Acknowledgement

"No man grows great alone." After that, I moved to the lab Groupement de la Recherche Eau, Sol, Environnement (GRESE) of Université de Limoges. Here, I met one of my co-promoters Prof. Gilles Guibaud and also my mentor Dr. Isabelle Bourven, who gave me the most support, help and encouragement on my PhD topic. Without them, I cannot finish this PhD study successfully, I would like to give my greatest appreciation to them. Special thanks to my BFFs Dr. Natalie Sleimane, future Dr. Anne Rapin, Dr Asmaa Rouwane, Dr Frank Fré mion, Dr. Lulu Zhao, Dr. Cheng Wang and Dr. Zhiji Ding. Because of their company, support and encouragement, I never feel alone and helpless in a foreign country, and I also started to enjoy the life there. And I love this city, Limoges. The people I met there, they were all very nice and kind to me, which made me so comfortable and feel like coming home although I cannot speak or understand too much French language.

Next, I moved to the Netherlands, the country full of surprise and no language boundary. I have stayed in the lab of UNESCO-IHE Institute for Water Education for six months. There, I feel so lucky to know another co-promoter Prof. Piet Lens, his way of thinking impressed me a lot. And my mentor, Dr. Eldon Raj, his serious working style also influenced me. With their help, I became more confident on my topic. And thank you my friends: Dr. Alessandra Marra, Dr.

Nirakar Pradhan, future Dr. Lea Chua Tan, and Dr. Joyabrata Mal. With your company and support, my life in Delft city became more colorful. Besides, I also would like to thank the lab technicians Mr. Ferdi Battes, Mr. Peter Heerings and Mr. Berend Lolkema in IHE. Because of their help with the analytical instruments, I could finish all my experiments successfully and on time.

I heartedly thank my family, especially my mom. There is no word can describe your endless love and great support to me, which also provide me the motivation to go through all those tough times. Because of you, I can pursue my dreams without barrier. 妈妈,谢谢 你,我永远爱你。 Last but no least, I would like to thank all the people I met during these three years. It's YOU make my life more complete.

曹斐姝

Feishu CAO

August 2017 @China Summary Extracellular polymeric substances (EPS) are not only the "house" for the microbial cells, but they also facilitate several important cellular functions of the microorganisms. The hydrophobic properties of EPS exert a profound influence on the cell surface properties. However, many factors such as EPS extractions methods and substrate type influencing EPS characteristics as well as limited information regarding the hydrophobic features of EPS are reported. The main aim of this study is to develop a proper method to determine EPS hydrophobicity, and then investigate the hydrophobic features of the extracted EPS.

The hydrophobic fractionation of EPS by Supelite™ DAX-8 resin was first applied on the EPS extracted from anaerobic granular sludge, two elution pH conditions i.e. pH 2 and 5 were tested. The impact of seven different EPS extraction methods on the hydrophobic features of EPS was also assessed. The results showed that the extraction methods and bulk solution pH dramatically influenced the EPS composition and the measured EPS hydrophobicity, respectively.

Besides, the EPS extracting reagents namely formaldehyde, ethanol, sodium dodecyl sulfate (SDS) and Tween 20 not only introduced extra carbon content during total organic carbon (TOC) measurement, but also interacted with the DAX-8 resin. By comparing the apparent molecular weight (aMW) distribution of the untreated and pH-adjusted EPS samples, more complete aMW information of EPS was preserved at pH 5. Thus, the hydrophobic fractionation by DAX-8 resin at pH 5 and physical EPS extraction methods were preferred in this study for the following analysis of EPS extracted from anaerobic granular sludge.

After identifying the proper conditions for DAX-8 resin fractionation, detailed qualitative analysis of the EPS hydrophobic features was investigated by size exclusion chromatography (SEC) and excitation and emission fluorescence matrix (EEM) techniques. The results showed that the humic-like substances (HS-like) were the major organic constituent of EPS extracted from the anaerobic granular sludge, and they were also the main molecular support of EPS hydrophobicity (> 50%). Those hydrophobic HS-like compounds were mainly small molecules ranging from 8 kDa to <1 kDa, and their hydrophobicity could be ascribed to their humic acidlike molecular structure. Proteins (PN) and polysaccharides (PS) contributed to the EPS hydrophobicity to a lesser extent (< 30%, respectively).

Although the hydrophobic features of HS-like compounds were demonstrated, the role of PN and PS in the EPS hydrophobicity was difficult to be shown. It is known that the major organic constituents of the EPS extracted from fungi are PN and PS. Ni 2+ is the essential metal for the microorganisms and it is required as micronutrients at nano-molar concentrations, and it is also identified as the trace element for various biological systems such as anaerobic biochemical reactions. To explore the hydrophobic features of PN and PS, as well as to investigate the effect of metal addition (i.e. Ni 2+ ) under sub-toxic concentration on the EPS hydrophobicity, fungus Phanerochaete chrysosporium was chosen as the model microorganism. The results showed that the contents of PN and PS in the extracted fungal EPS varied with the initial Ni 2+ concentration added in the growth medium. With an increase in the initial Ni 2+ concentration from 0 mg/L to 25 mg/L, the PN content was increased whereas the PS content eventually remained stable. The hydrophobicity of fungal EPS, determined by the DAX-8 resin treatment, was gradually decreased as the Ni 2+ concentration increased. The zeta-potential of the fungus pellets was reduced by the presence of Ni 2+ . Besides, the peak intensity of the PN-like molecules ranging from 0.5 kDa to 14 kDa detected by SEC were intensified by the increased Ni 2+ concentration, while the aMW distribution of the total organics in the EPS remained almost stable. These results indicated that, under sub-toxic concentration of Ni 2+ , the presence of Ni 2+ promoted the fungus pellets becoming less hydrophobic, and the PN content was improved without changing its intrinsic fluorescence characteristics. Nevertheless, the molecular features of PS were still difficult to be elaborated and lack of specific spectroscopic properties that can be detected by EEM or SEC techniques.

Overall, the EPS extraction methods and bulk solution pH influenced both composition and hydrophobicity of the EPS. For the more heterogeneous EPS extracted from anaerobic granular sludge, HS-like compounds were identified as the major organic component, as well as the main molecular support of the EPS hydrophobicity. By studying the hydrophobic features of the EPS extracted from the fungus Phanerochaete chrysosporium, it showed that the PN and PS in the fungal EPS played an active role in protecting the fungus under Ni 2+ stress.

Re śume ́

Les polymè res extracellulaires (PEC) sont non seulement la « maison» pour les cellules microbiennes, mais ils facilitent é galement plusieurs fonctions cellulaires importantes des microorganismes. Les proprié té s hydrophobes (l'hydrophobicité) des PEC exercent une influence profonde sur les proprié té s de la surface cellulaire. Cependant, de nombreux facteurs tels que les mé thodes d'extractions et le type de substrat influencent les caracté ristiques des PEC extraits.

Egalement les informations concernant des caracté ristiques hydrophobes des PEC sont rarement documenté es. L'objectif principal de cette é tude est de dé velopper une mé thode approprié e pour é tudier l'hydrophobicité des PEC, puis d'é tudier les caracté ristiques moleculaires des PEC hydrophobes.

Le fractionnement hydrophobe des PEC par la résine Supelite™ DAX-8 a d'abord é té appliqué sur les PEC extraits de boues granulaires anaé robies, deux conditions de pH d'é lution, pH 2 et 5, ont é té testé es. L'impact de sept diffé rentes mé thodes d'extraction sur les caracté ristiques hydrophobes des PEC a é té é valué . Les ré sultats ont montré que les mé thodes d'extraction et le pH de la solution extractrice ont considé rablement influencé la composition biochimique des PEC et leur hydrophobicité . En outre, les extraitants des PEC, par exemple le formaldé hyde, l'é thanol, le dodé cylsulfate de sodium (SDS) et Tween 20, ont non seulement introduit une quentité supplé mentaire de carbone pendant la mesure du carbone organique total (COT), mais ils ont é galement interagit avec la ré sine DAX-8. En comparant la ré partition du poids molé culaire apparent (aMW) des é chantillons des PEC non traité s et ajusté s à pH 2 ou 5, les PEC sont mieux pré servé s à pH 5. Ainsi, le fractionnement hydrophobe par la ré sine DAX-8 à pH 5 et les mé thodes physiques d'extraction PEC ont é té pré fé ré s dans cette é tude pour l'analyse suivante des PEC extraits de boues granulaires anaé robies.

Aprè s avoir identifié les conditions approprié es au fractionnement par la ré sine DAX-8, une analyse qualitative dé taillé e des caracté ristiques hydrophobes des PEC extraits a é té effectué par chromatographie d'exclusion sté rique (SEC pour size exclusion chromatography en anglais) et la matrice de fluorescence d'excitation-emission (EEM). Les ré sultats ont montré que les substances de type humique (HS-like) repré sentaient la majorité des composé s organiques des PEC extraits de la boue granulaire anaé robie et constituaient é galement le principal support molé culaire de l'hydrophobicité des extraits (> 50%). Ces composé s hydrophobes de type HS-like é taient essentiellement des molé cules petites tailles de 8 kDa à <1 kDa, et leur structure similaire à celle de l'acide humique contribue à leur hydrophobicité. La contribution à l'hydrophobicité par les proté ines (PN) et les polysaccharides (PS) est d'une moindre importance (< 30%, respectivement).

Bien que les caracté ristiques hydrophobes des composé s HS-like aient é té dé montré es, le rôle de PN et de PS dans l'hydrophobicité é tait difficile à é tablir. Il est notable que les principaux constituants organiques des PEC extraits des bacté ries, des algues et des micro-champignons sont PN et PS. Ni 2+ est le mé tal essentiel pour les microorganismes et il est né cessaire en tant que micronutriments aux concentrations nano-molaires, et il est é galement identifié comme l'oligoé lé ment pour divers systè mes biologiques tels que les ré actions biochimiques anaé robies. Par conséquent, afin d'explorer les propriétés hydrophobes de PN et de PS, ainsi é valuer l'impact de mé tal (i.e. Ni 2+ ) à concentration sous-toxique sur l'hydrophobicité des extraits des microchampignons, Phanerochaete chrysosporium a é té choisi. Les ré sultats ont montré que la teneur de PN et de PS dans les PEC extrait de ce type de micro-champignons variait en fonction de la concentration de Ni 2+ . Avec une augmentation de la concentration de Ni 2+ de 0 mg/L à 25 mg/L, la teneur en PN a augmenté alors que celle de PS a é té finalement resté stable. L'hydrophobicité des PEC du fongi, dé terminé e par le traitement de la ré sine DAX-8, a diminué alors que la concentration de Ni 2+ augmentait. Le potentiel zê ta des micro-champignons a é té ré duit par la pré sence de Ni 2+ . Par ailleurs, l'intensité du pic des empreintes SEC correspondant aux molé cules PN-like de 0,5 kDa à 14 kDa a é té augmenté e par l'addition Ni 2+ ; en mê me temps, la distribution d'aMW selon les empreintes SEC des composé s organiques totaux dans les PEC restait presque stable. Ces ré sultats ont indiqué que, sous une concentration sous-toxique de Ni 2+ , la pré sence de Ni 2+ a favorisé le dé veloppement du micro-champignon devenant moins hydrophobe et la teneur en PN amé lioré e sans changer ses caracté ristiques de fluorescence intrinsè que. Né anmoins, les caracté ristiques molé culaires de la PS é taient encore difficiles à é laborer, car elles n'avaient pas de caracté ristiques spectroscopiques spé cifiques à dé tecter par les techniques EEM ou SEC. Dans l'ensemble, les mé thodes d'extraction des PEC et le pH de la solution extractante ont influencé la composition et l'hydrophobicité des PEC. Dans l'extrait plus hé té rogè ne des PEC de boues granulaires anaé robies, des composé s HS-like repré sentaient le composant organique majeur, ainsi le principal support molé culaire de l'hydrophobicité des PEC. En é tudiant les caracté ristiques hydrophobes des PEC extrait du micro-champignon Phanerochaete chrysosporium, le PN et le PS des PEC jouaient un rôle actif dans la protection du microchampignon sous le stress Ni 2+ .

Sommario

Le sostanze polimeriche extracellulari (EPS) non sono solo la "casa" delle cellule microbiche, ma esse facilitano anche molte importanti funzioni cellulari dei microrganismi. Le proprietà idrofobiche delle EPS esercitano un'influenza profonda sulle proprietà della superficie cellulare.

Tuttavia, sono riportati molti fattori, come i metodi di estrazione delle EPS e il tipo di substrato, che influenzano le caratteristiche delle EPS così come limitate sono le informazioni riguardanti le caratteristiche idrofobiche delle EPS. Obiettivo principale di questo studio è quello di sviluppare un metodo adeguato per determinare l'idrofobicità delle EPS e, quindi, indagare le caratteristiche idrofobiche delle EPS estratte.

Il frazionamento idrofobico delle EPS mediante resina Supelite™ DAX-8 è stato applicato per la prima volta sulle EPS estratte da fanghi granulari anaerobi e sono state testate due condizioni di pH di eluizione, cioè pH 2 e pH 5. È stato inoltre valutato l'impatto di sette diversi metodi di estrazione delle EPS sulle caratteristiche idrofobiche delle stesse. I risultati hanno mostrato che i metodi di estrazione e il pH della soluzione madre hanno influenzato drasticamente la composizione delle EPS e l'idrofobicità misurata delle EPS, rispettivamente. Inoltre, i reagenti estrattori delle EPS, cioè formaldeide, etanolo, sodio dodecil solfato (SDS) e Tween 20, non solo hanno introdotto contenuti extra di carbonio durante la misura del carbonio organico totale (TOC), ma hanno anche interagito con la resina DAX-8. Confrontando la distribuzione apparente del peso molecolare (aMW) dei campioni di EPS non trattati e con pH regolato, informazioni più complete su aMW delle EPS sono state conservate a pH 5. Quindi, il frazionamento idrofobico mediante resina DAX-8 a pH 5 e i metodi fisici di estrazione delle EPS sono stati preferiti in questo studio per la successiva analisi delle EPS estratte da fanghi granulari anaerobi. Dopo aver individuato le condizioni adeguate per il frazionamento della resina DAX-8, un'analisi qualitativa dettagliata delle caratteristiche idrofobiche delle EPS è stata condotta mediante cromatografia ad esclusione dimensionale (SEC) e mediante tecniche di spettroscopia a matrice di eccitazione e di emissione di fluorescenza (EEM). I risultati hanno mostrato che le sostanze simili a quelle umiche (HS) erano il principale componente organico delle EPS estratte dai fanghi granulari anaerobici ed erano anche il principale supporto molecolare dell'idrofobicità delle EPS (> 50%). Questi composti idrofobici simili a HS erano principalmente piccole molecole che vanno da 8 kDa a <1 kDa e la loro idrofobicità potrebbe essere attribuita alla loro struttura molecolare simile all'acido umico. Le proteine (PN) ei polisaccaridi (PS) hanno contribuito in misura minore (<30%) all'idrofobicità delle EPS.

XII

Sebbene il carattere idrofobico dei composti simili alle sostanze HS sia stato dimostrato, il ruolo di PN e PS nell'idrofobicità delle EPS è stato difficile da mostrare. È noto che i principali componenti organici delle EPS estratte dai funghi sono PN e PS. Ni 2+ è il metallo essenziale per i microrganismi ed è richiesto come micronutriente a concentrazioni nano-molari ed è anche identificato come elemento in traccia per vari sistemi biologici come le reazioni biochimiche anaerobiche. Per esplorare le caratteristiche idrofobiche di PN e PS, oltre che per studiare l'effetto dell'aggiunta di metallo (ossia Ni 2+ ) in condizioni di concentrazione sub-tossica per l'idrofobicità delle EPS, il fungo Phanerochaete chrysosporium è stato selezionato come microrganismo modello. I risultati hanno mostrato che il contenuto di PN e PS nelle EPS estratte da fungo variava con la concentrazione iniziale di Ni 2+ aggiunta nel mezzo di crescita. Con un aumento della concentrazione iniziale di Ni 2+ da 0 mg/L a 25 mg/L, il contenuto di PN è aumentato mentre il contenuto di PS è rimasto stabile. L'idrofobicità delle EPS fungine, determinata mediante trattamento con resina DAX-8, è stata gradualmente diminuita con l'aumentare della concentrazione di Ni 2+ . Il potenziale zeta dei pellet dei funghi è stato ridotto dalla presenza di Ni 2+ .

Inoltre, l'intensità di picco delle molecole simili a PN che vanno da 0,5 kDa a 14 kDa rilevate mediante SEC è stata intensificata dall'aumento della concentrazione di Ni 2+ , mentre la distribuzione aMW dei composti organici totali è rimasta pressoché stabile nelle EPS. Questi risultati hanno indicato che, in condizioni di concentrazione sub-tossica di Ni 2+ , la presenza di Ni 2+ ha reso i pellets di funghi meno idrofobici e il contenuto di PN è migliorato senza modificarne le caratteristiche intrinseche di fluorescenza. Tuttavia, le caratteristiche molecolari dei PS erano ancora difficili da elaborare per la mancanza di specifiche proprietà spettroscopiche che possono essere rilevate da tecniche EEM o SEC.

Nel complesso, i metodi di estrazione delle EPS e il pH della soluzione madre hanno influenzato sia la composizione che l'idrofobicità delle EPS. Per le EPS più eterogenee estratte da fanghi granulari anaerobi, i composti simili a HS sono stati identificati come il principale componente organico, nonché il supporto molecolare principale dell'idrofobicità delle EPS. Lo studio delle caratteristiche idrofobiche delle EPS estratte dal fungo Phanerochaete chrysosporium, ha mostrato che le PN e i PS nelle EPS funginee hanno giocato un ruolo attivo nella protezione del fungo in presenza di concentrazioni di Ni 2+ .

Samenvattig

Extracellulaire polymere stoffen (EPS) zijn niet alleen het "huis" voor de microbië le cellen, maar vergemakkelijken ook verschillende belangrijke cellulaire functies van de micro-organismen. De hydrofobe eigenschappen van EPS hebben een grote invloed op de eigenschappen van het celoppervlak. Echter, veel factoren, zoals EPS-extractiemethoden en substraattype die EPSeigenschappen beï nvloeden, alsmede beperkte informatie over de hydrofobe eigenschappen van EPS, worden gerapporteerd. Het hoofddoel van deze studie is het ontwikkelen van een goede methode om EPS-hydrofobiciteit te bepalen en vervolgens de hydrofobe eigenschappen van het geë xtraheerde EPS te onderzoeken. In het algemeen beï nvloeden de EPS-extractiemethoden en bulkoplossing pH zowel de samenstelling als de hydrofobiciteit van het EPS. Voor de meer heterogene EPS geë xtraheerd uit anaerobe granulair slib werden HS-achtige verbindingen geï dentificeerd als de belangrijkste organische component, evenals de belangrijkste moleculaire ondersteuning van de EPS hydrofobiciteit. Door het bestuderen van de hydrofobe eigenschappen van het EPS, geë xtraheerd uit de schimmel Phanerochaete chrysosporium, bleek dat de PN en PS in de schimmel EPS een actieve rol speelden bij het beschermen van de schimmel onder Ni 2+ stress.

De hydrofobe fractionering van EPS door

Chapter I.

Introduction 1.1 Background

Microbial cells are surrounded by a slime-like matrix called extracellular polymeric substances (EPS), which originate from the microbial secretion, cell lyses and/or sorption from the environment [START_REF] Flemming | The biofilm matrix[END_REF]. EPS not only form the three-dimensional scaffold for the microbial aggregates such as biofilms or flocs, but also involve several cellular functions: microbial adhesion to a surface, binding of metal ions or organic compounds, water retention, etc.

EPS from bacteria, fungus and algae mainly composes of proteins (PN) and polysaccharides (PS), as well as small amounts of nucleic acid, lipids, etc. [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF][START_REF] Ravella | Extracellular polysaccharide (EPS) production by a novel strain of yeast-like fungus Aureobasidium pullulans[END_REF][START_REF] Turu | Synthesis and characterization of cryogel structures for isolation of EPSs from Botryococcus braunii[END_REF]. In a mixed culture system such as biofilms or sludge flocs, the composition of extracted EPS is more heterogeneous, which also contain high amounts of humiclike substances (HS-like) and mineral particles containing (calcium or ferric phosphates, carbonates, oxides, etc.) [START_REF] D'abzac | Metal binding properties of extracellular polymeric substances extracted from anaerobic granular sludges[END_REF].

Previous studies evidenced that microbial adhesion, a fundamental step for the formation of microbial aggregates, strongly depends on the hydrophobic-hydrophilic structure of the interacting surfaces. Increasing cell surface hydrophobicity could facilitate the cell surface approaching, and trigger the specific forces that are responsible for the irreversible adhesion (Ding et al., 2015a). The hydrophobic properties of EPS significantly influence the cell surface properties [START_REF] Lin | A critical review of extracellular polymeric substances (EPSs) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies[END_REF][START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF], and the hydrophobic areas in the EPS are also related to organic pollutants removal [START_REF] Spä Th | Sorption properties of biofilms[END_REF].

The relative ratio between the hydrophilic and the hydrophobic fraction in the EPS depends on the EPS composition, and the composition largely depends on the extraction methods.

EPS extraction is based on the interruption of the chemical interactions (such as hydrogen bonds, electrostatic, hydrophobic or van der Waals interactions) that are responsible for the stabilization of three-dimensional structure of EPS via physical forces or chemical reagents. Physical extraction methods such as centrifugation, heating and sonication breaks the low-energy bonds between the EPS molecules. Organic reagents like formaldehyde, sodium dodecyl sulfate (SDS) and Tween 20 are also used to extract EPS, and the extraction mechanism is primarily based on the interaction between the reagents and certain functional groups harbored in the EPS [START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF].

Hydrophobic fractionation by XAD/DAX resin is one of the frequently used technique to fractionate the natural organic matter (NOM) from the water. This technique isolates the hydrophobic/hydrophilic fractions of the NOM, and thus, facilitates the hydrophobicity studies on the NOM [START_REF] Bolto | Experimental evaluation of cationic polyelectrolytes for removing natural organic matter from water[END_REF][START_REF] Marhaba | Modified dissolved organic matter fractionation technique for natural water[END_REF][START_REF] Leloup | Organic matter produced by algae and cyanobacteria: quantitative and qualitative characterization[END_REF][START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF].

Classical XAD/DAX resin fractionation procedures include: (i) acidification of the samples to pH 2 in order to protonate the samples to an uncharged state; and (ii) hydrophobic/hydrophilic fractionation by the resin XAD-8/DAX-8 and XAD-4 in tandem use. Organic acids become protonated at acidic condition, and with the aid of certain hydrophobic carbon skeletons these acidic organic solvents are sorbed by the resin via hydrophobic interaction, and thus, considered hydrophobic [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF]. Generally, samples are separated into three fractions by passing through XAD/DAX resin series: hydrophobic fraction (HPO) is the part adsorbed on the XAD-8/DAX-8 resin at pH 2, transphilic fraction (THP) is the elute of XAD-8 which then adsorbs to XAD-4 at pH 2, and hydrophilic fraction (HPI) is the elute from both columns in series at pH 2 [START_REF] Aiken | Isolation of hydrophilic organic acids from water using nonionic macroporous resins[END_REF][START_REF] Leenheer | Comprehensive approach to preparative isolation and fractionation of dissolved organic carbon from natural waters and wastewaters[END_REF].

Trace amounts of metals (Fe, Zn, Ni, Cu, and Co) are simultaneously viewed as potential contaminants as well as micronutrients in the environment and biological processes [START_REF] Gikas | Single and combined effects of nickel (Ni(II)) and cobalt (Co(II)) ions on activated sludge and on other aerobic microorganisms: a review[END_REF][START_REF] Thanh | Trace metal speciation and bioavailability in anaerobic digestion: a review[END_REF]. The functional groups harbored in the EPS, such as carboxyl, phosphoric, hydroxyl, sulfhydryl and amino groups, can bind with the metal cations [START_REF] Azeredo | Methods to extract the exopolymeric matrix from biofilms: a comparative study[END_REF][START_REF] Liu | Extraction of extracellular polymeric substances (EPS) of sludges[END_REF][START_REF] Rudd | Complexation of heavy metals by extracellular polymers in the activated sludge process[END_REF]. The metal binding ability of EPS is affected by the EPS hydrophobic properties, as the hydrophilic fraction of EPS could provide more reactive sites for the immobilization of metal ions than the hydrophobic fraction [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF]Li et al., 2016;[START_REF] Vá Zquez-Juá Rez | Cell surface hydrophobicity and its relation to adhesion of yeasts isolated from fish gut[END_REF]. The immobilized metal cations could reduce the negative charges of the cell surface [START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF], and divalent cations like Ca 2+ and Mg 2+ are also indispensable bridging ions to stabilize the three-dimensional structure of EPS [START_REF] Flemming | The biofilm matrix[END_REF]. Since the EPS may determine the physicochemical properties and maintain the structural and functional integrity of the microbial aggregates, therefore, the cell surface properties as well as the wastewater treatment performance of the reactors are both influenced by the interactions between the metal ions and the EPS [START_REF] Li | Effects of Fe(III) on floc characteristics of activated sludge[END_REF][START_REF] Liu | Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge[END_REF]. Li et al. (2016a) claimed that PN were responsible for the cell surface hydrophobicity of anaerobic granules, and non-polar groups such as side chains of aromatic amino acids were pivotal factors impacting the EPS hydrophobicity. Several studies demonstrated that acetylated PS form the hydrophobic moiety in the EPS structure and support the EPS hydrophobicity [START_REF] Mayer | The role of intermolecular interactions: studies on model systems for bacterial biofilms[END_REF][START_REF] Neu | Structural studies of an emulsionstabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain[END_REF]. HS-like compounds are the major constituent of EPS extracted from the sludge used in wastewater treatment processes (Cao et al., 2017;[START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF][START_REF] Yuan | Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge[END_REF], but the hydrophobic features of these HS-like compounds in the EPS are usually neglected in the EPS-related studies. Some inconsistent conclusions regarding the role of EPS hydrophobicity in the microbial aggregates are reported in the literature. [START_REF] Zhang | Composition of EPS fractions from suspended sludge and biofilm and their roles in microbial cell aggregation[END_REF] found that after the EPS extraction, the hydrophobicity of cell suspensions was higher than before the extractions steps. In contrast, [START_REF] Zhao | Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS)[END_REF] showed that surface hydrophobicity of intact bacteria was higher than loosely bound EPS (LB-EPS) free bacteria according to the microbial adhesion to hydrocarbon (MATH) test.

Problem statement

When different EPS extraction methods are applied, different EPS composition as well as different EPS properties can be observed [START_REF] Zuriaga-Agustí | Influence of extraction methods on proteins and carbohydrates analysis from MBR activated sludge flocs in view of improving EPS determination[END_REF]Domí nguez et al., 2010a;Comte et al., 2006a). The difference in extraction methods also affects the possible intercomparison that could be made between the studies. The influence of extraction methods on the EPS hydrophobic properties should be taken as the preliminary step to develop the EPS studies.

When exposed to a toxic or inhibitory substrate, microorganisms are still able to regulate their cell surface properties. Many heavy metals or toxic hydrocarbons present as environmental pollutants, but they are also the potential substrates for the microorganisms. The tolerance to the metals or organic contaminants exhibited by the microorganisms are probably related to the specific nutritional requirements/mechanisms in the cells [START_REF] Anahid | Heavy metal tolerance of fungi[END_REF][START_REF] Valix | Adaptive tolerance behaviour of fungi in heavy metals[END_REF]. To increase the accessibility of those low bioavailability compounds, one specific adaptive mechanism developed by the microorganisms is the modification of cell surface properties i.e.

EPS production to permit direct interactions with those substrates [START_REF] Heipieper | Surface properties and cellular energetics of bacteria in response to the presence of hydrocarbons[END_REF].

Researches have focused on the biosorption/biodegradation capacity of EPS aiming at metal/organic pollutants removal. Nevertheless, when metals are added as trace elements for the microbial growth, the information related to the effect of metal on the surface properties of the microorganisms i.e. EPS hydrophobicity is limited.

In addition, aqueous NOM samples are commonly acidified to pH 2 before the hydrophobic fractionation by XAD/DAX resin, as it maximizes the hydrophobicity of NOM [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF]. Nevertheless, lower pH may disrupt EPS structure and cause more precipitation of the EPS molecules [START_REF] Bey | Structure and properties of native and unfolded lysing enzyme from T. harzianum: chemical and pH denaturation[END_REF]. Therefore, to maintain acidic conditions to protonate EPS charge for the DAX-8 resin fractionation, as well as to minimize the EPS denaturation, a proper elution pH condition should be considered.

Research objectives

The main aim of this study is to investigate the hydrophobic features of EPS by using Supelite ™ DAX-8 resin, and to explore the effect of metal ions i.e. Ni 2+ on the EPS hydrophobic features.

Therefore, this research was divided into three specific objectives, as follows:

• Establish an appropriate hydrophobic fractionation procedure by using DAX-8 resin to determine EPS hydrophobicity, and evaluate the impact of extraction methods on the determination of EPS hydrophobicity.

• Investigate the hydrophobic features of EPS in terms of molecular weight distribution, fluorescence characteristics, and the molecular support of EPS hydrophobicity.

• Study the hydrophobic features of EPS extracted from pure culture i.e. Phanerochaete chrysosporium, and investigate the evolution of EPS characteristics under sub-toxic Ni 2+ concentration.

Thesis structure

This thesis is presented in six chapters as follows:

• Chapter I: Introduction

In this chapter, the background and the difficulties regarding the study of EPS hydrophobicity are included. The thesis structure is also introduced.

• Chapter II: Literature review

In this chapter, the role of cell surface hydrophobicity in the microbial aggreagtion is illustrated, and the techniques applied in the hydrophobicity determination are also introduced. The role of EPS in the cell surface hydrophobicity is briefly explained.

• Chapter III: Hydrophobic fractionation of EPS extracted from anaerobic granular sludge by DAX-8 resin

The aim of this chapter is to identify a proper procedure to determine the hydrophobicity of EPS extracted from the anaerobic granular sludge, and to illustrate the influence of EPS extraction methods and bulk solution pH on the EPS hydrophobicity.

• Chapter IV: Hydrophobic features of EPS extracted from anaerobic granular sludge treating wastewater from a paper recycling plant

The aim of this chapter is to investigate the hydrophobic features of EPS extracted from the anaerobic granular sludge. The hydrophobic features investigate include organics constitution, apparent molecular weight, and fluorescence characteristics. The main molecular support of EPS hydrophobicity is also discussed.

• Chapter V: Evolution of EPS characteristics in the fungus Phanerochaete chrysosporium when exposed to sub-toxic concentration of nickel (II)

The aim of this chapter is to investigate the hydrophobic features of EPS extracted from a pure culture, and to evaluate the evolution of EPS characteristics in the presence of Ni 2+ .

• Chapter VI: General discussion and perspectives

In this chapter, the results concluded from Chapter III to V are comprehensively discussed. Some perspectives regarding the future study are introduced.

Chapter II.

Literature Review

This chapter is submitted to the book Microbial Biofilms in Bioremediation and Wastewater Treatment as: Cao F., Bourven I., Lens P.N.L., van Hullebusch E.D., Pechaud Y., Guibaud G. Role of extracellular polymeric substances (EPS) in cell surface hydrophobicity.

Introduction

Industrial activities, such as the manufacturing of textile, paper and pulp, iron-steel, petroleum, pesticide or pharmaceutics, generate large amounts of wastewater containing various hazardous heavy metals or organic micro-pollutants. Due to the non-biodegradable characteristics of the heavy metals as well as the potential mutagenicity and carcinogenicity of some organic micropollutants, biofilms or sludge are usually used in the wastewater treatment system to adsorb and remove those pollutants from the wastewater [START_REF] Aksu | Application of biosorption for the removal of organic pollutants: a review[END_REF][START_REF] Wang | Adsorption of p-chlorophenol by biofilm components[END_REF][START_REF] Wicke | Environmental influences on the partitioning and diffusion of hydrophobic organic contaminants in microbial biofilms[END_REF].

Microbial aggregation is one fundamental feature involved in the biofilm formation, granulation and sludge flocculation (Adav and Lee, 2008a;[START_REF] Dufrê Ne | Sticky microbes: forces in microbial cell adhesion[END_REF]. It can be regarded as a process starting with the individual cells adhering to each other via cell-to-cell interactions. The process of microbial aggregation is mediated by a multitude of molecular interactions including specific (i.e. molecular recognition between receptors and ligands) or non-specific (i.e. hydrogen bonding, hydrophobic, van der Waals, electrostatic, and macromolecular forces) interactions [START_REF] Busscher | Specific molecular recognition and nonspecific contributions to bacterial interaction forces[END_REF]. Hydrophobic interactions take place when particles or molecules are incapable of interacting electrostatically or forming hydrogen bonds with the water molecules [START_REF] Magnusson | The hydrophobic effect and how it can be measured with relevance for cell-cell interactions[END_REF]. These hydrophobic interactions between the cells are usually evaluated in terms of cell surface hydrophobicity [START_REF] Guo | Variations of both bacterial community and extracellular polymers: the inducements of increase of cell hydrophobicity from biofloc to aerobic granule sludge[END_REF][START_REF] Zita | Effects of bacterial cell surface structures and hydrophobicity on attachment to activated sludge flocs[END_REF]. In wastewater treatment processes, sludge settling, granulation, dewatering as well as membrane fouling are strongly related to the cell surface hydrophobicity [START_REF] Lin | A critical review of extracellular polymeric substances (EPSs) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies[END_REF][START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF].

Analytical techniques usually applied in the hydrophobicity measurement are contact angle measurements (CAM) [START_REF] Pen | Effect of extracellular polymeric substances on the mechanical properties of Rhodococcus[END_REF], hydrophobic interaction chromatography (HIC) [START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF], salt aggregation tests (SAT) [START_REF] Lindahl | A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells[END_REF][START_REF] Rozgonyi | Improvement of the salt aggregation test to study bacterial cell-surface hydrophobicity[END_REF], and microbial adhesion to hydrocarbon (MATH) tests [START_REF] Gao | Influence of extracellular polymeric substances on microbial activity and cell hydrophobicity in biofilms[END_REF][START_REF] Rosenberg | Adherence of bacteria to hydrocarbons: A simple method for measuring cell-surface hydrophobicity[END_REF].

Besides those techniques, hydrophobic/hydrophilic fractionation by XAD/DAX resin is frequently used to fractionate natural organic matter (NOM) in the water. This technique can isolate the hydrophobic/hydrophilic fractions of NOM according to their polarity and thus, facilitates hydrophobicity studies [START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF][START_REF] Thurman | Prediction of capacity factors for aqueous organic solutes adsorbed on a porous acrylic resin[END_REF].

Extracellular polymeric substances (EPS) are located outside the cells and account for the major part of the total organic carbon (TOC) in the biofilms or sludge [START_REF] Nielsen | Extraction of EPS[END_REF].

They exert a profound impact on the cell surface hydrophobicity (Liu et al., 2004a), and enhance the affinity of heavy metals or organic micro-pollutants to the microorganisms [START_REF] Jia | Degradation of pyrene in soils by extracellular polymeric substances (EPS) extracted from liquid cultures[END_REF][START_REF] Wei | Adsorption of Cu 2+ and Zn 2+ by extracellular polymeric substances (EPS) in different sludges: effect of EPS fractional polarity on binding mechanism[END_REF]. Two operationally defined fractions, soluble and bound EPS, constitute the EPS [START_REF] Nielsen | Conceptual model for production and composition of exopolymers in biofilms[END_REF]. Soluble EPS (colloids, slimes) reside in the surrounding of microbial cells, which are protecting the cells against severe conditions. However, it is difficult to distinguish between produced soluble microbial products (SMP) and soluble EPS [START_REF] Laspidou | A unified theory for extracellular polymeric substances, soluble microbial products, and active and inert biomass[END_REF]. Bound EPS closely associate with the cell walls and consist of two layers. The outer layer is a loose and dispersible slime layer without an obvious edge called loosely bound EPS (LB-EPS). While, the inner layer has a certain shape and binds tightly and stably with the cell surface are called tightly bound EPS (TB-EPS) [START_REF] Comte | Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods[END_REF][START_REF] Nielsen | Extraction of EPS[END_REF].

The main organic fraction determined in the EPS of multispecies microbial aggregates include proteins (PN), polysaccharides (PS), humic-like substances (HS-like), and small amounts of nucleic acids, lipids or uronic acids [START_REF] Comte | Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods[END_REF][START_REF] Sheng | Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review[END_REF]. Besides organic macromolecules, mineral particles (calcium or ferric phosphates, carbonates, oxides, etc.) are also embedded in the EPS matrix [START_REF] D'abzac | Metal binding properties of extracellular polymeric substances extracted from anaerobic granular sludges[END_REF]. The EPS composition is controlled by several factors, such as microbial type, nutrients and local environmental conditions (i.e. pH, temperature, and metal concentration) [START_REF] Sheng | Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review[END_REF]. There is no standard method to extract the EPS from biofilms and microbial aggregates. The extraction yield, organic composition, molecular structure as well as metal binding ability of the EPS are all affected by the extraction method used [START_REF] Comte | Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods[END_REF][START_REF] Domí Nguez Chabaliná | Characterization of soluble and bound EPS obtained from 2 submerged membrane bioreactors by 3D-EEM and HPSEC[END_REF][START_REF] Zuriaga-Agustí | Influence of extraction methods on proteins and carbohydrates analysis from MBR activated sludge flocs in view of improving EPS determination[END_REF].

The hydrophilic/hydrophobic properties of EPS are attributed to the different functional groups (i.e. carboxyl, hydroxyl, phosphate, amine, and sulfate) and non-polar regions (i.e. hydrophobic regions in O-methyl/acetyl polysaccharides; aromatics and aliphatic regions in proteins) [START_REF] Moran | Microbial Glycobiology: Structures, Relevance and Applications[END_REF]. Nevertheless, the role of EPS in the cell surface hydrophobicity is not well identified. [START_REF] Zhang | Composition of EPS fractions from suspended sludge and biofilm and their roles in microbial cell aggregation[END_REF] found that after LB-EPS and TB-EPS extraction, the hydrophobicity of the cell suspensions was higher than before the extractions steps. In contrast, [START_REF] Guo | Variations of both bacterial community and extracellular polymers: the inducements of increase of cell hydrophobicity from biofloc to aerobic granule sludge[END_REF] showed that after EPS extraction, the cell surface hydrophobicity of the aerobic granules decreased.

A better characterization of EPS from both quantitative and qualitative aspects could improve the understanding of the microbial aggregation (i.e. biofilm formation, granulation and sludge flocculation), as well as the monitoring of pollutants removal in wastewater treatment processes. Therefore, this chapter is focusing on the role of EPS in the cell surface hydrophobicity of pure cultures (bacteria, fungi and algae) and mixed-culture aggregates such as biofilms, aerobic/anaerobic granules and activated sludge. The role of cell surface hydrophobicity in the microbial aggregation, the relation between EPS and cell surface hydrophobicity, and the techniques applied in the characterization of hydrophobicity are aslo described.

Hydrophobicity in microbial aggregation

Hydrophobic interactions are viewed as the spontaneous tendency of non-polar groups to aggregate or cluster to minimize their contact with the water molecules (Fig. 2.1). Despite nonpolar solutes have low solubility in water, the hydrophobic interactions are responsible for the adhesion between the hydrophobic surfaces. These interactions are also the cause of rapid coalescence or flocculation that is commonly observed in colloidal systems of hydrophobic liquid droplets or solid particulates [START_REF] Meyer | Recent progress in understanding hydrophobic interactions[END_REF]. In a sense of process thermodynamics, microbial aggregation is driven by free energy decrease, thereby increasing the cell surface hydrophobicity would cause a corresponding decrease in excess Gibbs energy of the surface.

Therefore, cell surface hydrophobicity promotes the cell-to-cell interaction and further serves as a triggering force for the cells to aggregate in the hydrophilic liquid phase (Liu et al., 2004a).

Microbial adhesion

Microbial aggregation, such as biofilm formation and granulation, is initiated by the microbial adhesion. The early stage of microbial aggregation begins with the attachment of planktonic microorganisms to a biotic or abiotic surface. These first colonists initially adhere to the surface through non-specific, long-range attractive Lifshitz-van der Waals forces as well as electrostatic, acid-base and hydrophobic forces [START_REF] Chung | Interaction forces between spores and planar surfaces in aqueous solutions[END_REF][START_REF] Israelachvili | Interactions involving polar molecules[END_REF].

Hydrophobic interactions affect the adhesion process by removing the vicinal water film between the cell surface and the substratum. In another words, hydrophobic groups use their dehydrating capacity to remove the water and aid the cell appendages to attach to the surface [START_REF] Busscher | Interfacial rearrangement in initial microbial adhesion to surfaces[END_REF][START_REF] Busscher | Specific and non-specific interactions in bacterial adhesion to solid substrata[END_REF]. In low ionic strength environment, cell surface hydrophobicity plays an important role in determining the adhesion of microorganisms to the surface ( van Loosdrecht et al., 1987b), as the repulsion between the microorganisms and the surface increases with the decrease of the cell surface hydrophobicity [START_REF] Muadcheingka | Distribution of Candida albicans and nonalbicans Candida species in oral candidiasis patients: Correlation between cell surface hydrophobicity and biofilm forming activities[END_REF][START_REF] Pimentel-Filho | Bovicin HC5 and nisin reduce Staphylococcus aureus adhesion to polystyrene and change the hydrophobicity profile and Gibbs free energy of adhesion[END_REF][START_REF] Stenström | Bacterial hydrophobicity, an overall parameter for the measurement of adhesion potential to soil particles[END_REF]. Uniform, flat, and thin biofilms can rapidly form on the hydrophobic surface of polyvinylidene fluoride (PVDF), whilst the hydrophilic surface of polyvinyl alcohol (PVA) prevents the adhesion of Pseudomonas putida and the biofilm formation [START_REF] Saeki | Effect of hydrophobicity of polymer materials used for water purification membranes on biofilm formation dynamics[END_REF]. However, [START_REF] Parkar | Factors influencing attachment of thermophilic bacilli to stainless steel[END_REF], [START_REF] Flint | The influence of cell surface properties of thermophilic streptococci on attachment to stainless steel[END_REF] and [START_REF] Sorongon | Hydrophobicity, adhesion, and surface-exposed proteins of gliding bacteria[END_REF] demonstrated that there was little or no relationship between the cell surface hydrophobicity and the bacterial cell adhesion to the solid substratum.

The development of granular biofilms is a complex process and initiated by the formation of smaller microbial aggregates (Adav and Lee, 2008a). Cell surface hydrophobicity induced by the culture conditions could serve as the triggering force for both anaerobic and aerobic granulation (Liu et al., 2004a). With the increase of cell surface hydrophobicity, the granulation process is accelerated [START_REF] Liu | Physicochemical characteristics of microbial granules[END_REF]. [START_REF] Xu | Anaerobic granulation with methanol-cultured seed sludge[END_REF] used high surface hydrophobicity methanogen-enriched sludge to seed the upflow anaerobic sludge blanket (UASB) reactor. They found that the granulation process reached its post-maturation stage in about 15 -20 days ahead of the control reactor in which normal seed sludge was used.

One of the models to elaborate anaerobic granulation is the local dehydration and hydrophobic interaction model proposed by [START_REF] Wilschut | Membrane fusion: from liposomes to biological membranes[END_REF]. This model shows that irreversible adhesion will occur when the bacterial surface is strongly hydrophobic. Bacteria present in the different layers of anaerobic granules have different hydrophobicity: a surface layer with moderate hydrophobicity, a middle layer with extremely high hydrophobicity, and a core with high hydrophobicity [START_REF] Daffonchio | Contact angle measurement and cell hydrophobicity of granular sludge from upflow anaerobic sludge bed reactors[END_REF]. Non-granular sludge washed out from anaerobic reactors was more hydrophilic than the sludge retained in the reactors [START_REF] Mahoney | The effect of calcium on microbial aggregation during UASB reactor start-up[END_REF]. Likewise, aerobic granulation also follows a function of cell surface hydrophobicity over hydrophilicity, i.e. high cell surface hydrophobicity strongly favors microbial aggregation and results in a more compact granular structure (Liu et al., 2003). Therefore, a proper cell surface hydrophobicity is one of the important criteria to enhance sludge granulation performance [START_REF] Yu | Effects of added powdered and granular activated carbons on start-up performance of UASB reactors[END_REF].

Sludge flocculation, settling and dewatering

An effective wastewater treatment by using activated sludge is largely depending on the sludge flocculation and settling. However, it is difficult to manipulate sludge flocculation and settling processes, due to the complex nature and interactions among the microbial communities in the sludge [START_REF] Wilé N | Relationship between flocculation of activated sludge and composition of extracellular polymeric substances[END_REF]. [START_REF] Zita | Effects of bacterial cell surface structures and hydrophobicity on attachment to activated sludge flocs[END_REF] found that the cell surface hydrophobicity of Escherichia coli and the bacteria isolated from wastewater correlated well with the degree of the adhesion to the sludge flocs, and they postulated that a low level of cell surface hydrophobicity could be the reason why planktonic cells did not attach to the flocs. [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF] demonstrated that the sludge with higher cell surface hydrophobicity produced an effluent of lower turbidity, but it had no effect on the sludge volume index (SVI). Thus, they inferred that the cell surface hydrophobicity was crucial to the flocculation, but not the settling. Nevertheless, [START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF] reported that intrafloc hydrophobic interactions enhanced the sludge settling.

Sludge dewatering is the bottleneck in many wastewater treatment processes. The difficulty in monitoring sludge dewatering is also due to the physical, chemical and microbial complexity in the sludge [START_REF] Liu | Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge[END_REF]. Many efforts have been made to improve the sludge dewatering performance. For example, some hydrophobically modified polymers (e.g., cationic polyacrylamide) are often added as flocculants to improve the performance. One of the main mechanisms is that those macromolecular flocculants can span and reduce the gap between the particles through a bridging effect. This effect occurs when the flocculant polymer chains interact with the hydrophobic groups on the activated sludge surface through the hydrogen bonding, van der Waals forces, and hydrophobic interaction [START_REF] Sun | Evaluation of dewatering performance and fractal characteristics of alum sludge[END_REF]. [START_REF] Huang | Synthesis of hydrophobic cationic chitosan flocculant and its sludge dewatering property[END_REF] synthesized hydrophobic cationic chitosan (HTCC) flocculants by reacting chitosan with epoxy propyl trimethyl ammonium chloride (EPTAC) and (2,3-epoxy propyl) dodecyl dimethyl ammonium chloride (EDC). They found that with the enhancement of HTCC hydrophobicity, a better dewatering performance of the activated sludge was achieved. In addition, a good dewatering performance by using thermo-sensitive polymers (N,Ndimethylaminopropylacrylamide) in the activated sludge was observed by [START_REF] Sakohara | Dewatering of activated sludge by thermosensitive polymers[END_REF].

They ascribed the improvement to the hydrophobic interactions between the thermo-sensitive polymer molecules and the activated sludge.

Membrane biofouling

Membrane biofouling is a result of biofilm formation on the solid substratum, in which membrane surface properties i.e. hydrophobicity play an important role [START_REF] Van Der Bruggen | How a microfiltration pretreatment affects the performance in nanofiltration[END_REF]. It is generally believed that a hydrophilic membrane leads to a lower membrane fouling potential rather than a hydrophobic one [START_REF] Evans | The influence of hydrophobicity, roughness and charge upon ultrafiltration membranes for black tea liquor clarification[END_REF][START_REF] Qu | Ultrafiltration membrane fouling caused by extracellular organic matter (EOM) from Microcystis aeruginosa: effects of membrane pore size and surface hydrophobicity[END_REF][START_REF] Weis | The influence of morphology, hydrophobicity and charge upon the long-term performance of ultrafiltration membranes fouled with spent sulphite liquor[END_REF]. However, since the cell surface hydrophobicity is not enough to evaluate the extent of fouling caused by the interaction between broth components and membrane materials (Zhang et al., 2015a), some inconsistent results were also reported. More hydrophobic polyvinylidene fluoride (PVDF) membranes gave the lesser fouling tendency compared to polysulfone (PSF) and cellulose acetate (CA) membranes [START_REF] Choo | Effect of anaerobic digestion broth composition on membrane permeability[END_REF]. Although the hydrophobicity of membranes PVDF, CA and polyether sulfones (PES) follows the order of PVDF > PES > CA, experimental fouling data from [START_REF] Chen | Interaction energy evaluation of soluble microbial products (SMP) on different membrane surfaces: role of the reconstructed membrane topology[END_REF] showed that the CA membrane was fouled more severely than PVDF and PES membranes.

Characterization of microbial hydrophobicity

Techniques that are usually applied in the hydrophobicity determination are the contact angle measurement (CAM) [START_REF] Pen | Effect of extracellular polymeric substances on the mechanical properties of Rhodococcus[END_REF], hydrophobic interaction chromatography (HIC) [START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF], salt aggregation test (SAT) [START_REF] Lindahl | A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells[END_REF][START_REF] Rozgonyi | Improvement of the salt aggregation test to study bacterial cell-surface hydrophobicity[END_REF], microbial adhesion to hydrocarbons (MATH) test [START_REF] Gao | Influence of extracellular polymeric substances on microbial activity and cell hydrophobicity in biofilms[END_REF][START_REF] Rosenberg | Adherence of bacteria to hydrocarbons: A simple method for measuring cell-surface hydrophobicity[END_REF], and hydrophobic/hydrophilic fractionation by XAD/DAX resin [START_REF] Jorand | Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances[END_REF]. It is noticed that the development of HIC and SAT methods are based on the hydrophobic properties of PN, while CAM and MATH methods give the general description of microbial hydrophobicity (Table 2.2).

The comparison between the results obtained from SAT and HIC methods showed a good correlation (p < 0.025) for Staphylococcus aureus [START_REF] Jonsson | Cell surface hydrophobicity of Staphylococcus aureus measured by the salt aggregation test (SAT)[END_REF].

Contact angle measurement (CAM)

One of the advantages of CAM is that this technique can non-destructively characterize cell surface hydrophobicity (Table 2.2). The measurement procedures include: depositing a drop of liquid on a layer made from the studied cells, and then measuring the angle (θ) between the surface of the cells layer and the tangent (angle) to the drop at the solid-liquid-air meeting point (Fig. 2.2) [START_REF] Kwok | Contact angle measurement and contact angle interpretation[END_REF]. The liquid is often replaced by the deionized (DI) water droplets. CAM can also be applied to measure the contact angle of dried sludge cakes [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF].

θ liquid microbial cells layer
The preparation work follows the procedure described by [START_REF] Busscher | Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion[END_REF]. The washed cells are firstly filtered on a cellulose filter (normally 0.45 µm pore size) to form a layer of cells. Then, the filter with the deposited cells layer is transferred into a solidified agar plate and dehydrated for some time to ensure the constant moisture content. Since the water evaporation is expected to influence the contact angle, the dehydration process should be carried out under controlled atmosphere (i.e. 20 -22 °C, 50 -60% relative humidity) [START_REF] Mozes | Methods for measuring hydrophobicity of microorganisms[END_REF]. The following step is to place a drop of DI water on the cells layer, and a video image system is used to view the sessile drop from the top. The drop shape is captured as soon as no further shirking of the water drop is observed, and the image of which is used to estimate the contact angle values.

Considering the possible penetration of the water drop into the cell cake, the image recording is usually performed immediately (within 2 -7 s) after depositing the water drop [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF][START_REF] Busscher | Measurement of the surface free energy of bacterial cell surfaces and its relevance for adhesion[END_REF].

According to the water contact angle, cell surface hydrophobicity is roughly classified into three categories: hydrophobic surface with a contact angle greater than 90°, medium hydrophobic surface with a contact angle range between 50° and 60°, and hydrophilic surface with a contact angle below 30° [START_REF] Mozes | Methods for measuring hydrophobicity of microorganisms[END_REF]. Most activated sludge and bacteria have a contact angle ranging from 20° to 45°, while some are higher than 50° or lower than 15° (Table 2.1).

However, the cells layer on the filter consists not only packed individual microorganisms but also of some unavoidably void spaces. The deposited drops on this thin porous cells layer can behave in different ways, depending on many characteristics of both the liquid and the cells (Table 2.2), such as liquid viscosity, surface tension, porosity of the cells layer, ratio of the porous layer thickness to the drop radius and surface Gibbs energy of the individual particles [START_REF] Marmur | Penetration of a small drop into a capillary[END_REF]. (adapted from [START_REF] Kwok | Contact angle measurement and contact angle interpretation[END_REF]).

Therefore, the contact angle measured can only be viewed as a relative indicator of cell surface hydrophobicity [START_REF] Gallardo-Moreno | Insights into bacterial contact angles: difficulties in defining hydrophobicity and surface Gibbs energy[END_REF].

Hydrophobic interaction chromatography (HIC)

HIC differentiates the molecules based on their hydrophobic properties. It primarily adopts the reversible interactions that take place between the hydrophobic regions of protein-derived macromolecules and weakly hydrophobic ligands (i.e. butyl, octyl and phenyl) attached to the stationary phase (column packing matrix) under a high concentration of salt mobile phase. The working principle of HIC is the "salting-out chromatography" proposed by [START_REF] Shepard | The chromatography of proteins. The effect of salt concentration and pH on the adsorption of proteins to silica gel[END_REF]. In general, the salts in the mobile phase reduce the solvation of the sample solutes. As the solvation decreases, hydrophobic regions in the sample molecules become exposed and are adsorbed by the stationary phase. The more hydrophobic the molecules are, a longer elution time is needed [START_REF] Lienqueo | Current insights on protein behaviour in hydrophobic interaction chromatography[END_REF].

Usually a decreasing salt gradient is applied to elute samples from the column so that the hydrophobic interactions can be intensified. The salts frequently used are called anti-chaotropic salts (also kosmotropic or lyotropic salt), which include sodium, potassium, ammonium sulfate, sodium nitrate, and sodium chloride in phosphate buffer at pH 7 [START_REF] Baca | A comprehensive study to protein retention in hydrophobic interaction chromatography[END_REF]. These salts have a higher polarity to bind water tightly, and their presence in the mobile phase benefits the stabilization of protein structure [START_REF] Xia | Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis[END_REF]. By using these salts, the exclusion of water between the proteins and the ligand surface can be induced, and thus promotes the hydrophobic interactions and protein precipitation (salting-out effect) [START_REF] Kunz | Zur Lehre von der Wirkung der Salze" (about the science of the effect of salts): Franz Hofmeister's historical papers[END_REF]. However, studies have shown that the retention of hydrophobic molecules in HIC is affected by the interplay of different factors (Table 2.2), such as pH, salt type and its concentration, ligand type and density in the column material, protein folding upon adsorption and kinetic of protein spreading [START_REF] Haimer | Hydrophobic interaction chromatography of proteins IV: kinetics of protein spreading[END_REF][START_REF] Jungbauer | Hydrophobic interaction chromatography of proteins: III. Unfolding of proteins upon adsorption[END_REF][START_REF] Mahn | Optimal operation conditions for protein separation in hydrophobic interaction chromatography[END_REF][START_REF] Nfor | High-throughput protein precipitation and hydrophobic interaction chromatography: salt effects and thermodynamic interrelation[END_REF][START_REF] Perkins | Protein retention in hydrophobic interaction chromatography: modeling variation with buffer ionic strength and column hydrophobicity[END_REF][START_REF] Xia | Evaluation of selectivity changes in HIC systems using a preferential interaction based analysis[END_REF].

The hydrophobicity determined by HIC is usually evaluated by the dimensionless retention time (DRT) [START_REF] Lienqueo | An expert system for the selection and synthesis of multistep protein separation processes[END_REF]. The calculation of the DRT is conducted according to Eq. 2.1:

(Eq. 2.1) where: tR is the time corresponding to the peak on the chromatogram; t0 is the time corresponding to the start of the salt gradient and tf is the time corresponding to the end of the salt gradient.

When DRT equals to 1, it indicates the tested sample is extremely hydrophobic. [START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF] utilized this method to characterize the hydrophobic EPS separated from biofilms. Their results showed that 97% of the hydrophobic EPS were represented by two distinct fractions: DRT 0.3 and DRT 0.9 (Table 2.1). They concluded that hydrophobic EPS were more abundant in the cohesive layers of the biofilms than in the top layer.

Salt aggregation test (SAT)

The SAT method is originally developed by [START_REF] Lindahl | A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells[END_REF] and applied to measure the cell surface hydrophobicity of bacteria [START_REF] Basson | Evaluation of adherence, hydrophobicity, aggregation, and biofilm development of Flavobacterium johnsoniae-Like Isolates[END_REF][START_REF] Mozes | Methods for measuring hydrophobicity of microorganisms[END_REF][START_REF] Polak-Berecka | The effect of cell surface components on adhesion ability of Lactobacillus rhamnosus[END_REF]. This method is also based on the salting-out effect: precipitation of cells by dosing salts (i.e. (NH4)2SO4) at neutral pH, and highly hydrophobic cells are precipitated at low salt concentration. The grade of cell precipitation can be evaluated by comparing the absorbance of the supernatant of cell suspensions with/without ammonium sulfate at a certain wavelength (usually at 280 or 600 nm). The percentage of absorbance decrease can be used to reflect the cell surface hydrophobicity, and a greater absorbance decrease is corresponding to a more hydrophobic cell surface [START_REF] Lindahl | A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells[END_REF][START_REF] Martí N-Cereceda | Characterization of extracellular polymeric substances in rotating biological contactors and activated sludge flocs[END_REF][START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF].

SAT can also be applied to investigate the cell surface hydrophobicity of sludge flocs.

Martí n-Cereceda et al. ( 2001) demonstrated that the percentage of absorbance decrease of biofilm EPS (28 -38%) was twice higher than that of activated sludge EPS (around 15%), implying that the EPS extracted from the biofilm were two times more hydrophobic than those extracted from the activated sludge (Table 2.1). [START_REF] Jonsson | Cell surface hydrophobicity of Staphylococcus aureus measured by the salt aggregation test (SAT)[END_REF] used the same technique but expressed the results in terms of the lowest molarity of ammonium sulfate (in the mixture with cells) at which gave visual cell aggregation. The same expression is also seen in [START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF], [START_REF] Mozes | Methods for measuring hydrophobicity of microorganisms[END_REF] and [START_REF] Rozgonyi | Improvement of the salt aggregation test to study bacterial cell-surface hydrophobicity[END_REF] (Table 2.1). According to [START_REF] Balebona | Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish[END_REF], the lowest molarity of ammonium sulfate is below 1 M, cells are considered as highly hydrophobic; the value between 1 M and 2 M, cells are moderately hydrophobic; and the value is above 2 M, cells are weakly hydrophobic or hydrophilic.

However, many hydrophobic bacterial cells will clump when ammonium sulfate is absent.

Therefore, this technique only provides a qualitative estimation of the relative rank of microbial hydrophobicity [START_REF] Liss | Flocculation in Natural and Engineered Environmental Systems[END_REF]. Besides, electrostatic interactions affect SAT results more than other hydrophobicity measurement techniques (Table 2.2). Temperature, pH, reaction time and the cell concentration also effect the results [START_REF] Rosenberg | Microbial adhesion to hydrocarbons: twenty-five years of doing MATH[END_REF].

Microbial adhesion to hydrocarbons (MATH) test

MATH test is a simple and widely used method to determine the relative hydrophobicity (RH) of bacterial or fungal cells. When it is applied in mixed cultures like activated sludge or granules, a glass homogenizer or tissue grinder is often required to make homogeneous cell suspensions [START_REF] Xie | Surface properties of bacteria from activated sludge in relation to bioflocculation[END_REF][START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF]. In the assay, the washed cells are suspended in PUM buffer at pH 7.1 (per liter: 22.2 g K2HPO4•3H2O, 7.26 g KH2PO4, 1.8 g urea, 0.02 g MgSO4•7H2O), and then vortexed in the presence of liquid hydrocarbon (i.e. n-hexadecane, n-octane, p-xylene). During the vortex procedure, the liquid hydrocarbon is dispersed into droplets and then adhere hydrophobic microbial cells. When the adhesion takes place, the cell-coated droplets will rise to form a stable foam (Fig. 2.3) [START_REF] Chao | Hydrophobicity of diverse bacterial populations in activated sludge and biofilm revealed by microbial adhesion to hydrocarbons assay and high-throughput sequencing[END_REF].

Fig. 2.3 Phase separation of microbial cells by adding hydrocarbon

Phase separation is usually completed within one or two minutes. The extent of adhesion to the hydrocarbon droplets is simply ascertained by the decrease in the cell-mediated turbidity of the lower aqueous phase. To measure the decrease, the lower aqueous phase is withdrawn by using a Pasteur pipette and transferred to a cuvette for absorbance (Abs) measurements at a wavelength (λ) of 400 or 600 nm [START_REF] Rosenberg | Adherence of bacteria to hydrocarbons: A simple method for measuring cell-surface hydrophobicity[END_REF]. Therefore, the difference in the Abs values of the aqueous phase before (Absλ(before)) and after (Absλ(after)) adding the hydrocarbon can be used to calculate RH (Eq. 2.2). The proper value of Absλ(before) is usually controlled at 0.4 -0.6.

(Eq. 2.

2)

It is generally considered that a cell surface corresponding to a RH (%) above 50% is highly hydrophobic; the values ranges between 20% and 50%, the cell surface is moderately hydrophobic and the value is below 20%, the cell surface is considered mostly hydrophilic [START_REF] Balebona | Hydrophobicity and adhesion to fish cells and mucus of Vibrio strains isolated from infected fish[END_REF][START_REF] Lee | A comparison of three methods for assaying hydrophobicity of pathogenic vibrios[END_REF]. Nevertheless, the cells cannot only adhere to the hydrocarbons, they also adhere to each other. Therefore, the MATH test does not measure the absolute cell surface hydrophobicity, but the interplay of all the physicochemical forces including hydrophobic interactions, van de Waals forces and electrostatic interactions (Table 2.2). In addition, the interference of electrostatic interactions in the MATH test can be reduced by performing the test under ionic conditions in which either the cells or the hydrocarbon droplets (or

Aqueous phase

Hydrocarbon phase both) are uncharged [START_REF] Geertsema-Doornbusch | Microbial cell surface hydrophobicity: the involvement of electrostatic interactions in microbial adhesion to hydrocarbons (MATH)[END_REF]. For this reason, several authors have carried out the test at a pH closer to the isoelectric points (IEP) of the solvents or the bacterial cultures to minimize the electrical interactions involved in the adhesion process, as well as to obtain a better indication of the microbial surface hydrophobicity. Moreover, the kinetics of cell adhesion also depends on the initial cell concentration. In Candida albicans M7, the mixing of cells at low concentrations with n-hexadecane led to a coalesce within seconds after vortexing, whereas higher cell concentrations yield stable cell-coated droplets and relatively high adhesion levels [START_REF] Rosenberg | Microbial adhesion to hydrocarbons (math)[END_REF].

Hydrophobic/hydrophilic fractionation by XAD/DAX resins

Besides above-mentioned techniques, hydrophobic/hydrophilic fractionation by XAD/DAX resins is frequently used to fractionate natural organic matter (NOM). Classical XAD/DAX resin fractionation procedures are composed of: (i) acidification, as to protonate the samples to an uncharged state, and (ii) hydrophobic/hydrophilic fractionation by resin XAD-8/DAX-8 and XAD-4 in tandem use. As the production of Amberlite® XAD-8 resin (Rohm & Haas) has been ceased some years ago, Supelite™ DAX-8 resin (Sigma-Aldrich), also referred as poly(methyl methacrylate) resin, has substituted for XAD-8 resin and became popular in recent studies (Cao et al., 2017;[START_REF] Leloup | Organic matter produced by algae and cyanobacteria: quantitative and qualitative characterization[END_REF][START_REF] Marhaba | Modified dissolved organic matter fractionation technique for natural water[END_REF][START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF].

As water is a polar solvent, it forms strong hydrogen bond with polar organics and ionic groups which are, therefore, referred to as hydrophilic. Non-polar organics (termed hydrophobic) are unable to interact in this way and are partially separated from the aqueous phase. The resin fractionation exploits the differences in polarity to separate NOM into different fractions [START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF][START_REF] Thurman | Prediction of capacity factors for aqueous organic solutes adsorbed on a porous acrylic resin[END_REF].

Fig. 2.4

Interaction between organic acids/bases and XAD/DAX resins controlled by pH (adapted from [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF]).

Disassociation tendencies of the functional groups (e.g., -COOH, -NH3 + ) in the NOM can be controlled by pH. Organic acids (-COOH) become protonated in acidic condition, with the aid of certain hydrophobic carbon skeletons, these acidic organic solvents are sorbed by the resin via hydrophobic interactions (Fig. 2.4) and thus, considered hydrophobic [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF]. Generally, samples are separated into three fractions by passing through the XAD/DAX resin series: hydrophobic fraction (HPO) is the part adsorbed on the XAD-8/DAX-8 resin at pH 2, the transphilic fraction (THP) is the elute of XAD-8/DAX-8 which then adsorbs to XAD-4 at pH 2, and the hydrophilic fraction (HPI) is the elute from both columns in series at pH 2 (Fig. 2.5). One of the advantages of this technique is that the fractionation facilitates the post-analyses of the hydrophobic/hydrophilic properties of the NOM [START_REF] Aiken | Isolation of hydrophilic organic acids from water using nonionic macroporous resins[END_REF][START_REF] Leenheer | Comprehensive approach to preparative isolation and fractionation of dissolved organic carbon from natural waters and wastewaters[END_REF][START_REF] Thurman | Prediction of capacity factors for aqueous organic solutes adsorbed on a porous acrylic resin[END_REF]. Aqueous NOM samples are commonly acidified to pH 2, as it maximizes the hydrophobicity of NOM without causing precipitation of the humic acids [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF].

Nevertheless, when this method is applied in the EPS studies, the conformation of EPS molecules may be changed by adjusting pH, resulting in the deviations in the post-analyses [START_REF] Dogsa | Structure of bacterial extracellular polymeric substances at different pH values as determined by SAXS[END_REF]Wang et al., 2012a).

Leloup et al. (2013) characterized the algal organic matter (AOM) produced by algae

Euglena gracilis and cyanobacterium Microcystis aeruginosa by using DAX-8 and XAD-4 resins.

The results showed that the HPI was the major fraction in the AOM whatever the growth phase was, and it was almost the only fraction produced during the lag and exponential phases. TOF). By using this developed protocol, a structural scheme and organic matter composition of the algae were obtained [START_REF] Nicolau | Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) coupled to XAD fractionation: method to algal organic matter characterization[END_REF].

Factors influencing cell surface hydrophobicity

Microbial aggregation is also viewed as a result of the cell response to a stressful environment [START_REF] Liu | Contribution of extracellular polymeric substances (EPS) to the sludge aggregation[END_REF]. When the microbes enter an unfavorable environment, their cell surfaces will start a series of adaption changes. It is expected that the change of cell surface hydrophobicity can strengthen the cell-to-cell interactions and result in a stronger surface structure to protect the cells from the unfavorable conditions. The hydrophobicity change is influenced by many factors such as substrates, microbial growth phase, external conditions (pH, temperature, metal cations, organic pollutants), as well as the EPS production (Liu et al., 2004a). in AP-MEM. Anaerobic granules grown in protein-rich media were reported to have a lower cell surface hydrophobicity and slower granulation process, when compared with those grown in carbohydrate-rich media [START_REF] Thaveesri | Organic and ammonium nitrogen and oxygen in relation to granular sludge growth in lab-scale UASB reactors[END_REF]. Protein-rich substrates can lower the surface tension of the reactor liquid, which leads to the formation of anaerobic granules displaying low hydrophobicity [START_REF] Thaveesri | Granulation and sludge bed stability in upflow anaerobic sludge bed reactors in relation to surface thermodynamics[END_REF].

Substrates

The cell surface hydrophobicity of Corynebacterium glutamicum became higher under the phosphate-saturated growth conditions than under the phosphate-depleted conditions [START_REF] Büchs | Cell adsorption control by culture conditions[END_REF]. Phosphate limitations induce the synthesis of teichuronic acids instead of teichoic acids in Gram-positive bacteria [START_REF] Swoboda | Wall teichoic acid function, biosynthesis, and inhibition[END_REF], and in turn some additional changes could occur between these two hydrophilic polymers and the cell surface [START_REF] Büchs | Cell adsorption control by culture conditions[END_REF].

When exposed to a toxic or inhibitory substrate, microorganisms are still able to regulate their cell surface properties. Many toxic hydrocarbons that are present as environmental pollutants are also the potential substrates for the bacteria. To increase the accessibility to these low bioavailability compounds, one specific adaptive mechanism developed by the bacteria is the modification of cell surface hydrophobicity to permit direct hydrophobic interactions with those substrates [START_REF] Heipieper | Surface properties and cellular energetics of bacteria in response to the presence of hydrocarbons[END_REF]. The Gram-positive bacterium Bacillus licheniformis reduced the cell surface hydrophobicity when cultured with the organic solvent isoamyl alcohol, and exhibited little affinity towards 3-methylbutan-1-ol [START_REF] Torres | Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase[END_REF]. In contrast,

Mycobacterium frederiksbergense increased its cell surface hydrophobicity in the presence of anthracene. High affinity of the cells to anthracene, in case utilize it as unique carbon source, requires an augmented cell surface hydrophobicity. This augmentation was induced by a degradative aromatic pathway which was developed by the bacterium to metabolize anthracene [START_REF] Wick | Responses of Mycobacterium sp. LB501T to the low bioavailability of solid anthracene[END_REF][START_REF] Yamashita | Utilization of hydrophobic bacterium Rhodococcus opacus B-4 as whole-cell catalyst in anhydrous organic solvents[END_REF]. The mutants of the Gram-negative bacterium E. coli K-12 presented a less hydrophobic cell surface when compared to the parental cells, and displayed a higher tolerance to organic solvents [START_REF] Aono | Cell surface properties of organic solvent-tolerant mutants of Escherichia coli K-12[END_REF].

Microbial growth phase

In a study regarding the impact of brewing yeast cell age on fermentation performance, [START_REF] Powell | The impact of brewing yeast cell age on fermentation performance, attenuation and flocculation[END_REF] reported that the flocculation potential of cells and cell surface hydrophobicity increased in conjunction with the cell age. [START_REF] Malmqvist | Bacterial hydrophobicity measured as partition of palmitic acid between the two immiscible phases of cell surface and buffer[END_REF] showed that the cell surface hydrophobicity of the bacterium Staphylococcus aureus increased 4 -5 times during the exponential growth phase of the culture. Generally, cells in the stationary growth phase were more hydrophobic than those in the exponential growth phase [START_REF] Allison | Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms[END_REF][START_REF] Hazen | Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata[END_REF]. However, [START_REF] Van Loosdrecht | The role of bacterial cell wall hydrophobicity in adhesion[END_REF] observed that 23 bacterial strains became more hydrophobic during the exponential growth phase by studying their cell surface hydrophobicity.

Similar results were also obtained by [START_REF] Jana | Influence of growth and environmental conditions on cell surface hydrophobicity of Pseudomonas fluorescens in non-specific adhesion[END_REF] who concluded that the early-to mid-log exponential cells of 18 isolates from Pseudomonas fluorescens were more hydrophobic than those in the stationary phase. This contradictory observation could be ascribed to that the cell surface hydrophobicity is also influenced by other conditions such as temperatures, pH and the substrates type [START_REF] Blanco | Incubation temperatures affect adherence to plastic of Candida albicans by changing the cellular surface hydrophobicity[END_REF][START_REF] Correa | Cellular envelopes and tolerance to acid pH in Mesorhizobium loti[END_REF][START_REF] Mattarelli | Effect of growth temperature on the biosynthesis of cell wall proteins from Bifidobacterium globosum[END_REF][START_REF] Muda | Aggregation and surface hydrophobicity of selected microorganism due to the effect of substrate, pH and temperature[END_REF].

Some studies showed that starvation conditions could enhance cell surface hydrophobicity and in turn, facilitate microbial adhesion and aggregation [START_REF] Bossier | Triggers for microbial aggregation in activated sludge?[END_REF]. It is shown by [START_REF] Chiesa | Feast/famine growth environments and activated sludge population selection[END_REF] that the periodic starvation cycle increased the cell surface hydrophobicity of activated sludge. Nevertheless, some studies showed different effects that were caused by the starvation. [START_REF] Castellanos | Starvation-induced changes in the cell surface of Azospirillum lipoferum[END_REF] demonstrated that upon transferring from a rich growth medium to a starvation condition, the cell surface hydrophobicity of Azospirillum lipoferum dropped sharply but recovered its initial value within 24 -48 h. [START_REF] Sanin | Effect of starvation on the adhesive properties of xenobiotic degrading bacteria[END_REF] reported that the cell surface hydrophobicity of xenobiotic degrading bacteria (two Pseudomonas strains and Rhodococcus corallines) stayed constant during carbon starvation, whereas a significant decrease in the hydrophobicity was observed when all these cultures were starved for nitrogen.

Other external conditions

Cell surface hydrophobicity is also affected by some other external conditions, such as pH, temperature, oxygen and the presence of multivalent cations or phosphate. [START_REF] Palmgren | Influence of oxygen limitation on the cell surface properties of bacteria from activated sludge[END_REF] tested the influence of oxygen on the cell surface hydrophobicity of four bacteria isolated from activated sludge, and found that oxygen limitation caused the decrease in the cell surface hydrophobicity of the studied strains. [START_REF] Blanco | Incubation temperatures affect adherence to plastic of Candida albicans by changing the cellular surface hydrophobicity[END_REF] found that the majority number of 42 strains of Candida albicans were hydrophobic at 22 °C, but hydrophilic at 37 °C. An opposite trend was reported in Candida glabara, which demonstrated higher cell surface hydrophobicity at 37 °C than 25 °C [START_REF] Hazen | Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata[END_REF]. Sludge retention time (SRT) also influences sludge surface hydrophobicity. [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF] demonstrated that the activated sludge at higher SRT (12, 16, 20 d) was more hydrophobic than that at lower SRT (4, 9 d).

The negatively charged functional groups such as hydroxyl and carboxyl groups embedded in the EPS provide many sorption sites for the metal cations [START_REF] Flemming | Sorption properties of biofilms[END_REF]. The interactions between metal cations and these functional groups alter the cell surface properties by decreasing the negative charges of the cell surface [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF][START_REF] Li | Effects of Fe(III) on floc characteristics of activated sludge[END_REF][START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF]. Cell surface with less negative charges was observed to possess higher hydrophobicity [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF]. Divalent cations, such as Ca 2+ and Mg 2+ , also can influence the bacterial self-immobilization and microbial aggregation [START_REF] Ding | Amphiphilic exopolymers from Sagittula stellata induce DOM self-assembly and formation of marine microgels[END_REF][START_REF] Schmidt | Effects of magnesium on thermophilic acetate-degrading granules in upflow anaerobic sludge blanket (UASB) reactors[END_REF][START_REF] Yu | The roles of calcium in sludge granulation during UASB reactor start-up[END_REF], as well as the expression of cell surface hydrophobicity [START_REF] Fattom | Hydrophobicity as an adhesion mechanism of benthic Cyanobacteria[END_REF]. The removal of Ca 2+ results in a disintegration of anaerobic granular sludge (Grotenhuis et al., 1991). [START_REF] Yu | The roles of calcium in sludge granulation during UASB reactor start-up[END_REF] found that an increase of Ca 2+ concentrations from 150 to 300 mg/L enhanced the anaerobic granulation process, and [START_REF] Jiang | Ca 2+ augmentation for enhancement of aerobically grown microbial granules in sludge blanket reactors[END_REF] reduced the granulation time of seed activated sludge by dosing 100 mg/L of Ca 2+ . Besides, the presence of divalent cations Ca 2+ and Mg 2+ improved notably the cell surface hydrophobicity of Agrobacterium and

Citrobacter [START_REF] Khemakhem | Effect of environmental conditions on hydrophobicity of marine bacteria adapted to textile effluent treatment[END_REF]. However, [START_REF] Singh | Clumping characteristics and hydrophobic behaviour of an isolated bacterial strain from sewage sludge[END_REF] claimed that the expression of cell surface hydrophobicity of one clumping bacteria (identified as Pseudomonas sp.) from sewage sludge was not influenced by the presence of metal cations.

Role of EPS in cell surface hydrophobicity

At the early stage of biofilm formation, once the microorganisms have firmly attached to the surface, they undergo a series of physiological changes to adjust their life on the surface. One common adaption is the expression of large quantities of EPS to form the three-dimensional architecture for hosting the cells. The subsequent biofilm development and maintenance of structured multicellular microbial communities are also depending on the production and quantity of EPS [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] O'toole | Biofilm formation as microbial development[END_REF]. Since cells are embedded in the EPS matrix, their surface characteristics such as surface charge and hydrophobicity are monitored by the EPS. Besides, the change of those properties mediate cellular recognition and promote initial cell adhesion onto the surface [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF].

EPS production and composition are also dependent on different parameters such as microorganisms, growth phase, substrates type and other external conditions [START_REF] Sheng | Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review[END_REF].

EPS from the pure culture (bacteria, fungi and algae) are mainly composed by PN and PS [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF][START_REF] Ravella | Extracellular polysaccharide (EPS) production by a novel strain of yeast-like fungus Aureobasidium pullulans[END_REF][START_REF] Turu | Synthesis and characterization of cryogel structures for isolation of EPSs from Botryococcus braunii[END_REF]. In the mixed-culture aggregates, the composition of extracted EPS is more heterogeneous. LB-EPS from anaerobic granular sludge are found mainly contain HS-like [START_REF] Yuan | Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge[END_REF], while PN are determined as the major constituent of TB-EPS from anammox sludge [START_REF] Ni | Distribution of extracellular polymeric substances in anammox granules and their important roles during anammox granulation[END_REF].

EPS in the biological processes

Microbial adhesion

Tsuneda et al. ( 2003) investigated the impact of EPS on the bacterial adhesion onto glass beads by using 27 heterotrophic bacterial strains isolated from a wastewater treatment reactor. Their results

showed that when EPS content was relatively high, cell adhesion was enhanced by the hydrophobic interaction induced by the EPS. Meanwhile, four LB-EPS free bacteria (Bacillus subtilis, Streptococcus suis, Escherichia coli and Pseudomonas putida) exhibited fewer cell surface hydrophobicity values (26.1% -65.0%) when compared to the intact cells (47.4% -69.3%). Moreover, [START_REF] Parker | Increased cell surface hydrophobicity associated with possession of an additional surface protein by Aeromonas salmonicida[END_REF] reported that Aeromonas salmonicida possessed an additional surface protein, protein-A, and the cell surface of this strain was more hydrophobic than that of the strains devoid of this protein. [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF] found that the presence of PS promoted the adhesion strength of the EPS produced by Pseudomonas aeruginosa and Bacillus subtilis, while PN had a less adherence effect.

Granulation

During the aerobic granulation process, EPS production mainly occurs during the exponential phase, and they could serve as carbon and energy source in the starvation phase. Thus, the growth of bacteria in the interior and exterior of granules as well as the integrity of granules are regulated by the EPS [START_REF] Wang | Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors[END_REF]. It was further found that the outer layer of aerobic granules exhibited a higher hydrophobicity than the core of the granules. The insoluble EPS present in the outer layer of the granule would play a protective role with respect to the structure stability and the integrity of aerobic granules [START_REF] Wang | Distribution of EPS and cell surface hydrophobicity in aerobic granules[END_REF]. Generally, the production of EPS benefits the granulation process (Liu et al., 2004a), and a higher EPS content may result in higher cell surface hydrophobicity [START_REF] Wang | Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors[END_REF]. However, [START_REF] Yu | Enhanced aerobic granulation with extracellular polymeric substances (EPS)-free pellets[END_REF] noticed that the aerobic granulation process was enhanced by EPS-free pellets, they assumed that EPS initially embedded in the seed sludge prior to granulation may sterically slow down subsequent cell-cell contact, thereby, delaying aerobic granulation.

LB-EPS and TB-EPS exert different contributions to granulation. By analyzing the interaction energy curves of aerobic and anaerobic granules before and after the EPS extraction using extended DLVO theory, [START_REF] Liu | Contribution of extracellular polymeric substances (EPS) to the sludge aggregation[END_REF] concluded that the LB-EPS always display a positive effect on the granulation, while the role of TB-EPS in the granulation is dependent on the separation distance among the granules. In addition, with increased PN to PS ratio, the granule's surface became more hydrophobic after aerobic granulation than the flocculent seed sludge, which was observed by [START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF]. The molecular support in the EPS that contributes to the granulation and structural stability has not been well elucidated, and the conclusions are contradictory to each other. [START_REF] Mcswain | Composition and distribution of extracellular polymeric substances in aerobic flocs and granular sludge[END_REF] found that the stability of granules was dependent on a PN core, and [START_REF] Zhu | Specific component comparison of extracellular polymeric substances (EPS) in flocs and granular sludge using EEM and SDS-PAGE[END_REF] also claimed that PN significantly contributed to the formation of granular sludge via surface charge adjustment. In contrast, [START_REF] Seviour | Aerobic sludge granulation: a tale of two polysaccharides?[END_REF] and Wang et al. (2012a) found that PS were responsible for the aggregation of flocs into granules.

Flocculation

The surface properties of activated sludge are affected by the EPS composition more than the number of filaments, and the EPS content was negatively correlated with the relative cell surface hydrophobicity and flocculation ability [START_REF] Wilé N | Relationship between flocculation of activated sludge and composition of extracellular polymeric substances[END_REF]. [START_REF] Li | Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge[END_REF] claimed that excessive EPS production in the form of LB-EPS could weaken cell attachment and the floc structure, which led to poor flocculation and retarded sludge-water separation. Li et al. (2016) discovered that although the increase of EPS content was beneficial to the aggregation of larger flocs, the flocs with a high EPS content were susceptible to the large-scale fragmentation resulting in much smaller daughter-particles. However, [START_REF] Liao | Surface properties of sludge and their role in bioflocculation and settleability[END_REF] found that the sludge surface hydrophobicity was influenced by individual EPS constituents instead of the total EPS content.

Their results and other studies [START_REF] Jorand | Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances[END_REF][START_REF] Xie | Surface properties of bacteria from activated sludge in relation to bioflocculation[END_REF] demonstrated that PN had a positive influence on the sludge surface hydrophobicity, but not PS. In all, the cell surface hydrophobicity of the activated sludge increases with the PN concentration, and high cell surface hydrophobicity generally results in a better flocculation [START_REF] Liu | Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge[END_REF].

Other biological processes

It is also found that EPS play a key role in binding a large volume of water (i.e. bound water) and influence sludge dewatering [START_REF] Li | Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge[END_REF]. The breakdown of the EPS structure tends to reduce the relative hydrophobicity of the sludge, enabling the release of some water trapped within the floc to the bulk liquid phase, and thus, facilitate water removal [START_REF] Raynaud | Compression dewatering of municipal activated sludge: effects of salt and pH[END_REF].

There exists a certain EPS content at which the sludge dewatering reached a maximum [START_REF] Houghton | Municipal wastewater sludge dewaterability and the presence of microbial extracellular polymer[END_REF]. The findings of [START_REF] Murthy | Factors affecting floc properties during aerobic digestion: implications for dewatering[END_REF] showed that PN are generally more important in sludge flocculation and dewatering than PS, whereby a high PN concentration is detrimental for the dewatering process. Moreover, [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF] demonstrated that sludge dewatering correlated well with the PN/PS ratio in sludge EPS.

Abundant negative charged functional groups of the EPS provide various binding sites for the metal cations [START_REF] Aquino | Soluble microbial products formation in anaerobic chemostats in the presence of toxic compounds[END_REF]. This metal binding ability of EPS is also influenced by the hydrophobicity, as hydrophilic EPS fraction of anaerobic granular sludge adsorbed more Zn and Cu than the hydrophobic EPS fraction [START_REF] Wei | Adsorption of Cu 2+ and Zn 2+ by extracellular polymeric substances (EPS) in different sludges: effect of EPS fractional polarity on binding mechanism[END_REF]. Moreover, EPS are also considered as one of the membrane foulants [START_REF] Lin | A critical review of extracellular polymeric substances (EPSs) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies[END_REF], and the hydrophobic fraction in the EPS provides the sorption sites for the organic micro-pollutants such as polycyclic aromatic hydrocarbons (PAHs), or phenolic compounds [START_REF] Liu | Phenanthrene desorption from soil in the presence of bacterial extracellular polymer: observations and model predictions of dynamic behavior[END_REF][START_REF] Nguyen | Biofouling of water treatment membranes: a review of the underlying causes, monitoring techniques and control measures[END_REF][START_REF] Wang | Adsorption of p-chlorophenol by biofilm components[END_REF][START_REF] Yu | Hydrophobicity and molecular weight of humic substances on ultrafiltration fouling and resistance[END_REF]. The presence of metal cations or organic micro-pollutants significantly influence the overall hydrophobicity of microbial aggregates in bioreactors [START_REF] Liu | Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge[END_REF]. Generally, a higher EPS content may result in a higher cell surface hydrophobicity [START_REF] Wang | Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors[END_REF], and statistical analyses showed a significantly high correlation between the ability to degrade organic pollutants and cell surface hydrophobicity [START_REF] Obuekwe | Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment[END_REF].

EPS hydrophobicity

The relative ratio between the hydrophilic and hydrophobic fraction of the EPS largely depends on the extraction method (Cao et al., 2017). The extraction is based on interrupting the interactions that are responsible for the stabilization of the three-dimensional structure of the EPS (such as hydrogen bonds, electrostatic, hydrophobic or van der Waals interactions) via physical forces or chemical reagents (Fig. 2.6).

Physical extraction methods such as centrifugation, heating, and sonication breaks the low-energy bonds between the EPS and the cell surface. Cationic exchange resin (CER) method, usually DOWEX ® 50 × 8 (20 -50 mesh in the sodium form), extracts EPS from the biomass via the shear force provided by the shaking and the destabilization of the microbial aggregates structure by removing divalent cations such as Ca 2+ [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF]. Organic reagents like formaldehyde can bind with the functional groups (e.g., amine groups) on the cell walls and break the connection between EPS and these functional groups, which, as a result, has facilitated the extraction of EPS [START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF]. Hydrophobic extraction by using surfactants as sodium dodecyl sulfate (SDS) or Tween 20 are rarely reported in the literature, the extraction mechanism lies in the fact that these surfactants improve the solubility of EPS molecules by forming micelles via hydrophobic interactions to enhance the EPS extraction efficiency [START_REF] Ras | A high yield multi-method extraction protocol for protein quantification in activated sludge[END_REF].

Nevertheless, the breakage degree of those interactions between EPS molecules cannot be interfere with the determination of EPS hydrophobicity (Cao et al., 2017). Tourney and Ngwenya, 2014). Some authors concluded that during aerobic granulation process, a more hydrophobic cell surface corresponded to a higher PN/PS ratio [START_REF] Xie | Surface properties of bacteria from activated sludge in relation to bioflocculation[END_REF][START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF]. Limited information regarding to the molecular support of EPS hydrophobicity can be found in the literature. Studies showed that mainly PN and HS-like contributed to the EPS hydrophobicity, whereas carbohydrates contributed more to the hydrophilic nature [START_REF] Jorand | Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances[END_REF][START_REF] Yu | Hydrophobicity and molecular weight of humic substances on ultrafiltration fouling and resistance[END_REF]. Li et al. (2016a) claimed that PN were responsible for the cell surface hydrophobicity of anaerobic granules, and nonpolar groups such as side chains of aromatic amino acids were pivotal factors deciding the EPS hydrophobicity. Several studies demonstrated that acetylated PS form the hydrophobic moiety in the EPS structure and support the EPS hydrophobicity [START_REF] Mayer | The role of intermolecular interactions: studies on model systems for bacterial biofilms[END_REF][START_REF] Neu | Structural studies of an emulsionstabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain[END_REF]. The presence of N-acetylamino sugars in the wastewater and treated water was confirmed by pyrolysis, and those molecules were originating from bacterial cell walls (Dignac et al., 2000). Besides, intra-molecular hydrogen bonding of PS can inhibit interactions between the hydroxyl groups and water, and thus making the PS hydrophobic. Therefore, the gel-forming PS will influence the hydrophobicity of microbial aggregates as soon as they form a continuous network that potentially allows for hydrophobic components to be immobilized within the biofilm matrix as filler material [START_REF] Seviour | Aerobic sludge granulation: a tale of two polysaccharides?[END_REF].

In addition, HS-like compounds are also integral constituents of EPS extracted from the sludge used in wastewater treatment processes (Cao et al., 2017;[START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF][START_REF] Riffaldi | Humic substances in sewage sludges[END_REF][START_REF] Yuan | Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge[END_REF], but the hydrophobic features of these HS-like compounds in the EPS are usually neglected in the EPS-related studies.

Future Work

Attached growth processes i.e. biofilm formation or granulation and activated sludge start with microbial adhesion, where cell surface hydrophobicity acts as a triggering force when molecules are incapable of interacting electrostatically or establishing hydrogen bonds with water molecules (Liu et al., 2004a;[START_REF] Muadcheingka | Distribution of Candida albicans and nonalbicans Candida species in oral candidiasis patients: Correlation between cell surface hydrophobicity and biofilm forming activities[END_REF]. Since the secretion of EPS is a universal characteristic of microorganisms and abundant EPS molecules are found in-/outside granules or flocs, cell surface hydrophobicity of the microbial aggregates largely depends on the quantity and quality of EPS [START_REF] More | Extracellular polymeric substances of bacteria and their potential environmental applications[END_REF][START_REF] Zhang | Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics[END_REF]. Therefore, a thorough study of EPS hydrophobicity and the influencing factors in the EPS hydrophobicity become highly demanded.

Most of EPS studies are ex situ studies, in other words, investigating EPS characteristics after extracting them from the microorganisms. The EPS extraction methods are based on breaking the linkage between the cell surface and the EPS molecules, or within the EPS molecules [START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF]. This indicates that the extraction procedure may disrupt the original molecular structure of EPS and cell surface. Moreover, the leftover of EPS chemical extractants in the extracted EPS solution or on the cell surface cannot be predicted or evaluated. Therefore, the results obtained from ex situ studies can only reflect the EPS or the cell surface characteristics under certain conditions. In the more complicated microbial systems such as biofilms, granules or activated sludge, the distribution of the microorganisms in the systems is more heterogeneous.

The exposure of the nutrients or the wastes to the microorganisms is unevenly assigned between the inner and outer layer of these aggregates. Therefore, the production and the distribution of EPS in these microbial aggregates is also heterogeneous [START_REF] Guo | Variations of both bacterial community and extracellular polymers: the inducements of increase of cell hydrophobicity from biofloc to aerobic granule sludge[END_REF]. In turn, EPS extracted from these microbial aggregates can be considered as "average" EPS produced by all the microorganisms in the system. To make specific EPS characterization, different EPS extraction methods may be needed in the same study. The type of biomass and working conditions should be carefully controlled, as these two factors also influence the EPS production and their properties.

Thus, by comparing the characteristics of EPS molecules extracted by different methods, more representative information about EPS extracted from certain type of biomass can be revealed.

A better characterization of EPS hydrophobicity improves the understanding of microbial adhesion. Evidence showed that mainly PN and HS-like determined EPS hydrophobicity, and acetylated PS could form a hydrophobic "pocket" within the EPS structure and possibly contributed to the EPS hydrophobicity [START_REF] Jorand | Hydrophobic/hydrophilic properties of activated sludge exopolymeric substances[END_REF][START_REF] Yu | Hydrophobicity and molecular weight of humic substances on ultrafiltration fouling and resistance[END_REF][START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF].

As those organic matters are macromolecules and usually link with each other (Bourven et al., 2015a;Grotenhuis et al., 1991;[START_REF] Watanabe | Characterization of a large glycoprotein proteoglycan by size-exclusion chromatography combined with light and X-ray scattering methods[END_REF], the specific molecular structure in determining EPS hydrophobicity is still unknown, and thus, should be studied case by case. After knowing the molecular support of the EPS hydrophobicity, the synthesizing pathway of these molecules as a function of the microbial species react to the environmental conditions could be a subject of future study.

Metals such as Fe, Zn, Ni, Cu, and Co can act as micronutrients, and the microbial growth can be stimulated by dosing trace amounts of these metals [START_REF] Gikas | Single and combined effects of nickel (Ni(II)) and cobalt (Co(II)) ions on activated sludge and on other aerobic microorganisms: a review[END_REF][START_REF] Thanh | Trace metal speciation and bioavailability in anaerobic digestion: a review[END_REF].

Divalent cations like Ca 2+ and Mg 2+ are bridging ions to maintain the stability of the EPS structure [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF]. Moreover, the release of some extracellular molecules is carried out as a function of the cell membrane enzymes. The normal enzymatic activity required for proper energy metabolism in living cells is also influenced by those metallic co-factors [START_REF] Kosaric | Microbial aggregates in anaerobic wastewater treatment[END_REF]. It is therefore essential to investigate whether the presence of those metal ions could change EPS hydrophobicity, aiming at the manipulation of engineered bioprocesses for enhancing metal bioavailability in case of trace metallic element dosing [START_REF] Wei | Adsorption of Cu 2+ and Zn 2+ by extracellular polymeric substances (EPS) in different sludges: effect of EPS fractional polarity on binding mechanism[END_REF][START_REF] Thanh | Trace metal speciation and bioavailability in anaerobic digestion: a review[END_REF]. Likewise, many toxic hydrocarbons are present as environmental pollutants, but they are also potential substrates for the microorganisms [START_REF] Heipieper | Surface properties and cellular energetics of bacteria in response to the presence of hydrocarbons[END_REF][START_REF] Jia | Degradation of pyrene in soils by extracellular polymeric substances (EPS) extracted from liquid cultures[END_REF][START_REF] Torres | Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase[END_REF][START_REF] Xu | Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system[END_REF]. The removal of these organic compounds by the microorganisms is also influenced by the cell surface hydrophobicity [START_REF] Aksu | Application of biosorption for the removal of organic pollutants: a review[END_REF]. Since the EPS content positively correlated with the microbial cell surface hydrophobicity [START_REF] Obuekwe | Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment[END_REF], the interplay between EPS production and the removal ability of organic micro-pollutants by the EPS should also be investigated in the future.

Chapter III.

Hydrophobic fractionation of EPS extracted from anaerobic granular sludge by DAX-8 resin

This chapter is published as: Cao F., Bourven I., Lens P.N.L., van Hullebusch E.D., Pechaud Y., Guibaud G. (2017). Hydrophobic features of EPS extracted from anaerobic granular sludge: an investigation based on DAX-8 resin fractionation and size exclusion chromatography. Appl. Microbiol. Biotechnol. 101: 3427-3438.

Introduction

Most of the microorganisms are present in aggregated forms such as biofilms and flocs [START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF]. Previous studies evidenced that microbial adhesion, a fundamental step for biofilm formation, depends on the hydrophobichydrophilic structure of interacting surfaces. Increasing cell surface hydrophobicity could facilitate the surface approaching and trigger the specific forces responsible for their reversible adhesion (Ding et al., 2015a;[START_REF] Fletcher | Influence of substratum characteristics on the attachment of a marine Pseudomonad to solid surfaces[END_REF]Liu et al., 2004b).

Surrounding the microbial aggregates are three-dimensional matrixes called extracellular polymeric substances (EPS), which originate from microbial secretion, cell lyses and sorption of molecules from the environment. Hydrophobic properties of EPS are claimed to exert a profound influence on the cell surface hydrophobicity of these aggregates [START_REF] Lin | A critical review of extracellular polymeric substances (EPSs) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies[END_REF]. [START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF] reported the hydrophobic EPS content correlates better with cohesive properties of the biofilm layers than the total EPS content. However, the role of EPS on the cell surface hydrophobicity is not well identified and varies between the studies. [START_REF] Zhang | Composition of EPS fractions from suspended sludge and biofilm and their roles in microbial cell aggregation[END_REF] found that after loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) extraction, the hydrophobicity of cell suspensions without LB-EPS and TB-EPS was higher than before the extraction steps. In contrast, [START_REF] Guo | Variations of both bacterial community and extracellular polymers: the inducements of increase of cell hydrophobicity from biofloc to aerobic granule sludge[END_REF] showed that after EPS extraction, hydrophobicity of the cells from aerobic granules decreases. Moreover, [START_REF] Zhao | Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS)[END_REF] displayed that cell surface hydrophobicity of intact bacteria (Bacillus subtilis, Streptococcus suis, Escherichia coli and Pseudomonas putida)

was higher than LB-EPS free bacteria according to the microbial adhesion to hydrocarbon (MATH) test.

Proteins (PN), polysaccharides (PS) and humic-like substances (HS-like) are considered as the three main EPS organic components of multispecies microbial aggregates in the environment [START_REF] Flemming | The biofilm matrix[END_REF]. These EPS organic macromolecules harbored different functional groups (i.e. carboxyls, hydroxyls, phosphates, amines, and sulfates) as well as nonpolar regions (e.g., hydrophobic regions in O-methyl/acetyl polysaccharides, aromatics and aliphatic regions in proteins), which also contribute to the EPS hydrophilic/hydrophobic properties [START_REF] Moran | Microbial Glycobiology: Structures, Relevance and Applications[END_REF]. As different extraction methods were applied, different concentrations of these main organic constituents in the EPS are reported [START_REF] Domí Nguez | 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][START_REF] Zuriaga-Agustí | Influence of extraction methods on proteins and carbohydrates analysis from MBR activated sludge flocs in view of improving EPS determination[END_REF]. Therefore, the influence of EPS extraction methods on the hydrophobic properties of the extracted EPS should be taken as the preliminary step to develop the studies of EPS hydrophobicity.

Techniques that are usually applied in the hydrophobicity measurement are contact angle measurement (CAM) [START_REF] Pen | Effect of extracellular polymeric substances on the mechanical properties of Rhodococcus[END_REF], hydrophobic interaction chromatography (HIC) [START_REF] Ras | Distribution and hydrophobic properties of extracellular polymeric substances in biofilms in relation towards cohesion[END_REF], salt aggregation test (SAT) [START_REF] Lindahl | A new test based on "salting out" to measure relative surface hydrophobicity of bacterial cells[END_REF][START_REF] Rozgonyi | Improvement of the salt aggregation test to study bacterial cell-surface hydrophobicity[END_REF], as well as MATH test [START_REF] Gao | Influence of extracellular polymeric substances on microbial activity and cell hydrophobicity in biofilms[END_REF][START_REF] Rosenberg | Adherence of bacteria to hydrocarbons: A simple method for measuring cell-surface hydrophobicity[END_REF]. However, these methods can only provide a global view of the hydrophobic properties of the substances in the presence of their hydrophilic fraction, or are mainly based on the properties of specific constituents i.e. PN. Except the abovementioned techniques, XAD resin fractionation is one of the frequently used techniques to fractionate natural organic matter (NOM) from natural water according to its polarity. This technique isolates the hydrophobic/hydrophilic fractions of NOM separately and thus, facilitates hydrophobicity studies [START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF][START_REF] Thurman | Prediction of capacity factors for aqueous organic solutes adsorbed on a porous acrylic resin[END_REF]. Classical XAD hydrophobic/hydrophilic fractionation uses resins XAD-8 and XAD-4 in tandem. As the production of Amberlite® XAD-8 resin (Rohm & Haas) has been ceased some years ago, Supelite™ DAX-8 resin (Sigma-Aldrich), also referred as poly(methyl methacrylate) resin, has substituted the XAD-8 resin and became popular in related studies [START_REF] Bolto | Experimental evaluation of cationic polyelectrolytes for removing natural organic matter from water[END_REF][START_REF] Leloup | Organic matter produced by algae and cyanobacteria: quantitative and qualitative characterization[END_REF][START_REF] Marhaba | Modified dissolved organic matter fractionation technique for natural water[END_REF][START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF]. It has been reported that the DAX-8 resin is easier to handle, its wetting ability is better than that of XAD-8 resin and its fine content is below 1% (w/w) [START_REF] Peuravuori | Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: comparative study using pyrolysis gas chromatography[END_REF]. Before the fractionation, aqueous NOM samples are commonly acidified to pH 2 as it maximizes the hydrophobicity of NOM [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF].

In this study, seven different EPS extraction methods, including physical methods (centrifugation, heating and sonication) and chemical methods by using organic reagents (ethanol, formaldehyde, SDS and Tween 20), were applied to extract EPS from anaerobic granular sludge.

Since the hydrophobic properties of EPS were mainly concerned, only the DAX-8 resin was used to separate the hydrophobic fraction of EPS samples at pH 2. Besides, a higher pH i.e. pH 5 for the resin fractionation was also tested, as a compromise between the neutralization of the EPS surface charge to improve the fractionation performance and the minimization of EPS denaturation under stronger acidic condition. To investigate EPS hydrophobic properties, besides the quantification of the total organic carbon (TOC), PN, HS-like and PS concentrations, qualitative analyses by using size exclusion chromatography (SEC) coupled with UV detection was applied to study the apparent molecular weight (aMW) distribution of hydrophobic EPS molecules.

Materials and methods

Anaerobic granular sludge origin and characteristics

Samples were collected from an upflow anaerobic sludge blanket (UASB) operated by Smurfit Kappa paper recycling factory (Saillat-sur-Vienne, France). The treated influent was the wastewater from cardboard manufacturing. The color of the granules was dark grey. The volatile suspended solid (VSS)/total suspended solid (TSS) ratio of the sludge was around 0.85, in which the TSS and VSS concentration were 125 (± 2.7) g/L and 106 (± 2.6) g/L, respectively. After the collection, sludge samples were stored immediately at 4 °C. The collected sludge was washed twice by ultrapure water and re-suspended in ultrapure water prior to EPS extraction.

EPS extraction

Seven Four different chemical extraction methods were applied: (i) 0.3 mL formaldehyde (Prolabo, 36.5%) was added in per 50 mL sludge suspension, 1 h incubation at 4 °C, then heating in a water bath at 80 °C for 10 min [START_REF] Liu | Extraction of extracellular polymeric substances (EPS) of sludges[END_REF]; (ii) ultrapure water in per 50 mL sludge suspension was replaced by 0.01% (w/v) sodium dodecyl sulfate (SDS, Merck), 4 h shaking at 300 rpm under 4 °C [START_REF] Wu | Evaluation of different methods for extracting extracellular DNA from the biofilm matrix[END_REF]; (iii) ultrapure water was replaced by 0.25% (v/v) Tween 20 (Applichem) prepared in Phosphate Buffered Saline at pH 7.4 (PBS buffer: 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.76 mM KH2PO4), 1 h shaking at 150 rpm under 4 °C [START_REF] Ras | A high yield multi-method extraction protocol for protein quantification in activated sludge[END_REF]; and (iv) 90 mL ethanol (Prolabo, 99%) was added in 30 mL supernatant collected from the centrifugation method presented above, overnight incubation at 4 °C, the precipitates formed after adding ethanol were considered as extracted EPS [START_REF] Rä Ttö | Colanic acid is an exopolysaccharide common to many enterobacteria isolated from paper-machine slimes[END_REF].

At the end of each extraction procedure, extracted EPS were separated by means of centrifugation at 15,000 ×g for 20 min under 4 °C. In the ethanol method, precipitates were collected and dissolved in 30 mL ultrapure water again; as for other extraction methods, supernatant was collected and considered as extracted EPS.

EPS quantification and characterization

Dry weight (DW) and volatile dry weight (VDW) of all the extracted EPS samples (named EPSuntreated) were determined at 105 °C and 550 °C, respectively [START_REF] Guibaud | Relation between extracellular polymers' composition and its ability to complex Cd, Cu and Pb[END_REF]. The TOC concentration of EPSuntreated samples were determined by a TOC analyzer (TOC-L, Shimadzu).

Each EPSuntreated sample was diluted by ultrapure water to an estimated range of 2 -8 mg C/L for a better accuracy on the TOC analyzer. Each measurement was performed in duplicate on each EPS sample.

Quantification of PN and HS-like in the EPSuntreated samples were performed according to the modified Lowry method, bovine serum albumin (BSA, Sigma) and humic acid (Sigma) were used as standards [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF]. PS were quantified by the phenol-sulfuric acid method, glucose (Prolabo) was used as standard [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. Each quantification was carried out in triplicate on each EPS sample.

DAX-8 resin fractionation on EPS

The preparation of DAX-8 resin column was carried out according to the supplier's (Supelite™) instruction, and a schematic illustration of fractionation procedure is demonstrated in Fig.

Qualitative analyses on EPS by SEC at UV absorbance 210 nm

All the EPS fingerprints, including EPSuntreated, EPSbefore and EPSafter, were recorded by a Merck

Hitachi LA Chrom chromatograph equipped with diode array UV detector (L7455). The fingerprints monitored by UV absorbance at 210 nm were considered in this study, as they are corresponding to the whole organic and mineral compounds present in the extracted EPS [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][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][START_REF] Simon | Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges[END_REF]. To get a better resolution and improve the separation of EPS molecules, two columns were used in series [START_REF] Villain | The use of a new mobile phase, with no multivalent cation binding properties, to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment[END_REF]: Bio SEC 300Å and 100Å (Agilent), of which the theoretical size exclusion range limits are 5 -1,250 kDa and 0.1 -100 kDa, respectively. 150 mM phosphate buffer (75 mM Na2HPO4 and 75 mM NaH2PO4, Prolabo) at pH 7.0 (± 0.1) was used as mobile phase at a constant flow of 0.7 mL/min.

EPS samples were filtered through a 0.2 µm pore size membrane (Whatman ™ , GE Health) before the injection, and the injection volume of each sample was 100 µL.

The column calibration was carried out by using protein or amino acid standards [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]: ferritine (Sigma)-440 kDa; immunoglobulin G from human serum (Sigma)-155 kDa;

bovine serum albumin (Sigma)-69 kDa; ribonuclease A (Sigma)-13.7 kDa and thyrotropin releasing hormone (Sigma)-0.36 kDa. In the aMW calibration curve (Eq. 3.2), the logarithm of aMW in Da (log (aMW)) is plotted as a function of the elution volume (Ve, unit: mL):

(Eq. 3.2)

A total permeation volume was determined by NaNO3 at Ve = 22 mL. Peaks appearing after the total permeation volume were not taken into consideration, since the presence of those peaks might be partly due to EPS molecules that strongly interact with the column packing material [START_REF] Comte | Effect of extraction method on EPS from activated sludge: an HPSEC investigation[END_REF][START_REF] Villain | The use of a new mobile phase, with no multivalent cation binding properties, to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment[END_REF].

The aMW of hydrophobic EPS molecules retained by the DAX-8 resin can be obtained by comparing the fingerprints of EPSbefore and EPSafter. From this point of view, recorded EPS fingerprints were fractionated into several fractions according to the elution volume of different peaks. The peak area of each fraction was calculated by Origin 7.1 software. Therefore, the hydrophobicity of each molecular fraction can be displayed in terms of peak area difference ratio (Eq. 3.3).

(Eq. 3.3) where, Areabefore represents the area of the selected fraction attributed to EPSbefore fingerprints, whereas Areaafter is the area of the selected fraction attributed to EPSafter fingerprints.

Results

EPS characteristics

The DW, VDW, TOC and biochemical composition of different EPSuntreatedrawraw samples are summarized in Table 3.1. These results are significantly different depending on the extraction method used. The DW of different EPSunteated samples ranges from 5.3 to 34.7 mg/g sludge DW,

while VDW varies from 1.3 to 32.7 mg/g sludge VDW. The heating method gives both the highest DW and VDW values, 34.7 (± 0.1) mg/g sludge DW and 32.7 (± 0.1) mg/g sludge VDW, respectively, which is followed by the F.+Heat. method. In Table 3.1, when comparing the results among the centrifugation, ethanol and SDS extraction methods, the DW of EPSuntreated are almost the same, but the VDW are different. As for the Tween 20 method, the DW and VDW of EPSuntreated are both higher than those in the centrifugation method. The TOC content also varies in a wide range between different extraction methods (Table 3.1). The heating method induces the highest TOC level (396 ± 23 mg C/g EPSuntreated DW)

among the physical extraction methods. The lowest TOC value is observed in the sonication method (63 ± 5 mg C/g EPSuntreated DW).

Regarding the PN, HS-like and PS contents, the highest values are still reached by the heating method (185 ± 10 mg/g EPSuntreated DW for PN, 288 ± 8 mg/g EPSuntreated DW for HS and 133 ± 7 mg/g EPSuntreated DW for PS, respectively), which is followed by the F.+Heat. method (Table 3.1). In addition, no severe cell disruption during the extraction of EPS from multispecies microbial aggregates such as granules or biofilms was reported for the chosen extraction methods (Adav and Lee, 2008b;[START_REF] Zhang | Comparison of extraction methods for quantifying extracellular polymers in biofilms[END_REF].

Impact of bulk solution pH on EPS composition and fingerprints

Fig. 3.2 compares the impact of bulk solution pH on the biochemical composition of EPSuntreated and two pH-adjusted EPS samples (pH 5 and pH 2). In most cases, when a lower bulk solution pH (pH 2) is reached, lower concentrations of EPS components are measured. However, the situation is the opposite for the sonication and ethanol methods, as a decrease in the higher organics concentrations is observed at higher pH i.e. pH 5 (Fig. 3.2). When comparing the fingerprints between EPSuntreated and EPSbefore at pH 5, only the variation of peak intensity can be observed (Fig. 3.3). Besides, F1 and F2 can be detected in EPSuntreated and EPSbefore at pH5, but not in EPSbefore at pH 2, while F3 and F4 are detected in all three EPS samples. These phenomena indicate that the integrity of EPS aMW distribution is better preserved at pH 5 than at pH 2. Besides, more peaks can be observed in the fingerprint of EPSbefore at pH 5 than at pH 2 (Fig. 3.3), which could be that more EPS macromolecules are precipitating at lower pH.

Interferences induced by the organic reagents in TOC quantification and EPS hydrophobicity determination

The TOC concentrations of EPSbefore and EPSafter are displayed in Fig. 3.4. It can be seen from the results that the TOC concentration of EPSafter is obviously lower than that of EPSbefore. By comparing the TOC concentrations at two pH, higher values are determined in EPSbefore at pH 5 than at pH 2, indicating more organic constituents of EPSbefore molecules are preserved at pH 5.

However, the data from the ethanol method show the TOC concentrations of EPSbefore at both pH values are exceptionally above 1000 mg C/L (Fig. 3.4), which implies the carry-over of the organic reagents to the extracted EPS samples as previously demonstrated by D'Abzac et al.

(2010). In addition, the TOC concentrations of the organic reagents after the DAX-8 resin treatment are also decreased (Fig. 3.5), which evidenced the possible interactions between the organic reagents and the resin.

aMW features of EPS before and after resin treatment

Based on the SEC results analyzed before, the fingerprints of EPSbefore and EPSafter demonstrate in this section are mainly focusing on the results obtained at pH 5. The fingerprints are all displayed in Fig. 3.7. A significant difference observed in Fig. 3.7 is that F2 (aMW 175 -31 kDa) can be detected in EPSbefore but not in EPSafter. As for the other fractions (F1, F3, F4), only an intensity decrease can be identified (Fig. The peak area difference (%) is calculated and displayed in Table 3.2. Ethanol and Tween 20 extraction methods are excluded, due to the interference in the fingerprints (data not shown).

Table 3.2 shows that the area differences of each extraction method are different. Centrifugation, sonication and SDS demonstrated the similar performance, in which the area differences of F1, F2

and F3 are all above 50%. In contrast, the area differences of these three fractions are found below 50% in the heating and F.+Heat. methods. It should be noted that the area difference of F2

(aMW = 175 -31 kDa) is over 50% in all the displayed extraction methods (Table 3.2).

Discussion

Impact of bulk solution pH on EPS characteristics

The decrease in EPS component contents at both pH values investigated (Fig. 3.2) can be explained by the isoelectric point (IEP) of different organic compounds. The IEP is the pH at which a molecule carries no net electrical charge, and it affects the solubility of the molecule at a given pH. Such molecules at a given pH that corresponds to their IEP often precipitate out of the bulk solution [START_REF] Brown | Organic Chemistry[END_REF]. A few studies have determined the IEP values of bacterial surfaces or the bacterial EPS in terms of electrophoretic mobility (EM). Both intact and EPS-free Bacillus licheniformis S-86 cells have IEP values below 2 according to [START_REF] Tourney | The effect of ionic strength on the electrophoretic mobility and protonation constants of an EPS-producing bacterial strain[END_REF]. [START_REF] Harden | The isoelectric point of bacterial cells[END_REF] So far, no specific IEP value has been reported for the EPS extracted from anaerobic granular sludge. Since more precipitation of EPS was determined at pH 2 (Fig. 3.2), it can be inferred that pH 2 is more close to the IEP value of the extracted EPS. However, the IEP value of the EPS extracted by sonication and ethanol methods seems close to pH 5 (Fig. 3.2). Anaerobic granules are made up by different microbial consortia and also EPS extraction methods influence the extracted EPS properties [START_REF] Nielsen | Changes in the composition of extracellular polymeric substances in activated sludge during anaerobic storage[END_REF]. Therefore, different IEP values of EPS could be determined even for the same type of granular sludge.

In addition, IEP values are influenced by the acid dissociation constant (pKa) of given molecules [START_REF] Tourney | The effect of ionic strength on the electrophoretic mobility and protonation constants of an EPS-producing bacterial strain[END_REF], and the charge of certain functional group are depending on the bulk solution pH [START_REF] Behrens | Approximate calculation and experimental derivation of native isoelectric points of membrane protein complexes of Arabidopsis chloroplasts and mitochondria[END_REF]. Four distinct pKa values can be determined for the EPS extracted from anaerobic granular sludge by potentiometric titration: pKa1 4 -5 corresponding to the carboxyl groups; pKa2 6 -7 corresponding to the phosphoric groups; in bound EPS extracted from the same type of anaerobic granular sludge. The pKa value of sulfate group is known to be below 2.5 [START_REF] Braissant | Exopolymeric substances of sulfate-reducing bacteria: interactions with calcium at alkaline pH and implication for formation of carbonate minerals[END_REF]. pH 2 is commonly used in the hydrophobic/hydrophilic fractionation, as most of the functional groups (i.e. carboxyl, phenolic) become nonionic at this pH, and in turn, maximize the hydrophobicity of studied molecules [START_REF] Aiken | Humic substances in soil, sediment, and water: geochemistry, isolation, and characterization[END_REF][START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF].

In our study, pH 5 is chosen as a compromise between the conservation of EPS molecules structure and the protonation of certain functional groups. According to the reported IEP and pKa values of bacteria or EPS discussed above, when comparing the two tested pH values with the initial pH of EPSuntreated in this study (Table 3.1), more functional groups in EPS should be protonated at pH 2 than at pH 5. Higher pH (pH 5) indicates an ionized state of functional groups such as carboxyl and sulfate groups, which may result in less EPS molecules being retained by the resin. Thus, the EPS global hydrophobicity (%) determined at pH 2 should be higher than that at pH 5, as it is observed in Fig. 3.6. However, the EPS extracted by the heating method displays an opposite situation, which may due to the PN denaturation caused by the heat treatment (Petsko and Ringe, 2008). Therefore, the similar trend on EPS global hydrophobicity (%) at both pH values (Fig. 3.6) and the better conservation of EPS aMW features at pH 5 (Fig. 3.3) confirm the feasibility of elution at pH 5.

Impact of EPS extracting reagents on EPS characterization

In respectively) are almost five times higher than that of centrifugation method (Table 3.1), but its EPS global hydrophobicity (%) is below the one of centrifugation method (Fig. 3.6B).

In the SDS extraction method, although the final concentration of SDS (about 0.001%, w/v) is below its CMC in water (0.23%, w/v), the EPS global hydrophobicity (%) is still below the value determined for centrifugation method (Fig. 3.6B). For ionic surfactants like SDS, their CMC levels are changing with the ionic strength of the bulk solution [START_REF] Giehm | SDSinduced fibrillation of α-synuclein: an alternative fibrillation pathway[END_REF].

Besides that, even below their CMC levels, the formation of micelle-like surfactant clusters is induced by the presence of the proteins. Those clusters can interact with the proteins to enhance their solubility in the water [START_REF] Otzen | Protein-surfactant interactions: A tale of many states[END_REF].

Formaldehyde could cross-link between amino and sulfhydryl groups of PN, as well as enter cells and subcellular compartments to stabilize cellular architecture before gross and microscopic degradation of the native cellular components [START_REF] Chang | Reaction mechanisms of formaldehyde with endocyclic imino groups of nucleic acid bases[END_REF][START_REF] Sutherland | Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions[END_REF]. If the exposure of bacterial cells to the high temperature is poorly controlled, an abnormal cell lysis of intracellular components as well as a denaturation of a part of the EPS molecules (PN or enzymes) can be induced. Nevertheless, no severe cell disruption in microbial aggregates, such as granules¸ is caused by heating at 80 C for 10 min (Adav and Lee, 2008b;[START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF][START_REF] Zhang | Comparison of extraction methods for quantifying extracellular polymers in biofilms[END_REF]. Besides, the addition of formaldehyde during EPS extraction is to minimize the leakage of intracellular components [START_REF] Sutherland | Chemical extraction methods of microbial cells[END_REF]. Thus, the determined EPS biochemical contents by the F.+Heat. method should be similar to the heating method alone, which is in accordance with the results in Table 3.1.

The addition of ethanol is a common concentration step to precipitate polysaccharides [START_REF] Sutherland | Biofilm exopolysaccharides: a strong and sticky framework[END_REF]. When ethanol is applied in EPS extraction, EPS molecules that have a polarity different from the ethanol phase will become insoluble and precipitate. These precipitates are considered as extracted EPS. Since the addition of ethanol takes place after the centrifugation step, the EPS yield in the ethanol method should be theoretically similar or lower than the centrifugation method [START_REF] Wei | Influence of extracellular polymeric substances (EPS) on Cd adsorption by bacteria[END_REF][START_REF] Flemming | The biofilm matrix[END_REF][START_REF] Forster | The production of polymer from activated sludge by ethanolic extraction and its relation to treatment plant operation[END_REF]. However, the overwhelming TOC concentration of EPS in the ethanol method (beyond 1000 mg C/L) indicates the presence of ethanol in the dissolved EPS solution (Fig. 3.4). Besides, in Fig. 3.2, it is noticed in the ethanol method that more PN are determined in EPSbefore at pH 2 than in EPSuntreated. This might due to the residual ethanol interfering with the PN determination [START_REF] Lucarini | Comparative study of Lowry and Bradford methods: interfering substances[END_REF].

It can also be inferred that the extra organic carbon content provided by other reagents such as formaldehyde, SDS and Tween 20 and their presence in the extracted EPS should not be neglected [START_REF] Comte | Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods[END_REF]. The decrease of the TOC concentrations of those organic reagents after DAX-8 resin treatment (Fig. 3.5) has confirmed the interactions between these reagents and the resin. DAX-8 resin fractionation relies on the hydrophobic/hydrophilic interactions between the resin and the solutes, and exploits differences in polarity of organic matters. No related study has reported the interaction between the DAX-8 resin and those organic reagents. Nevertheless, since ethanol and formaldehyde are polar organic molecules, SDS and Tween 20 possess hydrophobic molecular moiety, the interaction between the resin and those organic reagents should be estimated. Moreover, it is not appropriate to subtract those interfering carbon concentrations (Fig. 3.5) from the TOC concentrations of EPSbefore and EPSafter to determine the global hydrophobicity (%), not only because the TOC concentration of these organic reagents before and after the resin treatment is beyond the values of the EPS which they extracted (Fig. 3.5), but also because the sorption of these reagents onto granules at an unknown concentration could occur during the EPS extraction.

In addition, it is noticed that the final concentration of Tween 20 used in the EPS extraction is above its CMC, which may generate the formation of EPS-surfactant micelles [START_REF] Johnson | Detergents: Triton X-100, Tween-20, and more[END_REF]. Owing to this fact, Tween 20 could interfere in the characterization of EPS aMW distribution in this study. This postulation is confirmed by the wide-ranged peak on the fingerprints of EPSbefore at pH 5 of the Tween 20 method (Ve = 8.5 -17 mL, Fig. 3.3) and Tween 20 at pH 5 (Ve = 14.5 -22 mL, data not shown). Similar interferences caused the organic reagents, i.e. formaldehyde and EDTA, in the EPS fingerprints was also demonstrated by [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].

It is therefore concluded that the DAX-8 resin fractionation with TOC measurement is not appropriate to determine the hydrophobicity of EPS extracted by organic reagents, since: (i) extra carbon content contained in organic reagents induces interference in the TOC determination; (ii) the reaction between EPS and the surfactants may change the EPS characteristics and influence the resin fractionation results; and (iii) the interaction between organic reagents and the DAX-8 resin exist, which indicates these organic reagents may compete with hydrophobic EPS molecules for the binding sites on the resin. To avoid these interferences caused by the organic reagents, this study shows that physical EPS extraction methods are preferred in the studies of the hydrophobic properties of EPS.

EPS hydrophobic features

Due to the improperness of using the TOC content to determine EPS global hydrophobicity (%), the sum of three main organic constituents of EPS (PN, HS-like and PS) quantified by the colorimetric methods is used in its determination. Fig. 3.6B shows that the highest EPS global hydrophobicity (%) is achieved by the centrifugation method at both two pH, which implies this extraction method has extracted a higher content of hydrophobic EPS molecules than any other method. High-speed centrifugation is known as a mild extraction method to extract LB-EPS from the biomass [START_REF] Ramesh | Membrane biofouling by extracellular polymeric substances or soluble microbial products from membrane bioreactor sludge[END_REF][START_REF] Zhao | Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS)[END_REF]. [START_REF] Zhao | Bacterial cell surface properties: role of loosely bound extracellular polymeric substances (LB-EPS)[END_REF] investigated the effect of LB-EPS on the surface properties of four bacteria (Bacillus subtilis, Streptococcus suis, Escherichia coli and Pseudomonas putida), and found that the four LB-EPS free bacteria all exhibited fewer hydrophobicity values as compared to the intact cells.

In Fig. 3.7, the peak intensity of each fraction is reduced after the hydrophobic fractionation, it is thus assumed that the peak area difference could be assimilated as the hydrophobic part of each molecular fraction. F2 is not present in the EPSafter fingerprints, indicating these EPS molecules are completely retained by the resin, and thus highly hydrophobic.

Besides, the peak area difference ratio of F2 in each extraction method is above 60% (Table 3.2), which implies that these highly hydrophobic EPS molecules can be extracted by all the chosen methods. Despite different values about EPS global hydrophobicity (%) were achieved (Fig.

3.6B

), from the qualitative aspect, EPS molecules featured by an aMW ranging from 175 to 31 kDa may play the major role in determining EPS hydrophobicity.

In addition, if an aMW fraction with a peak area difference ratio above 60% is considered highly hydrophobic, it is noticed that the peak area difference ratio of F3 in the centrifugation method is also above 60%, while others are not (Table 3.2). This observation suggests that the hydrophobicity of EPS extracted by the centrifugation method is also largely ascribed to the molecules distributed in F3 (aMW = 16 -1 kDa). As for the other extraction methods, EPS molecules in F3 demonstrate moderately (sonication and SDS) or weakly (heating and F.+Heat.) hydrophobic properties. This observation could explain why EPS extracted by centrifugation is displaying the highest global hydrophobicity (Fig. 3.6B).

Conclusion

The EPS extraction methods and bulk solution pH influenced both composition and hydrophobicity of the EPS. By comparing the aMW distribution of untreated and pH-adjusted EPS samples, more complete EPS aMW information was preserved at pH 5. Thus, elution at pH 5 was preferred for the qualitative analysis of EPS hydrophobic features. The hydrophobic fraction of EPS retained by the resin at pH 5 was ascribed to a wide aMW range ranging from >440 to 0.3 kDa. Within this range, EPS molecules ranging from 175 to 31 kDa were mostly retained by the resin, which indicates that these EPS molecules are highly hydrophobic.

Introduction

Pulping and paper-making activities generate the third largest amount of wastewater in the world [START_REF] Kallas | Post-treatment of pulp and paper industry wastewaters using oxidation and adsorption processes[END_REF]. This wastewater contains a variety of organic macromolecules that mostly originate from tannins, lignins, resins, and chlorine compounds [START_REF] Buzzini | Evaluation of a upflow anaerobic sludge blanket reactor with partial recirculation of effluent used to treat wastewaters from pulp and paper plants[END_REF].

Most of these organic pollutants are hydrophobic and considered potentially persistent because of their structural analogy to polycyclic aromatic hydrocarbons (PAHs) and biphenyls [START_REF] Terasaki | Organic pollutants in paper-recycling process water discharge areas: first detection and emission in aquatic environment[END_REF]. To remove or degrade these compounds from wastewater, anaerobic granular sludge systems have been successfully applied in the wastewater treatment of pulp and paper plants since the 1980s [START_REF] Savant | Anaerobic degradation of adsorbable organic halides (AOX) from pulp and paper industry wastewater[END_REF][START_REF] Lettinga | High-rate anaerobic waste-water treatment using the UASB reactor under a wide range of temperature conditions[END_REF].

Extracellular polymeric substances (EPS) cover the microbial cell surface and form the support matrix of biofilms and anaerobic granular sludge [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF]Li et al., 2016).

Two types of EPS are operationally defined: loosely bound EPS (LB-EPS) which are described as a loose and dispersible slime layer outside the cell walls, and tightly bound EPS (TB-EPS) that are closely associated with the cell walls [START_REF] Wingender | What are bacterial extracellular polymeric substances? In: Microbial Extracellular Polymeric Substances[END_REF]. EPS are responsible for the cell surface hydrophobicity and influence the sorption capacity of granular sludge [START_REF] Beer | Distribution of extracellular polysaccharides and flotation of anaerobic sludge[END_REF]Grotenhuis et al., 1991;[START_REF] Ni | Distribution of extracellular polymeric substances in anammox granules and their important roles during anammox granulation[END_REF]. Generally, a higher EPS content may result in a higher cell surface hydrophobicity [START_REF] Wang | Effects of extracellular polymeric substances on aerobic granulation in sequencing batch reactors[END_REF], and statistical analyses showed a significantly high correlation between the ability to degrade organic pollutants and cell surface hydrophobicity [START_REF] Obuekwe | Hydrocarbon degradation in relation to cell-surface hydrophobicity among bacterial hydrocarbon degraders from petroleum-contaminated Kuwait desert environment[END_REF].

The hydrophobic fraction in the EPS provides the sorption sites for organic pollutants such as phenanthrene and benzene, which is mainly due to the hydrophobic interactions between the EPS and the organic pollutants [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF][START_REF] Jia | Degradation of pyrene in soils by extracellular polymeric substances (EPS) extracted from liquid cultures[END_REF][START_REF] Pan | Binding of phenanthrene to extracellular polymeric substances (EPS) from aerobic activated sludge: a fluorescence study[END_REF][START_REF] Xu | Roles of extracellular polymeric substances (EPS) in the migration and removal of sulfamethazine in activated sludge system[END_REF]. These interactions alter the EPS hydrophobicity, and in turn significantly influence the overall cell surface hydrophobicity of microbial aggregates in the bioreactors [START_REF] Liu | Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge[END_REF]. The hydrophobic fraction in the EPS is largely ascribed to their main organic constituents i.e. proteins (PN), polysaccharides (PS) and humic-like substances (HS-like) [START_REF] Flemming | Sorption properties of biofilms[END_REF][START_REF] Jahn | Cell biomass and exopolymer composition in sewer biofilms[END_REF]. Studies showed that mainly PN contributed to the cell surface hydrophobicity [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF][START_REF] Jahn | Cell biomass and exopolymer composition in sewer biofilms[END_REF], and acetylated PS could form hydrophobic moieties and thus influence EPS hydrophobicity (Henriques [START_REF] Vieira | Role of hydrophobic extracellular polysaccharide of Aulacoseira granulata (Bacillariophyceae) on aggregate formation in a turbulent and hypereutrophic reservoir[END_REF][START_REF] Mayer | The role of intermolecular interactions: studies on model systems for bacterial biofilms[END_REF][START_REF] Neu | Structural studies of an emulsionstabilizing exopolysaccharide produced by an adhesive, hydrophobic Rhodococcus strain[END_REF]. HS-like compounds are also integral constituents of EPS extracted from the sludge used in wastewater treatment processes [START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF][START_REF] Riffaldi | Humic substances in sewage sludges[END_REF][START_REF] Yuan | Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge[END_REF]. Nevertheless, the hydrophobic features of these HS-like compounds in the EPS are usually neglected in EPS-related studies.

The differentiation between LB-EPS and TB-EPS largely depends on the extraction method:

LB-EPS can be extracted by mild extraction methods such as high-speed centrifugation, while more harsh conditions (e.g., sonication, heating or adding chemical reagents) are required to extract TB-EPS from the biomass [START_REF] Jahn | Cell biomass and exopolymer composition in sewer biofilms[END_REF]. Besides, the relative ratio between the hydrophilic and hydrophobic fraction of EPS lies on the extraction method, as different EPS extraction yields are achieved when applying different extraction methods [START_REF] Comte | Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods[END_REF][START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF]. To obtain an integral view on the hydrophobic features of extracted EPS, different EPS extraction methods should thus be considered.

Therefore, three physical (centrifugation, heating and sonication) and one chemical (sodium dodecyl sulfate (SDS)) extraction methods were applied to extract LB-and TB-EPS from anaerobic granular sludge [START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF][START_REF] Wu | Evaluation of different methods for extracting extracellular DNA from the biofilm matrix[END_REF]. A modified hydrophobic fractionation method developed from [START_REF] Thurman | Aquatic humic substances[END_REF] by using DAX-8 resin alone was applied to isolate the hydrophobic fraction of the extracted EPS (Cao et al., 2017). To characterize the hydrophobic molecular features of EPS from both quantitative and qualitative aspects, also classical colorimetric methods, size exclusion chromatography (SEC) coupled to UV and fluorescence detection, as well as fluorescence excitation and emission matrix (EEM) spectroscopy were applied.

Materials and methods

Source of anaerobic granular sludge

The studied anaerobic granules were sampled from an upflow anaerobic sludge blanket (UASB)

reactor, operated at the Smurfit Kappa paper recycling plant (Saillat-sur-Vienne, France). The treated wastewater is discharged from the cardboard manufacturing. The total suspended solid (TSS) and volatile suspended solid (VSS) of the sludge were 125 (± 2.7) g/L and 106 (± 2.6) g/L, respectively.

EPS extraction

Prior to EPS extraction, the collected sludge was washed by ultrapure water twice and then re- water bath; and (iv) ultrapure water was replaced by 0.01% (w/v) SDS, and then agitated at 300 rpm for 4 h at 4 °C.

At the end of each extraction process (except for centrifugation), EPS were harvested by a centrifugation procedure (15,000 × g, 20 min, 4 °C), the supernatant was collected and considered as extracted EPS. EPS extracted by centrifugation were considered as LB-EPS, whereas EPS extracted from a combination of other methods and centrifugation were considered as the sum of LB-EPS and TB-EPS [START_REF] Nielsen | Extraction of EPS[END_REF]. All the extracted EPS samples (EPSuntreated) were stored at -20 °C until subsequent analysis.

EPS characterization

Dry weight (DW) and volatile dry weight (VDW) of EPSuntreated were quantified according to [START_REF] Guibaud | Relation between extracellular polymers' composition and its ability to complex Cd, Cu and Pb[END_REF]. Total organic carbon (TOC) concentrations of all the EPSuntreated samples was determined by a TOC analyzer (TOC-L, Shimadzu). Each measurement was conducted in duplicate.

The quantification of the PN, PS and HS-like contents was carried out in triplicate by using colorimetric methods: a modified Lowry method was applied to quantify PN and HS-like compounds using, respectively, bovine serum albumin (BSA, 96%, Sigma-Aldrich) and humic acid (Sigma-Aldrich) as standards [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF]; the Dubois method by using phenol and sulfuric acid was applied for PS quantification, in which glucose was used as the standard [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF].

Hydrophobic fractionation by DAX-8 resin

The DAX-8 resin column was prepared according to the supplier's instruction (Supelite™, Sigma-Aldrich). Detailed EPS fractionation by the prepared column can be found in (Cao et al., 2017), and the fractionation of each EPS sample was performed in duplicate. Briefly, each EPSuntreated sample was adjusted to pH 5.0 (± 0.1) by 2 M HCl (Sigma-Aldrich) and filtered on a 0.45 µm cellulose nitrate filter (Whatman™, GE Healthcare) before uploading into the column.

The filtrate (EPSbefore) was pumped into the column at a speed of 50 mL/h. The eluted EPS fraction from the column was collected as EPSafter. Ultrapure water and 0.01% SDS (Merck) at pH 5.0 were also tested on the column as control group. The PN, HS-like and PS concentrations of both EPSbefore and EPSafter were determined using the methods described above.

Since the DAX-8 resin only retains hydrophobic molecules, the characteristic difference between EPSbefore and EPSafter can be ascribed to the hydrophobic EPS fraction sorbed onto the resin. Thus, the hydrophobicity (%) of a certain macromolecule class can be calculated by Eq. 4.1: used to estimate the involvement of the specific molecular structures in the hydrophobic interactions with DAX-8 resin.

(Eq. 4.2)

where: n represents the peak labelling in the region i.

Size exclusion chromatography (SEC)

A Merck Hitachi LA Chrom chromatograph equipped with a fluorescence detector (L7485) and diode array UV detector (L7455) were utilized to detect EPS fingerprints. For a better resolution of the apparent molecular weight (aMW) distribution of the EPS samples, Agilent Bio SEC 300Å and 100Å columns were used in series [START_REF] Villain | The use of a new mobile phase, with no multivalent cation binding properties, to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment[END_REF]. The theoretical size exclusion limits of those two columns are 5 -1,250 kDa and 0.1 -100 kDa, respectively. The excitation (Ex) and emission (Em) wavelengths chosen for the fluorescence detection were 222/300 nm (Ex/Em) for PN-like organics and 300/500 nm (Ex/Em) for HS-like organics [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][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]. Meanwhile, the fingerprints recorded at UV absorbance of 210 nm (UV/210 nm) were also considered in this study, as they correspond to all organic and mineral compounds present in the EPS [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][START_REF] Simon | Evaluation of size exclusion chromatography (SEC) for the characterization of extracellular polymeric substances (EPS) in anaerobic granular sludges[END_REF].

Before the injection, each EPS sample was filtered by 0.2 µm regenerated cellulose filter (Whatman ™ , GE Healthcare). The injection volume of each sample was 100 µL. 150 mM phosphate buffer (75 mM Na2HPO4 and 75 mM NaH2PO4, pH 7.0 ± 0.1) was used as the mobile phase, and its flow rate was controlled at 0.7 mL/min. 0.01% SDS at pH 5.0 was also tested as control.

The MW calibration was carried out by using several protein or amino acid standards [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]: thyroglobulin (Sigma) -660 kDa; ferritine (Sigma) -440 kDa;

immunoglobulin G from human serum (Sigma) -155 kDa; bovine serum albumin (Sigma) -69 kDa, ribonuclease A (Sigma) -13.7 kDa and thyrotropin releasing hormone (Sigma) -0.36 kDa.

Eq. 4.3 was obtained by plotting the logarithm of aMW (log (aMW)) versus the elution volume (Ve).

( Eq. 4.3) where: the units of aMW and Ve are Da and mL, respectively. The total permeation volume (Ve = 22 mL) was determined by NaNO3.

Results

Characterization of extracted EPS

Table 4.1 summarizes the biochemical characteristics of different EPSuntreated samples. HS-like compounds are the major organic constituent of all EPSuntreated samples, while PN and PS are less abundant. The organic composition of EPSuntreated extracted by the centrifugation method shows the lowest PN content (about 5 mg/g EPSuntreated DW) compared to the other three methods, while significant amounts of HS-like and PS can be quantified (Table 4.1). This suggests that the LB-EPS from the studied granules are mainly composed by the latter two organic constituents. For the other methods, higher PN contents than those in the centrifugation method are quantified (> 50 mg/g EPSuntreated DW) (Table 4.1). This also implies that PN are enriched in the TB-EPS. The PN, HS-like and PS content in these methods generally follows the descending order as: HS-like > PN ≥ PS.

When comparing between the centrifugation and SDS method, despite their respective DW and VDW values of EPSuntreated are almost the same, a higher content of PN, HS-like and PS are quantified in the SDS method (Table 4.1). This could be due to: (i) surfactant SDS has interfered with the measurement of colorimetric method, or (ii) the solubility preference of SDS for certain type of molecules has improved the EPS extraction yield.

Effect of DAX-8 resin treatment on the organic matter content of EPS

The PN, HS-like and PS contents quantified in, respectively, the EPSbefore and EPSafter samples are displayed in Fig. 4.1. Lower contents of these organic constituents are observed in the EPSafter samples (Fig. 4.1), implying that the DAX-8 resin has effectively retained the hydrophobic moiety in these organic constituents. The hydrophobicity (%) of each EPS organic constituent is calculated according to Eq. 4.1 and demonstrated in Fig. 4.2. The values vary with the extraction method used. Nevertheless, the hydrophobicity (%) of HS-like compounds is the most significant compared to PN and PS, regardless of the extraction method used. In general, the hydrophobicity (%) of these three main organic constituents follow the descending order as: HS-like > PS > PN (Fig. 4.2). A negative effect is observed in the PN content from the centrifugation method, it could be due to the lowest PN concentration in the EPS extracted by the centrifugation method (Table 4.2) which reaches the measurement limitation of the colorimetric method, and thus, causes relatively higher standard deviations. Based on the peak location, the obtained EPS fingerprints are generally divided into five aMW fractions for a better analysis: (i) F1, Ve = 9.5 -11.5 mL, corresponding to aMW >660 kDa;

Hydrophobic molecular features of EPS characterized by excitation & emission matrix (EEM) spectroscopy

(ii) F2, Ve = 14 -16 mL, corresponding to aMW 122 -31 kDa; (iii) F3, Ve = 18 -19.7 mL, corresponding to aMW 8 -2 kDa; (iv) F4, Ve = 19.7 -21 mL, corresponding to aMW 2 -1 kDa; and (v) F5, Ve = 21 -22.5 mL, corresponding to aMW <1 kDa.

A similar peak distribution is observed between the fingerprints of EPSbefore samples recorded for TOM (UV/210 nm) and PN-like compounds (Ex/Em 222/300 nm) (Fig. 4.5). When comparing these two fingerprints among different extraction methods, an obvious difference in the peak distribution can be seen between the centrifugation and other EPS extraction methods. In the heating, sonication and SDS methods, peaks assigned to the TOM and PN-like compounds occupy all five aMW fractions (Fig. 4.5b -d). It implies that the TOM and PN-like compounds in these EPSbefore samples have a wide aMW distribution, ranging from >660 kDa to <1 kDa. In the centrifugation method, the aMW fraction of F2 is not present in the EPSbefore fingerprints (Fig.

4.5a

). As for the EPSbefore fingerprints of HS-like compounds (Ex/Em 300/500 nm) (Fig. 4.5), the corresponding peaks occupy only three aMW fractions: F3 -F5, which shows that the HS-like compounds in all the EPSbefore samples are mainly composed by small molecules (aMW = 8 -<1 kDa).

When comparing the peak intensity of all types of EPSbefore and EPSafter fingerprints, peaks with a lower intensity are observed in aMW fractions F3, F4 and F5 of the EPSafter fingerprints compared to those of the EPSbefore fingerprints (Fig. 4.5). This observation indicates that the EPS molecules assigned to these four fractions all possess hydrophobic molecular moieties.

Meanwhile, it is also noticed that F1 in the centrifugation method and F2 in the other extraction methods cannot be found in the corresponding EPSafter fingerprints (Fig. 4.5). This indicates that the EPS molecules in F1 (aMW >660 kDa) detected in the centrifugation method and F2 (aMW = 122 -31 kDa) detected in the other extraction methods are completely retained by the DAX-8 resin, and thus, are considered highly hydrophobic.

Discussion

Hydrophobic molecular features of organic constituents in the EPS

Humic-like substances

Table 4.1 shows that the HS-like compounds are the major organic constituent of the EPS extracted from anaerobic granular sludge used to treat the wastewater from a paper recycling plant.

Their presence in the sludge EPS is due to (i) the sorption of humic substances (HS) in the aquatic environment by the microorganisms [START_REF] Wingender | What are bacterial extracellular polymeric substances? In: Microbial Extracellular Polymeric Substances[END_REF]; (ii) the consequence of partial enzymatic degradation of various biopolymers by the microorganisms; and (iii) condensation ("repolymerization") of refractive small organic breakdown products in the wastewater, such as lignin, tannins and secondary metabolites [START_REF] Huang | Formation mechanisms of humic substances in the environment[END_REF][START_REF] Wingender | What are bacterial extracellular polymeric substances? In: Microbial Extracellular Polymeric Substances[END_REF].

Besides, HS-like compounds are highly resistant to biodegradation when compared with PS and PN [START_REF] Kä Stner | Biodegradation of humic substances[END_REF]. Therefore, it is considered that the high content of HS-like compounds in the extracted EPS is a consequence of the interplay between the microbial activities and the wastewater composition.

In most cases, HS-like compounds have a higher hydrophobicity (%) than the other two constituents (Fig. 4.2) and contribute the most to the EPS global hydrophobicity (Fig. 4.3). This could be due to the molecular structure of HS-like compounds, which are more hydrophobic (contain a more abundant hydrophobic moiety) than PN and PS. HS-like compounds are usually described as phenolic compounds with more than two fused benzene rings, the corresponding fluorophores are usually detected in Region II by the EEM technique [START_REF] Bassandeh | Evaluation of ion exchange resins for the removal of dissolved organic matter from biologically treated paper mill effluent[END_REF][START_REF] Hudson | Fluorescence analysis of dissolved organic matter in natural, waste and polluted waters-a review[END_REF]. A slight decrease found in the CI of EPSafter (Table 4.2) indicates that the decrease of polycyclic aromatic fluorophores in Region II after the resin treatment is greater than that of the limited number aromatic rings fluorophores in Region I. Therefore, the HS-like molecules are the major support of the EPS hydrophobicity in the anaerobic granules used in the wastewater treatment system investigated.

The FI reduction (%) of Peak E (Ex/Em 270-285/380-430 nm) in Region II is significantly higher than that of Peak C (Ex/Em 225-235/370-400 nm) (Table 4.2), which indicates that the fluorophores at the Peak E position are more hydrophobic than the fluorophores at Peak C. The source of fluorophores detected in Region II from the paper mill effluents can be ascribed to the breakdown products of lignin (Ex/Em 230-275/400-520 nm), polyaromatic hydrocarbons such as phenanthrene, anthracene, pyrene (Ex/Em 220-300/370-430 nm), and humic acid (Ex/Em 220-320/400-550 nm) present in the paper mill effluents [START_REF] Carstea | Fluorescence spectroscopy for wastewater monitoring: a review[END_REF]. In the analysis of EPS extracted from the sludge treating those effluents, Peak C and Peak E are usually designated as fulvic acid-like and humic acid-like fluorophores, respectively [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][START_REF] Sun | Structural and functional properties of organic matters in extracellular polymeric substances (EPS) and dissolved organic matters (DOM) after heat pretreatment with waste sludge[END_REF][START_REF] Zhu | Specific component comparison of extracellular polymeric substances (EPS) in flocs and granular sludge using EEM and SDS-PAGE[END_REF]. Fulvic acid-like compounds are more water soluble (hydrophilic), as they contain more carboxylic and hydroxyl functional groups than humic acid-like compounds; whilst the latter are mostly characterized by compounds with an aliphatic structure [START_REF] Ishiwatari | Macromolecular material (humic substance) in the water column and sediments[END_REF][START_REF] Thurman | Aquatic humic substances[END_REF]. Thus, the hydrophobicity of HS-like compounds in the EPS is probably ascribed to the humic acid-like molecular structure.

SEC results reveal these HS-like compounds are mainly composed of small aMW molecules (8 -<1 kDa) detected in F3 -F5 fractions, and the decreased peak intensity observed in the EPSafter fingerprints has also confirmed their hydrophobic properties (Fig. 4.5). Similarly, [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] as well as [START_REF] Adav | Characterization of extracellular polymeric substances (EPS) from phenol degrading aerobic granules[END_REF] also concluded that the aMW of HS-like compounds in the EPS extracted from anaerobic or aerobic granules was lower than 6 kDa.

Therefore, the aMW of HS-like compounds involved in the EPS hydrophobicity is not impacted by the extraction method (except the heating method), and these HS-like compounds are all involved in the EPS hydrophobicity. Nevertheless, some PN-like molecules were present in the same low aMW fractions (F3 -F5) in the fingerprints (Fig. 4.5). It indicates that the HS-like compounds in the extracted EPS could be present in pure form but also as HS-PN-like complexes.

Treated EPS samples regarding to the heating extraction method demonstrated some different characteristics: it is noticed that there are no peaks in Region II present in the EEM spectra of the heating method (Fig. 4.4b). This could be due to a too high sample dilution factor

Proteins

PN are the second more abundant organic constituent in the extracted EPS (Table 4.1), but they demonstrate the lowest hydrophobicity (%) among the three components (Fig. 4.2). It may indicate that the molecular structure of PN is involved in the EPS hydrophobicity to a lesser extent compared to the other constituents. Li et al. (2016) concluded that nonpolar groups such as side chains of aromatic amino acids were important for the EPS hydrophobicity of the anaerobic granular sludge surface. Besides, PN can be related to catalytic and hydrolase activity in the anaerobic sludge [START_REF] Kim | Hydrolytic activities of extracellular enzymes in thermophilic and mesophilic anaerobic sequencingbatch reactors treating organic fractions of municipal solid wastes[END_REF], and these extracellular enzymes may promote the production of HS-like compounds within the anaerobic granules.

In the EEM spectra, the FI reduction (%) of Peak B (Ex/Em 225-230/285-330 nm) in Region I is significantly higher than that of peak A (Ex/Em 220-225/285 nm) and D (Ex/Em 255/279-295 nm) (Table 4.2), implying that the fluorophores at peak B are the most hydrophobic fluorophores in Region I. Peaks in Region I are usually considered as PN deprived peaks, and the fluorophores at the peak B position (Ex/Em 225-230/285-330 nm) can be described as tryptophan-like molecules [START_REF] Coble | Characterization of marine and terrestrial DOM in seawater using excitationemission matrix spectroscopy[END_REF][START_REF] Riopel | Fluorescence characterization of natural organic matter at a northern Ontario wastewater treatment plant[END_REF].

In aqueous environments, the PN structure includes a hydrophobic and a hydrophilic network. Amino acids such as tryptophan, leucine, isoleucine are considered as hydrophobic amino acids and form the hydrophobic network of the PN structure, whereas hydrophilic amino acids i.e. tyrosine, glycine, serine form the hydrophilic network. Those hydrophobic amino acids come close to each other and locate in the buried region of the folded structure; whereas the hydrophilic amino acids are present on the surface [START_REF] Rose | Hydrophobicity of amino acid residues in globular proteins[END_REF]. Heating may lead to PN denaturation, which involves the disruption and possible destruction of hydrogen bonds and nonpolar hydrophobic interactions in both secondary and tertiary structures of PN. Thus, a loose PN secondary structure facilitates a full exposure of the inner hydrophobic groups to express hydrophobicity [START_REF] Hou | Role of extracellular polymeric substance in determining the high aggregation ability of anammox sludge[END_REF]. This could explain why PN demonstrate a higher hydrophobicity than the HS-like compounds and PS in the heating method (Fig. 4.3).

It is noticed that the peak in F5 of EPSafter in the PN-like fingerprints using the SDS method demonstrates a higher intensity than that of EPSbefore (Fig. 4.5d). It could be ascribed to the protein-surfactant interactions: SDS denatures PN by strong binding to charged and hydrophobic side chains of PN, and some hemi-micelles could form on the PN surface [START_REF] Nielsen | Unfolding of β-sheet proteins in SDS[END_REF][START_REF] Otzen | Protein-surfactant interactions: A tale of many states[END_REF]. It is thus considered that the removal of some residual SDS in the EPS solution by DAX-8 resin has induced the higher intensity of PN-like peaks in F5 recorded for EPSafter (Cao et al., 2017).

Since the EPS samples extracted by the centrifugation method contain a minimal concentration of PN (Fig. 4.1) and the aMW distribution of HS-like compounds are only present in F3 -F5 (Fig. 4.5), it can be deducted that F1 (aMW >440 kDa) and F2 (aMW = 122 -31 kDa) in the EPS are mainly made up by PN and PS, or glycoproteins/proteoglycans. The presence of glycoprotein and sulfated proteoglycan-like in the EPS extracted from anaerobic granular sludge was evidenced by Bourven et al. (2015a), and these compounds are high molecular weight (HMW) molecules (aMW >150 kDa). Therefore, despite the PN molecules demonstrate the lowest hydrophobicity (%) in the EPS (Fig. 4.2) from a quantitative point of view, the possible links between PN and PS or HS-like molecules make it difficult to assign the exact role of PN in the EPS hydrophobicity.

Polysaccharides

PS are the least abundant organic components in the extracted EPS (Table 4.1), but they demonstrate a higher hydrophobicity (%) than the PN (Fig. 4.2). [START_REF] Zhang | Distribution of extracellular polysaccharides in the anaerobic granular sludges[END_REF] showed that the main constituents of extracellular PS in the methanogenic granules were glucose/mannose and N-acetyl-galactosamine, whereas a fewer content of fucose and galactose was also identified.

Some bacterial PS, in particular if acetylated, form hydrophobic pockets [START_REF] Mayer | The role of intermolecular interactions: studies on model systems for bacterial biofilms[END_REF]. [START_REF] Almond | Towards understanding the interaction between oligosaccharides and water molecules[END_REF] demonstrated that the opposite side of the α-(1→2) fucose linkage has a methyl moiety, and is predicted to form a hydrophobic pocket involving the nonpolar planar faces of nearby sugar residues. In other words, intra-molecular hydrogen bonding of PS can inhibit interactions between the hydroxyl groups and water, and then make the PS hydrophobic.

Therefore, the gel-forming PS will influence the hydrophobicity of microbial aggregates as soon as they form a continuous network that potentially allows for hydrophobic components to be immobilized within the biofilm matrix as filler material [START_REF] Seviour | Aerobic sludge granulation: a tale of two polysaccharides?[END_REF]. Besides, it seems that the hydrophobic features of PS are not influenced by the EPS extraction method. However, determining the hydrophobic features of PS molecules in the EPS by the EEM or SEC techniques presents significant analytical challenges, since they lack spectroscopic characteristics that enable them to be easily detected [START_REF] Arnosti | Fluorescent derivatization of polysaccharides and carbohydrate-containing biopolymers for measurement of enzyme activities in complex media[END_REF].

According to the discussion in the above section, F1 (aMW >660 kDa) and F2 (aMW = 122 -31 kDa) in the EPS are mainly made up by PN and PS, or glycoproteins/proteoglycans.

These conclusions are confirmed by a previous study by Bourven et al. (2015a), which confirmed the presence of high aMW (>150 kDa) proteoglycan-like and sulfated proteoglycan-like compounds in the EPS extracted from the same type of anaerobic granules. The carboxyl and hydroxyl groups in the PS molecules generate specific interactions with the PN or polypeptides via covalent bonds to form PN-PS composites [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF][START_REF] Ishiwatari | Macromolecular material (humic substance) in the water column and sediments[END_REF]. The link between PS and PN molecules could also induce the EPS hydrophobicity, as some hydrophobic properties were observed in the membrane N-glycan [START_REF] Fan | Identification of the hydrophobic glycoproteins of Caenorhabditis elegans[END_REF][START_REF] Krasowska | How microorganisms use hydrophobicity and what does this mean for human needs? Front[END_REF].

Hydrophobic properties of LB-EPS and TB-EPS in the anaerobic granular sludge

Table 4.1 displays that the LB-EPS extracted from the studied granules by centrifugation are mainly composed by HS-like compounds and PS, whereas PN are enriched in the TB-EPS. [START_REF] Yuan | Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge[END_REF] demonstrated that the LB-EPS from anaerobic granular sludge used in a municipal wastewater treatment plant (WWTP) were mainly comprised of HS-like compounds. [START_REF] Zhang | Extracellular protein analysis of activated sludge and their functions in wastewater treatment plant by shotgun proteomics[END_REF] investigated the extracellular PN composition of anaerobic, anoxic and aerobic sludge, which were also collected from a municipal WWTP. Their results showed that the PN with catalytic activity (i.e. hydrolase) were more widespread in the anaerobic sludge than in the anoxic and aerobic sludge, and that the PN content in the central section of the anaerobic granules is more abundant than in its periphery [START_REF] Quarmby | An examination of the structure of UASB granules[END_REF].

In this study, LB-EPS are found more hydrophobic than the TB-EPS, as the EPS extracted by the centrifugation method show a higher global hydrophobicity (%) than the EPS extracted by the other methods (Fig. 4.3). This is in accordance with the results reported for the sludge growing in a mesophilic anaerobic membrane bioreactor (Ding et al., 2015b), where the LB-EPS were also more hydrophobic than the TB-EPS. Besides, the LB-EPS hydrophobicity is mostly supported by HS-like and PS, whereas PN, besides HS-like and PS also contribute to the TB-EPS hydrophobicity (Fig. 4.3). Overall, HS-like compounds were always the most hydrophobic molecules in both LB-and TB-EPS.

Conclusion

The HS-like compounds were the major organic components in the extracted EPS, as well as the main molecular support of the EPS hydrophobicity. The hydrophobic HS-like compounds were mainly small molecules (aMW = 8 -<1 kDa), and their hydrophobicity could be ascribed to their humic acid-like molecular structure. PN and PS contributed to the EPS hydrophobicity to a lesser extent. PN compounds were enriched in the TB-EPS. LB-EPS displayed a higher global hydrophobicity (%) than TB-EPS. SEC analyses demonstrated that the LB-EPS with an aMW of >660 kDa and the TB-EPS molecules of which aMW range from 122 -31 kDa were highly hydrophobic.

Introduction

Nickel is widely used in the metallurgical industry to produce high quality iron-based alloys, and it is also applied as catalyst in the chemical and food industry, or as prime material for the production of paints and batteries [START_REF] Gikas | Single and combined effects of nickel (Ni(II)) and cobalt (Co(II)) ions on activated sludge and on other aerobic microorganisms: a review[END_REF]. Those manufacturing activities generate a considerable amount of wastewater containing nickel. In aqueous environments, nickel is often present as divalent cation i.e.

Ni 2+ . At micro-or millimolar concentrations, Ni 2+ may become toxic for microorganisms and hinder their activity. [START_REF] Thomas | Effects of heavy metals on the morphology of some marine phytoplankton[END_REF] reported that ~23 mg/L of Ni 2+ completely blocked the cell division in the coccolithophorida Cricosphaera carterae. Nevertheless, Ni 2+ is also an essential metal that is required as micronutrient at micro-molar concentrations for the microorganisms, and it is considered as the trace element for various biological systems [START_REF] Thanh | Trace metal speciation and bioavailability in anaerobic digestion: a review[END_REF]. For example, 0.4 mg/L of Ni 2+ was the minimum concentration required in the thermophilic methane fermentation of glucose [START_REF] Takashima | Minimum requirements for trace metals (iron, nickel, cobalt, and zinc) in thermophilic and mesophilic methane fermentation from glucose[END_REF].

Ni 2+ plays a fundamental role in the microbial metabolism and catalyzes the biochemical reactions in the active sites of metallo-enzymes such as urease, hydrogenase, acetyl-CoA synthase, and methyl-CoM reductase. In the living cells, the microbial growth and normal enzymatic activity require a proper energy metabolism, which are influenced by the metallic co-factors [START_REF] Can | Structure, function, and mechanism of the nickel metalloenzymes, CO dehydrogenase, and Acetyl-CoA synthase[END_REF][START_REF] Maroney | Nonredox nickel enzymes[END_REF]. Many studies have focused on the Ni 2+ removal aiming at its recovery, in which bioreactors inoculated with the fungus Phanerochaete chrysosporium have exhibited many advantages compared to bacteria inoculated reactors [START_REF] Espinosa-Ortiz | Fungal pelleted reactors in wastewater treatment: applications and perspectives[END_REF]Li et al., 2016). P.

chrysosporium is a metal-resistant fungus, but its growth rate is limited by the presence of 50 mg/L of Ni 2+ [START_REF] Falih | Influence of heavy-metals toxicity on the growth of Phanerochaete chrysosporium[END_REF]. Besides, this fungus is well-known for its unique ability to secrete ligninolytic enzymes, i.e. lignin peroxidase, manganese peroxidase, and laccase, by which a wide range of organic pollutants such as 2,4-dichlorophenol [START_REF] Chen | Simultaneous cadmium removal and 2,4-dichlorophenol degradation from aqueous solutions by Phanerochaete chrysosporium[END_REF] and sulfamethoxazole [START_REF] Guo | Biodegradation of sulfamethoxazole by Phanerochaete chrysosporium[END_REF]) can be degraded. Trace amounts of divalent metal cations like Cu 2+ or Mn 2+ in the fungal growth medium facilitate the production of ligninolytic enzymes in P. chrysosporium, and this characteristic affects the fungal pelletization and biodegradation process [START_REF] Baldrian | Interactions of heavy metals with white-rot fungi[END_REF].

Microbial survival in the presence of toxic compounds is perceived to be accomplished by the following five strategies: (i) active transport efflux systems, (ii) intra-and extra-cellular sequestration of the toxic compound, (iii) reduction of microbial membrane permeability, (iv) enzymatic alteration to a less toxic form, and (v) reduction in the sensitivity of cellular targets to metal ions [START_REF] Bruins | Microbial Resistance to Metals in the Environment[END_REF]. The production of extracellular polymeric substances (EPS) by the microorganisms plays an important role in the microbial tolerance of toxic metals through sequestration of metal ions outside the cells [START_REF] Clarke | Ferric hydroxide and ferric hydroxysulfate precipitation by bacteria in an acid mine drainage lagoon[END_REF]. The metal binding ability of EPS thus protects the fungal cells against heavy metal inhibition [START_REF] Sheng | Quantification of the interactions between Ca 2+ , Hg 2+ and extracellular polymeric substances (EPS) of sludge[END_REF]. A positive correlation is usually found between the metal tolerance capacity and the EPS production [START_REF] Mikes | The influence of heavy metals on the production of extracellular polymer substances in the processes of heavy metal ions elimination[END_REF].

The main organic constituents of EPS include proteins (PN), polysaccharides (PS), uronic acids, and nucleic acids as DNA [START_REF] Wingender | What are bacterial extracellular polymeric substances? In: Microbial Extracellular Polymeric Substances[END_REF]. They link with each other through electrostatic bonds, hydrogen bonds, van der Waals forces and hydrophobic interactions. These organic constituents are responsible for the maintenance of the multicellular community structure of microbial aggregates [START_REF] Busscher | Specific molecular recognition and nonspecific contributions to bacterial interaction forces[END_REF]. The metal binding ability of EPS is affected by the EPS hydrophobicity, as the hydrophilic fraction of EPS acts as reactive sites for the immobilization of metal ions [START_REF] Vá Zquez-Juá Rez | Cell surface hydrophobicity and its relation to adhesion of yeasts isolated from fish gut[END_REF] and can adsorb more metal ions than the hydrophobic fraction [START_REF] Wei | Adsorption of Cu 2+ and Zn 2+ by extracellular polymeric substances (EPS) in different sludges: effect of EPS fractional polarity on binding mechanism[END_REF]. When metal ions get attached to or enter fungal cells, they affect both the individual reactions and complex metabolic processes [START_REF] Baldrian | Interactions of heavy metals with white-rot fungi[END_REF]. The immobilized metal cations can reduce the negative charges of the cell surface [START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF], but also some divalent cations like Ca 2+ and Mg 2+ are indispensable bridging ions in stabilizing the threedimensional structure of EPS [START_REF] Wingender | What are bacterial extracellular polymeric substances? In: Microbial Extracellular Polymeric Substances[END_REF]. Therefore, the cell surface properties as well as the wastewater treatment performance of the reactors are influenced by the interactions between the metal ions and the EPS [START_REF] Li | Effects of Fe(III) on floc characteristics of activated sludge[END_REF]. Since the EPS may determine the physicochemical properties and maintain the structural and functional integrity of the fungus, it is therefore important to explore the evolution of EPS characteristics when P. chrysosporium is exposed to sub-toxic concentration of Ni 2+ (<50 mg/L) during its growth.

Materials and methods

Fungal pelletization

The fungus P. chrysosporium MTCC187 was provided by the Institute of Microbial Technology, Chandigarh (India). This fungal strain was cultivated for 3 d at 37 ℃ on malt agar plates. The fungal spore suspension was prepared by scrapping the culture grown in one agar plate into 50 mL sterile distilled water. The subsequent fungal pelletization process was carried out in 100 mL liquid medium with 10 % (v/v) inoculum of the fungal spore suspension (equivalent to about 1.78 g fungal spores (dry weight) in 1 L nutrient broth).

The original nutrient broth contained: 10 g/L glucose, 2 g/L KH2PO4, 0.5 g/L MgSO4• 7H2O, 0.1 g/L NH4Cl, 0.1 g/L CaCl2• 2H2O, 0.001 g/L thiamine and 5 mL/L trace elements solution (3 g/L MgSO4, 0.5 g/L MnSO4, 1 g/L NaCl, 0.1 g/L FeSO4• 7H2O, 0.1 g/L CoCl2, 0.01 g/L AlK(SO4)2• 12H2O, Na2MoO4• 2H2O, 1.5 g/L Nitrilotriacetate, 0.1 g/L ZnSO4• 7H2O, 0.1 g/L CuSO4, 0.01 g/L H3BO3) [START_REF] Tien | Lignin peroxidase of Phanerochaete chrysosporium[END_REF]. In order to study the effect of Ni 2+ on the alteration of fungal EPS, the trace elements solution in the above recipe was replaced by a proper volume of a 500 mg/L Ni 2+ stock solution (prepared from NiCl2• 6H2O). The final concentration of Ni 2+ in the broth was maintained at 0.5, 1, 5, 10, 25 mg/L, respectively.

The initial pH of the medium was adjusted to ~4.5 by adding 1M HCl aliquots. Fungal pellets cultivated in the absence of Ni 2+ were defined as the control group in this study. The inoculated broth was incubated at 30 ℃ shaking at 150 rpm for 48 h to produce the fungal pellets [START_REF] Espinosa-Ortiz | Removal of selenite from wastewater in a Phanerochaete chrysosporium pellet based fungal bioreactor[END_REF]. Six replicates of each group were incubated at one time in order to have enough biomass for EPS extraction and further characterization.

After 48 h of incubation time, the pH of the liquid medium was lowered to 4. The fungal pellets were harvested by vacuum filtration, and the dry weight (DW) of the collected biomass was measured (105 ℃, 24 h) after washing twice with ultrapure water. The Ni 2+ concentration in the growth medium, before and after the 48 h incubation time, was measured by flame atomic absorption spectroscopy (FAAS, A Analyst 200, PerkinElmer).

EPS extraction procedure

Four replicates of the washed fungal pellets from each group were used for the EPS extraction, and each replicate was re-suspended in 15 mL ultrapure water. The remaining two replicates were stored as intact biomass.

The EPS extraction method used in this study was modified from the protocol described by [START_REF] Hou | Extracellular polymeric substances from copper-tolerance Sinorhizobium meliloti immobilize Cu 2+[END_REF]. Briefly, each 15 mL suspension containing the fungal pellets was heated at 60 ℃ for 10 min in a water bath, and then centrifuged at 12,000×g at a temperature of 4 ℃ for 20 min.

Both the supernatant and the residual fungal pellets were collected and stored at -20 ℃ for further analysis. The supernatant was considered as extracted fungal EPS, whereas the residual fungal pellets were called the EPS-free biomass. The DW of the extracted EPS was measured after 24 h of heating at 105 °C [START_REF] D'abzac | Extraction of extracellular polymeric substances (EPS) from anaerobic granular sludges: comparison of chemical and physical extraction protocols[END_REF]. The Ni 2+ concentrations in both intact biomass and extracted EPS were measured by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Scientific Xseries 2) after an acid digestion treatment.

Zeta-potential measurement

The intact and EPS-free fungal pellets were washed twice with phosphate urea magnesium sulfate (PUM) buffer at pH 7.1 (22.2 g/L KH2PO4, 7.26 g/L KH2PO4, 1.8 g/L Urea, 0.2 g/L MgSO4• 7H2O).

After that, the fungal cell suspensions were dispersed in PUM buffer again and homogenized using a glass grinder. After allowing it to settle for 30 min under room temperature, the upper suspension which contains an abundance of well-dispersed fungal cells was collected. The collected suspension was diluted by PUM buffer to a concentration equivalent to the absorbance read at 600 nm (Abs600) of 0.4-0.5 using an UV/visible spectrophotometer (Lambda 365, PerkinElmer). 1 mL of the diluted suspension was used to measure the zeta-potential (Zetasizer Nano, ZS90, Malvern). All the analyses were carried out in triplicates.

Cell surface hydrophobicity (CSH)

The CSH (%) of the intact and the EPS-free fungal cells were determined by microbial attachment to hydrocarbon (MATH) test as described in [START_REF] Rosenberg | Microbial adhesion to hydrocarbons (math)[END_REF]. The fungal cell suspension was prepared in the same way as described above.

After the preparation, 10 mL of the diluted cell suspension and 5 mL n-hexane were added in a 50 mL screw-capped tube. The water and n-hexane phases were mixed by a vortex mixer at its maximum speed for 2 min, and phase separation was done for 30 min. 2 mL of the lower aqueous phase was collected and measured at Abs600. The CSH (%) was calculated according to Eq. 5.1:

(Eq. 5.1)

where, Absinitial and Abshexane-treated represents the Abs600 values of the dispersed fungal cells suspension before and after adding n-hexane, respectively. All the analyses were carried out in triplicate.

EPS characterization

EPS composition

The total organic carbon (TOC) of the extracted EPS was measured by a TOC-analyzer (TOC-L, Shimadzu, Japan). The protein (PN) content was quantified by the modified Lowry method [START_REF] Frølund | Extraction of extracellular polymers from activated sludge using a cation exchange resin[END_REF] where bovine serum albumin (BSA) was used as the standard. Polysaccharides (PS)

were determined by the phenol-sulfuric acid method with glucose as the standard [START_REF] Dubois | Colorimetric method for determination of sugars and related substances[END_REF]. Deoxyribonucleic acid (DNA) content was determined by using diphenylamine, where calf thymus DNA was used as the standard [START_REF] Burton | A study of the conditions and mechanism of the diphenylamine reaction for the colorimetric estimation of deoxyribonucleic acid[END_REF]. ICP-MS was also used to measure the Ni 2+ concentration in the extracted EPS. The standard deviations were calculated based on the data obtained from triplicate measurements.

Excitation and emission fluorescence matrix spectroscopy (EEM)

The EEM spectra of the extracted EPS were measured by spectrofluorometric analysis (Shimadzu RF-5301 PC, Japan). The spectra were recorded with the help of scanning emission (Em) spectra from 220 to 500 nm at an interval of 1 nm increase and varying the excitation wavelength (Ex) from 220 to 380 nm at 5 nm interval. All the EPS samples were diluted using ultrapure water to the desired TOC concentration (~ 1 mg C/L) if the sample had a maximum fluorescence emission intensity exceeding 1000 au (au: arbitrary unit) [START_REF] Chen | Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[END_REF]. The spectrum of ultrapure water was also collected as the blank. Each sample was measured twice. Panorama Fluorescence 3.1 software (Lab Cognition, Japan) was used to process the spectra data.

Fourier transform infrared (FT-IR) spectroscopy

The extracted fungal EPS were dried at 40 °C in an oven (PROLABO fabrication ASTEL, France).

The dried EPS samples were mixed with KBr at a ratio of 1:100, and homogenized in an agate grinder. Approximately 180 mg of the ground mixture was compressed and analyzed in a FT-IR spectrometer (Spectrum 1000, PerkinElmer). The scanning range for the wavenumber was 4000-400 cm -1 . The fungal EPS samples were measured twice and the spectral peak shift between the two measurements was not greater than 1 cm -1 .

Size exclusion chromatography (SEC)

The apparent molecular weight (aMW) of the fungal EPS were determined by a Merck Hitachi LA chromatograph equipped with a fluorescence detector (L7485) and diode array UV detector (L7455).

Agilent Bio SEC 300Å and 100Å columns were used in series [START_REF] Villain | The use of a new mobile phase, with no multivalent cation binding properties, to differentiate extracellular polymeric substances (EPS), by size exclusion chromatography (SEC), from biomass used for wastewater treatment[END_REF]. The theoretical size exclusion limits of the two columns were 5-1.250 kDa and 0.1-100 kDa, respectively. The selection of the excitation and emission wavelengths for the fluorescence detector was based on the maximum intensity detected in the EEM spectra. The fingerprint detected at an UV absorbance of 210 nm (UV/210 nm) was also considered in this study, as it reflects the total organics present in the EPS [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]. Each EPS sample was filtered using a 0.2 µm pore size membrane (Whatman™, GE Health) before the injection, and the injection volume was 100 µL for each sample.

150 mM sodium phosphate buffer (75 mM Na2HPO4, 75 mM NaH2PO4, pH 7.0 ± 0.1) was used as the mobile phase, and the flow rate was maintained at 0.7 mL/min. Each sample was analyzed twice.

The aMW calibration curve (Eq. 5.2) was obtained by plotting the logarithm of aMW (log (aMW)) versus the elution volume (Ve). Several protein or amino acid standards were used in the tests [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]: Thyroglobulin (Sigma)-660 kDa; Ferritine (Sigma)-440 kDa;

Immunoglobulin G from human serum (Sigma)-155 kDa; Bovine Serum Albumin (Sigma)-69 kDa;

Ribonuclease A (Sigma)-13.7 kDa and Thyrotropin releasing hormone (Sigma)-362.38 Da. Eq. 5.2 was used to quantify the aMW of all the fungal EPS samples.

(Eq. 5.2)

where, the units of aMW and Ve are Da and mL, respectively. The total permeation volume (Ve = 22 mL) was determined by NaNO3.

Hydrophobic fractionation by DAX-8 resin

The hydrophobic fractionation of fungal EPS was carried out using Supelite™ DAX-8 resin (Sigma-Aldrich), according to the procedure outlined in Cao et al. (2017). Briefly stating, the pH of each fungal EPS sample was adjusted to pH 2.0 (± 0.1) by 1M HCl, as the initial pH of the extracted fungal EPS was ~4.0. Thereafter, the pH-adjusted EPS samples were filtered with 0.45 µm pore size membrane (Whatman™, GE Health) before uploading into the DAX-8 resin column. The filtered EPS samples were named as EPSbefore, and the samples collected from the outlet of the column were named as EPSafter. Ultrapure water at pH 2.0 (± 0.1) was used as a control for the DAX-8 resin column. The resin treatment on each sample was performed in duplicates.

The hydrophobicity (%) of each pH-adjusted EPS sample was calculated by using the TOC concentration of EPSbefore and EPSafter, as shown in Eq. 5.3:

EPS hydrophobicity (%) = (Eq. 5.3)

where, TOCbefore and TOCafter represent the TOC concentration (mgC/L) of EPSbefore and EPSafter, respectively.

Results

Impact of Ni 2+ concentration on the metal binding ability of P. chrysosporium and the extracted EPS

Table 5.1 shows the Ni 2+ concentrations in the growth medium, intact biomass and extracted EPS, respectively. With an increase in the Ni 2+ concentration in the fungal broth, a higher content of Ni 2+

was measured in both intact biomass and extracted EPS, and the Ni 2+ content in the extracted EPS was found to be 3-30 times higher than in the intact biomass (compared in terms of µg/g DW). At a Ni 2+ concentration of 10 mg/L, the Ni 2+ content in the extracted EPS reached its maximum value of ~250 µg/g EPS DW, but no further increase was observed when the Ni 2+ concentration was increased to 25 mg/L. However, in the case of the intact biomass, its Ni 2+ content increased with an increase in Ni 2+ concentration in the broth.

Zeta-potential measurement

The zeta-potential of both intact biomass and EPS-free biomass in the PUM buffer showed negative values of -10 mV and -8 mV, respectively (Fig. 5.1). This indicates that they both carry negative charges in the PUM buffer (pH 7.1). When comparing the control group with the groups exposed to Ni 2+ (Ni 2+ groups), it is evident that the zeta-potential of the intact biomass decreased by 1-2 mV due to the presence of Ni 2+ in the fungal broth, while the value of EPS-free biomass remained nearly stable at -10 mV. In the control group, the EPS-free biomass demonstrated a higher zeta-potential than the intact biomass. In the Ni 2+ groups, with increasing Ni 2+ concentration, the difference between the intact biomass and the EPS-free biomass gradually reduced.

Cell surface hydrophobicity (CSH)

The CSH (%) of the intact and the EPS-free fungal pellets is shown in Fig. 5.2. The CSH (%) of the biomass in the control group, for the intact and EPS-free biomass, was found to be lower than those observed for the Ni 2+ groups. In the control group, the CSH (%) of the intact biomass and the EPSfree biomass also showed a similar value of ~40%. As in the Ni 2+ groups, the CSH (%) of the EPSfree biomass was higher than that of the intact biomass, and the value corresponding to the EPS-free biomass exceeded 50%. Nevertheless, the difference between the intact biomass and the EPS-free biomass in the Ni 2+ groups decreased with increasing concentrations of Ni 2+ . concentrations have an effect on the PN and PS contents. In the control group, the PN content (275 mg/g EPS DW) was higher than the PS content (175 mg/g EPS DW). At concentrations varying from 0.5 to 5 mg/L Ni 2+ , the PN and PS contents were almost similar at ~225 mg/g EPS DW. When the Ni 2+ concentration was ≥10 mg/L, the PN content in the extracted EPS increased, whereas the PS content decreased to values similar to that of the control group, i.e. 175 mg/g EPS DW.

Evolution of EPS characteristics

EPS hydrophobicity

FT-IR spectra of the extracted fungal EPS

The FT-IR spectra of all the EPS samples revealed similar features. Therefore, as examples, the spectra which correspond to 0, 0. components (Badireddy et al., 2010). The adsorption bands at ~1240 cm -1 , which could be associated with the deformation vibration of C=O of nucleic acids [START_REF] Breierová | Biosorption of cadmium ions by different yeast species[END_REF], were found to be relatively weak. Besides, the bands between 935 and 1,100 cm -1 could be attributed to the stretching of the phosphoryl group (-PO4 3-) [START_REF] Jiang | Elucidation of functional groups on gram-positive and gram-negative bacterial surfaces using infrared spectroscopy[END_REF].

Discussion

Ni 2+ binding ability of the fungal cells and the EPS

From Table 5.1 it is clearly evident that the Ni 2+ binding ability of the fungus and the extracted EPS increased with an increase in the Ni 2+ concentration. Moreover, the extracted EPS can bind Ni 2+ 3-30 times more than the intact biomass (Table 5.1). This characteristic distinctly shows that, when the fungus is exposed to sub-toxic concentrations of Ni 2+ , the EPS provide more metal binding sites than the fungal cells, and contribute significantly to the Ni 2+ binding ability of the fungal cells.

Nevertheless, when the Ni 2+ concentration in the broth reached values ≥10 mg/L, the metal uptake capacity of the extracted EPS appeared to reach its saturation level. In contrast, metal uptake by the intact biomass still increased irrespective of the increasing Ni 2+ concentrations (Table 5.1).

The metal binding ability of EPS produced by the fungus enables the microbial tolerance to a high concentration of metal ions in the extracellular space [START_REF] Breierová | Biosorption of cadmium ions by different yeast species[END_REF]. The behavior of the cells to the negative growth conditions is strain-dependent and it is also related to the type of metal ions [START_REF] Mikes | The influence of heavy metals on the production of extracellular polymer substances in the processes of heavy metal ions elimination[END_REF]. Considering the fact that the DW of the extracted EPS remained almost stable despite the varying concentrations of Ni 2+ in the broth (subsection 5. In addition, the pH not only affects the metal species in the solution, but also the surface properties of biomass such as the surface charge and the dissociation of binding sites [START_REF] D'abzac | Metal binding properties of extracellular polymeric substances extracted from anaerobic granular sludges[END_REF]. [START_REF] Say | Biosorption of cadmium(II), lead(II) and copper(II) with the filamentous fungus Phanerochaete chrysosporium[END_REF] showed that the maximum sorption capacities of Cd 2+ , Pb 2+ and Cu 2+ onto P.

chrysosporium was obtained at pH 6.0. It is therefore assumed that, at pH values <6, the metal binding ability of the fungus could be decreased.

Effect of Ni 2+ concentration on the surface properties of P. chrysosporium

Microbial cell surface properties are affected by the growth medium, culture conditions, i.e. pH, temperature, and the presence of multivalent cations [START_REF] Krasowska | How microorganisms use hydrophobicity and what does this mean for human needs? Front[END_REF]. When exposed to an inhibitory substrate, microorganisms are still able to regulate their cell surface properties. The metal tolerance exhibited by P. chrysosporium grown in the presence of Ni 2+ is probably related to the specific nutritional requirements and/or accumulation in the cells [START_REF] Anahid | Heavy metal tolerance of fungi[END_REF].

When compared to the control group, the zeta-potential of the intact biomass became more negative due to the presence of Ni 2+ (Fig. 5.1). This variation clearly indicates that the presence of Ni 2+ promoted the fungal cells to acquire more negative charges in order to bind Ni 2+ . When the Ni 2+ concentration increased from 0.5 to 10 mg/L, the zeta-potential of the intact biomass decreased (Fig. 5.1). Nevertheless, at the highest Ni 2+ concentration of 25 mg/L, no significant decrease in the zetapotential was observed in the intact biomass, suggesting that no more negatively charged binding sites are available for Ni 2+ . These observations are consistent with the varying metal binding abilities of the fungus shown in Table 5.1.

The FT-IR results (Fig. 5) evidenced the presence of some ionized functional groups, such as carboxyl and phosphoryl groups, in the extracted EPS at pH 4. Carboxyl groups (pKa ≈ 4) are mainly attributed to the PS (i.e. uronic acids), acidic amino acids in the PN (i.e. glutamic and aspartic acids), or free amino acids [START_REF] Baker | Role of extracellular polymeric substances in the surface chemical reactivity of Hymenobacter aerophilus, a psychrotolerant bacterium[END_REF]. The phosphoryl groups (pKa ≈ 6) are likely to be present in the PS or DNA [START_REF] Hong | Electrostatic behavior of the charge-regulated bacterial cell surface[END_REF], and indeed DNA was found to be present in very small quantities in this study (subsection 5.3.3). It was hypothesized that these ionized functional groups in the EPS contribute to the negative charges of the cell surface. In the absence of EPS, some phosphoryl and carboxyl groups were also present on the surface of the fungal cells due to phospholipids and lipopolysaccharides (LPS) in the outer membrane [START_REF] Phoenix | Characterization and implications of the cell surface reactivity of Calothrix sp. strain KC97[END_REF]. It was conceived that Ni 2+ could complex with the functional groups present in the EPS and/or cell surface.

Under such conditions, the EPS could act as the first barrier to protect the fungal cells from Ni 2+

stress. Besides, no more binding sites are available when the Ni 2+ concentration reaches values ≥10 mg/L and thus, the zeta-potential difference between the intact biomass and the EPS-free biomass is reduced (Fig. 5.1).

The EPS-free biomass demonstrated a higher CSH (%) than the intact biomass (Fig. 5.2).

This important characteristic clearly shows that the wrapped EPS promoted the fungal cells to become more hydrophilic, allowing them to utilize the hydrophilic substrates and/or to bind Ni 2+ [START_REF] Heipieper | Surface properties and cellular energetics of bacteria in response to the presence of hydrocarbons[END_REF]. This is consistent with the improved metal binding ability of P. chrysosporium displayed in Table 5.1. Evidently, the presence of Ni 2+ increased the CSH (%) of both intact and EPS-free biomass. This observation is similar to the results observed by [START_REF] Khemakhem | Effect of environmental conditions on hydrophobicity of marine bacteria adapted to textile effluent treatment[END_REF], who showed that the presence of divalent cations like Ca 2+ and Mg 2+ in the bacterial growth notably improved the CSH of Agrobacterium and Citrobacter sp.

However, it is noteworthy to mention that the measurement zeta-potential and CSH were carried out in PUM buffer at pH 7.1, whereas the metal binding ability of the different biomasses was measured at pH 4 (Table 5.1). The ionic strength and the pH value affect the zeta-potential and CSH measurements, and thus, minor deviations between the actual values and the measured zetapotential and CSH values could exist.

Evolution of EPS characteristics

As shown in Fig. 5.3, the EPS hydrophobicity decreased by 10-20% due to the presence of Ni 2+ . This is in accordance with the decreasing zeta-potential trend displayed in Fig. 5.1. Since the cells are embedded in the EPS matrix, their surface charge also largely depends on the EPS characteristics [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF]. FT-IR results identified the presence of some negatively charged functional groups, i.e. carboxyl and phosphoryl groups, in the EPS (Fig. 5.5). The interactions between the metal cations and the functional groups in the EPS could neutralize the negative charges on the EPS [START_REF] Li | Effects of Fe(III) on floc characteristics of activated sludge[END_REF]. Although the presence of Ni 2+ in the EPS could decrease the EPS hydrophobicity, the hydrophilic EPS fraction could still complex more metal cations than the hydrophobic EPS fraction [START_REF] Wei | Adsorption of Cu 2+ and Zn 2+ by extracellular polymeric substances (EPS) in different sludges: effect of EPS fractional polarity on binding mechanism[END_REF]. Thus, the EPS with decreased hydrophobicity should complex more Ni 2+ , which is supported by the results displayed in Table 5.1 and Fig. 5.3.

Considering this mechanism, Ni 2+ dosing in the fungal broth appears to be advantageous for improving the metal binding ability of the fungus P. chrysosporium, which in turn benefits the metal removal performance of fungal reactors.

At Ni 2+ concentrations ranging between 0.5 and 5 mg/L, the PN content in the extracted EPS was found to be slightly lower than those observed in the control group (Fig. 5.4). The metal ions could interact with the amino acid side chains of the PN via electrostatic attraction [START_REF] Hoa | The effect of nutrients on extracellular polymeric substance production and its influence on sludge properties[END_REF].

This interaction may alter the conformation of the PN and subsequently modify the orientation of their active sites by causing denaturation, aggregation or precipitation of the PN [START_REF] Andreini | Metal ions in biological catalysis: from enzyme databases to general principles[END_REF]. Thus, it can be conjectured that the interaction between the metal ions and the PN results in a lower PN content in the extracted EPS. Nevertheless, when the initial Ni 2+ concentration reaches ≥10 mg/L, the PN content seemed to reach a higher value than the control group (Fig. 5.4). A similar trend was also observed by [START_REF] Breierová | Biosorption of cadmium ions by different yeast species[END_REF], who showed that the presence of Cd 2+ affected the EPS composition of eight different yeast species. In their study, Cd 2+ seemed to promote the secretion of PN in the EPS, because the presence of Cd 2+ enhanced the glutamic acid content while the content of other amino acids in the PN moiety remained stable. However, this observation was not confirmed in the present study due to the lack of specific fluorescence characteristics for glutamic acid and its presence can thus not be detected by the EEM technique [START_REF] Lakowicz | Protein fluorescence[END_REF].

As shown in Fig. 5.4, the variation of the PS content in the extracted EPS is apparently opposite to that of the PN content. Comparing this trend with the control group, the secretion of PS increased whenever Ni 2+ addition to the broth was <5 mg/L. This observation is consistent with results of [START_REF] Ozturk | Modification of exopolysaccharide composition and production by three cyanobacterial isolates under salt stress[END_REF] where the authors showed that the presence of metal ions during the microbial growth phase increased the production of extracellular carbohydrates, possibly as a result of metal binding polymer production [START_REF] White | Accumulation and effects of cadmium on sulphate-reducing bacterial biofilms[END_REF]. In another study, during the sorption of Zn 2+ and Pb 2+ by the fungus Paecilomyces marquandii, [START_REF] Słaba | Efficient Zn 2+ and Pb 2+ uptake by filamentous fungus Paecilomyces marquandii with engagement of metal hydrocarbonates precipitation[END_REF] reported that the metal ions were crystallized as carbonates on the mycelium surface. The complexed metal ions can serve as nucleation sites that promoted the formation of crystals more intensively and rapidly [START_REF] Fortin | Nickel sulfide, iron-nickel sulfide and iron sulfide precipitation by a newly isolated Desulfotomaculum species and its relation to nickel resistance[END_REF]. In this study, when the Ni 2+ concentration was increased from 0.5 to 10 mg/L, the PN/PS ratio also increased from 0.9 to 2.2, whereas the ratio of the control group was 1.6.

Therefore, the adjustment of the PN and PS contents in the EPS can be considered as a metabolic adaptation strategy by the fungal cells to protect themselves against unanticipated stress conditions [START_REF] Huang | Degradation of lead-contaminated lignocellulosic waste by Phanerochaete chrysosporium and the reduction of lead toxicity[END_REF].

Although the PN and PS contents in the extracted EPS are influenced by the Ni 2+ concentration in the broth, the EEM spectra of all the EPS samples display a similar pattern (Fig. S1).

Mainly, the two protein-deprived peaks, i.e. peak A (Ex/Em = 225/341-346 nm) and peak B (Ex/Em = 275-280/345-347 nm) were detected (Fig. 5.6) [START_REF] Chen | Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[END_REF][START_REF] Lakowicz | Protein fluorescence[END_REF][START_REF] Carstea | Fluorescence spectroscopy for wastewater monitoring: a review[END_REF]. The fluorescence intensity of each peak remained almost stable despite the increase of Ni 2+ concentration in the broth (Table 5.2). This stable peak behavior clearly indicates that the fluorescence characteristics of the extracted EPS were not affected by the presence of Ni 2+ nor its concentration. A quenching effect introduced by the metal ions, especially paramagnetic ions like

Ni 2+ and Zn 2+ , should also be considered. These metal ions are able to quench the fluorescence of organic ligands by enhancing the rate of some non-radiative processes that compete with fluorescence, such as intersystem crossing [START_REF] Provenzano | Fluorescence behaviour of Zn and Ni complexes of humic acids from different sources[END_REF]. However, trace amounts of Ni 2+

were also detected in the control group (Table 5.1), and hence it is difficult to justify whether there is a genuine quenching effect or not.

In Besides, at the same Ni 2+ concentration, the peak location in the two types of fingerprints (UV/210 nm and PN-like) is different from each other, and a more significant difference was observed only in the fraction of Ve = 17-22 mL (aMW = 14-0.5 kDa) (Fig. 5.7). Fungal EPS are usually characterized as a rich matrix of macromolecules comprising of PN, PS and/or glycoproteins [START_REF] Breierová | Biosorption of cadmium ions by different yeast species[END_REF][START_REF] Huang | Fractionation, characterization and antioxidant activity of exopolysaccharides from fermentation broth of a Cordyceps sinensis fungus[END_REF]. Since the fingerprints detected at UV/210 nm could represent the total organics in the extracted EPS [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 difference between the two types of fingerprints probably ascribes to the change of PS moiety, i.e. uronic acid in the EPS [START_REF] More | Extracellular polymeric substances of bacteria and their potential environmental applications[END_REF]. However, due to the absence of spectroscopic characteristics of the PS, using the EEM or SEC techniques to determine the molecular features of PS in the extracted EPS is a significant analytical challenge [START_REF] Arnosti | Fluorescent derivatization of polysaccharides and carbohydrate-containing biopolymers for measurement of enzyme activities in complex media[END_REF].

Conclusion

The results from this study shows that, when P. chrysosporium was incubated with sub-toxic concentration of Ni 2+ (0.5-25 mg/L), the EEM spectra and aMW distribution of the total organics in the extracted EPS exhibited similar features regardless of the Ni 2+ concentration. At higher Ni 2+ concentrations in the broth, the zeta-potential of the fungus and the EPS hydrophobicity gradually decreased. In contrast, an opposite trend was observed for the PN and PS contents in the extracted EPS, and the peak intensity of PN-like molecules (aMW = 0.5-14 kDa) improved at higher Ni 2+

concentrations. The adjustment of the EPS composition, i.e. PN and PS contents, and enrichment of certain PN molecules can be considered as one of the defense mechanisms developed by P.

chrysosporium to withstand Ni 2+ stress.

General discussion

When the microorganisms are exposed to an inhibitory concentration of metals or organic pollutants, one specific adaptive mechanism developed by the microorganisms is the modification of cell surface properties i.e. EPS hydrophobic properties against the stress conditions [START_REF] Heipieper | Surface properties and cellular energetics of bacteria in response to the presence of hydrocarbons[END_REF]. Chapter III demonstrated that the measured hydrophobic properties of EPS was

Impact of EPS extraction method on the characterization of EPS hydrophobicity

EPS extraction is based on the interruption of the interactions that are responsible for the stabilization of the three-dimensional structure of EPS (such as hydrogen bonds, electrostatic, hydrophobic or van der Waals interactions) via physical forces or chemical reagents (Fig. 6.2).

Therefore, EPS extraction methods are generally divided into physical and chemical methods [START_REF] Nielsen | Extraction of EPS[END_REF]. Besides, the differentiation between loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) largely depends on the extraction method: LB-EPS can be extracted by mild extraction methods such as high-speed centrifugation, while more harsh conditions (i.e.

sonication, heating or adding chemical reagents) are required to extract TB-EPS from the biomass [START_REF] Jahn | Cell biomass and exopolymer composition in sewer biofilms[END_REF]. 3.1), and the extracted EPS samples also showed different properties such as sorption ability, molecular weight distribution, etc. [START_REF] Domí Nguez | 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][START_REF] Zuriaga-Agustí | Influence of extraction methods on proteins and carbohydrates analysis from MBR activated sludge flocs in view of improving EPS determination[END_REF]. This is because the breakage degree of those linkages between EPS molecules cannot be estimated during the extraction process (Fig. 6.2), resulting the unpredictable EPS extraction yield.

Considering the interaction between Supelite™ DAX-8 resin and the organic reagents (Fig. 3.5), physical extraction methods were used in Chapter IV to analyze the hydrophobic properties of the EPS extracted from anaerobic granular sludge. In Chapter IV, the highest EPS global hydrophobicity (%) at pH 5 was achieved by using the centrifugation method (>60%), whilst the lowest value at pH 5 was quantified in the heating method (~40%) (Fig. 4.3). It seems that the centrifugation method could extract more hydrophobic EPS molecules than any other methods, while the heat treatment could decrease the EPS global hydrophobicity (%).

Chapter IV also concluded that the HS-like compounds are the major molecular support of EPS hydrophobicity in the anaerobic granular sludge. The EPS extracted by the centrifugation method showed the highest percentage of HS-like in the extracted EPS, which occupy more than 70% of the quantified EPS composition (Table 3.1). This could be the reason why the EPS extracted by this method also have the highest global hydrophobicity (>60%) when excluding the interferences caused by the organic reagents (Fig. 3.6). Regarding the hydrophobic properties of the EPS extracted by the heating method, it was noticed that there were no fluorescent peaks present in Region II of the EEM spectra (Fig. 4.4b). This could be due to the too high sample dilution factor (1/357) that was used to detect the fluorophores, or a modification of the fluorescent properties of certain polycyclic aromatic molecules present in the EPS after the heat treatment. Since the lowest HS-like hydrophobicity was achieved by the heating method (~30%) (Fig. 4.2), it is assumed that the heat treatment could change the fluorescent properties or molecular structure of the extracted EPS i.e. HS-like compounds, and in turn, these EPS molecules become more hydrophilic. Besides, heating may lead to PN denaturation: the disruption and possible destruction of hydrogen bonds and non-polar hydrophobic interactions in both secondary and tertiary structures of PN. Thus, a loose PN secondary structure facilitates a full exposure of inner hydrophobic groups to express hydrophobicity [START_REF] Hou | Role of extracellular polymeric substance in determining the high aggregation ability of anammox sludge[END_REF]. This also could explain why PN demonstrated a higher hydrophobicity (~40%) than the HS-like compounds and PS in the heating method (Fig. 4.2).

Surfactants such as sodium dodecyl sulfate (SDS) and Tween 20 used in the EPS extraction are meant for increasing the solubility of hydrophobic molecules in the EPS extract to improve EPS extraction yield. Formaldehyde could cross-link between amino and sulfhydryl groups of PN, as well as enter cells and subcellular compartments to stabilize cellular architecture before gross and microscopic degradation of the native cellular components [START_REF] Sutherland | Utility of formaldehyde cross-linking and mass spectrometry in the study of protein-protein interactions[END_REF][START_REF] Chang | Reaction mechanisms of formaldehyde with endocyclic imino groups of nucleic acid bases[END_REF]. The addition of ethanol is a common strategy to precipitate polysaccharides [START_REF] Sutherland | Biofilm exopolysaccharides: a strong and sticky framework[END_REF]. When ethanol is applied in EPS extraction, EPS molecules that possess a polarity different from the ethanol phase become insoluble and be easily precipitated from the solution. The application of these organic reagents has facilitated EPS extraction. However, ethanol leads to the high TOC value of the EPS (beyond 1000 mgC/g EPS DW) (Fig. 3.4), which indicates the leftover problem of the organic reagents in the extracted EPS solution should be considered.

The residual organic reagents in the extracted EPS may cause interferences in the following EPS characterization [START_REF] Comte | Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and EPS complexation properties: Part I. Comparison of the efficiency of eight EPS extraction methods[END_REF]. Meanwhile, the decreased TOC values of the organic reagents after DAX-8 resin treatment (Fig. 3.5) suggests the interactions between the resins and the organic reagents. Therefore, the classical method to determine EPS hydrophobicity by using TOC content is not appropriate when organic reagents are used.

Moreover, a wide-ranged peak (Ve = 8.5 -17 mL) was detected in the EPSbefore fingerprints of Tween 20 method by size exclusion chromatography (SEC) (Fig. 3.7g), and the same type of peak was also detected for Tween 20 alone (data not shown). It was noticed that the final concentration of Tween 20 used in the EPS extraction was above its CMC level in water (about 0.01%, m/v), which may lead to the formation of EPS-surfactant micelles [START_REF] Johnson | Detergents: Triton X-100, Tween-20, and more[END_REF].

Owing to those facts, chemical EPS extraction methods should be carefully considered when study the EPS hydrophobicity.

Impact of bulk solution pH on the characterization of EPS hydrophobicity

pH 2 is commonly used in the hydrophobic/hydrophilic fractionation by XAD/DAX resin, because most of the functional groups (i.e. carboxyl, phenolic and amino groups) become nonionic at this pH. In Chapter III, pH 5 was chosen in the hydrophobic fractionation by DAX-8 resin as a compromise between the conservation of EPS molecular structure and the protonation of certain functional groups. The initial pH value of the extracted EPS was around 7 (Table 3.1).

However, when the pH value of the extracted EPS was lowered to pH 2 or 5, large quantities of EPS organic components were lost (Fig. 3.2). The "disappearance" of these EPS molecules can be ascribed to the isoelectric point (IEP) of different organic compounds at which the molecules carry no net electrical charge, and thus, be precipitated out of the solution [START_REF] Brown | Organic Chemistry[END_REF]. So far, no specific IEP value has been reported for the EPS extracted from anaerobic granular sludge, as the IEP values are influenced by the acid dissociation constant (pKa) value of given molecules [START_REF] Tourney | The effect of ionic strength on the electrophoretic mobility and protonation constants of an EPS-producing bacterial strain[END_REF].

According to [START_REF] Malcolm | Factors to be considered in the isolation and characterization of aquatic humic substances[END_REF] and [START_REF] Aiken | Humic substances in soil, sediment, and water: geochemistry, isolation, and characterization[END_REF], the hydrophobicity of natural organic matter (NOM) can be maximized at pH 2. In Chapter III, the EPS global hydrophobicity (%), determined at pH 2, was higher than that those observed at pH 5, and they demonstrated similar trends at both pH (Fig. 3.6). Nevertheless, the apparent molecular distribution (aMW) of the EPS at pH 5 was more closely to that of raw EPS (Fig. 3.3), which indicates that the integrity of EPS aMW distribution is better preserved at pH 5 than at pH 2. Considering this aspect, pH 5 was preferred in Chapters III and IV for monitoring hydrophobic aMW of the EPS molecules extracted from the anaerobic granular sludge. In Chapter V, pH 2 was still chosen in the hydrophobic fractionation of the fungal EPS, as the initial pH of the fungal EPS was ~4.

Hydrophobic properties of the EPS

EPS from bacteria, fungus and algae are mainly composed of PN and PS [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF][START_REF] Ravella | Extracellular polysaccharide (EPS) production by a novel strain of yeast-like fungus Aureobasidium pullulans[END_REF][START_REF] Turu | Synthesis and characterization of cryogel structures for isolation of EPSs from Botryococcus braunii[END_REF]. In the mixed culture system, such as biofilms and sludge flocs, the composition of extracted EPS is more heterogeneous because the presence of HS-like and some minerals i.e. calcium or ferric phosphates (Fig. 6.3). Besides, LB-EPS from anaerobic granular sludge are found mostly contain HS-like [START_REF] Yuan | Effects of solution conditions on the physicochemical properties of stratification components of extracellular polymeric substances in anaerobic digested sludge[END_REF], while PN are the major constituents of TB-EPS from Annamox sludge [START_REF] Ni | Distribution of extracellular polymeric substances in anammox granules and their important roles during anammox granulation[END_REF].

Many studies have demonstrated the relationship between the EPS and the cell surface hydrophobicity: PN and HS-like in the EPS mainly contribute to the cell surface hydrophobicity, while carbohydrates contribute more to the hydrophilic nature [START_REF] Yu | Hydrophobicity and molecular weight of humic substances on ultrafiltration fouling and resistance[END_REF]. Some authors also concluded that PN/PS ratio of EPS extracted from aerobic granules positively correlated with the cell surface hydrophobicity [START_REF] Xie | Surface properties of bacteria from activated sludge in relation to bioflocculation[END_REF][START_REF] Zhang | Role of extracellular protein in the formation and stability of aerobic granules[END_REF]. Nevertheless, various conditions such as microbial type, growth phase and substrates type lead to the generation of EPS displaying different physicochemical characteristics [START_REF] Sheng | Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review[END_REF].

EPS extracted from anaerobic granular sludge

Based on the hydrophobic fractionation by DAX-8 resin at pH 5, it is clear that the HS-like compounds were the major organic constituent of the EPS extracted from anaerobic granular sludge (Table 3.1). In most cases, those HS-like compounds demonstrated higher hydrophobicity (%) than PN and PS (Fig. 4.2), and the HS-like compounds also contributed the most to the EPS global hydrophobicity (%) (Fig. 4.3).

HS-like compounds are usually described as phenolic compounds with more than two fused benzene rings (Fig. 6.3), and the corresponding fluorophores are usually detected in Region II of the EEM spectra [START_REF] Bassandeh | Evaluation of ion exchange resins for the removal of dissolved organic matter from biologically treated paper mill effluent[END_REF][START_REF] Hudson | Fluorescence analysis of dissolved organic matter in natural, waste and polluted waters-a review[END_REF]. According to [START_REF] Carstea | Fluorescence spectroscopy for wastewater monitoring: a review[END_REF], the fluorophores detected in Region II can be ascribed to the breakdown products of lignin (Ex/Em 230-275/400-520 nm), polyaromatic hydrocarbons such as phenanthrene, anthracene, pyrene (Ex/Em 220-300/370-430 nm), and humic acid (Ex/Em 220-320/400-550 nm) present in the paper mill effluents. Therefore, the molecular features of HS-like compounds in the EPS could be the consequence of the interplay between the microbial activities and the organic matter present in the wastewater. The results from size exclusion chromatography (SEC) showed that the hydrophobic HS-like compounds were small molecules ranging from 8 kDa to <1 kDa (Fig. 4.5).

It was also found that the HS-like compounds may contain PN-like moiety (Fig. 4.5). Thus, the hydrophobic HS-like compounds in the extracted EPS could be present in pure form but also as HS-PN-like complexes.

PN showed the lowest hydrophobicity (%) among the three main components (Fig. 4.2). It may indicate that the molecular structure of PN is involved in the EPS hydrophobicity to a lesser extent compared to the other constituents. Li et al. (2016) considered that the non-polar groups such as side chains of aromatic amino acids were important for the EPS hydrophobicity of the anaerobic granular sludge. There is no specific spectroscopic characteristics that can be used to characterize PS, thus, therefore, determining the hydrophobic features of PS molecules in the EPS by the EEM or SEC techniques presents significant analytical challenges [START_REF] Arnosti | Fluorescent derivatization of polysaccharides and carbohydrate-containing biopolymers for measurement of enzyme activities in complex media[END_REF].

Nevertheless, the carboxyl and hydroxyl groups in the PS molecules could generate specific interactions with the PN or polypeptides via covalent bonds to form PN-PS composites i.e.

glycoproteins or proteoglycans [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF][START_REF] Ishiwatari | Macromolecular material (humic substance) in the water column and sediments[END_REF]. The compounds like glycoproteins or proteoglycans could also contribute to the EPS hydrophobicity, as some hydrophobic properties were observed in the membrane N-glycan [START_REF] Fan | Identification of the hydrophobic glycoproteins of Caenorhabditis elegans[END_REF][START_REF] Krasowska | How microorganisms use hydrophobicity and what does this mean for human needs? Front[END_REF]. Besides, a previous study confirmed the presence of high aMW (>150 kDa)

proteoglycan-like and sulfated proteoglycan-like compounds in the EPS extracted from the same type of anaerobic granules (Bourven et al., 2015a). Therefore, it is inferred that F1 (aMW >660 substrates or to bind metal cations [START_REF] Zhang | Composition of EPS fractions from suspended sludge and biofilm and their roles in microbial cell aggregation[END_REF][START_REF] Heipieper | Surface properties and cellular energetics of bacteria in response to the presence of hydrocarbons[END_REF]. This conclusion is also proved by the improved metal binding ability of the fungus showed in Table 5.1.

The Fourier transform infrared spectroscopy (FT-IR) results evidenced the negative zetapotential value was supported by the negatively charged functional groups, such as hydroxyl and carboxyl groups, in the extracted EPS (Fig. 5.5). Since cells are embedded in the EPS matrix, their surface charge also relies on the EPS characteristics [START_REF] Harimawan | Investigation of extracellular polymeric substances (EPS) properties of P. aeruginosa and B. subtilis and their role in bacterial adhesion[END_REF]. It is believed that these functional groups in the EPS could contribute to the negative charges on the cell surface, and the interactions between the metal cations and those functional groups could reduce the negative charges of the cell surface [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF][START_REF] Li | Effects of Fe(III) on floc characteristics of activated sludge[END_REF][START_REF] Urbain | Bioflocculation in activated sludge: an analytic approach[END_REF].

The PN and PS in the extracted EPS showed an opposite variation trend with the increase of Ni 2+ concentration (Fig. 5.4): PN content was increased, whereas the PS content eventually dropped to the same level as the control group. It is known that the microorganisms under stress conditions could adjust their extracellular excretion and surface properties, which could eliminate the toxic effect induced by the metal ions [START_REF] Jaenicke | The stability of proteins in extreme environments[END_REF]. The adjustment of the PN and PS content in the EPS under the exposure to different initial Ni 2+ concentrations could be considered as the protection role exerts by the EPS when the fungus involves in a stress condition [START_REF] Huang | Degradation of lead-contaminated lignocellulosic waste by Phanerochaete chrysosporium and the reduction of lead toxicity[END_REF]. Nevertheless, the way how the cells answered on the negative growth conditions is different at varied microorganisms strains and it is also related to the type of metal ions [START_REF] Mikes | The influence of heavy metals on the production of extracellular polymer substances in the processes of heavy metal ions elimination[END_REF].

Despite those above-mentioned alterations in the EPS characteristics, all the EEM spectra (Fig. 5.6) and the aMW distribution of the total organics in the EPS (Fig. 5.7) showed similar features. It was noticed that the variation trend of peak intensity of the fraction Ve = 17 -22 mL (aMW = 14 -0.5 kDa) in the PN-like fingerprints (Fig. 5.7) were in accordance with the variation of PN content demonstrated in Fig. 5.4. Since the fingerprints detected at UV/210 nm could represent the total organics in the extracted EPS [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 difference between the two types of fingerprints could probably due to the change of PS moiety i.e. uronic acid in the EPS [START_REF] Ozturk | Metal removal of cyanobacterial exopolysaccharides by uronic acid content and monosaccharide composition[END_REF]. However, since the PS lack spectroscopic features that enable them to be easily detected, determining the molecular features of PS in the extracted EPS by the EEM or SEC techniques presents significant analytical challenges [START_REF] Arnosti | Fluorescent derivatization of polysaccharides and carbohydrate-containing biopolymers for measurement of enzyme activities in complex media[END_REF].

Despite the hydrophobicity (%) of the fungal EPS undergoes some variations as the Ni 2+ concentration increases, it seems that the fungal pellets could adjust their surface hydrophobic properties to adapt to different stress conditions, but in the meantime, maintain the molecular size and fluorescence characteristics of the EPS stable.

Perspectives

Cells are embedded in the EPS matrix, and their surface characteristics, such as the charge and the hydrophobicity, are controlled by the EPS. The adjustment of those characteristics mediates the cellular recognition, and promotes the initial cell adhesion onto other surfaces (Harimawan and Table 6.1 Main conclusions drawn from this study and their implications in the future work.

Main conclusion Suggestions for the future work

Implication for the process industries

Chapter III pH 5 is the better elution pH for the hydrophobic fractionation of EPS pH should be carefully considered in the EPS characterization

Chapter IV HS-like compounds are the major molecular support of the EPS hydrophobicity the interplay between the microorganisms and environmental conditions.

A better understanding of the sludge flocculation, settling and dewatering process, and benefit in solving membrane fouling problems.

the synthesize pathway and the chemical structure of hydrophobic HS-like compounds; the sorption of organic pollutants by such hydrophobic molecules.

The chemical synthesis of hydrophobic molecules, and their application in the organic pollutants removal.

Chapter V

Phanerochaete chrysosporium is a hydrophobic fungus, and its EPS are highly hydrophobic.

The application of fungal EPS in the waste sludge treatment to improve sludge dewatering process.

The hydrophobicity of fungal EPS is decreased by adding Ni 2+ in the growth medium

The improvement of sorption/degradation performance of the fungal pelleted reactor

Sludge dewatering is the bottleneck in many wastewater treatment processes. [START_REF] Murthy | Factors affecting floc properties during aerobic digestion: implications for dewatering[END_REF] showed that PN were generally more important in sludge dewatering than PS, whereby a high PN concentration was detrimental for the dewatering process. [START_REF] Higgins | Characterization of exocellular protein and its role in bioflocculation[END_REF] demonstrated that sludge dewatering correlated well with the PN/PS ratio in sludge EPS. It is claimed that EPS in the sludge can bind a large volume of water (i.e. bound water) [START_REF] Li | Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge[END_REF], and the breakdown of EPS facilitates the water removal [START_REF] Raynaud | Compression dewatering of municipal activated sludge: effects of salt and pH[END_REF].

Besides, the metal binding ability of EPS and organic pollutants removal are both related to the EPS hydrophobicity [START_REF] Liu | Phenanthrene desorption from soil in the presence of bacterial extracellular polymer: observations and model predictions of dynamic behavior[END_REF][START_REF] Wei | Adsorption of Cu 2+ and Zn 2+ by extracellular polymeric substances (EPS) in different sludges: effect of EPS fractional polarity on binding mechanism[END_REF]. Therefore, EPS hydrophobicity is one of the factors that influences the wastewater treatment performance of the bioreactors [START_REF] Liu | Influences of extracellular polymeric substances (EPS) on flocculation, settling, and dewatering of activated sludge[END_REF]. Since the results from this study showed that the HS-like compounds were the major molecular support of EPS hydrophobicity, the role of HS-like compounds in those biological processes should be investigated.

Some hydrophobically modified polymers (e.g., cationic polyacrylamide) are often added as flocculants to enhance the cell surface hydrophobicity of sludge, by which the sludge dewatering performance is improved [START_REF] Sakohara | Dewatering of activated sludge by thermosensitive polymers[END_REF][START_REF] Sun | Evaluation of dewatering performance and fractal characteristics of alum sludge[END_REF]. However, there are many disadvantages of using chemical polymers, as they are expensive, toxic and corrosive. The development of more sustainable and non-hazardous materials becomes highly demanding in the waste sludge treatment. Besides, some experiences are already gained by using the fungal pellets in the researches [START_REF] Mannan | Use of fungi to improve bioconversion of activated sludge[END_REF][START_REF] More | Potential use of filamentous fungi for wastewater sludge treatment[END_REF]. Chapter V shows that EPS extracted from fungus Phanerochaete chrysosporium, even without the presence of large quantities of the HS-like compounds, demonstrated high hydrophobicity. Considering this, the possibility of applying these fungal EPS in the waste sludge treatment to improve dewatering process is worth investigating.

In chapter V, the EPS hydrophobicity (%) of the fungus was decreased by adding Ni 2+ in the growth medium. Phanerochaete chrysosporium has an ability to degrade various organic pollutants by its ligninolytic enzymes [START_REF] Chen | Simultaneous cadmium removal and 2,4-dichlorophenol degradation from aqueous solutions by Phanerochaete chrysosporium[END_REF][START_REF] Guo | Biodegradation of sulfamethoxazole by Phanerochaete chrysosporium[END_REF]. Whether the decreased EPS hydrophobicity will affect the sorption/degradation of the organic pollutants in fungal pelleted reactor can be studied as a topic of practical interest. Moreover, besides the organic pollutants, how the decreased EPS hydrophobicity influences the metal uptake by the fungal pellets in the bioreactor is also interesting.

Firstly

  , I would like to thank the program Environmental Technology for Contaminated Solids, Soils and Sediments (ETeCoS 3 ) from Erasmus Mundus Joint Doctorate (EMJD) program for providing me the position and scholarship to carry out my PhD study. With this opportunity, then I could explore this scientific world deeply, and be one part of it. Because of its mobility, I spent my PhD years in three different countries (France, the Netherlands and Italy), and I got the chance to know different people and places which has broadened my horizon. I would like to express my sincere gratitude to my promoter, Hab. Dr. Eric van Hullebusch, for giving me an opportunity to conduct my PhD research under his supervision. In the first eight months of my PhD life, I have stayed in the lab Gé omaté riaux et Environnement (LGE) of Université Paris-Est. One of my mentors, Dr. Yoan Pechaud, helped and taught me a lot on the research methods. I would like to thank him so much. During the stay, I met my officemates Dr. Lucian Staicu, and Dr. Manivanan Sethurajan. I also learned a lot from them, and their way of doing research. Thanks to those people, I became get to used to the PhD life in a foreign country.

  Supelite™ DAX-8 hars werd eerst toegepast op EPS geë xtraheerd uit anaeroob granulair slib, twee elutie pH-omstandigheden, d.w.z. pH 2 en 5 werden getest. De impact van zeven verschillende EPS-extractiemethoden op de hydrofobe eigenschappen van EPS werd ook beoordeeld. De resultaten toonden aan dat de extractiemethoden en bulkoplossing pH de EPS-samenstelling en de gemeten EPS-hydrofobiciteit respectievelijk beï nvloedden. Daarnaast hebben de EPS-extractieve reagentia, met name formaldehyde, ethanol, natriumdodecylsulfaat (SDS) en Tween 20, niet alleen extra koolstofgehalte geï ntroduceerd tijdens de totale organische koolstof (TOC) meting, maar ook met de DAX-8-hars geinteractieerd. Door de schijnbare molecuulgewicht (aMW) verdeling van de onbehandelde en pH-aangepaste EPS-monsters te vergelijken, werd meer volledige aMWinformatie van EPS bewaard bij pH 5. Zo werd de hydrofobe fractionering door DAX-8 hars bij pH 5 en fysieke EPS-extractiemethoden. In deze studie werden de voorkeur gegeven voor de volgende analyse van EPS, geë xtraheerd uit anaeroob granulair slib. Na het identificeren van de juiste omstandigheden voor DAX-8 hars fractionering, werd gedetailleerde kwalitatieve analyse van de EPS hydrofobe eigenschappen onderzocht door middel van exclusie chromatografie (SEC) en excitatie-en emissie-fluorescentiematrix (EEM) technieken. Uit de resultaten blijkt dat de humic-achtige stoffen (HS-achtig) het belangrijkste organische bestanddeel van EPS uit het anaerobe granulair slib waren, en ze waren ook de belangrijkste moleculaire ondersteuning van EPS hydrofobiciteit (>50%). Die hydrofobe HSachtige verbindingen waren voornamelijk kleine moleculen varië rend van 8 kDa tot <1 kDa, en hun hydrofobiciteit kan toegeschreven worden aan hun humane zuurachtige moleculaire structuur. Proteï nen (PN) en polysacchariden (PS) hebben in mindere mate bijgedragen tot de EPS hydrofobiciteit (<30%). XIV Hoewel de hydrofobe eigenschappen van HS-achtige verbindingen werden aangetoond, was de rol van PN en PS in de EPS-hydrofobiciteit moeilijk te laten zien. Het is bekend dat de belangrijkste organische bestanddelen van EPS die uit schimmels worden geë xtraheerd, PN en PS zijn. Ni 2+ is het essentië le metaal voor de micro-organismen en het is nodig als micronutrië nten bij nano-molaire concentraties en wordt ook geï dentificeerd als het spoorelement voor diverse biologische systemen, zoals anaerobe biochemische reacties. Om de hydrofobe eigenschappen van PN en PS te onderzoeken, evenals het effect van metaaltoevoeging (d.w.z. Ni 2+ ) onder subtoxische concentratie op de EPS-hydrofobiciteit te onderzoeken werd Phanerochaete Chrysosporium schimmel gekozen als het model micro-organisme. De resultaten toonden aan dat de inhoud van PN en PS in de geë xtraheerde schimmel-EPS varieerde met de initië le Ni 2+ concentratie die in het groeimedium werd toegevoegd. Met een toename van de initië le Ni 2+ concentratie van 0 mg/L tot 25 mg/L, werd het PN-gehalte verhoogd terwijl het PS-gehalte uiteindelijk stabiel bleef. De hydrofobiciteit van schimmel-EPS, bepaald door de DAX-8 harsbehandeling, is geleidelijk afgenomen als de Ni 2+ concentratie toegenomen. Het zetapotentieel van de schimmelpellets werd verminderd door de aanwezigheid van Ni 2+ . Bovendien werd de piekintensiteit van de PN-achtige moleculen varië rend van 0.5 kDa tot 14 kDa gedetecteerd door SEC versterkt door de verhoogde Ni 2+ concentratie, terwijl de aMW-verdeling van de totale organische stoffen in de EPS bijna stabiel bleef. Deze resultaten wijzen erop dat, onder subgiftige concentratie van Ni 2+ , de aanwezigheid van Ni 2+ die de zwampellets bevorderde minder hydrofobe worden, en het PN-gehalte werd verbeterd zonder zijn intrinsieke fluorescentieeigenschappen te veranderen. Niettemin waren de moleculaire eigenschappen van PS nog steeds moeilijk te worden uitgewerkt en gebrek aan specifieke spectroscopische eigenschappen die gedetecteerd kunnen worden door EEM of SEC-technieken.

Fig. 2 . 1

 21 Fig. 2.1 Schematic illustration of hydrophobic interactions in the water.

Fig. 2 . 2

 22 Fig. 2.2 Schematic illustration of a sessile-drop contact angle system

Fig. 2 . 5

 25 Fig. 2.5 Classical hydrophobic/hydrophilic fractionation by XAD/DAX resins.

  Jorand et al. (1998) fractionated the EPS extracted from activated sludge by XAD-8 and XAD-4 resins. The results demonstrated that at least 7% of the dissolved carbon and 12% of the proteins were hydrophobic (Table2.1).XAD/DAX fractionation coupled to other techniques, such as mass spectroscopy and size partition, allows determining a specific fingerprint of targeted organic matter. To obtain a comprehensive understanding of the fouling potential of SMPs in a membrane bioreactor(MBR) and their molecular size,[START_REF] Shen | A systematic insight into fouling propensity of soluble microbial products in membrane bioreactors based on hydrophobic interaction and size exclusion[END_REF] fractionated MBR supernatant into different hydrophilic/hydrophobic fractions by using DAX-8 resin with joint size partition (ultrafiltration membrane). Results showed that hydrophilic fraction was the dominant foulant and responsible for the flux deterioration in the MBR. The apparent molecular weight (aMW) of this hydrophobic fraction was above 100 kDa.Cao et al. (2017) investigated the hydrophobic properties of EPS extracted from anaerobic granular sludge by DAX-8 resin, the EPS fingerprints recorded by size exclusion chromatography (SEC) demonstrated that the highly hydrophobic EPS molecules that retained by the resin with an aMW of 31 -175 kDa. After separation by XAD resins (XAD-8 and XAD-4), the algal (Euglena gracilis) organic fractions were studied by laser desorption ionization (LDI) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-

  [START_REF] Hazen | Influence of growth conditions on cell surface hydrophobicity of Candida albicans and Candida glabrata[END_REF] compared the cell surface hydrophobicity of both Candida albicans and Candida glabrata among different growth media, and a greater cell surface change was found in Auto-Pow minimum essential medium with Earle salts supplemented with biotin, glucose, glycine and HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (AP-MEM). They ascribed the change of cell surface hydrophobicity to the cell surface compositional modifications that occur during the form transition, as a low percentage of cells had germ tubes when incubated 24h

  estimated during the extraction resulting in unpredictable EPS extraction yields (Domí nguez et al., 2010b; Zuriaga-Agustí et al., 2013), and the left-over of organic reagents in the EPS solution can

Fig. 2 . 6

 26 Fig. 2.6 Schematic illustration of various interactions to maintain the EPS structure

  methods were chosen to evaluate the influence of the extraction procedure on EPS hydrophobicity determination. Each extraction was undertaken on 50 mL homogenized sludge suspension. Three physical extraction methods modified from D'Abzac et al. (2010) were applied: (i) centrifugation at 15,000 ×g for 20 min at 4 °C; (ii) sonication (Sonoplus GM 70, Bandelin) at 40 W for 1 min in an ice bath; and (iii) heating in a water bath at 80 °C for 10 min.

  3.1. In brief, 5.0 mL of prepared DAX-8 resin was filled in a stainless-steel column (50×15 mm) and connected to a peristaltic pump to maintain a constant flow rate at 50 mL/h. Before the resin treatment, 100 mL of EPSuntreated solution (TOC < 400 mg C/L, diluted by ultrapure water) of each extraction were acidified to pH 2.0 (± 0.1) or pH 5.0 (± 0.1) by 2 M HCl (Prolabo), and then filtered by a 0.45 µm cellulose nitrate membrane (Sartorius) to remove any possible precipitates, the filtrate was named EPSbefore. 40 mL of EPSbefore solution was uploaded in one DAX-8 column.All the eluted EPS fraction (40 mL) from the column was collected and named EPSafter. The TOC, PN, HS-like and PS contents of both EPSbefore and EPSafter samples were quantified according to the procedures described above. Ultrapure water and the organic reagents (ethanol, 0.6% formaldehyde, 0.01% SDS and 0.25% Tween 20 in PBS) used in the EPS extraction were also acidified into pH 2 and pH 5, respectively, and tested on the DAX-8 column as control group. The resin treatment on each sample was performed in duplicate.

Fig. 3 . 1

 31 Fig. 3.1 Schematic illustration of DAX-8 resin fractionation on EPS.

Fig. 3 . 2

 32 Fig. 3.2 Comparison of the biochemical EPS composition of untreated EPS and pH-adjusted EPS samples.

Fig. 3 . 3

 33 Fig. 3.3 pH effect on the SEC/UV 210 nm fingerprints of EPS extracted by different methods: (a) -centrifugation (d (dilution) 1/1); (b) -heating (untreated EPS, EPSbefore at pH 5 and pH 2, d 1/50, 1/5 and 1/4, respectively; (c) -sonication (EPSuntreated, d 1/3); (d) -ethanol (d 1/1); (e) -F.+heat. (EPSuntreated, EPSbefore at pH 5 and pH 2, d 1/50, 1/5 and 1/4, respectively); (f) -SDS (EPSuntreated, d 1/2); and (g) -Tween 20 (EPSuntreated, d 1/2).

Fig. 3 .

 3 Fig. 3.4 TOC concentration of EPS extracted by different methods before and after DAX-8 treatment at pH 5 and pH 2.

Fig. 3 .

 3 Fig. 3.5 TOC concentration of the organic reagents before and after DAX-8 treatment at pH 5 and pH 2.

Fig. 3 . 6

 36 Fig. 3.6 Global hydrophobicity (%) of EPS extracted by different methods: (A) calculated from TOC concentration; (B) calculated from the sum of three main constituents (proteins, polysaccharides and humic-like substances) concentrations.

Fig. 3 . 7

 37 Fig. 3.7 Effect of DAX-8 resin treatment at pH 5 on the SEC/UV 210 nm fingerprints of EPS: (a) -centrifugation (d 1/1); (b) -heating (before and after, d 1/5 and 1/4, respectively); (c) -sonication (d 1/1); (d) -ethanol (d 1/1); (e) -F.+heat. (before and after, d 1/ 5 and 1/4, respectively); (f) -SDS (d 1/1); and (g) Tween 20 (d 1/1).

  have determined IEP values of 31 different gram negative and positive bacteria, their results showed that most of the tested bacterial cells have IEP values ranging from 1.75 to 3.5. Wang et al. (2012b) determined the IEP value of EPS from Bacillus megaterium TF10, they concluded that the IEP value of the studied EPS extracted by EDTA was about 4.8.

pKa3 8 -

 8 10 and pKa4 ≈10 corresponding to the phenolic, hydroxyl, and amino groups (D'Abzac et al., 2013). More recently, Bourven et al. (2015a) identified the presence of sulfate groups (-SO3ˉ)

  suspended in ultrapure water. Each extraction was carried out on a 50 mL homogenized sludge suspension. Three physical EPS extraction methods modified from D'Abzac et al. (2010) and one chemical method (Wu and Xi, 2009) were chosen: (i) centrifugation at 15,000 × g for 20 min under 4 °C; (ii) sonication at 40 W for 1 min in an ice-bath; (iii) heating at 80 °C for 10 min in a

Fig. 4 . 1

 41 Fig. 4.1 Effect of DAX-8 resin treatment on the organic constituents of EPS (PN, HS-like and PS).

Fig. 4 . 2

 42 Fig. 4.2 Hydrophobicity (%) of the organic constituents in the EPS (PN, HS-like and PS).

Fig. 4 . 3

 43 Fig. 4.3 Global hydrophobicity (%) of EPS and the contribution (%) of each EPS constituent (PN, HS-like and PS).

Fig. 4 .

 4 Fig. 4.4 displays the EEM spectra of all the EPSbefore (1) and EPSafter (2) samples at pH 5. For the spectra of EPSbefore, similar peak numbers and peak location are observed in Region I for all the extraction methods: Peak A (Ex/Em 220-225/285 nm), Peak B (Ex/Em 225-230/285-330 nm) and Peak D (Ex/Em 255/279-295 nm); whilst a different situation is observed in Region II: mainly two peaks, Peaks C (Ex/Em 225-235/370-400 nm) and E (Ex/Em 270-285/380-430 nm) aredetected in the centrifugation, sonication and SDS methods; but no apparent peaks could be detected when using the heating method. This could be due to the sample dilution factor that is

Fig. 4 . 5

 45 Fig. 4.5 Comparison of SEC/UV 210 nm, PN-like and HS-like fingerprints of EPSbefore and EPSafter: (a) -centrifugation (d 1/1); (b) -heating (d 1/4); (c)sonication (d 1/1); and (d) -SDS (d 1/1).

  (1/357) to detect the fluorophores, or a modification of the fluorescent properties of certain polycyclic aromatic molecules present in the EPS after the heating treatment. Peaks detected in Region I could be assigned as fluorophores containing a limited number of aromatic rings, such as lignin phenols, vanillic acid (Ex/Em <380/326 nm), syringic acid (Ex/Em <380/338 nm), toluene (Ex/Em 266/300-400 nm), indole (Ex/Em 230/330-350 nm), DNA (267/327 nm), and aromatic amino acids such as tryptophan (Ex/Em 275/304 nm), and tyrosine (Ex/Em 295/353 nm) are present in the wastewater discharged from the paper mills[START_REF] Carstea | Fluorescence spectroscopy for wastewater monitoring: a review[END_REF]. Besides, in the fingerprints of HS-like compounds (Fig. 4.5b), several peaks are observed in F3 -F5; while in other extraction methods, only one (Fig. 4.5a and c) or two (Fig. 4.5d) peaks are detected and they are centered in F3. Since the lowest HS-like hydrophobicity and EPS global hydrophobicity (%) are both achieved by the heating extraction method (Fig. 4.2 and 4.3), it is assumed that the heating treatment could change the fluorescent properties or molecular structure of the extracted EPS i.e. HS-like molecules, and in turn, these EPS molecules become more hydrophilic.

Fig. 4 .

 4 Fig. 4.5 shows that F2 (aMW = 122 -31 kDa) is not present in the fingerprints of LB-EPS, but both LB-and TB-EPS have a similar aMW distribution of HS-like compounds. This implies that the LB-EPS and the TB-EPS also have a different aMW distribution, especially the hydrophobic PN-like/PS-like molecules. In addition, although the EPS (LB-EPS + TB-EPS) extracted by heating, sonication and SDS methods demonstrate a similar aMW distribution (Fig. 4.5b-d), some different EPS hydrophobic features demonstrated before have evidenced the influence of the EPS extraction method on the EPS characterization.

Fig. 5 . 1

 51 Fig. 5.1 Comparison of zeta-potential between the intact and the EPS-free fungal pellets in the PUM buffer (pH 7.1).

Fig. 5 . 2

 52 Fig. 5.2 Cell surface hydrophobicity (%) of the intact and the EPS-free biomass.

Fig. 5 .

 5 Fig. 5.3 shows the hydrophobicity (%) of different EPS samples at pH 2. The highest EPS hydrophobicity (%) was identified in the control group (~82%), and this value gradually decreased due to increased Ni 2+ concentrations in the fungal broth. At Ni 2+ concentrations of 0.5, 1 and 5 mg/L, their corresponding EPS hydrophobicity (%) decreased to a similar value of ~70%. When the Ni 2+ concentration in the broth reached ≥10 mg/L, the EPS hydrophobicity (%) reached its lowest value of 63%.

Fig. 5 . 3

 53 Fig. 5.3 EPS hydrophobicity (%) characterized by DAX-8 resin treatment at pH 2.

Fig. 5 . 4

 54 Fig. 5.4 The content of PN and PS in the EPS extracted from the fungus cultivated at different Ni 2+concentrations.

  5 and 25 mg/L of Ni 2+ addition are shown in Fig. 5.5. The strong and broad bands at around 3200-3400 cm -1 are assigned to the stretching vibration of -NH in amide I and II, or -OH in alcohols or phenols. The bands near 2900-2800 cm -1 can be assigned to the stretching vibration of the -OH group in the carboxylic acids, or -CH groups. The bands at ~1630-1650 cm -1 and 1550-1560 cm -1 are assigned to the stretching vibration of C=O and -NH. Bands near 1400 cm -1 were attributed to C=O stretching of the carboxylic acids and deformation stretching of -OH (alcohols and phenols) and/or N-C-H (PN), which could indicate the acidic nature of EPS

  Fig. 5.5 FT-IR spectra of the EPS extracted from the fungus exposed to sub-toxic concentration of Ni 2+ : (a) 0 mg/L, (b) 0.5 mg/L, and (c) 25 mg/L.

  different extracted EPS samples are shown in Fig.5.7. The EPS fingerprints recorded at UV/210 nm can be generally divided into two fractions, i.e. Ve = 10-17 mL (aMW= >660-14 kDa) and Ve = 17-22 mL (aMW = 14-0.5 kDa). The peak intensities of the former fraction were much lower than those of the latter fraction. Besides, with increasing Ni 2+ concentrations in the broth, no significant difference of peak distribution was observed in those two fractions (Fig.5.7). This corroborates the fact that the aMW of total organics in the extracted EPS was not influenced by the Ni 2+ concentration.The fingerprints of PN-like compounds recorded by the fluorescence detector at Ex/Em = 225/345 nm show different trends (Fig.5.7). At a Ni 2+ concentration of 0.5 mg/L, the PN-like fingerprint demonstrated the same distribution as that of the control group, and the peaks detected in these two PN-like fingerprints were mainly located in the fraction of Ve = 17-22 mL (aMW = 14-0.5 kDa) (Figs. 5.7a and b). However, when the Ni 2+ concentration in the broth reached values >1 mg/L, those PN-like fingerprints showed a similar wide distribution in the fraction of Ve = 10-22 mL (aMW = >660-0.5 kDa), and the peak intensity of this fraction was higher than that of the control group. Besides, enhanced peak intensities were also observed in the fraction Ve = 17-22 mL (aMW = 14-0.5 kDa) when the Ni 2+ concentration in the broth reached 10 and 25 mg/L (Figs. 5.7cf).

Fig. 5 .

 5 Fig. 5.7 aMW distribution of the EPS extracted from the fungus exposed to sub-toxic concentrations of Ni 2+ : (a) 0 mg/L, (b) 0.5 mg/L, (c) 1 mg/L, (d) 5 mg/L, (e) 10 mg/L, and (f) 25 mg/L.

  3.3), the possible metal binding saturation observed in the extracted EPS could be due to one of the following mechanisms: (i) limited metal binding sites in the EPS, because the binding sites present in the EPS also reached their saturation regardless of the presence of Ni2+ , or (ii) the presence of Ni 2+ engenders the alteration of surface properties of the fungal cells, and thus, the metal binding ability of the EPS is limited.

  Fig. 5.7, unlike the fingerprints detected by SEC at UV/210 nm demonstrating a similar aMW distribution regardless of the presence of Ni 2+ , the fingerprints of the PN-like compounds in the EPS clearly witnessed two different trends with an increase in the Ni 2+ concentration. When the Ni 2+ concentration in the broth was <0.5 mg/L, the two fingerprints showed a similar trend, whereas a different, yet resembling trend was observed at Ni 2+ concentrations of 1, 5, 10 and 25 mg/L, respectively (Fig. 5.7). It was also observed that the varying trend of the peak intensity for the fraction Ve = 17-22 mL (aMW = 14-0.5 kDa) in the PN-like fingerprints is in accordance with the variations of the PN content shown in Fig. 5.4.

  affected by the EPS extraction methods and bulk solution pH. Based on the DAX-8 resin technique, Chapter IV revealed that the humic-like substances (HS-like) were the main molecular support for the hydrophobicity of EPS extracted from anaerobic granular sludge, while the proteins (PN) and polysaccharides (PS) took a secondary position to contribute to the EPS hydrophobicity. In Chapter V, under sub-toxic concentration of Ni 2+ (0 -25 mg/L), PN and PS in the fungal EPS extracted from fungus Phanerochaete chrysosporium played an active role in supporting the EPS hydrophobicity by varying their contents.

Fig. 6 . 1

 61 Fig. 6.1 The interlink between the research chapters.

Fig. 6 . 2

 62 Fig. 6.2 EPS extraction methods are based on the interruption of chemical interactions that are responsible for the EPS stabilization.

  kDa) and F2 (aMW = 122 -31 kDa) in the PN-like fingerprints detected by SEC (Fig. 4.5) are mainly made up by PN and PS, or glycoproteins/proteoglycans.

Fig. 6 . 3 A

 63 Fig. 6.3 A proposed schematic of the floc structures of anaerobic granular sludge (modified from Zhang et al. (2015b)).

Table 2 .1

 2 Techniques applied for the determination of microbial hydrophobicity

	Type	Technique	Conditions	Relative hydrophobicity	Reference
	Bacteria Rhodococcus RC291	CAM		20° -65°	Pen et al., 2015
	Sludge from sequencing batch reactor (SBR)	CAM		10° -45°	Liao et al., 2001
	Bacteria
	Streptococcus
	salivarius HB, Streptococcus sanguis CH3, Streptococcus	CAM	Static sessile method using de-ionized water	10° -65°	Busscher et al., 1984
	mitior T6, and
	Veillonella alcalescens
	Vl
	Thermophilic
	spores from bacteria	CAM		10° -30°	Seale et al., 2008
	Geobacillus spp.
	EPS extracted from a heterotrophic biofilm and a mixed autotrophic-heterotrophic biofilm	HIC	UV absorbance at 280 nm; elution gradient started off with 100% salt buffer (buffer B: 0.05 M KH2PO4/K2HPO4 with 3 M ammonium sulfate, pH 7) and KH2PO4/K2HPO4, pH 7) ended with 100% elution buffer (buffer A: 0.05 M	Hydrophobic EPS from the two biofilms were mainly represented by two time (DRT) 0.3 and DRT (dimensionless retention distinct fractions	Ras et al., 2013
				0.9)
	EPS extracted from Pseudomonas and Sagittula stelleta fluorescens Biovar II	HIC	UV absorbance at 214 nm; eluted by 20 mM Tris buffer (0.25, 0.5, 0.75 and 1.0 M) (pH 8.0) plus different ammonium sulfate concentrations	-	Xu et al., 2011

Table 2 .2

 2 Comparison of the techniques applied in the hydrophobicity determination

	Technique	Advantages	Limitations	References
			Extremely experimental care is needed.
		Simple and rapid; non-	Very minor vibrations can cause	Busscher et al.,
	CAM	destructive; provides information related to surface	advancing contact angles to decrease, resulting in errors of several degrees:	1984; Marmur, 1988; Pen et al.,
		energetic; hydrated samples can	surface roughness; contacting time; liquid	2015; Seale et
		be observed.	penetration; swelling of a solid by a liquid	al., 2008
			can change the chemistry of the solid.
			Based on the difference in the
		Samples with high ionic strength	hydrophobicity of amino acids; significant
		can be used; the elution	baseline changes during gradient elution
	HIC	conditions promote the retention of the tertiary conformation and	and a requirement for non-volatile mobile phases; type and concentration of ligands,	Alpert, 1988; Ras et al., 2013
		the biological activity of most	column packing materials, salts influence
		proteins; good selectivity.	the performance, as well as pH, and
			temperature.
				Lindahl et al.,
	SAT	A rapid and reproducible screening test.		1981; Ljungh and Wadström,
				1982
	MATH	Simple, no special equipment is needed; can be used for observing mixed cultures of the same species; bound bacteria to hexadecane can be desorbed by 16 °C. solidify at temperatures below allowing the hexadecane to	Under low ionic strength buffer, otherwise the hydrophobic interactions play a lesser and hydrocarbon. physicochemical forces between the cells role and electrostatic interactions increase; low discriminating power for hydrophilic strain; it measures the interplay of all the	Rosenberg, 2006
			This hydrophobic-hydrophilic distinction
		Samples are fractionated	is artificial, since the pH of the tested
	XAD/DAX resin	according to their hydrophobicity; allows characterization of separated	samples is pre-adjusted; possible physical and chemical alteration during pH adjustment and fractionation process;	Malcolm, 1991
	fractionation	fractions by determination of their average structural and	irreversible adsorption of the samples onto the resins; contamination from the resin
		functional groups	bleeding; size-exclusion effect induced by
			the resins.

Hydrophobic bacterial cells will clump when ammonium sulfate is absent; poor visualization of the bacterial aggregates without a dark background; electrostatic interactions between the cells, temperature, pH, time and the bacterial cell concentration affect the results.

Table 3 .1

 3 Characteristics of untreated EPS (EPSuntreated) extracted from anaerobic granular sludge (F.: formaldehyde; SDS: sodium dodecyl sulfate).

	Extraction Method	pH	DW (mg/g sludge DW)	VDW (mg/g sludge VDW)	TOC content (mg C/g EPSuntreated DW)	Biochemical composition (mg/g EPSuntreated DW) Humic-like Polysacchari Proteins substances des
	Centrifugation 7.9	5.3 ± 0.7	1.9± 0.1	184 ± 3	5 ± 1	104 ± 5	31 ± 2
	Heating	8.4	34.7 ± 0.1 32.7± 0.1	396 ± 23	185 ± 10	288 ± 8	133 ± 7
	Sonication	7.2	8.2 ± 0.1	3.4± 0.1	63 ± 5	60 ± 3	98 ± 6	50 ± 2
	Ethanol	7.1	5.3 ± 0.1	1.3 ± 0.1	> 1000	9 ± 1	147 ± 3	46 ± 3
	F.+Heat.	6.9	27.0 ± 0.7 20.4 ± 0.1 418 ± 23	153 ± 8	167 ± 10	109 ± 3
	SDS	6.8	5.3 ± 0.2	2.2 ± 0.1	81 ± 12	58 ± 2	118 ± 5	56 ± 2
	Tween 20	8.6	26.7 ± 0.1	8.8± 0.1	93 ± 9	27 ± 2	26 ± 3	11 ± 1

  Thus, fingerprints obtained at pH 5 are preferred in monitoring aMW of

	(e)									(f)
	(a)	0.085								(b)		0.045
		0.025										0.1
	UV absorbance 210 nm (au) UV absorbance 210 nm (au)	0.005 0.01 0.015 0.02 0.017 0.034 0.051 0.068					F1	F2	F3	F4	UV absorbance 210 nm (au) UV absorbance 210 nm (au)	0.02 0.04 0.06 0.08 0.036 0.009 0.018 0.027	F1	F2	F3	F4
		0										0
		0	6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27			6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
			6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Ve (mL) Ve (mL)		0	6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Ve (mL)
													Ve (mL)
							untreated	pH5	pH2
	(c)	0.04								(d)
		0.12										0.024
	UV absorbance 210 nm (au) UV absorbance 210 nm (au)	0.03 0.06 0.09 0.008 0.016 0.024 0.032									UV absorbance 210 nm (au)	0.006 0.012 0.018
		0
			6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
		0	6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Ve (mL)		0	6	7	8	9	10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
								Ve (mL)					Ve (mL)
	(continued)
	EPSbefore and EPSafter.

Table 3 .2

 3 Peak area difference ratio (%) of different fractions in the SEC chromatograms.

	Extraction Method	Fraction 1 Ve = 9.5 -11 mL (aMW> 440 kDa)	Fraction 2 Ve = 13.5 -16 mL (aMW= 175-31 kDa)	Fraction 3 Ve = 17 -21 mL (aMW= 16 -1 kDa)	Fraction 4 Ve = 21 -23 mL (aMW= 1-0.3 kDa)
	Centrifugation	56.9	64.8	71.4	39.3
	Heating	16.7	64.2	7.3	2.7
	Sonication	51.1	60.2	57.4	39.8
	F.+Heat.	34.0	96.6	14.7	8.5
	SDS	51.7	68.8	50.9	35.3

  most EPS studies, chemical EPS extraction methods are preferred due to their higher extraction efficiency compared to physical EPS extraction methods (D'Abzac et al., 2010). The application of surfactants such as SDS and Tween 20 in the EPS extraction is meant for increasing the

	solubility of hydrophobic molecules in the EPS to improve EPS extraction yield. At
	concentrations above the critical micelle concentration (CMC) level, surfactants have the ability
	of solubilizing higher amounts of hydrophobic organic molecules compared to a surfactant-free
	water solution. The final concentration of Tween 20 in this study (0.1%, w/v) is far beyond its
	CMC in water (about 0.01%, w/v), which could explain that the DW and VDW of EPSuntreated
	extracted by Tween 20 (26.7 ± 0.1 mg/g sludge DW and 8.8 ± 0.1 mg/g sludge VDW,

Table 4 .1

 4 Characteristics of EPS extracted from anaerobic granules by different methods.

					Biochemical composition
	Extraction Method	EPS Type	(g/L) DW	(g/L) VDW	(mg/g EPSuntreated DW)
					PN	HS-like	PS
	Centrifugation	LB	0.7 ± 0.1	0.2 ± 0.1	5 ± 1	104 ± 5	31 ± 2
	Heating	LB+TB	4.3 ± 0.3	3.5 ± 0.6	185 ± 10	288 ± 8	133 ± 7
	Sonication	LB+TB	1.0 ± 0.3	0.4 ± 0.1	60 ± 3	98 ± 6	50 ± 2
	SDS	LB+TB	0.7 ± 0.1	0.2 ± 0.1	58 ± 2	118 ± 5	56 ± 2

Table 4 .

 4 2 presents the fluorescence intensity (FI) and reduction (%) of each peak in the spectra, as well as the corresponding CI values of EPSbefore and EPSafter samples at pH 5, respectively. According to the Student's paired t-test (p = 0.05) analysis, the FI reduction (%) of Peak B and Peak E respectively detected in Region I and II are the most significant. In addition, the CI values are all decreased after the resin treatment, and a dramatic decrease is observed in the SDS extraction, in which the CI value is decreased from 1.1 to 0.4 (Table4.2). It suggests that complex polycyclic aromatic molecules are more involved in the hydrophobic interactions with the resin than the molecules containing a limited number of aromatic rings.

Table 4 .2

 4 Average fluorescence intensity (au/TOC) and corresponding reduction rate (%) of the peaks in the EEM spectra (relative standard deviation (RSD) < 10 %).

				Peak A	Peak B	Peak C	Peak D	Peak E
	Extraction Method	Dilution factor	TOC concent-ration	(Ex/Em 220-225/285)	(Ex/Em 225-230/285-330)	(Ex/Em 225-235/370-400)	(Ex/Em 255/279-295)	(Ex/Em 270-285/380-430)	CI
			(mg C/L)	Maxima Intensity (au/TOC)	Reduction (%)	Maxima Intensity (au/TOC)	Reduction (%)	Maxima Intensity (au/TOC)	Reduction (%)	Maxima Intensity (au/TOC)	Reduction (%)	Maxima Intensity (au/TOC)	Reduction (%)
	Before		3.3	45.6		86.1		84.2		48.3		74.0	1.1
	Centrifugation	1/13			0.6		58.1		16.8		4.0	37.3
	After		1.0	45.3		36.1		70.1		46.4		46.4	0.9
	Before		0.2	881.6		2845.2				367.7
	Heating	1/357			66.9		75.2	-			22.3
	After		0.2	291.6		705.7				285.8
	Before		2.0	67.9		206.5		170.9		54.7		160.2	1.0
	Sonication	1/25			21.3		65.9		41.0		2.3	53.0
	After		0.8	53.4		70.4		100.8		53.4		75.3	0.9
	Before		1.4	176.7		434.1		293.9		319.1		446.7	1.1
	SDS	1/36			38.0		74.3		68.2		76.0	63.4
	After		0.8	109.6		111.4		93.3		76.6		163.7	0.4

4 aMW distribution of EPSbefore and EPSafter characterized by SEC Fig

  . 4.5 compares the fingerprints of EPSbefore (1) and EPSafter (2) at pH 5 in two detection modes by SEC: (i) UV/210 nm for the total organic matter (TOM) present in the EPS, and (ii) fluorescence detection for PN-like (Ex/Em = 222/300 nm) and HS-like (Ex/Em = 300/500 nm) compounds. Only peaks appearing below the total permeation volume (Ve = 22 mL) are considered, and no significant peaks were detected in the control group (data not shown).

	(d)	(b) (c) 0.05	0.1 0.04
	UV absorbance 210 nm PN-like fluorescence	0 0.005 0.01 0.015 0.02 0.025 (au) 10 20 30 40 50 60 intensity (au) 0 0.02 0.04 0.06 0.08 UV-absorbance 210 nm (au) UV absorbance 210 nm PN-like fluorescence 0 100 200 300 400 500 0.01 0.02 0.03 UV absorbance 210 nm (au) 0.01 (a) 0.02 0.03 0.04 (au) 0 600 6 0 120 160 PN-like fluorescence intensity (au) 14 0 20 40 60 80 100 intensity (au) 0 20 40 60 80 100 120 140 intensity (au) PN-like fluorescence 6 30	6 6 6 7 6 7	7 7 7 7 8 8	8 8 8	9 8 9	9 9 9 10 9 10		10 10 10 11 10 11		11 11 11 12 11 12	12 12 12 13 12 13	13 13 13 14 Ve (mL) 13 14	14 14 14 15 14 15	15 15 15 16 15 16	16 16 16	17 16 17	17 17 17	18 17 18	18 18 18	19 19 18	19 19 19	20 20	19	20 20 20	21 21	20	21 21 21	22 22	21	22 22 22	22
	HS-like fluorescence	0 0 2 4 6 8 10 12 14 (continued) HS-like fluorescence intensity (au) 0 2 4 6 8 10 12 HS-like fluorescence Intensity (au) 0 3 6 9 12 15 18 6 0 HS-like fluorescence intensity (au) 5 10 15 20 25 Intensity (au)	6 6 6 6 6 7	7 7 7 7 7 8	8 8 8	8 8 9	9 9 9 10 9 9	F1	10 10 10 10 F1 F1 10 11	F1	11 11 Before DAX-8 12 12 Before DAX-8 13 13 11 12 13 Before DAX-8 11 12 13 11 12 13 Before DAX-8 Ve (mL) Ve (mL) 14 14 14 Ve (mL) 14 14 Ve (mL) 15 15 15 F2 15 15 F2 12 13 14 15 16 F2	16 16 After DAX-8 17 17 After DAX-8 18 18 16 17 18 After DAX-8 16 17 16 17 After DAX-8 17 18 F3 19 18 18	19 19 F3 19 F3 F3 19 19 20 F4	20 20 20 F4 F4 20 20 21	21 21 21 F4 F5	21 21 22	22 22 22 F5 F5	22 22 F5
		(continued) (continued)

Table 5 .1

 5 Amount of Ni 2+ in the growth medium, intact biomass and extracted EPS.

	Growth medium
			Intact biomass	EPS
	Initial	After 48 h incubation	(µg/g biomass DW)	(µg/g EPS DW)
	(mg/L)	(mg/L)
	0	-	<1.6	<12.5
	0.5 ± 0.1	0.4 ± 0.1	3 ± 1	98 ± 9
	1.0 ± 0.1	0.8 ± 0.1	6 ± 1	188 ± 13
	5.0 ± 0.2	3.8 ± 0.2	42 ± 6	222 ± 19
	10.0 ± 0.4	8.0 ± 0.2	40 ± 5	250 ± 26
	25.0 ± 0.3	21.2 ± 0.1	92 ± 10	245 ± 22
	5.3

.2 Effect of Ni 2+ concentration on the cell surface properties of P. chrysosporium

Table 5 .2

 5 Location and fluorescence intensity of each peak in the EEM spectra.

	Initial Ni 2+	Peak A		Peak B
	concentration (mg/L)	Location (Ex/Em, nm)	Intensity (au/mgC)	Location (Ex/Em, nm)	Intensity (au/mgC)
	0	225/344-348	8 ± 1	275/341-347	8 ± 1
	0.5	225/342-346	7 ± 1	275/342-345	7 ± 1
	1	225/341-347	7 ± 2	275/344-345	6 ± 1
	5	225/342-348	6 ± 2	280/342-346	6 ± 1
	10	225/341-347	5 ± 2	280/342-350	7 ± 1
	25	225/345-348	6 ± 1	280/343-348	7 ± 1

Chapter IV.

Hydrophobic molecular features of EPS extracted from anaerobic granular sludge treating wastewater from a paper recycling plant

This chapter is published as: Cao F., Bourven I., Lens P.N.L., van Hullebusch E.D., Pechaud Y., Guibaud G. (2017). Hydrophobic molecular features of EPS extracted from anaerobic granular sludge treating wastewater from a paper recycling plant. Process Biochem. 58: 266-275. (Eq. 4.1) where: Conc.before and Conc.after represent the PN, HS-like or PS concentration (mg/L) of EPSbefore and EPSafter, respectively.

Besides, for obtaining the global hydrophobicity (%) of EPS, the Conc.before and Conc.after in Eq. 4.1 were replaced by the sum of PN, HS-like and PS concentrations (mg/L) quantified in the EPSbefore and EPSafter fractions, respectively (Cao et al., 2017).

Excitation and emission fluorescence matrix (EEM) spectroscopy

EEM spectra of both EPSbefore and EPSafter samples were recorded by Shimadzu RF-5301 PC spectrofluorometry. The spectra were collected with the emission (Em) wavelengths from 220 to 500 nm at 1 nm increases by varying the excitation (Ex) wavelength from 220 to 380 nm at 5 nm intervals. The fluorescence signals were processed by Panorama Fluorescence 3.1 software (Lab Cognition, Japan). All the EPS samples were diluted by ultrapure water to a proper TOC concentration if the sample had maximum fluorescence emission intensity exceeding 1000 au [START_REF] Chen | Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[END_REF]. For a better comparison of the results, the same sample dilution time was applied for EPSbefore and EPSafter in the analyses. Ultrapure water and 0.01% SDS at pH 5.0 were also tested as control. To partially account for Raleigh scattering, the fluorescence intensity detected for the ultrapure water was subtracted from the peak intensity recorded for the EPS samples [START_REF] Chen | Fluorescence excitationemission matrix regional integration to quantify spectra for dissolved organic matter[END_REF]. Each sample was measured twice.

The fluorescence spectra were mainly divided into two regions according to [START_REF] Carstea | Fluorescence spectroscopy for wastewater monitoring: a review[END_REF]: Em < 380 nm (Region I) and Em > 380 nm (Region II). The region Em < 380 nm is associated with fluorophores containing a limited number of aromatic rings and the indole moiety of free tryptophan, whilst the region Em > 380 nm is associated with polycyclic aromatic fluorophores [START_REF] Li | Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi-excitation/emission scan: a comparative study[END_REF]. Detected peaks were labelled as Peak A, B, C, etc. and the reduction (%) in the fluorescence intensity (FI) of each peak was calculated by using the same equation as described in Eq. 4.1, in which the concentration was replaced by the FI assigned to each peak. All the FI values are normalized to a TOC concentration of 1 mg C/L for comparison of different EPS samples. The TOC-normalized intensity value has a unit of au/TOC (au: arbitrary unit). The Student's paired t-test (p = 0.05) was applied to compare the FI reduction (%) among the peaks.

The complexity index (CI) of each EPS sample (Eq. 4.2) was defined as the ratio of the sum of the FI of the peaks in Region II over the sum of FI of the peaks in Region I [START_REF] Muller | Combining chemical sequential extractions with 3D fluorescence spectroscopy to characterize sludge organic matter[END_REF][START_REF] Wang | Fluorescent dissolved organic matter variations in a submerged membrane bioreactor under different sludge retention times[END_REF]. The comparison between the CI values of EPSbefore and EPSafter can be

EEM spectra of the extracted fungal EPS

The EEM spectra of all the EPS samples also showed similar features, as observed from the two main fluorescence peaks (Fig. 5.6): peak A, at a position of Ex/Em = 225/341-348 nm and peak B, at a position of Ex/Em = 275-280/342-350 nm. The fluorescence intensities (in au/mgC) of each peak are listed in Table 2. Despite the increase of Ni 2+ concentration from 0 to 25 mg/L, the two peaks were detected at similar locations, and the fluorescence intensity of each peak was maintained at a similar value (Table 5.2). Chapter VI.

(a)

General discussion and perspectives

Ting, 2016). The EPS hydrophobicity plays a key role in the biological processes such as biofilm formation, sludge flocculation/granulation, sludge dewatering, and membrane fouling [START_REF] Lin | A critical review of extracellular polymeric substances (EPSs) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies[END_REF][START_REF] Li | Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge[END_REF]. Besides, the hydrophobic fraction of the EPS provides sorption sites for the organic pollutants such as phenanthrene, benzene, etc [START_REF] Flemming | Biofilms: an emergent form of bacterial life[END_REF][START_REF] Liu | Phenanthrene desorption from soil in the presence of bacterial extracellular polymer: observations and model predictions of dynamic behavior[END_REF], whilst the hydrophilic fraction is responsible for the sorption of metal ions (Li et al., 2016).

Therefore, the characterization of EPS hydrophobicity provides better understanding of the sludge formation, as well as in finding solutions for the problems encountered in biological wastewater treatment process (Table 6.1).

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