A Cabassud

C Didi

Hplc- Guigui

Chengcheng Li
email: lichengcheng_1002@hotmail.com

Corinne Cabassud
email: corinne.cabassud@insa-toulouse.fr

Christelle Guigui
email: christelle.guigui@insa-toulouse.fr

Bernard Reboul
email: bernard.reboul@insa-toulouse.fr

Aurore Didi

Effects of carbamazepine in peak injection on fouling propensity of activated sludge from a MBR treating municipal wastewater

Keywords: BAM, micropolluants pharmaceutiques, colmatage, proté ines MBR, pharmaceutical micropollutants, fouling, protein-like substances membrane bioreactor, carbamazepine, peak injection, fouling propensity, activated sludge fouling of a submerged MBR treating municipal wastewater: case MBR, HPLC-SEC-fluorescence, fouling, SMP, protein-like substances

Ré sumé

Le colmatage des membranes reste la principale limitation pour le dé veloppement du bioré acteur à membrane (BAM). Dans cette thè se, l'objectif principal se concentre sur les effets des micropolluants pharmaceutiques qui se retrouvent dans les eaux usé es domestiques sur le colmatage de la membrane du BAM. Carbamazé pine (CBZ), un mé dicament antié pileptique, a é té choisi. Les effets de la CBZ sur le colmatage du BAM ont é té é tudié s de deux maniè res: un pic de pollution pour é tudier les effets des pics de CBZ à court terme sur le pouvoir colmatant et une pollution continue pour examiner les effets de CBZ à long terme sur le colmatage du BAM. Les ré sultats ont montré que, pendant 3 heures contact avec CBZ de 100 µg L -1 , le pouvoir colmatant des boues activé es a augmenté en raison de l'augmentation des proté ines de 100-1000 kDa dans le surnageant, ce qui pourrait ê tre complè tement retenu par la membrane du BAM et les membranes utilisé es dans les essais de filtrabilité .

L'augmentation des proté ines de 100-1000 kDa dans le surnageant peut probablement ê tre causé par la dé composition bacté rienne face aux mé dicaments. L'effet ré duit a é té observé e pour les boues provenant du BAM fonctionné sous la charge organique plus é levé e. Pendant le contact continue, la vitesse du colmatage plus é levé e a é té observé aprè s l'addition en continu de CBZ dans le BAM (90 µg L -1 dans l'alimentation), qui pourrait ê tre lié à l'augmentation importante des proté ines de 10-100 kDa dans le surnageant. Des proté ines de 10-100 kDa s'accumulent dans le dé pôt de la membrane du BAM, modifient de la structure de dé pôt et changent les caracté ristiques de ré tention de BAM. L'augmentation des proté ines de 10-100 kDa a probablement é té causé e par la ré action de dé fense des bacté ries face en permanence des mé dicaments. Lé gè re inhibition de l'activité microbienne a é té trouvé e plusieurs jours aprè s l'addition de la CBZ dans le BAM, puis il a é té stabilisé à un certain niveau en raison de l'acclimatation des boues au stress pharmaceutique. Similaire, l'augmentation significative de la concentration en proté ine a é té observé e au dé but plusieurs jours aprè s l'addition de la CBZ dans le BAM, puis retourne à son niveau initial. Aucun changement significatif de la taille des flocs des boues et de la concentration en polysaccharides dans le surnageant n'a é té constaté au cours de la pé riode de contact continu à long terme. Cette é tude pourrait contribuer à amé liorer la compré hension des interactions complexes entre les micropolluants pharmaceutiques, boues activé es et le colmatage du BAM.

Background

Nowadays, water scarcity is still a big challenge facing humanity in many places around the world. To solve this problem, municipal wastewater has been considered to be an alternative water source for various applications after proper treatment. Among all kinds of treatment processes, membrane bioreactor (MBR) has been increasingly used in municipal wastewater reuse and reclamation applications due to its high effluent quality. MBR process is a hybrid process that couples the activated sludge process with membrane separation, i.e. microfiltration (MF) or ultrafiltration (UF). The coupling of membrane separation with the biological process enables the MBR to possess many advantages, which may include high effluent quality (especially the high removal of pathogenic microorganisms), small footprint, less sludge production, and flexible process design (independent control of solids and hydraulic retention time). However, the membrane fouling remains the major limitation for the development of MBR.

Membrane fouling in MBRs

Membrane fouling means the decline of permeability during MBR operation. For MBRs operated at constant flux, it also means the increase of transmembrane pressure (TMP) during MBR operation. Membrane fouling was caused by the biocake, which is formed on membrane surface due to the convective flow of activated sludge and microbial growth, by the pore blocking due to small particles, colloids and solute adsorption [1][2][3]. Normally, three steps (see Fig. 1) of fouling are proposed for MBRs when they are operated at constant flux operation: conditioning fouling (step 1) caused by initial pore blocking and adsorption of solutes, steady fouling (step 2) occurring due to biofilm formation and further pore blocking, and the TMP jump (step 3) occurring due to the following different mechanisms or a combination of them: (i) due to a progressive closure of pores, local flux starts to exceed the critical value and particles begin to deposit at an ever increasing rate; (ii) coagulation occurs in the first layers at a critical pressure; (iii) percolating colloids inside the cake reduce the size of the cake voids until connections are lost [2]. Fig. 1. Schematic illustration of the long-term TMP profile (adopted from Meng et al. [4] )

The supernatant of activated sludge, which contains colloids and solutes, was shown to play a more important role in membrane fouling of MBR than the biological flocs in many studies [5][6]. Due to the gas-liquid two-phase cross flow generated by aeration in submerged MBRs or liquid cross flow generated by a recirculation pump in side-stream MBRs, the low flux operation and the relatively easy removal of biomass deposit by the shear force, the biomass deposit is of minor importance on MBR fouling in long-term operation. Thus, soluble microbial products (SMPs), the major organic constituents of sludge supernatants, have been acknowledged as the major foulants that lead to membrane fouling in MBRs [7][8]. SMPs are identified as soluble extracellular polymeric substances (EPS) and are composed of proteins, polysaccharides, humic substances etc. [9][10][11]. Besides, SMPs could be further divided into two categories: utilization-associated products (UAPs), which are produced during substrate metabolism, and biomass-associated products (BAPs), which are formed from decay of biomass. Factors affecting the SMP production may include operating conditions such as organic loading rate [12][13], sludge retention time (SRT) [14][15], hydraulic conditions like aeration and mechanical stirring [16][17], and the presence of environmental stress (for bacteria) in the wastewater such as heavy metals [18][19][20], osmotic pressure (salts) [21][22], and industrial compounds like pharmaceuticals [23][24]. However, simply correlating the MBR fouling with protein-like or polysaccharide-like SMP concentrations in the supernatant or with their retention in MBRs could not explain the contribution of each component in membrane fouling, since some of the SMPs may be retained by the cake layer and may not be attached on membrane surface or in membrane pores [25]. Thus, in MBRs, fouling is also affected by the molecular weight (MW) distribution of SMPs [26][27]. Besides, the physiochemical properties like relative hydrophobicity of SMP components may also significantly affect the MBR fouling [28]. Proteins were reported to dominate in foulants desorbed from the fouled membranes of MBRs due to its relatively high hydrophobicity [29].

Fouling propensity of biological fluids is currently estimated by a calculation of fouling resistance, permeability or fouling rate through an analysis of data (permeate flux, TMP) that are obtained (i) during operation of the MBR [30][31]; (ii) from batch stirred dead-end filtration cells [32][33][34]; (iii) from batch tangential modules (ex situ) operated under standardized operating conditions [35][36]; and (iv) from some submerged modules placed directly inside the MBR (in-situ) and using aeration to generate cross-flow conditions [37][38], which could avoid the problems like the change of sludge quality during storage period. In the case of a fouling occurring as a biocake deposit, some recent works have also proposed in-situ fouling characterization methods thanks to some filtration apparatus equipped with acoustic or optical sensors [39][40][41][42]. Still, there is a great interest in the industry to find some simple and cheap methods that could allow estimating the fouling propensity of biological fluids such as MBR supernatants. Thus, many analytical tools, including three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy and high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) equipped with different detectors (UV, refractive index, fluorescence and organic carbon), have been proposed to characterize SMPs in supernatants [43][44]. Among these techniques, HPLC-SECfluorescence, which integrates the advantages of EEM fluorescence spectroscopy with HPLC-SEC analysis, could achieve an accurate detection of protein-like substances in different molecular weight ranges, especially for the compounds in biopolymer range (MW bigger than 10 kDa) [44][45][46], which could help to improve understanding the effects of protein-like substances and their molecular weight distribution on membrane fouling.

Problematics of pharmaceutical micropollutants in MBRs

Removal of pharmaceutical micropollutants in MBRs

In the last decade, great attention has been paid to some emerging trace organic contaminants, also called micropollutants, such as endocrine-disrupting compounds, pharmaceuticals, personal care products etc., found in aquatic environment [47][48]. Among these micropollutants, pharmaceutical micropollutants got concerns since the late 1990s [49][50][51], as pharmaceuticals are designed to have some biological effects on living organisms and may have potential adverse effects on human health or aquatic organisms [52]. Also, concerns have been raised regarding that continuous discharge of antibiotics to aquatic environment may facilitate the development or proliferation of resistant strains of bacteria [53]. Moreover, chronic toxicity effects have been reported for aquatic organisms exposed to human pharmaceuticals at trace concentration [54][55][56]. So far, there is evidence of occurrence of some 160 different drugs in the effluents of wastewater treatment plants (WWTPs), and also of plants for the production of potable water from surface waters and groundwaters [57][58].

Pharmaceuticals found in water resources are classified into different therapeutic categories: analgesics and anti-inflammatory drugs, lipid regulators, antiepileptic drugs, beta-blockers, antibiotics, cytostatic drugs etc. Many pharmaceutical micropollutants, such as analgesics like ibuprofen, diclofenac, naproxen and ketoprofen, lipid regulators like bezafibrate and gemfibrozil, the antiepileptic drug carbamazepine, and antibiotics such as trimethoprim and sulfamethoxazole, have been frequently detected at concentration up to or higher than 1.0 µg/L in effluents of WWTPs around the world [59]. Pharmaceutical residues are discharged to sewers through a mixture of urine and faeces as the initial molecules or their metabolites after absorption by human bodies. Thus, pharmaceuticals mainly enter WWTPs via sewer networks after human release. Due to the incomplete removal or degradation of pharmaceutical micropollutants in conventional WWTPs, effluents discharged from WWTPs become a major source of pharmaceutical micropollutants entering into the water surface or underground resources [60][61].

Extensive studies have been carried out to investigate the removal efficiency and removal mechanisms of pharmaceutical micropollutants in MBR process, due to its potential advantages (such as higher SRT, accumulation of enzymatic biopolymers in the biological reactor) on removal of persistent organic pollutants as compared to the activated sludge process employed in conventional WWTPs. MBR was found to significantly enhance the removal of some partial degradable pharmaceutical micropollutants such as diclofenac, mefenamic acid, ketoprofen, clofibric acid, gemfibrozil etc. as compared to conventional activated sludge process (CAS) [62][63][64]. According to their removal efficiency in MBRs reported in the literature, pharmaceutical micropollutants can be classified into different groups of molecules [59], depending on their propensity to be removed in MBRs: (i) easily, with an average removal efficiency higher than 90 %, such as ibuprofen and bezafibrate; (ii) moderately, with an average removal efficiency of 50%-80%, such as naproxen, ketoprofen, gemfibrozil, trimethoprim, and sulfamethoxazole; (iii) poorly, with an average removal efficiency lower than 40%, such as diclofenac and carbamazepine. Thus, most pharmaceutical micropollutants are moderately removed in MBRs. In particular, carbamazepine is removed with an average removal efficiency of only 7% in MBRs [59].

The mechanisms involved for the removal of pharmaceutical micropollutant in MBRs may include physical retention by the membrane, sorption onto the activated sludge and biotransformation. Since the molecular size of most pharmaceuticals (molecular weight between 100 and 1000 g mol -1 ), is at least 100 times smaller than the pore size of the membranes that are used in MBRs, no direct physical retention of the compounds by a MBR membrane itself can be expected [65]. However, Urase et al. [66] suggested that the deposit formed on the membrane surface might act as an additional barrier for the micropollutants and contribute to an enhanced removal of pharmaceutical micropollutants in an MBR. Further studies need to be performed to investigate the role of deposits or preferentially of biofilm on the removal of pharmaceutical micropollutants. Since the value of sorption coefficient Kd (L kg -1 ), defined as the partition of a molecule between the activated sludge and the water phase, of many pharmaceutical micropollutants, is very low (less than 500 L kg -1 ) [67], as compared with that of some other micropollutants such as the fragrance tonalide (18000 L kg -1 ) and the steroid hormone 17β-estradiol (35000 L kg -1 ) [68], the removal of pharmaceutical micropollutants via sorption to the activated sludge in an MBR is often of minor importance [69]. Some studies identified the Kd value of 500 L kg -1 to be the critical value, below which sorption is not relevant for the removal of organic micropollutants in biological wastewater treatment processes including MBRs [67,70]. In particular, carbamazepine has a Kd value of only 1.2 L kg -1 [70], indicating that it is not significantly sorbed into the activated sludge However, attention should be paid to some antibiotics like ciprofloxacin and norfloxacin. As the major mechanism relevant for their removal is sorption into the activated sludge, due to their high Kd value (higher than 15000 L kg -1 ) [71]. Taking into account the difficulty in the precise quantification of pharmaceuticals in the sludge phase and also the different ways used to determine the Kd value (for example, sludge samples could be sampled from batch experiments or sampled from full-scale WWTPs), the role of sorption on removal of pharmaceutical micropollutants in MBR may be underestimated. Thus, in the literature it is often considered that the main mechanism involved in the removal of most pharmaceutical micropollutants in MBR is biodegradation [72].

All above discussion concerned MBRs that were used for treating domestic wastewaters, which will be the focus of our study. For other kinds of waste waters, for example, effluents of hospitals or drug production facilities, since they may contain very high concentration of some pharmaceuticals, especially the antibiotics [73][74][75], a specific adapted biomass may be required for the treatment of these very specific effluents.

The effects of pharmaceutical micropollutants on MBR process

Only a few studies examined the effects of the presence of pharmaceutical micropollutants on the MBR process, the majority of which focused on the effects on sludge microbial activity or the evolution of microbial community in the sludge. In batch experiments that used the activated sludge sampled from an MBR, Aubenneau et al. [76] examined the effect of trace concentration of carbamazepine on the mixed microbial communities and observed an inhibition of the exogenous respiration rate, a higher endogenous respiration rate, and a smaller floc size in presence of 1 µg L -1 carbamazepine in the sludge. Delgado et al. [77] reported that the continuous introduction of 5 µg L -1 of the cytostatic drug cyclophosphamide and its principal metabolites (CPs) in a MBR caused the inhibition of the exogenous respiration rate and increased the mean floc size of the activated sludge. Through the denaturing gradient gel electrophoresis (DGGE) analyses of the sludge community, Lay et al. [23] observed shifts in microbial community and a reduced microbial diversity in the activated sludge several days after adding a mixture of carbamazepine, diclofenac, ibuprofen and naproxen in the feed tank (20-25 µg L -1 of each pharmaceutical in the feed) of an Osmotic Membrane Bioreactor (OMBR). Through the terminal restriction fragment length polymorphism (T-RFLP) analyses of the bacterial 16S rRNA genes, Kraigher et al. [78] observed shifts in the bacterial community structure in the activated sludge of a bioreactor continuously fed with a synthetic wastewater containing pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid) at a concentration of 50 μgL -1 , as compared to a control reactor, which was operated without addition of pharmaceuticals.

It is known that a chemical stress can induce the activated sludge to produce extracellular polymeric substances (EPSs) and soluble microbial products (SMPs), which are often identified as the main responsible for MBR fouling. The release of EPSs or SMPs may result from (i) the defensive response of bacteria to form a protective layer or (ii) decay (lysis) of bacteria and release of EPSs or SMPs inside the bacterial cells [79][80]. Still, very few studies reported the effects of pharmaceutical micropollutants on SMP production. In the literature, the effects of chemical stress on SMP production were examined in two ways:

(i) Long-term continuous contact. Lesage et al. [81] reported that after continuous addition of a toxic molecule, 2,4 dimethylphenol (DMP) in an MBR feed (90 mg/L in the feed), a significant increase in both protein and humic concentrations was observed. In contrast, the authors found no significant effect on polysaccharide concentration. Zhou et al. [20] reported that an MBR fed with a municipal wastewater containing 0.4 mg L -1 Cr (VI) had a higher concentration of SMPs in the supernatant, especially in proteins compared with the MBR fed with the same municipal wastewater without addition of Cr (VI). Lay et al. [23] observed a significant increase in the protein and polysaccharide proportions for both soluble and bound EPS in the activated sludge after adding carbamazepine, diclofenac, ibuprofen and naproxen in the feed tank (20-25 µg L -1 of each pharmaceutical in the feed) of an Osmotic Membrane Bioreactor (OMBR).

(ii) Short-term discontinuous contact. Wang et al. [22] reported that a high osmotic pressure (5% NaCl) induces the production of SMPs with a relative strong hydrophilicity and protein-like characteristics in batch experiments. Sheng et al. [19] reported that in the presence of toxic chemicals, such as 30 mg l -1 Cu (II), 40 mg l -1 Cr (VI), 5 mg l -1 Cd (II) for a suspension of the bacteria strains Rhodopseudomonas acidophila, the EPS content increased by 5.5, 2.5, and 4.0 times, respectively. Moreover, the authors reported that under these toxic conditions, the increase in the protein content far exceeded the one of the other EPS constituents.

The objectives of this thesis

From the literature, most studies focused on the removal of pharmaceutical micropollutants in MBRs, and only a few studies investigated the effects of pharmaceutical micropollutants on MBR process, most of which dealt with the effects of pharmaceutical micropollutants on microbial activities or microbial community structure of activated sludge in MBRs. Taking into account the effects of pharmaceutical micropollutants on the SMP production in the MBR supernatant and the role of SMPs in the fouling, there is a lack of knowledge on the effects of pharmaceutical micropollutants on MBR fouling, which remains a key issue.

The main objective in this thesis was to examine the effects of pharmaceutical micropollutants, which are frequently found in domestic wastewaters, on the MBR fouling. Carbamazepine (CBZ), an anti-epileptic drug, was selected as a representative of pharmaceutical micropollutants in this study. It was chosen because it has been frequently detected in influent as well as effluents of domestic WWTPs [82] and it is almost not biodegraded in biological wastewater treatment processes including MBRs, which makes it become a possible anthropogenic marker in the aquatic environment [83]. In addition, CBZ was found to have some inhibition effects on sludge microbial activity [76,84].

After a bibliographic review on the removal of pharmaceutical micropollutants in MBRs, including the effects of pharmaceutical micropollutants on MBR process (see paper 1), the thesis proposes to investigate the effects of CBZ on MBR fouling in two ways:

(i) a discontinuous contact (see paper 2), where CBZ was added in a batch reactor that uses the sludge from a lab-scale MBR to study the short term peak effects of CBZ on the fouling propensity of both activated sludge and its supernatant;

(ii) a continuous contact (see paper 3), where CBZ was added continuously in a lab scale MBR via the feed to examine the long term effects of CBZ on MBR fouling.

The objectives are to be able to relate the short-term as well as long-term effects of CBZ on sludge fouling propensity or MBR fouling with the change in the characteristics of the sludge and supernatant after adding CBZ in batch reactors or MBR to understand the mechanisms of interactions among the CBZ, activated sludge and fouling at different durations of interaction.

Fouling propensity of the sludge and supernatants (sampled both in the MBR and in the batch reactors) will be characterized using some standardized filterability tests performed with a stirred dead-end filtration cell. Fouling will also be considered at the scale of the MBR for the mixture of flocs and supernatant during MBR operation and characterized by the transmembrane pressure (TMP) time-variation. In order to explain the effects of CBZ on the fouling comportments, it is necessary to also obtain for the two ways (continuous/discontinuous) some information:

(i) on the biological activity: Respirometric experiments were performed to examine the effects of CBZ on sludge microbial activities during the short term period as well as long term period of contact with CBZ. Finally, the possibility of using HPLC-SEC-fluorescence as a tool for rapid estimation of the modification of fouling in MBRs, when some variations in fouling conditions happen, was proposed (see draft paper 4). In real full scale applications, the concentrations of some chemicals, including pharmaceutical micropollutants, in raw waters and the operating conditions such as organic loading rates may vary a lot, which may affect the characteristics of MBR sludge and supernatant. In both situations, the fouling in MBRs may be unsteady.

Thus, there is a great interest to find some simple and cheap methods that could allow estimating the fouling propensity of MBR supernatants under unsteady conditions. The organization of this thesis is shown in Fig. 2.

Materials and methods

This part gives the main details on materials and methods used in the following papers.

Membrane bioreactor set-up

A lab-scale submerged MBR set-up consisting of an anoxic reactor (5.4 L) and an aerobic reactor (12.6 L) was used in this study (see Fig. 3). A flat sheet microfiltration membrane

(Lp 0 = 1110 L h -1 m -2
bar -1 at 20 °C, Kubota, Japan) was submerged inside the aerobic reactor.

It has an effective filtration area of 0.1 m 2 , with an average pore size of 0.2 µm. Two aeration systems were used in the aerobic reactor. An aeration system with big air bubble was installed below the membrane to reduce fouling and it was operated continuously at flow rate of 1 The MBR was operated in two steps (see table 1). In the first step, the MBR was operated at a sludge retention time (SRT) of 40 days, with a sludge concentration in the aerobic reactor at 4-6 g/L. In the second step, the MBR was operated at a 30 days SRT, with a sludge concentration in aerobic reactor at 6-8 g/L. The MBR reached high removal of COD, more than 95%, during both two steps. Whereas, a limited removal of TN, of about 40%, was observed during step 1 when the MBR was operated at an organic loading rate (C m ) of about 0.1 kg COD/kg MLSS/d, due to the insufficiency of the organic source for heterotrophic denitrification [85].

Physico-chemical properties of selected pharmaceutical micropollutant

Batch reactors

Batch reactors were used to characterize the short-term effects of CBZ on MBR sludge.

Figure 4 shows the protocol used for batch experiments. Firstly, quantities of the concentrated mother solution sufficient to yield 100 µg L -1 final concentrations of CBZ were added in one of the 2 batch reactors (reactor-with CBZ). As comparison, the same quantity of pure ethanol was added in the other batch reactor (control-reactor). Then the ethanol in the two batch reactors was blown off using a gentle stream of air, leaving only the CBZ in the reactors to rule out the impact of ethanol [87]. Finally, a same volume of sludge samples taken from the MBR aerobic tank was added in the two batch reactors and the mixed liquids were stirred using the aeration in the bottom of each reactor for 3 hours to dissolve CBZ and to make the sludge contact with CBZ in aerobic condition. Then the sludges in both reactors were sampled and the two corresponding supernatants were prepared by centrifugation of the sludge samples at 4000 g during 10 minutes at room temperature at the same time. 3 hours of contact time was chosen since it takes about 3 hours before that all the readily degradable substances are consumed by the activated sludge [77]. Besides, no significant difference was found concerning the effects of CBZ on characteristics of sludge and supernatant between contact time of 3 hours and 5 hours. Too long time, for example 24 hours, for sludge in batch reactors without exogenous substrate would cause the decay of bacteria and impact the quality of activated sludge. Also, no significant difference was found concerning the effects of CBZ on characteristics of sludge and supernatant when the peak concentration of CBZ was modified to 500 µg L -1 , as compared to the effects of CBZ at concentration of 100 µg L -1 . Besides, no significant difference was observed between the sludge obtained from control-reactor and blank reactor where no ethanol was added (see Fig. 4) in terms of biomass and supernatant quality, which confirms that the ethanol did not affect the quality of sludge after being evaporated by the gentle stream of air.

Filterability tests

Fouling propensity of sludge and supernatant sampled from batch reactors (reactor-with CBZ and control-reactor) was characterized by filterability test performed with a stirred dead-end filtration cell (Amicon 8050, Millipore). In the study of long-term CBZ effect, ultrafiltration membranes (Microdyn-Nadir GmbH, Germany), made of polyethersulfone (PES), with a molecular weight cutoff (MWCO) 150 kDa (Lp 0 = 400-600 L h -1 m -2 bar -1 at 20 °C) were used for the filterability tests of both sludge and supernatant. For the 150 kDa PES membranes, the same protocol for the filterability test was used except that prior to filtration test, they were rinsed by filtration of ultra pure water at 0.7 bar during 15 min and all filtration tests were carried out only at constant TMP of 0.5 bar.

Membrane fouling resistance was determined according to Eq. ( 1).

() mf TMP J RR    (1)
In this equation, J is the permeate flux (m 3 s -1 m -2 ) at 20 °C, with R m the membrane resistance (m -1 ), R f the fouling resistance (m -1 ), TMP the transmembrane pressure (Pa) and μ is the dynamic viscosity at 20 °C (Pa s).

In the dead-end filtration cell, C can be determined by cake deposit model as expressed in Eq. ( 2)

f V RC A  (2)
In this equationis the specific cake resistance (m kg -1 ), C is the deposited mass per filtered permeate volume (kg m -3 ), V is the cumulated permeate volume (m 3 ) and A is the effective membrane area for filtration (m 2 ). For supernatant filtration, C could be calculated by the retained DOC (kg m -3 ). For sludge filtration, C is assumed to be MLSS value of sludge.

Characterization of sludge microbial activity

Sludge microbial activity was characterized by the respiration rate, i.e. oxygen uptake rate (OUR). It was measured in respirometric batch experiments as defined by Sperandio and Paul [88]. During study on short-term effect, sludge samples were collected from the aerobic tank of the MBR. Firstly, the collected sludge was aerated for at least 3 h to reach the endogenous condition. Because CBZ was prepared in pure ethanol, pure ethanol was used as exogenous substrate in the respirometric experiments. Then pure ethanol was added to respirometric reactor to test the biological activity of sludge before adding CBZ. Then quantities of the concentrated mother solution sufficient to yield 100 µg L -1 final concentrations of CBZ were added in the respirometric reactor to test the inhibition effect of CBZ on the activated sludge.

During the long-term effect study, the sludge samples were collected from the aerobic tank of the MBR before and after addition of CBZ in the MBR. Then, the collected sludge was aerated for at least 3 h to reach the endogenous condition. Then, sodium acetate (NaAC), a readily biodegradable compound, was used as exogenous substrate to measure the maximum respiration rate. Then the exogenous respiration rate (see Fig. 5) was calculated as the difference between the maximum respiration rate and the endogenous respiration rate (measured in the endogenous condition). The specific exogenous respiration rate is defined as the exogenous respiration rate divided by the concentration of mixed liquor suspended solids (MLSS).

Analytical methods

The concentration of mixed liquor suspended solids (MLSS) was measured according to the standard method AFNOR NFT 90-105 Polysaccharide concentration in supernatant, expressed in mg equivalent of glucose per litre, was measured by the Anthrone method [90]. Carbamazepine concentration was analyzed using LC-MS/MS method by the Departmental Water Laboratory in Toulouse, France.

High performance liquid chromatography-size exclusion chromatography (HPLC-SEC)

analysis was performed by an Akta Purifer (GE Healthcare, USA), equipped with a column Protein Kw804 (Shodex, Japan), a silica-based column with an exclusion limit of 1000 kDa, and a fluorescence detector (Varian, USA). The eluent solution for the mobile phase was prepared with 25 mM Na 2 SO 4 and a phosphate buffer (2.4 mM NaH 2 PO 4 and 1.6 mM Na 2 HPO 4 at pH 6.8). The flow rate of eluent passing through the column was 1.0 mL/min.

Excitation/emission wavelengths were set at 280/350 nm, which were confirmed as the required values by EEM fluorescence results [44,46], to detect protein-like substances. The column was calibrated using standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) from GE Healthcare. Prior to analysis of HPLC-SEC-fluorescence, each sample was prefiltered on a 0.45 μm membrane. 6 shows result of the filterability tests of sludges, sampled from batch reactors using sludge from the MBR operated under organic loading rate of 0.1 kg COD/kg MLSS/d, at 1.0 bar. The result showed that with addition of CBZ in the sludge the fouling propensity was significantly higher (32%). Reduced CBZ effect (12%) on fouling resistance was observed for sludge from MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d (see Fig. 7). Concerning the long term CBZ effect, sludge and supernatant filterability tests using sludge and supernatant sampled from MBR before and after addition of CBZ were performed at 0.5 bar, detailed in paper 3. Fig. 8 shows that the continuous addition of CBZ in MBR increased the fouling propensity of sludge. However, result from Fig. 9 shows that the addition of CBZ in MBR decreased the fouling propensity of supernatant. -Before the addition of CBZ in the MBR quite steady fouling conditions were reached, where TMP increases very slightly.

-1 day after addition of CBZ in MBR, a significant increase of TMP was observed.

Thanks to the addition of CBZ the TMP jump was very rapidly created and it clearly appears as an answer of the bioseparation process to the effect of a stress on the biomass.

-Between 1 day and 8 days after addition of CBZ, TMP increased gradually while still at a higher rate as compared to that during the steady fouling conditions before addition of CBZ.

-Starting from 8 days after addition of CBZ, a continuous TMP jump started to occur, where TMP increased continuously at a significantly higher rate. In conclusion, the contact of activated sludge with CBZ increased the sludge fouling propensity and MBR fouling. Three assumptions can be proposed to explain the increased sludge fouling propensity and MBR fouling after addition of CBZ: i) a change in microbial activity linked to a stress on the microorganisms ii) a change in biomass physico-chemical properties, like mean floc size distribution, or iii) a change in the supernatant quality. These three assumptions will be discussed in the following paragraphs that aim to evaluate the effects of CBZ on the supernatant and biomass properties.

Effects of CBZ on biomass characteristics

Effect of CBZ on sludge microbial activity

Effect of CBZ on sludge microbial activity was characterized by respirometric experiments.

During the short-term contact in batch reactor, no inhibition of sludge microbial activity with presence of 100 µg L -1 CBZ in the sludge was observed (see paper 2). Whereas, during the long term contact (see paper 3), the continuous addition of CBZ in the MBR via feed (90 µg L -1 in the feed) caused about 20% inhibition of the sludge microbial activity, expressed as specific exogenous respiration rate in the MBR, at the beginning several days, as shown in Fig. 11. No further inhibition was observed between 4 days and 11 days after addition of CBZ in the MBR, which indicates that some extent of adaption or acclimation of the activated sludge was reached from 4 days after addition of CBZ in the MBR. However, during the longterm contact, after addition of CBZ in MBR, no significant change of the COD and TN removal was observed, as compared to that before addition of CBZ. This suggests that the inhibition is also not significant in the long-term contact condition. In conclusion, the effect of CBZ on sludge microbial activity is not significant in both shortterm and long-term situations and the modification of sludge fouling propensity and MBR fouling could not be attributed to the modification in sludge microbial activity.

Effect of CBZ on sludge floc size distribution

No significant effects of CBZ on sludge floc size distribution were observed during shortterm peak contact as well as during the long-term continuous contact (see paper 2 and paper

3). This may indicate that no significant bacterial decay (lysis) occurred when facing CBZ stress in both situations. Thus, the modification of sludge fouling propensity and MBR fouling could not be attributed to the modification in sludge floc size distribution.

Effects of CBZ on supernatant characteristics

Effect of CBZ on SMP composition in supernatant

The short term effects of CBZ on supernatant composition were examined on supernatants sampled from the batch reactors (see paper 2). Fig. 12 shows the result of protein and polysaccharide concentrations in the supernatants from batch reactors using the sludges from the MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d. Slight increase in protein concentration (about 20%) was observed in supernatant from the reactor-with CBZ, as compared with that from the control-reactor. In contrast, no significant difference in polysaccharide concentration was found in supernatant from both reactors. The long term effects of CBZ on supernatant composition were examined on supernatants sampled from MBR before and after addition of CBZ in MBR (see paper 3). Fig. 13 shows that a significant increase in the protein concentration in supernatant was observed just 1 day after addition of CBZ, then the protein concentration kept almost constant for several days, and then reduced effect on protein production was observed after about 7 days after the addition of CBZ in MBR. However, no significant increase in retention of protein in MBR was found, which indicates that the protein with a higher concentration were mainly relatively small molecules that could pass through the MBR membrane and biocake. In contrast, no significant change was observed for the polysaccharide concentration in supernatant after addition of CBZ in MBR. In conclusion, consistent results were found concerning the effects of CBZ on SMP composition in supernatants in both short-term and long-term contact situations, i.e. an increase of protein SMPs and no significant change of polysaccharide SMPs after addition of CBZ in the sludge. The result found in this study indicates that protein-like SMPs seemed to play a more important role for microorganisms when facing a pharmaceutical stress. Thus, the increase in sludge fouling propensity and MBR fouling with addition of CBZ could be attributed to the modification of protein-like SMPs in supernatant, since no significant effect was observed in floc size distribution and microbial activity under short-term as well as longterm continuous contact conditions.

Effect of CBZ on SMP molecular size distribution in supernatant

To further investigate the role of protein-like SMPs in fouling, the molecular size distribution of protein-like SMPs was characterized by HPLC-SEC-fluorescence with excitation/emission wavelengths set at 280/350 nm. In case of the short-term contact (see paper 2), HPLC-SEC analysis of supernatants prepared from the batch reactors using sludges that were taken from the MBR operated under low organic loading rate (0.1 kg COD/kg MLSS/d) was shown in Fig. 14. The molecular weight of protein-like substances in MBR supernatants showed bimodal distribution. Indeed, two peaks were observed in the HPLC-SEC results which represent two groups of protein-like molecules, i.e. molecular size of 100-1000 kDa and 10-100 kDa, respectively. A significantly higher peak of protein-like substances corresponding to100-1000 kDa molecules in the supernatant prepared from reactor-with CBZ was observed as compared to that in the supernatant prepared from the control-reactor. In contrast, no significant difference of detected peak heights of protein-like substances with a molecular size of 10-100 kDa between the two supernatants that were prepared from the reactor-with CBZ and the control-reactor was observed. Taking into account that the 100-1000 kDa protein-like SMPs were completely retained by 0.2 µm PS membranes during the sludge filtration tests (see paper 2), the increased sludge fouling propensity with addition of CBZ in short-term contact condition (see Fig. 6) could be attributed to the increase of 100-1000 kDa protein-like SMPs in supernatant. To investigate the impact of organic loading rate on CBZ effects, supernatants coming from batch reactors using sludges that were taken from the MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d were analyzed by HPLC-SEC, as shown in Fig. 15.

Comparing with the HPLC-SEC result of supernatants from batch reactors that using sludges that were taken from MBR operated under low organic loading rate of 0.1 kg COD/kg MLSS/d (see Fig. 14), much less significant effect of CBZ on 100-1000 kDa protein-like SMPs was observed, which could explain the reduced effect of CBZ on fouling propensity of sludge that was from the MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d (see Fig. 7). Concerning the long-term CBZ effect (see paper 3), MBR supernatants before and after adding CBZ in the MBR were analyzed by HPLC-SEC, as shown in Fig. 16. The result from Fig. 16 showed that the presence of 90 µg L -1 CBZ in MBR during a long-term period could cause two measurable effects on protein-like SMPs:

-a decrease of the concentration of 100-1000 kDa protein-like SMPs. More specifically, 1 day after addition of CBZ in the MBR, a significant decrease (about 16%) of 100-1000 kDa protein-like substances, was observed in the MBR supernatant. Whereas, only a slight decrease of 100-1000 kDa protein-like SMP was observed between 1 day and 7 days after addition of CBZ in MBR.

-a more significant increase of 10-100 kDa protein concentration. More specifically, 1 day after addition of CBZ in the MBR, a quite significant increase of the 10-100 kDa protein-like substances, about 23%, was observed in the MBR supernatant.

Whereas, only a slight increase of 10-100 kDa protein-like substances was observed in MBR supernatant between 1 day and 7 days after addition of CBZ. To investigate the role of different molecular size protein-like SMPs on the sludge and supernatant fouling propensity, the retention of protein-like SMPs in the MBR supernatant during sludge and supernatant filtration tests was analyzed by HPLC-SEC, as shown in Fig. 17 and Fig. 18. The results showed that the 100-1000 kDa protein-like SMPs could be almost completely retained by 150 kDa PES membranes during both sludge and supernatant filtration tests. However, concerning the retention of 10-100 kDa protein-like SMPs, different retention behavior was observed. No retention of 10-100 kDa protein-like SMPs was found during the supernatant filtration test as shown in Fig. 17, whereas partial retention of 10-100 kDa protein-like SMPs was observed during the sludge filtration test, which indicates that the fouling layer formed during the sludge filtration is more compact than that formed during the supernatant filtration. The results also suggests that the supernatant fouling propensity mainly depends on the 100-1000 kDa SMPs, whereas the both 10-100 kDa SMPs and 100-1000 kDa SMPs could affect the sludge fouling propensity.

Thus, the increase in sludge fouling propensity during long-term contact (see Fig. 8) could be attributed to the significant increase in 10-100 kDa protein-like SMPs in supernatant after addition of CBZ (see Fig. 16). Whereas, the decrease in supernatant fouling propensity (Fig. 9) could be attributed to the decrease in 100-1000 kDa protein-like after addition of CBZ in MBR (see Fig. 16). This further demonstrated that the 10-100 kDa protein-like SMPs in supernatant also played an important role in sludge fouling propensity. The difference between the effect of CBZ on sludge filterability and supernatant filterability could be attributed to the different deposits formed on the membrane surface during the filtration tests, since the same membranes were used for the sludge and supernatant filterability tests. Thus, the increased sludge fouling propensity could be attributed to the significant increase in 10-100 kDa protein-like SMPs in supernatant that may reduce the voids in the biocake when passing through the biocake and modify the structure of biocake formed on the membrane surface. In conclusion, the effect of CBZ on molecular weight distribution of protein-like SMPs in supernatant depends on the contact time. During short-term contact, the CBZ induced augmentation of 100-1000 kDa protein-like SMPs. In contrast, during long-term contact, the CBZ induced augmentation of 10-100 kDa protein-like SMPs and a decrease of 100-1000 kDa protein-like SMPs. Both 10-100 kDa and 100-1000 kDa protein-like SMPs in supernatant contribute to the increase in sludge fouling propensity. The 100-1000 kDa protein-like SMPs could be retained by the biocake on membrane surface and thus enhance formation of biofilm in the biocake, while the 10-100 kDa protein-like SMPs could reduce the voids in the biocake and modify the biocake structure.

Effect of CBZ on characteristics of MBR biocake

The characteristics of MBR biocake could be examined by its retention capacity for SMPs in supernatant, detailed in paper 3. Fig. 19 shows the HPLC-SEC analysis of supernatant and permeate obtained in the MBR on the day just before adding CBZ. The result showed that before adding CBZ, the MBR membrane retained almost all the 100-1000 kDa protein-like substances. In contrast, almost all the 10-100 kDa protein-like substances passed through the MBR membrane. Fig. 20 shows that the 10-100 kDa protein-like that were present in the MBR supernatant were partially retained by the MBR membrane 11 days after addition of CBZ. Taking into account the role of 10-100 kDa in modification of biocake structure, it could be assumed that the significant increase of 10-100 kDa protein-like SMPs in MBR supernatant after addition of CBZ induced an enhanced accumulation of these small biopolymers inside the biocake, which caused the loss of voids in the biocake, and finally led to a more "tight" retention characteristics of the MBR "membrane". Thus, the TMP plateau before addition of CBZ in MBR could be attributed to the retention of 100-1000 kDa SMPs in supernatant and thus to the formation of the biofilm and fouling layer on the membrane surface by protein bridges and bindings, since no retention of 10-100 kDa protein-like substances in supernatant was found. The TMP jump that appeared 1 day after addition of CBZ may be caused by the significant increase of 10-100 kDa protein-like SMPs in the supernatant at that time. The sudden significant increase of 10-100 kDa protein-like SMPs may cause a sudden loss of voids inside biocake, which increased the resistance of permeate passing through the MBR membrane. The gradual TMP increase, not the continuous TMP jump, between 1 day and 8 days after addition of CBZ could be attributed to the reduced effect of CBZ on protein-like SMP production (see Fig. 16). The higher increase rate in TMP during this period could be explained by the continued accumulation of 10-100 kDa proteinlike SMPs inside biocake. The TMP jump that appeared after the gradual increase of TMP may be due to that the accumulation of 10-100 kDa protein-like SMPs, i.e. loss of voids inside biocake, reached a critical point, leading to that local flux starts to exceed the critical value.

The result from Fig. 20 could support this assumption, where partial retention of 10-100 kDa protein-like substances was observed during this period.

As compressibility of fouling layer may also contribute to the MBR fouling, biocake compressibility test was performed by filtration of ultra-pure water through a formed biocake at different TMP in filtration cell, as shown in Fig. 21. Firstly, biocake was formed after filterability test of MBBR sludge (sampled at 11 days after addition of CBZ) at 0.5 bar. Then, Ultra-pure water was filtrated through the formed biocake at different TMP to examine the compressibility of biocake. Since no organic foulants were introduced into the formed biocake during the filtration of ultra-pure water, the increase in the fouling resistance could be attributed to the compressing effect of biocake. Moreover, slight higher fouling rate was observed at higher TMP, which further indicates that the fouling resistance increased more quickly with increasing TMP. Thus, the biocake formed by the MBR sludge is compressible and its compressibility may contribute to the continuous TMP jump, since the compressing effect could aggravate the MBR fouling when TMP increases significantly. In conclusion, consistent results concerning the CBZ effects on sludge fouling resistance were found in both short-term and long-term contact conditions, whereas caused by different fouling mechanisms due to the different effects of CBZ on molecular size distribution of protein-like SMPs between these two conditions. For filterability tests of sludges sampled from batch reactors during short term contact, the increase in fouling rate of sludge with addition of CBZ originated from the increased retention of 100-1000 kDa protein-like SMPs.

Whereas for MBR fouling during long-term CBZ contact, the increase in MBR fouling rate, i.e. the TMP increase rate, after addition of CBZ was due to the increase of 10-100 kDa protein-like SMPs in the MBR supernatant, which accumulated inside the biocake that was formed on the MBR membrane, thus modified the biocake structure, and finally together with the compressing effect of biocake resulted in the TMP jump. As the peak height is a simple and effective indicator to stand for the fluorescence intensity and to quantify the constituent content in the HPLC-SEC-fluorescence analysis [44] and almost complete retention of protein-like compounds with molecular size of 100-1000 kDa in supernatant is obtained by MBR membrane (see Fig. 19), the fluorescence peak height of 100-1000 kDa protein-like compounds could be considered as fouling indicator to estimate fouling in MBRs. To relate the peak height of protein-like substances in 100-1000 kDa range with fouling propensity of supernatant samples (sample 1, 2, 3 and 4), the peak heights of 100-1000 kDa protein-like compounds in supernatants and calculated C values of supernatants obtained in filterability tests using 0.01 μm PES membranes at three different TMP (0.5 bar, 0.8 bar, 1.0 bar) are plotted in Fig. 24. The result showed good linear relation between the peak height and C value at all three TMP for the selected samples, which further confirmed the feasibility of using HPLC-SEC-fluorescence as a tool to estimate fouling propensity of MBR supernatants. In this case, peak heights of protein-like substances in 100-1000 kDa range and calculatedC values in filterability tests of 4 supernatants sampled from MBR before and after addition of CBZ in MBR, using 150 kDa PES membranes at TMP 0.5 bar, were plotted in Fig. 25. The result also showed good linear relation between the peak height of 100-1000 kDa protein-like SMPs and C value. Remind that after addition of CBZ in MBR, a decrease of protein-like substances in 100-1000 kDa was observed (see Fig. 16) and a significant increase of TMP occurred in MBR (see Fig. 10). This means in case that fouling situations were modified by

5 HPLC-SEC-fluorescence for estimation of fouling in MBRs

Conclusions and perspectives

The presence of pharmaceutical micropollutants in domestic wastewater raised the questions like if the presence of pharmaceutical micropollutants may affect the MBR process, especially the fouling of MBR. This study investigated the effects of CBZ on sludge fouling propensity in batch experiments as well as on MBR fouling in a continuously operated lab-scale MBR.

The effects of CBZ on activated sludge of MBR depend on contact time (continued or discontinued mode) due to the different microbial reactions in these two modes. The short term contact of CBZ with sludge led to increase in 100-1000 kDa protein-like SMPs due to decay (lysis) of bacteria. In contrast, the continuous contact of CBZ with sludge in MBR induced to production of 10-100 kDa protein-like SMPs due to the defensive responses of bacteria facing CBZ stress. Table 3 summarized the effects of CBZ on characteristics of MBR sludge and supernatant in these two situations. Due to the different effects of CBZ on the characteristics of MBR sludge and supernatant in the two contact ways, the fouling mechanisms involved in these two situations are quite different. Both 100-1000 kDa and 10-100 kDa protein-like SMPs have potential to contribute to sludge fouling propensity or membrane fouling in MBR. The 100-1000 kDa SMPs could be completely retained by MF/UF membrane or MBR membrane during sludge filtration, leading to formation of biofilm in biocake, which contributes to higher sludge fouling propensity and steady increase of MBR fouling. The 10-100 kDa protein-like SMPs could be partially retained by MF/UF membrane or MBR membrane during sludge filtration, leading to the reduction of the voids of biocake and result in change of biocake structure, which could lead to higher sludge fouling propensity and higher fouling rate and TMP jump in MBR. This study paves ways to understand the complex interactions among pharmaceutical micropollutants, activated sludge and membrane fouling in different contact durations. The important role of the protein-like SMPs in these interactions has been demonstrated. The use of HPLC-SEC-fluorescence as a useful tool to study the phenomena involved in these interactions has also been demonstrated. However, many questions are remained unanswered.

More study need to be performed to distinguish the two stress effects involved: the decay of cells and the defensive responses by the viable bacteria. Also, studies concerning the effects of pharmaceutical micropollutants on the change of microbial community structures and the corresponding effects on biofouling potential should be investigated.

To improve the capacity of MBR on removal of pharmaceutical micropollutants, more studies should be carried out to identify the bacterial species that favor removal of CBZ as well as other pharmaceuticals in MBR and determine the optimal operation conditions that help to enrich these bacterial species. In addition, the mass transfer and biodegradation of pharmaceuticals in sludge phase and also in the deposits of the membrane surface need to be examined to better understand the removal mechanisms of pharmaceutical micropollutants in MBR.

More tools such as LC-OCD (liquid chromatography with organic carbon detector), CLSM (confocal laser scanning microscopy), in-situ fouling layer analysis like LSGI (laser sheet at grazing incidence) etc. need to be employed to further analyze the biocake composition and structure to validate the hypotheses made in this study.

Moreover, future studies with the objectives to increase removal of pharmaceutical micropollutants and at the same time reduce fouling of MBR in presence of pharmaceutical stress need to be performed. For example, powdered activated carbon (PAC) could be added in MBR to increase removal of CBZ via adsorption and thus reduce the induced stress effects.

Moreover, the process like integrated MBR-RO-Ozonation could be examined, which uses ozone for RO (reverse osmosis) concentrate treatment and recycles RO concentrate to MBR reactor after the treatment. The ozonation could reduce the toxic effects and also increase biodegradability of persistent substances in MBR.

In the last, more studies concerning the risk assessment of pharmaceutical micropollutants on human health and aquatic organisms should be conducted to evaluate the necessity of advanced treatment process like MBR to be employed in the wastewater treatment and reuse schemes.

P P u u b b l l i i c c a a t t i i o o n n 1 1 54

Introduction

Water scarcity is still a big challenge facing humanity in many places around the world. Thus, municipal wastewater has been considered to be an alternative water source for various applications after proper treatment [1][2]. Membrane bioreactor (MBR) which couples the activated sludge process and membrane separation, i.e. microfiltration (MF) or ultrafiltration (UF), has a significant potential to become a key element in municipal wastewater reclamation and reuse schemes worldwide, since it greatly improves the treated effluent quality as compared to conventional activated sludge (CAS) process, especially for removal of pathogenic microorganisms and micropollutants [3][4]. Besides, when higher effluent quality is required in some reuse applications, activated carbon could be added into MBR to further improve removal of organic contaminants [5]. Moreover, MBR could be employed as pre-treatment process for reverse osmosis (RO) which is served as a secondary barrier for removal of pathogenic bacteria, viruses and hazardous chemicals [6][7].

In the last decade, great attention has been paid to some emerging trace organic pollutants, also called micropollutants, such as endocrine-disrupting compounds, pharmaceuticals, personal care products etc., found in aquatic environment [8][9]. Among these micropollutants, pharmaceuticals got special concerns since late 1990s [10][11][12][13], as pharmaceuticals are designed to have some biological effect and to be persistent to avoid being metabolized before having a curing effect [14]. Besides, concerns have been raised regarding the possibility that continuous discharge of antibiotics to aquatic environment may facilitate the development or proliferation of resistant strains of bacteria [15]. Moreover, chronic toxicity effects have been reported for aquatic organisms exposed to human pharmaceuticals at trace concentration [16][17][18]. Thus, the existence of pharmaceutical micropollutants in aquatic environment may pose a potential danger on human health as well as aquatic organisms.

Therefore, extensive studies have been carried out to remove pharmaceutical micropollutants in municipal wastewater by all kinds of treatment approaches. In the literature, several reviews have been performed on removal of pharmaceutical micropollutants in municipal wastewater treatment [19][20][21][22][23][24][25], however, no review has been conducted specially on removal of pharmaceutical micropollutants by MBR or MBR related processes, such as activated carbon assisted MBR and combined MBR-RO process. This paper gives an overview on removal of pharmaceutical micropollutants by MBR or MBR related processes in order to evaluate the status of membrane bioreactor in wastewater treatment and reuse schemes.

Sources and occurrence of pharmaceutical micropollutants

Nowadays, about 3000 substances are registered in the EU for pharmaceutical purposes alone [23,26]. Also, there is evidence of occurrence of some 160 different drugs in effluent of wastewater treatment plants (WWTPs), surface water and groundwater [27]. Pharmaceuticals found in aquatic environment can be divided into different therapeutic categories: analgesics and anti-inflammatory drugs, lipid regulators, antiepileptic drugs, beta-blockers, antibiotics, cytostatic drugs etc. [28][29]. After consumed by human body, pharmaceutical residues are discharged to sewers through urine and faeces as unchanged compounds or metabolites. For unused or expired drugs, usually they are flushed down the drain or disposed of in the trash [27]. Thus, after domestic use pharmaceuticals mainly enter WWTPs. Concerning the contribution of hospitals to concentration of pharmaceuticals detected in WWTP influents, normally it is low for most pharmaceuticals except for some specific pharmaceuticals, for example, some antibiotics [30][31][32].

Due to incomplete removal of pharmaceuticals in conventional WWTPs, WWTP effluents become a major source of pharmaceutical micropollutants entering aquatic environment [29,33]. Many pharmaceutical micropollutants have been detected at concentration up to µg/L level in conventional WWTP effluents around the world. Table 1 summarizes the occurrence of typical pharmaceutical micropollutants in conventional WWTP effluents at concentration higher than 1.0 µg L -1 . Miè ge et al. [34] also confirmed these pharmaceutical micropollutants as among the most investigated pharmaceuticals in WWTPs. Since the pharmaceutical micropollutants listed in Table 1 have been frequently detected in WWTP effluents at relatively high concentration, they might be insufficiently removed in conventional WWTPs. Therefore, these pharmaceutical micropollutants are selected in this review to examine the capacity of MBR or MBR related processes on removal of pharmaceutical micropollutants. ), is at least 100 times smaller than the pore size of membrane used for MBR process, no direct physical retention of the compounds by MBR membrane can be expected [67]. However, Sahar et al. [71] and Urase et al. [72] suggested the deposits formed on the membrane surface might act as an additional barrier and thus contributed to an enhanced removal of pharmaceutical micropollutants in MBR. Further investigations should be carried out to confirm this assumption. As for removal by volatilization or stripping, the Henry coefficient (dimensionless, µg L -1 air / µg L -1 wastewater ) of a compound should be higher than 0.005 to have a significant removal (5%) as found in a EU project POSEIDON [26,67]. Because most of pharmaceutical micropollutants have a Henry coefficient smaller than 10 -5 [73] (also see table 2), removal of pharmaceutical micropollutants via stripping is negligible. Since the turbidity of wastewater blocks most sunlight and no secondary clarifier is employed in MBR, the photo-degradation of pharmaceutical micropollutants in MBR is not significant. Thus, the main possible mechanisms for removal of pharmaceutical micropollutants in MBR are sorption and biodegradation. Sorption of organic micropollutants to activated sludge of MBR depends on two main mechanisms, absorption and adsorption: Absorption is the hydrophobic interactions of the aliphatic and aromatic groups of a compound with the lipophilic cell membrane of the microorganisms or with the lipid fraction of the sludge; Adsorption is the electrostatic interactions of positively charged groups of a compound with the negatively charged surface of the microorganisms [23,67]. Since sorption coefficient K d (L kg SS -1 ) is defined as the partition of a compound between the sludge and the water phase [23], K d does not only depend on hydrophobicity of a compound, but also depends on the presence of positively charged groups (e.g., amino groups) in its structure. In addition, the relation between K d and LogKow is not obvious for acidic pharmaceutical micropollutants ibuprofen, diclofenac, naproxen, ketoprofen and gemfibrozil as indicated in a study [80]. their removal by sorption to activated sludge in biological process was found to be negligible [77,81]. Since the K d values of most pharmaceutical micropollutants, including all compounds listed in Table 2, are less than 500 L kg -1 and sludge production in MBR is generally smaller than conventional activated sludge process, the removal of pharmaceutical micropollutants via sorption in MBR is of minor importance. In particular, carbamazepine has a K d value of 1.2 L kg -1 , far away from the critical value of 500 L kg -1 , indicating that it is not sorbed to activated sludge to a significant degree. For antibiotics trimethoprim and sulfamethoxazole, although their K d values are relatively high (higher than 200 L kg -1 ), their sorption to activated sludge was not significant [63,82]. However, attention should be paid for the antibiotics ciprofloxacin and norfloxacin, the major mechanism relevant for their removal is sorption to activated sludge, due to their high K d values (higher than 15000 L kg -1 ) [83][84]. Ciprofloxacin and norfloxacin are not listed in table 1 due to their relatively low detected concentration in WWTP effluents.

MBR process for removal of pharmaceutical micropollutants

Removal mechanisms of pharmaceutical micropollutants in MBR

Therefore, the main mechanism for removal of most pharmaceutical micropollutants in MBR is via biodegradation. Due to their trace level concentration in municipal wastewater, cometabolism probably occurs for biological transformation or degradation of pharmaceutical micropollutants, in which case the bacteria accidentally break down or partially convert the micropollutant and do not use it as a carbon source for their growth [23]. In addition, the biodegradability of pharmaceutical micropollutants in MBR varies greatly in the range from zero to complete biotransformation [85]. Joss et al. [26] divided the pharmaceutical micropollutants into 3 different classes according to their degradation constant k biol values in municipal wastewater treatment process: compounds with k biol < 0.1L g SS -1 d -1 are not removed to a significant extent (<20%), compounds with k biol > 10 L g SS -1 d -1 are transformed by more than 90% and for compounds with k biol in between, moderate removal efficiency is expected.

Therefore, the remarkably poor removal of carbamazepine and diclofenac in biological wastewater treatment process is due to their low biodegradability with k biol values less than 0.1L g SS -1 d -1 as well as their low sorption potential to activated sludge with K d values less than 20 L kg -1 [69]. Based on the degradation of a heterogeneous group of 35 compounds, Joss et al. [26] concluded that state of the art biological treatment schemes, including MBR, for municipal wastewater treatment are not efficient in degrading pharmaceuticals: only 4 out of 35 compounds are degraded by more than 90% while 17 compounds are removed by less than 50%. proposed carbamazepine as a possible anthropogenic marker in the aquatic environment.

Removal of typical pharmaceutical micropollutants in MBR

Moreover, it is interesting to note that negative removal efficiency of diclofenac and carbamazepine in MBR was observed. This could be attributed to enzymatic cleavage of the glucuronic conjugates of those pharmaceuticals and consequently the release of the parent compounds in the treated effluent [59,69], or analytical uncertainty and sampling uncertainty occurred during the analysis process [87]. Similarly, Göbel et al. [88] attributed the observed high variability of sulfamethoxazole elimination to the possible transformation of N 4acetylsulfamethoxazole, main human metabolite of sulfamethoxazole, back to sulfamethoxazole and a simultaneous elimination of sulfamethoxazole itself during biological treatment. Thus, majority of pharmaceutical micropollutants are moderately biodegraded in MBR. In addition, degradation constant k biol could be used as an effective parameter to predict the removal of pharmaceutical micropollutants in MBR. The k biol values of the poorly removed compounds diclofenac and carbamazepine are less than 0.1 L g SS -1 d -1 ; the k biol values of moderately removed compounds (naproxen, ketoprofen, gemfibrozil, erythromycin, trimethoprim, and sulfamethoxazole) are between 0.1 L g SS -1 d -1 and 2 L g SS -1 d -1 ; the k biol value of easily removed compound (bezafibrate) is in range from 2 L g SS -1 d -1 to 5 L g SS -1 d -1 . The k biol value of very easily removed compound (ibuprofen) is higher than 5 L g SS -1 d -1 .

Factors affecting removal of pharmaceutical micropollutants in MBR

SRT

Solid retention time (SRT) seems to be the most important parameter affecting the removal of pharmaceutical micropollutants in MBR. Table 4 summarizes the effect of SRT on removal of selected pharmaceutical micropollutants in MBR.

Removal of diclofenac, ketoprofen, gemfibrozil, trimethoprim and erythromycin in MBR was found to be significantly affected by SRT [47,88,90,[START_REF] Kimura | Elimination of selected acidic pharmaceuticals from municipal wastewater by an activated sludge system and membrane bioreactors[END_REF][START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF][104]]. Clara et al. [47] reported that no removal of diclofenac was observed with a SRT of approximate 10 days, while with increasing SRT a partial removal was observed. Kimura et al. [START_REF] Kimura | Elimination of selected acidic pharmaceuticals from municipal wastewater by an activated sludge system and membrane bioreactors[END_REF] reported that the MBR operated with a longer SRT of 65 days significantly improved the elimination of ketoprofen and diclofenac as compared to the MBR with a shorter SRT of 15 days, from 82% to 98% and from 50% to 82%, respectively. Bernhard et al. [90] observed that the removal efficiency of diclofenac in a lab scale MBR was between 8% and 38% with an SRT increased from 20 to 48 days, and 59% with an SRT of 62 days. Maeng et al. [START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF] found that the removal efficiencies of gemfibrozil and ketoprofen were increased from 41% to 88% and from 64% to 90%, respectively, when SRT was increased from 20 days to 80 days. Göbel et al. [88] reported that for trimethoprim and erythromycin, a two to three times higher removal rate was seen for these compounds at a SRT of 60-80 days, up to 87-90% removal, as compared with the removal rate observed at SRT of 16±2 and 33±3 days.

No significant effect of SRT on removal of ibuprofen, bezafibrate, naproxen, carbamazepine and sulfamethoxazole was observed in MBR [23,52,88 As for sulfamethoxazole, Göbel et al. [88] reported removal efficiency of about 40% for sulfamethoxazole and about 80% for sulfamethoxazole together with its main human metabolite N 4 -acetylsulfamethoxazole in MBR, with no dependence on SRT from 16 days to 80 days.

High SRT allows the enrichment of slow-growing microorganisms (e.g. nitrifying bacteria)

and consequently the establishment of a more diverse bacteria population, which favors removal of pharmaceutical micropollutants in MBR [52]. Thus, the effect of SRT on removal of some moderately removed pharmaceutical micropollutants is quite significant, for example, ketoprofen, gemfibrozil, trimethoprim and erythromycin. Terne et al. [23] concluded that the biological transformation of a pharmaceutical compound depended on the age of the activated sludge; Bezafibrate, sulfamethoxazole, and ibuprofen required a sludge age of 2-5 days for significant degradation; diclofenac needed 5-15 days; Carbamazepine was not degraded even at a sludge age >20 days. Similarly, Clara et al. [52] defined a critical SRT for effective removal of pharmaceutical micropollutants, e.g. amounting to about 5 days for ibuprofen and to about 10 days for bezafibrate. Thus, SRT of higher than about 15 days is recommended to significantly improve the removal of pharmaceutical micropollutants in municipal wastewater treatment system [23,52,76].

pH

Although sorption of most pharmaceutical micropollutants to activated sludge in MBR is negligible at neutral pH, enhanced sorption was observed for some acidic pharmaceuticals at lower pH. Urase et al. [72] reported that the removal rate of acidic pharmaceuticals gemfibrozil, ibuprofen, ketoprofen, and diclofenac in MBR was much higher at pH of 4.3-5.0 than that at pH of 6.8-7.6 and 7.5-8.0. On the other hand, the removal of neutral pharmaceutical carbamazepine was not significantly affected by pH. The authors explained that in the neutral pH condition, these acidic pharmaceuticals were ionized; however, in the acidic pH condition, these pharmaceuticals were not ions and their hydrophobicity was increased, resulting in their sorption onto activated sludge. Moreover, it was found that the target substances attached to the sludge were not accumulated in the sludge phase, and they were biologically degraded. Tadkaew et al. [START_REF] Tadkaew | Effect of mixed liquor pH on the removal of trace organic contaminants in a membrane bioreactor[END_REF] studied the removal of sulfamethoxazole, carbamazepine, diclofenac, ibuprofen and ketoprofen in a batch scale MBR at different pH (pH 5-9). The results showed that the influence of mixed liquor pH on the removal of pharmaceuticals was quite dramatic for all four ionisable compounds (sulfamethoxazole, diclofenac, ibuprofen and ketoprofen), with highest removal at pH 5; in contrast, no apparent variation in removal efficiency of the neutral compound carbamazepine was observed.

Redox conditions

Monsalvo et al. [START_REF] Monsalvo | Removal of trace organics by anaerobic membrane bioreactors[END_REF] observed a poor removal, lower than 15%, for diclofenac, carbamazepine, ibuprofen, gemfibrozil and ketoprofen, partial removal for trimethoprim and naproxen, i.e. 35.4% and 70.3%, respectively, and high removal of 95.2% for sulfamethoxazole in anaerobic MBR, suggesting the poor capacity of anaerobic MBR on removal of most pharmaceutical micropollutants. In contrast, Hai et al. [START_REF] Hai | Removal of carbamazepine and sulfamethoxazole by MBR under anoxic and aerobic conditions[END_REF] observed that during near-anoxic operation with dissolved oxygen concentration in the bioreactor about 0.5 mg L -1 , an exceptionally high removal (68 ± 10%) of carbamazepine was achieved in MBR as compared to its low removal efficiency (12 ± 11%) under aerobic conditions; On the other hand, an average removal efficiency of 65% for sulfamethoxazole was achieved irrespective of the DO concentrations.

Nitrifying biomass

The enrichment of nitrifying bacteria was reported to enhance removal of pharmaceutical micropollutants in biological wastewater treatment system [79,[START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF][START_REF] Tran | Insight into metabolic and cometabolic activities of autotrophic and heterotrophic microorganisms in the biodegradation of emerging trace organic contaminants[END_REF][START_REF] Fernandez-Fontaina | Influence of nitrifying conditions on the biodegradation and sorption of emerging micropollutants[END_REF][113]]. Maeng et al. [START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF] found that the removal of gemfibrozil, diclofenac, bezafibrate, and ketoprofen was enhanced by ammonia oxidizing bacteria in MBR. Suarez et al. [113] reported the removal efficiency of diclofenac, from zero to74%, in the aerobic reactor was positively correlated with nitrifying biomass concentration rather than SRT. Tran et al. [79] reported that the enriched nitrifier culture enhanced degradation of gemfibrozil, ketoprofen, naproxen, diclofenac, and carbamazepine with their increased biodegradation constant k biol , especially for carbamazepine and diclofenac. Besides, the authors found that removal efficiency of these compounds by enriched nitrifier culture increased with the increase of initial ammonium concentration in batch reactors. Similarly, Fernandez-Fontaina et al. [START_REF] Fernandez-Fontaina | Influence of nitrifying conditions on the biodegradation and sorption of emerging micropollutants[END_REF] reported high biodegradation efficiencies of ibuprofen, naproxen, trimethoprim, and erythromycin were obtained with nitrifying activated sludge working at high nitrogen loading rates.

Molecular structures

Some authors related the removal of pharmaceutical micropollutants in MBR to their compound structures. Tadkaew et al. [START_REF] Tadkaew | Removal of trace organics by MBR treatment: the role of molecular properties[END_REF] reported that the removal of pharmaceuticals possessing only electron donating groups (like hydroxyl groups and primary amine groups), such as ibuprofen, ketoprofen, and sulfamethoxazole, was usually higher than 70%, while the removal of pharmaceuticals possessing only electron withdrawing groups (like a chlorine atom or amide group), such as carbamazepine and diclofenac, was usually lower than 20%.

Kimura et al. [START_REF] Kimura | Removal of pharmaceutical compounds by submerged membrane bioreactors (MBRs)[END_REF] attributed the poor removal of diclofenac in MBRs to the presence of chlorine in their structures. Bouju et al. [START_REF] Bouju | Perspectives of persistent organic pollutants (POPS) removal in an MBR pilot plant[END_REF] attributed high removal of ibuprofen to its simple molecular structure.

Effects of pharmaceutical micropollutants on MBR process

The presence of pharmaceutical micropollutants in MBR may affect microbial activity and microbial community structure of microorganisms in activated sludge [START_REF] Aubenneau | Membrane bioreactor for pharmaceutically active compounds removal: Effects of carbamazepine on mixed microbial communities implied in the treatment[END_REF][START_REF] Wang | Effects of pharmaceutically active compounds on a mixed microbial community originating from a municipal wastewater treatment plant[END_REF][START_REF] Delgado | Effect of cytostatic drugs on microbial behaviour in membrane bioreactor system[END_REF]. Besides, the presence of pharmaceutical micropollutants in MBR may also induce protection mechanism of microorganisms to produce some extracellular polymeric substances (EPS) [START_REF] Delgado | Effect of cytostatic drugs on the sludge and on the mixed liquor characteristics of a cross-flow membrane bioreactor: Consequence on the process[END_REF][START_REF] Avella | Effect of cytostatic drug presence on extracellular polymeric substances formation in municipal wastewater treated by membrane bioreactor[END_REF],

which could affect membrane fouling of MBR [START_REF] Delgado | Effect of cytostatic drugs on the sludge and on the mixed liquor characteristics of a cross-flow membrane bioreactor: Consequence on the process[END_REF]. 3. 4.1 Effects on microbial activity and microbial community structure Aubenneau et al. [START_REF] Aubenneau | Membrane bioreactor for pharmaceutically active compounds removal: Effects of carbamazepine on mixed microbial communities implied in the treatment[END_REF] examined the effect of trace concentration of carbamazepine on mixed microbial communities of activated sludge taken from MBR. The authors reported that in presence of 1 µg L -1 carbamazepine, higher endogenous respiration rates, lower exogenous respiration rates, and smaller flocs size were observed. The authors explained that the increase in endogenous respiration rates suggested an increase in maintenance requirements of bacteria in order to manage the chemical stress induced by carbamazepine; and the decrease of exogenous respiration rates indicated a change in the metabolic pathways of the substrate or a change in the active bacterial species. Delgado et al. [START_REF] Delgado | Effect of cytostatic drugs on the sludge and on the mixed liquor characteristics of a cross-flow membrane bioreactor: Consequence on the process[END_REF] also reported an increase in the endogenous respiration of heterotrophic micro-organisms after continuous addition of about 5 µg L -1 cytostatic drug cyclophosphamide and its principal metabolites (CPs) in MBR. The authors attributed the high microbial activity in endogenous conditions to their adapting to the presence of CPs. Wang et al. [START_REF] Wang | Effects of pharmaceutically active compounds on a mixed microbial community originating from a municipal wastewater treatment plant[END_REF] reported a 39%, 39%, and 19% decrease, respectively, in specific oxygen uptake rate (SOUR) when presence of 10 μM naproxen, ketoprofen, and carbamazepine in activated sludge in batch experiments. The results indicated that the presence of some pharmaceuticals may inhibit microbial activity of some activated sludge microorganisms. The authors also found shifts in microbial community structure in the presence of ketoprofen and naproxen via DGGE analysis. Through T-RFLP analyses of the bacterial 16S rRNA genes, Kraigher et al. [START_REF] Kraigher | Influence of pharmaceutical residues on the structure of activated sludge bacterial communities in wastewater treatment bioreactors[END_REF] observed a minor but consistent shift in the bacterial community structure in activated sludge of the bioreactor supplied with pharmaceuticals (ibuprofen, naproxen, ketoprofen, and diclofenac) at concentration of 50 μgL -1 , compared to the control reactor operated without addition of pharmaceuticals;

Moreover, a greater structural divergence was observed in the reactors operated with higher concentration of pharmaceuticals.

Effects on MBR performance and MBR fouling

Delgado et al. [START_REF] Delgado | Effect of cytostatic drugs on the sludge and on the mixed liquor characteristics of a cross-flow membrane bioreactor: Consequence on the process[END_REF] studied the effects of continuous addition of 5 µg L -1 CPs in MBR on characteristics of activated sludge and membrane fouling. In presence of CPs, formation of small particles and higher soluble EPS concentration in activated sludge were observed.

Meanwhile, higher transmembrane pressure and higher specific cake resistance of activated sludge were observed, which indicated faster membrane fouling. However, no effect on removal of COD and total nitrogen by MBR was observed in presence of CPs. Lay et al. [START_REF] Lay | Effect of pharmaceuticals on the performance of a novel osmotic membrane bioreactor (OMBR)[END_REF] observed increase in the protein and polysaccharide ratio for both soluble and bound EPS in the activated sludge after adding 20-25 µg L -1 carbamazepine, diclofenac, ibuprofen and naproxen in the feed tank of a Novel Osmotic Membrane Bioreactor (OMBR). The authors attributed the increase in protein content to a natural microbial response or occurrence of cell lysis and release of intracellular polymers under the pharmaceutical stress. Avella et al. [START_REF] Avella | Effect of cytostatic drug presence on extracellular polymeric substances formation in municipal wastewater treated by membrane bioreactor[END_REF] reported that the continuous addition of trace concentration CPs in MBR induced an increase in soluble EPS (mainly proteins of about 18 KDa and polysaccharides of about 6 KDa) in bulk solution and to a much lower degree in bound EPS in the sludge. The authors attributed the increase of these macromolecular species to a protection mechanism of microorganisms and attributed the more important membrane fouling to retention of these macromolecular species by the membrane. However, Jacob [START_REF] Jacob | Ré utilisation des eaux usé es é puré es par association de procé dé s biologiques et membranaires[END_REF] observed stable operation of MBR using real municipal wastewater that contained pharmaceutical micropollutants (diclofenac 0.7 µg L -1 , naproxen 0.7 µg L -1 , ketoprofen 1.6 µg L -1 , bezafibrate 0.6 µg L -1 and carbamazepine 0.5 µg L -1 ) during 164 days, which indicated that acclimation of microorganisms to pharmaceutical stress may happen in this situation and no significantly additional fouling was induced by the presence of these pharmaceutical micropollutants in raw wastewater.

MBR related processes for removal of pharmaceutical micropollutants

Since MBR alone is not sufficient to completely eliminate all pharmaceutical micropollutants in municipal wastewater, hybrid or combined processes based on MBR get much attentions.

Among MBR related processes, the capacity of activated carbon assisted MBR and combined MBR-RO process on removal of pharmaceutical micropollutants have been well investigated.

Table 5 summarizes the removal efficiencies of selected pharmaceutical micropollutants in activated carbon assisted MBR and combined MBR-RO process. Other MBR related processes, such as MBR-Ozone [START_REF] Huber | Oxidation of pharmaceuticals during ozonation of municipal wastewater effluents: a pilot study[END_REF], MBR-UV [START_REF] Nguyen | Removal of emerging trace organic contaminants by MBR-based hybrid treatment processes[END_REF], MBR-TiO 2 [START_REF] Laera | An integrated MBR-TiO2 photocatalysis process for the removal of Carbamazepine from simulated pharmaceutical industrial effluent[END_REF] and OMBR [START_REF] Lay | Effect of pharmaceuticals on the performance of a novel osmotic membrane bioreactor (OMBR)[END_REF][START_REF] Alturki | Performance of a novel osmotic membrane bioreactor (OMBR) system: flux stability and removal of trace organics[END_REF] were also reported to significantly enhanced the removal of pharmaceutical micropollutants, however, more studies are required to fully understand their capacity.

Activated carbon assisted MBR system can be further divided into two categories: MBR-PAC hybrid process and combined MBR-GAC process. In MBR-PAC hybrid process, powdered activated carbon (PAC) is added into MBR, while in combined MBR-GAC process, granular activated carbon column is used as post polishing unit after MBR. The coupling of activated carbon with MBR enables the activated carbon assisted MBR system to increase removal of some pharmaceutical micropollutants that are originally difficult to be removed in MBR, such as carbamazepine and diclofenac [START_REF] Nguyen | Coupling granular activated carbon adsorption with membrane bioreactor treatment for trace organic contaminant removal: Breakthrough behaviour of persistent and hydrophilic compounds[END_REF][START_REF] Nguyen | Removal of trace organic contaminants by a membrane bioreactor-granular activated carbon (MBR-GAC) system[END_REF][START_REF] Li | Simultaneous activated carbon adsorption within a membrane bioreactor for an enhanced micropollutant removal[END_REF][START_REF] Nguyen | Enhancement of removal of trace organic contaminants by powdered activated carbon dosing into membrane bioreactors[END_REF][START_REF] Serrano | Removal of persistent pharmaceutical micropollutants from sewage by addition of PAC in a sequential membrane bioreactor[END_REF]. Serrano et al. [START_REF] Serrano | Removal of persistent pharmaceutical micropollutants from sewage by addition of PAC in a sequential membrane bioreactor[END_REF] reported that after a single addition of 1 g L -1 of powdered activated carbon (PAC) directly into the aeration tank of MBR an immediate and sharp removal increase was observed for the recalcitrant pharmaceutical micropollutants such as carbamazepine, diclofenac and trimethoprim, with removal efficiencies in the range of 93%-99%. Besides, the moderately degraded substance erythromycin was completely removed after PAC addition (97%-99%). Moreover, microbial ecology present in the biomass showed a higher abundance of ammonium oxidizing bacteria after PAC addition, which may enhance degradation of pharmaceutical micropollutants in MBR. Nguyen et a. [START_REF] Nguyen | Enhancement of removal of trace organic contaminants by powdered activated carbon dosing into membrane bioreactors[END_REF] reported that the removal of hydrophilic and biologically persistent pharmaceutical micropollutants (ketoprofen, naproxen, diclofenac and carbamazepine) was immediately improved to above 95%, after the addition of only 0.1 g/L PAC into MBR.

While, the gradual decrease in removal underscored the requirement for the addition of fresh PAC during the continuous operation. Li et al. [START_REF] Li | Simultaneous activated carbon adsorption within a membrane bioreactor for an enhanced micropollutant removal[END_REF] reported that the removal efficiencies of sulfamethoxazole and carbamazepine increased to 82 ± 11% and 92 ± 15% from the levels of 64 ± 7%, and negligible removal, respectively, when the PAC dosage was raised from 0.1 g L - 1 to 1.0 g L - 1 . Moreover, it is interesting to note that after the PAC addition the MBR membrane achieved significant additional removal of both micropollutants, especially for carbamazepine, as compared to the situation in MBR without addition of PAC. The authors attributed the enhanced removal to their sorption onto membrane cake layer. Nguyen et a. [START_REF] Nguyen | Removal of trace organic contaminants by a membrane bioreactor-granular activated carbon (MBR-GAC) system[END_REF] reported that the granular activated carbon (GAC) post-treatment could significantly improve removal of the pharmaceutical micropollutants which were poorly removed by MBR. For example, the compounds, which were removed by MBR with efficiencies below 40% (ketoprofen, naproxen, diclofenac and carbamazepine), achieved overall removal efficiencies of 98% or above following GAC treatment. Since some pharmaceutical micropollutants could not be biodegraded in MBR, replacement or regeneration of activated carbon should be required to maintain high removal of pharmaceutical micropollutants during the long term operation for activated carbon assisted MBR systems.

The combined MBR-RO process is one of the dual membrane systems often used for production of reclaimed water besides the CAS-MF/UF-RO process [START_REF] Tam | A pilot study for wastewater reclamation and reuse with MBR/RO and MF/RO systems[END_REF]. Alturki et al. [74] reported that the combination of MBR and a low pressure RO membrane resulted in more than 95% removal or removal to below the analytical detection limit of all 40 trace organic compounds including most typical pharmaceutical micropollutants. Dolar et al. [START_REF] Dolar | Removal of emerging contaminants from municipal wastewater with an integrated membrane system, MBR-RO[END_REF] reported that the combination of MBR and RO treatment showed excellent overall removal of 20 detected pharmaceutical micropollutants in WWTPs with removal efficiencies above 99%.

Cartagena et al. [START_REF] Cartagena | Reduction of emerging micropollutants, organic matter, nutrients and salinity from real wastewater by combined MBR-NF/RO treatment[END_REF] also found that the post RO process can significantly improve the removal of pharmaceutical micropollutants like carbamazepine and diclofenac after MBR treatment in real municipal wastewater. However, the total removal of carbamazepine and diclofenac by MBR-RO process was not complete, 82.1-93.1% and 88.3-95.9%, respectively.

Similarly, Joss et al. [START_REF] Joss | Water reuse:> 90% water yield in MBR/RO through concentrate recycling and CO2 addition as scaling control[END_REF] also reported the incomplete removal of carbamazepine and diclofenac by MBR-RO process. Snyder et al. [START_REF] Snyder | Role of membranes and activated carbon in the removal of endocrine disruptors and pharmaceuticals[END_REF] reported almost complete removal of all detected pharmaceuticals in the tested MBR-RO pilots. While, Sahar et al. [START_REF] Sahar | The use of RO to remove emerging micropollutants following CAS/UF or MBR treatment of municipal wastewater[END_REF] reported that RO cannot serve as an absolute barrier for pharmaceutical removal in MBR-RO process since ibuprofen and diclofenac were detected in the effluent in the range of 28-223 ng L -1 , although most pharmaceutical micropollutants investigated were removed in high efficiencies, higher than 93%, by MBR-RO process. Kimura et al. [START_REF] Kimura | Influence of residual organic macromolecules produced in biological wastewater treatment processes on removal of pharmaceuticals by NF/RO membranes[END_REF] reported that in the filtration tests with MBR effluent no significant variance of rejection of the pharmaceutical micropollutants was observed for RO membrane as compared with deionized pure water spiked with the pharmaceutical micropollutants, suggesting that the main mechanism for rejection of pharmaceutical micropollutants by RO membrane is size exclusion.

Conclusions

The presence of pharmaceutical micropollutants in the aquatic environment has become a serious problem for municipal wastewater reclamation and reuse around the world. MBR process with high SRT is a good choice to replace the conventional activated sludge process in water reuse projects due to its better removal of micropollutants. Although the MBR process alone could not assure the complete removal for the majority of pharmaceuticals, the possibility of MBR to be coupled with other processes, such as activated carbon adsorption and RO membrane filtration, enables it to significantly improve removal of pharmaceutical micropollutants. Thus, MBR would play a more important role in wastewater treatment and reuse applications in the future.

It can be concluded that the main mechanism for removal of most pharmaceutical micropollutants in MBR is biodegradation; In terms of operation conditions, higher SRT, lower pH, higher nitrogen loading rate and anoxic condition favor removal of some pharmaceutical micropollutants in MBR; while, no evidence was found concerning the effect of MBR configuration (flat sheet or hollow fiber) on removal of pharmaceutical micropollutants in MBR; In addition, the presence of pharmaceutical micropollutants in MBR could affect the microbial activity and microbial community structures of microorganisms in activated sludge and cause more serious membrane fouling. However, the difficulty to compare data from different scales of MBR or MBR related processes (lab scale, pilot scale and full scale), the difficulty in quantifying sorped mass content of pharmaceutical micropollutants in sludge phase, the uncertainty in analysis of pharmaceutical micropollutants and their metabolites in liquid phase, and even sampling procedures make it hard to draw reliable conclusions from the literature.

Still, many questions are remained unanswered. Regarding the mechanisms for removal of pharmaceutical micropollutants, distinguish between sorption and biodegradation should be further investigated, taking into account the fact that the sorped pharmaceutical micropollutants in sludge phase may be further biodegraded by activated sludge and sorption may play an important role in biodegradation of pharmaceutical micropollutants in terms of mass transfer. Also, the function of deposits on membrane surface as an additional barrier for removal of pharmaceutical micropollutants should be further examined. In addition, more studies should be performed to identify the bacterial species that favor removal of pharmaceutical micropollutants in MBR and determine the optimal operation conditions that help to enrich these bacterial species. Last but not least, it is recommended that more studies concerning the risk assessment of pharmaceutical micropollutants on human health and aquatic organisms be conducted to evaluate the necessity of advanced wastewater treatment processes, like MBR and MBR related processes, to be employed in the wastewater treatment and reuse schemes.

P P u u b b l l i i c c a a t t i i o o n n 2 2

Introduction

The presence of micropollutants, notably the pharmaceuticals, in aquatic environment has been paid great attention since the late 1990s due to their potential adverse effects on human health [1][2][3][4]. Since then, pharmaceutical micropollutants have been frequently detected in domestic wastewater treatment plants (WWTPs) and also in the plants that produce potable water from surface waters or ground waters [5][6][7]. Many pharmaceutical micropollutants, including analgesics like ibuprofen, diclofenac, naproxen and ketoprofen, lipid regulators like bezafibrate and gemfibrozil, the antiepileptic drug carbamazepine, and antibiotics such as trimethoprim and sulfamethoxazole, have been frequently detected at concentration higher than 1.0 µg/L in WWTP influents around the world [8][9][10][11][12].

As membrane bioreactors (MBRs) get increasing importance in the field of municipal wastewater treatment and reuse [13][14][15], the removal of pharmaceutical micropollutants in MBRs has been thoroughly investigated [16][17]. The removal efficiency of pharmaceutical micropollutants in MBR varies greatly due to the difference in their biodegradability. For example, ibuprofen and bezafibrate are easily biodegraded with more than 90% elimination efficiency, carbamazepine is hardly biodegraded with an elimination efficiency lower than 10%, and the majority of pharmaceutical micropollutants (such as ketoprofen, gemfibrozil, trimethoprim, sulfamethoxazole etc.) are moderately biodegraded with elimination efficiency varying between 50% and 80% [18]. Thus, sludge retention time (SRT) is believed to be capable of affecting the biodegradation of pharmaceutical micropollutants in MBRs. High SRT allows the enrichment of slow-growing microorganisms (e.g. nitrifying bacteria) in the sludge and consequently the establishment of a more diverse bacteria population, which may favor removal of pharmaceutical micropollutants in MBR [19]. In particular, removal of diclofenac, ketoprofen, gemfibrozil, trimethoprim and erythromycin in MBR was found to be significantly affected by SRT [20][21][22]. For other kinds of wastewaters, for example, effluents of hospitals or drug production facilities, a specific acclimated biomass may be required for MBRs treating these kinds of effluents, since they may contain very high concentration of pharmaceuticals, especially the antibiotics [23][24][25].

Taking into account that the concentration of pharmaceutical micropollutants in influents of domestic WWTPs varies greatly due to their therapeutic consumptions for treatment of seasonal diseases [26][27][28][29] or inappropriate disposal of unused pharmaceuticals [30], it is possible that peak concentration of some pharmaceutical micropollutants occur in domestic wastewaters during short periods, which may affect the performance of biological process such as MBRs. For example, ibuprofen was detected at concentration of 319 µg/L in the influent of a Spanish WWTP [31]. Also, naproxen was detected at concentration of 611 µg/L in the influent of a Canadian WWTP [32]. In the literature, a few studies have been carried

out to investigate the effects of peak chemical stress, including pharmaceutical micropollutants, on the microbial activity of MBR sludges. Henriques et al. [33] observed that a cadmium shock load (20 mg/L) caused about 50% inhibition of heterotrophic respiration (through measurement of specific oxygen uptake rates) on MBR sludge. Lesage et al. [34] found that a peak concentration (130 mg/L) of a toxic compound, 2,4 dimethylphenol (DMP), could cause 50% inhibition of respiration on MBR sludge. Aubenneau et al. [35] examined the effect of trace concentration of carbamazepine on mixed microbial communities of activated sludge taken from MBR. The authors reported inhibition of exogenous respiration rate of activated sludge in presence of 1 µg L -1 carbamazepine. Wang et al. [36] reported a 39%, 39%, and 19% decrease, respectively, in specific oxygen uptake rate (SOUR), defined as sludge oxygen uptake rate divided by the concentration of volatile suspended solid in the sludge, when presence of 2.3 mg L -1 naproxen, 2.5 mg L -1 ketoprofen, and 2.4 mg L -1 carbamazepine in the same activated sludge in batch experiments.

However, to our knowledge the bibliography doe not report the peak effect of domestic pharmaceutical micropollutants on MBR fouling. It is known that a chemical stress can induce the production of soluble microbial products (SMP) or extracellular polymeric substances (EPS), which are identified as main foulants for MBR fouling [37][38][39]. For example, Reid et al. [40] reported that a salt shock, introduced by dosing directly 30 g L -1

NaCl solution during 10 min in MBR, resulted in significant increase of SMP and EPS concentrations in MBR. Similar results have been recently observed by Wang et al. [41], who reported that a high osmotic pressure (5% NaCl) induces the production of SMPs with a relative strong hydrophilicity and protein-like characteristics in batch experiments. Sheng et al. [42] reported that in presence of toxic chemicals, such as 30 mg l -1 Cu(II), 40 mg l -1 Cr(VI), 5 mg l -1 Cd(II), in the bacteria strain Rhodopseudomonas acidophila, the EPS content increased by 5.5, 2.5, and 4.0 times, respectively. Moreover, the authors reported that under these toxic conditions, the increase in the protein content far exceeded than that of other constituents in EPS. Lay et al. [43] observed a significant increase in the protein and polysaccharide ratio for both soluble and bound EPS in the activated sludge after adding carbamazepine, diclofenac, ibuprofen and naproxen in the feed tank (20-25 µg L -1 of each pharmaceutical in the feed) of an Osmotic Membrane Bioreactor (OMBR).

The objective of this study is to investigate the peak effects of pharmaceutical micropollutants that are currently found in domestic wastewater on fouling propensity and physico-chemical characteristics of the activated sludge from a MBR treating domestic wastewater.

Carbamazepine (CBZ), an anti-epileptic drug, was selected as the representative of pharmaceutical micropollutants found in domestic wastewater because of the following reasons: i) it has been frequently detected in influents as well as effluents of WWTPs in concentration up to or higher than 1.0 µg L -1 [44]; ii) it is almost not biodegradable and its sorption to activated sludges is negligible in biological activated sludge processes [45][46]; iii)

it was found to have some inhibition effects on biological activity of microorganisms in activated sludge [35][36].

Material and Methods

Peak effects of carbamazepine on MBR sludge

Table 1 introduces the physicochemical properties of carbamazepine. A concentrated mother solution was prepared by dissolving carbamazepine (from Sigma-Aldrich) in pure ethanol (from VWR International) at a concentration of 1 g/L, stored at 4 °C and used within a week. The peak effects of CBZ on sludge were characterized in batch reactors with sludge taken from the aerobic tank of a lab-scale MBR due to the easier manipulation for repeatability examination, as compared with the tests conducted in MBR reactor. Sludges from MBR operated under different organic loading rates were examined, since the SMP composition and microbial activity of MBR sludge was significantly affected by the organic loading rates [49][50]. Two batch reactors were operated in parallel in presence of CBZ (at concentration of 100 µg L -1 in the same sludge) or without CBZ for 3 hours. Fig. 1 shows the protocol used for batch experiments. Firstly, quantities of the concentrated mother solution sufficient to yield 100 µg L -1 final concentrations of CBZ were added in the reactor-with CBZ. As comparison, the same quantity of pure ethanol was added in the control-reactor to rule out the impact of ethanol. Then the ethanol in the two batch reactors was blown off using a gentle stream of air, leaving only the CBZ in the reactor-with CBZ [51]. Finally, a same volume of sludge samples taken from MBR aerobic tank was added in the reactor-with CBZ and control-reactor, and the mixed liquids were stirred using the aeration in the bottom of each reactor for 3 hours to dissolve CBZ and to make the sludge contact with CBZ in aerobic condition. Then the sludges in both reactors were sampled and the two corresponding supernatants were prepared by centrifugation of the sludge samples at 4000 g during 10 minutes at room temperature at the same time. 3 hours of contact time was chosen since it takes about 3 hours before all the readily degradable substances are consumed by the activated sludge [52]. Indeed, no significant difference was found concerning the effects of CBZ on characteristics of sludge and supernatant between contact time of 3 hours and 5 hours. Too long time, for example 24 hours, for sludge in batch reactors without exogenous substrate, would cause the decay of bacteria and impact the quality of activated sludge. And no significant difference was found concerning the effects of CBZ on characteristics of sludge and supernatant when the peak concentration of CBZ was modified to 500 µg L -1 , as compared to the effects of CBZ at concentration of 100 µg L -1 . Moreover, no significant difference was observed between the sludge obtained from control-reactor and blank reactor where no ethanol was added (see Fig. 1) in terms of biomass and supernatant quality, which confirms that the ethanol did not affect the quality of sludge after evaporated by the gentle stream of air. Temperature was measured and flux was normalized to the flux at 20 °C. For supernatant filtration, no stirring was performed, while for sludge filtration, the sludge was stirred at 200 rpm. After filtration the membrane was turned face down and was backwashed with ultra pure water at 1 bar for 15 min in order to investigate flux recovery by permeability measurement.

Membrane fouling resistance was determined according to Eq. ( 1).

() mf TMP J RR    (1)
In this equation, J is the permeate flux (m 3 /s/m 2 ) at 20 °C, with R m the membrane resistance (m -1 ), R f the fouling resistance (m -1 ), TMP the transmembrane pressure (Pa) and μ the dynamic viscosity at 20 °C (Pa s).

Then in the dead-end filtration cell, C can be determined by cake deposit model as expressed in Eq. ( 2).

f V RC A  (2)
In this equationis the specific cake resistance (m kg -1 ), with C the deposited mass per filtered permeate volume (kg m -3 ), V the cumulated permeate volume (m 3 ) and A the effective membrane area for filtration (m 2 ). For supernatant filtration, C could be calculated by the retained DOC (kg m -3 ). For sludge filtration, C is assumed to be MLSS value of sludge.

Characterization of sludge microbial activity

Sludge microbial activity was characterized by the microbial respiration rate. It was measured in respirometric batch experiments defined by Spé randio and Paul [53]. Sludge samples were collected from the aerobic tank of the MBR. Firstly, the collected sludge was aerated for at least 3 h to reach the endogenous condition. Then pure ethanol was added to respirometric reactor to test the sludge microbial activity before addition of CBZ. Pure ethanol was used as exogenous substrate in the respirometric experiments, because CBZ was prepared in pure ethanol. Then, quantities of the concentrated mother solution sufficient to yield 100 µg L -1 final concentration of CBZ were added in the respirometric reactor to test the effect of CBZ on the sludge microbial activity.

Sludge

Sludge for batch experiments was sampled from the aerobic tank of a lab-scale submerged MBR set-up consisting of an anoxic reactor (5.4 L) and an aerobic reactor (12.6 L). A flat sheet microfiltration membrane (Lp 0 = 1110 L h -1 m -2 bar -1 at 20 °C, Kubota, Japan) was submerged inside the aerobic reactor. It has an effective filtration area of 0.1 m 2 , with an average pore size of 0.2 µm. Two aeration systems were used in the aerobic reactor. An aeration system with coarse air bubble was installed below the membrane to reduce fouling and it was operated continuously at flow rate of 1.5 L/min, i.e. SAD p of 90 m 3 air/m 3 permeate. Besides, another aeration system with fine air bubbles was used to maintain the dissolved oxygen concentration in the sludge at about 2-3 mg/L and automatically controlled by an oxygen sensor. The sludge recirculation ratio was fixed at 4. The MBR was fed with a municipal wastewater taken from a wastewater treatment plant close to Toulouse, France, after a primary physical treatment. The MBR was operated in two steps (see Table 2). In the first step, the MBR was operated at a SRT of 40 days, with a sludge concentration in the aerobic reactor at 4-6 g/L. In the second step, the MBR was operated at a 30 days SRT, with a sludge concentration in aerobic reactor at 6-8 g/L. The MBR reached high removal of COD, more than 95%, during both two steps. Whereas a limited removal of TN, about 40%, was observed during step 1 when the MBR was operated at an organic loading rate (C m ) of about 0.1 kg COD/kg MLSS/d.

Analytical methods

The concentration of mixed liquor suspended solids (MLSS) was measured according to the Three assumptions can be proposed to explain the increased sludge fouling propensity after addition of CBZ: i) a change in microbial activity linked to a stress on the microorganisms ii) a change in biomass physico-chemical properties, like mean floc size distribution, or iii) a change in the supernatant quality. These three assumptions will be discussed in the following paragraphs that aim to evaluate the effects of CBZ on the supernatant and biomass properties.

Effect of CBZ on sludge microbial activity

The effect of CBZ on sludge microbial activity was investigated in a respirometric reactor.

The sludge microbial activity was characterized by the microbial respiration rate. Fig. 3 shows the result of respirometric tests for the sludge sampled from aerobic tank of the MBR operated under organic loading rate of 0.1 kg COD/kg MLSS/d. As shown in Fig. 3, the activated sludge was in the endogenous condition before adding ethanol in the respirometric reactor.

Then an immediately significant increase of respiration rate was observed after addition of pure ethanol in the respirometer due to the degradation of ethanol. Then the respiration rate kept constant for a short period, during which time the bacteria may adapt to the presence of ethanol. After this period, increased respiration rate was observed until the exhaustion of ethanol, which indicates that the microbial growth occurred in this period. Then after the activated sludge reached endogenous condition in absence of exogenous substrate, quantities of the concentrated CBZ solution sufficient to yield 100 µg L -1 final concentrations of CBZ in sludge were added in the respirometric reactor to test the effect of CBZ on the sludge microbial activity. The result showed no inhibition of sludge microbial activity with presence of 100 µg L -1 CBZ in the sludge, since after addition of CBZ ethanol solution in the respirometric reactor the respiration rate was immediately increased to the same level that was reached before exhaustion of ethanol when pure ethanol was added in the respirometric reactor. Moreover, respiration rate continued to increase without adaptation period to the chemical stress induced by CBZ until the exhaustion of ethanol, which again indicated the microbial growth without inhibition in presence of exogenous substrate. This result indicates that the presence of 100 µg L -1 CBZ in the sludge during a short period contact time did not change the degradation and growth behavior of bacteria in activated sludge or the growth of bacteria offset the small inhibition effect induced by CBZ in presence of exogenous substrate.

This result is consistent with the result of Delgado et al. [52], who found no inhibition on microbial activity of activated sludge taken from MBR in presence of pulse of cytostatic compounds (about 200 µg L -1 cyclophosphamide and its principal metabolites) during the short contact time in the respirometric reactor. No inhibition effect of CBZ on microbial activity was also found for the sludge sampled from the MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d. Thus, the modification of sludge fouling propensity could not be attributed to the modification in sludge microbial activity.

Effect of CBZ on supernatant composition

Fig. 5 shows the result of protein and polysaccharide concentration in the supernatants of sludges sampled from the batch reactors. Slight higher protein concentration (about 20%) was observed in supernatant from the reactor-with CBZ, as compared with that from the controlreactor. However, no significant difference in polysaccharide concentration was found. This result suggests that for sludge under CBZ stress the released SMPs are mainly proteins, or the released polysaccharides were easily biodegraded. Thus, the proteins may play a more important role in the CBZ effect. Then, the effect of CBZ on production of protein-like SMPs was further examined by HPLC-SEC that uses fluorescence detector with excitation/emission wavelengths set at 280/350 nm to obtain the information on the molecular weight distribution, which is important to understand fouling propensity of sludge. Fig. 6 shows the result of HPLC-SEC analysis of supernatants prepared from the batch reactors using sludges that were taken from the MBR operated under low organic loading rate (0.1 kg COD/kg MLSS/d). The molecular weight of protein-like substances in MBR supernatants showed bimodal distribution. Indeed, two peaks were observed in the HPLC-SEC results which represent two groups of protein-like molecules, i.e.

molecular size of 100-1000 kDa and 10-100 kDa, respectively. As shown in Fig. 6, a significantly higher peak of protein-like substances corresponding to100-1000 kDa molecules in the supernatant prepared from the reactor-with CBZ was observed, as compared to that in the supernatant prepared from the control-reactor. This result indicates that only 3 hours contact of 100 µg L -1 CBZ with activated sludge could cause the sludge to release some protein-like SMPs, which could be attributed to the defensive response of bacteria when facing chemical stress [58] or to the decay (lysis) of some bacteria and release of SMPs inside the bacterial cells. The latter assumption is consistent with the results of Ni et al. [59][60] who found that the molecular size of biomass associated products (BAPs), which is associated with biomass decay, is bigger than 100 kDa. In contrast, Fig. 6 showed no significant difference of detected peak heights of protein-like substances with a molecular size of 10-100 kDa between the two supernatants prepared from reactor-with CBZ and the control-reactor. In conclusion, the change in fouling propensity of sludge could be mainly attributed to the increase in the quantity of protein-like substances with a molecular size of 100-1000 kDa in the supernatant, since no significant change of floc size distribution and microbial activity was observed after addition of CBZ in the sludge.

Effect of CBZ on fouling layer characteristics

The fouling layer of a membrane developed during sludge filtration is related to the retained components in the sludge, thus it includes biomass cake layer and biofilm layer. Fig. 7 shows one example of HPLC-SEC analysis of a MBR supernatant (prepared from the MBR sludge) and the corresponding permeate obtained on a 0.2 μm PS membrane. As shown in Fig. 7, the 100-1000 kDa protein-like SMPs in supernatant could be almost completely retained by the 0.2 μm PS membrane, indicating that the fouling layer formed on the 0.2 μm PS membrane surface behaves as an additional barrier and made the effective pore size of the fouled MF membrane be more in the UF range. Moreover, the 0.2μm PS membrane could partially retain 10-100 kDa protein-like SMPs in the supernatant during the filtration of activated sludge, which indicates that some small flocs may cause pore blocking of the MF membrane, thus reducing the pore size and allowing the partial retention of small molecules in the 10-100 kDa range. Particularly, with addition of CBZ, the α value increased more significantly at higher TMP.

These results may suggest that the increase of 100-1000 kDa protein-like SMPs in supernatant with addition of CBZ (see Fig. 6) could modify the structure of fouling layer formed during the sludge filtration and the effect of CBZ was more significant for the fouling layer formed at higher TMP (1.0 bar). In addition, higher flux recovery by backwash was observed in filtration tests that were performed using the sludge with addition of CBZ at each TMP (see table 3), which suggests that the more compact fouling layer formed on the 0.2 μm PS membrane during filtration of the sludge with addition of CBZ could reduce the irreversible fouling caused by adsorption of small molecules or pore blocking due to colloids, and also suggests that the increased fouling resistance during filtration of the sludge with addition of CBZ may be caused by the modification of fouling layer on the membrane surface instead of the modification of fouling inside the membrane pores. The fouling layer of a membrane developed during filtration of a supernatant, i.e. the biofilm, is related to the retained components in the supernatant. Fig. 8 shows one example of HPLC-SEC result for a MBR supernatant (used as feed for supernatant filtration) and its corresponding permeate obtained on a 0.01 μm PES membrane. The result showed that the 0.01 μm PES membrane retained almost all 100-1000 kDa protein-like SMPs and let pass almost all the 10-100 kDa protein-like SMPs. This could be explained by the fact that a 0.01 μm pore size is often considered as corresponding to the 100 kDa molecular weight cut-off (MWCO) for ultrafiltration membranes. This result also emphasizes the importance of protein-like compounds in 100-1000 kDa range to the supernatant fouling propensity and indicates that the biofilm formed could not retain the 10-100 kDa SMPs. Table 4 shows the calculated results of α values obtained from filterability tests of supernatants coming from the batch reactors using sludges that were taken from the MBR operated under low organic loading rate (0.1 kg COD/kg MLSS/d). Similar to the case of sludge filtration, table 4 indicates that the increase of 100-1000 kDa protein-like SMPs in supernatant with addition of CBZ (see Fig. 6) could modify the structure of fouling layer formed during the supernatant filtration and the effect of CBZ was more significant for the fouling layer formed at higher TMP (0.8 bar or 1.0 bar) as compared to that formed at lower TMP (0.5 bar). Table 4 also showed slight higher flux recovery after backwash for filtration of supernatant with addition of CBZ, compared with that without addition of CBZ, indicating fouling layer formed on the 0.01 μm PES membrane during filtration of the supernatant with addition of CBZ could slightly reduce the irreversible fouling caused by adsorption of small molecules or pore blocking due to colloids.

Effect of MBR organic loading rate on CBZ effects

The effects of CBZ was examined for sludges sampled from MBR operated under two different organic loading rates, i.e. 0.1 kg COD/kg MLSS/d and 0.2 kg COD/kg MLSS/d, due to the significant differences in the SMP composition and microbial activity of MBR sludge under different organic loading rates. Fig. 9 shows result of the filterability tests of sludges, sampled from batch reactors using sludges taken from the MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d at 1.0 bar. The result showed that fouling resistance was slightly higher during filtration of the sludge with presence of 100 µg L -1 CBZ.

Compared to the filterability test result of sludges sampled from batch reactors using sludges taken from the MBR operated under organic loading rate of 0.1 kg COD/kg MLSS/d (see Fig. 2), a significantly reduced effect of CBZ on sludge fouling propensity was observed. To elucidate the difference of CBZ effects on sludge fouling propensity, the HPLC-SEC analysis of supernatants coming from batch reactors that used sludges taken from the MBR operated under organic loading rate of 0.2 kg COD/kg MLSS/d was performed, as shown in Fig. 10. Compared with the result obtained using sludges from MBR operated under organic loading rate of 0.1 kg COD/kg MLSS/d (see Fig. 6), much less significant effect of CBZ on 100-1000 kDa protein-like SMPs in supernatant was observed, which suggests that under higher organic loading rate condition, the bacteria in the activated sludge may more easily adapt to the pharmaceutical stress. Thus, the higher organic loading rate condition may protect the bacteria from decay (lysis) when facing pharmaceutical stress. Or this effect may be caused by the difference in bacterial species that were developed in MBRs operated under different organic loading rates. However, further studies need to be performed to verify these assumptions. Thus, the reduced effect of CBZ on sludge fouling propensity for sludge from MBR operated under higher organic loading rate could be attributed to the reduced effect of CBZ on the increase of 100-1000 kDa protein-like SMPs in supernatant.

Conclusions

To study the interactions between peak concentration of pharmaceutical micropollutants and fouling in MBRs, this study investigated the effects of presence of peak concentration carbamazepine in sludge on the fouling propensity and physico-chemical characteristics of activated sludge from a submerged MBR.

The results showed that the presence of 100 µg L -1 CBZ in the sludge caused no inhibition on the sludge microbial activity, no significant change of floc size distribution and no significant change of polysaccharide SMPs in supernatant. However, a significant increase of proteinlike substances with a molecular size of 100-1000 kDa in supernatant was observed for the sludge sampled from the MBR operated under organic loading rate of 0.1 kg COD/kg MLSS/d, which led to higher fouling propensity during filtration of sludge and supernatant.

The increased fouling resistance of sludge and supernatant mainly originated from the increased retention of 100-1000 kDa protein-like SMPs, which led to modification of fouling layer on membrane surface. In addition, higher flux recovery by backwash was observed in the filtration tests of sludge sampled from batch reactor with addition of peak CBZ, which indicates that the "CBZ-modified" fouling layer could reduce the irreversible fouling that is caused by the adsorption of soluble organics or pore blocking due to colloids. Moreover, the CBZ effects are reduced for sludge from MBR operated under higher organic loading rate (0.2 kg COD/kg MLSS/d), which suggests that for MBR under higher organic loading rate condition, the bacteria in the activated sludge may more easily adapt to the environmental stress and under this condition less decay (lysis) of bacteria may happen.

This study sheds some light on fouling mechanisms of MBR when facing some accidental pharmaceutical stress and helps to understand the effect of organic loading rate on performance of MBR under pharmaceutical stress. Long-term study concerning effects of pharmaceutical stress on MBR performance needs to be carried out by addition of CBZ directly in MBR to better understand the interaction between pharmaceutical micropollutants and MBR process and to also examine the possibility of using MBR as pre-treatment process of reverse osmosis (RO) when RO concentrate was recycled to the MBR, in which case the accumulation of pharmaceutical micropollutants occurs in the biological reactor. -Biofouling is compressible and this effect is measurable until a limit pressure [START_REF] Massé | Bioré acteur à membranes immergé es pour le traitement des eaux ré siduaires urbaines: spé cificité s physico-chimiques du milieu biologique et colmatage[END_REF].

Many studies showed that the supernatant of activated sludges, which contains colloids and solutes, plays a more important role in membrane fouling of MBR than the biological flocs (Wisniewski and Grasmick, 1998;[START_REF] Bouhabila | Fouling characterisation in membrane bioreactors[END_REF]. Thus, soluble microbial products (SMPs), the major organic constituents of sludge supernatants, have been acknowledged as The main objective of this study was to examine the effects of one pharmaceutical micropollutant, which is frequently found in domestic wastewaters, on the MBR fouling.

Carbamazepine (CBZ), an anti-epileptic drug, was selected in this study. Because it has been frequently detected in domestic wastewater [START_REF] Zhang | Carbamazepine and diclofenac: removal in wastewater treatment plants and occurrence in water bodies[END_REF] and it is almost not biodegraded in biological wastewater treatment processes including MBRs, which makes it become a possible anthropogenic marker in the aquatic environment [START_REF] Clara | Carbamazepine as a possible anthropogenic marker in the aquatic environment: investigations on the behaviour of Carbamazepine in wastewater treatment and during groundwater infiltration[END_REF]). In addition, CBZ was found to have some inhibition effects on sludge microbial activity (Wang et al., 2008;[START_REF] Aubenneau | Membrane bioreactor for pharmaceutically active compounds removal: Effects of carbamazepine on mixed microbial communities implied in the treatment[END_REF]. In this study, CBZ was added in the feed of MBR to a relatively high concentration of 90 µg L -1 to have an intensified effect to represent the seasonal or accidental variations in concentration of mixtures of pharmaceutical micropollutants present in real municipal wastewater and continuously introduced to MBR via the feed after a steady operation of MBR was achieved. Thus, the objectives are to be able to relate the effects of CBZ on MBR fouling with the change in the characteristics of the biomass and supernatant after adding CBZ in MBR in order to understand the mechanisms of interactions among the CBZ, activated sludge and MBR fouling. Fouling propensity of the sludge and supernatant sampled from the MBR was characterized using some standardized filterability tests performed with a stirred dead-end filtration cell. Fouling was also considered at the scale of the MBR for the mixture of flocs and supernatant during MBR operation and characterized by the time-variation of transmembrane pressure (TMP). In order to explain the effects of CBZ on the fouling comportments, it was thus necessary to obtain some information:

(i) on the biological activity: Respirometric experiments were performed to examine the effects of CBZ on sludge microbial activity;

(ii) on the physico-chemical properties of the sludge (particle size distribution, soluble proteins and polysaccharides, molecular weight distribution of SMPs in the supernatant): Floc size distribution analysis was performed on the activated sludge to study the effects of CBZ on physico-chemical characteristics of biomass.

Concentrations of proteins and polysaccharides were analyzed in supernatant and MBR permeate to study the effects of CBZ on the change of SMP compositions in supernatant and their retention in MBR. HPLC-SEC-fluorescence was used to characterize protein-like SMPs in supernatant and permeate of filterability tests or the MBR, with the information on molecular size distribution.

Material and Methods

Membrane bioreactor set-up

A lab-scale submerged MBR set-up consisting of an anoxic reactor (5.4 L) and an aerobic reactor (12.6 L) was used in this study (see Fig. 1). A flat sheet microfiltration membrane (Lp 0 = 1110 L h -1 m -2 bar -1 at 20 °C, Kubota, Japan), made of chlorinated polyethylene, was submerged inside the aerobic reactor. It has an effective filtration area of 0.1 m 2 , with an average pore size of 0.2 µm. This MBR was operated at the high SRT of 30 days, with a sludge concentration in the aerobic reactor between 6-8 g/L and an organic loading rate at about 0.

Selected pharmaceutical micropollutant

Table 1 introduces the physicochemical properties of carbamazepine. A concentrated mother solution was prepared by dissolving carbamazepine (from Sigma-Aldrich) in pure ethanol (VWR International) at a high concentration of 10 g/L to rule out the effect of ethanol on the organic content in the feed. Then, quantities of this concentrated mother solution sufficient to yield about 90 µg L -1 final concentrations of CBZ were added in the feed tank after the steady state of the MBR reached, i.e. on 84 th day, which is higher than 2 times of SRTs (30 days).

Characterization of sludge microbial activity

Sludge microbial activity was characterized by the specific exogenous respiration rate. The respiration rate, i.e. oxygen uptake rate (OUR), was determined in respirometric batch experiments, defined by Spé randio and Paul (2000), by measuring the dissolved oxygen concentration in activated sludge in an air-tight vessel. The sludge samples were collected from the aerobic tank of the MBR before and after addition of CBZ in MBR. Firstly, the collected sludge was added in the respirometric vessel and aerated for at least 3 h to reach the endogenous condition. Then, sodium acetate (NaAC), a readily biodegradable compound, was used as exogenous substrate, and added in the respirometric vessel to measure the maximum respiration rate. Then the exogenous respiration rate was calculated as the difference between the maximum respiration rate and the endogenous respiration rate (see Fig. 2). The specific exogenous respiration rate is defined as the exogenous respiration rate divided by the concentration of mixed liquor suspended solids (MLSS).

Filterability tests

Filterability tests of sludges and supernatants sampled from MBR were performed by a stirred dead-end filtration cell (Amicon 8050, Millipore). Ultrafiltration membranes (Microdyn-Nadir GmbH, Germany), made of polyethersulfone (PES), with a molecular weight cut-off (MWCO) 150 kDa (Lp 0 = 400-600 L h -1 m -2 bar -1 at 20 °C), were used for the filterability tests because in MBRs that employ microfiltration membranes, the deposits formed on the membrane surface and inside the pores constitute an additional barrier and make the apparent pore size of the fouled microfiltration membrane be in the ultrafiltration (UF) range (Judd, 2010). This 150 kDa PES membrane was chosen also because its permeability is comparable to that of MBR membrane used in this study and its membrane surface is permanently hydrophilic like the MBR membrane, i.e., chlorinated polyethylene, used in this study (Hai et al., 2013). A new membrane sample was used for each filtration test. All membranes were soaked in ultra pure water over night to maintain pores wetted. Then they were rinsed by filtration of ultra pure water at 0.7 bar during 15 min prior to filtration test. All filtration tests were carried out at constant TMP of 0.5 bar. For each filtration test, the sludge or supernatant was filtered through the selected membrane until 70 ml permeate was produced (corresponding to a filtered permeate volume of about 50 L m -2 ); temperature was measured and flux was normalized to the flux at 20 °C. During sludge filtration, the sludge was stirred at 200 rpm. For supernatant filtration, no stirring was applied. Membrane fouling resistance was determined according to Eq. ( 1).

() mf TMP J RR    (1)
In this equation, J is the permeate flux (m 3 /s/m 2 ) at 20 °C, with R m the membrane resistance (m -1 ), R f the fouling resistance (m -1 ), TMP the transmembrane pressure (Pa) and μ the dynamic viscosity at 20 °C (Pa s).

Analytical methods

The concentration of mixed liquor suspended solids (MLSS) was measured according to the standard method AFNOR NFT 90-105. Supernatants were prepared by centrifugation of the sludge samples at 4000 g during 10 minutes at room temperature. Chemical oxygen demand (COD) and total nitrogen (TN) were measured by spectrometry with reagent kits. Floc size distribution was measured by Malvern Mastersizer 2000. Carbamazepine concentration was analyzed using LC-MS/MS method by the Departmental Water Laboratory in Toulouse, France. Quantification of proteins and polysaccharides was performed on the supernatants.

Protein content, expressed in mg equivalent of bovine serum albumin per liter, was measured by the acid bicinchoninic method [START_REF] Smith | Measurement of protein using bicinchoninic acid[END_REF]. Polysaccharide concentration, expressed in mg equivalent of glucose per liter, was measured by the Anthrone method (Dreywood, 1946).

High performance liquid chromatography-size exclusion chromatography (HPLC-SEC)

analysis was performed by an Akta Purifier (GE Healthcare, USA), equipped with a column Protein Kw804 (Shodex, Japan), a silica-based column with an exclusion limit of 1000 kDa, and a fluorescence detector (Varian, USA). The eluent solution for the mobile phase was prepared with 25 mM Na 2 SO 4 and a phosphate buffer (2.4 mM NaH 2 PO 4 and 1.6 mM Na 2 HPO 4 at pH 6.8). The flow rate of eluent passing through the column was 1.0 mL/min.

Excitation/emission wavelengths were set at 280/350 nm, which were confirmed as the required values by EEM fluorescence results [START_REF] Her | Characterization of DOM as a function of MW by fluorescence EEM and HPLC-SEC using UVA, DOC, and fluorescence detection[END_REF]Teychene et al., 2011), to detect protein-like substances. The column was calibrated using standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) from GE Healthcare. Prior to analysis of HPLC-SEC-fluorescence, each sample was pre-filtered on a 0.45 μm membrane.

Results and Discussion

CBZ removal in MBR

As the MBR was fed with a real domestic wastewater, CBZ may be present in the raw wastewater. Actually, the average concentration of CBZ in the raw domestic wastewater was about 0.2-0.3 µg L -1 , which is negligible as compared to the concentration of about 90 µg L -1 in the feed after adding CBZ, as shown in Fig. 3. No removal of CBZ was achieved in the MBR before and after addition of extra CBZ in the feed, which is consistent with the literature [START_REF] Radjenović | arceló, ate and distribution of pharmaceuticals in wastewater and sewage sludge of the conventional activated sludge (CAS) and advanced membrane bioreactor (MBR) treatment[END_REF]. This result also suggests that CBZ itself does not participate to membrane fouling via adsorption onto membrane material. The low removal of the added CBZ could be attributed to its low sorption and biodegradation capacities. Indeed: i) Its sorption constant K d , defined as the partition of a compound between the sludge and the water phase, is 1.2 L/kg ss (see table 1), much lower than the critical value 500 L/kg ss identified for a significant removal by sorption (Ternes et al., 2004;[START_REF] Joss | Removal of pharmaceuticals and fragrances in biological wastewater treatment[END_REF]. Thus the removal of CBZ via sorption to the activated sludge is expected to be of minor importance.

ii)

The biodegradability of CBZ is also very low since its degradation constant k biol , defined as a pseudo first-order reaction constant, is less than 0.01 L/g ss /d (see table 1). Based on the measurement of k biol in batch experiments, [START_REF] Joss | Biological degradation of pharmaceuticals in municipal wastewater treatment: proposing a classification scheme[END_REF] concluded that the pharmaceutical micropollutants with k biol < 0.1 L/g ss /d are not removed to a significant extent (<20%).

Instead, higher average concentration of CBZ was found in the MBR permeate than that in the feed before addition of extra CBZ. According to [START_REF] Vieno | Elimination of pharmaceuticals in sewage treatment plants in Finland[END_REF], who also reported that carbamazepine was constantly found at higher concentrations in the treated water than in the raw water of WWTPs, the elevated concentration in the permeate could be attributed to an enzymatic cleavage of the carbamazepine glucuronides and thus to a release of carbamazepine in the treatment process. However, only a few studies reported the k biol value of CBZ. Thus, the biodegradation of CBZ in MBR remains to be further investigated. For example, Hai et al.

(2011) observed that during near-anoxic operation with a dissolved oxygen concentration in the bioreactor of about 0.5 mg L -1 , an exceptionally high removal (68 ± 10%) of carbamazepine was achieved in the MBR as compared to its low removal efficiency (12 ± 11%) under aerobic conditions.

Effects of CBZ on MBR fouling

Membrane fouling in MBR was characterized by the change of TMP in MBR. Fig. 4 presents the time-variation of TMP during operation of the MBR before and after addition of CBZ in MBR. Three different periods and comportments can be distinguished:

-Before the addition of CBZ in the MBR, quite steady fouling condition (a TMP plateau) was reached, where TMP increases very slightly.

-Between the day when CBZ was added and 8 days after addition of CBZ: 1 day after addition of CBZ in MBR, a significant increase of TMP was observed.

Thanks to the addition of CBZ, the TMP jump was very rapidly created and it clearly appears as a response of the bioseparation process to the effect of a chemical stress on the biomass. Between 1 day and 8 days after addition of CBZ, TMP increased gradually while at a higher rate as compared to that during the steady fouling condition, i.e. before addition of CBZ.

surface and inside the pores. In contrast, almost all the 10-100 kDa protein-like substances passed through the MBR membrane. Therefore, the TMP plateau before addition of CBZ (see Fig. 4) was attributed to the retention of 100-1000 kDa SMPs in MBR and thus to the formation of the gel layer on the membrane surface by protein bridges and bindings, as earlier proposed by [START_REF] Masse | Comparison of sludge characteristics and performance of a submerged membrane bioreactor and an activated sludge process at high solids retention time[END_REF], since no retention of 10-100 kDa protein-like substances in supernatant was found. Concerning to the TMP jump and subsequent increased fouling rate that appeared in MBR after addition of CBZ, three assumptions could be proposed: i) a change in biological activity linked to a stress on the microorganisms ii) a change in biomass physico-chemical properties, like mean floc size distribution, or iii) a change in the supernatant quality. These three assumptions will be discussed in the following paragraphs that aim to evaluate the effects of extra CBZ on the supernatant and biomass properties.

Dissertation on Assumption 1 to explain CBZ enhanced fouling: change in microbial activity

Sludge microbial activity was characterized in a respirometric batch reactor. Fig. 6 shows that the continuous addition of CBZ in the MBR caused about 20% inhibition of the sludge microbial activity, which is expressed as specific exogenous respiration rate, at the beginning several days. The exogenous respiration rate means the degradation rate of exogenous substrate. Thus, the inhibition of exogenous respiration rate caused by CBZ indicates that the presence of 90 µg L -1 CBZ in the MBR changed degradation behavior of bacteria or modified the community structure of bacteria in activated sludge. Whereas, no further inhibition was observed between 4 days and 11 days after addition of CBZ in the MBR, which indicates that some extent of adaption or acclimation of the activated sludge to CBZ stress was reached 4 days after addition of CBZ in MBR. The results indicate that the presence of CBZ caused some stress on the bacteria in the sludge, thus bacterial lysis or bacterial protective responses, which can lead to release of EPS (as shown later in Fig. 8), may occur during the bacterial acclimation to CBZ stress. In this case, deterioration in sludge filterability might happen. The inhibition of sludge microbial activity induced by CBZ give rise to the question like if the inhibition would affect the removal of COD and TN in the MBR. No significant variation was observed for COD and TN removal before and after addition of CBZ in MBR, which suggests that the presence of 90 µg L -1 CBZ in MBR did not significantly affect the biodegradation, nitrification and denitrification processes for an MBR operated at the organic loading rate of 0.2 kg COD/kg MLSS/d, in spite of a slight inhibition of microbial activity in the sludge.

In conclusion, the addition of CBZ in MBR induced a slight inhibition of sludge microbial activity, which may indirectly cause the enhanced membrane fouling via modification of EPS concentration in sludge. However, taking into account that no significant change in COD biodegradation or in TN removal was observed after addition of CBZ in the MBR, the effect of CBZ on the sludge microbial activity was probably not significant. to hinder access of the toxin to the bacterial cells. The increase of protein content in sludge (see Fig. 8) may increase the hydrophobicity of flocs and lead to deterioration in sludge filterability. In addition, the possibility of increase in small flocs after addition of CBZ could not be completely ruled out, since deflocculation may also happen for activated sludge exposed to toxic chemicals [START_REF] Henriques | The role of extracellular polymeric substances in the toxicity response of activated sludge bacteria to chemical toxins[END_REF], and some small flocs, for example, flocs at size of near micro or sub-micro level, are difficult to be detected. In this case, the deterioration in sludge filterability might also happen.

Dissertation on

Dissertation on Assumption 3 to explain CBZ enhanced fouling: change in supernatant characteristics

The effects of CBZ on concentration of proteins and polysaccharides in the MBR supernatant

were examined due to their potential fouling capacity. Fig. 8 shows that the addition of CBZ caused a significant increase in the protein concentration in supernatant at the beginning several days as compared to concentration level before addition of CBZ, and then reduced effect on protein production was observed after about 7 days after the addition of CBZ in MBR. In contrast, no significant change was observed for the polysaccharide concentration in supernatant. This result indicates that microorganisms mainly released protein-like SMPs when facing a pharmaceutical stress or the released polysaccharides were easily biodegraded.

Similar results were observed concerning the effects of other chemical stress. Zhou et al.

(2014) reported that the MBR fed with municipal wastewater containing 0.4 mg L -1 Cr (VI) had a higher concentration of SMPs in the supernatant, especially the protein content, as compared with the MBR (MBR-control) fed with municipal wastewater without addition of Cr (VI). The reduced protein production after about 7 days after the addition of CBZ may be caused by the acclimation of activated sludge to the pharmaceutical stress, as shown in Fig. 6.

No significant increase in retention of protein in MBR was also observed after the significant increase of protein concentration in supernatant (see Fig. 8), which indicates that the increased protein were mainly relatively small molecules that could pass through the MBR membrane and biocake layer. To further investigate the effects of CBZ on the release of protein-like SMPs, HPLC-SECfluorescence analysis of supernatants was performed. Fig. 9 shows the results of HPLC-SEC analysis of MBR supernatants 1 day (83 th day) before, the day (84 th day) just before and 1 day (85 th day) after adding CBZ in the MBR. The results showed that the continuous presence of 90 µg L -1 CBZ in MBR could cause two measurable effects on SMPs just 1 day after addition of CBZ:

-a decrease of the concentration in 100-1000 kDa protein-like SMPs. More specifically, 1 day after addition of CBZ in the MBR a significant decrease, about 16%, of 100-1000 kDa protein-like SMPs was observed in the MBR supernatant, as compared to that in the MBR supernatant that was obtained 1 day before addition of CBZ and the one obtained the day just before addition of CBZ in the MBR.

-a more significant increase in 10-100 kDa protein-like SMPs. More specifically, for the 10-100 kDa protein-like SMPs, a quite significant increase, about 23%, was observed in the MBR supernatant 1 day after addition of CBZ as compared to that in the MBR supernatant that was obtained 1 day before addition of CBZ and the day just before addition of CBZ. Elution volume (mL) Fig. 9. HPLC-SEC analysis of MBR supernatants before and 1 day after addition of CBZ Taking into account that inhibition of the microbial activity occurred after addition of CBZ in the MBR (see Fig. 6), the decrease of 100-1000 kDa protein-like substances in the MBR supernatant 1 day after addition of CBZ might be explained by an inhibition of the microbial activity. [START_REF] Ni | Fractionating soluble microbial products in the activated sludge process[END_REF] reported that BAP, which was produced from decay of bacteria, could be further classified into the growth-associated BAP (GBAP), which are produced in the microbial growth phase, and the endogeny-associated BAP (EBAP), which are generated in the endogenous phase. Therefore, the inhibition of microbial activity might reduce the production of growth-associated BAPs in the supernatant which is considered as having a molecular weight higher than 100 kDa. Moreover, [START_REF] Zhou | Role of microorganism growth phase in the accumulation and characteristics of biomacromolecules (BMM) in a membrane bioreactor[END_REF] reported that the MBR sludge in the rapid growth phases with high microbial activities produced significantly higher BAPs than the MBR sludge in stationary growth phase and more large molecular weight compounds (higher than 100 kDa) were observed in MBR supernatant in the rapid growth phase. Whereas, the significant increase of 10-100 kDa protein-like substances in the MBR supernatant 1 day after addition of CBZ may be caused by the defensive response of microorganisms to release SMPs when facing a chemical stress, since molecules of this size are not in the range of BAPs. Similarly, [START_REF] Avella | Effect of cytostatic drug presence on extracellular polymeric substances formation in municipal wastewater treated by membrane bioreactor[END_REF] found that the continuous addition of 5 µg L -1 cytostatic drug cyclophosphamide and its main metabolites in a MBR caused a significant increase of proteins in supernatant, with molecular weight of about 18 kDa.

Fig. 10 shows the results of HPLC-SEC analysis of MBR supernatants at 1 day and 7 days after adding CBZ in the MBR. Reduced CBZ effects on production of both 100-1000 kDa and 10-100 kDa protein-like SMPs in supernatant were observed between 1 day and 7 day after addition of CBZ in MBR, i.e. a slight decrease of 100-1000 kDa protein-like SMPs and a slight increase of 10-100 kDa protein-like SMPs in the MBR supernatants, as compared to that in the MBR supernatant 1 day after addition of CBZ (see Fig. 9). This also suggested the adaptation of MBR biomass to the pharmaceutical stress, as indicated in Fig. 6. SMPs was obtained by the MBR membrane 1 day after addition of CBZ. Reminding that no retention of 10-100 kDa protein-like SMPs by the MBR membrane was observed on the day (84th day) just before addition of CBZ in MBR (see Fig. 5), this result indicated that the retention characteristics of M "membrane" did not change significantly just 1 day after addition of CBZ. However, Fig. 12 showed that enhanced retention of 10-100 kDa proteinlike SMPs by the MBR membrane was obtained 11 days after addition of CBZ, as compared to that at 1 day after addition of CBZ. Thus, it could be hypothesized that the significant increase of 10-100 kDa protein-like SMPs in MBR supernatant after addition of CBZ induced an enhanced accumulation of these small biopolymers inside the biocake that was formed on MBR membrane surface, which caused the loss of voids in the biocake, and finally led to a more "tight" retention characteristics of the M "membrane" and enhanced membrane fouling. In conclusion, the continuous introduction of CBZ in MBR caused significant variation in 10-100 protein-like SMPs, which could be related to the change in membrane fouling. The TMP jump occurring just 1 day after the addition of CBZ in MBR could be probably attributed to the significant increase of 10-100 kDa protein-like SMPs in the supernatant. And the higher increase rate of TMP, not the continuous TMP jump, after 1 day after addition of CBZ in MBR as compared to that before addition of CBZ could be probably attributed to the reduced increase in 10-100 kDa protein-like SMPs in the supernatant. The continuous TMP jump starting from 8 days after addition of CBZ might be due to the convective flow of biomass, which could be caused by the enhanced fouling that leads the local flux to exceed the critical value, and to the compressing effect of biocake at increasing local TMP.

Effects of CBZ on sludge and supernatant filterability

In order to further understand the effect of CBZ on MBR fouling, the sludge and supernatant filterabilities before and after addition of CBZ in MBR were examined in filterability tests using a dead-end filtration cell. Fig. 13 shows that the fouling propensity of sludge, expressed as the fouling resistance produced when the fixed amount of permeate volume was filtered, increased after the continuous addition of CBZ in the MBR. This result further demonstrated that the modification in the biomass physico-chemical characteristics and supernatant quality mentioned in the above sections after addition of CBZ in sludge could lead to the deterioration in sludge filterability. Since after addition of CBZ in MBR, a decrease in 100-1000 kDa protein-like SMPs and an more significant increase in 10-100 kDa protein-like SMPs occurred at the same time (see Fig. 9), this results also suggests the important role of small biopolymer in the sludge fouling propensity. )

Permeate volume (m 3 /m 2 ) Fig. 13. Effect of CBZ on sludge filterability Fig. 14 shows the change of supernatant filterability after addition of CBZ in MBR. The result showed that the fouling propensity of supernatant decreased after the continuous addition of CBZ in the MBR. This could be attributed to the decrease of 100-1000 kDa protein-like SMPs in the supernatant after addition of CBZ in MBR (see Fig. 9), since in supernatant filtration, the 10-100 kDa protein-like SMPs in supernatant could pass through the 150 kDa PES membrane. Thus, the fouling mainly originated from the retention of 100-1000 kDa SMPs in supernatant by the 150 kDa PES membranes and thus the formation of the gel layer on the membrane surface. This result further suggests that, when considering the change induced in supernatant quality after addition of CBZ, the increase in sludge fouling propensity was not originated from the change in 100-1000 kDa protein-like SMPs, but from the increase in 10-100 kDa protein-like SMPs.

Conclusions

Understanding fouling mechanisms of membranes in MBRs, and particularly in presence of domestic pharmaceutical micropollutants is still a key issue. In this study, the effects of a pharmaceutical micropollutant, carbamazepine, on membrane fouling of a submerged MBR treating domestic wastewater were investigated. CBZ was continuously added in the MBR via feed (90 µg L -1 in the feed). A TMP jump occurred just 1 day after the addition of CBZ in MBR and a significantly higher increase rate of TMP was also observed after 1 day after addition of CBZ in MBR, as compared to that before addition of CBZ. This indicates that the pharmaceutical stress induced by CBZ causes more severe membrane fouling, which could be related to the change in biomass and supernatant characteristics. The continuous introduction of CBZ in the MBR led to a significant increase of protein content in the MBR supernatant, whereas no significant difference of polysaccharide content in supernatant was observed.

Besides, the increased protein-like compounds were mainly in the 10-100 kDa range, which may be caused by the protection mechanisms of microorganisms when facing a pharmaceutical stress. In contrast, a decrease of 100-1000 kDa protein-like compounds in the supernatant was observed after addition of CBZ, which may be due to the reduced microbial activity. The increase of protein-like SMPs in 10-100 kDa range may cause the accumulation of 10-100 kDa protein-like compounds inside the biocake formed on MBR membrane surface.

In addition, the continuous presence of CBZ in the activated sludge caused a slight inhibition in sludge exogenous respiration rate and a slight increase in mean sludge floc size. Thus, the modification of biomass biological and physico-chemical characteristics together with the change in supernatant quality could modify the composition and structure of biocake and lead to a significant increase of TMP in MBR.

This study may help to understand the effects of mixtures of pharmaceutical micropollutants on MBR fouling, since with mixtures of pharmaceutical micropollutants enhanced effects are expected on the sludge microbial activities, biomass physico-chemical characteristics and on the release of SMPs by the bacteria under stress, as compared to the effect of CBZ alone.

More studies should also be carried out with the objectives to avoid the TMP-jump induced by pharmaceutical stress, for example by modifying operating operations to develop sludge that is less sensitive to the pharmaceutical stress. Or hybrid MBR processes, like MBR-PAC process where powdered activated carbon (PAC) is added in MBR to enhance removal of pharmaceutical micropollutants via adsorption, could be proposed to reduce the contact of activated sludge with pharmaceutical micropollutants, thus reducing the stress effect induced by pharmaceutical micropollutants.

Introduction

Membrane bioreactors, which couple the activated sludge process with membrane filtration, greatly increase the treated effluent quality as compared to conventional activated sludge process. Thus, they have been increasingly used for wastewater treatment and reuse applications [1][2]. The other advantages of MBRs include smaller footprint or less excess sludge production, depending on the operating conditions, and flexibility of process design, i.e. independent control of sludge retention time (SRT) and hydraulic retention time (HRT) [3]. However, membrane fouling is still a key scientific issue and a limitation for the development of the MBR process, because of the increase in operating costs and also the potential decrease in membrane lifetime.

The decline of permeability in MBR could be caused by clogging, which involves filling of the channels between the membranes with sludge solids, or membrane fouling [4][5].

Membrane fouling in MBR was caused by biomass deposit, biofilm and pore blocking due to particles, colloids and solute adsorption [6][7][8]. Due to gas-liquid two-phase cross flow generated by aeration in submerged MBRs or liquid cross flow induced by recirculation pump in side-stream MBRs, the low flux operation and the relatively easy removal of cake deposit, cake deposit formation is of minor importance on MBR fouling in long-term operation. Many studies showed that the supernatant of activated sludges (which contains colloids and solutes) plays a more important role in membrane fouling of MBR than biological flocs [9][10].

Soluble microbial products (SMP), the major organic constituents of sludge supernatants [11],

have been acknowledged as major foulants that lead to membrane fouling in MBRs [12][13][14].

SMP have been identified as soluble extracellular polymeric substances (EPS) which are composed of proteins, polysaccharides, humic substances, nucleic acids etc. [11,[15][16]. SMP could be divided into two categories: utilization-associated products (UAP), which are produced during substrate metabolism, and biomass-associated products (BAP) which are formed from decay of biomass [11,17]. The composition and concentration of SMP is significantly affected by operating conditions, for example, organic loading rates [18][19],

sludge age [20][21][22] or hydraulic conditions [23][24], and by the presence of chemicals in the feed that can affect the biological activity [25][26][27].

Conflicting observations were often reported in the literature concerning the relative importance of SMP components on fouling in MBRs. Some authors attributed protein-like substances to be the main foulant responsible for fouling [14,22,[28][29]. By HPLC-SEC and fluorescence analysis, Teychene et al. [30] demonstrated foulant species of MBR to be protein-like substances. On the basis of statistical analysis of sample analytical results, protein-like substances were also found in a study to be highly correlated to the fouling potential of two MBR supernatants [31]. In contrast, some other authors related a preponderant MBR fouling with polysaccharide-like substances in supernatant [12,32].

Besides, some studies showed that proteins were observed to dominate in foulants obtained from the fouled membranes of MBRs [33] and are preferentially attached to the membrane surface and membrane pores, whereas carbohydrates are enriched in the intermediate layer of cake deposit [34].

However, simply correlated the fouling with protein or polysaccharide concentration in the supernatant or their retention in MBR could not explain the contribution of each component in fouling, since some SMP components may be retained by the cake layer, and may not reach to the membrane surface or the membrane pores [34]. Thus, in MBRs, fouling is not only affected by the SMP concentration in the sludge supernatant, but is also affected by molecular weight (MW) distribution. Some studies found that the major responsible for fouling are the SMP compounds with a molecular size higher than 100 kDa [12,35], whereas in another study the compounds with a molecular size 10-100 kDa were observed to cause the main contribution the fouling propensity [36]. Similarly, the UAP was reported to have higher specific cake resistance and pore blocking resistance than BAP, although their retention percentage was lower than BAP due to their higher percentage of low molecular weight molecules [37]. The common shared point is that the SMP compounds that cause the major fouling are in biopolymer range (MW higher than 10 kDa) and the fouling is caused by biofilm (gel) layer formation or pore blocking. The divergences concerning the contribution of biopolymers in different MW range to membrane fouling may be caused by the differences in the pore size of membranes used for the fouling propensity tests and the methods used for biopolymer MW distribution analysis.

Fouling propensity of biological fluids is currently estimated by a calculation of fouling resistance, permeability or fouling rate obtained from an analysis of operating data (permeate flux, TMP) [38][39][40]; by batch stirred dead-end filtration cells fed with samples of the sludge or supernatants [35,[41][42][43]; by batch tangential modules (ex situ) operated under standardized operating conditions and continuously fed with wastewater effluents [44] or the sludge [45];

and by some submerged modules placed directly inside MBR (in-situ) and using aeration to generate cross-flow conditions [46][47], which could avoid the problems like the change of sludge filterability during store period. In the case of a fouling occurring as a cake deposit, some recent works have proposed in-situ fouling characterization filtration apparatus equipped with acoustic or optical sensors [48][49][50][51]. The later method was studied at lab scale with diluted sludges, but has not been yet applied to in-line detection of membrane fouling in MBR [52]. Still, there is a great interest in the industry to find some simple and cheap methods that could allow estimating the fouling propensity of biological fluids such as MBR Membrane fouling resistance was determined according to Eq. ( 1).

() mf TMP J RR    (1)
In this equation, J is the permeate flux (m 3 /s/m 2 ) at 20 °C, with R m the membrane resistance (m -1 ), R f the fouling resistance (m -1 ), TMP the transmembrane pressure (Pa) and μ is the dynamic viscosity at 20 °C (Pa s).

Then in the dead-end filtration cell, C can be determined by cake deposit model as expressed in Eq. (2).

f V RC A  (2)
In this equationis the specific cake resistance (m kg -1 ), C is the deposited mass per filtered permeate volume (kg m -3 ), V is the cumulated permeate volume (m 3 ) and A is the effective membrane area for filtration (m 2 ). For supernatant filtration, C could be calculated by the retained DOC (kg m -3 ).

Results

Fouling in MBR versus HPLC-SEC results

Fouling in MBR was characterized by the decline of membrane permeability during MBR operation. Fig. 3 presents the time-variation of permeability during operation of the MBR with the numbers in the figure to indicate the sampling of supernatants (sample 1, 2, 3 and 4).

The MBR was operated under varied organic loading rates. Between every two successive sets, membrane cleaning was performed to recover the permeability. As shown in Fig. 3, when the organic loading rate was increased from 0. To investigate the MW range of biopolymer that is responsible for MBR fouling, HPLC-SECfluorescence analysis of a supernatant and permeate obtained from the MBR was performed, as shown in Fig. 4. The molecular weight of protein-like substances in MBR supernatants showed bimodal distribution. Indeed, two peaks were observed in the HPLC-SEC results which represent two groups of protein-like molecules, i.e. molecular size of 100-1000 kDa and 10-100 kDa, respectively. Since Her et al. [54] and Huber et al. [62] identified components of SMP with molecular weight higher than 10 kDa as the biopolymer fraction, the two detected peaks are both in the range of biopolymers. As shown in Fig. 4, the proteinlike substances in supernatant with molecular size of 100-1000 kDa could be almost completely retained by the MBR membrane, whereas almost all of the protein-like substances with molecular size of 10-100 kDa passed through MBR membrane. This indicates that the 100-1000 kDa protein-like SMPs may play an important role in MBR fouling through the biofilm formation. To investigate the relation between 100-1000 kDa protein-like SMPs and MBR fouling, HPLC-SEC-fluorescence analysis of supernatants sampled from MBR under different organic loading rates was performed. As shown in Fig. 5, a significant increase of protein-like substances with molecular size of 100-1000 kDa was observed in MBR supernatant, from sample 1 and sample 2 to sample 3 when the organic loading rate of MBR was increased, from 0.1 kg COD/kg MLSS/d to 0.2 kg COD/kg MLSS/d, and also from sample 3 to sample 4 when 0.2 kg COD/kg MLSS/d to 0.4 kg COD/kg MLSS/d. This may be explained by a growth of the microbial activity in activated sludge when the organic loading rate was increased. Zhou et al. [63] reported that the MBR sludge in the rapid growth phases with high microbial activities produced significantly higher BAP than the MBR sludge in stationary growth phase and more large MW compounds (higher than 100 kDa) were observed in MBR supernatant in the rapid growth phase. Ni et al. [64] reported that BAP could be further classified into the growth-associated BAP (GBAP), which are produced in the microbial growth phase, and the endogeny-associated BAP (EBAP), which are generated in the endogenous phase. Therefore, the growth of microbial activity might increase the growthassociated BAPs in the supernatant which is considered as having a molecular size between 100-1000 kDa. Taking into account the almost complete retention of 100-1000 kDa proteinlike SMPs in supernatant by MBR membrane (see Fig. 4), the significant increase in proteinlike 100-1000 kDa SMPs may lead to the higher fouling rate of MBR membrane, as indicted in Fig. 3. Thus, HPLC-SEC-fluorescence could characterize the variation of 100-1000 kDa protein-like SMPs when the change in operating conditions of MBR occurs and help to estimate the fouling in MBR that is caused by variation of 100-1000 kDa protein-like SMPs in MBR supernatant. In Fig. 5, a peak of protein-like compounds with elution volume between 14 mL to 16 mL was also observed in sample 4. The molecular size of compounds in this elution volume is less than 10 kDa and their effect on fouling of MBR is of minor importance. As the peak height is a simple and effective indicator to stand for the fluorescence intensity and to quantify the constituent content by the HPLC-SEC-fluorescence analysis [54] and almost complete retention of protein-like compounds with molecular size of 100-1000 kDa in supernatant is obtained by MBR membrane as indicated in Fig. 4, the fluorescence peak height of 100-1000 kDa protein-like compounds could be considered as a fouling indicator to estimate fouling in MBRs.

Fouling propensity of supernatants versus HPLC-SEC results

To further validate the relation between fouling propensity of supernatants and fluorescence peak height of the protein-like compounds with molecular size of 100-1000 kDa detected by HPLC-SEC-fluorescence, filterability tests of the same supernatant samples obtained from MBR under different fouling conditions were performed together with HPLC-SECfluorescence analysis. To investigate the fouling propensity of supernatant samples, the filterability tests of supernatant samples (sample 1, 2, 3 and 4) obtained from MBR under different fouling situations using 0.01μm PES membranes were performed at 1.0 bar, as shown in Fig. 7.

Considering the content of 100-1000 kDa protein-like SMPs in these supernatant samples (see Fig. 5), the results from Fig. 7 indicates that supernatant with higher peak height of 100-1000 kDa protein-like substances had higher fouling rate, defined as the fouling resistance produced for a fixed volume of permeate filtered during the filtration tests. Notably, the fouling rate of supernatant samples from set 3 and set 4 were significantly higher than that from set 1 and set 2. -Une diminution de la concentration de proté ines de taille molé culaire de 100-1000 kDa.

Plus pré cisé ment, un jour aprè s l'addition de la CBZ dans le BAM, une diminution importante (environ 16%) de proté ines de 100-1000 kDa a é té observé e dans le surnageant du BAM.

Ensuite, seulement une lé gè re baisse de 100-1000 kDa proté ines a é té observé e entre 1 jour et 7 jours aprè s l'addition de la CBZ.

-Une augmentation plus importante de proté ines de taille molé culaire de 10-100 kDa.

Plus pré cisé ment, un jour aprè s l'addition de la CBZ dans le MBR, une augmentation trè s significative de proté ines de 10-100 kDa, environ 23%, a é té observé e dans le surnageant du 201 BAM. Seule une lé gè re augmentation de 10-100 kDa proté ines a é té observé e dans le surnageant du BAM entre 1 jour et 7 jours aprè s l'addition de la CBZ dans le BAM.

Par ailleurs, é tant donné que la ré tention des proté ines de taille molé culaire de 100-1000 kDa est quasi-complè te, que celle des proté ines de taille molé culaire de 10-100 kDa est partielle lors des essais de filtrabilité de boues, et que une diminution de proté ines avec une taille molé culaire de 100-1000 kDa et une augmentation plus important de proté ines avec une taille molé culaire de 10-100 kDa est observée après l'ajout de Z, on peut conclure que l'augmentation de la ré sistance de colmatage est issue d'une modification par les proté ines de 10-100 kDa de la structure du dé pôt formé lors de la filtration de la boue.

Effets de CBZ sur les caracté ristiques de dé pôt du BAM

Les caracté ristiques de dé pôt du BAM pourraient ê tre examiné es par la ré tention des PMS dans le surnageant. Comme repré senté sur la Fig. 8, l'analyse HPLC-SE a montré qu'avant d'ajouter la CBZ, la membrane du BAM retient presque toutes les proté ines de 100-1000 kDa.

En revanche, presque toutes les proté ines de 10-100 kDa ont traversé la membrane du BAM.

Cependant, 11 jours aprè s addition de la CBZ dans le BAM, les proté ines de 10-100 kDa dans le surnageant ont é té partiellement retenues par la membrane du BAM, comme le montre dans la Fig. 9. On peut supposer que l'augmentation importante des proté ines de 10-100 kDa dans le surnageant du BAM aprè s addition de la CBZ induit une accumulation de ces petits biopolymè res à l'inté rieur du dé pôt, qui ré duit donc sa porosité et induit finalement une ré tention plus importante de ces composé s dans le BAM.

(

  ii) on the physico-chemical properties of the sludge (particle size distribution, soluble proteins and polysaccharides, molecular weight distribution of SMPs in the supernatant): Floc size distribution analysis was performed on the activated sludge to study the effects of CBZ on physico-chemical characteristics of biomass. HPLC-SECfluorescence was used to characterize protein-like SMPs in supernatant and permeate of filterability tests or MBR, with the information on the fluorescence intensity in different molecular size ranges. Concentrations of proteins and polysaccharides were analyzed in supernatant and MBR permeate to study the effects of CBZ on the change of SMP composition.

Fig. 4 .

 4 Fig. 4. Schema of batch experiments

Fig. 5

 5 shows the schematic diagram of the filterability test. In the study of short-term CBZ effect, microfiltration membranes (from Alfa Laval, FR), made of polysulphone (PS), with a pore size of 0.2 μm (Lp 0 = 200 l h -1 m -2 bar -1 at 20 °C), were used for the filterability tests of sludge, because a pore size of 0.2 μm is in the range of pore size of microfiltration membranes used in the flat sheet MBRs. Whereas, ultrafiltration membranes (from Orelis, FR), made of polyethersulfone (PES), with a nominal pore size of 0.01 μm (Lp 0 = 50-80 L h -1 m -2 bar -1 at 20 °C) were used for the filterability tests of supernatant to retain biopolymer components in the MBR supernatant, behaving like the second layer on MBR membrane surface. A new membrane sample was used for each filtration test. All membranes were soaked in ultra pure water over night to maintain pores wetted. Then they were rinsed by filtration of ultra pure water at 1 bar during 15 min prior to filtration test. All filtration tests were carried out in an Amicon cell (Millipore), with an effective membrane filtration surface of about 13.4 cm2 . The filtration tests were performed at three constant transmembrane pressures (TMP): 0.5 bar, 0.8 bar, and 1.0 bar. For each filtration test, sludge or supernatant was filtered through selected membranes until 70 ml permeate was produced (corresponding to a filtered permeate volume of about 50 L m -2 ); temperature was measured and flux was normalized to the flux at 20 °C. For supernatant filtration, no stirring was performed, and for sludge filtration, the sludge was stirred at 200 rpm. After filtration the membrane was turned face down and was backwashed with ultra pure water at 1 bar for 15 min, in order to investigate flux recovery by permeability measurement.

Fig. 5 .

 5 Fig. 5. Schematic diagram of respirometric analysis

Fig. 6 .Fig. 7 .

 67 Fig. 6. Short-term effect of CBZ on sludge fouling propensity (C m = 0.1 kg/kg/d, CBZ = 100 µg L -1 )

Fig. 8 .Fig. 9 4 . 1 . 2

 89412 Fig. 8. Long-term CBZ effect on sludge fouling propensity (C m = 0.2 kg/kg/d, CBZ = 90 µg L -1 )

Fig. 10 .

 10 Fig. 10. Effect of CBZ on TMP of MBR

Fig. 11 .

 11 Fig. 11. Long-term effect of CBZ on sludge specific exogenous respiration rate

Fig. 12 .

 12 Fig.12. Short-term effect of CBZ on SMP composition in supernatant

Fig. 13 .

 13 Fig.13. Long-term effect of CBZ on SMP concentration in supernatant and permeate of MBR

Fig. 14 .

 14 Fig. 14. Short-term effect of CBZ on protein-like SMPs in supernatant (C m = 0.1 kg COD/kg MLSS/d)

Fig. 15 .

 15 Fig. 15. Short-term effect of CBZ on protein-like SMPs in supernatant (C m = 0.2 kg COD/kg MLSS/d)

Fig. 16 .

 16 Fig.16. Long-term effect of CBZ on protein-like SMPs in supernatant of MBR

Fig. 19 Fig. 20

 1920 Fig. 19 HPLC-SEC analysis of MBR supernatant and permeate before adding CBZ in MBR

Fig. 21 .

 21 Fig. 21. Biocake compressibility test (MLSS=6.2 g/L)

4. 5 . 1

 51 In case of fouling situations modified by organic loading rates In this case (see paper 4), supernatants sampled from MBR operated under different organic loading rates were analysed by HPLC-SEC-fluorescence. Then the modifications in HPLC-SEC-fluorescence results were compared with the membrane fouling rate in the MBR. Fig. 22 presents the time-variation of permeability during operation of the MBR with the numbers in the figure to indicate the sampling of supernatants. Result from Fig. 22 showed when the organic loading rate was increased from 0.1 kg COD/kg MLSS/d (set 1 and set 2) to 0.2 kg COD/kg MLSS/d (set 3), and to 0.4 kg COD/kg MLSS/d (set 4), increased average fouling rate, expressed as the decrease rate of permeability, in MBR was observed, i.e. from about 40 L/h/m 2 /bar/d (set 1 and set 2) to about 90 L/h/m 2 /bar/d (set 3) and to about 140 L/h/m 2 /bar/d (set 4).

Fig. 22 .

 22 Fig. 22. Permeability of MBR operated under varied organic loading rates

Fig. 23 .

 23 Fig.23. HPLC-SEC analysis of supernatants of MBR under different organic loading rates

Fig. 24

 24 Fig. 24 Peak heights versus the C values of supernatants

Fig. 25

 25 Fig. 25 Peak heights versus the C values of supernatants

Fig. 1 . 2 . 2

 122 Fig. 1. Schema of batch experiments

standard method AFNOR NFT 90 - 105 .Fig. 2 .

 901052 Fig. 2. Effect of CBZ on sludge fouling propensity (C m = 0.1 kg COD/kg MLSS/d)

Fig. 3 . 3 . 1 . 2 Fig. 4 .

 33124 Fig. 3. Effect of CBZ on sludge microbial activity

Fig. 5 .

 5 Fig. 5. Effect of CBZ on supernatant composition

Fig. 6 .

 6 Fig. 6. Effect of CBZ on protein-like SMPs in supernatant (C m = 0.1 kg COD/kg MLSS/d)

Fig. 7 .

 7 Fig.7. HPLC-SEC analysis of sludge supernatant and permeate from a sludge filterability test

110

 110

  Fig.8HPLC-SEC analysis of supernatant and permeate from a supernatant filterability test

Fig. 9 .

 9 Fig. 9. Effect of CBZ on sludge fouling propensity (C m = 0.2 kg COD/kg MLSS/d)

Fig. 10 .

 10 Fig. 10. Effect of CBZ on protein-like SMPs in supernatant (C m = 0.2 kg COD/kg MLSS/d)

  Membrane bioreactors (MBRs), which couple the activated sludge process with membrane separation, have been increasingly used in wastewater treatment and reuse due to their high effluent quality. However, membrane fouling remains a major limitation for the development of MBR process. Membrane fouling in MBRs involves i) biocake fouling on the membrane surface due to the deposit of suspended solids in relation with the convective flow of biological fluids and to the microbial growth and to the excretion of biopolymers, ii) pore blocking by particles, colloids and iii) adsorption of solutes onto the membrane material (Ognier et al., 2002; Le-Clech et al., 2006; Meng et al., 2009). Very often, three stages of fouling are proposed for MBRs when they are operated under constant flux condition: conditioning fouling caused by initial pore blocking and adsorption of solutes, steady fouling occurring due to biocake formation and further pore blocking, and TMP jump. This last phenomenon is observed in MBRs and the mechanisms involved in its occurrence are not completely elucidated. Different possible causes for TMP jump have been suggested: -Due to a successive closure of pores, local flux starts to exceed the critical value and particles begin to deposit (Ognier et al., 2004); -Coagulation occurs in the first layers of the gel layer at a critical pressure (Chang et al., 2006); -Percolating colloids reduce the size of the biocake voids until connections are lost (Drews, 2010);

  major foulants that lead to membrane fouling in MBRs (Hernandez Rojas et[START_REF] Lesage | Etude d'un procédé hybride dsorption/ ioréacteur à membranes pour le traitement des effluents industriels[END_REF][START_REF] Rosenberger | Impact of colloidal and soluble organic material on membrane performance in membrane bioreactors for municipal wastewater treatment[END_REF]. Recently, SMPs were identified as soluble extracellular polymeric substances (EPS) and are mainly composed of proteins, polysaccharides and humic substances[START_REF] Liang | Soluble microbial products in membrane bioreactor operation: behaviors, characteristics, and fouling potential[END_REF]. Among these components, humic SMPs are generally believed to be not relevant to MBR fouling due to their small molecular size as compared with that of proteins and polysaccharides. In addition, SMPs could be further divided into two categories: utilization-associated products (UAPs), which are produced during substrate metabolism, and biomass-associated products (BAP), which are produced from decay of biomass. However, simply correlating the MBR fouling with protein or polysaccharide concentration in the supernatant or even their retention in MBR could not explain the contribution of each component in MBR fouling, since some SMP components may be retained by the cake layer and may not be attached on membrane surface or in membrane pores[START_REF] Metzger | Characterisation of polymeric fouling in membrane bioreactors and the effect of different filtration modes[END_REF]. Thus, MBR fouling is also affected by molecular weight (MW) distribution of SMPs[START_REF] Arabi | Impact of molecular weight distribution of soluble microbial products on fouling in membrane bioreactors[END_REF][START_REF] Jiang | Characterization of soluble microbial products and their fouling impacts in membrane bioreactors[END_REF]. Besides, the physiochemical properties like relative hydrophobicity of SMP components may also significantly affect the MBR fouling. For example, proteins were reported to dominate in foulants desorbed from the fouled membranes of MBRs due to their hydrophobic properties (Yamato et al., 2006). In the literature, factors affecting the SMP production in MBR supernatant may include operating conditions such as organic loading rate (Kimura et al., 2005; Trussell et al., 2006), sludge retention time (SRT) (Masse et al., 2006; Al-Halbouni et al., 2008) and hydraulic conditions like aeration and mechanical stirring (Menniti et al., 2009; Stricot et al., 2010), and the presence of environmental stress in the wastewater such as heavy metals (Zhou et al., 2014), osmotic pressure (salts) (Sun et al., 2010), and industrial compounds like pharmaceuticals (Lay et al., 2012), due to the defensive response or decay (lysis) of bacteria when facing chemical tress (Holakoo et al., 2006; Henriques and Love, 2007).In the last decade, great attention has been paid to the presence of pharmaceutical micropollutants in the domestic wastewater due to their potential adverse effects on human health(Verlicchi et al., 2012). Many pharmaceutical micropollutants, such as analgesics like ibuprofen, diclofenac, naproxen and ketoprofen, lipid regulators like bezafibrate and gemfibrozil, and the antiepileptic drug carbamazepine, have been frequently detected in domestic wastewater at concentration higher than 1.0 µg/L and their removal in MBR process has been extensively investigated[START_REF] Li | Evaluation of membrane bioreactor on removal of pharmaceutical micropollutants: a review[END_REF]. However, few studies were carried out to examine the effects of pharmaceutical micropollutants on MBR fouling. Nevertheless, some studies reported that the presence of pharmaceutical micropollutants could induce some microbial effects on activated sludge.Lay et al. (2012) observed a significant increase in the protein and polysaccharide ratio in supernatant after addition of carbamazepine, diclofenac, ibuprofen and naproxen in the feed of an Osmotic Membrane Bioreactor (20-25 µg L -1 of each pharmaceutical in the feed). Delgado et al. (2010) reported the continuous introduction of 5 µg L -1 cytostatic drug cyclophosphamide and its principal metabolites (CPs) in MBR caused the inhibition in exogenous respiration rate of activated sludge and increased its mean floc size. Through the terminal restriction fragment length polymorphism (T-RFLP) analysis of the bacterial 16S rRNA genes, Kraigher et al. (2008) observed shifts in the bacterial community structure in activated sludge of a bioreactor continuously fed with synthetic wastewater containing pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid) at a concentration of 50 μg L -1 , as compared to the control reactor, which was operated without addition of pharmaceuticals.

FeedFig. 1 .

 1 Fig. 1. MBR set-up used in this study

Fig. 2 .

 2 Fig. 2. The principle of respirometric experiments

Fig. 3 .

 3 Fig.3. The CBZ concentration in the feed and permeate of MBR before and after addition of extra CBZ

Fig. 5 .

 5 Fig.5. HPLC-SEC analysis of supernatant and permeate of MBR before adding CBZ in MBR

Fig. 6 .

 6 Fig.6. Effect of CBZ on sludge exogenous respiration rate

  Assumption 2 to explain CBZ enhanced fouling: change in biomass physico-chemical properties Sludge floc size distribution represents an important indicator of biomass characteristics and sludge filterability. Fig. 7 shows that after adding CBZ in the MBR, the mean floc size increased slightly from 128 µm to 161 µm. Similar result was observed by Delgado et al., (2010), who reported the continuous introduction of 5 µg L -1 cytostatic drug cyclophosphamide in MBR caused increase in mean floc size of activated sludge. This means that the presence of CBZ caused flocculation of activated sludge flocs and produced larger flocs, which may be induced by the released EPS when facing the CBZ stress, as shown later in Fig. 8. EPS may behave as glue that binds cells, particles, colloids together to form sludge flocs. Henriques and Love (2007) also pointed out the released EPS when facing toxic chemicals could function as a protective barrier for the bacteria inside activated sludge flocs

Fig. 7 .

 7 Fig. 7. Effect of CBZ on sludge floc size distribution

Fig. 8 .

 8 Fig. 8. Effects of CBZ on SMP concentration in supernatant and permeate of MBR

Fig. 10 .

 10 Fig.10. HPLC-SEC analysis of MBR supernatants at 1 day and 7 days after addition of CBZ

Fig. 11 .

 11 Fig. 11. HPLC-SEC analysis of supernatant and permeate of MBR 1 day after addition of CBZ

Fig. 12 .

 12 Fig.12. HPLC-SEC analysis of supernatant and permeate of MBR 11 days after addition of CBZ

Fig. 14 .

 14 Fig. 14. Effect of CBZ on supernatant filterability

  supernatants. Many analytical tools, including three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy and high performance liquid chromatography-size exclusion chromatography (HPLC-SEC) equipped with different detectors (UV, refractive index, fluorescence and organic carbon), have been used to characterize SMP in supernatant.

FeedFig. 2 .

 2 Fig. 1. Schematic diagram of study plan

Fig. 3 .

 3 Fig. 3. Permeability of MBR operated under varied organic loading rates

Fig. 4 .

 4 Fig.4. HPLC-SEC analysis of supernatant and permeate of MBR

Fig. 5 .

 5 Fig.5. HPLC-SEC analysis of supernatants of MBR under different organic loading rates

Fig. 6

 6 Fig.6shows one example of HPLC-SEC-fluorescence results for a MBR supernatant (used as

Fig. 6 .

 6 Fig.6. HPLC-SEC analysis of MBR supernatant of and permeate of 0.01 μm PES membrane

Fig. 7

 7 Fig. 7 Filterability tests of selected MBR supernatants at 1.0 bar

  Cependant, il y a un manque de connaissances sur les effets des micropolluants pharmaceutiques sur le colmatage du BAM.Ainsi, l'objectif principal de cette thè se é tait d'examiner les effets sur la performance de filtration de la pré sence de micropolluants pharmaceutiques qui sont fré quemment trouvé s dans les eaux usé es domestiques. La carbamazé pine (CBZ), un mé dicament antié pileptique, a é té choisi comme repré sentant des micropolluants pharmaceutiques dans cette é tude. Il a é té choisi en raison de sa pré sence dans les eaux usé es domestiques et sa persistance aprè s certains traitements. En outre, la CBZ a é té reconnu pour avoir des effets d'inhibition sur l'activité microbienne des boues. Dans cette é tude, les effets de la CBZ sur le colmatage du BAM ont é té é tudié s de deux maniè res: un pic de pollution (la CBZ a é té ajouté dans un ré acteur discontinu qui utilise de la boue à partir d'un BAM) pour é tudier les effets des pics de CBZ à court terme sur le pouvoir colmatant et une pollution continue (la CBZ a é té ajouté en continu dans un BAM) pour examiner les effets de CBZ à long terme sur le colmatage du BAM. L'étude se veut faire un lien entre le colmatage de la membrane et le changement des caracté ristiques de la boue et du surnageant aprè s l'ajout CBZ. Enfin, la possibilité d'utiliser HPLC-SEC-fluorescence comme un outil pour une estimation rapide de la filtrabilité du colmatage des boues a é té proposé .

2. Effets de la CBZ sur le colmatage 2 . 1 Fig. 1 .

 211 Fig. 1. L'effet à court terme de la CBZ sur le pouvoir colmatant des boues activé es

3 .

 3 Effets de la CBZ sur les caracté ristiques de la biomasse L'effet de la CBZ sur l'activité microbienne de la boue dé pend du temps de contact. Pendant une courte pé riode de contact avec 100 µg L -1 de CBZ dans le ré acteur respiromé trique, aucune inhibition de l'activité microbienne des boues n'a été observée, ce qui indique que les bacté ries n'ont pas modifié leur mode de dé gradation ou encore que la croissance bacté rienne compense le faible effet d'inhibition induit par la CBZ. En revanche, l'addition continue de CBZ (90 µg L -1 CBZ) dans le BAM a causé environ 20% d'inhibition de l'activité microbienne des boues aprè s plusieurs jours. Cette inhibition peut ê tre causé e par un changement de comportement de la croissance bacté rienne ou par l'é volution de la population de la communauté microbienne. 4 jours aprè s l'ajout de la CBZ, pas plus de l'inhibition (c'est toujours 20%) n'a é té observé e, ce qui indique que l'adaptation ou acclimatation des boues activé es a é té atteint. Cependant, aprè s l'addition de CBZ dans le BAM aucun changement significatif des performances d''élimination de la D et de l'azote n'a été observé, par rapport à celle avant l'addition de CBZ, ce qui aussi indique que l'inhibition n'est pas significative. Concernant l'effet de la CBZ sur la distribution de taille des flocs, aucun effet significatif n'a é té observé lors d'un pic d'injection ainsi que pendant l'injection continu. Cela peut indiquer 197 qu'aucune décomposition bactérienne importante n'a eu lieu en ré ponse au stress induit par la pré sence de la CBZ que ce soit sous forme d'un pic ponctuel ou d'une pollution continu, ce qui est en accord avec les ré sultats de l'analyse de l'activité microbienne obtenue.

4 .Fig. 4 .

 44 Fig. 4. Effet à court terme de la CBZ sur la composition de PMS dans le surnageant

Fig. 5 .

 5 Fig. 5. Effet à long terme de la CBZ sur PMS dans le surnageant et permé at du BAM

Fig. 6 . 7 .

 67 Fig.6. Effet à court terme de la CBZ sur des proté ines dans le surnageant

Fig. 7 .

 7 Fig. 7. Effet à long terme de la CBZ sur des proté ines dans le surnageant

Fig. 8 .Fig. 9 .

 89 Fig. 8. Analyse de surnageant et permé at du BAM par HPLC-SEC avant d'ajouter CBZ dans le BAM

  The sludge recirculation ratio was fixed at 4. The MBR was fed with a municipal wastewater taken from a wastewater treatment plant close to Toulouse, France, after a primary physical treatment.

					Permeate
				400%
					MF flat sheet
				Anoxic tank	Membrane
					Aerobic tank
							.5
	L/min, i.e. SAD	m of 0.9 m 3	air/h/m 2	and SAD	p of 90 m 3	air/m 3	permeate. Besides, another

aeration system with fine air bubbles was used to maintain the dissolved oxygen concentration in the sludge at about 2-3 mg/L and automatically controlled by an oxygen sensor. Feed

Fig. 3. Schematic diagram of the membrane bioreactor set-up

Table 1 .

 1 Experimental conditions and MBR performances in this study

	Step	organic loading rate (kg COD/kg MLSS/d)	Aerobic MLSS (g/L)	SRT (days)	Filtration/ relaxation cycles (min/min)	Net flux (L/h/m 2 )	Average COD removal (%)	Average TN removal (%)
	1	0.1	4-6	40	8/2	10	96.7 ±2.2	41.4 ±8.4
	2	0.2	6-8	30	8/4	10	97.1 ±1.6	82.5 ±6.2

Table 2

 2

introduces the physicochemical properties of carbamazepine. During short-term batch experiments, a concentrated mother solution was prepared by dissolving carbamazepine (from Sigma-Aldrich) in pure ethanol at a concentration of 1 g/L, stored at 4 °C and used within a week. During long-term experiments, a concentrated mother solution was prepared by dissolving carbamazepine in pure ethanol at a high concentration of 10 g/L to rule out the effect of ethanol on the organic content in the feed. Then, quantities of this concentrated mother solution sufficient to yield about 90 µg L -1 final concentrations of CBZ were added in the feed tank of the MBR in step 2 after the steady state of the MBR reached, i.e. in 84 th day, which is higher than 2 SRTs (30 days). The final concentration of ethanol in the feed was less than 0.001% (v/v).

Table 2 .

 2 Physico-chemical properties of carbamazepine[70, 82, 86]

	Structure
	Formula

  . Chemical oxygen demand (COD) and total nitrogen (TN) were measured by spectrometry with reagent kits. Dissolved organic carbon (DOC) was measured by a Total Organic Carbon analyser (Shimadzu, FR). Prior to analysis of DOC, each sample was prefiltered on a 0.45 μm membrane. Floc size distribution was measured by Malvern Mastersizer 2000. Protein content in supernatant, expressed in mg equivalent of bovine serum albumin per litre, was measured by the acid bicinchoninic method[89].

4 Main results of the work 4.1 Effects of CBZ on fouling 4

  .1.1 Effect of CBZ on sludge fouling propensity Fouling propensity is defined as the fouling resistance produced, which is measured by filterability test that uses a dead-en filtration cell, when a fixed volume of permeate was filtered. The short term effect of CBZ on sludge fouling propensity was examined by the filterability tests using sludges sampled from batch reactors (reactor-with CBZ and controlreactor), detailed in paper 2. Due to the significant differences in the SMP composition and

microbial activity of activated sludge in MBRs operated under different organic loading rates, sludge from MBR operated under two different organic loading rates, i.e. 0.1 kg COD/kg MLSS/d and 0.2 kg COD/kg MLSS/d, respectively, were tested in batch reactors. Fig.

Table 3 .

 3 The comparison between the effects of CBZ on characteristics of MBR sludge

		Short-term peak effects	Long-term continuous effects
	Sludge microbial activity	No significant effect	Slight inhibition
	Mean floc size	No significant effect	Slight increase
	Polysaccharide SMPs	No significant effect	No significant effect
	Protein SMPs	Increase	Increase
	100-1000 kDa protein-like SMPs	Increase	Decrease
	10-100 kDa protein-like SMPs	No significant effect	Increase

Table 1 .

 1 Occurrence of typical pharmaceutical micropollutants in conventional WWTP effluents at concentration

	higher than 1.0 µg L -1
	Compound	Concentration (µg L -1 )	Reference
	Analgesic
	Ibuprofen	1.7-55.0	[28, 35-49]
	Diclofenac	1.2-5.4	[28, 36, 38, 41, 43, 47, 50-53]
	Naproxen	1.0-8.0	[35-40, 42, 48-49, 51, 54]
	Ketoprofen	1.1-3.9	[36-37, 39-40, 49]
	Lipid regulator
	Bezafibrate	1.0-4.8	[28, 36, 38, 47, 52]
	Gemfibrozil	1.3-5.5	[28, 36, 38, 43, 48, 51, 55-56]
	Antiepileptic
	Carbamazepine	1.1-6.3	[28, 36-37, 43, 47-50, 52-53, 57-60]
	Antibiotic
	Erythromycin-H 2 O	2.0-6.0	[57, 61-62]
	Trimethoprim	1.2-3.0	[56-57, 63-65]
	Sulfamethoxazole	1.5-2.0	[61, 65-66]

Table 2

 2 lists the physico-chemical properties of selected pharmaceutical micropollutants that relate to their removal in MBR. The mechanisms involved in MBR for pharmaceutical micropollutant removal may include physical retention of membrane, biotransformation, air

stripping, sorption and photo-transformation

[19, 22,[67][68][69][70]

. Since the molecular size of most pharmaceuticals (molecular weight between 100 and 1000 g mol

-1

Table 2 .

 2 Physico-chemical properties of selected pharmaceutical micropollutants

	Compound	MW	H a	pK a	LogK ow	K d	k biol
	Analgesics
	Ibuprofen	206.3	6.1 × 10 -6	4.91	3.79-3.97	7	9-22
	Diclofenac	296.15	1.9 × 10 -10	4.15	4.02-4.51	16	<0.1
	Naproxen	230.3	1.4 × 10 -8	4.15	3.10-3.18	13	0.4-0.8
	Ketoprofen	254.3	8.7 × 10 -10	4.45	3.0-3.12	16	0.68-1.59 *
	Lipid regulator
	Bezafibrate	361.8	8.7 × 10 -14	3.61	4.25	20	3.4-4.5
	Gemfibrozil	250.3	4.9 × 10 -7	4.75	4.77	75	0.5-1.8
	Antiepileptic
	Carbamazepine	236.27	4.4 × 10 -9	13.9	2.25-2.45	1.2	<0.1
	Antibiotics
	Erythromycin	734.0	2.2 × 10 -27	8.88	2.48-3.06	165	<1.1
	Trimethoprim	290.3	9.8 × 10 -13	7.2	0.73-0.91	200	0.22
	Sulfamethoxazole	253.3	3.9 × 10 -11	5.6	0.89-0.91	260	0.2
	MW, molecular weight (g/mol); H, Henry coefficient (µg L -1 air/ µg L -1 wastewater ); pK a , dissociation constant; Log
	K

ow , octanol-water partition coefficient; K d , sorption coefficient for activated sludge (L kg SS -1 ); k biol , degradation rate constant in MBR (L g SS -1 d -1 ); all data from

[20, 22, 26, 68,[74][75][76][77][78][79]

; * data obtained from batch experiments using enriched nitrifier culture

[79]

.

  Because of the carboxyl functional group in their structures, these compounds are negatively charged (see their pK a values in table 2) at neutral pH. Thus, they are hydrophilic in the ionic state at neutral pH with their low K d values (less than 100 L kg SS -1 ) even though their Log Kow values are high (see their K d and Log Kow values in table 2). For compounds with K d value less than 500 L kg SS

	-1

Table 3

 3

	easily biodegraded in MBR with average removal efficiency higher than 95% (ibuprofen); (2)
	compounds easily biodegraded in MBR with average removal efficiency higher than 90%
	(bezafibrate); (3) compounds moderately biodegraded in MBR with average removal
	efficiency of 50%-80% (naproxen, ketoprofen, gemfibrozil, erythromycin, trimethoprim, and
	sulfamethoxazole); and (4) compounds poorly biodegraded in MBR with average removal
	efficiency less than 40% (diclofenac and carbamazepine). Notably, carbamazepine is removed
	with average removal efficiency of only 7% in MBR. Due to the persistency of
	carbamazepine in the biological treatment process and aquatic environment, Clara et al. [86]

summarizes the removal efficiency of typical pharmaceutical micropollutants in aerobic MBR. The removal efficiency range as well as average removal efficiency together with standard deviations is presented. According to the data from Table

3

, these typical pharmaceutical micropollutants could be classified into 4 groups: (1) compounds that are very

Table 3 .

 3 Removal of selected pharmaceutical micropollutants in MBR

	Compound	Removal efficiency (%)	Average removal ±SD (%)	Reference
	Analgesics
	Ibuprofen	90-100	96±3	[47, 52, 77, 85, 89-103]
	Diclofenac	-19-87	32±27	[47, 52, 77, 85, 89-103]
	Naproxen	16-99	69±27	[77, 85, 89, 91-98, 100-104]
	Ketoprofen	44-100	75±20	[85, 91, 93-98, 100-101, 103-104]
	Lipid regulator
	Bezafibrate	77-98	91±6	[47, 52, 85, 91, 93-96, 98, 103]
	Gemfibrozil	28-99	70±29	[85, 93-95, 98, 100-103]
	Antiepileptic
	Carbamazepine	-24-58	7±18	[47, 52, 77, 85, 89-90, 92-95, 98-
				103, 105-106]
	Antibiotics
	Erythromycin	25-91	60±29	[71, 85, 88-89, 94, 105]
	Trimethoprim	17-95	62±30	[71, 85, 88-89, 101-102, 104,
				107]
	Sulfamethoxazole	24-92	63±16	[47, 71, 85, 88-89, 92, 94, 101-
				102, 104-106, 108]

  , 90, 97, 103-104]. Ibuprofen and bezafibrate were effectively removed when SRT is higher than a critical value, about 10 days[23, 52,[START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF]. While, carbamazepine was found to be recalcitrant to degradation in MBR regardless of the change in SRTs and microbial activity[90,[START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF]. Concerning naproxen,Tambosi et al. [104] found that removal of naproxen in MBR was in the range of 85%-90% at SRT of 15 days and 30 days. Kimura et al.[START_REF] Kimura | Elimination of selected acidic pharmaceuticals from municipal wastewater by an activated sludge system and membrane bioreactors[END_REF] observed removal efficiency of naproxen higher than 95% in MBR operated at SRT of 15 days and 65 days. Maeng et al.[START_REF] Maeng | Influences of solid retention time, nitrification and microbial activity on the attenuation of pharmaceuticals and estrogens in membrane bioreactors[END_REF] found that the removal efficiency of about 25% for naproxen in MBR at SRT of 20 days and 80 days.

Table 4 .

 4 Effect of SRT on removal of selected pharmaceutical micropollutants in MBR

	Compound	Significantly affected by SRT	Not significantly affected by SRT	Reference
	Analgesics
	Ibuprofen		×	[22-23, 52, 103]
	Diclofenac	×		[47, 90, 97]
	Naproxen		×	[97, 103-104]
	Ketoprofen	×		[97, 103-104]
	Lipid regulator
	Bezafibrate		×	[23, 52, 103]
	Gemfibrozil	×		[103]
	Antiepileptic
	Carbamazepine		×	[23, 90, 103]
	Antibiotics
	Erythromycin	×		[88]
	Trimethoprim	×		[88, 104]
	Sulfamethoxazole		×	[23, 88, 104]

Table 5 .

 5 Removal of selected pharmaceutical micropollutants in MBR related processes

	Compound	Removal efficiency (%) in MBR-PAC a	Removal efficiency (%) in MBR-GAC b	Removal efficiency (%) in MBR-RO c
	Analgesics
	Ibuprofen	>95	>98	>95
	Diclofenac	>93	>98	>88
	Naproxen	>95	>98	>95
	Ketoprofen	>95	>98	>95
	Lipid regulator
	Bezafibrate	n.a.	n.a.
	Gemfibrozil	>95	>98	>95
	Antiepileptic
	Carbamazepine	>92 *	>98	>82
	Antibiotics
	Erythromycin	>97	n.a.	>95
	Trimethoprim	>97	n.a.	>95
	Sulfamethoxazole	82 *	n.a.	>95

a

[START_REF] Li | Simultaneous activated carbon adsorption within a membrane bioreactor for an enhanced micropollutant removal[END_REF][START_REF] Nguyen | Enhancement of removal of trace organic contaminants by powdered activated carbon dosing into membrane bioreactors[END_REF][START_REF] Serrano | Removal of persistent pharmaceutical micropollutants from sewage by addition of PAC in a sequential membrane bioreactor[END_REF]

; b

[START_REF] Nguyen | Removal of trace organic contaminants by a membrane bioreactor-granular activated carbon (MBR-GAC) system[END_REF]

; c

[74,[START_REF] Kim | Occurrence and removal of pharmaceuticals and endocrine disruptors in South Korean surface, drinking, and waste waters[END_REF][START_REF] Cartagena | Reduction of emerging micropollutants, organic matter, nutrients and salinity from real wastewater by combined MBR-NF/RO treatment[END_REF][START_REF] Snyder | Role of membranes and activated carbon in the removal of endocrine disruptors and pharmaceuticals[END_REF][START_REF] Dolar | Removal of emerging contaminants from municipal wastewater with an integrated membrane system, MBR-RO[END_REF][START_REF] Nguyen | Removal of emerging trace organic contaminants by MBR-based hybrid treatment processes[END_REF][START_REF] Joss | Water reuse:> 90% water yield in MBR/RO through concentrate recycling and CO2 addition as scaling control[END_REF][START_REF] Sahar | The use of RO to remove emerging micropollutants following CAS/UF or MBR treatment of municipal wastewater[END_REF]

; * The influent concentration in the experiment is 750 µg L -1 ; n.a. = not available.

Table 1 Physico

 1

	Structure
	Formula

-chemical properties of carbamazepine

[44,[47][48]

Table 2 .

 2 Experimental conditions and MBR performances in this study

	Step	Experiment duration (days)	organic loading rate (kg COD/kg MLSS/d)	Aerobic MLSS (g/L)	SRT (days)	Filtration/ relaxation cycles (min/min)	Net flux (L/h/m 2 )	Average COD removal (%)	Average TN removal (%)
	1	161	0.1	4-6	40	8/2	10	96.7 ±2.2	41.4 ±8.4
	2	102	0.2	6-8	30	8/4	10	97.1 ±1.6	82.5 ±6.2

Table 3 .

 3 Effect of CBZ on fouling propensity of sludges (C m = 0.1 kg COD/kg MLSS/d)

	ΔP (bar)	Flux recovery (%)	α x 10 -13 (m -2 )	α x 10 -12 (m kg -1 )
	0.5 bar	79±6	4.8±0.36	8.1±0.61
	Sludge
	0.8 bar	82±3	5.0±0.41	9.6±0.79
	without CBZ
	1.0 bar	71±4	8.2±0.62	15.2±1.15
	0.5 bar	94±2	5.4±0.45	9.2±0.77
	Sludge
	0.8 bar	86±4	6.2±0.48	11.9±0.92
	with CBZ
	1.0 bar	90±5	10.8±0.91	20.0±1.68

Table 4 .

 4 Effect of CBZ on fouling propensity of supernatants (C m = 0.1 kg COD/kg MLSS/d)

	ΔP (bar)	Flux recovery (%)	α x 10 -14 (m -2 )	α x 10 -16 (m kg -1 )
	0.5 bar	81±5	0.91±0.07	2.6±0.20
	Supernatant
	0.8 bar	84±4	1.2±0.10	3.5±0.29
	without CBZ
	1.0 bar	85±6	1.3±0.12	3.8±0.35
	0.5 bar	86±4	1.3±0.09	3.1±0.21
	Supernatant
	0.8 bar	87±5	2.0±0.16	4.7±0.38
	with CBZ
	1.0 bar	87±3	2.1±0.19	5.0±0.45

Table 1

 1 Physico-chemical properties of carbamazepine (Joss et al., 2005; Zhang et al., 2008; Abegglen et al.,

	2009)
	Structure
	Formula

Table 1

 1

	summarizes the advantages and disadvantages of different techniques for SMP
	characterization.

Table 1 .

 1 SMP characterization methods and their features

	Method	Principle	Advantages	Disadvantages	Reference
				No molecular weight
	EEM fluorescence	Fluorescence spectroscopy	Sensitive and selective detection of proteins and humic substances	distribution analysis; Not for polysaccharide
				detection.

  De nos jours, le stress hydrique est un grand dé fi auquel l'humanité dans de nombreux endroits à travers le monde doit faire face. Pour ré soudre ce problè me, les eaux usé es municipales ont é té considé ré es comme une source d'eau alternative pour diverses applications aprè s un traitement approprié . Parmi tous les types de procé dé s de traitement des eaux usé es, le bioré acteur à membrane (BAM) occupe une place de choix dans un contexte de ré utilisation de ces eaux usé es traité es en raison de sa haute qualité de l'effluent. Cependant, le colmatage de la membrane inhé rent à ses proprié té s de ré tention reste encore la principale limitation pour le dé veloppement du BAM. Le colmatage des membranes se traduit par le dé clin de la permé abilité en cours de fonctionnement du BAM. Le colmatage de la membrane est un phé nomè ne complexe. On peut y retrouver un dé pôt, formé sur la surface de la membrane, un blocage des pores en raison des plus petites particules, colloï des et soluté , des composés adsorbés sur la surface ou encore le développement d'une couche de gel. Le surnageant de la boue activé e, qui contient des colloï des et des soluté s, s'est ré vé lé jouer un rôle plus important dans le colmatage de la membrane du BAM que les flocs biologiques dans de nombreuses é tudes. Ainsi, les produits microbiens solubles (PMS), les principaux constituants organiques du surnageant de boues, ont é té reconnus comme les principaux responsables du colmatage des membranes du BAM. Plusieurs facteurs peuvent influer sur la production de PMS : les conditions de fonctionnement du BAM telles que la charge organique traité e, l'âge de boues, les conditions hydrodynamique comme l'aé ration ou l'agitation mé canique, ou encore la pré sence de stress environnementaux (pour les bacté ries) dans les eaux usé es comme les mé taux lourds, la pression osmotique (sels), et les micropolluants comme les produits pharmaceutiques. Dans la derniè re dé cennie, une grande attention a é té accordé e aux micropolluants pharmaceutiques pré sents dans les eaux usé es domestiques en raison de leurs effets né gatifs potentiels sur la santé humaine ou les organismes aquatiques.

	1. Introduction
	R R é é s s u u m m é é l l o o n n g g e e n n f f r r a a n n ç ç a a i i s s

Acknowledgements

This work was carried out at the Laboratory of Biological systems and Process Engineering, INSA Toulouse.

Evaluation of membrane bioreactor on removal of pharmaceutical micropollutants: a review

+33 05 61 55 [START_REF] Kimura | Elimination of selected acidic pharmaceuticals from municipal wastewater by an activated sludge system and membrane bioreactors[END_REF] [START_REF] Quintana | Pathways and metabolites of microbial degradation of selected acidic pharmaceutical and their occurrence in municipal wastewater treated by a membrane bioreactor[END_REF] ; Email : chengcheng.li@insa-toulouse.fr.

Abstract

Municipal wastewater reclamation and reuse has become an important solution in many places around the world to deal with water scarcity problems. Among the available treatment approaches, membrane bioreactor (MBR) has a great potential to become a key element in municipal wastewater reclamation and reuse schemes due to its significantly higher treated effluent quality as compared to the conventional activated sludge process. As great concerns have been raised to some emerging trace pollutants found in aquatic environment in the last decade, notably the pharmaceuticals, removal of pharmaceutical micropollutants by MBR or MBR related processes should be evaluated to further understand the status of MBR in different wastewater treatment and reuse schemes.

This paper gives an overview on removal of pharmaceutical micropollutants by MBR or MBR related processes, such as activated carbon assisted MBR, combined MBR-RO (membrane bioreactor and reverse osmosis) process etc.

Key words: pharmaceutical micropollutants; membrane bioreactor; activated carbon assisted MBR; MBR-RO; wastewater treatment and reuse liter, was measured by the acid bicinchoninic method [54]. Polysaccharide concentration, expressed in mg equivalent of glucose per liter, was measured by the Anthrone method [55].

High performance liquid chromatography-size exclusion chromatography (HPLC-SEC) analysis was performed by an Akta Purifier (GE Healthcare, USA) equipped with a column Protein Kw804 (Shodex, Japan), a silica-based column with an exclusion limit of 1000 kDa, and a fluorescence detector (Varian, USA). The eluent solution for the mobile phase was prepared with 25 mM Na 2 SO 4 and a phosphate buffer (2.4 mM NaH 2 PO 4 and 1.6 mM Na 2 HPO 4 at pH 6.8). The flow rate of eluent passing through the column was 1.0 mL/min.

The excitation/emission wavelengths were set at 280/350 nm, which were confirmed as the required values by EEM fluorescence results [56][57], to detect protein-like substances. The column was calibrated using standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) from GE Healthcare. Prior to analysis of HPLC-SEC-fluorescence, each sample was pre-filtered on a 0.45 μm membrane.

Results and Discussion

Effect of CBZ on sludge fouling propensity

Sludge fouling propensity was characterized by filterability test. Fig. 2 shows the result of filterability tests of sludges sampled from the batch reactors (reactor-with CBZ and controlreactor), using sludges taken from the MBR operated under low organic loading rate (0.1 kg COD/kg MLSS/d), at 1.0 bar. As shown in Fig. 2, for the sludge from the reactor-with CBZ, the fouling propensity, expressed as the fouling resistance produced when a fixed volume of permeate was filtered, was higher as compared with the sludge from the control-reactor.

Abstract

Membrane bioreactor (MBR) is increasingly used for municipal wastewater treatment and reuse and great concerns have been raised to some emerging trace pollutants found in aquatic environment in the last decade, notably the pharmaceuticals. As a consequence the removal of pharmaceutical micropollutants by MBRs has been extensively investigated. But there is still a lack of knowledge on the effects of the current presence of pharmaceutical micropollutants in domestic wastewaters on MBR fouling. Among the different pharmaceuticals, it was decided to focus on carbamazepine (CBZ), an anti-epileptic drug, because of its occurrence in domestic wastewaters and persistency in biological processes including MBRs. This paper focuses on the effects of continuous carbamazepine pollution on MBR fouling. A continuous introduction of CBZ into the MBR via the feed (about 90 µg L -1 CBZ in the feed) provoked a TMP jump. It occurred just 1 day after the addition of CBZ in MBR and a significantly higher increase rate of TMP was also observed after 1 day after addition of CBZ in MBR, as compared to that before addition of CBZ. This indicates that the pharmaceutical stress induced by CBZ causes more severe membrane fouling. Addition of CBZ was shown to induce a significant increase of the concentration of proteins in the supernatant at the beginning several days then stabilized to original level whereas no significant change was found for polysaccharides. HPLC-SEC analysis showed that addition of CBZ induced a decrease of 100-1000 kDa protein-like SMPs and a more significant increase of 10-100 kDa proteinlike SMPs in the supernatant. Moreover it was found that addition of CBZ in the MBR affected the sludge microbial activities, as a slight inhibition (about 20%) of the exogenous respiration rate was observed. The increased membrane fouling could be related to the change in biomass characteristics and supernatant quality after addition of CBZ in MBR. This study allows also suggesting that 10-100kDa protein-like SMPs might accumulate inside the biocake that was formed on the membrane surface during MBR operation and play an important role in the TMP jump phenomenon.

Key words: membrane bioreactor, pharmaceutical micropollutants, carbamazepine, fouling, protein-like SMPs -Starting from 8 days after addition of CBZ, a continuous TMP jump started to occur, where TMP increased continuously at a significantly higher rate as compared to that during the period between 1 day and 8 days after addition of CBZ. Thus, the HPLC-SEC analysis of supernatant and permeate sampled from the MBR on the day (84 th day) just before adding CBZ was examined, as shown in Fig. 5. The molecular weight of protein-like substances in MBR supernatants showed bimodal distribution. Indeed, two peaks were observed in the HPLC-SEC results which represent two groups of protein-like molecules, i.e. molecular size of 100-1000 kDa and 10-100 kDa, respectively. The results

showed that before adding CBZ, the MBR membrane could retain almost all the 100-1000 kDa protein-like substances. Taking into account that the average pore size of the membrane used in the M is 0.2 μm (corresponding to the retention of molecules bigger than 1000 kDa), the 100-1000 kDa molecules may be retained by the deposits formed on the membrane The objective of this study was to evaluate the possibility of HPLC-SEC-fluorescence as a tool for rapid estimation of the modification of fouling in MBRs, when some variations in fouling conditions happen. For example, the operating conditions of MBRs such as organic loading rates may vary a lot in real industrial applications. It was then decided to obtain information on HPLC-SEC fluorescence and on fouling propensity using the same biological fluids. During different operational periods, various fouling situations in a lab-scale MBR

Materials and methods

Membrane bioreactor set-up

A lab-scale submerged MBR set-up consisting of an anoxic reactor (5.4 L) and an aerobic reactor (12.6 L) was used in this study (see Fig. 2). A flat sheet microfiltration membrane (Lp 0 = 1110 L h -1 m -2 bar -1 at 20 °C, Kubota, Japan) was submerged inside the aerobic reactor.

It has an effective filtration area of 0.1 m 2 , with an average pore size of 0.2 µm. This MBR was operated at the high sludge retention time (SRT) of 40 days, with a sludge concentration in the aerobic reactor between 4-6 g/L. The instantaneous permeate flux was fixed at 12.5 L/h/m 2 with filtration/relaxation cycles of 8 min/2 min, leading to a net permeate flux of 10 L/h/m 2 . Two aeration systems were used in the aerobic reactor. An aeration system with big air bubble was installed below the membrane to reduce fouling and it was operated continuously at flow rate of 1.5 L/min, i.e. SAD m of 0.9 m 3 air/h/m 2 and SAD p of 90 m 3 air/m 3 permeate. Besides, another aeration system with fine air bubbles was used to maintain the dissolved oxygen concentration in the sludge at about 2-3 mg/L and automatically controlled by an oxygen sensor. The sludge recirculation ratio was fixed at 4. The MBR was fed with a municipal wastewater taken from a wastewater treatment plant close to Toulouse, France, after a primary physical treatment.

Analytical methods

The concentration in mixed liquor suspended solids (MLSS) was measured according to the standard method AFNOR NFT 90-105. Chemical oxygen demand (COD) and total nitrogen (TN) were measured by spectrometry with reagent kits. Dissolved organic carbon (DOC) was measured by a Total Organic Carbon analyser (Shimadzu, FR). Prior to analysis of DOC, each sample was prefiltered on a 0.45 μm membrane. Supernatants were prepared by centrifugation of the sludge samples at 4000 g during 10 minutes at room temperature.

High performance liquid chromatography-size exclusion chromatography (HPLC-SEC)

analysis was performed by an Akta Purifer (GE Healthcare, USA), equipped with a column Protein Kw804 (Shodex, Japan), a silica-based column with an exclusion limit of 1000 kDa, and a fluorescence detector (Varian, USA). The eluent solution for the mobile phase was prepared with 25 mM Na 2 SO 4 and a phosphate buffer (2.4 mM NaH 2 PO 4 and 1.6 mM Na 2 HPO 4 at pH 6.8). The flow rate of eluent passing through the column was 1.0 mL/min.

Excitation/emission wavelengths were set at 280/350 nm, which were confirmed as the required velues by EEM fluorescence results [43,54], to detect protein-like substances. The column was calibrated using standard proteins: thyroglobulin (669 kDa), ferritin (440 kDa), aldolase (158 kDa), conalbumin (75 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa), ribonuclease A (13.7 kDa) and aprotinin (6.5 kDa) from GE Healthcare. Prior to analysis of HPLC-SEC-fluorescence, each sample was prefiltered on a 0.45 μm membrane.

Filterability tests

Ultrafiltration membranes (from Orelis, FR), made of polyethersulfone (PES), with a nominal pore size of 0.01 μm (Lp 0 = 50-80 L h -1 m -2 bar -1 at 20 °C) and ultrafiltration membranes (Microdyn-Nadir GmbH, Germany), made of polyethersulfone (PES), with a molecular weight cutoff (MWCO) 150 kDa (Lp 0 = 400-600 L h -1 m -2 bar -1 at 20 °C) were used for the filterability tests of supernatant, because the 0.2 μm PS membranes could retain very little components in the MBR supernatant. In addition, in MBRs that employ microfiltration membranes, the deposits formed on the membrane surface and inside the pores constitute an additional barrier and makes the apparent pore size of the fouled microfiltration membrane be in the ultrafiltration (UF) range [61]. A new membrane sample was used for each filtration test. All membranes were soaked in ultra pure water over night to maintain pores wetted.

Then they were rinsed by filtration of ultra pure water at 1 bar during 15 min prior to filtration test. All filtration tests were carried out in an Amicon cell (Millipore), with an effective

Conclusions

Since membrane fouling remains the major limitation for MBR. Understanding fouling is still a key issue for a more sustainable operation of MBRs. There is a need for simple and cheap methods that could allow estimating the fouling in MBRs when some modification in fouling situations occurs. This study related the HPLC-SEC-fluorescence analysis to membrane fouling rate in MBR as well as fouling propensity (as indicated by the calculated C value) of MBR supernatants. The results showed good linear relation between fluorescence peak height of protein-like substances in 100-1000 kDa range and fouling propensity of MBR supernatants sampled. Also, HPLC-SEC-fluorescence was found to be a simple and useful tool to relate the fouling in MBRs operated under varied organic loading rates with the peak height of 100-1000 kDa protein-like SMPs in supernatant. Thus, the peak height of proteinlike compounds in 100-1000 kDa range was identified as a fouling indicator. More studies still need to be performed to validate the use of HPLC-SEC-fluorescence as a tool for estimating fouling of MBR in different fouling situations caused by modified operating conditions such as SRT and MLSS concentration. Also, the possibility of using HPLC-SECfluorescence analysis to optimize the MBR fouling conditions should be further investigated.

En ce qui concerne les effets à long terme, des essais de filtrabilité des boues provenant du BAM avant et aprè s addition de la CBZ ont é té ré alisé s. Comme montre la Fig. 2, l'addition continue de la CBZ dans le BAM augmenté le pouvoir colmatant des boues. -Avant l'ajout de la CBZ dans le BAM, des conditions de filtrabilité stable ont é té atteintes, et la PTM augmente trè s lé gè rement.

-1 jour aprè s l'addition de la CBZ dans le BAM, une augmentation significative de la PTM a é té observé e : Il apparaî t clairement comme une ré ponse du processus de biosé paration à l'effet d'un stress sur la biomasse.

-Entre 1 jour et 8 jours aprè s l'addition de la CBZ, la PTM a augmenté progressivement avec une vitesse d'augmentation plus é levé e par rapport à celle avant l'addition de la CBZ.

-8 jours aprè s l'addition de la CBZ, un saut de PTM se produit, et la PTM augmente continuellement à une vitesse significativement plus é levé e.