A new laccase-mediator system facing the biodegradation challenge: Insight into the NSAIDs removal

Chemosphere 215 (2019) 535e542

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A new laccase-mediator system facing the biodegradation challenge: Insight into the NSAIDs removal Azzurra Apriceno, Maria Luisa Astolfi, Anna Maria Girelli*, Francesca Romana Scuto Department of Chemistry, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy

h i g h l i g h t s

g r a p h i c a l a b s t r a c t


Laccase is immobilized via oxidized carbohydrate moiety on chitosan beads.
NSAIDs compounds in water can be efficiently eliminated by a batch laccase reactor.
Pharmaceuticals removal progresses until products mineralization.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 22 May 2018 Received in revised form 11 October 2018 Accepted 14 October 2018 Available online 15 October 2018

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely found pollutants in the aquatic environment and the currently available treatments for their removal are usually associated with some drawbacks. The aim of this research was to apply a laccase-mediator system for the degradation of some commonly used NSAIDs, namely diclofenac (DCF), naproxen (NAP) and ketoprofen (KP). The biocatalyst was obtained by direct immobilization on chitosan beads of a periodate-oxided laccase from Trametes versicolor. A preliminary study aimed to optimize DCF degradation in the presence of 2,2-azinobis (3ethylbenzothiazoline-6-sulfonicacid) diammonium salt (ABTS) as mediator. It turned out that pH 3 and a 1:1 M ratio for ABTS:drug were the best experimental conditions under which DCF was degraded at 90% after 3 h. In addition, an efficient reuse of the biocatalyst for up to 5 cycles emerged. DCF was further mixed with naproxen and ketoprofen to test whether laccase was still able to eliminate DCF and eventually act on the other compounds. At just 0.02 U of laccase activity, diclofenac was completely degraded within 3 h, while an almost complete removal for naproxen (~90%) and a partial removal for ketoprofen (30%) occurred in 7 d when drugs were added at high concentrations (78.5 mM, 98 mM and 108 mM, respectively). After 7 d of degradation, transformation products of diclofenac, identified as hydroxylated compounds, disappeared. Naproxen products were, instead, reduced to very small amounts. © 2018 Published by Elsevier Ltd.

Handling Editor: Klaus Kümmerer Keywords: Immobilized laccase Chitosan support NSAIDs degradation ABTS

1. Introduction The non-steroidal anti-inflammatory drugs (NSAIDs), which are

* Corresponding author. E-mail address: [email protected] (A.M. Girelli). https://doi.org/10.1016/j.chemosphere.2018.10.086 0045-6535/© 2018 Published by Elsevier Ltd.

classified as emerging contaminants in superficial and wastewater, are a concern of considerable interest in the technical-scientific area, because of their toxic effects. Indeed, according to their chemical-physical characteristics, some of them are able to affect the hormonal system of aquatic organisms and fauna in general. Hundreds of tons of pharmaceutical substances flow to sewage treatment plants every year and NSAIDs are one of the main sources

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of contamination for superficial waters (Schwaiger et al., 2004). The ineffective conventional activated sludge treatment systems additionally contribute to their environmental diffusion. Notably, no regulation nor assessment of their limits have been so far reported. According to literature data (Carballa et al., 2004; Castiglioni et al., 2006; Fent et al., 2006), in wastewater treatment plants drugs removal varies from 0% (carbamazepine) to 20%e70% (diclofenac, ibuprofen) with effluent concentrations within the pharmacologically active range of these compounds. Quintana et al., 2005, investigated the removal of acidic pharmaceuticals by using activated sludge as inoculums under aerobic conditions. The study reported no degradation of diclofenac, partial cometabolically conversion of naproxen and partial mineralization of ketoprofen within 28 d. A further confirmation of the partial removal of ketoprofen in most of the sewage treatment plants was reported by Radjenovic et al. (2009). Hence, in order to improve the removal efficiency for these contaminants, the implementation of purification plants with advanced treatments is necessary. Nowadays, wastewater treatment processes are the subject of several studies, even if they are rarely used at full-scale due to excessive investment costs and management. Some good examples of these treatments are the so-called advanced oxidation processes (AOPs), that represent a valid alternative to the conventional approaches for waters depuration. These systems are based on the combination of oxidant reagents (ozone, hydrogen peroxide, transition metals and metals oxides) with photocatalytic, oxidation or ultrasonic processes or adsorption on active carbon. This arrangement produces highly reactive and low selective species, like hydroxyl radicals, that are able to degrade completely molecules to CO2 and H2O. These technologies have shown to be capable of improving the removal of emerging contaminants. Diclofenac in urban wastewater can be, in fact, totally detoxified and mineralized by photocatalytic ozonation (Moreira et al., 2015). However, if AOPs are not adequately conducted, they can determine the formation of intermediate reaction products often more toxic than the starting compounds, as reported for naproxen by Isidori et al. (2005). In the ongoing research of valid solutions, the use of enzymebased oxidations turned to be a powerful alternative (Pandey et al., 2017; Husain, 2006). Specifically, in the last decades, the interest of biotechnological research has been focused on a particular class of oxidoreductase glycoproteins, known as laccases (Apriceno et al., 2017). Thanks to the high redox potential, laccases are able to oxidize a wider range of substrates when compared with the same low- and medium-redox potential species (Mate and Alcade, 2017). The variety of redox potentials for these biomolecules is strictly related to the sources (bacterial, plant or white-rot fungi). This diversification is basically due to difference in the aminoacid residues composition around the copper of the first reaction site, the so-called T1Cu (Rodgers et al., 2009). The redox potential at the copper site (E0T1) measured versus normal hydrogen electrode 0 (NHE) is low for bacterial and plant laccases (E0 T1 <0.460 mV) which have a methionine residue as axial ligand. Fungal laccases, e.g., laccases from ascomycetes and basidiomycetes, with a leucine 0 residue as the non-coordinating axial ligand, have an E0 T1 ranging from 0.460 to 0.710 mV. Laccases from white-rot fungi have the 0 highest E0 T1 between 0.730 and 0.790 mV with a phenylalanine as the non-coordinating axial ligand at the T1Cu site (Rodgers et al., 2009). As shown in literature (Naghdi et al., 2018), the white-rot fungi oxidoreductase have the ability to remove pharmaceuticals under mild conditions producing non-toxic co-products. However, the application of enzymes in the free/soluble form is limited by the low stability and high cost of production due to the impossibility of the recovery. The immobilization on the surface of solid supports is one of the most effective methods to increase

activity and stability of enzymes for prolonged periods (Girelli and Mattei, 2005; Datta et al., 2013). In this study, a white-rot fungi laccase was covalently immobilized on chitosan and exploited for the degradation of the NSAIDs most commonly found within aquatic environment, namely diclofenac (DCF), naproxen (NAP) and ketoprofen (KP). The used approach for the immobilization recently proved to be advantageous (Apriceno et al., 2018) and it relies on the chemical oxidation with KIO4 of the laccase carbohydrate moieties. These enzymatic portions are usually not directly involved in the active site, and particularly not in enzymatic catalytic activity (Price and Knell, 1942). The removal of drugs was performed in "batch" mode in the presence of the mediator 2,20 -azino-bis (3-ethylbenzothiazolin6-sulfonic acid) (ABTS). Different experimental parameters have been tested in order to get the optimal experimental condition conditions for the best degradation results. The evaluation of oxidation and elimination of the target compounds was monitored by high-resolution chromatography (HPLC) coupled with a diodearray detector.

2. Experimental 2.1. Reagents Pure water obtained with the MilliQ system (Millipore Inc. Bedford, Massachusetts, USA) was used for the preparation of all solutions. Laccase from Trametes versicolor (136 U mg1), 2,2-azinobis (3ethylbenzothiazoline-6-sulfonicacid) diammoniumsalt (ABTS), Chitosan from crab shells (highly viscous), 2,4dinitrophenylhydrazine (DNPH), Diclofenac sodium salt (DCF), Naproxen (NAP), Ketoprofen (KP) and methyl alcohol for HPLC were from Sigma-Aldrich (Italy). The citrate-phosphate buffer was prepared mixing aliquots of 0.1 M citric acid and 0.2 M Na2HPO4 in order to reach the prefixed pH.

2.2. Chitosan macro beads preparation Chitosan macro-beads (3.3 mm ± 0.07 of diameter) have been prepared as previously reported (Apriceno et al., 2017). Specifically, 0.5 g of chitosan were dissolved in 20 mL of an acetic acid solution (5% v/v) at room temperature under slight stirring. The chitosan solution was later inserted into a syringe and added drop by drop to a 2 M NaOH solution through a needle (0.33 mm diameter). Beads obtained were filtered, washed with deionized water in order to remove the residual sodium hydroxide and finally stored in 0.05 M phosphate buffer pH 7.

2.3. Laccase oxidation with potassium periodate 1.3 mg of laccase were, firstly, dissolved in 2 mL of 0.05 M phosphate buffer at pH 6.5 (~8 U) and then mixed with 2 mL of a 0.02 M KIO4 solution for 30 min at 4  C under dark conditions. Then, 3 mL of ethylene glycol were added for 10 min in the dark at 4  C in order to remove the unreacted periodate. The resultant solution, was transferred in a cellulose membrane (cut-off 12e14 KDa) (Wolfe and Hage, 1995) and successively dialyzed against a 0.05 M phosphate buffer solution pH 6.5 for 24 h, at 4  C. The estimation of formed aldehyde groups was spectrophotometrically determined at 490 nm using 2,4-dinitrophenylhydrazine (DNPH) as reagent (Apriceno et al., 2018).

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2.4. Immobilization of laccase oxidized on chitosan beads As reported from Apriceno et al. (2018), laccase was covalently immobilized onto the chitosan beads, after KIO4 oxidation of enzyme carbohydrate moieties. The reaction mixture was composed of 100 mg of beads, 1 mL of 0.05 M phosphate buffer at pH 7, and 1 mL of laccase. All the reactants were left in contact at 4  C and the reaction was followed for 24 h.

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to be the best tested experimental conditions. 5 mL of DCF, KP and NAP (all 5 mg mL1 in methanol) were added for a final concentration of 78.5 mM, 98 mM and 108 mM, respectively. Quantitative determination of the analytes was carried out in a chromatographic system as above reported with some slight modification: mobile phase flow was set at 0.6 mL min1 and its composition contained H2O (with 1.3% of formic acid)-MeOH (40:60, v/v). The results were evaluated in comparison with blank samples and were conducted in triplicate.

2.5. Determination of laccase activity For laccase activity determination, 1 immobilized laccase bead (10 mg) or 0.01 mL of soluble enzyme, were mixed with 2 mL of ABTS and a proper volume of 0.05 M citrate-phosphate buffer pH 3, in order to reach a final volume of 2.7 mL. The oxidation reaction was followed spectrophotometrically measuring the increasing absorbance at 420 nm (ε ¼ 3.6  104 cm1 M1) at 30 s intervals on a spectrophotometer (PG Instrument Limited, Leicester, United Kingdom). One activity unit was defined as the amount of enzyme needed to oxidize 1 mM of ABTS per minute. 2.6. Diclofenac degradation tests For degradation purposes, diclofenac has been chosen as model drug to be tested, in order to first optimize the contaminant removal conditions. To this aim, laccase activity, pH and mediator amount have been varied during the reactions. The parameters were studied one at a time, maintaining fixed, each time, the other experimental conditions. Particularly, the enzymatic oxidation of 5 mL of a DCF standard solution (5 mg mL1 in methanol) was carried out at room temperature under dark conditions varying the citrate-phosphate buffer pH (3e7), the immobilized laccase activity (0.002e0.02 U) and the ABTS concentration (0e240 mM). Once optimized, the reuse of the biocatalyst was tested at fixed conditions with the aim to study how many times the immobilized enzyme could be possibly used for DCF degradation. The test was carried out on 5 repeated cycles and the variation of activity was measured comparing the activity to the initial one. After each assay, the bioreactor was washed with 0.05 M phosphate buffer pH 7.0 and then catalytic activity was measured again by adding fresh DCF solution. Quantitative analysis of DCF transformation was performed on a High Performance Liquid Chromatograph (Shimadzu, LC-6A) coupled to a UVeVis diode-array detector (Shimadzu, SPD-M6A). The system was equipped with a reversed-phase column C-18 (250  4.6 mm DI, particle size 5 mm) and a pre-column from Alltech. Injection volume and mobile phase flow were set at 20 mL and 0.9 mL min1, respectively. An isocratic mobile phase containing H2O (with 1.3% of formic acid)-MeOH (30:70, v/v) was used and the diode array detector was operated at 277 nm. In order to evaluate the exclusive effect of the enzyme, the oxidative action of the biocatalyst was compared with the action of the free enzyme (0.01 U) and with two blank samples: chitosan beads with no enzyme and ABTS alone. Blank samples were prepared according to the same protocols and tested on the same reaction mixtures. All experiments were performed in triplicate and results are reported as average values. 2.7. Evaluation of FANS conversion The degradation ability of immobilized laccase was tested on a pharmaceuticals cocktail where diclofenac was mixed with naproxen and ketoprofen. The study was conducted for 7 d at room temperature, under dark conditions, in citrate-phosphate buffer pH 3, 270 mM of ABTS and 0.02 U of immobilized laccase, that resulted

2.8. Analysis of DCF main degradation product by mass spectrometry The main degradation product of diclofenac was collected by HPLC-DAD, concentrated under nitrogen flow and analyzed by a triple quadrupole mass (TQM) spectrometer (Perkin Elmer SCIEX API 3000 Ms system), equipped with a Turbo Ion Spray (TIS) source. Nitrogen was used as collision gas (4 mTorr), air as nebulizing (1 L min1) and drying gas (7.5 L min1) for the TIS source. The gas temperature and nitrogen collision energy were 300  C and 3000 V, respectively. The acquisition parameters were optimized by direct infusion of the compound through a syringe pump with a flow rate of 10 mL/min in full scan mode (50e600 m/z) with negative ions. 3. Results 3.1. Diclofenac degradation by immobilized laccase A new enzyme-based system is proposed for studying the degradation of some emerging contaminant drugs found in superficial and wastewater. In order to ensure the best NSAIDs degradation efficiency, a preliminary study on the optimal removal conditions (pH, enzyme concentration, mediator concentration) has been conducted using diclofenac as a model of NSAIDs, since it is the most frequently revealed drug in wastewater. A chromatographic analysis of DCF shows a well resolved peak with a retention time of 13.9 min. A decrease of DCF is obtained after the laccase oxidative reaction with the concomitant formation of two peaks at retention times of 4.2 (product 1, P1) and 6.1 min (product 2, P2). Time-course degradation experiments of DCF are shown in Fig. 1. Particularly, the action of immobilized laccase is reported in comparison with free laccase at the same activity (0.01 U) and with two different controls, pure chitosan beads and ABTS alone. As shown in Fig. 1, ABTS alone in the reaction mixture is not able to oxidize the diclofenac. Not surprisingly, chitosan beads have a small adsorption effect on the drug and, in addition, although reaction kinetic is favored with free laccase, both laccase-systems (free and immobilized) degraded the drug for ~ 90%. 3.2. Effect of immobilized laccase activity The effect of immobilized laccase activity on diclofenac degradation was explored in the range 0.002e0.02 U. The reaction mixture consisted of a 0.05 M citrate-phosphate buffer pH 3 and 240 mM of ABTS as mediator. The supplementary data in Fig.S1, depicts the percentage of DCF degradation after a fixed degradation time of 250 min. Results clearly show that the enzyme is able to act on the compound of interest reaching a 30% of DCF degradation even if immobilized laccase activity on chitosan is low (0.002 U). In addition, the degradation rises linearly with the activity, as reported in the graph. This evidence suggests that the amount of enzyme required to get great removal results is not huge. As a matter of fact, 90% of degradation is reached with only 0.02 U of enzyme, that is quite a low value of enzymatic activity if compared with literature data (a proper comparison is reported in Table 1).

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Fig. 1. Time-course degradation of diclofenac by laccase from Trametes versicolor. In the main figure are depicted the effect on DCF degradation by pure chitosan beads (:), ABTS solution (-), free (◊) and immobilized (A) laccase. In the insert, the timeecourse of DCFP1 (continuous line) and DCFP2 (dashed line) is reported when free (◊) and immobilized (A) laccase were used. Experimental conditions: laccase activity 0.01 U, pH 3, ABTS 240 mM and DCF 157 mM.

Table 1 Comparison of diclofenac degradation with literature results for free and immobilized laccase. Laccase sources

Laccase amount

Support

Trametes versicolor

0.02 U 2000e6000U L1 2000 U L1 1 mgmL1

Chitosan beads ABTSa 160 mM e ABTSa/HBTb 1 mM

Myceliphthora Termophila Pleorotus Florida

2000 U L1 730 U L1 2000 U L1 2000 U L1 4U

Pycnoporus Sanguineus 100 U L1 a b c d e f g

e -PVA/CS/ MWNTsc e e e e PLGAfnanofiber e

Mediator/mediator amount

DCF amount

Conditions (pH,T)

Degradation yield (%)

Time (h)

References

50 mg L1 10 mg L1

pH 3; room T pH 4.5; 30  C

90 100

4 3

Present study Tran et al., 2010

HBTb 1 mM

5 mg L1 pH 4; 25  C 12.5 mgL1 pH 4; 50  C

99 100

0.5 6

Lloret et al., 2013 Xu et al., 2014

e e VA/Vd 1 mM FAe 1 mM SAg 250 mM

40 mg L1 20 mg L1 5 mg L1 5 mg L1 50 ppm

pH 4.5; 25  C pH 4; 25  C pH 5; 22  C pH 5; 22  C pH4; 30  C

>95 90 100 83 e

4.5 5 24 24 e

Marco-Urrea et al., 2010a Margot et al., 2013 Lloret et al., 2010 Lloret et al., 2010 Sathishkumar et al., 2012

e

10 mg L1

pH 5; room T

50

8

Rodriguez-Rodriguez et al. 2010

ABTS ¼ 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonicacid) diammoniumsalt. HBT ¼ 1-hydroxybenzotriazole. PVA/CS/MWNTs ¼ polyvinyl alcohol chitosan/multi-walled carbon nanotubes. VA/V ¼ Violuric acid/Vanilin. FA ¼ ferulic acid. PLGA-nanofiber ¼ poly(lactic-co-glycolic-acid). SA ¼ syringaldeyde.

3.3. pH effect The pH impact on DCF degradation was explored. Certainly, it is important to test the catalytic effect of the laccase-mediator system on a target substrate when different environmental conditions might need to be employed in practical applications. In particular,

pH differently influences time stability and the laccase activity. On one hand, the longer the reaction time, the higher the loss of laccase activity by inactivation, especially in acidic media, since laccase is poorly stable in these experimental conditions. On the other hand, the highest catalytic activity for laccase is widely recognized to be at pH 3 (Apriceno et al., 2018). Thus, pH effect was studied in

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order to find the best compromise for degradation purposes. A pH range between 3 and 7 was investigated at 0.02 U of laccase activity and 240 mM of ABTS. Fig. 2 shows DCF timeecourse degradation at various pH. Results prove the pH medium affects the final degradation: the reaction rate, related to the slope of the initial linear section of degradation curves, increases as the pH decreases, reaching the optimum removal in the acidic condition (pH 3). This profile suggests that, when long reaction time is employed, the advantage associated with higher laccase stability at higher pH is overcome by the loss of activity at these pH values. At acidic pH, laccase is less stable for long times but more active. In addition, at pH < DCF pKa (4.15), the drug results to be protonated (Suarez et al., 2008), facilitating its interaction with the anionic mediator. Thus, pH 3 was chosen as optimal condition for further analyses. 3.4. Mediator amount effect It has been widely reported that the low-redox potentials of laccases only allow direct degradation of low-redox-potential phenolic compounds. Mediators are small molecules that act as electron shuttles between enzymes and target compounds and enhance substrate conversion. Therefore, the presence of a suitable mediator during the reaction is fundamental, as it allows the oxidation of various organic compounds that would not be adequate substrates of laccase, such as drugs. In the present work, acid 2,20 -azino-bis (3ethylbenzothiazolin-6-sulfonic) diammonium salt was chosen as mediator Thus, at pH 3 and room temperature with 0.02 U of laccase activity, the effect of ABTS concentration (range 0e240 mM) was investigated, in order to check how mediator amount can affect diclofenac oxidation. Results, reported in Fig. 3, DCF (160 mM) is most effectively removed by laccase only in the presence of high ABTS concentrations. In fact, within 1400 min, the diclofenac degradation rate shows a five-folder increase from 20 to 98% as the ABTS concentration rises from 0 to 160 mM. Beyond 160 mM there were not significant variations. For further analyses, an equimolar amount of DCF and mediator have been used. 3.5. Biocatalyst reuse for the DCF degradation The advantage of confining an enzyme onto a support is basically represented by the working performances of the biomolecule that is possible to achieve, mainly in terms of storage time and reusability. Thus, the immobilized laccase on chitosan beads was

Fig. 3. Effect of mediator amount on diclofenac oxidation. Experimental conditions: pH 3, DCF 157 mM, immobilized laccase activity 0.02 U, ABTS concentration of ( ) 0 mM, (A) 40 mM, (;) 120 mM, (✕) 160 mM and (C) 240 mM.

▪

employed for the study of DCF oxidation on repeated cycles, in order to test the resistance of the catalyst under stressed conditions. Results (data non reported), referred to a 5-h incubation process, evidence that the biocatalyst is able to keep the degradation activity at promising levels after repeated uses. As a matter of fact, at the first cycle, the DCF oxidation was 92% and this result remained unchanged up to the third cycle. This effect suggested that the enzyme is perfectly ‘recharged’ after the reaction and no inactivation phenomena occur for a repeated use. The degradation capacity reaches 40% at the fifth use. 3.6. Comparison with literature data A comparison with main works from literature regarding the exclusive DCF degradation is summarized in Table 1. The table includes all types of free and immobilized laccases that have been used for DCF oxidation and most of them are exploited in the free form. A salient consideration has to be pointed out on laccase activities, since just the current work is proposing an approach in which less than 1 U is used (0.02 U). This amount is enough wellmaintained on the support, and it is sufficient for reaching good degradation efficiency (>90%) with catalytic activity four orders of magnitude lower than literature data. Most of the works confirm the Laccase-Mediator-System set, in which different compounds are used to act as electron shuttle between the enzyme and the analyte. In all cases presented, an efficient degradation grade (more than 50%) is reported and these data confirm the great potential of laccase for an action towards pharmaceutical pollutants independently from the source. A complete removal of the drug, however, was so far obtained just in specific experimental conditions. In fact, when the degradation was carried out at room temperature, a 24 h’ treatment was necessary while, shortening the reaction time, the environment needed to be warmed up to 50  C. Here, a perfect combination of rapid degradation and mild operative conditions has been obtained, with a 90% of degradation from a massive DCF solution (50 mg mL1) in just 4 h at room temperature. 3.7. Analysis of DCF degradation products

Fig. 2. Effect of pH on drug removal. Diclofenac degradation is reported at pH 3 (:), 4 (A), 5 (-), 6 (✕) and 7 (C). Experimental conditions: ABTS 240 mM, DCF 157 mM, immobilized laccase activity 0.02 U.

As already mentioned above, it appears evident that DCF removal leads to the formation of the same products with a similar kinetic profile, regardless of the form of the laccase (free or

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immobilized). It can be also hypothesized that the two obtained products (tr 4.2 and 6.1 min) are strictly related and dependent on each other. In fact, in both laccase forms, when the area of DCFP1 decreases, DCFP2 area increases (Fig. 1). Actually in literature, detailed studies are focused on DCF transformation mechanism even if it is not still elucidated. Lloret et al., 2010, detected a decarboxylated compound while MarcoUrrea et al., 2010b and Lonappan et al., 2017, proposed that degradation mechanism involved hydroxylation of DCF forming products less toxic than the precursor. In addition, Lonappan et al., 2017, evidenced that after 24 h of laccase action, no transformation products were identified and DCF was subjected to a ring opening and final mineralization to CO2, NH3, H2O and HCl were obtained. In order to obtain more information about laccase mediated DCF degradation pathway, an HPLC-MS study of the eluted main product (DCFP1) was performed employing a triple quadrupole mass (TQM) analyzer. Although a low mass resolution is associated to the use of this instrument, TQM approach is low cost and highly versatile. Moreover, the structural identification of small molecules is reliable. The analysis conducted in negative mode showed the presence of three signals with a m/z ratio of 311, 325 and 339 (Fig. 4). The first signal (m/z 311) can be probably associated with a hydroxylated form of DCF, 3-OH-DCF, 4-OH-DCF or 5-OH-DCF, since they have 16 mass units more than diclofenac (PM ¼ 296). This correspondence could be also justified by the fact that the amino group is a strong electron donating group and acts as ortho-para directing groups. These phenolic products could be formed by the addition of the electrophilic hydroxyl radical to the aromatic ring, forming a resonance-stabilized carbon-centered radical with subsequent addition of oxygen and elimination of a hydroperoxyl radical (Cooper and Song, 2003). Hydroxylated products are obtained even when chloro peroxidase was used for degradation. Li et al., 2017, have shown, through a two-dimensional NMR analysis (COSY), that the main product of DCF degradation was 5-hydroxy-DCF. For what concerns the signal at m/z 325, Li et al., 2017, hypothesize that it was formed for a subsequent oxidation of the 5OH-DCF or 4-OH-DCF during the ionization process within the mass spectrometer. In fact, they did not detect it in the NMR analysis. The product with m/z 339, however, has never been found in DCF degradation studies with laccase and the only data of HPLCMS are insufficient to identify the structure even if a further oxidation inside the mass spectrometer could be hypothesized. On the other hand, DCFP2 has a UV spectra with lmax at 270 nm but it was not possible to carry out a mass analysis on the eluted product because of its too small amount. Based on the hypothesized structures of the obtained degradation product, a putative transformation pathway can be proposed. Presumably, laccase oxidizes the mediator (ABTS) to a radical cation. This species promotes, in turn, hydroxylation of diclofenac via radical intermediates using oxygen-donors molecules. Further

steps include C-N-cleavage and ring-cleavage and, for long degradation time, products tend to disappear (Fig. 5) for mineralization process (NH3, H2O, CO2, HCl). 3.8. NSAIDs degradation with immobilized laccase The efficiency of the biocatalyst has been further demonstrated in a more realistic scenario testing a polluted solution of the analyte of interest. Then, DCF was mixed with two different NSAIDs, particularly naproxen and ketoprofen. In this way, the added compounds played both the roles of contaminants and interferents since an action of laccase towards these substrates has been

Fig. 5. Chromatograms of diclofenac, ketoprofen and naproxen degradation at t ¼ 0 (a), t ¼ 3 h (b), t ¼ 48 h (c) and t ¼ 168 h (d) time points. Experimental conditions: pH 3, 270 mM of ABTS and 0.02U of immobilized laccase.

Fig. 4. HPLC-MS spectrum of the diclofenac product (P1). Experimental conditions: flow rate 10 mL/min, full scan mode (50e600 m/z) with negative ions.

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reported. (Marco-Urrea et al., 2010a; Rodriguez-Rodriguez et al., 2010). From a preliminary chromatographic analysis (Fig. 5a), it is evidenced that KP, NAP and DCF have the correspondent peaks separated and well resolved at 9.6 min (lmax of 270 nm), 11.9 min (l1max ¼ 273 nm and l2max ¼ 265 nm) and 25.9 min (lmax ¼ 277 nm), respectively. Thus, it was possible to study, at the fixed experimental conditions, the fate of the drugs during the degradation reaction through the evolution of their respective peaks. After 3 h of treatment, a total disappearance of DCF (Fig. 5b) emerges and the presence of two degradation products, DCFP1 and DCFP2 at retention time 7.2 min and 8.4 min, come up. Their identification was confirmed by their absorption spectra, having maxima values at 306 and 270 nm, respectively. However, no change in NAP and KP peaks was detected. After about 48 h of reaction (Fig. 5c), the total disappearance of the DCFP1 peak and a decrease in the DCFP2 product is observed. Naproxen decreases of 63% and two new peaks at retention times of 5.89 min and 14.6 min are associated as NAP products (NAPP1 and NAPP2) respectively, thanks to a degradation study of NAP alone (data not reported). No variation of the ketoprofen peak area is observed. An action of the enzyme for KP removal is seen after 7d treatment, when just a 23% of degradation is reached (Fig. 5d). Naproxen, after the same time-course, is almost totally reduced (92%) and its products are still present in very small amount. What clearly emerges from NSAIDs overall oxidation process, is the great potential of the developed system. The already promising abilities of the enzyme to degrade DCF alone were preserved even when contaminants were present in the reaction environment, since the drug was completely removed after just 3 h of treatment. The occurrence of other substances does not affect laccase activity, but rather, if they are susceptible of being oxidized by the enzyme, their concrete removal can be disclosed. In this case, a consequential degradation process can be pointed out, in fact, naproxen degradation starts when diclofenac disappears (first reaction hours), while ketoprofen degradation starts when naproxen is completely oxidized. This is probably due to the different chemical structures of the three drugs that consequently influence their potential redox. In fact, generally, electron donor groups (EDG) are more favorable to oxidative attacks than electron attractor groups (EWG). This difference can result in poor degradation efficiency of organic contaminants (Zeng et al., 2017; Yang et al., 2013). Furthermore, it should be pointed out that all three degradation processes lead to the formation of products which also tend to disappear as the reaction progresses until probably their complete mineralization. 4. Conclusion A trametes versicolor laccase has been immobilized on chitosan according to a recent developed strategy. The immobilization approach avoids the activation step of the support and prevent the catalytic site from the inactivation after the bond formation between the biomolecule and the carrier. Thanks to this procedure, the developed system was able to maintain the catalytic activity of the enzyme after the immobilization and it proved to be suitable for the removal of non-steroidal anti-inflammatory drugs. Diclofenac was chosen as model drug in order to carry out a preliminary study of degradation in which experimental conditions were optimized. This exploratory tests revealed that small amounts of laccases (0.002 U) operated toward DCF in presence of ABTS. The mediator is not responsible of any degradative action on the drug so, an effective removal is only possible when the enzyme is

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present in the mixture reaction. At the best conditions (pH 3, equimolar ratio ABTS-drug and 0.02 U of laccase), DCF was degraded at 90% in 4 h at room temperature. In addition, the laccase-mediator DCF degradation generates the formation of hydroxylated-species (4-OH-DCF or 5-OH-DCF) as main products. For prolonged degradation times, a complete mineralization of the products is probably obtained (NH3, H2O, CO2, HCl). A deepest investigation of a more complex environment was carried out and diclofenac was mixed with two drugs, naproxen and ketoprofen. The presence of other substances revealed not to affect degradation extent: laccase (0.02 U) was still active and DCF was removed at 100% in 3 h. An additive effect of the enzyme appeared: a consequential process of degradation on NAP and KP occurred and 90% and 23% of removal were obtained in 7 d, respectively. This is probably due to their different chemical structures, which affect redox potentials and then, determine the oxidative efficacy of the laccase-mediator system. Oxidative degradation of NSAIDs and products formation were monitored by high-level chromatography resolution (HPLC) coupled with a diode array UV detector that, unlike the common UVevis detectors, allows simultaneous recording of the chromatogram and absorption spectra of analytes. In this way it was possible to follow the course of reaction and to observe that the enzymatically formed products of DCF and NAP tend to totally disappear as the reaction progresses. The developed biocatalyst is a powerful, low cost and effective solution to fulfill biodegradation goals. With just 0.02 U of immobilized laccase activity, a successful oxidation of some of the most commonly used NSAIDs was achieved, when the analytes were at significantly higher concentration (mg L1) than values usually found in the aquatic environment (mg L1). In addition, a highly maintained structure of the enzyme can guarantee a perfect reuse of the system up to 5 times. In this way, it can be considered a really new promising alternative for the treatment of waters containing emerging pollutants. A future perspective is to apply this approach in real waste water samples analysis in order to verify its use in industrial scale. Conflicts of interest None of the authors have a financial or personal relationship with other people or organizations which could inappropriately influence or bias this publication. Acknowledgements The authors are grateful to the support from University Sapienza of Rome, Italy. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2018.10.086. References Apriceno, A., Bucci, R., Girelli, A.M., 2017. Immobilization of laccase from Trametes versicolor on chitosan macro beads for anthracene biodegradation. Anal. Lett. 50, 2308e2322. Apriceno, A., Girelli, A.M., Scuto, F.R., 2018. Design of a heterogeneous enzymatic catalyst on chitosan: investigation of the role of conjugation chemistry in the catalytic activity of a Laccase from Trametes versicolor. J. Chem. Technol. Biotechnol. 93, 1413e1420. Carballa, M., Omil, F., Lema, J.M., Llompart, M., García-Jares, C., Rodríguez, I., mez, M., Ternes, T., 2004. Behavior of pharmaceuticals, cosmetics and horGo mones in a sewage treatment plant. Water Res. 38, 2918e2926. Castiglioni, S., Bagnati, R., Fanelli, R., Pomati, F., Calamari, D., Zuccato, E., 2006.

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