heterogeneity, the crushed aggregates (CA1 and CA2) displayed better phosphate removal rates; however, the efficiencies also declined to under 60% after 120 days of operations.

The organic matter distributions indicate that surface retention (0~15cm) in the fine river sand and the enrichment of heterotrophic bacteria are also found in this "cake"-like layer. On the other hand, both crushed aggregates showed a deeper insertion of organic matter and attached bacteria due to their grain size heterogeneity and the angularity of their shapes. A higher nitrifying bacteria content is found at 10~15cm in the fine sand (RS1) and at 10~30cm in the crushed aggregates (CA1 and CA2). La même méthodologie a été appliquée pour la biomasse totale et les EPS. L'évolution de la matière organique a été suivie pendant la mise en place du procédé, jusqu'à 360 jours. De plus, à l'issue de l'expérimentation, la répartition des EPS en fonction de la hauteur dans le biofiltre a aussi été réalisée.

La caractérisation de la matière organique est principalement basée sur des dosages colorimétriques des protéines, polysaccharides, substances humiques, acides nucléiques mais aussi sur la répartition en taille des matières organique assimilées à des protéines (protéinelike) et des substances humiques-like. Ces dernières caractérisations sont réalisées par chromatographie d'exclusion stérique couplée à une détection par spectrofluorescence.

Par ailleurs, au préalable nous avons voulu vérifier que l'empreinte des matières organiques de l'effluent était différente de celle de la biomasse totale ou de celle des EPS.

Experimental procedures of characterization of biomass, EPS and feeding water

The biomass developed onto the filter material is one of the main subjects in this study. It is the key to the purification performance of filtration rectors. The biomass/biofilm is characterized by its cells and EPS.

Sampling and total organic matters

Sampling

During the operation period, the river sands and the crushed aggregates were sampled at different layers in the reactors from time to time. 5 sampling ports provided in the filtration reactors of 70cm height the access to the inside at following layers : 5, 10, 15, 30, 45cm, and top layer (0-2cm) could be sampled freely. About 20g materials were collected from each layer and 200g were sampled at the top layer. The accessibility to the center part of column in the depth is difficult to achieve. The extractions of biomass and EPS constituents should be carried out at once or under short term (within 24 hours) cold temperature storage (at 0-4°C).

The freezing create microorganism die-off, cell lyse and release of intracellular proteins.

Dry weight and total organic matters (volatile dry weight)

The totality of organic matters contained in sand samples is characterized by volatile dry weight (VDW). This parameter can be considered as the total biomass and expressed by mg/g dry material. About 5g material was taken from the sample, dried at the 105°C in ventilated oven during 24 hours and the dry weight is calculated as:

Equation 11: DW (%) = S a 3S 'a½°¿ S a × 100
The dried samples were taken from ventilated oven and calcinated in the furnace at 550°C.

The weight loss between 550°C and 105°C represents the total organic matters in the sample and the rests are minerals. The results are expressed on mg/g dry material.

. Extractions of biomass and EPS constituents

The extractions of biomass and EPS constituents from the sand medium were carried out mainly by sonication or by heating. The working conditions are summarized in Table 2 The extraction efficiencies were not expected to be high. It is important to realize that use of even well-standardized extraction procedure is still qualitative in nature, and perhaps only a minor part is extracted (Wingender et al., 1999). The thermal treatment for biomass or EPS extraction largely interferes in the results of HPSEC fingerprints: the heat denatures the biochemical structure of macromolecules, such as proteins (Bourven et al, 2012). During the first time of extraction, the top layer samples were collected from the two places: side and center. The results showed in the same scales: with a difference in VDW <0.22mg/g dried material and in biochemical components sum <6mg/kg material.

Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 128

After the extractions, the samples were centrifuged at 4000g for 5min in order to separate the settable materials with liquid phase, than the samples were filtrated through 0.22µm (acetate cellulose) to remove bacterial cells. The prepared samples were frozen at -20°C.

Biochemical compositions of Biomass and EPS

Colorimetric assays

The quantification of extracted biomass and EPS constituents was carried out by biochemical analysis, in another word, the colorimetric assays for four major components: proteins, polysaccharides, humic substances, and nucleic acids. Some methods have been revisited in the literature review section. The methods used in this study and the analytic parameters are summarized in Table 2

3D Excitation-Emission Matrix (3D EEM) of fluorescence

The extracted biomass and EPS samples were characterized by 3D EEM which provides the characteristics of the fluorophores. With the EEM spectra, the detection wavelengths of protein-like substances and humic-like substances could be determined for the use of SEC (steric exclusion chromatography) detection. The typical peaks and their corresponding Excitation/Emission wavelength couples have been classified by several previous studies (Figure 2.9 and Table 2.3.3): To obtain EEM spectra, the samples were diluted with 50mM phosphate buffer at pH=7.0±0.1. The spectra were measured at 20°C, using Shimazu RF-5301 PC spectrofluorophotometer. The emission was scanned from 300 to 500nm after excitation ranged from 220 to 450nm with 15nm increments.

SEC coupled with fluorescence detection fingerprints

HPLC instrument and Separation

The separations of extracted biomass and EPS were performed by High Molecular Weight (HMW) Agilent Bio SEC300Å (5-1250kDa) column followed by Low Molecular Weight (LMW) Agilent Bio SEC 100Å (0.1-100kDa) column. Samples were processed by Merck

Hitachi LA Chom chromatograph equipped with a L7200 autosampler, a L7100 quaternary pump, a D7000 interface, a diode array UV detector (L7455), and a fluorescence detector (L7485).

Mobile phase

The columns functioned with a mobile phase of 150mM NaCl and 50mM phosphate buffer (25mM Na 2 HPO 4 and 25mM NaH 2 PO 4 ) at pH=7.0 ± 0.1 with a flow rate 0.7mL/min. 100µL of filtered samples (0.2µm, acetate cellulose) was injected for each analysis.

Detection

The UV detection was performed in the range of 210-300nm and the fingerprints were recorded at 210nm and 254nm. The protein-like fluorescence fingerprints were detected with 3 wavelength couples, Ex/Em 220nm/300nm for tyrosine-like, Ex/Em 220nm/330nm for tryptophan-like and Ex/Em 220nm/350nm also for tryptophan-like. The humic substance (HS)-like fluorescence fingerprints were recorded at Ex/Em 350nm/460nm.

Calibration

The apparent MW for association of columns was calibrated using six proteins or amino acids

Introduction

Onsite wastewater treatment (OWTS) systems affect the health of local residents and their living environments in decentralized areas throughout the world. Household wastewater contains high concentrations of pollutants with an organic discharge of about 77g BOD/day per capita [1]. Common configurations involve septic tanks for use as pre-treatment, with the application of natural or reconstructed secondary treatment, e.g. soil adsorption fields or trenches, constructed wetlands or sand filters [2][3][START_REF] Molle | Effect of reeds and feeding operations on hydraulic behaviour of vertical flow constructed wetlands under hydraulic overloads[END_REF]. Sand filtration has become more popular for treating domestic wastewater in light of its efficiency, ease of maintenance and limited energy costs. This application requires suitable materials, such as river sands. However, natural deposits have been more heavily exploited, to a point of even being lacking in some areas, hence requiring the search for similar materials, such as crushed aggregate. The question then gets raised over the risks and consequences of altering packing materials.

Sand filters function like a fixed, porous biomass reactor for the medium that includes mechanical retention acting through the sand grain network with biological degradations due to the biomass developing around these grains. Suspended solids are physically removed by straining [START_REF] Mcdowell-Boyer | Particle transport through porous media[END_REF]. The retained organic carbon pollutants undergo hydrolysis and biologically degrade under the aerobic reactions of heterotrophic bacteria [START_REF] Van Cuyk | Hydraulic and purification behaviors and their interactions during wastewater treatment in soil infiltration systems[END_REF][START_REF] Rodgers | Organic carbon removal and nitrification of high strength wastewaters using stratified sand filters[END_REF].

This filter enables a gradual process of pore space reduction, in comparison with the initial state of clean beds, which is introduced by the accumulation of suspended solids, precipitates and the colonization of biomass [START_REF] Gill | Nutrient loading on subsoils from on-site wastewater effluent, comparing septic tank and secondary treatment systems[END_REF][START_REF] Cooke | Modeling biochemically driven mineral precipitation in anaerobic biofilms[END_REF][START_REF] Vandevivere | Effect of bacterial extracellular polymers on the saturated hydraulic conductivity of sand columns[END_REF]. The non-cellular particulates may originate from both the feed effluent and fine mineral particles dissociated from the packing materials. The biomass system reaches a steady state whereby biomass growth and organic abatement occur after a given operating period [START_REF] Hirata | Evaluation of kinetic parameters of biochemical reaction in three-phase fluidized bed biofilm reactor for wastewater treatment[END_REF]. The biomass is produced by cells attached to the filter medium and plays an important role as biodegradation agent. The biomass also contributes to physically changing the porous medium [START_REF] Kristiansen | Sand filter trenches for purification of septic tank effluent: I. The clogging mechanism and soil physical environment[END_REF][START_REF] Liu | Biomass accumulation and carbon utilization in layered sand filter biofilm systems receiving milk fat and detergent mixtures[END_REF][START_REF] Zhao | Clogging processes caused by biofilm growth and organic particle accumulation in lab-scale vertical flow constructed wetlands[END_REF]; it is a complex spatial network of organic matter composed mainly of bacterial cells. For the most part, its constituents are proteins, sugars, lipids and nucleic acids, i.e. extracellular molecules produced by cells (in the case of proteins and sugars) and captured molecules such as humic-like substances, minerals [START_REF] Jahn | Cell biomass and exopolymer composition in sewer biofilms[END_REF][START_REF] Flemming | Relevance of microbial extracellular polymeric substances (EPSs)--Part I: Structural and ecological aspects[END_REF][START_REF] D'abzac | Characterization of the mineral fraction associated to extracellular polymeric substances (EPS) in anaerobic granular sludges[END_REF].

When a non-clogging condition is active, the non-polysaccharide fractions (e.g. proteins, humic-like substances, lipids) are found in greater quantities than polysaccharides in the biomass [START_REF] Le Bihan | Monitoring biofilter clogging: biochemical characteristics of the biomass[END_REF]. Protein is a component of all living cells [START_REF] Paul | Production of dissolved DNA, RNA, and protein by microbial populations in a Florida reservoir[END_REF]. Some of these extracellular molecules act as "cement" that binds other particles and potentially causes "pore sealing"

Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 134 [START_REF] Mauclaire | Sand filtration in a water treatment plant: biological parameters responsible for clogging[END_REF]. The proteins serve important functions in the biomass, namely structural (capsular peptides) and enzymatic (biodegradation) [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF][START_REF] Wingender | Microbial extracellular polymeric substances: characterization, structure, and function[END_REF].

In considering the new materials for sand filter packing, studies conducted have shown that crushed aggregate must be widely graded in size, rather than being heterogeneous in form and surface, and more varied in mineral composition than natural aggregates [START_REF] Linard | Choice of the sand for sand filters used for secondary treatment of wastewater[END_REF][START_REF] Tsomokos | Effect of grain shape and angularity on the undrained response of fine sands[END_REF][START_REF] Wanko | Distribution des temps de séjour en infiltration-percolation. Performances de deux types de matériaux Techniques sciences méthodes[END_REF]. The authors also suggest that the biomass growth rate would tend to be slightly lower for crushed aggregate compared to river sand of a similar size during the feeding period [START_REF] Wanko | Biological Processing Capacities and Biomass Growth in Waste Water Treatment by Infiltration On two Kinds of Sand[END_REF]. The roughness of the medium surface also influences biomass maintenance [START_REF] Alnnasouri | Influence of surface topography on biofilm development: Experiment and modeling[END_REF]. The characteristics of crushed aggregate can modify the hydrodynamics and oxygen distribution relative to natural sands, and these differences may lead to a difference in filter operations.

In this study, two types of packing materials will be studied and compared: natural sand stemming from alluvial deposits (Loire River sand), set as the reference material; and two crushed aggregates resulting from quarry production. This work will seek to determine the impact of packing materials on biomass development throughout the operating period, in terms of quantity and especially the evolution in main biochemical components.

Materials and methods

Batch experiments (cylindrical filtration reactors)

Filtration reactors 70cm high and 30 cm in diameter with sampling ports were packed with river sand and crushed aggregate, respectively (see Fig. 1). These reactors were fed with septic effluent that had been stored in a mixed tank and replenished every 7 days. The reactors were fed discontinuously, at a hydraulic loading rate of 12cm/day in 10 daily batches.

Packing material characteristics

The filter materials were analyzed before column packing since the river sand and two crushed aggregates are of different compositions and involve different treatment processes in the quarries. The three aggregates were studied and compared in terms of particle distribution, mineralogical, physical and hydrodynamic characteristics.

The particle size distribution analysis was conducted by means of mechanical shaking with a series of sieves with the following opening diameters: 0.08, 0.16, 0.2, 0.25, 0.315, 0.4, 0.5, 0.63, 0.8, 1, 1.6, 2, 2.5, 4, and 5mm. The fine particle content is defined as the percentage of the sample with diameters less than 80µm.

A water retention analysis was performed using columns 8cm in diameter and 70cm high, fed

with clear water at a flow rate of 10mL/min until reaching steady state. The ratio of the retained water volume to the material volume was set as the volumetric retention capacity (%). After draining the columns, the weight loss at 105°C was measured with the wet filter materials, and the ratio of weight loss to packing material weight in the column was evaluated as the mass retention capacity of static water (%). Hydraulic conductivities were estimated by infiltration tests using the columns 8cm in diameter and 20cm high [START_REF] Linard | Choice of the sand for sand filters used for secondary treatment of wastewater[END_REF]. The hydraulic residence time (HRT) was measured via instant injections with lithium chloride solution(1g Li/L), and the lithium concentrations were identified by atomic absorption spectroscopy (SpectrAA 220, VARIAN). These characteristics are listed in Table 1.

Feed water: Septic effluent characteristics

The feed water was collected from the septic effluent settling tank, with all main characteristics being monitored throughout the operating period. The average and extreme values of each characteristic are reported in Table 2.

Biomass extraction

Three samplings were carried out during some 12 months of operations. The samples were collected from 5 different depths inside the reactors (5, 10, 15, 30 and 45cm) and from the top layer (0-2 cm). The total organic matter contents of each layer were assessed by the volatile dry weight (VDW, in mg/g of material). The biomass was extracted as quickly as possible (<24h) by heating to 80°C during 30min for the colorimetric analysis and by sonication at 4°C during 60min for the size exclusion chromatography (SEC) fingerprint analysis [START_REF] Zhang | Comparison of extraction methods for quantfying extracellular polymers in biofilm[END_REF][START_REF] Bhatia | Fluorescence detection to determine proteins and humic-like substances fingerprints of exopolymeric substances (EPS) from biological sludges performed by size exclusion chromatography (SEC)[END_REF]. The SEC fingerprint analysis actually reveals the evolution of molecular weight (MW) and how a heat treatment should modify the MW fingerprints [START_REF] Bourven | Influence of extraction method on size exclusion chromatography fingerprints of EPS from wastewater sludges[END_REF].

Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 136

Biomass characterization

Total organic contents were determined by the weight loss of sampled materials, as measured by incineration between 105°C and 550°C [START_REF] Tanner | Accumulation of organic solids in gravel-bed constructed wetlands[END_REF].

The biochemical components of biomass were quantified using colorimetric methods:

proteins and humic substances by the modified Lowry method in introducing Folin's reagent [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF]; polysaccharides by Dubois' method; and nucleic acids by Burton's method [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF][START_REF] Burton | A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid[END_REF].

The MW distributions of biomass proteins and humic substances were analyzed by HPSEC (Merck Hitachi LA Chrom Chromatograph) coupled with fluorescence detection. The high MW separation was performed by the Agilent column, bioSec, 300A (5-1250kDa) and the low MW separation by the Agilent column, bioSec, 100A (0.1-100kDa). All columns were placed in series for the separation improvement step [START_REF] Bourven | Effect of various size exclusion chromatography (SEC) columns on the fingerprints of extracellular polymeric substances (EPS) extracted from biological sludge[END_REF]. The mobile phase was composed of 150mM NaCl and 50mM phosphate buffer at pH 7.0. The apparent MW for associating the columns was calibrated using six proteins or amino acids with MW values of: 440000, 155000, 69323, 5777, 362 and 181 Da (respectively ferritine (Sigma), immunoglobulin G from human serum (Sigma), albumin from bovine serum (Sigma), insulin from porcine pancreas (Fluka), thyrotropin-releasing hormone (Fluka), and tyrosine (Fluka)). For mass calibration curves, the logarithm of the molecular mass (log (MW)) is plotted as a function of the elution volume (mL), i.e.:

Log (MW) = -0.3164 Ve (mL) + 9.4676 (R2= 0.982)

With: MW: molecular weight (in Da); and Ve: elution volume (in mL). The permeation volume determined with NaN 3 is 22mL. The Ex/Em wavelength fluorescence detection for protein tryptophan-like substances was found to be 222nm/330nm, while for humic-like substances it was 350nm/460nm. These wavelengths were evaluated after a three-dimensional spectrofluorimetric analysis [START_REF] Chen | Fluorescence excitation-emission matrix regional integration to quantify spectra for dissolved organic matter[END_REF].

The chromatograms are established from both the fluorescent intensity (volts) and elution volume (mL). Several fractions were identified as the interval of elution volume (e.g.

Treatment efficiency with two types of materials

The filter medium physically removed the suspended solids retained them within the medium.

Organic carbon pollutants are removed biologically through the heterotrophic bacterial uptake.

Removal efficiency results on both TSS and COD are shown in Figure 2. The suspended solid removals stabilized after some 60 days of colonization for both river sand (RS) and crushed aggregate (CA1). Variations were observed due to the feed water, though all removals typically exceeded 95%. The organic (COD) removals also stabilized after 60 days of operations, although the river sand demonstrated more stable efficiencies than CA1. The COD abatements by river sand were generally between 95%and 98%, while crushed aggregates showed similar efficiencies albeit with greater variations (92% ~ 98%).

Similar trends were observed with the TSS removals. Both filter media were sufficiently effective for suspended solids and organic pollutant removals; moreover, the efficiencies were in line with previous studies [3,[START_REF] Van Cuyk | Hydraulic and purification behaviors and their interactions during wastewater treatment in soil infiltration systems[END_REF][START_REF] Rodgers | Organic carbon removal and nitrification of high strength wastewaters using stratified sand filters[END_REF][START_REF] Ellis | Penetration of solids and biological activity into slow sand filters[END_REF]. The TSS removals were primarily governed by a mechanical filtration process. During the first month, the reactor with river sand showed quicker improvement of TSS removals. After 1 month of colonization, efficiency was optimized due to grain size fineness and regularity (see Table 1). The authors also pointed out that grain sizes do exert a great impact on suspended solids removal [START_REF] Rolland | Influence of the physical and mechanical characteristics of sands on the hydraulic and biological behaviors of sand filters[END_REF]. For both of these materials, the most noteworthy change took place around 8 weeks (50-60 days in Fig. 2) when comparing the performance before and after. This early behavior was assumed to be due to TSS filtration, which caused pore blockage and increased the filtration area being utilized [START_REF] Van Cuyk | Hydraulic and purification behaviors and their interactions during wastewater treatment in soil infiltration systems[END_REF][START_REF] Healy | Treatment of dairy wastewater using constructed wetlands and intermittent sand filters[END_REF]. COD removals by two materials indicated this improvement with respect to TSS removals. The elimination of organic pollutants depended on both the hydraulic residence time and development of a heterotrophic biomass. For starters, CA1 displayed higher HRT (93hours) than river sand (35hours); this observation may be explained by the clayey fine particles in crushed aggregate that tend to retain the solutes, which was not observed in river sand. The river sand, on the other hand, showed rapid abatement above 90% after roughly 10 days of operations; this finding may be due to the effect of filtration. The significant improvement in COD removals at around 8 weeks of colonization in both media could be ascribed to biomass growth, especially in the surface layer (0~2cm), where the retentions of both bacterial cells and the substrate were greatest [START_REF] Rodgers | Organic carbon removal and nitrification of high strength wastewaters using stratified sand filters[END_REF][START_REF] Kristiansen | Sand filter trenches for purification of septic tank effluent: III. The microflora[END_REF]. Accordingly, the organic contents inside several filter layers revealed increases for the two media, both of which were assessed by VDW during operations (Fig. 3).

Fig. 3. VDW distributions along the depth of two types of filter media, i.e. river sand (RS1)

and crushed aggregate(CA1), over the entire operating period Throughout the operating period, the accumulations of organic matter increased in all sampling layers for both media. Higher VDW in the 0~5cm surface layers of both filter media reflected higher organic accumulations compared to the VDW in deeper layers. The river sand has shown relatively greater retention of organic matter at the reactor surface, in accordance with grain size [START_REF] Liu | Biomass accumulation and carbon utilization in layered sand filter biofilm systems receiving milk fat and detergent mixtures[END_REF]. Except for the higher accumulations on the surface layer, the VDW distributions below 10cm were quite homogeneous with respect to depth. the accumulations of organic matter were more pronounced at the surface, especially in the presence of particulate matter [START_REF] Zhao | Clogging processes caused by biofilm growth and organic particle accumulation in lab-scale vertical flow constructed wetlands[END_REF][START_REF] Lloréns | Nitrogen transformation in two subsurface infiltration systems at pilot scale[END_REF]. At the beginning of operations, CA1 showed higher organic accumulations with depth, unlike the river sand. This finding might also be related to the grain size heterogeneity and higher porosity of CA1, which allows particulates or dissolved organic substances and bacterial cells to reach the deeper layers [START_REF] Ellis | Penetration of solids and biological activity into slow sand filters[END_REF]. Over the next 50 days, the river sand increased more substantially in every layer than did the CA1, which could be due to the colonization of bacterial cells and excretion of extracellular polymers.

Small increases were found from 210 to 360 days in the river sand across all layers. On the other hand, the VDW in CA1kept increasing after 210 days, especially in the top 0~10cm.On the higher parts of the reactors, organic accumulation included matter from the feed water and biomass growth within the filter media. At the same time, the increases in organic degradation efficiencies for both materials indicated the presence of biological activity in heterotrophic bacteria under aerobic conditions [START_REF] Liu | Biomass accumulation and carbon utilization in layered sand filter biofilm systems receiving milk fat and detergent mixtures[END_REF]. With a focus on accumulated organic matter, the evolution of biochemical fractions of top layers in two filter media was studied from time to time with respect to feed water characteristics.

Influence of feed water filtration on organic matter accumulation

As evidenced by the VDW value, organic accumulation was more abundant in the top 0~5cm.

The biochemical fractions of extract from solid or liquid samples were quantified by employing colorimetric methods. The results from river sand extracts were compared to the feed water (septic effluent), as displayed in Figure 4. The proportions of these components vs.

time are shown in Figure 5. retention of organic matter from the influent since at the time; the biomass was of a relatively small quantity. HS-like dominated the biochemical composition of the river sand extracts, which were assumed to have been derived from the re-polymerization of biologically or chemically-degraded environmental organic matter [START_REF] Francioso | Spectroscopic study of humic acids fractionated by means of tangential ultrafiltration[END_REF]. During the second extraction time (210 days) however, the biomass evolved: an increase in proteins as well as nucleic acids indicated that colonization by biomass had become a dominant origin. The 360-day extraction showed slight increases in proteins, HS-like and polysaccharides. The ratio between proteins and polysaccharides (PN/PS) rose from 0.75 to 2.44 vs. time and remained nearly constant between 210 and 360 days of operating time. This increase in PN/PS ratio is considered an indication of biomass strength; for ratio values around 1, the biomass is fragile and loose [START_REF] Nguyen | Organic matter composition, microbial biomass and microbial activity in gravel-bed constructed wetlands treating farm dairy wastewaters[END_REF][START_REF] Characklis | Biofilms[END_REF][START_REF] Lazarova | Biofilm performance of a fluidized bed biofilm reactor for drinking water denitrification[END_REF]. Figure 5 shows that the proteins and HS-like represent respectively about 45% (±5%) of the major compositions for the 210 and 360-day extractions, while the polysaccharide fractions decreased from 30% to less than 20%. This result was in agreement with several sewer biomass studies suggesting that proteins and HS-like were in fact the predominant components in natural biomass [START_REF] Jahn | Cell biomass and exopolymer composition in sewer biofilms[END_REF][START_REF] Le Bihan | Monitoring biofilter clogging: biochemical characteristics of the biomass[END_REF][START_REF] Ragusa | Indicators of biofilm development and activity in constructed wetlands microcosms[END_REF]. Given that the HS-like are environmentally generated, the evolution in protein-like compounds was considered as a present indicator of biomass: the PN/HS ratio increased from 0.47 to 1.44 until 360 days of colonization [START_REF] Francioso | Spectroscopic study of humic acids fractionated by means of tangential ultrafiltration[END_REF]. A further qualitative assessment of MW distributions by HPSEC fingerprints between the organic matter of the river sand extraction and the feed water could thus be described.

Figure 6 shows the protein-like SEC fingerprints of biomass from river sand extractions at 60 and 210 days and of the feed water. In comparing the results of 60-day biomass extraction and the feed water organic composition, certain peak locations in the protein-like fingerprints overlapped, despite their peak areas not being the same. Four groups of peaks or four MW fractions can be distinguished on the chromatograms displayed in Figure 6. represented over 35% of the total sample; Fraction 2 tended to stabilize at a level below that of Fraction 1. Compared to feed water, the evolution in chromatographic fractions suggests that these extracted protein-like molecules of a biomass of a system that has not reached stationary phase (60 days) might be polymers, derived by bacterial synthesis (Fraction 1) or partially conveyed by septic effluent, retained by the top layer with a similar level (Fraction 2).

The increase in Fraction 1 compared to Fraction 2 suggests change in molecular configuration during the biomass maturation process (from 60 to 210 days). This configuration change could be detected due to either the analytical method or the actual presence in the biomass of very high MW compounds. Such compounds could be molecular associations, e.g. polymers found in an extracellular matrix in the biofilm of the biomass [START_REF] Bourven | Effect of various size exclusion chromatography (SEC) columns on the fingerprints of extracellular polymeric substances (EPS) extracted from biological sludge[END_REF]. Based on this observation, the biomass in two crushed aggregates was also assessed by comparison to the reference material: river sand, using CA1 as the primary material; and CA2 as a supplemental input (S1, S2). Their respective characteristics are summarized in Table 1. moreover has not reached stabilization by the end of the process, or else colonization seems to take more time in crushed aggregates than in river sand. This difference in the biomass dynamic in the presence of different media appears to be dictated by aggregate size, i.e.

average diameter(0.82mm for RS, 1.36mm for CA1, 1.6 mm for CA2) and porosity(approx.

30% for RS and 40% for CA1 and CA2). Larger aggregate grains offer less specific surface area for bacterial adhesion, nutrient availability and biomass colonization [START_REF] Stevik | Retention and removal of pathogenic bacteria in wastewater percolating through porous media: a review[END_REF][START_REF] Picano | Influence of porosity and composition of supports on the methanogenic biofilm characteristics developed in a fixed bed anaerobic reactor[END_REF]. The accumulation of suspended solids and biomass helps reduce pore space and provides adsorption sites for upcoming colonization. The study by Wanko et al. also showed an early increase (at 30 days) of biomass growth in river sand and a slower growth in crushed aggregate [START_REF] Wanko | Biological Processing Capacities and Biomass Growth in Waste Water Treatment by Infiltration On two Kinds of Sand[END_REF]. Another significant difference between the two supports was the variation in O2 gas within the systems (Table 1). Results indicate that crushed aggregate was still in an unsaturated condition at the top layer, even at the time of batch inlet. This finding could be explained by the greater size heterogeneity and porosity from CA1 and CA2. As such, the top river sand filter layer accounted for less O 2 gas as the feed water flowed. If this process were being continuously fed, the oxygen content could keep dropping; however, in this study, the filters were discontinuously fed and oxygen could be replenished at rest, hence no clogging was observed during the process [START_REF] Rolland | Influence of the physical and mechanical characteristics of sands on the hydraulic and biological behaviors of sand filters[END_REF]. The temporary saturation conditions in RS1 lead to a more uniform distribution of nutrients and bacterial cells in the river sand, which resulted in their rapid growth and early balance.

As observed in biochemical composition analyses, the biomass in crushed aggregate shows a difference in terms of both quantity and stabilization time. Similarly, the protein-like compounds in crushed aggregates (CA1 and CA2) were also studied by the HPSEC, and four major fractions were also recorded. The major group fractions were found to be comparable to (35% vs. 27%) could be due to the lower biomass protein production, which enhanced the protein-like molecules from septic effluent, but this could also be due to the protein-like synthesis during biomass growth.

CA2 at 210 days showed similar proportions to CA1 at 60 days, suggesting a more heavily delayed biomass development in this material, which may be due to its coarseness compared to that of the other materials. The similar proportions between RS1 and CA1 were observed at around 210 days of colonization, suggesting a similar evolution as in RS1, i.e. a period of stabilization for these quantities, though the biomass indicator (protein-like compounds) may endure a similar process during biomass development. HPSEC may be a suitable method for tracking the evolution/stabilization of the protein-like biomass in such processes. This method seems to be more sensitive than the percentage of biochemical compounds.

Conclusion

This study has been based on comparative experiments conducted between traditional filter media during 360 days: river sand and two crushed aggregates stemming from quarry output.

The characteristics, especially grain size distributions, showed marked differences among these materials. CA1 exhibited considerable size heterogeneity and fine particle content. Even though great differences were observed in these materials, the purification behavior study suggested that both river sand and CA1 were effective and stable enough to remove the main pollutants of septic effluent (>95% of TSS, >90% of COD) as of Day 60.

The organic accumulations in various materials displayed their highest contents on the top layer (0~5cm), where the nutrients and bacterial cells were most abundant and where the accumulations included organic substances originating from either feed water or biomass. The subsequent biochemical study indicated an evolution in the biochemical composition of extracted organic matter, notably the increases in protein-like compounds and in PN/PS and PN/HS ratios. The HPSEC protein-like fingerprints revealed an orientation towards higher MW compositions after 210 days of colonization for the RS and two CA extracted biomass.

With the crushed aggregates tested herein (namely CA1 and CA2), the main difference observed was the decrease in quantities of biochemical components and the longer time required to establish biomass stability, especially for CA2. The biomass stability monitoring tool consists of: the PN/PS and PN/HS ratios and the percentages of >1000kDa fraction with

Introduction

On-site wastewater treatment systems (OWTS) are typically located in rural areas without connections to a municipal wastewater treatment network. The most common configuration involves a septic tank offering pretreatment by anaerobic digestion, whereby the pretreated (septic) effluent is spread over soil infiltration fields, or more commonly, filtration beds packed with river sands or similar materials. Biomass exits the filtration reactors in the form of a biofilm, which protects the microbial cells that are irreversibly attached to the substratum by embedment in a matrix of extracellular polymeric substances (EPS) (Donlan and Costerton, 2002). The biomass or biofilm population serves an important role as purification agent of organic nitrogenous pollutants. The structural characteristics of biofilm (i.e. the EPS matrix) however also take into account the environment of a porous medium and tend to reduce the limited space between pores until biofilm development reaches stabilization. Yet the presence of biofilm is also considered to "clog" porous media, as provided by river sand or other materials in filtration reactors, e.g. polysaccharides (Zhao et al., 2009;[START_REF] Kim | Biofilm morphology as related to the porous media clogging[END_REF].

The extracellular matrix of biofilm contains a "gel"-like structure and is composed of major biochemical components either tightly or loosely bound to the cells, such as proteins, polysaccharides, nucleic acids, humic-like substances (Frølund et al., 1996;Flemming and Wingender, 2001a), metabolic wastes, and absorbed substrates and minerals (Wingender et al., 1999;Tsai et al., 2008;D'Abzac et al., 2011b). The roles of EPS are believed to include, without being limited to, the following: structural formation and maintenance of aggregates or biofilm; increase in substrate diffusivity; and aggregate morphology in correlation with EPS hydrophobicity. It can still be argued whether EPS production reflects higher or lower microbial activity. The excretion of intracellular material might be a survival mechanism under unfavorable conditions, such as enhanced extracellular enzymatic activity and/or other mechanisms facilitating cell aggregation (Liu et al.,2004).

EPS proteins constitute a group of molecules that perform critical functions in the biofilm [START_REF] Arnosti | High activity and low temperature optima of extracellular enzymes in Arctic sediments: implications for carbon cycling by heterotrophic microbial communities[END_REF]Frølund et al., 1996;Pell and Nyberg, 1989). Proteins also possess a high molecular weight (45~670kDa) and may be associated with other macromolecules, such as polysaccharides (Görner et al., 2003;Bourven et al., 2014). The molecular weight partition of the EPS protein changes depending on the state of the biofilm [START_REF] Martinez | Role of exopolymeric protein on the settleability of nitrifying sludges[END_REF][START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF]. The polysaccharides in an EPS matrix exist in the form of either a capsule covalently associated with a cellular membrane (lipopolysaccharides)

or slime weakly coupled with the cells (Kumar et al., 2007). Polysaccharides are hydrophilic molecules that tend to retain water and assist the microbial cells in retaining nutrients (Wingender et al., 1999;[START_REF] Imai | Advanced startup of UASB reactors by adding of water absorbing polymer[END_REF]. The humic-like substances (HS-like) are believed to be exogenous compounds captured in the biofilm before undergoing repolymerization (Francioso et al., 2002). However, Guo et al. (2011) found that humic acid-like substances detected by means of three-dimensional spectrofluorimetry appear during aerobic granulation.

Some authors have pointed out that another pathway exists for the formation of these HS-like:

following the degradation of macromolecules (like carbohydrates and proteins) under microbial attack, the refractory compounds or biopolymers are selectively transformed to produce the high MW precursor of humin, subsequent to which the molecules become smaller during the additional oxidation process [START_REF] Vanloon | Environmental Chemistry: a Global Perspective[END_REF]. Nucleic acids used to be considered as the indicator of cell lysis during extraction; later research however has found that many microorganisms secrete extracellular nucleic acids (Steinberger and Holden, 2005).

Protein and polysaccharide EPS contents had been tracked as a potential indicator of biofilm state: the protein content increases with the formation and stability of aerobic granules [START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF], whereas [START_REF] Ahimou | Effect of Protein, Polysaccharide, and Oxygen Concentration Profileson Biofilm Cohesiveness[END_REF] showed that the level of biofilm cohesive energy is strongly correlated with polysaccharide content.

Studies on EPS from bioprocess systems have been extensively conducted in an attempt to establish a connection between reactor performance and EPS physicochemical features. This performance includes sludge settling and flocculation capability (Liu and Fang, 2003), biofilm and granular formation (Liu et al., 2004), and membrane fouling (Her et al., 2007).

Accordingly, the EPS study on OWTS is focused on two topics: biofilm formation, and clogging prevention. However, research carried out on biofilm extracted from filtration reactors has mainly dealt with biological clogging tied to biomass expansion and the microbial community on the filter medium, i.e. total organic accumulation [START_REF] Campos | Biomass development in slow sand filters[END_REF]Zhao et al., 2009), in including the main biofilm components. Proteins and polysaccharides exhibit a linear correlation with operating time and a decreasing abundance with depth (Regusa et al., 2004). Among these organics, the loose "slime"-like exopolymers appear to cause the drop in hydraulic conductivity while the cells exert no effect on clogging (Vandevivere and Baveye, 1992;[START_REF] Ronner | Characterization of microbial clogging in wastewater infiltration systems[END_REF]. None of these studies have ever covered the evolution of EPS composition and characteristics. The aim of this work therefore is to provide in-depth information regarding EPS characteristics in relation to the various stages of the biofilter enrichment process as well as to bed thickness. For this study, two types of packing materials, river sand and crushed aggregate, have been used.

Materials and methods

Batch experiments (cylindrical filtration reactors)

Two filtration reactors, 70 cm high and 30 cm in diameter with sampling ports, were packed with river sand and crushed aggregate, respectively (see Fig. 1). These reactors were then fed with septic effluent that had been stored in a mixed tank with replenishment every 7

days. The reactors were fed discontinuously, at a hydraulic loading rate of 12 cm/day in 10 daily batches. The feed-rest condition induces liquid passage, which may involve varying the O 2 level. The O 2 gas variations in each reactor were estimated by means of an optical oxygen sensor (OXROB10, Fire Sting O2, with Pyroscience sensor technology). A saturated/unsaturated gas phase alternation was found with the river sand reactor, but the reactor with crushed aggregate showed less variation in oxygen gas.

Figure 1 : Filtration reactor with sampling ports

Packing material characteristics

The filter materials were analyzed before column packing, given that the river sand and crushed aggregate differed in composition and involved distinct treatment processes at the quarries. The two materials were studied and compared in terms of particle distribution, and both mineralogical and physical characteristics. Their hydraulic and hydrodynamic properties were assessed using two post-packing filtration reactors (Liénard et al., 2001). These characteristics are listed in Table 1.

Feed water: Septic effluent characteristics

The feed water was collected from the septic effluent settling tank, with all main characteristics being monitored throughout the operating period. The average and extreme values of each characteristic are reported in Table 2.

Total biomass and EPS extraction

Three samplings and extractions were carried out on Days 60, 210 and 360 for the top layer of the medium. At the end of the operating period (i.e. 360 days), extractions were also performed on the various layers in the two filtration reactors (5 cm, 10-15 cm and 30 cm).

The total biomass was extracted as quickly as possible (< 24 h) both by heating to 80°C during 30 min for the colorimetric analysis (proteins, HS-like, polysaccharides and nucleic acids) and by sonication at 4°C during 60 min for the Size Exclusion Chromatography (SEC) fingerprint analysis (Zhang et al., 2009;Bhatia et al., 2013). The EPS was extracted by sonication at 4°C over 5 min for both the colorimetric analysis and SEC fingerprint analysis.

The nucleic acids were assessed with a series of ultrasound extractions of increasing duration: after 5 min, the nucleic acid contents exhibited a significant increase within the filter media samples.

Total biomass, feed water and EPS characterization

The biochemical components of biomass, EPS and feed water were quantified by employing colorimetric methods: proteins and humic-like (HS-like) substances using the modified Lowry method in introducing Folin's reagent (Frølund et al., 1996); polysaccharides using Dubois' method; and nucleic acids using Burton's method (Burton, 1956;Dubois et al., 1956).Nucleic acids are present for the purpose of EPS extraction control (Liu et al., 2003).

Considering the relatively weak content of nucleic acids in the extracted EPS, it appears that the EPS extracted during this study was not contaminated by significant amounts of intracellular materials (Comte et al., 2006).

The apparent molecular weight (aMW) distributions of biomass proteins and humic substances were analyzed by means of High Pressure Size Exclusion Chromatography (HPSEC) (Merck Hitachi LA Chrom Chromatograph), coupled with fluorescence detection.

The high molecular weight separation was performed with the Agilent column (BioSec, 300A, 5-1,250 kDa), while the low MW separation made use of the BioSec 100A Agilent column (0.1-100 kDa). All columns were placed in series for the separation improvement step (Bourven et al., 2014). The mobile phase was composed of a 150-mM NaCl and 50-mM phosphate buffer at pH 7.0. The MW were calibrated using six proteins or amino acids, with MW values of: 440000, 155000, 69323, 5777, 670, 362 The feed water (effluent) was responsible for importing a variety of organic matter, some of which was caught by the filtration medium. This organic matter will subsequently be used as substrate; however, its presence is inevitable and influences biofilm component characterization. The EPS were extracted and the fractions of each major biochemical component were compared to those of the feed water (Fig. 2). For this comparison with feed water, EPS extracted from RS has been used.

Quantitative results indicate that biomass and EPS display a distribution of biochemical components different from that of feed water: protein proportions in the biomass or EPS show a rise (20% in feed water vs. approx. 40% and 50% respectively in EPS and total biomass).

The EPS from river sand is composed of a similar ratio between proteins and HS-like compounds at both 210 days (7 mg proteins vs. 6 mg HS-like/kg material) and 360 days (30 mg protein vs. 38 mg HS-like/kg material). Both the nucleic acids and polysaccharides are less significant components in the EPS than in feed water (Fig. 2a). The proportions of biochemical components (Fig. 2b) also exhibit distinct distributions between EPS and total biomass, with higher proteins and lower HS-like proportions for biomass extraction, since the total biomass includes intracellular as well as extracellular proteins. In EPS on the other hand, the proportion of HS-like exceeds proteins at 360 days. This increase in EPS proteins may be due to bacterial production or protein-like molecules; also, it might stem from the environment or from cell lysis (Wingender et al., 1999;Flemming and Leis, 2001). In Figure 3a, the HPSEC protein-like fingerprints present similar MW distributions between the EPS and biomass of the river sand sample: the aMW of protein-like substances (PN-like) spikes from < 6 kDa to > 1000 kDa, although this distribution differs in comparison to feed water. Several fractions can be described, namely: The percentages of the fingerprint fractional area have been calculated and displayed in Figure 4. These characterizations reveal a similar distribution between biomass and EPS: a higher VHMW fraction with aMW > 1000 kDa, while the feed water contains mainly very low MW PN-like eluted after the permeation volume (VLMW fraction). The biomass and EPS exhibit similar proportions of VHMW fraction, which could result from the polymerization of PNlike compounds due to their association with other types of molecules within the extracellular biofilm matrix (Bourven et al., 2014). The biomass PN-like fingerprint reveals higher HMW and LMW fractions when compared with those of EPS: these fractions may be PN-like molecules located in cells (intracellular protein-like).

In contrast, compared to proteins, the HS-like are primarily molecules with a weak MW (Ve > 18 mL: aMW < 6 kDa) (Fig. 3b). Bhatia et al. found that the HS-like in EPS of an aerobic granule was less than 6 kDa in the MW fraction (Bhatia et al., 2013). The HS-like also present similar fingerprints between EPS and biomass extracted from river sand. Since humic compounds are not produced by biofilm cells but rather captured from the environment, the presence of HS-like compounds should be extracellular. The similarity between EPS and biomass HS-like fingerprints, along with the differences in PN-like fingerprints of EPS and biomass, has confirmed that the biomass extraction includes both intracellular and extracellular components. The shape and peak maxima of the HS-like fingerprints of EPS and biomass on Day 210 however differ from those of feed water (feed water: Ve maximum peak = 20 mL; EPS and biomass: Ve maximum peak = 21 mL). The hypothesis may thus be forwarded that the river sand biofilm selectively adsorbed humic-like compounds F(VLMW) (18-22mL) from the feed water at a different developmental state, and/or the HS-like modification (metabolism) took place in the biofilm due to degradation (catabolism) (Volks et al., 1999;[START_REF] Vanloon | Environmental Chemistry: a Global Perspective[END_REF].

The above findings show that the biochemical components of total biomass and EPS differ from those of feed water, with a higher PN content, in particular very high MW PN-like compound (> 1000 kDa) percentages. Furthermore, the total biomass contains two slightly higher MW fraction percentages (HMW fraction: 1000 kDa ~ 109 kDa and LMW fraction:

109 kDa ~ 25 kDa) than the corresponding EPS percentages. These fractions could correspond to intracellular proteins.

The evolution in EPS has resulted from the filtration reactors at two scales, i.e.: evolution during process implementation, and evolution as a function of the vertical profile (depth) of the reactors. Two materials from validated biofilter treatment processes (river sand (RS) and crushed aggregate (CA)) can yield observations of biofilm evolution, due to the fact that EPS is essential to biofilm survival.

Evolution of the biochemical characteristics of EPS vs. operating time

1. Biochemical composition:

The evolution of EPS percentages in the biomass extracted from the top layer of each filtration reactor on Days 60, 210 and 360 (as calculated from the data available in Fig. 5) is presented in Table 4. During the implementation process, total biomass increased in the two materials, by factors of 3 and 2 for RS and CA respectively. This biomass increase corresponds to the protein content evolution (95% and 71% factors for RS and CA, respectively), which serves as the biomass activity indicator (Di Iaconi et al., 2006) (Fig. 4).

Biomass implementation seems to reach stabilization for RS with a growth factor of 1.3 and 1.8 respectively for biomass and especially for protein. This same trend is observed in the two materials with an increase of EPS percentage in the biomass, from approx. 16% to 30% in RS and from 21% to 32% in CA. It is still open to debate whether EPS production reflects higher or lower microbial activity, though it is usually reported that during the exponential growth phase, EPS content increases with time yet decreases once the stationary phase has been reached (Jia et al.,1996). Both EPS percentages exhibit a slight decrease between Days 60 and 210 (Table 4).This decrease is simply due to the drop in HS-like substances (Fig. 4) from 7.1 to 5.9mg/kg material in RS and from 15.1 to 7.1mg/kg material in CA. As explained in the previous section, humic substances, which are generated from effluent, do not get synthesized but might be metabolized by biofilm cells around Day 210 (Volks et al., 1999).

The EPS percentage in the biomass however remains somewhat higher for CA, especially at the beginning of the implementation step; at this same time, a higher biomass increase for RS than for CA is revealed. The biomass content evolution could be due to a higher specific surface area for RS than for CA (Table 1) or to a higher speed of development. As observed from the material characteristics (Table 1), the CA reactor displays an extended hydraulic residence time and water retention capacity, both of which may due to the fine particle content. The static liquid phase may lead to microenvironments with a weak renewal of nutrients and/or accumulated residues. The hydraulic and hydrodynamic variations could result in low biomass activity (i.e. total protein content in CA) but a high proportion of EPS in the reduced biomass (as is the case with CA). This low substrate content could favor EPS synthesis (Gao et al., 2008;Nichols et al., 2004). Starvation would be the preferred hypothesis over hydrodynamic influence for the increase in EPS content from aerobic granules growing on a zeolite material biofilter (Di Iaconi et al., 2006). Moreover, changes in environmental conditions could induce a shift in microbial community and, subsequently, more EPS-producing content (Gao et al., 2008;Liu et al., 2004). Throughout all days of the process, total -N removal (%) is greater (by a factor of 1.5) for RS than for CA, which means that denitrifying bacteria are less active or less present in the CA biofilter. The biochemical contents of EPS and biomass extracted from the top layer of the two reactors on Days 60, 210 and 360 are presented in Figures 5 and6:

composed of proteins and humic compounds (Fig. 6), which are found in greater proportions in active sludge (Frølund et al., 1996). [START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF] observed that the stability of aerobic granules during biofilm granulation is mediated by PN, while for Ahimou et al.

(2007) cohesiveness is correlated with PS.

When comparing the evolution of proportions in biochemical components (Fig. 5), this same trend is identified with increasing PN percentages (RS: 10~33%; CA: 24~31%) and decreasing HS-like (RS: 47~43%; CA: 50~33%). [START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF] described such enrichment in PN content of EPS during aerobic granule formation and proposed that an increase in PN might enhance neighboring microbial cells and form a cross-linked network by attracting organic and inorganic material (Liu et al., 2004). The percentage of PS however becomes inverted, with an increase (18~33%) and decrease (30~18%) for CA and RS, respectively. Hence, EPS in biofilm does not necessarily develop in the same way on the two filter materials. The difference in EPS biochemical composition may be explained by the differing conditions generated in the materials, i.e. porosity (both external and internal), fine particle contents and reactor hydrodynamics. Moreover, the mineralogical composition (Table 1) differs substantially with crushed aggregate: higher calcium content leads to a greater presence of divalent cations at the material surface, with the possibility that divalent cations bind with extracellular PS-alginate-like via an ionic link, thus resulting in a complex "eggbox" configuration (Sobeck and Higgins, 2002;Lin et al., 2010). Stabilized PS are less influenced by effluent and/or less metabolized.

In light of the above discussion, similar trends can be observed for PN-and HS-like, as recorded in both materials. A follow-up qualitative comparison of PN-like and HS-like substances was conducted by examining the MW distribution.

Protein and HS-like HPSEC fingerprints:

In examining the major biofilm components, a further qualitative study on the MW distribution by HPSEC fingerprints of protein-like substances between filter materials will be described and the chromatogram displayed in supplementary data. Moreover, Figure 7will present the fractional area percentages.

As observed in the PN-like fingerprints (S1, supplementary data), their peak number and elution volume differ with incubation time. This finding indicates that diverse molecular Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 169 structures have occurred as a result of biomass enrichment. Similarly, in Figure 7, the increase in the VHMW fraction and decrease in the VLMW fraction of PN-like fingerprints were noted in both filter materials after 60 days of operations. This MW distribution shift may be due to the production of new PN-like and their polymerization with other organic molecules during the enrichment step. Since extracellular proteins contain a considerable amount of enzyme, it is assumed that the extracellular enzymatic activity might change with the increased biofilm implementation activity. This increase in the VHMW fraction may be correlated with the aggregation of bacteria in biofilm during biomass enrichment.

The RS showed a relatively stable evolution in fractional area percentages from Day 60 to Day 210. A decrease in the VHMW fractional proportion coupled with an increase in the LMW fractional proportion could be noticed on Day 360. The decrease in the VHMW fractional proportions may be explained by a degradation in existing compounds at the mature biofilm state (Nielson et al., 1996). For CA on the other hand, the VHMW fraction is increasing during all 360 days of biomass enrichment, thus suggesting a late increase in molecular polymerization. This late evolution in the MW shift distribution of PN-like molecules in CA may be caused by somewhat delayed biofilm development in such material; this pattern was also observed in the biochemical component evolution for total biomass (Fig. 4). Let's notice that between Days 210 and 360, in both materials, the HMW fractional distributions became smaller, which may be due to the degradation of PN-like for cell catabolism.

Evolution of EPS biochemical composition with bed thickness

To observe the impact of packing materials on biofilm evolution in the deeper part of the filtration reactors, i.e. where the environment differs from that of the top layer (less rich in substrates), a follow-up study of the vertical EPS distribution in filtration beds with different filter materials was conducted after 360 days of operations using the same analytical tools.

Biochemical composition:

The evolution of biomass and EPS percentages in the biomass extracted from layers 0-2 cm, 5 cm, 10-15 cm and 30 cm in each biofilter on Day 360 (as calculated from the data available in Fig. 9) is shown in lower biomass at 30 cm compared to CA were observed. This same trend had been identified

for both types of material packing: increasing EPS percentage in biomass, coupled with a declining biomass over the depth. The EPS percentage remained constant in the same layer and on the same order of magnitude for both materials: about 30% for the top, and increasing with depth (up to 38%). The biomass also decreased with depth due to fewer cells when less of the substrate had penetrated into the depth. Like for the previous study (EPS vs. time of evolution process), total biomass components appear in higher contents from the top to the 10-15 cm layer in RS than in CA. EPS components show a similar scale, except with slightly more polysaccharides in CA. The increase in EPS percentage above this level, compared to its total biomass over the filter medium depth, could be due to: i) the downward shift through the depth of the weakly bound soluble EPS; ii) less substrate and/or bacteria community shift (more nitrifying bacteria, which are autotrophic); and iii) a new C/N ratio over the deeper part of the beds. Gao et al. (2008) also described how nitrifiers (which appear due to the new C/N ratio) tend to produce more EPS than heterotrophs within the upper media of an aerobic biofilter.

Moreover, a low C/N ratio (as is expected with depth in our study) was reported by Durmaz et al. (2001) to induce a low EPS content when compared with a ratio of 40. The fine particles in CA should be more noticeable over the depth than in the top layer, which may also lead to a higher EPS percentage by structurally influencing the biofilm [START_REF] Vieira | Effect of clay particles on the behavior of biofilms formed by Pseudomonas fluorescence[END_REF].

The proportions of biochemical components have changed in the deeper part of the filtration beds (Fig. 10, 30 cm). CA reveals lower quantities in proteins than in polysaccharides of EPS (in both the absolute and relative fractions), with the PN/PS ratio decreasing from 1 in the top layer to 0.1 at 30 cm. For the RS material, PN/PS ratios were typically higher, with a value of 2 in the top layer, around 1 from 5 to 15 cm and 0.5 at 30 cm.

Vertical evolution of proteins and HS-like fingerprints:

The MW evolution of PN-like compounds in the 10-15 cm and 30 cm depths was also studied by means of HPSEC fingerprints. These results, presented in S2 (supplementary data),

show similar fractions of PN-like EPS to those in the top layer; moreover, the four major fractions were identified as in Figure 3. The corresponding fractional percentage calculations are shown in Fig. 11. These low aMW compounds (<6kDa) may be amino acids-like or small peptides-like or molecules of similar configurations conveyed by the feed water, then transported and sorbed onto the deep medium. The LMW and VLMW fractions may also be associated with low MW PN-like molecules, resulting from the degradation of substrate by means of microorganism metabolism [START_REF] Ni | Soluble microbial products and their implications in mixed culture biotechnology[END_REF].

The HS-like fingerprint evolution over the depth of two materials has also been compared and summarized in Figure 12. These fingerprint chromatograms reveal similar shapes to the two filter materials and feed water, which suggests that HS-like compounds stem from the retention of exogenic organic matter. The HS-like fingerprints also display overlapping peaks with the 10-15 cm and 30 cm samples of both materials and moreover indicate that HS-like may be captured from the feed water and distributed relatively homogeneously inside the RS CA filter medium, despite the fact that the types of filter materials and these exogenic HS-like compounds possess similar and low MW (< 6 kDa).

Figure 12: Evolution in HS-like fingerprints of extracted EPS and biomass over time in both river sand (RS) and crushed aggregate (CA)

Conclusion

This paper has sought to provide in-depth information regarding the EPS extracted from a biofilter at various enrichment stages during a 360-dayperiod and for different bed thicknesses. Two types of packing materials, river sand and crushed aggregate, were used and a series of quantitative and qualitative analysis conducted to investigate the differences in EPS characteristics. The following conclusions could be drawn:

Overall, the percentages of EPS in biomass increase with biomass enrichment despite a decrease due to humic-like substances as of Day 210. During biomass enrichment, the humic- An increase in the very high MW fraction may be correlated with bacterial aggregation.

Within the bed depths, the very high MW fraction decreases, thus suggesting less aggregation.

This fraction decreases after Day 210, but only for the RS material, which is perhaps connected to a more mature biofilm.

The SEC fingerprint applied with detection for HS-like also provided valuable

Conclusion:

Les études montrent clairement que la matière organique du biofiltre n'est pas une accumulation de la matière organique de l'effluent mais bien une production par le biofilm (pour la biomasse totale et l'EPS). La teneur en protéines est ainsi beaucoup plus faible pour l'effluent ainsi que la fraction de protéine-like de forte masse moléculaire (MM).

Au cours de la mise en place du procédé, la biomasse totale et les EPS augmentent et en particulier les teneurs en protéines de la biomasse.

Les résultats montrent une augmentation plus forte de la biomasse pour RS1 mais aussi une hétérogénéité dans la taille, avec un contenu de particules fines plus élevée et une surface spécifique plus faible (4.04 m 2 /kg pour RS1 vs 2.78 m 2 /kg pour CA1 et 2.88 m 2 /kg pour CA2). Le sable RS1 est par sa taille le plus proche de la norme NFDTU64.1 et est considéré comme référence pour l'étude. Au contraire, RS2 peut être considéré comme un sable grossier et doit permettre de différencier l'effet de taille. CA2 ne présentait pas de caractéristiques extrêmes comme CA1, mais une minéralogie très hétérogène a été observée. Les principales caractéristiques de ces matériaux sont résumées dans le Tableau 3.1. Les rendements d'élimination en phosphate sont relativement instables dans les systèmes de filtration, cependant les granulats concassés montrent une meilleure élimination des phosphates. De plus, les rendements diminuent à moins de 60% après 120 jours de fonctionnement. La minéralogie du support qui a été estimée par lixiviation du support, pourrait expliquer cette élimination des phosphates par précipitation ainsi que le comportement des filtres dans le temps avec une diminution de la concentration en cations (Zanini et al., 1998).

Les résultats précédents montrent que des éléments majeurs peuvent impacter les rendements épuratoires sur des filtres biologiques: la taille des grains (moyenne et distribution) est un paramètre essentiel dans le choix des matériaux de garnissage; la présence de particules fines dans les granulats concassés est un facteur défavorable au bon fonctionnement du filtre.

Si les premiers bilans effectués sur les rendements épuratoires ont montré un comportement différent selon les matériaux, une meilleure compréhension de l'impact de la nature des bactéries [START_REF] Rose | A quantitative study of calcium binding and aggregation in selected oral bacteria[END_REF], ou spécifiquement avec les EPS (Higgins & Novak, 1997). Les cations divalents relargués (Ca 2+ ) par les agrégats concassés sont peut être à l'origine de la variation de la composition biochimique des EPS. Le Ca 2+ permet ainsi de former des complexes appelés « boîte à oeuf » qui sont composés de polysaccharides et de Ca 2+ (Sobeck & Higgins 2002).

Comparaison des EPS en fonction de la profondeur du réacteur en fonction du matériau :

La concentration en substrat de l'effluent diminue avec la profondeur puisque les substrats commencent à être dégradés dès la couche superficielle du biofiltre. La distribution de la biomasse totale correspond au profil décroissant en disponibilité de substrats pour les deux

Part ΙΙΙ: Conclusion générale matériaux. La masse d'EPS diminue aussi mais une proportion plus importante d'EPS est observée dans les couches profonde (RS1: 30% au sommet vs. 39% à 30cm et CA1: 32% vs. 37%). Le stress généré par la diminution de substrat disponible en profondeur peut expliquer l'augmentation de la proportion en EPS, de même que le changement de la flore microbienne.

En Effet, en profondeur, les bactéries nitrifiantes autotrophes sont plus favorisées que les autotrophes des couches superficielles, la nature et la quantité des EPS produits par une autre communauté microbienne peuvent alors varier.

III.6. Conclusion technique

L'étude de la matières organique du biofilm et en particulier celle de l'EPS a permis de Résumé français :

L'assainissement non collectif concerne 12 à 15 millions de personnes en France. La filière classique de ce mode d'assainissement se compose généralement d'un prétraitement anaérobie par une fosse septique recevant l'ensemble des eaux usées domestiques suivi d'un système d'infiltration dans le sol ou d'un filtre à sable. Le filtre à sable vertical drainé met à profit le pourvoir épuratoire qui est principalement lié à la présence d'une biomasse sous forme d'un biofilm. Cette dynamique de la croissance de la biomasse ou du biofilm est soumise à l'impact de la nature de matériaux filtrants. L'écoulement insaturé dans ces systèmes conditionne également cette croissance du biofilm.

Dans ce contexte, l'objectif du travail de la thèse est d'appréhender les mécanismes mis en jeu et particulièrement l'impact des matériaux dans le fonctionnement des filtres en comparant notamment deux types de matériaux: les sables de rivière et les agrégats concassés. Pour cela, une étude expérimental sur une unité pilote composé des réacteurs de filtration du diamètre de 30cm et différents épaisseurs de garnissage (15, 30 et 70cm) a été construite. Les réacteurs garnis de deux sables roulés et deux agrégats concassés, sont alimentés en effluent septique avec une charge volumique 12cm/jour par 10 bâchés par jour. Suite des matériaux, une étude de la performance épuratoire avec le suivi des composants biochimiques de la biomasse totale et de la matrice extracellulaire du biofilm est réalisée en comparant notamment les deux types de matériaux filtrants.

L'étude des matériaux et des propriétés des réacteurs filtrants ont montré que les agrégats concassés présentaient une hétérogénéité des tailles et des formes des grains, ainsi qu'une teneur élevée en particules fines (<0.08mm). L'hydrodynamique dans le réacteur garnis avec l'agrégat concassé possédant la teneur en particules fines le plus élevée est caractérisé par un temps de séjour moyen plus important. A la différence du sable roulé qui forme un environnement très uniforme, les agrégats concassés avec la présence des particules fines et des grains très anguleux peuvent créer des microenvironnements avec de plus forte turbulences, ou inversement des endroits peu accessibles à l'air ou au liquide.

La capacité épuratoire des matériaux filtrants est gouvernée principalement par la taille des grains, surtout pour la pollution particulaires et organiques, ainsi que pour l'élimination des ions ammoniacs. Un meilleur rendement de l'élimination de l'azote total est observé dans le réacteur garnis de 70cm du sable roulé fin, sous la charge hydraulique 12cm/jour. Une alternance de la phase aérobie/anoxique apportée par les bâchés dans ce fin médium favorise la dénitrification. L'effet des microenvironnements provoqué par l'agrégat concassé dans un massif filtrant diminue également la dénitrification. Une augmentation de la charge hydraulique réduit cette condition par une diminution en temps de séjours.

Les évolutions de la biomasse totale et de la matrice extracellulaire se différentient également entre le sable roulé et les agrégats concassés lors du suivie des composants biochimiques (protéines, humic-like substances, polysaccharides and acides nucléiques) et les empreintes HPSEC des protéines-like. Le sable roulé fin a présenté une stabilisation de la croissance en biomasse totale le plus tôt avec une production des composants extracellulaires par unité de la biomasse moins forte que les agrégats concassés. La teneur en particules fines dans les agrégats concassés peut-être à l'origine de microenvironnements pauvres en substrat ou en oxygène, ou des différentes propriétés de surface de grains impactées par la composition minéralogique modifiant la production des composants extracellulaires, surtout les polysaccharides.

L'impact des différents matériaux filtrants est principalement lié à la taille de grains qui gouverne la rétention mécanique des polluants et en même temps gère l'établissement de la biomasse. L'effet des particules fines dans l'agrégat concassé en réduisant le temps de séjour et en créant des microenvironnements hétérogènes, peut provoquer le colmatage du filtre sur le long terme. De plus, les agrégats concassés ont montré des formes des grains anguleuses et irrégulières, avec pour conséquence un comportement hydrodynamique moins homogène et ramener une distribution en nutriment et en air non uniforme dans la massive. Ainsi, l'évolution de la biomasse et surtout la composition en exsudats extracellulaires sont particulièrement différenciée entre un sable roulé et un agrégat concassé. La composition minéralogique d'agrégats impacte l'élimination des phosphates et modifie également l'excrétion des composants extracellulaires: la proportion des polysaccharides dans les EPS est plus élevés dans l'agrégat concassé qui montre une composition plus riche en calcium.

Cette étude, en comparant deux types de matériaux de nature très différente, contribue par deux aspects à une meilleure compréhension du fonctionnement des réacteurs de filtration: le pouvoir épuratoire et la composition biochimique des composants de la biomasse totale et de la matrice extracellulaire. De cette étude, des éléments techniques peuvent être retirés pour l'utilisation alternative de matériaux non traditionnels. De bonnes conditions de fonctionnement peuvent être attendues avec une granulométrie similaire à celle des sables roulé d'une taille effective inférieure à 0.4mm et un coefficient d'uniformité le plus faible possible.

Des études plus spécifiques sur une période de temps plus longue permettraient d'approfondir les connaissances sur certains aspects : par exemple, la distribution des communautés bactériennes dans le temps et dans la profondeur des réacteurs, la répartition des phases et l'hydrodynamique du liquide et de l'air à l'intérieure du massif filtrant de grains anguleux avec l'utilisation de traceurs. L'intérêt d'une étude sur une échelle de temps très longue est la compréhension des dysfonctionnements d'un filtre réel, par exemple, le colmatage.

2. 1 . 4 .

 14 Cells extraction and assessment ........................................................................ 100

Chapter 3 :

 3 Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances ....................................................... 125 3.1. Experimental procedures of characterization of biomass, EPS and feeding water .. 126 3.1.1. Sampling and total organic matters ................................................................... 126 3.1.2. Extractions of biomass and EPS constituents ................................................... 127 3.1.3. Biochemical compositions of Biomass and EPS ............................................... 128 3.1.4. SEC coupled with fluorescence detection fingerprints ..................................... 130 3.2. (Article 2): Evolution of biochemical compositions of biomass developed in different aggregates for the use of filter materials of onsite wastewater treatment systems ............ 132 3.3. (Article 3): Production of the evolution of extracellular polymeric substances (EPS) from the biofilm in onsite wastewater filtration reactors ................................................... 155 Part ΙΙΙ: Conclusion générale ................................................................ 184 III.1. Différentiation des matériaux ............................................................................... 185 III.2. Impact des matériaux sur le fonctionnement du réacteur: hydrodynamique et transfert de l'O 2 .................................................................................................................. 186 III.3. Impact des matériaux sur l'épuration des eaux ..................................................... 187 III.4. Biomasse et matériaux du massif filtrant .............................................................. 189 III.5. Evolution des EPS en fonction des matériaux de garnissage ............................... 190 III.6. Conclusion technique ............................................................................................ 192 III.7. Perspectives .......................................................................................................... 193 References ................................................................................................ 195

Figure 1 .

 1 Figure 1.1: Global view of OWTS system based on sand filtration fields ...............................

1 :

 1 Figure 1.1: Global view of OWTS system based on sand filtration fields ...............................

Figure 1 . 2 :

 12 Figure 1.2 : Overview of current knowledge on sand filter functioning ..................................

Figure 1 . 3 :

 13 Figure 1.3 : Simplified schema of clogging development........................................................

Figure 1 . 4 :

 14 Figure 1.4 : 7 months growth of biofilm on quartz sand grains ...............................................

Figure 1 . 5 :

 15 Figure 1.5 : A simplified description of the development of the biofilm onto a mineral surface ..................................................................................................................................................

Figure 1 . 6 :

 16 Figure 1.6 : Main interactions involved in the reversible adhesion .........................................

Figure 1 . 7 :

 17 Figure 1.7 : Governing interactions of bacterial cell adhesion.................................................

Figure 1 . 8 :

 18 Figure 1.8 : Spatial organizations of EPS ................................................................................

Figure 2 . 1 :

 21 Figure 2.1: Image processing of form parameters analysis for filter materials ........................

Figure 2 . 2 :

 22 Figure 2.2: Simplified overall view of the experimental pilot .................................................

Figure 2 . 3 :

 23 Figure 2.3 : Sampling ports positions of 70cm columns ..........................................................

Figure 2 . 4 :Figure 2 . 5 :

 2425 Figure 2.4: Experimental device for hydraulic conductivity determination by Grant method Figure 2.5: Schematic figure of water retention test ................................................................

Figure 2 . 6 :

 26 Figure 2.6: Size distribution curves of river sands (RS1 and RS2) ..........................................

Figure 2 . 7 :

 27 Figure 2.7: Size distribution curves of crushed aggregates (CA1 and CA2) ...........................

Figure 2 . 8 :

 28 Figure 2.8: profil de répartition de l'eau retenue .....................................................................

Figure 2 . 9 :

 29 Figure 2.9: Location of EEM peaks for five EEM regions ....................................................

Figure 3 . 1 :

 31 Figure 3.1:vue d'ensemble des pilotes de filtration ...............................................................

Figure 3 . 2 :

 32 Figure 3.2: Proposition d'un schéma conceptuel reprenant les deux structures de grains (sable de rivière et agrégats concassés) et la distribution de l'air et de l'eau dans le lit. .................

Figure 3 . 3 :

 33 Figure 3.3: Concept de microenvironnements au sein des filtres garnis de sable ou d'agrégats concassés ................................................................................................................................

  ............................................................................ Table 2.1.1: Quarries and natures of materials .........................................................................Table 2.1.2: Granulometric parameter definitions ................................................................... Table 2.1.3: Experimental conditions for released cations of selected sands .......................... Table 2.1.4: Granulometric characteristics of filter materials .................................................. Table 2.1.5: Photos of material grains of different size taken by binocular or microscope ..... Table 2.1.6: Physical and form characteristics of filter material (Average value of 5 tests for physical characterization) ......................................................................................................... Table 2.1.7: Mineral compositions and calcareous (limestone: CaCO 3 ) contents of filter materials ................................................................................................................................... Table 2.1.8: Released cations in water from the filter materials .............................................. Table 2.1.9: Infiltration time and estimated hydraulic conductivity at saturation and water retention capacities of filter materials ......................................................................................Table 2.1.10: Average HRT in 30cm and 70cm reactors ......................................................... Table 2.2.1: Hydraulic loading, packing materials and feed-rest frequencies ......................... Table 2.2.2: Analytic methods of physico-chemical parameters of feeding water and treated effluents ....................................................................................................................................

Table 3 . 1 :

 31 Principales caractéristiques chimiques et physiques des matériaux de filtration choisis ..................................................................................................................................... l'assainissement autonome (OWTS) représente une voie d'assainissement lorsque les habitations ne sont pas en mesure d'être connectées au réseau collectif de traitement des eaux usées. Ce type d'assainissement représente environ 15 millions de personnes et 100 000 à 150 000 installations sont construites chaque année avec un nombre total qui ne cesse d'augmenter. Au-delà de la législation française sur la qualité de l'eau et des milieux aquatiques N ° 2006-1772 en vigueur depuis le 30 décembre 2006, les objectifs de traitement des eaux usées au niveau européen ont été fixées par la direction du Conseil Européen (directive N ° 91/271 / CEE). Cette loi fixe des exigences strictes sur la qualité des rejets d'eaux usées dans l'environnement ; les petites collectivités de plus de 2000 équivalents-habitants (PE) sont obligées d'organiser la collecte des eaux usées et de mettre en place un système de traitement. Dans les districts ruraux de moins de 2000PE, les collectivités sont également tenues d'organiser et de contrôler les systèmes autonomes de traitement des eaux usées avec la mise en place de SPANC (Service Publique d'Assainissement Non Collectif). La configuration commune à tous les systèmes d'assainissement autonomes est la suivante : i) un système de collecte des eaux usées ii) une fosse toutes eaux pour le traitement primaire des eaux usées iii) un traitement secondaire par infiltration dans le sol ou par injection discontinue dans des systèmes de filtre à sable. Depuis mars 2007, les implémentations de ces dispositifs et d'autres installations de traitement sont décrites par XP DTU 64.1. Le choix et la conception des dispositifs autonomes sont souvent basés sur un savoir-faire et des règles empiriques. La connaissance scientifique et le développement technologique sont nécessaires pour améliorer la fiabilité et la durabilité de ces systèmes.

  différents matériaux et pour différentes conditions opératoires. Cette partie est présentée sous forme d'un article : • Evolution of purification efficiencies of different filter materials and impact of filtration packing height and hydraulic loading (Soumis le 6 Novembre 2015 à Journal of the Taiwan Institute of Chemical Engineers: JTICE-D-15-0153) -La dernière partie est consacrée à l'observation et à la discussion des effets des matériaux sur le développement du biofilm avec une approche sur la biomasse totale et sur les composants majeurs de la matrice extracellulaire en fonction du temps de fonctionnement. La composition du biofilm dans la profondeur des réacteurs est également abordée. Cette partie est présentée à travers 2 articles : • Evolution of biochemical compositions of biomass developed in different aggregates for the use of filter materials of onsite wastewater treatment systems, (Soumis le 25 Avril 2015 à Biochemical Engineering Journal: BEJ-D-15-00418) • Production of the evolution of extracellular polymeric substances (EPS) from the biofilm in onsite wastewater filtration reactors (Sera soumis à Bioresource Technology) Une conclusion générale développée et rédigée en français reprend les principaux résultats et propose une discussion autour des observations et des hypothèses importantes liées aux études expérimentales. Les perspectives des futures études sur le réacteur filtrant sont

Figure 1 . 1 :I. 2 . 2 )

 1122 Figure 1.1: Global view of OWTS system based on sand filtration fields

(

  The migration of solutes by molecular diffusion is caused by Brownian motion due to their thermal energy. The mechanical dispersion is resulted from the microscopic fluctuation of advection velocity.The one dimensional equation of non-absorbing, non-degradable solute transport in a homogeneous unsaturated porous media generally accepted is Convection-With θ is water content; q is Darcy velocity [LT -3 ] and D is the dispersion coefficient of Fick's Law[L 2 T -1 ] which takes account both mechanical dispersion and molecular diffusion.

  of non-Brownian particles and axial diffusion are overlooked comparing to the axial movement (flow direction), the Convection dispersion equation can be expressed:

Pe

  is defined as the Péclet number with H: the length of media, u: the flow velocity of mobile phase and D: the dispersive coefficient of mobile phase.

Figure 1 . 2 :

 12 Figure 1.2 : Overview of current knowledge on sand filter functioning

Figure 1 . 3 :

 13 Figure 1.3 : Simplified schema of clogging development[START_REF] Maillard | Les filtres à sable verticaux en assainissement autonome regroupé: rôle des conditions opératoires, des écoulements et des transferts gazeux sur les performances épuratoires et le vieillissement Université de Renne[END_REF]

Figure 1 . 4 :

 14 Figure 1.4 : 7 months growth of biofilm on quartz sand grains[START_REF] Jean | Comparative endoscopic and SEM analyses and imaging for biofilm growth on porous quartz sand[END_REF]

Part Ι :

 : Literature review metabolism, shear forces, and environmental stress. The shear forces of the biofilm detachment are erosion (losing small parts of biofilm caused by liquid flow), abrasion (caused by collision between the biofilm and small solid particles) and sloughing (losing large part of biofilm caused by brutal changes of environment).

Figure 1 . 5 :

 15 Figure 1.5 : A simplified description of the development of the biofilm onto a mineral surface Resource: [Center for Biofilm Engineering Montana State University, 2003]

Figure 1 . 6 :

 16 Figure 1.6 : Main interactions involved in the reversible adhesion

Figure 1 . 8 :

 18 Figure 1.8 : Spatial organizations of EPS(Wingender et al., 1999)

  Biodiversity: microbial genomic level characterization of the cells in biofilm (molecular techniques): PCR based molecular techniques: DGGE, PFGE, RISA and Pyrosequencing TM … Non-PCR based molecular techniques: FISH, GFP… Totality, surface and interface of biofilm characterizing techniques: Total amount (VDW, C or N contents of biofilm) Visualization of biofilm by microscopic techniques (SEM, CLSM, STXM…) Scattering (spectroscopy) techniques (ATR-IR, NMR, X-ray…) Nondestructive techniques: Microsensors (electrochemical, fiberoptic microsensors) EPS matrix: biochemical level characterization of the extracellular fractions in biofilm (separation techniques+ analytical techniques): EPS extraction (physical, chemical or combined methods) Molecular separation (GC, LC, SEC, FFF…) Quantitation of biochemical fractions (spectrophotometry) Analytical detection (MS, atomic spectrometry, electrochemical, optical detectors) Microbial activities: physiological level characterization of the cells in biofilm (specific compounds detections): Enzymatic activities (ATP, hydrolysis, dehydrogenase…) Cells extraction -culture -count… I.6.1. Biofilm observation (microscopic techniques)

  Attempts have been made to identify extracellular protein of activated sludge:[START_REF] Park | Evaluation of the extracellular proteins in full-scale activated sludges[END_REF] submitted 11 intense bands of polypeptides resulted from Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to liquid chromatography coupled with mass spectroscopy (LC-MS/MS) and the positive hits indicated that the diverse origins of protein in EPS matrix, besides the presence of the extracellular proteins resulted from bacterial defense, cell surface outer membrane and cell appendage, proteases from influent remained in the EPS matrix.[START_REF] Flemming | Relevance of microbial extracellular polymeric substances (EPSs)--Part I: Structural and ecological aspects[END_REF]

  fluorescence spectroscopy has been employed to characterize the fluorophores contents in natural organic matters. The protein-like and humic-like compounds are two great categories of the fluorophores in EPS matrix. Several studies have applied the 3D fluorescence excitation-emission matrix (3D-EEM) to characterize the presence of proteins and humic substances in EPS extract of activated sludge: the spectra obtained are generally composed of 3 main peaks: 2 peaks of emitted fluorescence of protein-likes compounds and 1peak of humic-like substances[START_REF] Esparza-Soto | Fluorescence spectroscopy and molecular weight distribution ofextracellular polymers from full-scale activated sludge biomass[END_REF][START_REF] Sheng | Characterization of extracellular polymeric substances of aerobic and anaerobic sludge using three-dimensional excitation and emission matrix fluorescence spectroscopy[END_REF]

Chapter 1 :

 1 le développement de la biomasse et une partie spécifique de celle-ci : les EPS Dans ce sens, 3 chapitres chacun comportant les résultats et la méthodologie adoptée sont présentés:Le premier chapitre sera consacré à la caractérisation des matériaux de remplissage du réacteur et leurs conséquences directes sur le comportement du réacteur: hydrauliques et hydrodynamique.Le second chapitre sera consacré à l'observation des effets éventuels des matériaux de garnissage sur le fonctionnement épuratoire du filtre. L'étude s'est focalisée sur la mise en place du procédé jusqu'à l'obtention d'un état stationnaire apparent soit sur une période de 360 jours.Enfin dans le dernier chapitre nous nous intéresserons au comportement de la biomasse développée dans le réacteur ainsi qu'aux EPS en fonction des matériaux mis en oeuvre. Cette approche est réalisée en suivant l'évolution de la matière organique du biofilm pendant le procédé.Pour cette étude, le pilote mis en place est constitué d'un ensemble de réacteurs de 70cm de profondeur qui respecte la recommandation propre à l'assainissement individuelle autonome(DTU 64.1).Part ΙΙ-Chapter 1: Study of filter materials and filtration reactor Study of filter materials and filtration reactors Introduction:Quatre matériaux de remplissage seront étudiés : deux sont des sables de rivières (RS1 et RS2) et deux sont des agrégats concassés (CA1 et CA2). RS1 est prévu pour être « le sable type » pour un fonctionnement optimal car répondant à une répartition et à une taille moyenne reconnue pour son efficacité (Cemagref,Liénard et al., 2001).En effet, la répartition des tailles des matériaux de remplissage des biofiltres est un paramètre majeur et classiquement pris en compte dans le choix des matériaux (NF DTU 64.1).Le choix des différents matériaux a donc été guidé par ce paramètre dont les limites pour un procédé de filtration en assainissement autonome sont définies par la norme NF DTU 60.1. A côté de ce paramètre de distribution des tailles, d'autres paramètres de caractérisation physiques (masse volumique apparente et réelle, porosité, surface spécifique) et chimique (composition minérale et comparaison des cations relargués) ont été mis en place.En effet, les caractéristiques physiques influencent l'hydrodynamique des réacteurs alors que les caractéristiques chimiques et morphologiques influenceront directement la mise en place du biofilm (adhésion et formation). L'excès de cations monovalents par exemple induit une désagrégation du biofilm(Higgins & Novak, 1997). L'hydrodynamique et l'hydraulique influenceront d'une part les échanges de substrats dont le plutôt la distribution des apports en substrats (matière organique, eau, O 2 ) au biofilm(Liu et al., 2004), mais également le développement du biofilm par des effets mécaniques plus ou moins prononcés dans le réacteur (zones de cisaillement ou zones d'échange).Les conséquences directes (les efficacités du traitement) sur le réacteur seront évaluées dans ce chapitre à l'aide d'un suivi classique des paramètres physico-chimiques.The samples have been collected from various quarries in France. The alluvial sands originate from the river Loire. The crushed aggregates are originated from sandstones and sandstones are mixture of mineral grains and rock fragments. Among these samples, not all the aggregates fitted the required granulometric zone, even though they were indicated by quarries as for filter usage. 2 river sands and 2 crushed aggregates have been chosen to pack the columns. During the filling, the materials were compressed by water in order to avoid the compression during the operation, and this step also washed the aggregates by eliminate the dusts. The natures and quarries of chosen aggregates are summarized in

  base for material selection in this study. According to the Standard NF EN 933-1(AFNOR, 1997), the principle of the analysis is passing a representative quantity of sample through a series of sieves with decreasing opening by mechanical shaking. The distribution of grains size is represented by the mass retained between two consecutive sieves. The openings of nominal sieves usually used are: 0.08, 0each sample was dried at 105°C in ventilated oven over night, in order to eliminate the humidity. The dried sample was placed in the first sieve (largest opening) and was sieved through the decreasing opening during 15-20min shaking. The distribution curve Part ΙΙ-Chapter 1: Study of filter materials and filtration reactor was established by the percentages of the refused mass of each sieve to the total dried sample quantity. This curve provides the access to several characteristics: effective size (D 10 ), average size, uniformity, and fine particles percentage, summarized in

Figure 2 . 1 :

 21 Figure 2.1: Image processing of form parameters analysis for filter materials

Figure 2 . 2 :

 22 Figure 2.2: Simplified overall view of the experimental pilot

Figure 2

 2 Figure 2.3 : Sampling ports positions of 70cm columns

  .exp is the material layer height in experimental condition (m); V s.exp is the water volume poured in the sand (m 3 );D exp is the diameter of sand layer surface (m); and t exp is the passing time of this volume of water (s).

Figure 2 . 4 :

 24 Figure 2.4: Experimental device for hydraulic conductivity determination by Grant method

Figure 2 . 5 :

 25 Figure 2.5: Schematic figure of water retention test

Part ΙΙ-Chapter 1 :

 1 Study of filter materials and filtration reactor 84 Establishment of residence time distribution (RTD) curves and average residence The establishment of RTD curves is based on the calculation of E (t) = C/C 0 as function of time t. The calculation of the function C/C 0 is shown in the following equation:

Figure 2 . 6 :Figure 2 . 7 :

 2627 Figure 2.6: Size distribution curves of river sands (RS1 and RS2)

1. 4 . 1 . 2 .

 412 photos indicated that the river sands (RS1 and RS2) are generally round, regular particles and have smooth edges, even for the smaller particles. On the contrary, the crushed aggregates present very irregular, oval or long forms and have angular and rough edges. The smaller particles of crushed aggregates present more imperfections. The differences on particle forms between two kinds of materials presumably lead to different arrangements among the grains, thus may impact the filter functioning or biomass development onto these materials. The results of physical and morphological parameters are summarized inTable 2.1.6:

Figure 2 . 8 :

 28 Figure 2.8: profil de répartition de l'eau retenue

-

  Afin d'approfondir les comparaisons d'efficacité d'abattement, différentes conditions opératoire sont mises en place : Différentes hauteur de réacteur : 15, 30 et 70 cm (30 étant la hauteur relative à l'étude du suivi des abattements sur l'ensemble de la période des 360 jours) -Différentes charges hydraulique pour le réacteur de 70 cm de hauteur : 12 et 20 cm/jour. Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials 97

Fig. 1 :

 1 Fig.1 : Overview of the pilot batch experiment at a laboratory scale

Fig. 2 :

 2 Fig.2: Treatment efficiencies of 30-cm reactors with four filter materials during 360 days of operations (a: TSS removals, in %; b: COD removals, in %; c: N-NH 4 removals, in %; d: N-NO 3 concentration, in mgN/l; e: Tot-N removals, in %; and f: P-PO 4 removals, in %)

. 3 :

 3 Fig.3 : Increase in nitrate ions, total nitrogen and phosphate removal efficiencies with 70-cm reactors for two materials under different hydraulic loadings

Part ΙΙ-Chapter 2 :Chapter 3 :

 23 Study of purification efficiencies with different filter materials Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials 123 Conclusion: Au-delà de la comparaison des sables de rivière avec les agrégats concassés, nous avons montré que le paramètre « taille des grains » est le plus impactant sur l'efficacité du biofiltre. L'agencement des grains de petite taille semble être le plus efficace pour arrêter les MES (matières en suspension) mais aussi en terme de colonisation (plus de MVS et plus de flore hétérotrophe). Avec une faible granulométrie, le réacteur est efficace dès une faible hauteur . Pour des grains de taille plus importante , l'abattement est augmenté pour la hauteur plus importante: de bons abattements des MES, et de la DCO (demande chimique en oxygène) ne sont observés qu'avec l'augmentation de la hauteur du réacteur. Ceci est corrélé avec le fait que le biofilm est de moindre importance en surface du réacteur mais plus présent dans les profondeurs du réacteur. Les efficacités sont d'une manière générale liée à l'ensemble de paramètres souvent interdépendants. Il est difficile de distinguer ces facteurs si les réacteurs présentent une efficacité similaire pour l'abattement des MES et DCO, comme pour les réacteurs RS1 et CA1. Le RS1 montre un effet important de filtration en surface, ce qui implique un abattement fort en MES et DCO pour les réacteurs ayant de faibles épaisseurs (15 et 30cm). Mais le cas particulier de RS2 (grains de tailles grossières), une augmentation des efficacités avec le temps de fonctionnement (MES et DCO) observée indique une mise en place de biomasse sur la hauteur du filtre. Cependant, cette distribution longitudinale de la biomasse permet une épuration sur toute la hauteur et ainsi une épuration moins importante avec des hauteurs de 15 et 30cm qu'avec une hauteur de lit de 70cm. Les agrégats concassés, avec une distribution des tailles des grains située entre le RS1 (taille petite) et RS2 (taille grossière), doivent présenter également une colonisation en profondeur du massif filtrant, puisque les réacteurs avec CA1 et CA2 présente les capacités similaires d'abattement en MES et DCO que RS1. Les mesures des MVS et les dénombrements de la flore aérobie hétérotrophe sur des couches à différentes profondeurs permettent une mise en évidence de l'importance de la biomasse dans la profondeur par rapport à celle de la couche supérieure. Le comportement vis-à-vis de l'azote est différent de celui des MES et de la DCO. La présence d'une phase anoxique avec un temps de séjour assez long pour qu'une partie du massif soit immergée n'est observée que pour le réacteur de 70cm en RS1 ce qui favorise la dénitrification, (environ 40% d'élimination N est réalisée). Part ΙΙ-Chapter 2: Study of purification efficiencies with different filter materials 124 L'élimination du phosphore peut être principalement mise en relation avec la composition minérale du matériel (phénomènes d'adsorption et de précipitation), même si une assimilation biologique par les bactéries au début du procédé peut être envisagée. L'alternance aérobie/anaérobie n'existe pas dans les conditions du réacteur filtrant insaturé ce qui est défavorable à une assimilation biologique du phosphate. La taille des grains semble impacter plus particulièrement la colonisation des couches superficielles du biofiltre. La diminution de la biomasse en fonction de la profondeur du filtre peut être mise en relation avec le % d'humidité initiale du filtre. En effet la rétention en eau qui est observée dans les profondeurs du filtre pour les agrégats concassés expliquerait la moins forte diminution des teneurs en matière sèche volatile ainsi que celle de la flore hétérotrophe. Il semble à l'issue de cette étude que la biomasse/biofilm soit un indicateur suffisamment sensible pour observer au cours du temps un comportement différentié du procédé en fonction des matériaux de garnissage Par ailleurs, la présence des particules fines (en quantité plus importante pour les agrégats concassés) augmente le temps de séjour. Nous pouvons penser que les particules fines restent «coincées» dans le massif filtrant du réacteur d'autant plus que celui-ci a une hauteur importante; ce qui signifierait que l'environnement du biofiltre sera d'autant plus impacté par la présence des particules fines que la hauteur du réacteur est importante. Dans cette hypothèse ce nouvel environnement qui est différent selon la hauteur des couches et qui est généré par les particules fines pourraient impacter le biofilm et les EPS. Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances Introduction :Dans ce chapitre, il s'agit de différencier les matériaux du biofiltre (sables roulés RS1 et agrégats concassés CA1 principalement) par le profil de la biomasse (biomasse totale qui est assimilée au biofilm) ainsi qu'une partie majoritaire du biofilm, les EPS (extracellular polymeric substances). En effet comme observé au chapitre précédent, le matériel de garnissage impacte la teneur en matière sèche volatile mais aussi la quantité de flore hétérotrophe dans la couche superficielle et en profondeur du biofiltre. Les EPS sont considérées selon[START_REF] Flemming | The EPS matrix: "House of biofilm cells[END_REF] comme "house of the biofilm cells". Les EPS déterminant les conditions immédiates de vie des cellules, ils peuvent être, eux aussi un indicateur pertinent pour la différenciation des matériaux dans le biofiltre.

  Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric

Figure 2 . 9 :

 29 Figure 2.9: Location of EEM peaks for five EEM regions(Chen et al., 2003)

Centre

  with MW values of440000, 155000, 69323, 5777, 362 and 181 Da (ferritine (Sigma), immunoglobulin G from human serum (Sigma), albumin from bovine serum (Sigma), insulin from porcine pancreas (Fluka), thyrotropin-releasing hormone (Fluka), and tyrosine (Fluka), Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 131 respectively). For mass calibration curves, the logarithm of the molecular mass (log (MW)) is plotted as a function of the elution volume (mL). Log (MW) = -0.3164 Ve (mL) 9.4676 (R2= 0.982) With MW: molecular weight in Da and Ve: elution volume in mL. The permeation volume determined with NaN 3 is 22mL. The chromatograms are established of the fluorescence intensity (volts) and the elution volume (mL). Several fractions were identified as the interval of elution volume (ex: 12~18mL). The calculation of fraction area percentage is based on the ratio between the chromatogram area of one fraction and the total area of chromatogram. The calculation was accomplished by the software Origin 6.0. The fraction area percentage is defined as: F% = 100×Area of fraction/Total area of chromatogram Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric Scientifique et Technique du Bâtiment, 11, Rue Henri Picherit, 44323, Nantes, Pays de la Loire, France Groupement de Recherche Eau, Sol et Environnement (GRESE), 123, av Albert Thomas 87060, Limoges, Limousin, France Abstract Due to the overuse of alluvial deposits, river sands are lacking in a number of areas, leaving crushed aggregate as a candidate filter material for onsite wastewater treatment systems. This study compares the evolution of the biochemical composition of biomass developed in river sand and crushed aggregate over 360 days of colonization by means of conducting batch experiments with septic effluent. By drawing comparisons with the biochemical composition of septic effluent, the results herein show an increase with operating time in the protein fraction of organic matter accumulating in the top layer: the protein/polysaccharide ratio rises from approx. 1 to 2.44 for river sand and to 1.98 or 3.37 for crushed aggregate. The HPSEC protein-like fingerprints show a similar trend across the three aggregates with operating time: enrichment of the high molecular weight protein-like fraction (>1,000kDa). This fractional percentage however is clearly higher for crushed sand after 210 days of colonization, and the percentage of the 6kDa-1,000kDa fraction still increases over time for a crushed aggregate, whereas for river sand it sharply decreases. Moreover, the quantities of biomass and biochemical components indicate a delayed stabilization in the presence of crushed aggregates due to their different physical characteristics, e.g. size, porosity. Keywords: packed bed bioreactors; filtration; biomass; protein; river sand; crushed aggregate (Soumisle 25 Avril 2015 à Biochemical Engineering Journal: BEJ-D-15-00418)

Fig. 1 .

 1 Fig.1. Filtration reactor with sampling ports

  12~18mL). The fraction area percentage calculation is based on the ratio of the chromatogram area of one fraction to the total chromatogram area. This calculation was achieved using the software package Origin 6.0. The fraction area percentage is defined as follows: F% = 100×Area of fraction/Total area of chromatogram Part ΙΙ-Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric

Fig. 2 .

 2 Fig.2. TSS and COD removal efficiencies with river sand (RS1) and crushed aggregate (CA1) in 70-cm filtration reactors

  Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 139

  Zhao et al. and Lloréns et al. have made similar observations on both the laboratory and field scales,

Fig. 4 .Fig. 5 .

 45 Fig.4. Biochemical compositions of biomass: (left) in the top layer of river sand throughout the operating period; and (right) of the septic effluent as feed water

Fig. 6 .Fraction 1 (

 61 Fig.6. HPSEC protein-tryptophan-like fingerprints of the biomass extracted from river sand after 60 and 210 days of operations and of the feed water (septic effluent)

Fig. 7 .

 7 Fig.7. Evolution in the fractions of major group areas relative to total chromatographic areas of septic effluent and river sand biomass extraction at 60 and 210 days

RS1.Fig. 10 .Fig. 11 .

 1011 Fig.10. Comparison of major group fractions among various materials at 60 days

Fraction 1 :

 1 <11mL (aMW ≥ 1000kDa) Fraction 2: 11-18mL (1000kDa > aMW > 6kDa) Fraction 3: 18-22mL (aMW < 6kDa) Fraction 4: > 22mL

3 . Results and discussion 3 . 1 .

 331 and 181 Da (matching respectively ferritine (Sigma), immunoglobulin G from human serum (Sigma), albumin from bovine serum (Sigma), insulin from porcine pancreas (Fluka), thyroglobulin, thyrotropin-releasing hormone (Fluka), and tyrosine (Fluka)). For the mass calibration curves, the logarithm of molecular mass (Log (MW)) has been plotted as a function of the elution volume (mL), i.e.: Log (MW) = -0.3164 Ve + 9.4676 (R 2 = 0.982) with MW being the molecular weight (Da), and Ve the elution volume (mL). The permeation volume determined with NaN 3 equaled 22 mL. The Excitation/Emission (Ex/Em) wavelength fluorescence detection for protein-like (protein tryptophan-like) substances was found to be 222/330 nm, while for humic-like substances (HS-like) the value amounted to 350/460 nm.These wavelengths were derived with the Shimadzu RF-5301 PC spectrofluorometer. The two coupled Ex/Em wavelengths corresponded to the maximal fluorescence in the protein tryptophan-like and HS-like, as defined byChen et al. (2003).The chromatograms were established from both the fluorescent intensity (volts) and elution volume (mL). Several fractions were identified as the elution volume interval (e.g. 12~18 mL). The fractional area percentage calculation was based on the ratio of the chromatogram area of one fraction to the total chromatogram area. This calculation was performed using the Origin 6.0 software package. The fractional(F) area percentage is defined as follows: F% = 100 × Area of fraction / Total area of chromatogram Comparison of biochemical characteristics of organic matters of effluent and of EPS extracted from filter material

Figure 2 :

 2 Figure 2 : Biochemical component contents (A) and repartition (B) of EPS, biomass from the top layer of the biofilter (RS) at both 210 and 360 days of colonization and feed water

--

  Chapter 3: Study of biomass development in different filter materials and evolution of biochemical compositions of total biomass and extracellular polymeric substances 163 Fraction of very high MW (VHMW) (10mL<Ve<12mL): one prior peak with an apparent MW (aMW) ≥1000kDa; -Fraction of high MW (HMW) (12mL<Ve<14mL): groups of peaks not well distinguished from one another and often with two peaks shown (1000kDa>aMW> 109kDa); Fraction of low MW (LMW) (14mL<Ve<16mL): groups of peaks distinct from one another (109kDa>aMW> 25kDa); -Fraction of very low MW (VLMW) (18mL<Ve<22mL): one or several peaks depending on the sample (aMW<6kDa). For an elution volume >22mL, the molecules are eluted beyond the total permeation volume of the column.

Figure 3 :

 3 Figure 3: Protein-like (PN-like) (A) and Humic-like (HS-like) substances (B) HPSEC fingerprints of EPS and biomass extracted from biofilter (RS) at 210 days compared to the feed water

Figure 4 :

 4 Figure 4: Protein-like SEC fingerprint fraction area percentage (%) of biomass and EPS from the river sand (RS) on Day 210, compared to the feed water percentage

Figure 7 :Figure 8 :

 78 Figure 7: Percentage of areas for PN-like fingerprint fractions of the EPS extracted from RS and CA during the operating period

Figure 11 :

 11 Figure 11: Percentage of areas of PN-like fingerprint fractions of the EPS, extracted from RS and CA over the different filtration bed depths

  information. It was shown that the HS-like from EPS have weak MW and result from the adsorption of feed water, perhaps initiated either by the preference for smaller humic molecules (aMW<720 Da) or by an HS-like molecule being metabolized by biofilm cells at the beginning of the process (up to 210 days). On Day 360, HS-like SEC fingerprints are the same as those from the feed water (aMW<6kDa) in the biofilter. Reference: Ahimou, F., Semmens, M.J., Haugstad, G., Navak, P.J. (2007) Effect of Protein, Polysaccharide, and Oxygen Concentration Profileson Biofilm Cohesiveness.Applied and environmental microbiology, 73, 2905-2910. Arnosti, C. & Jørgensen, B.B. (2003) High activity and low temperature optima of extracellular enzymes in Arctic sediments: implications for carbon cycling by heterotrophic microbial communities. Mar. Ecol. Prog. Ser. 249: 15-24. Bhaskar, P. &Bhosle, N.B. (2005) Microbial extracellular polymeric substances in marine biogeochemical processes. Current Science, 88, 45-53.

1 .

 1 développement de la biomasse avec un focus particulier sur les substances polymériques extracellulaires. Une interprétation du fonctionnement des filtres en fonction de la nature des matériaux est proposée. Part ΙΙΙ: Conclusion générale III.Différentiation des matériaux Une méthodologie concernant la mise en oeuvre de matériaux filtrants et une caractérisation de ces matériaux granulaires ont été proposées : Définition d'un procès expérimental : Pour répondre aux objectifs, la première étape a consisté à définir et à construire un dispositif expérimental à l'échelle du laboratoire. À cet effet, le pilote de filtration a été composé de 12 réacteurs de filtration de 30 cm de diamètre et de 3 hauteurs différentes de garnissage : 15, 30 et 70 cm. La hauteur de 70cm étant la hauteur de matériau recommandée par la norme NF, DTU 64.1 (AFNOR, 2011) alors que les hauteurs de 15cm et 30cm sont considérés comme les couches actives du processus de filtration. Un aperçu de l'unité pilote de filtration est proposé sur la Figure 3.1.

Figure 3 . 1 :

 31 Figure 3.1:vue d'ensemble des pilotes de filtration

Figure 3 .

 3 Figure 3.2 propose une représentation conceptuelle de cette différence de comportement entre les deux classes de matériaux.

Figure 3 . 2 :III. 3 .

 323 Figure 3.2: Proposition d'un schéma conceptuel reprenant les deux structures de grains (sable de rivière et agrégats concassés) et la distribution de l'air et de l'eau dans le lit.

Figure 3 . 3 :

 33 Figure 3.3: Concept de micro-environnements au sein des filtres garnis de sable ou d'agrégats concassés

III. 4 .III. 5 .

 45 matériaux notamment les agrégats concassés s'est avérée nécessaire. Nous nous sommes donc intéressés au comportement interne des réacteurs avec une approche quantitative et qualitative concernant la biomasse/ biofilm et les EPS. Pour cela, deux filtres respectivement garnis avec le sable de rivière RS1 et de l'agrégat écrasé CA1 ont été comparés pour des conditions d'effluent standard et avec une charge volumique 12cm/jour. Ces conditions ont été choisies car les rendements épuratoires sur les deux matériaux apparaissaient comparables pour des caractéristiques de matériaux très différentes. CA2 a été utilisé comme matériel supplémentaire en raison de ses caractéristiques intermédiaires entre RS1 et CA1. Biomasse et matériaux du massif filtrant La biomasse développée sur les couches supérieures des deux types de matériaux (RS1 et CA1, CA2) a été suivie en termes de quantités et de caractéristiques. L'évolution du rapport protéines (PN) /polysaccharides (PS) (les protéines étant considérées comme un indicateur de biomasse récente), l'évolution des masses moléculaires des protéines (ou assimilées -like en anglais) par HPSEC couplée à une détection par spectrofluorescence. En comparaison avec la qualité de l'eau d'alimentation, la biomasse totale du filtre montre à travers les différents indicateurs une augmentation de la biomasse récente: augmentation des protéines (à la fois la quantité et les proportions de la biomasse) et du rapport PN/PS ; via les chromatogrammes de HPSEC, l'augmentation de la fraction des composés protéiniques (PN-like) de haute PM (> 1000kDa) est aussi observée. Les différents matériaux ne semble pas modifier la nature de la biomasse, mais par contre modifie les quantités totales de biomasse (plus faibles quantités de biomasse avec les granulats concassés) ainsi que le temps de stabilisation (pas de stabilisation en fin de l'étude dans les colonnes garnies de granulats concassés).Il semble que la surface spécifique joue un rôle important dans l'établissement de la biomasse et une surface spécifique plus faible conduirait à un ralentissement de la production de biomasse. Plus le temps de séjour moyen et la porosité sont importants et plus l'organisation structurelle de la biomasse sera modifiée.La biomasse totale (biofilm) comprend non seulement des cellules bactériennes avec leurs composants intracellulaires, mais aussi la matrice extracellulaire qui contribue à l'organisation structurelle du biofilm. Ainsi, l'étude ciblée des EPS peut compléter cette première approche avec une production et une composition en EPS peut être plus sensible à la nature de l'environnement du biofilm. Evolution des EPS en fonction des matériaux de garnissage EPS du matériau de référence, RS1 et effluent :Le pourcentage d'EPS augmente en fonction du temps du procédé: RS1: 9~30%. Les protéines extracellulaires augmentent aussi entre le jour 60 et le jour 360 (<20mg/kg à 60mg/kg), et représentent environ 40% des EPS. Au jour 360, les empreintes HPSEC des protéines-like montrent des évolutions similaires entre la biomasse et les EPS et se différencient progressivement des empreintes SEC de l'effluent via la diminution de la fraction de faible masse moléculaire (MM apparente = MMa<6kDa) et l'augmentation de la fraction de très forte masse (>1000kDa). De 60 à 210 jours la proportion de forte MM augmente de 30% à 40% sur l'empreinte SEC de protéine-like. Ces résultats confirment que la matière organique présente dans le biofiltre n'est pas liée à une accumulation d'effluent mais en partie au moins à une production microbienne. Les HS-like (substance humiques-like) seraient d'origine exogène à l'EPS. Or, même si au jour 250 la majorité des HS-like présente une MM légèrement inférieure à celle de l'effluent (Mma<6000Da) leur empreinte SEC est identique à l'empreinte HPSEC des substances humiques (HS-like) de l'effluent au jour 360. Ces résultats confirmeraient que les substances humiques des EPS sont des molécules venant de l'environnement dans le cas d'un procédé biofiltre.Comparaison des EPS pour différents temps de procédé en fonction du matériau :Certaines évolutions de la matière organique des EPS sont similaires entre RS1 et CA1, ainsi, la proportion de protéines augmente au cours du procédé (20% au jour 60 et 30% au jour 360). Les empreintes SEC des protéines-like ont des évolutions similaires entre les deux matériaux mais la fraction de très forte MM apparait moins rapidement pour CA1 comparé à RS1. La biomasse est moins présente et moins active (moins de protéine dans la biomasse totale) dans le réacteur avec CA1. Ce déficit serait généré par l'environnement moins homogène et la présence de zones de courts-circuits dont l'origine serait la teneur plus importante en particules fines (Figure3.3). Cet environnement pourrait induire un stress responsable d'une plus forte production d'EPS soit par une flore microbienne similaire à celle trouvé dans RS1, soit parce que ces nouvelles conditions induisent un changement de communautés microbiennes, à l'origine d'un autre type d'EPS. Le pourcentage d'EPS dans le CA1 augmente en effet légèrement par rapport à RS1 au cours du temps (20 à 32% aux jours 60 et 360 pour CA1). Les EPS présentent aussi une plus forte proportion en polysaccharides avec CA1 (18 à 33% pour CA1 contre 30 à 17 pour RS1 du jour 60 au jour 360). Les SH-like ne sont pas impactées par le type de matériau. La minéralogie du support peut aussi influencer les caractéristiques des surfaces des grains qui intervient dans le mécanisme d'adhésion des

Figure 3 . 2 ,

 32 Figure 3.2, les formes des particules doivent jouer un rôle important sur le comportement de la phase liquide et l'hydrodynamique du système, ainsi que sur la distribution de la phase gazeuse. La distribution inégale des substrats ou de l'air peut créer une accumulation hétérogène de la biomasse et des obturations locales du lit; les particules fines : comme proposé sur la Figure 3.3, la présence de particules fines est un paramètre majeur qui influence les propriétés hydrauliques et hydrodynamiques des lits de filtration. Les particules fines jouent un rôle "d'éponge" en créant un milieu favorable au développement du biofilm) mais réduit également le taux d'infiltration et provoque un risque de colmatage; la composition minéralogique: elle est impliquée dans le processus d'élimination des anions (tels que les phosphates) par production de cations complexants. Elle pourrait influencer la mise en place du biofilm.

  .T.,Maratea, D. & Bishop, P.L. (1983) Electron microscopic examination of wastewater biofilm formation and structural components.Applied and Environmental Microbiology, 45, 1921-1931. Elimelech, M. (1995) Particle deposition and aggregation: measurement, modelling, and simulation. Butterworth-Heinemann. Ellis, B., Butterfield, P., Jones, W., McFeters, G. & Camper, A. (1999) Effects of carbon source, carbon concentration, and chlorination on growth related parameters of heterotrophic biofilm bacteria. Microb Ecol, 38, 330-347. Frølund, B., Palmgren, R., Keiding, K. & Nielsen, P.H. (1996) Extraction of extracellular polymers from activated sludge using a cation exchange resin. Water Research, .M., Paterson, E., Kemp, J.S., Cresser, M.S. & Killham, K. (1992) Factors affecting the movement of microorganisms in soils. Soil biochemistry,7, 263-305.

Table 1 .

 1 1 : Data (average values) of critical contaminants in domestic wastewater...............

Table 1 .

 1 2 : Main characteristics of septic effluent ..................................................................

Table 1 .

 1 3 : Values of permeability coefficient K of different textures of soil ........................

Table 1 .

 1 4 : Differential equations and parameters of biodiffusion model................................

Table 1 .

 1 5 : Studies in purification efficiencies of sand filters and influencing factors ............

Table 1 .

 1 6 : Factors influencing clogging process .....................................................................

Table 1 .

 1 7 : Summary of governing interactions in bacterial adhesion to solid surface ...........

Table 1 .

 1 8 : Summary of functions of EPS ................................................................................

Table 1 .

 1 9 : Biofilm investigation techniques overview ............................................................

Table 1 .

 1 10 : Studies on EPS biochemical composition of wastewater biofilms ......................

Table 1 .

 1 11 : Colorimetric assays of certain EPS components ..................................................

Table 1 .

 1 12 : Strategy of experimental study.

Table 2 .

 2 2.3: Septic effluent characteristics during long-term monitoring ...............................

Table 2 .

 2 2.4: Liquid medium additives for aerobic autotrophic flora count ...........................

Table 2 .

 2 3.1: Experimental conditions of biomass and EPS extractions .................................

Table 2 .

 2 3.2: Analytic parameters of colorimetric assays of biomass and EPS constituents ..

Table 2 .

 2 3.3: Corresponding compounds types to EEM spectra regions and Ex/Em ranges ..

Table 1 .

 1 1 : Data (average values) of critical contaminants in domestic wastewater

	.1):

Table 1

 1

	Authors	Van Cuyk et al. 2001	PhD thesis: Chabaud. 2007	Lakel. 2009	Kauppinen et al., 2014	Siegrist et al.2014
	Average values: Suspended solids [mg/L] and organic characteristics [mgO/L]
	TSS	69	99	74	37	26
	BOD 5	227	231	-	-	158
	COD	386	290	343	320	226
		Nitrogen and phosphorus characteristics [mgN/L]
	N-NH 4	47	71	48	-	52
	Total N	57	66	-	72	51
	Total P	4.6	3	-	9.2	23
		Microbiological characteristics [CFU/100mL]
	Total coliform	-	6.4×10 4	-	-	-
	Thermotolerant coliform	5.4×10 5	-	-	-	3.9×10 5

.2 summarizes the literature data of the main physico-chemical characteristics and indicator bacteria levels in septic effluent:

Table 1.2 : Main characteristics of septic effluent

Table 1 .

 1 3 : Values of permeability coefficient K of different textures of soil (NF DTU 64.1)

	Texture of soil	Permeability coefficient K (mm/h)	Permeability
	Clayish soil	<6	Impermeable
	Clay-silty soil	6-15	Weakly permeable
	Silty soil	15-30	Poorly permeable
	Sandy-silty soil	30-50	Correctly permeable
	Sandy soil	>50	Strongly permeable

table (

 (

				Part Ι: Literature review
	Table 1.4 : Differential equations and parameters of biodiffusion model Transport phenomena Liquid phase Biofilm phase Dispersion: K 6 K m ∂ c -Equation 19: G s, f, Pe, η = 2R * e t2 e t2/ p t2 m -‹ H t2 e 3‹ -e ‹ + e ‹ OE t2 + R• -t2 + Re 3‹ s ∂y Convection: K m ∂u -∂y Molecular diffusion: K K K m • ∂u ∂ƺ • ƺ = 0 ∂u ∂ϑ = K K m R is expressed by following equation: ∂ u ∂ƺ Coefficients Equation 20: R = Pef 6/ Žs + • • + t2 m• -• H ' '"' 5"• '"' -6/
	And:	c:		Ratio between the actual tracer concentration and input tracer concentration
	u: K1: ƺ: Equation 21: η =	O r -r } | ˜™:	Ratio between the tracer concentration in biofilm and input tracer concentration K 6 = D vH = Pe Inverse of the Peclet number 1 Distance in the biofilm from biofilm surface š : , f = š G: 7š :
		K2: Table1.4): K3: K4:		K = K = HD R vL Lω ε K m = ε ‡ ε = T ˆ ‡ T ˆ	Ratio between the actual retention time and the characteristic time for molecular diffusion into biofilm Geometric constant with ε is the actual porosity Ratio between the water volume without biofilm and actual water volume
	Based on the similar theory, Lakel et al. (1998) gave the resolution in Laplace domain as a
	function G(s) for a pulse input:

Table 1 .

 1 5 : Studies in purification efficiencies of sand filters and influencing factors

	Analyzed parameter	Influencing factors	Reference
		Grain size: larger grain size results deeper
	TSS	retention of solids	Ellis & Aydin, (1995)
		Presence of biofilm growth: induces higher
	Organics	organic removals at initial state of sand columns; Depth of filter media: great depth of filter	Rodgers et al, (2005) Lloréns et al, (2011)
		has higher removals;
		T°C: nitrification is inhibited at low T°C;
		Presence of clogging: anoxic zone may	Kristiansen, (1981 ΙΙ)
		induce the denitrification; slight clogging also improves the nitrification;	Van Cuyk et al., (2001)
	Nitrogen	Organic loads: higher organic loads reduces	Rodgers et al., (2005)
		the activities of nitrifying bacteria; Presence of plants: in combination with	Gill et al., (2009)
		nitrification;	Lloréns et al, (2011)

Phosphorus Mineralogy of filter media and the clay contents: both impact the P removal; Reactivity of filter media: alkaline

		materials under low organic loads results	Gill et al., (2009)
		better P removal;
		Hydraulic and organic loads: under
		unsaturated conditions, no effect of hydraulic
		loads on the removal; higher organic loads
		leads to higher bacteria removal
		Presence of slight clogging: the utilization
	Microorganisms	of the media increased resulting in the improvements of removals;

Distribution mode: uniform pressure dosing results better removals; T°C (seasonal operation): virus

	Van Cuyk et al., (2001)
	Ausland et al., (2002)
	removals
	diminished during colder weather

Table 1

 1

	.6:

Table 1 .6 : Factors influencing clogging process Influencing factor Mechanisms of impact Reference

 1

		Higher organic content in feed	Reneau et al., 1989;
	Organic content (feed water)	water leads to higher microbial
		activities and EPS production	Liu et al., 2003
		Grain size impacts directly the
	Grain size (filter media)	physical clogging and also the biofilm development at higher	Kristiansen, 1981a
		organic content
	Surface roughness or angularity of sand grains (filter media)	Rough surfaces might be favorable of bacterial deposition thus biofilm	Jacob et al., 2007 Wanko et al., 2005
	Hydrophobicity of cell surface (bacteria species)	Bacteria with hydrophobic sand surface surface are easier to adhere to	Jacob et al., 2007

Table 1 .

 1 7 : Summary of governing interactions in bacterial adhesion to solid surface

	Figure7 Legend	Interaction	(Un) favorable + / -
	(1)	Electrostatic	Repulsion
	(2)	Acid-base	Dependence of cell surface molecules
	(3)	Hydrophobicity	Attractive for hydrophobic cells and surface
	(4)	Van der Waal forces	Always attractive, but acts when close enough
	(5)	Hydrogen bonds	Cell surface molecules
	(6)	Calcium bridges	Favorable to adhesion

Table 1 .

 1 Studying the EPS requires the separation from the biofilm sample first, which is commonly known as the extraction, then further investigation can be done by analytical techniques. As the extraction of EPS is destructive to biofilm samples, the following-up of EPS evolution throughout time is quite unlikely. The main forces involved in binding EPS are Van der Waal force, electrostatics interactions mediated or not with multivalent cations, hydrogen bond, and hydrophobic interactions. The main dominating force varies from biofilm to another, thus the extraction method should be chosen according to each case, and no universal extraction method exists for a quantitative extraction of bound EPS compounds from microorganisms growing in suspensions or in aggregates or attached surfaces[START_REF] Sardin | Modeling the nonequilibrium transport of linearly interacting solutes in porous media: A review[END_REF]. The EPS yields depend on extraction time, shear force and the dependences are different for various EPS compounds. The extraction methods are classified into 2 categories by their shears force 10 : Studies on EPS biochemical composition of wastewater biofilms

	they provide:
	Physical methods: a shear applied to extract EPS by shaking, mixing, centrifugation,
	and sonication or heat…

I.6.3.1. EPS Extraction methods

Either soluble EPS or bound EPS need to be separated from the matrix or the biomass to carry out further investigations, and certain EPS components are divided purified from the extracts for more detailed analysis. The main difficulty in EPS extraction procedure is to obtain high extraction efficiency without unwanted cell lyses and disruption of macromolecules.

Chemical methods: treatments include addition of various chemicals that can break the linkage in the EPS matrix, such as: alkaline treatment with the addition of NaOH, complexing agent like ethylenediaminetetraacetic acid (EDTA), cation exchange resin (CER) (this method was actually a mix between physical (shaking with resin) and chemical (exchange with Na + and multivalent cations from the biofilm) extractions)…

The combination of physical-chemical extraction is more and more applied. Furthermore, the biofilms in wastewater treatment systems are highly heterogeneous. The matrix of EPS contains water, proteins, polysaccharides, nucleic acids, lipids and also bound minerals…Their biochemical fractions depends the nature of biomass, the extraction and analysis techniques, and also the type of substrates. Many studies have been carried out with wastewater EPS by comparing different extraction procedures and the advantages and shortcoming of some of these methods are summarized in the Table

1

.10:

  and bath (Sonicator, bath capacity of 2L, 40W-100W, Liu et al., 2009) are largely used for soil and activated sludge EPS extraction. The study of Yu et al. showed that ultrasound probe treatment extracted more types of extracellular enzymes than ultrasound bath treatment, but

Table 1 .

 1

		11 : Colorimetric assays of certain EPS components
	Component	Method (References)	Reagent	Target functional group	Standard	Calibration sensibility
		Lowry (Lowry et al., 1951)	Folin-Ciocalteu, 5% CuSO 4	Peptide links (>2) and phenols	Bovineseru malbumin (BSA)	0.04-0.2 g/L
	Proteins	Bradford (Bradford, 1976)	CoomassieBrilli ant Blue	Peptide links (>8) rich in arginine	BSA	0.04-0.2g/L
		BCA	Bicinchoninic	Protein-BCA-
		(Smith et al.,	acid (BCA),	Cu +1 complexion:	BSA	0,2-1 g/L
		1985)	CuSO 4	biuret reaction
	Humic-like substance	Modified Lowry method (Frølund et al., 1996)	Folin-Ciocalteu	Phenols	Humic acid	0.04-0.2 g/L
	Polysaccharides	Dubois (Dubois et al., 1956)	5% Phenol	Monosaccharide with>5 C(H 2 O)	Glucose	0.02-0.1 g/L
	Nucleic acids	Burton (Burton, 1956)	0.6% Diphenylamine	2-desoxyribose	Calf thymus DNA	0.005-0.05 g/L
	Molecular weight and HPSEC
	EPS are composed of molecules of variable molecular weight (MW). The determination
	of the apparent MW (aMW) distribution is achieved by high-pressure size exclusion

chromatography (HPSEC). This method provides a separation of crude EPS extracts based partly on molecular hydrodynamic size and partly based on non-size exclusion separation mechanisms. The signal is generally detected by UV absorbance or rafractiometric detection

Table 1 .

 1

		12 : Strategy of experimental study
		Comparative study of crushed sands and alluvial sands
	1 st phase	2 nd phase	3 rd phase
	Conception and	Experimental study of	Monitoring study of purification efficiencies of main pollutants by two types of sands and at different depths
	construction of	material	Monitoring study of biofilm components distribution
	sand filtration	characteristics	in the profile of filter depth and their evolutions
	columns system	as foundation of	throughout operating period: biomass quantification;
		comparison	evolution of the size of macromolecules; microbial
			activities…

Table 2

 2

	.1.1:

Table 2 .

 2 1.1: Quarries and natures of materials

	Filter materials	RS1	RS2	CA1	CA2
	Quarry name	Vritz	Les Alleuds	Mouen	Vaubadon
				Crushed	Crushed
	Nature	Alluvial (Loire)	Alluvial (Loire)	(Feldspathic	(Precambrian
				sandstone)	sandstone)

Table 2

 2 .1.2.

	Table 2.1.2: Granulometric parameter definitions
	Parameters (unit)	Symbol	Definition
	Effective diameter (mm)	D 10

Table 2

 2

	.1.3:

Table 2 .

 2 1.3: Experimental conditions for released cations of selected sands

	Sample treatments	Dried sieved aggregates (>0.2mm)
	Temperature	Ambient, 25°C
	Solid/liquid	5g/20mL; 10g/20mL; 15g/20mL
	Mixing condition	Mixing table, 200tr/min, overnight
	Liquid phase sampling	Settled for 2 hours, 15mL filtrate of 0.45µm
	Detected cations by ICP-ES (Shimadzu)	K + , Na + , Mg 2+ , and Ca 2+

Table 2 .

 2 1.4: Granulometric characteristics of filter materials

		RS1	RS2	CA1	CA2
	Material nature	Loire river	Loire river	Crushed	Crushed
	Effective size D 10 (mm)	0.38	1.60	0.17	0.44
	Average
	diameter D m	0.82	2.26	1.36	1.6
	(mm)
	Uniformity
	coefficient	2.78	1.75	10	5
	(D 60 /D 10 )
	Fine particles (<0.08mm)%	0.4%	0.5%	5%	2.4%

Table 2

 2

	.1.6:

Table 2

 2

	.1.7; four

Table 2 .

 2 1.7: Mineral compositions and calcareous (limestone: CaCO 3 ) contents of filter materials

	mg/kg	RS1	RS2	CA1	CA2
	Ca	<5	<5	795	7002
	Mg	74	17	1535	11022
	Na	<5	<5	295	537
	K	1370	139	2356	4323
	Fe	<1000	<1000	5576	32978
	Al	17075	1507	38800	59383
	Si	431166	460834	391779	256906
		RS1	RS2	CA1	CA2
	CaCO 3 (%)	<0.5	<0.5	1.3	1.7

Table 2 .

 2 1.8: Released cations in water from the filter materials

		RS1	RS2	CA1	CA2
	Ca (mg/kg)	4.30	2.28	10.43	28.72
	Mg (mg/kg)	0.52	0.33	1.37	3.64
	Na (mg/kg)	0.98	0.97	6.91	4.61
	K (mg/kg)	1.47	0.78	7.53	4.63

Table 2 .

 2 1.9: Infiltration time and estimated hydraulic conductivity at saturation and water retention capacities of filter materials

	Hydraulic characteristics	RS1	RS2	CA1	CA2
	t s (s)	62	20	170	67
	K s (m/s)	8.95×10 -4	2.77×10 -3	3.25×10 -4	8.25×10 -4
	Water retention capacity	RS1	RS2	CA1	CA2
	Cv (%)	14.6%	2.1%	20.0%	15.5%
	Cm (%)	8.4%	1.3%	12.7%	10.0%

Table 2 .

 2 1.10: Average HRT in 30cm and 70cm reactors

	Average HRT (hour)	RS1	RS2	CA1	CA2
	12cm/day, 30cm	23	8	32	20
	12cm/day, 70cm	35	-	93	-
	20cm/day, 70cm	12	-	48	-
	Oxygen gas
	variation	[11; 20]%	-	[19.2; 19.8]%	-
	(oxy% in air)

  partir de cette étude comparative des caractéristiques propres des matériaux et des réacteurs, les agrégats concassés se différencient surtout des sables roulés par leur distribution

	de tailles de grains. D'autres paramètres peuvent éventuellement influencer les propriétés
	hydrauliques et hydrodynamiques des réacteurs.
	Les différences en répartition des tailles entre les deux types des matériaux sont provoquées
	par la nature de la source du matériel et la procédure de l'exploitation et de la production en
	carrières. Les agrégats issus du concassage de la roche massive montrent une distribution des
	tailles hétérogènes (Coefficient d'uniformité ≥5, CU<3 pour les sables roulés). Ils possèdent
	également des particules fines du diamètre <0,08mm qui sont induites par des broyages entre

des grains pendant le concassage et le transport (Fines%>2%). Par contre, les sables issus de l'érosion en rivières, montrent une stabilité structurelle (Fines%<0,5%). Des différences sont également observées sur la forme des grains. Les agrégats concassés présentent des formes anguleuses, allongées et irrégulières. L'hétérogénéité des grains en taille et en forme est donc liée à la source des matériaux. Les sables roulés ont la même origine (La Loire) et à base de silice. Les agrégats de roche présentent différentes compositions, ils sont notamment plus riche en Ca, Fe, Al… Par conséquence, l'hydraulique du milieu se différentie pour les types des matériaux. La présence de particules fines, surtout pour CA1, induit une conductivité hydraulique plus faible que d'autres matériaux, et en même temps, une rétention en eau plus importante : le profil de la répartition de l'eau sur la longueur du lit montre des différences de répartition de la phase liquide :

Table 2 .

 2 .2.2: 2.2: Analytic methods of physico-chemical parameters of feeding water and treated effluents

	Physico-chemical parameters	Method	Testing range
				pH	Multimeter	1-14
	Conductivity (µS/cm)	Multimeter
	COD (mgO/L)	Merck COD Vials Kit	150-1500mgO/L; 10-150mgO/L
	N total (mgN/L)	Merck Total N Vials Kit	10-150mgN/L
	NH 4	+ (mgN/L)	Merck Ammonium 100 Tests	2.6-19.3 mgNH 4 /L
	NO 3	-(mgN/L)	Merck Nitrate 200 Tests	0.4-110.7mg NO 3 /L
	PO 4	3-(mgP/L)	Merck Phosphate 100 Tests	1-100mgP/L

Table 2 .

 2

				2.3: Septic effluent characteristics during long-term monitoring
			Reactor height	River sands	Materials	Crushed aggregates
				15cm	RS1	CA1
				30cm	RS1	RS2	CA1	CA2
				70cm	RS1	RS1	CA1	CA1
	Dosing conditions	12cm/day or 20cm/.day fractionated by 10 batches/day
					Septic effluent characteristics (Average value and [min, max])
				Parameters	Average value	[min; max]
				pH (5 tests)	7.1	[6.6; 7.5]
	TSS (mg/L, 18 tests)	39 (±11)	[20; 66]
	VSS (mg/L, 1 test)	23	-
	COD (mgO/L, 18 tests)	372 (±100)	[231; 572]
	Tot-N (mgN/L)	81	[58; 95]
	NH 4	+ (mgN/L, 22 tests)	46 (±21)	[20;77]
	NO 3	-(mgN/L, 22 tests)	<2.3	[0; 2.3]
	PO 4	3-(mgP/L, 14 tests)	9	[7.2; 12.7]
	Revivifiable aerobic flora*	5.1×10 5	[9.5×10 4 ; 1.1×10 6 ]
	(37°C) (CFU*/100mL)
	*Revivifiable aerobic flora (NF EN ISO 6222); *CFU=Colony Forming Unit

Table 2 .

 2 2.4: Liquid medium additives for aerobic autotrophic flora count (MPN*)

	Additives	Concentration (g/L)
	Liquid medium	Mineral water Contrex® (1L)
	NH 4 Cl	1
	FeSO 4	0.005
	KHCO 3	0.1
	Na 2 HPO 4	0.1
	NaOH	pH 7.2

*MPN: most probable number

Table 1

 1 Packing material characteristics

	Filter material	River Sand 1 (RS1)	River Sand 2 (RS2)	Crushed Aggregate 1 (CA1)	Crushed Aggregate 2 (CA2)
	Photograph
	Effective size D 10 (mm)	0.38	1.60	0.17	0.44
	Average diameter D m (mm)	0.82	2.26	1.36	1.6
	Uniformity coefficient (D 60 /D 10 )	2.78	1.75	10	5
	Circularity	0.849 (±0.060) 0.819 (±0.037) 0.824 (±0.088)	0.800 (±0.109)
	Roundness	0.74 (±0.11)	0.76 (±0.10)	0.73 (±0.15)	0.67 (±0.15)

Table 3

 3 Feed water (septic effluent) characteristics

	Parameter	Average value	[min; max]
	pH (5 tests)	7.1	[6.6; 7.5]
	TSS (mg/l, 18 tests)	39 (±11)	[20; 66]
	VSS (mg/l, 1 test)	23	-
	COD (mgO/l, 18 tests)	372 (±100)	[231; 572]
	Tot-N (mgN/l)	81	[58; 95]
	+ (mgN/l, 22 tests) -(mgN/l, 22 tests) NO 3 NH 4 3-(mgP/l, 14 tests) PO 4 Revivifiable aerobic flora	46 (±21) <2.3 9 5.1×10 5	[20;77] [0; 2.3] [7.2; 12.7] [9.5×10 4 ; 1.1×10 6 ]
	(37°C) (CFU*/100mL)
	* CFU=Colony Forming Unit

Table 4

 4 Treatment efficiencies of filtration reactors with two selected materials

			(RS1 and CA1) under different operating conditions
	Operating conditions	TSS%	COD%	N-NH 4 %	NO 3 (mgN/l)	Tot-N%	P-PO 4 %
	HLR (cm/day)	Thickness (cm)		RS1 Average value [min, max]%
	12	15cm	86 [60, 91]	71 [58, 84]	69 [6, 95]	30 [6, 55]	19 [8, 28]	33 [11, 55]
	12	30cm	94 [90, 96]	86 [73, 96]	90 [53, 100]	40 [4, 66]	21 [12, 39]	50 [29, 61]
	12	70cm	97 [80, 100]	96 [85, 99]	98 [74, 100]	29 [7, 57]	39 [21, 58]	44 [12, 85]
	20	70cm	94 [75, 100]	92 [81, 98]	94 [67, 100]	32 [5, 47]	23 [7, 46]	57 [16, 85]
	HLR (cm/day)	Thickness (cm)		CA1 Average value [min, max]%
	12	15cm	78 [45, 90]	64 [51, 80]	62 [8, 92]	31 [8, 61]	11 [2, 23]	40 [18, 57]
	12	30cm	91 [84, 100]	82 [66, 91]	85 [39,100]	41 [3, 68]	16 [10, 25]	54 [28, 75]
	12	70cm	96 [80, 100]	94 [80, 98]	95 [71, 100]	44 [6, 65]	22 [4, 36]	65 [33, 91]
	20	70cm	90 [65, 98]	90 [81, 98]	91 [63, 100]

Table 5

 5 Distribution of total organic matter in four materials at 360 days

	Layer: VDW (mg/g aggregate)	RS1	RS2	CA1	CA2
	0~5cm	12.49	3,69	10.03	8.01
	10~15cm	4.10	2,19	4.81	5.21
	30cm	2.98	1,63	3.39	3.56
	VDW decreasing from 0~30cm (%)	76%	56%	66%	56%

Table 6

 6 Distributions of heterotrophic and nitrifying bacteria throughout the depth of

		three materials at 360 days
	Heterotrophic flora (CFU*/g material)	RS1	CA1	CA2
	0~5cm	4.7×10 7 [2.5×10 7 ; 6.9×10 7 ]	4.28×10 6 [2.2×10 6 ; 6.3×10 6 ]	2.30×10 6 [1.2×10 6 ; 3.4×10 6 ]
	10~15cm	4.98×10 7 [2.6×10 7 ; 7.3×10 7 ]	2.98×10 6 [1.6×10 6 ; 4.4×10 6 ]	1.25×10 6 [6.6×10 5 ; 1.9×10 6 ]
	30cm	5.05×10 5 [2.7×10 5 ; 7.5×10 5 ]	2.93×10 6 [1.5×10 6 ; 4.3×10 6 ]	1.66×10 6 [8.7×10 5 ; 2.4×10 6 ]
	Nitrifying flora (MPN*/g material)	RS1	CA1	CA2
	0~5cm	20 [12; 48]	130 [78; 312]	50 [30; 120]
	10~15cm	6.00×10 3 [3.6×10 3 ; 1.4×10 4 ]	1.80×10 3 [1.1×10 3 ; 4.3×10 3 ]	3.50×10 3 [2.1×10 3 ; 8.4×10 3 ]
	30cm	N/A	9.00×10 2 [5.4×10 2 ; 2.2×10 3 ]	6.00×10 3 [3.6×10 3 ; 1.4×10 4 ]
	*CFU=colony forming unit (uncertainty: 95%, variation [min; max])
	*MPN=most probable number (uncertainty: [min 40%; max 140%])

Table 2 .

 2 .3.1: 3.1: Experimental conditions of biomass and EPS extractions

	Experimental conditions: Biomass exactions
	Extraction method: Sonication for HPSEC analysis
	Solid/liquid	30g sample / 20mL ultrapure water
	Temperature	4°C
	Ultrasound conditions	Ultrasound bath, 100W input (Branson, 1.9L)
	Duration	60min
	Extraction method: Heating for biochemical composition assessment
	Solid/liquid	10g sample / 20mL ultrapure water
	Temperature	80°C
	Heating conditions	Water bath
	Duration	30min
	Experimental conditions: EPS extractions
	Extraction method: Sonication
	Solid/liquid	30g sample / 20mL ultrapure water
	Working temperature	4°C
	Ultrasound conditions	Ultrasound bath, 100W input (Branson, 1.9L)
	Duration	5min with 2.5min resting to avoid over heating

Table 2 .

 2 .3.2: 3.2: Analytic parameters of colorimetric assays of biomass and EPS constituents

	Target constituent	Method	Wavelength (nm)	Detecting range (g/L)	Standard
	Proteins	Modified Lowry method	650	0.04-0.2	Bovine serum albumin
	Humic-like substances	Modified Lowry method	650	0.04-0.2	Humic acid
	Polysaccharides	Dubois method	492	0.02-0.1	Glucose
	Nucleic acids	Burton method	600	0.005-0.05	DNA of calf thymus

Table 1 .

 1 Characteristics of river sand (RS1) and crushed aggregate (CA1) as main packing materials, with CA2 providing a supplementary material

	Material	River sand (Reference: RS1)	Crushed aggregate (CA1)	Crushed aggregate (supplemental: CA2)
		Particle size distribution characteristics
	Effective size D10 (mm)	0.38	0.17	0.44
	Average diameter D m (mm)	0.82	1.36	1.60
	Uniformity coefficient (D60/D10)	2.8	10.0	5.0
	Fine particles% (<0.08mm %)	0.4%	5.0%	2.4%
	Physical and chemical characteristics (average values; 5 tests)
	Real density (kg/m 3 )	2525 (±42)	2438 (±111)	2546 (±51)
	Porosity (%) [min; max]	[30%; 33%] (±1.5%)	[38%; 41%] (±1.7%)	[38%; 44%](±3%)
	Specific surface (m 2 /kg)	4.04	2.78	2.88
		Hydraulic characteristics (average values; 5 tests)
	Estimated hydraulic conductivity (m/s)	[8.25~9.53] ×10 -4	[2.79~2.88]×10 -4	[7.49~9.73] ×10 -4
	Water retention capacity
	after drainage (static	8.4%	12.7%	10.0%
	water %)
	Hydraulic Residence Time (hours) (1 test)	35	93	-
	Variation of O 2 gas level
	at 10cm	[11, 20]%	[19.2, 19.8]%	-
	[min, max%]

Table 2 .

 2 Feed water characteristics

	Parameter	Average value	[min; max]
	pH (5 tests)	7.1	[6.6; 7.5]
	TSS (mg/L, 18 tests)	39 (±11)	[20; 66]
	VSS (mg/L, 1 test)	23	-
	COD (mgO/L, 18 tests)	372 (±100)	[231; 572]
	Revivifiable aerobic flora	5.1×10 5	[9.5×10 4 ; 1.1×10 6 ]
	(37°C) (CFU*/100mL)
	CFU*=Colony-forming unit

Table 1 :

 1 Characteristics of river sand (RS) and crushed aggregate (CA) as the main packing

	materials
	Material	River sand (ref. RS)	Crushed aggregate (CA)
	Particle size distribution characteristics
	Effective size D 10 (mm)	0.38	0.17
	Average diameter D m (mm)	0.82	1.36
	Uniformity coefficient (D 60 /D 10 )	2.8	10.0
	Fine particles % (< 0.08 mm %)	0.4%	5.0%
	Physical and chemical characteristics (average values; n=5)
	Real density (kg/m 3 )	2525 (±42)	2438 (±111)
	Porosity (%) [min; max]	[30%; 33%] (±1.5%)	[38%; 41%] (±1.7%)
	Specific surface area (m 2 /kg)	4.04	2.78
	Hydraulic characteristics (average values; n=5)
	Estimated hydraulic conductivity (m/s)[min, max]	[8.25~9.53] ×10 -4	[2.79~2.88]×10 -4
	Water retention capacity after drainage (static water %)	8.4%	12.7%
	Hydraulic Residence Time (hours) (n=1)	35	93
	Variation of O 2 gas level at 10 cm [min, max%]	[11, 20]%	[19.2, 19.8]%

Table 2 :

 2 Feed water characteristics

	Parameter	Average value	[min; max]
	pH (5 tests)	7.1	[6.6; 7.5]
	TSS (mg/L, 18 tests)	39 (±11)	[20; 66]
	VSS (mg/L, 1 test)	23	-
	COD (mgO/L, 18 tests)	372 (±100)	[231; 572]

Table 4 :

 4 Biomass evolution and EPS percentage in the biomass extracted from

		the top layer of each filtration reactor on Days 60, 210 and 360
	Material	River sand (RS) -top layer	Crushed aggregate (CA) -top layer
	Time	60 days	210 days	360 days	60 days	210 days	360 days
	Biomass (mg/kg)	96	238	303	123	141	251
	EPS%	16	9	30	21	17	32

Table 5 .

 5 The biomass in both materials decreased with filter medium depth; moreover, a higher biomass in RS from the top to the 10-15 cm layer and a

				HS-like Day 60 RS1				HS-like Day 210 RS1
				HS-like Day 360 RS1			Feeding water (Septic effluent)
		18
		16
	HS-like fluorescence Arbitrary unit (volts)	14 16 18 0 2 4 6 8 10 12 14	12	HS-likeDay 60 CA1 HS-like Day 360 CA1 14 16 18	20	22	HS-like Day 210 CA1 Feeding water (Septic effluent) 24 26 28 30
		12
		10
		8
		6
		4
		2
		0
			12	14	16	18	20	22	24	26	28	30
							Ve (mL)

Table 5

 5

			: Evolution of biomass and EPS% from biomass extracted
				over the depth of each reactor
	Material	River sand (RS) on Day 360	Crushed aggregate (CA) on Day 360
	Depth (cm)	0-2 cm	5 cm	10-15 cm	30 cm	0-2 cm	5 cm	10-15 cm	30 cm
	Biomass (mg/kg)	303	169	125	45	251	166	71	63
	EPS%	30	25	27	39	32	30	44	37

like fluorescence Arbitrary unit (volts) Ve (mL)

  substance/protein ratio decreases. Meanwhile, within the bed thickness, the polysaccharide/protein ratio rises by Day 360. Moreover, a correlation between the higher percentage of polysaccharide in EPS and less favorable environment for microorganisms potentially found in the CA biofilter has been proposed. The packing material mineral characteristic does affect polysaccharide implementation in EPS, in considering the divalent cations possibly released with CA.A clear difference in the SEC protein-like fingerprints during biomass enrichment, in terms of both intensity and number of peaks, was noticed. This finding indicates that diverse molecular structures have occurred as a result of the enrichment; a variation in the four fractional distributions (i.e. very high, high, low and very low MW) was also shown. When decreasing, the high MW fraction can reveal bacterial growth and aggregation in the biofilm.

		EPS HS-like Day 360 RS1 Top		EPS HS-like Day 360 RS1 10-15cm
		EPS HS-like Day 360 RS1 30cm		Feeding water (Septic effluent)
	14	16	18	20	22	24	26	28	30
	HS-EPS HS-like Day 360 CA1 Top EPS HS-like Day 360 CA1 30cm		EPS HS-like Day 360 CA1 10-15cm Feeding water (Septic effluent)
	14	16	18	20	22	24	26	28	30

like

  stabilisation pour RS1. Pour le réacteur contenant CA1, le développement de la biomasse semble plus long et semble encore en pleine croissance à l'issu des 360 jours de procédé. Le % d'EPS augmente dans le temps avec une teneur légèrement plus importante tout le long du procédé pour CA1. La répartition protéine/sucre est différente en fonction du type de matériaux ; le ratio augmente au cours du temps pour RS1 alors qu'il diminue pour CA1. Les futures études devront notamment se focaliser sur les protéines et le rapport protéines/sucre ainsi que sur l'évolution au cours du temps des empreintes SEC des protéines-like. Une étude du fonctionnement de différents filtres sur une longue période (360 jours) a été adoptée afin d'observer des différences de comportement de la phase de colonisation à l'état stationnaire. Le travail a consisté à caractériser au mieux les matériaux, puis à observer l'impact des matériaux sur les différents rendements et sur le

	Part ΙΙΙ: Conclusion générale
	Part ΙΙΙ: Conclusion générale
	Dans les systèmes d'assainissement autonome, une étape de filtration biologique
	intervient après le traitement anaérobie de l'effluent. L'environnement du lit filtrant avec ses
	conditions de milieu insaturé est un milieu complexe et hétérogène qu'il est difficile
	d'appréhender. Des mécanismes très différents permettent d'éliminer des compartiments très
	différents de la contamination des eaux usées : filtration physique des solides en suspension
	et de microorganismes, précipitation chimique de minéraux, dégradation biologique et
	conversion de contaminants du carbone et de l'azote. Les processus biologiques sont le
	moins bien connus car ils impliquent aussi bien l'assimilation/dégradation de la matière
	organique que le développement de la biomasse/biofilm.
	Les empreintes SEC des protéines-like de la biomasse montrent au bout de 210 jours une
	tendance vers une augmentation des fractions de fortes masses molaires (MM) même si la
	répartition est un peu différente pour RS1 et les CA. Pour les EPS, l'évolution des fractions
	des protéines like est par contre opposée : alors que la fraction de très haute MM tend à
	diminuer au cours du temps pour RS1, cette fraction augment pour CA1.
	A 360 jours le rapport protéines/sucres diminue en fonction de la profondeur du biofilm
	et ce d'autant plus pour le biofiltre contenant CA1. La fraction de forte MM des protéines like
	tend à diminuer avec la profondeur dans le biofiltre et ceci pour les deux types de matériaux.

L'étude par une méthode de chromatographie d'exclusion stéarique tend à démontrer que les substances humiques présentes majoritairement dans la biomasse et l'EPS seraient issues d'une accumulation de l'effluent au niveau de la matrice extracellulaire, bien que des empreintes de substance humique-like soient identiques entre l'effluent et les EPS après 210 jours de fonctionnement.

Ces résultats montrent que la caractérisation de la matière organique de la biomasse et des EPS est un critère potentiel de choix pour différencier les deux types de matériaux dans les biofiltres, mais plus d'études sont nécessaires.

Le sable de rivière est généralement utilisé comme matériau de garnissage et comme support de biomasse dans les filtres. L'exploitation importante du sable de rivière rend la ressource fragile et nécessite la recherche de matériaux de substitution. Les agrégats concassés disponibles et économiquement avantageux sont envisagés en substitution du sable. Quelques études ont montré la possibilité d'utiliser des supports concassés comme matériaux de garnissage dans les lits filtrants mais les conséquences de cette substitution sur l'épuration des eaux ne semblent pas bien maitrisées. Une étude des impacts de la nature des matériaux de garnissage et de leurs caractéristiques physiques et chimiques sur le fonctionnement des filtres est donc nécessaire. Notre approche a consisté à rechercher des éléments de différenciation du comportement des filtres. En plus de l'efficacité épuratoire des filtres sur le carbone, l'azote et le phosphore, le développement et la stabilisation de la quantité et de la qualité du biofilm ont été suivis au cours du temps.

Table 3 .

 3 1: Principales caractéristiques chimiques et physiques des matériaux de filtration choisis

	Matériel	RS1	RS2	CA1	CA2
	Nature	La Loire	La Loire	carrière	carrière
	Photo de l'échantillon
	Effectivesize:D10(mm)	0.38	1.60	0.17	0.44
	diamètre moyen : Dm(mm)	0.82	2.26	1.36	1.6
	Coefficient d'uniformité (D60/D10)	2.78	1.75	10	5
	Particule fines (<0.08mm)%	0.4%	0.5%	5%	2.4%
	Rondeur (-)	0.74 (±0.11)	0.76 (±0.10)	0.73 (±0.15)	0.67 (±0.15)
	Ca relargué (mg/kg)	4.30	2.28	10.43	28.72
	III.2

. Impact des matériaux sur le fonctionnement du réacteur : hydrodynamique et transfert de l'O 2

  Plus le temps de séjour moyen dans les colonnes de filtration est important (HRT) (93h pour CA1 vs. 38h pour RS1) et plus le volume de rétention d'eau est fort (13% for CA1 and 8% for RS1). Cette rétention réduit également la conductivité hydraulique (coefficient de perméabilité) avec des valeurs très différenciées entre CA1 et RS1 (CA1: 3.25×10-4 m/s et Part ΙΙΙ: Conclusion générale

% Operating time (days)-70cm reactors RS1-20cm/day CA1-20cm/day

Remerciements

support technique à l'étude expérimentale.

SEC fingerprints. This difference should be exposed by the various material characteristics: increase in porosity and grain size, along with size heterogeneity during the process resulting in a longer biomass stabilization period.

Abbreviation

AB:

Lewis acid-base interactions aMW: apparent molecule weight (kDa)

Presence of EPS

The presence of EPS covering on a cell surface alters the physiochemical aspects of bacterial cell adhesion onto solid surface. The extracellular protein and amino acids increase the surface hydrophobicity thus favorite the attachment (Pell & Nyberg, 1989). The uronic acids enhance the anionic properties of some Gram negative bacteria and allow the association with the divalent cations to increasing the binding force (Molle et al., 2005).

Anionic polysaccharides and amphiphilic cell coatings both exhibit steric repulsion at hydrophilic and hydrophobic solid surface [START_REF] Rijnaarts | DLVO and steric contributions to bacterial deposition in media of different ionic strengths[END_REF]. The Figure 1.7 and Table 1.7 summarize the interactions involved in the bacterial adhesion to a solid surface.

(5)

Part Ι: Literature review

Impact of filter medium on the qualitative characteristics of biomass

As indicated by the total organic contents (VDW) in Section 3.1, the CA1 showed only slightly lower quantities at the top layer, yet these remained of the same scale both after 210 days of colonization (11mg/g RS1vs. 8mg/g CA1) and at the end of the process (13 mg/g RS1 vs. 10mg/g CA1). The CA2 exhibited a slightly lower final VDW (8mg/g aggregates, see supplemental data, S1). A more specific analysis was needed to investigate the impact of materials with different characteristics on biomass quality. The evolution of biochemical compositions in CA1 was also compared to the reference material. These results (using river sand as the reference) are displayed in Figures 8 and9. The biomass extracted from CA1 showed a similar organic retention behavior at the beginning of the process. Like for the river sand, increases in protein fraction have been observed, and the trend in biochemical fraction evolution did not deviate significantly (Fig. 9). In accordance with the VDW, the biochemical compositions of CA1 (Fig. 7) yielded smaller quantities than RS1 with all components except for polysaccharides. The PN/PS ratio increased from 1.07 to 1.98 (river sand: 0.75~2.44), while the PN/HS ratio moved from 0.54 to 1.57 (river sand: 0.47~1.44). The CA2 (see supplemental data, S2) provided a similar percentage change (PN/PS: from 0.77 to 3.37; PN/HAS: 0.24 to 1. 22), yet with even lower quantities of biochemical components. The similar trend in biochemical fraction evolution suggests that the biomass growth mechanism undergoes the same process as in river sand, despite the crushed aggregate characteristics differing significantly.

However, the quantities of biochemical components showed a biomass stabilization in river sand from 210 to 360 days (e.g. proteins only rose from 120 to 140 mg/kg) that was not replicated in CA1. The latter exhibited much lower components around 210 days and then significantly increased around 360 days (proteins jumping from 60 to 120mg/kg, see supplemental data, S2), while the CA2 did not increase noticeably at the end of the process (proteins moving from 60 to 90mg/kg). The lower PN/PS ratio in CA1 (1.98 for CA1 vs. 2.44

for RS) or smaller increases in biochemical components of CA2 suggests that the biomass metabolism has not equilibrated, even under the same conditions as in river sand, and (Sera soumis à Bioresource Technology) The evolution in biochemical component quantities of extracted EPS and biomass over time in both river sand (RS) and crushed aggregate (CA) (Fig. 5) suggests that the evolution in extracellular biochemical components undergoes the same process for both materials.

Enrichments have been indicated in: PN-like (with growth factors of 19 and 3 for RS and CA respectively), HS-like (by factors of 5 and 2 for RS and CA respectively), and PS (factors of 3 and 5 respectively) (Fig. 5). The majority of organic matter in an extracellular matrix is The HS-like fingerprints of the EPS extracted from the two materials have also been described from Day 60 to Day 360. These results are presented in Figure 8.

As mentioned in the previous section, HS-like were confirmed to possess weak MW (< 6 kDa) on Day 360 in both materials. The majority (i.e. major peak) fraction varied similarly between the two filter materials over the operating period. The small peaks located between 20 and 22 mL on Day 60 had moved to between 20 and 21 mL by Day 210 and wound up being positioned at 20 mL on Day 360. Let's note that, for CA especially, the area under peak evolution exhibits the same trend as the HS-like content observed in Figure 4 over time;

during this process, the aMW of HS-like increased. As mentioned in the previous section, the HS-like first absorbed in the biofilm might thus be the humic compounds of very weak MW or the HS-like modified by the biofilm; furthermore, the HS-like fingerprints of EPS in the two materials were similar to those of the feed water. During the first process interval, either: i) in considering the hypothesis of selective adsorption of HS-like from the effluent, smaller molecules are perhaps more easily and quickly adsorbed onto the biofilm; or ii) in considering the hypothesis of metabolism of HS-like by cells, HS-like from the environment are no longer used after implementation (hence no longer being degraded).

In deeper parts, bacteria feature less substrate. Furthermore, the proportions of polysaccharides in EPS, which increase with medium depth in CA and RS, might be due to the lower accessibility of nutrients or change in bacterial community, or a new C/N ratio, or various physical environments. Durmaz et al. (2001) however showed that a lower C/N ratio induces an increase in PN content and a decrease in PS content.