Ana Luisa

Parra Guardado

Marie-Pierre Belleville

Magdalena De Jesús

Rostro Alanis

Roberto Parra Saldivar

Jose Sanchez-Marcano
email: jose.sanchez-marcano@univ-montp2.fr

Effect of redox mediators in pharmaceuticals degradation by laccase: a comparative study

Keywords: Laccase, pharmaceutical micropollutants, redox mediators, bio degradation

Bio-catalytic processes have recently attracted attention as an interesting option for the degradation of persistent pollutants as they are capable to catalyze specific reactions at mild conditions and low environmental impact. In this work the potential to transform pharmaceutical micropollutants of a novel laccase from Pycnoporus sanguineus CS43 was compared to the commercial laccases Trametes versicolor and Myceliophtora thermophila. In the absence of redox mediators micropollutants were resistant to degradation, except for the antibiotic amoxicillin that was transformed by all laccases. The influence of natural and synthetic redox mediators (syringaldehyde, pcoumaric acid and ABTS) on the laccase oxidation system was investigated. Results showed the degradation of a complex mixture of pharmaceuticals is both compound and redox mediator dependent. Syringaldehyde resulted as the best redox mediator allowing the highest degradation yields of the antibiotics amoxicillin (80%), sulfamethoxazole (100%) and ciprofloxacin (40%) within 3 h treatment. Overall, commercial laccases showed better catalytic performance in comparison to P. sanguineus CS43 laccase especially in the presence of redox mediators. The successful transformation of pharmaceuticals by the combined action of different laccases and redox mediators demonstrate the potential of these systems for the removal of complex pollutant matrices.

INTRODUCTION

Pharmaceuticals are synthetic or natural chemicals found in prescription medicines, therapeutic and veterinary drugs with a worldwide annual consumption estimated to 100 000 tons per year [START_REF] Naghdi | Removal of pharmaceutical compounds in water and wastewater using fungal oxidoreductase enzymes[END_REF]. The broad use of pharmaceuticals leads to its continuous release into the environment as intact substances or metabolites that are ineffectively removed by conventional wastewater treatment plants [START_REF] Bu | Pharmaceuticals and personal care products in the aquatic environment in China: A review[END_REF][START_REF] Luo | A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment[END_REF]. Hence, pharmaceuticals are detected in water bodies at concentrations from µg L -1 to ng L -1 representing a threat to aquatic ecosystems and human health [START_REF] Baquero | Antibiotics and antibiotic resistance in water environments[END_REF][START_REF] Voulvoulis | Pharmaceutical Residues in Sewage Treatment Works and their Fate in the Receiving Environment[END_REF].

The degradation of pharmaceutical compounds implies an important challenge due to their low bioavailability and complex structure. To date, several processes for the removal of these pollutants have been studied, including conventional techniques (activated sludge, filtration, coagulation, flocculation, sedimentation) advanced oxidation processes (ozonation, UV irradiation, Fenton oxidation), adsorption and membrane processes [START_REF] Luo | A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment[END_REF][START_REF] Yang | Occurrences and removal of pharmaceuticals and personal care products (PPCPs) in drinking water and water/sewage treatment plants: A review[END_REF]. Recently, the use of "hybrid processes", which are a combination of two or more treatment processes, was reviewed since it seems to be an effective way to remove micropollutants [START_REF] Grandclément | From the conventional biological wastewater treatment to hybrid processes, the evaluation of organic micropollutant removal: A review[END_REF]. Nonetheless, the high cost of operation, the inconstant efficiencies obtained and the possible formation of hazardous by-products are major drawbacks to their successful application of these technologies [START_REF] Luo | A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater treatment[END_REF].

A promising alternative treatment is the use of ligninolytic enzymes obtained from white rot fungi cultures (laccases, peroxidases, manganese peroxidases) since they catalyze specific reactions under moderate operational conditions preventing undesired side-reactions [START_REF] Couto | Industrial and biotechnological applications of laccases: A review[END_REF][START_REF] Yang | Understanding the factors controlling the removal of trace organic contaminants by white-rot fungi and their lignin modifying enzymes: A critical review[END_REF].

Particullarly, Laccases (1.10.3.2) are able to catalyze single electron oxidation of phenolic moieties via phenoxyl radicals with the reduction of oxygen to water. Furthermore, laccases catalyze the oxidization phenolic compounds, amines, methoxysubstituted phenols and some inorganic compounds [START_REF] Fernández-Fernández | Recent developments and applications of immobilized laccase[END_REF]. Numerous studies have indicated that laccases present a great potential in the bioremediation of contaminated effluents with dyes [START_REF] Salazar-López | Induced Degradation of Anthraquinone-Based Dye by Laccase Produced from Pycnoporus sanguineus (CS43), Water, Air[END_REF], phenolic compounds [START_REF] Mohammadi | Immobilization of laccase on epoxy-functionalized silica and its application in biodegradation of phenolic compounds[END_REF], pesticides [START_REF] Jin | Conditions Optimizing and Application of Laccase-mediator System (LMS) for the Laccase-catalyzed Pesticide Degradation[END_REF], pharmaceuticals and personal care products [START_REF] Ji | Cross-linked carbon nanotubes-based biocatalytic membranes for micro-pollutants degradation: Performance, stability, and regeneration[END_REF][START_REF] Shao | Immobilization of laccase on hollow mesoporous carbon nanospheres: Noteworthy immobilization, excellent stability and efficacious for antibiotic contaminants removal[END_REF][START_REF] Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] Barrios-Estrada | Potentialities of active membranes with immobilized laccase for Bisphenol A degradation[END_REF][START_REF] Gamallo | Sequential reactors for the removal of endocrine disrupting chemicals by laccase immobilized onto fumed silica microparticles[END_REF]. The oxidation effectiveness of substrates by laccase depends on factors such as the difference of redox potential between laccase T1 copper and substrate, pH of the medium and chemical structure of substrates [START_REF] Polak | Fungal laccases as green catalysts for dye synthesis[END_REF]. However, non-phenolic compounds like pharmaceuticals are not always suitable for laccase oxidation since their redox potential is usually higher than the redox potential of most laccases produced by fungi [START_REF] Gasser | Laccases to take on the challenge of emerging organic contaminants in wastewater[END_REF]. Moreover, the presence of certain functional groups in the structure of the substrate can greatly influence its transformation. Functional groups like hydroxyl and amines, better known as electron donating groups (EDG) make substrates more susceptible to laccase attack. Whereas electron withdrawing functional groups including amides, nitro and carboxylic groups will prevent laccase oxidation resulting in a more difficult process [START_REF] Yang | Understanding the factors controlling the removal of trace organic contaminants by white-rot fungi and their lignin modifying enzymes: A critical review[END_REF].

Laccase catalysis can be extended by the inclusion of redox mediators that act as electron shuttle between the enzyme and substrate. This mechanism involves the oxidation of the mediator by laccase, which results in the production of highly reactive and stable radical species that diffuse away from enzyme's active site to react with other substrates [START_REF] Baiocco | Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccasemediator systems[END_REF]. Thereby, it is possible to oxidize chemical compounds that in principle are not substrates of laccase. Mediators differ from each other in terms of optimal reaction conditions, substrate specificity and the mechanism followed to oxidize substrates (electron transfer, hydrogen atom transfer and ionic oxidation) [START_REF] Baiocco | Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccasemediator systems[END_REF][START_REF] Majeau | Laccases for removal of recalcitrant and emerging pollutants[END_REF]. Hence, understanding the role of mediators in laccase catalyzed reactions is an important matter. Recently, the evaluation of critical aspects such as type and dose of mediator and their influence on laccase stability and effluent toxicity for the removal of trace organic compounds was reported [START_REF] Ashe | Impacts of redox-mediator type on trace organic contaminants degradation by laccase: Degradation efficiency, laccase stability and effluent toxicity[END_REF]. However, this study is only focused on the performance comparison of a laccase with different mediator combinations, similarly to most of the previous investigations evaluating laccase-mediator systems for the removal of environmental pollutants [START_REF] Kurniawati | Efficacy of mediators for enhancing the laccase-catalyzed oxidation of aqueous phenol[END_REF][START_REF] Margot | Sulfamethoxazole and isoproturon degradation and detoxification by a laccase-mediator system: Influence of treatment conditions and mechanistic aspects[END_REF]. Only few investigations have studied and compared the effect of different type of oxidative enzymes on the treatment of organic pollutants mixtures alone or in combination with several redox mediator compounds [START_REF] Camarero | Lignin-Derived Compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes[END_REF][START_REF] Stadlmair | Mass spectrometry based in vitro assay investigations on the transformation of pharmaceutical compounds by oxidative enzymes[END_REF]. Therefore, research on the performance of different laccase-mediator combinations under identical operational conditions for the removal of pharmaceutical pollutants is still limited. A study with this focus would allow properly comparing and understanding the synergy between different laccase formulations and mediators and their effect in the oxidation of pharmaceutical compounds.

In the present work the potential of three different laccases (Myceliophthora thermophila, Trametes versicolor and Pycnoporus sanguineus CS43) for the degradation of three antibiotics (amoxicillin, ciprofloxacin and sulfamethoxazole) and one antiepileptic (carbamazepine) in free enzyme systems was evaluated. Firstly specific aspects such as the effect of pH and temperature were assessed for the potential improvement of the treatment. Then the performance of each laccase with selected natural (syringaldehyde and p-coumaric acid) and synthetic (ABTS) redox mediators was compared in terms of degradation efficiency. In this sense, a cyclic voltammetry study was carried out in order to understand the interactions between redox mediators and substrates and the mechanism involved for the oxidation of each compound.

MATERIALS AND METHODS

Chemicals and enzymes

The pharmaceuticals (amoxicillin (AMX), ciprofloxacin (CIP), carbamazepine (CBZ) and sulfamethoxazole (SMX)), mediators (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)) (ABTS), p-coumaric acid (PCA), syringaldehyde (SYR)) and all other chemicals were purchased from Sigma-Aldrich. The structure of pharmaceuticals and redox mediators is shown in Table . 1. Stock solutions (100 mg L -1 ) of AMX, CIP and SMX were prepared in citrate-phosphate buffer 0.1 M, pH 7. Considering the lack of solubility in water of CBZ the stock solution of this compound was prepared in pure ethanol at a concentration of 15625 mg L -1 . ABTS solution (5 mM) was prepared just before use in buffer citrate-phosphate pH 7. Stock solutions of SYR and PCA (114.9 mM) were prepared in pure ethanol.

Three different laccase preparations were tested. Laccases from Pycnoporus sanguineus CS43 (PSL) were obtained from a tomato medium according to Ramirez-Cavazos et al. [START_REF] Ramírez-Cavazos | Enhanced production of thermostable laccases from a native strain of Pycnoporus sanguineus using central composite design[END_REF] with some modifications. In short, mycelia were removed from the culture supernatant by filtration using two tangential flow filters in series, with respective pore sizes of 0.5 and 0.2 µm. The 0.2 µm filtrate (laccase cocktail) was then filtered on a 10 kDa ultrafiltration membrane. The resulting enzymatic preparation is a laccase cocktail containing at least two isoenzymes. A complete characterization of such laccase cocktail has been reported by Orlikowska et al. [START_REF] Orlikowska | Structural studies of two thermostable laccases from the white-rot fungus Pycnoporus sanguineus[END_REF]. Laccase powder from Trametes versicolor (ref 51639, activity ≥10 U mg -1 ) (TVL) was purchased from Sigma-Aldrich. Commercial laccase produced by submerged fermentation from Myceliophthora thermophila (59.5 g L -1 of pure laccase) (MTL) was provided by Novozymes (Denmark).

Laccase activity assay

The laccase activity was determined by measuring the oxidation of 0.5 mM ABTS solution prepared in citrate-phosphate buffer (0.1 M, pH 4). The reaction was monitored at 420 nm (Ɛ 420 = 36000 M -1 cm -1 ) with a spectrophotometer (Shimadzu UV-2401PC). One enzyme activity unit (U) was defined as the amount of enzymes that oxidized 1 μmol of ABTS per min.

Pharmaceuticals oxidation by laccases

Degradation experiments were run in 100 or 250 ml Erlenmeyer flasks at 25°C under dark conditions to avoid light oxidation and continuous stirring to ensure O 2 saturation of the reaction medium. Reaction mixtures (50 or 100 ml) contained one or a mixture of all pollutants in buffer citrate-phosphate (0.1 M) pH 7; each pollutant concentration was fixed at 20 mg L -1 (molar concentration: 55 µM AMX, 79 µM SMX and 60 µM CIP) except in the case of CBZ where it was 10 mg L -1 (molar concentration: 85µM). Some experiments were also carried out at different pH (citrate-phosphate buffer 0.1 M pH in the range of 3-7) and different temperatures (15 -40 °C).

To assess the effectiveness of redox-mediator for enhancing the bio-oxidation of micropollutants, experiments were carried out as described above after adding one of the following mediators (ABTS, PCA and SYR) at a concentration of 520 µM to the reaction medium. Mediator:substrate molar ratios up to 9:1 (depending on the pharmaceutical) were tested in order to ensure the effective removal of the micropollutants due to the mediator action. Reactions were initiated by adding one of the three enzyme preparations at the concentration of 1 200 U L -1 . To monitor the degradation process, samples of 1 mL were withdrawn every 24 h when there was no redox-mediator in the reaction medium. When using mediators, samples were taken every 20 minutes during the first three hours and then every hour until 8 h. It is worth noting that the number of samples taken was limited in order to keep the variation of reaction volume below 15%. After sampling, aliquots were inactivated in a water bath at 100 °C for 5 minutes and filtered with CHROMAFIL Xtra H-PTFE-20/30 filters to immediately be analyzed by HPLC-MS. In order to verify the repeatability of the treatments all experiments were carried out several times and at least duplicated. The results reported in all figures correspond to the average of the experiments with their corresponding standard deviations that are given in figures or captions. Controls without enzyme neither mediator as well as controls with mediator but no enzyme were run in parallel to assess the role of enzymes on the removal of the pharmaceuticals.

HPLC-MS quantitative analysis

Micropollutants concentration during enzymatic treatment was analyzed using a HPLC Alliance -Waters e2695 separations module equipped with a C18 Raptor column (150 x 2.1 mm, 5µm) coupled to a MS Micromass Quatro micro API (triquadripole) detector. 5 µl of sample were injected and two eluents, namely eluent A (95% water -5% methanol) and eluent B (100% methanol), were passed through the column at a flow rate of 0.25 ml min -1 with the following gradient program: 0-3 min, 100% (A), 3-8 min, 100% (B) and 8-15 min, 100% (A).

Redox potential determinations

Cyclic voltammetry experiments were carried out in sodium phosphate buffer 0.1 M, pH 7, using a VERSASTAT3 voltammetry analyzer (Princeton Applied Research).

A cell of 250 mL was used in all voltammetry studies. The electrochemical cell consisted of an Ag/AgCl reference electrode, a platinum wire counter electrode and a graphite working electrode. Scan rates of 0.5 V s -1 were applied whereas the concentration of substrates and mediators were in the range of 0.2 -3 Mm according to molar concentrations used in degradation reactions.

RESULTS AND DISCUSSION

Oxidation of pharmaceuticals by laccases

The potential of each one of three laccases to transform pharmaceutical active compounds was initially evaluated without mediators. Firstly, each pharmaceutical was treated individually with 60 U of each laccase. After 72 h of enzymatic oxidation no sign of laccase-catalyzed degradation for SMX, CIP and CBZ was found by any of the enzymes tested (results not shown). In contrast the antibiotic AMX was effectively transformed (Fig. 1). From the enzymes tested, PSL showed the highest affinity for AMX reaching 72% removal. Commercial laccases TVL and MTL presented similar performance with 58% degradation at the end of the reaction. In the case of PSL the first signs of oxidation were visible after 24 h of reaction.

No conversion was detected in controls without enzyme, except for AMX where a decrease in concentration was observed. Belonging to the β-lactam penicillins, AMX is an antibiotic known to be unstable in aqueous solutions due to its fast hydrolysis and degradation to various sub-products [START_REF] Gozlan | Amoxicillin-degradation products formed under controlled environmental conditions: Identification and determination in the aquatic environment[END_REF]. Nonetheless, the self-degradation of AMX was much lower in comparison to reactions where enzymes were added, evidencing the effective catalytic action of laccases on the antibiotic removal (Fig. 1).

Affinity of laccase towards substrates is influenced mainly by the redox potential of the specific laccase and the presence of strong electron donating functional groups (EDG) or electron withdrawing functional groups (EWG) [START_REF] Ashe | Impacts of redox-mediator type on trace organic contaminants degradation by laccase: Degradation efficiency, laccase stability and effluent toxicity[END_REF].

Functional EDG prone to be attacked by laccase include hydroxyl (-OH), amines (-NH 2 ), alkoxy (-RO), alkyl (-R) and acyl (-COR). On the other hand, EWG such as carboxylic (-COOH), amide (-CONR 2 ), halogen (-X) and nitro (-NO 2 ) may prevent electron abstraction from occurring by forming a steric shield [START_REF] Yang | Understanding the factors controlling the removal of trace organic contaminants by white-rot fungi and their lignin modifying enzymes: A critical review[END_REF]. Hence, removal efficiencies obtained in this study can be attributed to the chemical structure of the pharmaceuticals. Effective oxidation of AMX can be explained by the presence of a group hydroxyl attached to an aromatic ring that enables the catalytic action of laccases. The slight difference in removal efficiency among laccases may be due to the redox potential of the enzyme that varies depending on the fungal source. Indeed, the AMX degradation results discussed above are in good agreement with the removal of AMX by TVL previously reported by Becker et al. [START_REF] Becker | Removal of antibiotics in wastewater by enzymatic treatment with fungal laccase -Degradation of compounds does not always eliminate toxicity[END_REF] who observed that even if the self-degradation of AMX in blanks runs was obvious, the observed complete oxidation of this antibiotic in an enzymatic membrane reactor after 24 h of treatment was the result of the enzymatic oxidation.

The antibiotic SMX contains an amine (EDG) and a sulfonamide (EWG) group in its structure. The influence of both groups on the degradation has not been clearly established. In some studies effective removal of the pharmaceutical was reported [START_REF] Guo | Biodegradation of sulfamethoxazole by Phanerochaete chrysosporium[END_REF], whereas in others the sulfone group has been related with its recalcitrance to degradation [START_REF] Nguyen | Continuous adsorption and biotransformation of micropollutants by granular activated carbonbound laccase in a packed-bed enzyme reactor[END_REF]. Guo et al. [START_REF] Guo | Biodegradation of sulfamethoxazole by Phanerochaete chrysosporium[END_REF] reported that white-roth fungus P. chrysosporium could oxidize SMX. In contrast, Margot et al. [START_REF] Margot | Sulfamethoxazole and isoproturon degradation and detoxification by a laccase-mediator system: Influence of treatment conditions and mechanistic aspects[END_REF] observed SMX was recalcitrant to purified TVL oxidation in absence of redox mediators. Interestingly, higher removal of the antibiotic was obtained by fungal cultures in comparison to purified laccase as demonstrated by Gao et al. [START_REF] Gao | Simultaneous removal of ciprofloxacin, norfloxacin, sulfamethoxazole by co-producing oxidative enzymes system of Phanerochaete chrysosporium and Pycnoporus sanguineus[END_REF] who observed High oxidation yields of SMX by using P. chrysosporium and P. sanguineus fungal cultures; but visible lower catalytic effects were obtained when the corresponding purified enzyme extracts were used. Possibly, the effect of other enzymes or compounds present in fungal cultures enhanced SMX removal. In addition, low oxidation of SMX would be also attributed to the redox potential of selected laccases. The redox potential of sulfonamides is estimated to be in the range of 0.858-1.158 V which is considerable high in comparison to redox potential of most laccases (0.5-0.8 V). Indeed, these compounds are recalcitrant to the oxidation by laccase [START_REF] Weng | The implication of mediators for enhancement of laccase oxidation of sulfonamide antibiotics[END_REF]. In the case of CIP and CBZ both pharmaceuticals possess strong EWG. The amide group in CBZ and the presence of the carboxyl and halogen group in CIP make them highly resistant to laccase oxidation [START_REF] Nguyen | Continuous adsorption and biotransformation of micropollutants by granular activated carbonbound laccase in a packed-bed enzyme reactor[END_REF][START_REF] Ding | Simultaneous removal 33 and degradation characteristics of sulfonamide, tetracycline, and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorption[END_REF]. The lack of oxidation observed in this study agrees with the work of Ji et al. [START_REF] Ji | Biocatalytic degradation of carbamazepine with immobilized laccase-mediator membrane hybrid reactor[END_REF] who reported less than 5% removal of CBZ by free laccase treatment.

Considering the limited oxidative performance of laccases towards pharmaceuticals in single substrate assays, simultaneous conversion of AMX, SMX, CIP and CBZ in mixtures, was carried out to study potential substrate mediation/competition effect in the reaction. Investigation on the effect of substrate mixtures on enzyme specificity is of high interest to assess the applicability of enzymes as a treatment option since treated wastewaters generally contain a complex mixture of pollutants. In these experiments the concentration of SMX, CIP and CBZ remain unchanged in the reaction medium, confirming their recalcitrance to enzymatic oxidation (data not shown). Laccase affinity towards these substrates was not influenced by the presence of others micropollutants in the reaction. Nonetheless, their presence results also on a very slight decrease of the AMX concentration.

However, this decrease is difficult to be considered as result of the enzymatic degradation because of the self-oxidation of this substrate is of the same order of magnitude.

The simultaneous presence of micropollutants in a reaction media can affect the removal efficiency in the system. The results presented until now in literature are sometimes contradictory and depend on the mixture of pharmaceuticals studied. For example, Ji et al. [START_REF] Ji | Cross-linked carbon nanotubes-based biocatalytic membranes for micro-pollutants degradation: Performance, stability, and regeneration[END_REF] who studied the free enzymatic removal of five representative pharmaceuticals (bisphenol-A (BPA) , diclofenac (DCF), clofibric acid, ibuprofen (IBP) and CBZ) observed that degradation yield in mixtures of three molecules was doubled respect to samples with only one pharmaceutical. The increase in degradation was attributed to the presence of BPA, which is a phenolic compound and then after enzyme oxidation would form phenoxyl radicals that can act as redox mediator between laccase and other micropollutants more recalcitrant to enzymatic degradation. On the contrary, Margot et al. [START_REF] Margot | Influence of treatment conditions on the oxidation of micropollutants by Trametes versicolor laccase[END_REF] observed that removal yield was four times lower in the presence of mefenamic acid and diclofenac compared to single compound solution. Authors suggest that a competitive effect between substrates takes place thus reducing removal yields. Nguyen et al. [START_REF] Nguyen | Continuous adsorption and biotransformation of micropollutants by granular activated carbonbound laccase in a packed-bed enzyme reactor[END_REF] studied the simultaneous removal of , SMX, CMZ, DCF and BPA with a commercial laccase. No effect of the mixture of micropollutants on the removal was observed. These authors attributed the resistance (SMX and CBZ) or the vulnerability (DCF and BFA) to laccase oxidation to the chemical structure of each compound. Seems like the cocktail effect of micropollutants mixtures in laccase catalytic system depends on the presence of more reactive compounds that could promote the removal of recalcitrant ones [START_REF] Ji | Direct immobilization of laccase on titania nanoparticles from crude enzyme extracts of P. ostreatus culture for micro-pollutant degradation[END_REF]. Indeed, the presence of different compounds in the reaction media is strongly dependent on the mixture studied. In the present work the degradation of AMX observed could have an influence of the degradation of other micropollutants in the mixture. Furthermore, effective removal of pharmaceutical mixtures directly depends on the type of enzyme and compound as demonstrated by Stadlmair et al. [START_REF] Stadlmair | Mass spectrometry based in vitro assay investigations on the transformation of pharmaceutical compounds by oxidative enzymes[END_REF]. These authors tested different types of oxidative enzymes for the multiple conversions of acetaminophen, DCF and sotalol and they observed that inhibition or enhancement effect in case of horseradish peroxidase did not occur with laccase from Pleurotus ostreatus. The poor degradation performance obtained in this investigation could be also related to the operational conditions which were not optimized. Indeed, enzyme reactivity generally depends on conditions such as pH and temperature. The effect of these parameters was studied only for AMX since it was the most sensitive compound to enzymatic degradation.

Effect of pH and temperature in the oxidation of AMX by laccases

Removal of AMX by enzymatic treatment was investigated at different pH.

Laccase produced by P. sanguineus, M. thermophila and T. versicolor were used for this purpose. In order to evaluate AMX self-degradation, similar experiments were carried out by replacing the enzymatic solution by the same amount of corresponding buffer.

Results obtained are reported in Fig. 2a.

As observed in the control reactions, AMX in aqueous solution was less stable at acidic conditions in comparison to neutral pH values. After 48 h at pH 3 around 80% of the compound was disappeared. At higher pH values, self-degradation decreased and stabilized at around 30% (pH 5-7). It is important to note that after enzyme addition, no AMX removal was observed at pH 3 by any of the laccases used.

Moreover, AMX transformation yield significantly variated depending on the type of laccase considered. According to Fig. 2a the addition of MTL leads to a slight decrease of AMX concentration in comparison to the control as long as pH is less or equal to 5. However, at pH values higher than 5, no considerable transformation was observed regarding to the control curve.

The TVL presented a completely different reactivity towards AMX in function of pH. No oxidation occurred when pH was in the range from 3 to 5. Nonetheless, when pH was higher than 5, AMX concentration significantly decreased in comparison to control reactions (residual concentrations at pH 6 was 52% versus 73% and 34% versus 69% at pH 7). The best performance of TVL was thus achieved between pH6 and 7. PSL was definitely the best enzyme for the AMX degradation. In comparison to controls this enzyme allowed to improve AMX degradation from pH 4 to 7. The best performance was observed between pH 5 and 6. In such conditions AMX was almost completely removed (residual concentration less than 1% and 4% at pH 5 and 6 respectively).

Optimal pH depends on the laccase properties as well as on the substrate properties, since pH can alter the charge of the compound and the configuration shape of the enzyme which is fundamental for the access or the binding of substrates to the active site [START_REF] Margot | Influence of treatment conditions on the oxidation of micropollutants by Trametes versicolor laccase[END_REF]. These results are in good agreement with the conversions reported by Lloret et al [START_REF] Lloret | Laccase-catalyzed degradation of anti-inflammatories and estrogens[END_REF]. and Margot et al. [START_REF] Margot | Influence of treatment conditions on the oxidation of micropollutants by Trametes versicolor laccase[END_REF], who reported an optimal pH around 5.5 for the degradation of several micropollutants. Even though in some cases oxidation can be observed at low pH values, acidic conditions may lead to complete enzyme deactivation. According to the results presented in this work pH 6 would represent the balance between laccase activity and stability to maximize AMX oxidation capacity; therefore, pH 6 was chosen to evaluate the influence of temperature in the catalytic system.

The effect of temperature on the removal of AMX was evaluated at 15, 25 and 40°C. From Fig. 2b it can be noticed that the increase of reaction temperature leads to an increase of AMX self-degradation from 6% at 15 °C to nearly 40% at 40°C. This is not surprising, since most of chemical reactions are favored by temperature. AMX contains an EDG prone to laccase catalytic activity; hence laccase properties will be determinant to find the best reactivity conditions. Regardless of the tested temperature, MTL did not improve considerably the removal of AMX. These results were expected since this enzyme presented little activity at pH 6. After the addition of TVL, residual content of AMX decreased as temperature increased. The highest AMX removal (around 50%) was obtained at 40°C. However, if self-degradation is taken into account, no significant bio-oxidation increase was observed when passing from 25°C to 40°C. The best oxidation performance was obtained with PSL reaching removal values up to 80% and 90% after only 24 h at 25 and 40°C respectively. Afterwards reaction slowed downprobably due to the decrease of substrate concentration-even so the final AMX concentrations were less than 4 and 3% respectively. It is also important to remark that PSL showed good oxidation performance even at 15 °C. After 24 h 65% of AMX was successfully transformed.

Similar behavior was also reported by Margot et al. [START_REF] Margot | Influence of treatment conditions on the oxidation of micropollutants by Trametes versicolor laccase[END_REF] for the removal of diclofenac and mefenamic acid by T. versicolor laccase, removal increased from 10 to 25 °C but then no additional removal was observed at higher temperatures. Authors suggested that optimal temperature depends on the substrate properties since laccase denaturation is less likely to occur in the temperature range tested. The three laccases tested in this work are produced by different strains of white rot fungi; therefore, they are expected to display different catalytic properties. Moreover, commercial TVL and MTL are purified or partially purified enzymes contrary to the laccase cocktail from PSL. The better performance of PSL could be attributed to the presence of at least two laccase isoforms in the culture supernatant as well as some impurities. All these components in PSL supernatant could have protected the active center from pH and temperature variations making the laccase cocktail the same or more stable than the purified isoforms as suggested by Ramirez-Cavazos et al. [START_REF] Ramírez-Cavazos | Purification and characterization of two thermostable laccases from Pycnoporus sanguineus and potential role in degradation of endocrine disrupting chemicals[END_REF].

Oxidation of pharmaceuticals by laccases in the presence of redox mediators

In this section the mediated action of two natural mediators: p-coumaric acid and syringaldehyde was compared with the influence of the synthetic mediator ABTS for the oxidation of the pharmaceutical active compounds in mixtures. As mentioned in section 1 factors such as molecular structure of the micropollutant, redox potential of the enzyme, type and concentration of mediator can strongly influence the degradation process. In this sense we chose to work with mediator compounds that follow different oxidation mechanisms: hydrogen atom transfer (SYR and PCA) and electron transfer (ABTS). Mediator concentration was fixed at 520 µM to provide a high mediator:substrate molar ratio to allow the effective removal of the studied pharmaceuticals by laccase-mediator systems as demonstrated by Murugesan et al. [START_REF] Murugesan | Enhanced transformation of triclosan by laccase in the presence of redox mediators[END_REF]. Finally, all degradation reactions as well as control reactions were followed for at least 3 h.

Effect of p-coumaric acid addition

Degradation yields obtained after PCA addition variate in function of the substrate-enzyme couple as shown in Fig. 3. It is important to note that no effect of PCA addition on the transformation of pharmaceuticals was observed in control reactions, suggesting that degradation was carried out effectively by the laccasemediator system. The same observation can be done for all the couples enzymemediator studied.

In the case of TVL, addition of PCA leads to a slow but continuous oxidation of SMX and CIP (Fig. 3a and3c). Respectively 31% and 40% of these compounds were removed after 2 h of reaction; afterwards their concentrations remained nearly constant. However, TVL was still unable to oxidize AMX contrary to PSL which allowed removing 40% of AMX in less than 40 minutes of reaction. Again, the increase of reaction time did not lead to an enhancement of AMX removal; the residual concentration after 2 h stayed constant at 60% of the initial concentration. Regarding to CBZ, no sign of degradation was observed after PCA addition. In fact, it was not transformed by any couple laccase-mediator studied confirming the recalcitrance of this antiepileptic to biodegradation (data not shown).

The mechanism of reaction followed by PCA for substrate oxidation is based on hydrogen atom transfer (HAT) in which mediator remove a hydrogen atom to create a phenoxyl radical. Nevertheless, the effectiveness of this oxidation process is also related to the redox potential of PCA which altogether with the affinity of the enzyme for the mediator and the reactivity/stability of radicals formed will control the yield of laccase-mediator reactions [START_REF] Lloret | Laccase-catalyzed degradation of anti-inflammatories and estrogens[END_REF][START_REF] Camarero | Recalcitrant Compounds[END_REF]. Following this mechanism PCA has been reported as an excellent laccase mediator, especially for the removal of recalcitrant pollutants such as polycyclic aromatic hydrocarbons (PAH) [START_REF] Camarero | Recalcitrant Compounds[END_REF][START_REF] Cañas | Transformation of polycyclic aromatic hydrocarbons by laccase is strongly enhanced by phenolic compounds present in soil[END_REF]. In this way, PCA was reported to enhance the removal of the recalcitrant CBZ in hybrid membrane reactors, both in single [START_REF] Ji | Biocatalytic degradation of carbamazepine with immobilized laccase-mediator membrane hybrid reactor[END_REF] and mixture micropollutant solutions [START_REF] Ji | Direct immobilization of laccase on titania nanoparticles from crude enzyme extracts of P. ostreatus culture for micro-pollutant degradation[END_REF]. The improved degradation of CBZ in the mentioned works could be attributed to the improved stability of immobilized enzyme used in the bioreactors compared with free laccase. Although in the present investigation PCA did not enhance CBZ oxidation, its effect on the removal of other persistent pollutants was validated.

In this study from all the enzymes, TVL showed higher affinity to PCA since better degradation yields were observed specially towards recalcitrant pharmaceuticals such as CIP. Although redox potential of PCA was high enough to start oxidizing the pharmaceuticals, most likely phenoxyl radicals formed were not stable to continue with the process. For instance, visible effects were observed at the end of three hours with relatively low oxidation ratios (10-40%).

Effect of ABTS addition

The effects of ABTS addition on the transformation of pharmaceuticals are presented in Fig. 4. The presence of ABTS in the reaction medium allowed the oxidation of AMX and SMX by TVL and MTL. Degradation percentage observed for AMX by TVL was higher than the obtained with MTL; a complete removal AMX was observed after 2 h of reaction with TVL whereas about 90% of removal was achieved after 3 h with MTL (Fig. 4a). Moreover, it is worth noting that during the first 90 minutes of reaction, ABTS did not favor the oxidation of SMX by any of both laccases. 20 Visible effects were only observed after 2 h with removal efficiencies up to 30 and 50% by MTL and TVL respectively (Fig. 4b).

Few studies have reported the degradation of SMX by laccase-mediator systems [START_REF] Margot | Sulfamethoxazole and isoproturon degradation and detoxification by a laccase-mediator system: Influence of treatment conditions and mechanistic aspects[END_REF][START_REF] Gao | Simultaneous removal of ciprofloxacin, norfloxacin, sulfamethoxazole by co-producing oxidative enzymes system of Phanerochaete chrysosporium and Pycnoporus sanguineus[END_REF][START_REF] Yang | Degradation of tetracycline by immobilized laccase and the proposed transformation pathway[END_REF]. Margotr et al. [START_REF] Margot | Sulfamethoxazole and isoproturon degradation and detoxification by a laccase-mediator system: Influence of treatment conditions and mechanistic aspects[END_REF] obtained similar removal yields to those obtained in this work (30%) with T. versicolor laccase in presence of ABTS in pure compound solutions but at longer reaction times (10 h). ABTS is an azino mediator that undergoes oxidation by means of electron transfer mechanism and has been proposed for the transformation of compounds with relatively weak C-H bonds [START_REF] Cantarella | Free radical versus electron-transfer routes of oxidation of hydrocarbons by laccase/mediator systems: Catalytic or stoichiometric procedures[END_REF]. Nonetheless, in general, the reactivity of mediators towards substrates in laccasemediator systems varies depending on the functional groups in the substrate [START_REF] Baiocco | Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccasemediator systems[END_REF]. SMX presents a phenylamine in its structure whereas AMX shows a phenol group. Since both groups are susceptible for laccase degradation, phenol may be an easier substrate to be transformed due to the presence of electron-donating substituents at the benzene ring which decreases the electrochemical potential [START_REF] Cañas | Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes[END_REF].

Last assumption was confirmed by means of the electrochemical analysis carried out with substrates and mediators. When AMX or SMX were added to a solution of ABTS an increase in the oxidation current for both cation (ABTS •+ ) and dication (ABTS •2+ ) forms was observed. ABTS dication current increased from 11.5 to 23 mA in presence of AMX and from 11.5 to 13.5 mA for SMX. This phenomenon was previously reported by Bourbonnais et al. [START_REF] Bourbonnais | Electrochemical analysis of the interactions of laccase mediators with lignin model compounds[END_REF] for the oxidation of veratryl alcohol with ABTS. According to these authors, during cyclic voltammetry determinations the mediator is oxidized at the electrode and then radicals formed diffuse in the solution to oxidize the substrate. In the case of ABTS the reaction relies on the two electrons oxidation of substrate that regenerates the cation radical at the electrode, resulting in a current increase compared to the oxidation of ABTS alone. The fact that the increase of oxidation current for AMX is significantly higher than for SMX suggest that radicals formed during AMX oxidation are more stable and regenerates faster at the electrode making easier AMX transformation in comparison to SMX. Independently of the enzyme tested, the presence of ABTS did not allow CIP oxidation. These results contrast with those reported by Prieto et al. [START_REF] Prieto | Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products[END_REF] where almost complete degradation of CIP (i.e. 97%) by T. versicolor-ABTS system was achieved but in their case the reaction was carried out for 30 h. In addition to the difference of reaction times, the absence of CIP biodegradation could be related to the difference of fungal species used to produce the laccases. It may also result from a competition with AMX and SMX oxidation. ABTS cation radicals reacted first with AMX and SMX during 3 h, by that time radicals may not be stable or available anymore to continue with the oxidation which would limit the reaction [START_REF] Kurniawati | Efficacy of mediators for enhancing the laccase-catalyzed oxidation of aqueous phenol[END_REF]. Further research would be necessary to determine the optimal reaction parameters as well as the factors limiting the reaction (i.e. availability of ABTS and laccase stability).

On the contrary to the results obtained for TVL and MTL; ABTS did not significantly react with PSL since lower degradation percentages were obtained with this couple. A different behavior was reported by Gao et al. [START_REF] Gao | Simultaneous removal of ciprofloxacin, norfloxacin, sulfamethoxazole by co-producing oxidative enzymes system of Phanerochaete chrysosporium and Pycnoporus sanguineus[END_REF] who tested a laccase extract from P. sanguineus for the degradation of antibiotics, among them CIP and SMX, in single and mixture solution. They carried out reactions at 30 °C in the presence of ABTS (1 mM) obtaining high transformation yields (85-100%) but 72 h were necessary to achieve these removal yields. The high transformation yields obtained by Gao could be influenced by the operational conditions used in their experiments, higher temperatures and mediator concentration as well as longer reaction times may favor the transformation of pharmaceuticals.

Effect of syringaldehyde addition

The addition of SYR to the pharmaceuticals mixture resulted on a high enzymatic degradation (50-100%) (Fig. 5). Among all pharmaceuticals studied, SMX was removed with the highest efficiency by all laccases. MTL showed the best performance with almost 100% degradation within the first 10 minutes of reaction (Fig. 5b). TVL and PSL also completely transformed SMX after 1.5 and 2.5 h. These results are better to those reported by Shi et al. [START_REF] Shi | Removal of sulfonamide antibiotics by oriented immobilized laccase on Fe3O4 nanoparticles with natural mediators[END_REF] who observed almost a complete degradation of SMX after 30 min with a laccase from Echinodontium taxodii but in the presence of higher SYR concentration (1 mM).

Similarly, AMX was well removed by the couple MTL-SYR attaining 80% removal within the first 30 minutes. After this time the reaction stabilized and no significant increase in degradation was observed before the end of the experiment. In the case of TVL and PSL moderate transformation yields of AMX were obtained; 50% of removal was achieved at the end of 3 h (Fig. 5a). Increasing the reaction time up to 8 h showed no significant enhancement in degradation; after 4h AMX removal leveled off around to 70%. Degradation of AMX in presence of different concentrations of SYR (0.1 and 1 mM) was described by Becker et al. [START_REF] Becker | Removal of antibiotics in wastewater by enzymatic treatment with fungal laccase -Degradation of compounds does not always eliminate toxicity[END_REF]. These authors reported removal yields from 89-95% depending on the mediator concentration. Interestingly, authors observed that the use of SYR slightly reduced removal yield of AMX in comparison with the treatment with only laccase. Such negative effect was not observed in this study, on the contrary, enhanced oxidation of AMX was observed for all laccases tested. SYR was able to oxidize CIP altogether with MTL and TVL attaining 40% removal after 3h of reaction and up to 60% at the end of 8 h. To our knowledge, results obtained in this study represent the highest CIP degradation yield reported for a laccase catalyzed oxidation by free enzymes using SYR as mediator. Laccase-SYR system was previously tested for the degradation of CIP with very low removal yields [START_REF] Ding | Simultaneous removal 33 and degradation characteristics of sulfonamide, tetracycline, and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorption[END_REF][START_REF] Blanquez | The degradation of two fluoroquinolone based antimicrobials by SilA, an alkaline laccase from Streptomyces ipomoeae[END_REF]. For example, Ding et al. [START_REF] Ding | Simultaneous removal 33 and degradation characteristics of sulfonamide, tetracycline, and quinolone antibiotics by laccase-mediated oxidation coupled with soil adsorption[END_REF] observed negligible removal of CIP by free laccase. Moreover, the degradation was not improved by the presence of SYR even at concentrations as high as 2 mM.

Mediator SYR belongs to the group of substituted phenols compounds that have been proved as suitable laccase mediators [START_REF] Camarero | Lignin-Derived Compounds as Efficient Laccase Mediators for Decolorization of Different Types of Recalcitrant Dyes[END_REF][START_REF] Nguyen | Laccase-syringaldehyde-mediated degradation of trace organic contaminants in an enzymatic membrane reactor: Removal efficiency and effluent toxicity[END_REF]. The mechanism of reaction followed by SYR for the oxidation of non-phenolic substrates is the generation of phenoxyl radicals by HAT [START_REF] Torres-Duarte | Syringaldehyde a true laccase mediator: Comments on the Letter to the Editor from[END_REF]. Structurally, SYR presents a phenol group which is easily oxidized by laccase and two methoxy substituents in ortho position that increase the stability of phenoxyl radicals. Therefore, the efficiency of SYR as mediator in terms of speed and conversion yield is related to the high concentration and stability of the phenoxyl radicals formed during oxidation reactions [START_REF] Cañas | Laccases and their natural mediators: Biotechnological tools for sustainable eco-friendly processes[END_REF]. This actually explains the high and fast conversion yields obtained in this study.

Overall, from all mediators studied the best results were obtained with SYR.

The presence of SYR accelerates degradation of the pharmaceuticals within the first 3 h of reaction, after this time no further transformation was observed. Various hypotheses could explain the threshold observed: loss of mediators and/or laccase activity, enhancement of the concentration of oxidation products etc. All of these possibilities could have a negative influence on the degradation rate. For example, some authors have demonstrated that an excess of mediators, as is the case in this work, can result on the acceleration of the loss of laccase activity by the attack of the radicals formed during mediator oxidation can attack enzyme's catalytic sites [START_REF] Kurniawati | Efficacy of mediators for enhancing the laccase-catalyzed oxidation of aqueous phenol[END_REF][START_REF] Ji | Biocatalytic degradation of carbamazepine with immobilized laccase-mediator membrane hybrid reactor[END_REF][START_REF] Nguyen | The effects of mediator and granular activated carbon addition on degradation of trace organic contaminants by an enzymatic membrane reactor[END_REF]. However, in this work samples taken from the supernatant still showed enzymatic activity for the three laccases after 7h of reaction (results not presented). Indeed, it is also possible a competition for the oxidative process between the pharmaceuticals and oxidation products formed. De Cazes [START_REF] De Cazes | Design and optimization of an enzymatic membrane reactor for tetracycline degradation[END_REF][START_REF] De Cazes | Erythromycin degradation by EreB esterase in enzymatic membrane reactors[END_REF] has already noticed his effect for the degradation of tetracycline and erythromycin with immobilized enzymes. The authors observed a decrease of the degradation yield with time up to stabilization of antibiotics concentration after 24h of reaction. Then substrates solutions were replaced by fresh ones and the initial degradation activity was reached again. They were able to repeat the same cycles during more than 200 hours.

As mentioned at the beginning of this section, CBZ was not transformed by any laccase-mediator system assayed. Poor removal of CBZ was previously reported even in the presence of redox mediators like SYR or ABTS [START_REF] Ji | Biocatalytic degradation of carbamazepine with immobilized laccase-mediator membrane hybrid reactor[END_REF][START_REF] Nguyen | The effects of mediator and granular activated carbon addition on degradation of trace organic contaminants by an enzymatic membrane reactor[END_REF]. As previously discussed, the low removal amounts can be attributed to the presence of strong EWG in its structure making the pharmaceutical not suitable for biodegradation. Moreover, recent evidence suggests that reaction conditions could play a determinant role in CBZ degradation. Naghdi et al. [START_REF] Naghdi | Biotransformation of Carbamazepine by Laccase-Mediator System: Kinetics, Byproducts and Toxicity Assessment[END_REF] observed that addition of ABTS increased degradation of CBZ from 30% to 82% in 24 h by a laccase extract from T. versicolor, but when reaction conditions were optimized to 35 °C, pH 6, with 60 U L -1 of enzyme concentration and 18 μM of mediator up to 95% removal was obtained. Further investigation should be done in order to find the conditions allowing CBZ degradation in the matrix.

Generally, the addition of redox mediators resulted in the increase of pharmaceuticals degradation. However, the degradation yield depends on the mediator type and laccase used. The best combination of mediator/laccase leading to the highest removal of AMX was TVL in combination with ABTS. In the case of SMX the antibiotic was better removed by MTL and SYR. Similarly, CIP highest removal was obtained by both MTL and TVL in presence of SYR.

From the three redox mediators SYR showed as the most effective mediator allowing the degradation of AMX, SMX and CIP in short periods of time. The effectiveness of SYR to mediate the oxidation of pharmaceuticals was followed by ABTS and PCA which presented the slowest reaction yields. MTL and TVL were the most reactive enzymes showing high affinity to SYR (Table 2).

CONCLUSIONS

Results of this work show that without mediators, laccases only oxidized amoxicillin. Operational conditions had an important influence in the removal yield of the antibiotic that was favored at acidic conditions (pH 5-6).

In presence of redox mediators, pharmaceuticals degradation was both mediator and laccase dependent. Syringaldehyde and ABTS showed as the best redox mediators allowing the highest transformation yields (50-100%) in less than 3 h. However, there is not a broad spectrum mediator since carbamazepine was not transformed by any laccase-mediator system. From our knowledge this is the most detailed study presenting the enzymatic degradation of amoxicillin in laccase-mediator systems that was better degraded by laccase in presence of ABTS but closely followed by syringaldehyde. Results suggest that amoxicillin oxidation occurs by the ET mechanism of reaction but analysis of the reaction products will be needed to confirm this assumption. The best degradation yields of ciprofloxacin and sulfamethoxazole were reached by using a commercial laccase and syringaldehyde as mediator. These results show that pharmaceuticals containing phenolic groups are removed depending on the redox potential of the mediator and the stability of the radicals formed. Overall, commercial laccases presented better performance for the degradation of micropollutants in laccase-mediator systems. Nonetheless, the data obtained by the comparison of performance among the three different laccase formulations in the model study under the same controlled conditions offer valuable information of the potential use of this biocatalyst for the treatment of complex pollutant matrices where different oxidative characteristics are often needed.

This study demonstrates the potential utility of laccase-mediator systems for treatment of complex micropollutant mixtures. Notwithstanding the progress realized, many drawbacks like stabilization or decay of the enzymatic activity are still present.

Further studies to improve these enzymatic processes are being carried out, they include the study of their ecotoxicity. Moreover, the study of the immobilization of different laccase-mediator systems in order to reuse the enzymes is also under way.

Figure captions

Fig. 1 .

 1 Fig. 1. Oxidation of AMX in single substrate by laccases from M. thermophila ( ), P. sanguineus ( ) and T. versicolor ( ), control without enzyme ( ) at 25 °C and pH 7.

Fig. 2 .

 2 Fig. 2. (a) Oxidation of AMX at different pH by laccases from M. thermophila (•), P. sanguineus ( ) and T. versicolor ( ), control without enzyme (+) after 48 h at 25 °C. (b) Effect of temperature on the oxidation of AMX at pH 6 after 48 h by laccases from M. thermophila ( ), P. sanguineus ( ) and T. versicolor ( ), control without enzyme ( ).

Fig. 3 .

 3 Fig. 3. Residual concentration of (a) AMX, (b) SMX and (c) CIP in mixture after 170 minutes treatment with laccase-PCA mediator system: M. thermophila (•), P. sanguineus ( ) and T. versicolor ( ). Control without enzyme (+) at 25 °C and pH 7. Standard deviation less than 10% from duplicate experiments.

Fig. 4 .

 4 Fig. 4. Time course degradation of AMX (a), SMX (b) and CIP (c) by laccases in presence of ABTS as mediator at pH 7 and 25 °C. M. thermophila (•), P. sanguineus ( ) and T. versicolor ( ). Control without enzyme (+). Standard deviation less than 10% from duplicate experiments.

Fig. 5 .

 5 Fig. 5. Oxidation of AMX (a), SMX (b) and CIP (c) by laccases in presence of SYR as mediator at pH 7 and 25 °C. M. thermophila (•), P. sanguineus ( ) and T. versicolor ( ). Control without enzyme (+). Standard deviation less than 10% from duplicate experiments.

Table 1

 1 Chemical structure of pharmaceuticals and mediators used in this work ANTIBIOTICS ANTIEPILEPTIC Amoxicillin (AMX) Ciprofloxacin (CIP) Sulfamethoxazole (SMX) Carbamazepine (CBZ) MEDIATORS p-coumaric acid (PCA) Syringaldehyde (SYR) 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS)

  Fig.2

Table 2

 2

	Reactivity of selected laccases towards mediators
			LACCASE
	PHARMACEUTICAL _________________________________________________________
		M. thermophila	T. versicolor	P. sanguineus
	AMX	SYR (++)	ABTS (+++)	SYR (+)
	SMX	SYR (+++)	SYR (++)	SYR (+)
	CIP	SYR (+++)	SYR (+++)	-
	CBZ	-	-	-

(+++) very reactive; (++) reactive; (+) less reactive.

Acknowledgements

The Agence Nationale de la Recherche (ANR, France) is acknowledged for the support provided in the frame of the project POLPHARMA (ANR-15-CE04-0007). The first author acknowledges the CONACYT (Mexico) for the support provided in the form of PhD studies fellowship No. 410752. The authors thankfully acknowledge Mr. Robin Boiron, Mrs. Laurence Soussan and Mr. Eddy Petit for their technical assistance.


N Pagnoux

T Richet

Chartier

L Guironnet

P.-M Geffroy

F Jouay

C Pagnoux

N Richet

T Chartier

Pierre-Marie Geffroy
email: pierre-marie.geffroy@unilim.fr

δ electrophoretic coating for oxygen transport membranes

Keywords: electrophoretic coating, oxygen semi-permeation, porous layer, mixed conductor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The oxygen transport membrane (OTM) are dense ceramic membranes produced from mixed ionic and electronic conducting materials. One of the advantages of this technology is the high purity of the gaseous oxygen produced (higher than 99%). The technology could be interesting to use for laser cutting process [START_REF] Powell | Laser-oxygen of mild steel: the thermodynamics of the oxidation reaction[END_REF]. However, oxygen transport membranes must have a high oxygen semi-permeation for industrial applications. The electromotive force of the oxygen transport through the membranes comes from the difference of oxygen partial pressure of the two atmospheres on both membrane sides. Unfortunately, the oxygen flux through the membrane is usually limited by the oxygen surface exchanges [START_REF] Geffroy | Identification of the ratedetermining step in oxygen transport through La(1-x)SrxFe(1-y)GayO3-δ perovskite membranes[END_REF][START_REF] Geffroy | Understanding and identifying the oxygen transport mechanisms through mixed-conductor membranes[END_REF], and a coating of submicronic particles on the dense membrane can lead to improve the kinetics of the oxygen surface exchanges and then improve oxygen flux [START_REF] Hayamizu | Effects of surface modification on the oxygen permeation of Ba 0.5 Sr 0.5 Co 0.8 Fe 0.2 O 3-δ[END_REF][START_REF] Xu | Oxygen permeation rates through ion-conducting perovskite membrane[END_REF].

In this context, this work is focused on the impact of the thickness of a surface porous layer deposited by electrophoretic deposition on a membrane, using submicronic particles, on the oxygen flux performance. La 0.5 Sr 0.5 Fe 0.7 Ga 0.3 O 3-δ (LSFG 5573 ) perovskite is used as dense membrane material because of its high oxygen diffusion coefficient, good chemical stability and mechanical properties [Sunaro 2008[START_REF] Geffroy | Rational selection of membrane materials of MIEC materials in energy production precesses[END_REF]]. La 0.6 Sr 0.4 Fe 0.8 Co 0.2 O 3-δ (LSFCo 6482 ) perovskite, with very high kinetics for oxygen surface exchanges [START_REF] Guironnet | Improvement of oxygen flux through perovskite membranes using a coating of ultra-divided particles[END_REF]] but low mechanical properties, is used as coating materials onto the membrane, in order to improve the kinetics of oxygen surface exchanges. The coating on the dense membrane is manufactured by electrophoretic deposition (EPD). This coating method has a great interest because it can be used for many different materials.

The main advantages of electrophoretic deposition is a short coating time, easy control of films thickness (from few microns to several hundred microns), low cost, possibility of mass production and suitable for substrates with complex shape as tubular [START_REF] Zhang | Preparation and characterization of mixed-conducting thin tubular membrane[END_REF], Tan 210, Schulz 2010].

Electrophoresis coating method requires to prepare a dispersed suspension of ceramic particles. These ceramic particles should exhibit electrical surface charge to move in a solvent under an electric field applied between the electrode and the substrate. Then, particles coagulate onto the substrate to form a layer. The nature of the surface charges on the ceramic particles determine if the coating is cathodic or anodic. The properties of the coating mainly depend on the characteristics of the ceramic suspension [START_REF] Boccaccini | Application of electrophoretic and electrolytic deposition techniques in ceramics processing[END_REF][START_REF] Zhitomirsky | Electrophoretic deposition of ceramic materials for fuel cell applications[END_REF][START_REF] Heavens | Electrophoretic deposition as a processing route for ceramics[END_REF], Hammaker 1940].

The electrophoretic coating shows recently a great interest for the preparation of thin porous coating in electrochemical systems, in particular for the elaboration of electrode in solid oxide fuel cell [START_REF] Majhi | Anode supported solid oxide fuel cells (SOFC) by electrophoretic deposition[END_REF]]. This work shows the impact of electrophoretic coating on the electrochemical properties of mixed conductor membranes.

Experimental

Materials

The powders used in this study are synthesized by a solid state reaction synthesis [START_REF] Etchegoyen | An architectural approach to the oxygen permeability of a La 0.6 Sr 0.4 Fe 0.9 Ga 0.1 O 3-δ perovskite membrane[END_REF]] for La 0.5 Sr 0.5 Fe 0.7 Ga 0.3 O 3-δ (LSFG 5573 ) powder and by a citrate route [START_REF] Xu | Citrate method synthesis, characterization and mixed electronic-ionic conduction properties of La0.6Sr0.4Co0.8Fe0.2O3-d perovskite-type complex oxides[END_REF]] for La 0.6 Sr 0.4 Fe 0.8 Co 0.2 O 3-δ (LSFCo 6482 ) powder.

LSFG 5573 powder used for the membrane preparation is synthetized from pure oxides (La 2 O 3 (99.99 %, Alfa Aesar), Fe 2 O 3 (98 %, Alfa Aesar), Ga 2 O 3 (99.999 %, Alfa Aesar)) dehydrated at 100°C. Then oxides and carbonate (SrCO 3 (99.9 %, Sigma-Aldrich)) precursors are weighed in proper amount, mixed and attrition-milled at 600 rpm in ethanol. The attrition is performed using zirconia balls of 800 µm diameter. After 3 h, the mixture is separated from the balls with a 200 µm sieve, dried and calcined 5h at 1100°C until perovskite phase formation. Then, the powder is once again attrition-milled in ethanol. Each 15 minutes during the attrition milling, a granulometry analysis is done using a laser granulometer (Horiba LA950), in order to obtain the desired monomodal grain size distribution with a mean diameter close to 1 µm.

LSFCo 6482 powder used for the electrophoretic suspension is produced by citrate route [START_REF] Xu | Citrate method synthesis, characterization and mixed electronic-ionic conduction properties of La0.6Sr0.4Co0.8Fe0.2O3-d perovskite-type complex oxides[END_REF]]. The stoichiometric amount of metal nitrate (Sr(NO 3 ) 2 (≥ 99.0 %, Sigma-Aldrich), Fe(NO 3 ) 3 .9H 2 O (98+ %, Alfa Aesar), La(NO 3 ) 3 .6H 2 O (99.9 %, Alfa Aesar) and Co(NO 3 ) 2 .6H 2 O (98.0-102.0 %, Alfa Aesar)) are mixed and dissolved in deionized water. Then, solid citric acid (99+% Alfa Aesar) is introduced into the mixed solution and under magnetic stirring. The molar ratio of citric acid to metal nitrate is fixed at [CA]/[M]=1.5. After 30 minutes, a 28% ammonia solution is introduced in the mixed solution in order to reach a pH around 9.5 (the precipitation point is exceeded). The solution is heated at 120 °C until a gel is obtained which is then heated at 320 °C to provoke the auto-combustion. The powder obtained after auto-combustion is calcined at 1100 °C for 4 h to have an as pure as possible perovskite phase. The powder produced is characterized by Scanning Electron Microscopy (SEM, Cambridge instrument) and by laser granulometer (Horiba LA950). The mean grain size (Diffusion Laser Scattering, DLS) and the density of powders (gas pycnometer, Accupyc II 1340, Micormeritics) are reported in Table 1. . For each powder, the formation of a perovskite phase is confirmed by X-ray diffraction (Siemens D5000, Cu-K α1 ) and chemical composition is verified by inductively coupled plasma atomic emission spectrometer.

Preparation of the substrate by tape casting

Dense membranes are elaborated by tape casting [START_REF] Chartier | Tape casting[END_REF]]. An LSFCo 6482 slurry is prepared from the powder obtained by solid state synthesis, an organic solvent, a dispersant (phosphate ester), a binder and a plasticizer. Powder, organic solvent and dispersant are first ball-milled for 1 h, then the binder and the plasticizer are added to obtain a slurry with an adapted rheology for tape casting. A green tape of 150 µm thickness is tapecast, dried and punched into disks of 30 mm diameter. Then, those disks are stacked (to obtain 1 mm thick membrane after sintering) and thermo-laminated under 50MPa at 70 °C. The green membranes are debinded and sintered to 1350 °C at a rate of 5 °C/min with a dwell of 4 h to obtain a relative density higher than 95%. The relative density of the membrane is measured using Archimedes method.

Electrophoretic coating procedure

The suspension for electrophoretic deposition is prepared by adding to the LSFCo 6482 powder to the ethanol solvent and 1.5 wt.% of dispersant (phosphate ester). The LSFCo 6482 powder concentration is 5 wt.% in slurry.

The suspension is ball-milled at 260 rpm for 1 h to homogenize the suspension. Then, 2 wt.% of binder (Degalan 51/07, Degussa, Germany) is added to the suspension and ball-milled again 12 h at 130 rpm. The addition of binder improves the cohesion of the green electrophoretic coating after drying.

The electrophoretic cell presented Figure 1 consists in an anodic substrate centered between two parallel copper cathodic electrode plates (50.1*41.4*0.4 mm 3 ). The electrodes are movable, they can be spaced with distances from 0.5 to 5 cm. For this experiment, the distance is set at 3 cm and the submerged surface of the electrodes is 18.5 cm². A very slow stirring by magnetic stirrer prevents from the sedimentation of the suspension. A generator allows to adjust the voltage from 0 to 84 V between the electrodes. After coating, each membrane is taken out of the suspension and the deposit is dried at room temperature at least during 4 h. Then, the membrane with the dried coating is heated to 1150 °C for 2 h at a rate of 1 °C/min to remove all organic components and to further consolidate the porous coating. The remaining porosity in the coating is favorable to the diffusion of the gas through the thickness of the coating, which makes it possible oxygen adsorption on each perovskite particle on the pore surface in the thickness of coating.

Powders, substrate and coatings are observed by SEM to evaluate the morphology and the size of the grains but also the uniformity and the thickness of the coating.

Electrochemical characterizations

Oxygen semi-permeation measurements of membranes

Electrochemical performances i.e. oxygen semi-permeation flux and oxygen activities at the membrane surfaces are measured using a specific setup reported in Figure 2 and described in previous work [START_REF] Vivet | Influence of glass and gold sealants materials on oxygen permeation performances in La 0.8 Sr 0.2 Fe 0.7 Ga 0.3 O 3-δ perovskite membranes[END_REF]].

The membrane sealed between two alumina tubes by gold rings separates three chambers. In the first chamber, recombined air is injected (100 mL/min, oxygen-rich atmosphere), in the second chamber, argon is injected (200 mL/min, oxygen-lean atmosphere). The two different gas flows generate an oxygen partial pressure gradient through the membrane which is the driving force for oxygen diffusion through the membrane. The last chamber is used to avoid transversal leak. An argon flux is also injected in this chamber. The tightness between the chambers are checked at high temperature (900°C) before the measurements of oxygen semi-permeation.

Measurements are performed from 970 °C to 600°C with an interval of 10 °C between each measure.

Oxygen semi-permeation measurement

The oxygen flux through the membrane is calculated using the following equation:

= ( ) ( ) (Eq. 1)
Where is the oxygen flux through the membrane in mol.m -2 .s -1 , the Argon flow in l.s -1 , ( ) the molar volume of argon in l.mol -1 , the apparent membrane surface in m².

and ! are the oxygen partial pressure in the outlet argon flux and the oxygen partial pressure in the argon flux (in atm), respectively. They are evaluated from the electromotive force E (g) 2out , thanks to YSZ-oxygen sensors, following the Nernst law:

" (#) = $% &' () * (Eq. 2)
Where R is the universal gas constant, F the Faraday constant and T the temperature and P O2 is the oxygen partial pressure in the air (= 0.21 atm). The bulk mean grain size is around 2 µ m and a density of 95%. The grain size has a significant impact on the electrochemical performances. Indeed, recent work shows that large grain size (up to 1-2 microns) is usually favorable to ionic conduction through the membrane bulk [START_REF] Reichmann | Impact of microstructure on oxygen semipermeation performance of perovskite membranes: understanding of oxygen transport mechanisms[END_REF]].

Results and discussion

Characterization of the perovskite powders and of the dense membrane

Figure 4 shows the X-ray diffraction patterns of LSFCo 6482 powders produced by citrate route, LSFG 5573 powder produced by solid state reaction and LSFG 5573 sintered membrane. The LSFCo 6482 powder has a well-defined perovskite phase without apparent secondary phase. The LSFG 5573 powder synthesized by solid state reaction has the expected perovskite phase but also two secondary phases determined has La 2 O 3 and SrLaGa 3 O 7 . After the shaping of the powder by tape casting and the sintering of the obtained pellets, the La 2 O 3 -secondary phase disappears but the SrLaGa 3 O 7 -secondary phase still remain. This secondary phase has a low impact the oxygen diffusion coefficient, but it could have a large impact on the oxygen surface exchange kinetics. The impact of secondary phases on oxygen transport mechanism through the La 1-x Sr x Fe 1-y Ga x O 3-δ perovskite membrane the will be discussed in a further study.

Impact of the electrophoretic parameters on the coating

Impact of the applied voltage on the coating thickness

Figure 5 and 6 shows the influence of the applied voltage on the coating thickness for an electrophoretic coating time of 120 s. From 0 to 5 V, a very few particles are scattered (patchy) on the membrane surface, and the coating thickness is less than 10 µm. From 5 to 60 V, there is a linear relation between the coating thickness and the voltage. Above 60 V, the growth rate of the coating highly decreases with the voltage. Besides, the thickness homogeneity of the deposits decreases when the applied voltage increases in agreement with the results reported by Besra et al. [START_REF] Besra | A review on fundamentals and applications of electrophoretic deposition (EPD)[END_REF]]. The homogeneity of the coating is likely linked to the migration speed of particle in suspension. When the applied electrical field is high, the migration speed of particles is high in the suspension, that is likely not favorable to obtain a well-packed layer. However, if the voltage is low (below 2-3 V), there is not electrophoretic coating because the electrostatic forces on the ceramic particles are too low in comparison to the forces linked to mechanical agitation (or Brownien agitation) in the suspension during the electrophoretic coating.

Then, the voltage will be fixed to 20 V in the following sections of this study, which correspond to a good compromise between a compact layer and suitable migration rate of ceramic particles in the suspension.

Impact of the coating time on the deposit thickness

Figure 7 shows the evolution of the coating thickness in relation with the electrophoretic coating time. The coating thickness linearly increases with the time from 30 to 300 s. Above 300 s, the depletion of perovskite particles in suspension leads to a significant slowdown in the growth of the electrophoretic coating [START_REF] Besra | A review on fundamentals and applications of electrophoretic deposition (EPD)[END_REF]].

The maximum thickness of electrophoretic coating obtained in this study cannot be exceed 220 µm, because the growth of coating is too low above 200 µm due to the depletion of perovskite particles in the suspension.

Electrochemical performances

Electrochemical performances were studied for a dense LSFG 5573 membrane without coating, and three dense LSFG 5573 membranes coated with different thickness of LSFCo 6482 porous layer on both surfaces of LSFG 5573 dense membrane. Arrhenius plot and activation energies of the four different membranes are reported Figure 9 and Table 3, respectively. The activation energy (Ea) decreases when the thickness of the electrophoretic coating increases.

This trend is linked to the evolution of the limiting step of the oxygen transport through the membrane when the thickness of porous coating increases. Indeed, a recent work shows that the oxygen flux through a dense LSG 5573 membrane is mainly governed by the kinetics of oxygen surface exchanges [START_REF] Xu | Oxygen permeation rates through ion-conducting perovskite membrane[END_REF]]. Then, the kinetic of oxygen surface exchanges increases with the coating thickness or the surface developed by the porosity of electrophoretic coating. An increasing of the coating thickness leads to an evolution of the limiting step and then the apparent energy activation of oxygen flux through the membrane. This suggests that the oxygen flux is mainly governed by bulk diffusion through the membrane when the thickness of porous coating is large (up to 100 microns in this work).

Besides, a previous work [START_REF] Vivet | New route for high oxygen semi-permeation through surface-modified dense La 1-x Sr x Fe 1-y Ga y O 3-δ perovskite membranes[END_REF]] reported an activation energy of oxygen flux governed by oxygen diffusion in the bulk through La 0.6 Sr 0.4 Fe 0.6 Ga 0.4 O 3-δ (LSFGa 6464 ) dense membrane close to 70 kJ mol -1 , suggesting that LSFGa 5573 material has similar oxygen transport properties than LSFGa 6464 material. Also, the energy activation of the oxygen flux through LSFGa 5573 dense membrane with a porous coating of 115 µm in thickness is close to 60 kJ.mol -1 , and corresponds to the energy activation of the oxygen flux through LSFGa 6464 membrane when the oxygen flux is governed by oxygen diffusion the bulk (i.e. close to 70 kJ. mol -1 ). Table 3 shows that the evolution of activation energy in relation with the range of temperature. The activation energy increases at lower temperature. The first assumption is that the evolution of energy activation is linked to the evolution of the predominant limiting step with the temperature. Indeed, the oxygen flux through the membrane is mainly governed by surface exchange at low temperature and by oxygen bulk diffusion at high temperature (usually up to 1000°C), as previously reported [START_REF] Vivet | Influence of glass and gold sealants materials on oxygen permeation performances in La 0.8 Sr 0.2 Fe 0.7 Ga 0.3 O 3-δ perovskite membranes[END_REF]].

The coating thickness does not have the same impact on the kinetic coefficient at the oxygen-rich side and at the oxygen-poor side because the oxygen concentration of the gases and consequently the migration kinetic of the oxygen through the coatings are very different between the two faces of the ceramic membrane Indeed, the oxygen migration flux in the gaseous phase through the coating is proportional to its concentration as expressed by the Fick law (Eq. 8):

J , = D , ∆ / 0 1 (Eq. 8)
With e the thickness of the porous coating, D O2 the oxygen permeation coefficient (depending on the porous coating) and ∆P O2 the gradient of oxygen partial pressure.

In opposite, the oxygen migration flux in the bulk of the dense membrane is proportional to the gradient of lnP O2

(and not to P O2 ), which is expressed by the Wagner law (Eq. 9):

J , = 2 0 3 0 & ∆ 45/ 0 6 (Eq. 9)
With L the thickness of the dense membrane, C O the oxygen concentration in the material of the dense membrane D O the oxygen diffusion coefficient. ∆ln P O2 corresponds to the gradient of oxygen chemical potential.

The Fick law assumes that the gradient of oxygen partial pressure through the porous coating on the rich and poor-oxygen sides are similar, because the oxygen flux is constant through the membrane and the thickness of the porous coating is similar on both side of the membrane (see eq.8). However, the similar gradient of oxygen partial pressure has not the same impact on the driving force of oxygen flux or gradient of oxygen chemical potential through the membrane. Indeed, the oxygen flux is proportional to the gradient of oxygen chemical potential through the dense membrane, as reported previously by the Wagner law in eq. 9.

For instance, the oxygen partial pressures on the poor and rich-oxygen sides are 10 -3 and 0.21 atm. at 900°C, respectively. The gradients of oxygen partial pressure through both porous coatings are similar, estimated here to ∆P =5. 10 -4 atm. This leads to a very low gradient of oxygen chemical potential through porous coating at richoxygen side ( ∆µ O2 (rich) = RT ln 0.21/(0.21-∆P))= 23 J.mol -1 ) and large gradient of oxygen chemical potential through the porous coating at poor-oxygen side ( ∆µ O2 (poor) = RT ln (10 -3 -∆P)/(10 -3 ) = 3.9 10 3 J.mol -1 >> ∆µ O2 (rich) ), as reported on figure 10. It is then possible to conclude that the oxygen migration step through the porous coating at the poor-oxygen side can limit the oxygen flux through the dense membrane, when the porous coating is very thick. The higher coefficient of oxygen surface exchanges is obtained for the membrane with the thicker porous layer in the thickness range tested (i.e. up to 115 µm) but an optimum thickness will likely exist.

Conclusions

A dense LSFG 5573 membrane has been coated by electrophoretic coating method, with LSFCo 6482 particles of 0.45 µm diameter. This selection of materials was chosen wisely because it combines high oxygen diffusion coefficient for LSFG 5573 material used as the dense membrane and high kinetics of oxygen surface exchanges with LSFCo 6482 materials used as porous coating. This association of the two materials leads to a very high oxygen semi-permeation, i.e. 2.6×10 -3 mol.m -1 .s -1 at 900 °C, which corresponds to the high oxygen flux performances in the literature. However, these oxygen flux performances are lower than the oxygen fluxes obtained with Ba 1-x Sr x Fe 1-y Co y O 3-δ perovskite membranes, i.e. from 1 to 20×10 -3 mol.m -2 .s -1 at 900 °C, which are among the best performances ever reported in the literature for membrane materials [START_REF] Ge | Oxygen selective membranes based on B-site cation-deficient (Ba0.5Sr0.5)(Co0.8Fe0.2)yO3-8 perovskite with improved operational stability[END_REF][START_REF] Ge | Evaluation of Mixed-Conducting Lanthanum-Strontium-Cobaltite Ceramic Membrane for Oxygen Separation[END_REF]].

However, La 0.5 Sr 0.5 Fe 0.7 Ga 0.3 O 3-δ perovskite membranes have a better chemical stability and mechanical properties than Ba 1-x Sr x Fe 1-y Co y O 3-δ perovskite membranes in working conditions.

The impact of the electrophoretic voltage on the migration rate of particles has been established. However, when the coating thickness is large (up to 100 microns), the migration rate of particle decreases significantly. Then, the maximum coating thickness obtained by electrophoretic coating is limited to 100-120 microns due to the delamination of coating during the calcination step.

The higher oxygen flux is obtained with the membrane coated with the thickest coating possible to obtain, i.e.

115 µm. This suggest that the oxygen flux is mainly governed by the oxygen surface exchange at the membrane, but not by the oxygen bulk diffusion through the dense membrane.

Finally, this study shows that the electrophoretic coating is interesting method to obtain very homogenous porous coating from 2-5 µm to 115 µm thickness. The microstructure of electrophoretic coating is adapted to obtain a perovskite coating with high electrochemical performances. This should be particularly interesting for the elaboration of cathode materials for SOFC application.

Figure 8 A

 8 Figure 8 A shows the evolution of the oxygen semi-permeation flux versus the temperature for different thicknesses of the porous layer deposited on the LSFG 5573 dense membrane surfaces. Figure 8 B shows the oxygen semi-permeation flux at 900°C versus the thickness of the porous layer. The oxygen flux increases with

Figure captions Figure 1 .Figure 2 .Figure 3 .

 captions123 Figure captions Figure 1. Electrophoretic coating cell Figure 2. Oxygen semi-permeation setup Figure 3. Micrographs of A) LSFG 5573 perovskite powders (solid state reaction) and B) LSFCo 6482 perovskite powders (citrate route), of the LSFG 5573 dense membrane C1) surface and C2) cross section.

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .

 456789 Figure 4. XRD patterns of LSFCo 6482 citrate powder, LSFG 5573 dense membrane and LSFG 5573 solid state reaction powder Figure 5. Micrographs of the coating obtained by electrophoretic coating during 120s with a voltage of A) 5V, B) 40V and C) 80V Figure 6. Impact of the applied voltage on the thickness and on the homogeneity of coating for an electrophoretic coating time of 120s Figure 7. Impact of the coating time on the coating thickness, for an applied voltage of 20V Figure 8. Evolution of the oxygen semi-permeation A) as function of the temperature and B) as function of the thickness of the porous layer at 900°C Figure 9. Arrhenius plot of oxygen flux trough LSFG 5573 dense membrane without and with porous coating obtained by electrophoretic method.

Figure 10 .

 10 Figure10. Hypothesis on the profile of oxygen chemical potential (RT lnP 02 ) through the dense membrane with porous coating on poor and rich oxygen side.

Table 1 and Figure 3 A and B show

 1 the main characteristics of the LSFG 5573 powder produced by solid state reaction and LSFCo 6482 powder produced by citrate route before milling. The mean grain size of LSFG 5573 powder is close to 1 micron which is adapted to tape casting process (Table1). The mean grain size of LSFCo 6482 powder (i.e. 0.4 µm) is smaller than that of LSFG 5573 powder. The low grain size of LSFCo 6482 powder is favorable to a high exchange surface of electrophoretic coating to improve the kinetics of oxygen surface exchange of the membrane. Powder stoichiometry checked by ICP-AES analysis (Table2) show a chemical composition of LSFG 5573 and LSFCo 6482 powders close to the chemical compositions expected,

Table 2 .Table 1 .

 21 Characterization of perovskite powders

		Synthesized	Mean grain	Powder density
		route	size (µm)	(g.cm -3 )
	LSFG 5573	Solid state reaction	0.9	5.94
	LSFCo 6482	Citrate route	0.4	6.00

Table 2 .

 2 Chemical elementary analysis of LSFG 5573 and LSFCo 6482 powders obtained by Induced Coupling Plasma (ICP) Figure 3 C1 and C2 shows SEM micrographs of the surface and fracture section of LSFG 5573 dense membrane.

		stoichiometry	stoichiometry
	Elements	measured by ICP	desired
		LSFG 5573 (solid state reaction)
	La	0.52	0.5
	Sr	0.48	0.5
	Fe	0.71	0.7
	Ga	0.29	0.3
		LSFCo 6482 (citrate route)
	La	0.59	0.6
	Sr	0.41	0.4
	Fe	0.78	0.8
	Co	0.22	0.2

Table 3 .

 3 Activation energy calculated from Arrhenius plot of oxygen flux through LSFG 5573 dense membrane without and with porous coating.

		Temperature (°C)	Ea (kJ.mol -1 )	Temperature (°C)	Ea (kJ.mol -1 )
	0 µm	970 to 820	120	820 to 640	155
	5 µm	965 to 835	130	835 to 695	160
	75 µm	966 to 790	100	790 to 620	129
	115 µm	960 to 800	51	800 to 620	60

Table captions Table 1 .

 captions1 Characterization of perovskite powders

Table 2 .

 2 Chemical elementary analysis of LSFG 5573 and LSFCo 6482 powders obtained by Induced Coupling Plasma (ICP)

Table 3 .

 3 Activation energy calculated from Arrhenius plot of oxygen flux trough LSFG 5573 dense membrane without and with porous coating

Acknowledgements

The authors wish to express their gratitude to Air Liquide Company for financial and technical support and the ADEME (French Environment and Energy Management Agency) for financial support of this study.