Enzymatic removal of estrogenic activity of nonylphenol and octylphenol aqueous solutions by immobilized laccase from Trametes versicolor

Journal of Hazardous Materials 248–249 (2013) 337–346

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Enzymatic removal of estrogenic activity of nonylphenol and octylphenol aqueous solutions by immobilized laccase from Trametes versicolor Maria Catapane a,b , Carla Nicolucci b,c , Ciro Menale b,c , Luigi Mita b,c , Sergio Rossi a , Damiano G. Mita a,b,c,∗ , Nadia Diano a,b,c a

Institute of Genetics and Biophysics “ABT”, Via P. Castellino, 111, 80131 Naples, Italy National Institute of Biostructures and Biosystems (INBB), Viale Medaglie d’Oro, 305, 00136 Rome, Italy c Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli, 16, 80138 Naples, Italy b

h i g h l i g h t s     

Endocrine disruptors cause adverse effects in living organisms. Nonylphenol and Octylphenol are alkylphenols recognized as endocrine disruptors. It is necessary to remove or reduce their presence in the environment. Waters polluted by these pollutants have been bioremediated by immobilized laccase from Trametes versicolor. Laccase treated solutions were found to have lost any estrogenic activity.

a r t i c l e

i n f o

Article history: Received 21 November 2012 Received in revised form 7 January 2013 Accepted 16 January 2013 Available online 24 January 2013 Keywords: Bioremediation Fluidized bed Bioreactors Alkylphenols Laccase Estrogenic activity

a b s t r a c t A fluidized bed reactor, filled with laccase-based beads, has been employed to bioremediate aqueous solutions polluted by endocrine disruptors belonging to the alkylphenols (APs) class. In particular Octylphenol and Nonylphenol have been studied. The catalytic activity of free and immobilized laccase from Trametes versicolor has been characterized as a function of pH, temperature and substrate concentration in the reaction medium. In view of practical applications for each substrate concentration the removal efficiency (RE), the time to halve the initial concentration ( 50 ), and the tc=0 , i.e. the time to reach complete pollutant removal, have been calculated. The immobilized laccase exhibited a lower affinity for octylphenol (Km = 1.11 mM) than for Nonylphenol (Km = 0.72 mM), but all the other parameters of applicative interest resulted more significant for octylphenol. For example, the times to reach the complete removal of octylphenol compared to those for nonylphenol at the same concentration is shorter of about 15% (at low concentrations) up to 40% (at high concentrations). The study of cell proliferation with MPP89 cells, a human mesothelioma cell line, and the assay with the YES test indicated the loss of estrogenic activity of the APs solutions after laccase treatment. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Many surfactants are environmental pollutants confirming concerns owing to their poor biodegradability, toxicity and their potential as endocrine disruptor chemicals (EDCs). They, indeed, are known to interfere with the endocrine system and, in consequence, to cause serious health problems in animals, humans included. There is an extensive literature concerning the adverse effects induced by the exposure to these compounds on the development and reproduction of several species of fish, molluscs and

∗ Corresponding author at: Institute of Genetics and Biophysics “ABT” of CNR, Via P. Castellino, 111, 80131 Naples, Italy. Tel.: +39 081 6132608; fax: +39 081 6132608. E-mail address: [email protected] (D.G. Mita). 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.01.031

other animals [1,2]. Even if EDCs possess various chemical structures and physical properties, there are two main mechanisms of their action on eukaryotic organisms: one is mimicking the occurrence of natural hormones and the other is blocking normal hormonal functions [3,4]. The most common EDCs in the aquatic environment are the alkylphenols ethoxylates (APEs) and in particular the nonylphenol ethoxylates (NPE) and the octylphenol ethoxylates (OPE), used in the formulations of detergents and other auxiliaries such as dispersing agents, emulsifiers, spinning lubricants. Their hydrophilic ethylene oxide chains are easily degraded to nonylphenol (NP) or octylphenol (OP) in the environment. Nonylphenol and octylphenol are more persistent and noxious than their parent compounds [5,6] and accumulate in the environment owing to their relatively high hydrophobicity [7,8]. In particular, octylphenol had higher

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Table 1 Schematic representation of the structure of studied alkyphenols and some of their chemical and physical characteristics. Characteristics

Octylphenol

Nonylphenol

CAS number

1806-26-4

104-40-5

Structural formula Molecular weight Appearance Water solubility at 25 ◦ C Density at 25 ◦ C Log Kow

206.32 g/mol Light yellow viscous liquid 12.60 mg L−1 0.961 g/mL 4.12

220.35 g/mol White to off-white solid 5.43 mg L−1 0.953 g/mL 4.48

estrogenic activity than nonylphenol [9]. Beside to their endocrine activity, the APs, in high concentrations, are toxic to aquatic organisms [10]. The wastewater treatment plants are one of the most important sources of alkylphenols in the environment since the common wastewater treatments of sewage containing APEs usually lead to their incomplete degradation and to the occurrence of NP and OP. There are a lot of data concerning their effects on wildlife [10–14]. It has been also found that these compounds are able to induce hormone dependent breast tumour cells proliferation [15], by imitating the natural feminine estrogen 17-␤ estradiol (E2) and competing with the receptor for the binding site for the natural hormone [4]. The removal of APs from polluted wastewaters is considered an important issue, thus the investigations of their biodegradation mechanisms are of fundamental interest for understanding their fate [16–19]. Some microbial strains have been found to possess the ability to degrade APs [20,21]. Bacterial biodegradation has been studied extensively in the case of EDCs, but there are only some data concerning the ability of fungal cultures to degrade APs. Mostly of the investigated fungal strains belong to the ligninolytic or mitosporic fungi with the ability to produce laccase [22–24]. Only few papers [25–29] have reported APs degradation by purified laccase. From this point of view new strategies for APs removal are requested, also to reduce their concentration in fish, whose consumption for humans represents one of the main routes of exposition to EDCs. In this paper we have extended to NP and OP our previous studies on bioremediation of waters polluted by endocrine disruptors such as bisphenol A and its congeners [30] or organochlorine compounds [31] or phthalates [32]. Laccase has been covalently immobilized on polyacrylonitrile (PAN) beads working in a fluidized bed reactor in which synthetic aqueous solutions containing different concentrations of NP or OP were recirculated. Once characterized the catalytic system in function of pH and temperature, the catalytic beads were used to test the removal efficiency at different substrate concentrations and the results indicated that the exploitation of laccase-based beads is useful in the bioremediation of waters polluted by alkylphenols. Moreover the absence of estrogenic activity in reaction products and in the reaction medium has been ascertained by studying, before and after the enzyme treatment, the proliferation of the human mesothelioma cell line MPP89. 2. Experimental 2.1. Chemicals Laccase (EC. 1.10.3.2) [23.7 U/mg of solid towards 1 ␮mole of catechol] from Trametes versicolor, nonylphenol [4-(2,4-dimethylheptan-3-yl)-phenol] and octylphenol [4-(1,1,3,3tetramethylbutyl)-phenol] were purchased from Sigma–Aldrich (Sigma, Milano, Italy). The chemical and physical characteristics, as well as the structural formula of each substrate, are summarized

in Table 1. Their octanol–water partition coefficient Kow , inversely related to the solubility of the compound in water, has also been reported as an indication of the compound hydrophobicity. All other chemicals, including PAN powder, purchased from Sigma–Aldrich, were of analytical grade and used without further purification. 2.2. Methods 2.2.1. PAN beads preparation and activation 18 g of PAN powder, 1 g of LiNO3 and 3 g of glycerine were dissolved in 78 mL of dimethylformamide (DMF). The homogenized mixture was pipetted and precipitated in a volume of 300 mL of distilled water. The obtained beads were washed with distilled water and immersed for 24 h in a 30% (v/v) glycerine aqueous solution. After this step, the beads were dried at 70 ◦ C to reach a constant weight. A total of 20 cm3 (12 g) of PAN beads was activated at 50 ◦ C for 60 min by treatment with 15% (w/v) NaOH aqueous solution. After thorough washing with distilled water, the beads were treated for 60 min at room temperature with a 10% (v/v) aqueous solution of 1,2-diaminoethane (15 mL). At this point the beads were thoroughly washed once more with distilled water. 2.2.2. Enzyme immobilization Laccase has been immobilized on PAN beads through a process that involves the diazotization of the phenolic groups of its tyrosine residues. The immobilization procedure for diazotization was chosen as the tyrosine residues are not present in the catalytic site of the laccase. In the catalytic site, instead, histidine residues are present; consequently it is not advisable to perform an immobilization procedure based on a condensation process. The PAN beads were treated at room temperature for one hour with a 2.5% (v/v) aqueous solution of glutaraldehyde used both as a spacer arm and as a bifunctional reagent to bind covalently the aminoaril derivatives useful for enzyme immobilization. To generate the amino aril derivatives on beads, these were treated with a solution of 2% phenylenediamine (PDA) for 90 min in 0.1 M sodium carbonate buffer pH 9. After washing the beads with water, the aminoaril derivatives were treated for 40 min at 0 ◦ C with an aqueous solution containing 2 M HCl and 4% (w/v) NaNO2 . At the end of this treatment the beads were washed at room temperature in a citrate buffer solution at pH 5 and then treated for 16 h at 4 ◦ C in enzyme solution (3 mg/mL of laccase) in buffer citrate at pH 5. At the end of this step the beads were further washed with 0.1 M buffer citrate at pH 5 to remove the unbound enzyme. The whole scheme of the immobilization procedure is illustrated in Fig. 1. The amount of immobilized enzymes was calculated by subtracting the amount of laccase recovered in the solution at the end of the immobilization process and in the washing solutions from the amount of laccase initially used for the immobilization. The laccase concentration was measured using the Lowry method [33]. Under the experimental conditions reported above, the amount of

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339

Fig. 1. Schematic representation of PAN beads activation and laccase immobilization.

immobilized laccase was 3.33 ± 0.4 mg, i.e. about 0.28 mg of protein per gram of support. 2.2.3. The bioreactor In order to remove the APs from a buffered solution, 12 g (20 cm3 ) of catalytic PAN beads were employed in a fluidized bed reactor (see Fig. 2), constituted by a glass pipe, with an inner diameter of 1.7 cm and length of 20 cm. In order to uniformly suspend the PAN beads, the bioreactor was fed with 40 mL of substrate solution, duly thermostated, recirculating at a flow rate of 140 mL/min by means of a peristaltic pump. The bioreactor was, also, equipped with an aerator operating with an air flow of 100 cm3 min−1 continuously supplying the oxygen consumed in the reaction, so ensuring that this substrate, although limiting, allows the APs degradation by immobilized laccase. 2.2.4. Catalytic activity determination The sequence of the catalytic reaction by which laccase reduces the APs is illustrates in the Scheme 1. To calculate the laccase initial catalytic activity towards each substrate and to measure its catalytic efficiency in their removal, the decrease of nonylphenol or octylphenol concentration in the reaction mixture was followed during the time. The APs concentration in the reaction solution was measured by HPLC (LC-20AT Shimadzu) equipped with an UV–Visible Diode Array detector (SPDM20A). The separation of compounds was obtained with a Nucleodur Sphinx RP (25.0 cm × 4.6 mm ID, 5 ␮m) column (Macherey–Nagel GmbH & Co. KG, Germany) at a flow-rate of 1.3 mL/min. The mobile phase consisted of acetonitrile/water (30:70 v/v). The chromatographic determination was performed by using the following linear gradients: acetonitrile from 60 to 90% (from 0 to 5 min); acetonitrile 90% (from 5 to 10 min); acetonitrile from 90 to 60% (10–11 min). All analyzed samples were pre-filtered by means of a 0.2 ␮m MCE (Cellulose Mixed Esters) syringe filter (Macherey–Nagel GmbH & Co. KG, Germany).

Once known the substrate concentration changes (␮mol mL−1 ) during the time (min), the initial reaction rate (expressed in ␮mol min−1 ) was obtained by multiplying the value of (d[C]/dt) at t = 0 by the volume of the treated solution (mL). Just to give one example in Fig. 3 the OP concentration values are reported as a function of the enzyme treatment time by using a 40 mL of 1 mM solution in citrate buffer pH 5.0 and 40% ethanol, at the temperature of 25 ◦ C. At the same time the removal efficiency at t minutes, REt , defined as the percentage of APs removed from the solution after t minutes of treatment under the used experimental conditions, was calculated according to the formula REt (%) =

[C]0 − [C]t × 100 [C]t

where [C]0 and [C]t are the values of the substrate concentrations at the beginning of the run and at t minutes of treatment, respectively. To ensure that the removal efficiency was attributed only to laccase catalysis and not to adsorption on the carrier, some blank experiments were carried out under the same operational conditions by using the same beads volume but without the immobilized enzyme. All reported data are net of the contribution of carrier adsorption which was found to be about 5% of the initial concentration value. 2.2.5. Removal of estrogenic activity in APs aqueous solutions after enzyme treatment To assess the removal of estrogenic activity in APs aqueous solutions after laccase treatment we have followed two approaches: the first one concerning the study of the proliferation index of cells responsive to estrogenic stimuli in presence of NP or OP solutions, enzyme treated or untreated; the second one concerning the answer of engineered S. cerevisiae (YES test), to untreated and bioremediate solutions.

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Scheme 1. Schematic representation of oxidation reaction of APs by laccase.

To the first aim, human mesothelioma cell line MPP89 was used. Cells were obtained by the American Type Culture Collection (Rockville, MD, USA) and cultured as monolayer in culture dishes using RPMI-1640 medium, supplemented with 10% FBS (foetal bovine serum), penicillin 50 UI/mL and streptomycin 0.05 mg/mL, 1 mM Sodium Pyruvate and 10 mM Hepes. Cultures were main-

Fludized bed reactor

tained in a humidified atmosphere containing 5% CO2 at 37 ◦ C. Proliferation experiments were performed in the presence of phenol red-free RPMI-1640. The cells were seeded into 6 well plates at 1 × 105 cells/well in phenol red-RPMI-1640 with 10% FBS and allowed to attach for 16 h. Then cells were treated for 24, 48 and 72 h with OP and NP, or with OP and NP enzyme-treated or with E2. At the indicated times, cells were harvested and stained with trypan blue to evaluate cell viability using the dye exclusion method [34]. Untreated MPP89 cells were used as control of the cell growth. To the second aim, the yeast strain used was S. cerevisiae RMY326.This strain contains the human estrogen receptor alfa (hER␣) and an estrogen-responsive element (ERE) bound to the reporter gene lacZ encoding for the enzyme ␤-galactosidase [35]. The activation of the receptor due to the formation of a complex receptor–ligand causes expression of the reporter gene lacZ. The produced enzyme is secreted into the medium, where it metabolizes the chromogenic substrate, orto-nitrophenyl ␤Dgalactopyranoside (ONPG), which is normally colourless, into ortonitrophenol (ONP), a yellow substance that can be measured at 420 nm. Results are normalized with the number of cells assayed

Air

Thermostated bath

Thermostated bath

Peristaltic Pump Fig. 2. Schematic reproduction of the fluidized bed bioreactor.

Fig. 3. Concentration change in the bioreactor as a function of enzyme treatment time.

M. Catapane et al. / Journal of Hazardous Materials 248–249 (2013) 337–346

(OD420 × 1000) (t × V × OD600)

where t is the duration of the incubation time (min); V is the volume of culture used in the assay (mL); OD420 and OD600 are the absorbencies at 420 and 600 nm, respectively. The yeast cells were grown overnight at 26 ◦ C by shaking in minimal medium (Yeast Nitrogen Base in sterile water) and enriched with a solution of amino acids and glucose. After 24 h, an aliquot of the culture was diluted in fresh minimal medium to reach again, in further 16–18 h, a cell concentration of 2 × 107 cells/mL (exponential phase) in the presence of the samples to be tested. Different concentrations for each sample and 17␤-estradiol, as positive control, were assayed. Then, yeast cells were collected by serial centrifugations at 4000 rpm for 5 min and resuspended in Zbuffer (30 mM Na2 HPO4 –12H2 O, 20 mM NaH2 PO4 –H2 O, 5 mM KCl, 0.5 mM MgSO4 –7H2 O) plus a 0.025% ␤-mercaptoethanol, CH2 Cl2 , SDS 0.1%. The ␤-galactosidase activity was determined by the addition of 700 ␮L of ONPG (4 mg/mL in Z-buffer). The chromogenic reaction was stopped by the addition of 500 ␮L of Na2 CO3 1 M. Then, the cell debris was removed by centrifugation at 14,000 rpm for 2 min, and the absorbance at 420 nm of the sample was measured. 3. Results and discussion In order to characterize the catalytic system towards the two substrates, the catalytic activity of laccase, free or immobilized, has been assessed as a function of pH, temperature and substrate concentration in the reaction medium.

(a 100

Relative activity (%)

MU =

120

80

60

40

20

0

2

3

4

5

6

7

8

pH 120

(b 100

Relative activity (%)

and expressed as Miller Units (MU) using the following formula [36]:

341

80

60

40

3.1. pH dependence In Fig. 4 the relative activity of immobilized laccase is reported as a function of pH of the reaction medium kept at 25 ◦ C. The activity assays were carried out at pH in the range 3.0–7.0, by using 0.1 M citrate buffer for the pH from 3.0 to 6.0, or 0.1 M phosphate buffer for the pH from 6.0 to 7.0. Fig. 4a refers to OP (0.5 mM), while Fig. 4b to NP (0.5 mM). In both figures, for comparison, the relative activity of the free laccase (0.1 mg/mL) has been also reported. In the figures it is evident that the free and immobilized laccase have the same value of optimum pH at 5.0, indicating that the PAN beads do not have a net surface electric charge able to modify the microenvironment where the immobilized enzyme is operating. Calling optimal pH range the range where the enzyme retains up to 80% of its maximum relative activity, in Table 2 this range has been reported, for the free and immobilized laccase, for either OP or NP. From Table 2 and Fig. 4 it appears clear that the immobilization procedure confers higher resistance to the pH inactivation for the immobilized enzyme. At pH 3.0, indeed, the free laccase retains about 20% of its maximum activity towards OP and NP, while the immobilized laccase retains 35 and 32% towards OP and NP, respectively. These differences are more marked at pH 7.0 where the free laccase retains 15% towards OP and 35% towards NP, while the immobilized laccase retains 42 and 65% towards OP and NP, respectively. 3.2. Temperature dependence When the dependence on the temperature of the activity of immobilized laccase is studied, one obtains the results reported in Fig. 5. Fig. 5a refers to OP, while Fig. 5b refers to NP. The experimental conditions were the same that those reported in 3.1, with the exception that now the pH is kept constant at 5.0, while the temperature of the reaction medium is changed from 20 to 80 ◦ C for OP,

20

0

2

3

4

5

6

7

8

pH Fig. 4. Relative activity of free () and immobilized () laccase as function of pH: (a) OP and (b) NP. Standard experimental conditions: 0.5 mM of substrate concentration in buffered solution pH 5.0 at T = 25 ◦ C.

and from 20 to 60 ◦ C for NP. In both figures, for comparison, the relative activity of the free laccase has been also reported. In Table 2 the values of optimum temperature and optimum temperature range for the free and immobilized laccase have been reported. Results in Table 2 and Fig. 5 show that the optimum temperature shifts towards higher temperatures when the enzyme is immobilized and that the width of the optimum temperature range for OP increases from T = 17 ◦ C (from 33 to 50 ◦ C) for the free laccase to T = 20 ◦ C (from 42 to 62 ◦ C) for the immobilized laccase. Similarly the optimum temperature range for NP increases from T = 15 ◦ C (from 27 to 42 ◦ C) to T = 28 ◦ C (from 28 to 57 ◦ C) for the free and immobilized laccase, respectively. All together these results indicate that the immobilization procedure confers to the enzyme higher resistance to thermal inactivation. This is better evidenced considering that for OP at 30 ◦ C the residual activity of soluble laccase is about 30% and for immobilized laccase is 75%, while at 70 ◦ C the residual activities are 27.5% and 60% for the free and immobilized laccase, respectively. Similarly, when the activity towards NP is considered at 20 ◦ C the residual activities for the free and immobilized laccase are 52% and 60%, respectively, and at 60 ◦ C they become 30% and 70%.

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Table 2 Chemical-physical and biochemical parameters for the free and immobilized laccase using OP and NP as substrates. Chemical-physical and biochemical parameters

Optimun pH Optimum pH range Optimum temperature (◦ C) Optimum temperature range (◦ C) Activation energy (kJ mol−1 ) Km (mM) Vmax (␮mol min−1 mgenz −1 )

Free laccase

Immobilized laccase

OP

NP

OP

NP

5.0 4.6–5.4 40 33–50 32 0.70 2.5

5.0 4.6–5.6 35 27–42 34 0.42 1.32

5.0 4.1–5.6 50 42–62 50 1.11 1.25

5.0 4.2–6.3 45 28–57 55 0.72 0.70

At each temperature the substrate concentration in the reaction medium decreases during the time in a similar manner to that represented in Fig. 3, i.e. following an expression of the type Ct /C0 = e−kt , where C0 and Ct are the substrate concentration at the beginning (t = 0) and after t minutes and k is a constant proportional to the reaction rate. According to the Arrhenius equation and plotting lg(k) versus 1/T, it is possible to obtain the values of the activation energy reported in Table 2. From these values it is possible to see that the activation energies for the free enzymes are lower than those of the immobilized one and that the activation energy

towards the two substrates is practically identical either when the soluble enzyme or the immobilized one are considered.

Fig. 5. Relative activity of free () and immobilized () laccase as function of temperature: (a) OP and (b) NP. Standard experimental conditions: 0.5 mM of substrate concentration in buffered solution pH 5.0.

Fig. 6. Initial activity of free () and immobilized () laccase as function of substrate concentration: (a) OP and (b) NP. Standard experimental conditions: substrate in buffered solution pH 5.0 at T = 25 ◦ C.

3.3. Kinetic constants In Fig. 6 the initial reaction rates of immobilized laccase are reported as a function of substrate concentration in the reaction medium kept at 25 ◦ C, which represents the average temperatures of environmental surface waters needing bioremediation, and at pH 5.0. Fig. 6a refers to OP, while Fig. 6b refers to NP. For

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343

Table 3 Calculated time to reach substrate complete removal at different initial concentrations. Substrate concentration (mM)

(tc=0 )OP (min)

(tc=0 )NP (min)

0.10 0.20 0.30 0.40 0.50 0.75 1.00 1.50 2.00

118 139 159 172 220 260 300 398 510

110 160 200 225 250 330 400 551 720

comparison in both figures the initial reaction rates of soluble laccase are reported. Results in the both Fig. 6 allow the following observations: i) all the kinetics curves display a Michaelis–Menten behaviour; ii) the activities of immobilized laccase, as expected having the beads net neutral electrical surface charge, are lower than those of the free counter part; iii) at each substrate concentration the absolute activity towards the OP is higher than the activity towards NP, either for the immobilized or soluble laccase. Plotting the results in Fig. 6a and b according to the Lineweaver–Burk plot, the values of kinetic constants Km and vmax are obtained (Table 2). As it is well known, vmax represents the maximum reaction rate achieved by a determined enzyme system, i.e. when all the available catalytic site are engaged, while Km is the substrate concentration at which the reaction rate is half of vmax . High Km values indicate small affinity of the enzyme towards a substrate. These values are indicative to know if and how the immobilization process affects the enzyme reaction rate. Analyzing the Km and vmax values in Table 2, it is possible to observe that: i) the enzyme affinities of the free catalyst are higher than the ones for the immobilized laccase; ii) the laccase affinities for OP are smaller than those for NP, either for the free enzyme or for the immobilized one. 3.4. Biodegradation efficiency In view of practical applications some parameters of the process must be taken in consideration: the RE, the  50 and the tc=0 , the latter being the time to reach the zero concentration of the substrate, i.e. the complete pollutant removal. In Fig. 7 the RE values are reported as a function of the enzyme treatment time. Fig. 7a refers to OP, while Fig. 7b refers to NP. To avoid overlapping and overcrowding of results only those obtained at substrate concentration 0.1, 0.5 and 1.0 mM have been reported. Results in Fig. 7 clearly indicate that: i) for both substrates the values of percentage removal at each concentration are inverse function of the initial concentration; ii) the OP removal is faster than that of NP. Both these observation are better evidenced in Fig. 8 where, for all the concentration used, the values of percentage removal a t = 90 min (Fig. 8a) and the  50 , i.e. the time to halve the initial concentration, have been reported. To calculate the treatment time to reach the complete substrate removal, it has been extrapolated to c = 0 the curves similar to that reported in Fig. 3. We are aware that this methodology is not orthodox, but, also in presence of high error margin, it is possible to estimate the time to reach complete removal of substrates under the experimental condition used. In Table 3 the estimate tc=0 values have been reported for both substrates. 3.5. Time stability of the catalytic beads To test the time stability of the catalytic beads, their catalytic power has been assayed in a solution 1 mM of OP (Fig. 9). For comparison also the activity of the free laccase has been tested with a solution 1 mM of OP taking the same amount of enzyme solution

Fig. 7. Percentage of pollutant removal as a function of enzyme treatment time: (a) OP and (b) NP. Symbols: () 0.1 mM; () 0.5 mM; (♦) 1 mM. Standard experimental conditions: substrate in buffered solution pH 5.0 at T = 25 ◦ C.

from a stock aqueous solution preserved in a refrigerator at 4 ◦ C. It can be seen that the immobilized laccase retains over 95% of its original activity after 50 days. After the same time, the free laccase has zero activity. The results indicate that the laccase immobilized on PAN beads exhibits a good operational stability. 3.6. Estrogenic activity of OP and NP solutions, untreated or laccase treated All results above reported proved that a fluidized bed reactor filled with catalytic PAN beads is a useful tool for OP and NP removal from polluted aqueous solutions. But if these results are satisfactory from the chemical point of view, concern still remains about the presence of estrogenic activity in the enzyme treated solutions, since it is well known that in some cases the reaction products can be also more estrogenic or toxic than their parents. To answer to this concern two biological approaches have been carried out: the proliferation index of cells responsive to EDCs stimuli and the YES test assay. With the first approach, human mesothelioma cell line MPP89 was used. Cells (1 × 105 /well) were allowed to proliferate in presence of the natural estrogen 17␤-estradiol (E2 at the concentration 1 × 10−6 M), of OP and NP,

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120

1.6.106

(a

Percentage of substrate removal (%)

110

(a 1.4.106

100

1.2.106

90

80

1.0.106

Cell number

70

60 50

40

8.105 6.105 4.105

30 20

2.105

10

0 0

0.5

1.5

1.0

0 0

2.0

10

20

30

50

60

70

80

1.6.106

100

(b

(b

90

1.4.106

80

1.2.106

70

1.0.106

Cell number

60 50

τ 50

(min)

40

time (hours)

[Substrate] (mM)

40 30

8.105 6.105 4.105

20

2.105

10

0 0

0 0

0.5

1.0

1.5

10

20

2.0

40

50

60

80

70

time (hours)

[Substrate] (mM) Fig. 8. a) Percentage of substrate removal as a function of its initial concentration. b)  50 as a function of initial substrate concentration. Symbols: () OP; () NP.

30

Fig. 10. Cell growth as function of time when exposed at different stimuli: (䊉) only culture medium; () APs at 1 × 10−5 M; () E2 at 1 × 10−6 M. (a) OP and (b) NP.

separately, at the concentration of 1 × 10−5 M. The enzyme treated solutions before to be put in contact with the cell were filtered to avoid any contamination from the beads. In Fig. 10 the number of cells is reported as a function of the growth time. As usual, Fig. 10a refers to OP, while Fig. 10b refers to NP. Each experimental point in the figures is the average of 4 experiments. Each curve, of the type Ni (t) = N0 e(ki t), represents the curve best fitting the average points of four experiments. Ni (t) is the cells number measured at time “t” under the different experimental conditions and ki (h−1 ) is a constant proportional to the growth rate of the initial cells number. In Table 4 the ki values are reported for all the growth curves. From the data shown in Fig. 10 and as reported in Table 4 it is possible to derive that: i) the E2 has the highest growth rate notwithstanding its concentration is one order smaller than those

Table 4 ki constant calculated by the growth rates induced by E2 or APs. ki (h−1 )

Experimental conditions

Fig. 9. Time stability of free () and immobilized () laccase.

Control E2 Untreated APs Solutions Laccase treated APs Solutions

OP

NP

0.030 0.037 0.035 0.029

0.033 0.030

M. Catapane et al. / Journal of Hazardous Materials 248–249 (2013) 337–346 Table 5 Estrogenic response obtained by YES test at different concentrations of analyte solution. Analyte

Concentration (mM)

Miller units

E2

1 × 10−11 1 × 10−10 1 × 10−9

30.2 ± 1.8 200.4 ± 15.3 700.3 ± 53.2

OP

1 × 10−6 0.5 × 10−6

144.7 ± 13.5 0

B-OP

N.D. N.D.

NP

1 × 10−6 0.5 × 10−6

B-NP

N.D. N.D.

0 0

Buffer Distilled water

Dilution 1:100 –

0 0

0 0 199.8 ± 12.9 0

Note: B-OP and B-NP stay for bioremediated OP and NP, respectively. N.D. means not detectable.

of OP and NP; ii) the growth rate induced by OP is higher than that induced by NP; iii) the growth rates in the control medium or in presence of treated solution are quite identical, as expected. The last observation is the more interesting since clearly indicates that the estrogenic effects displayed by the OP and NP solutions disappear when the polluted solution are treated with laccase, at least in the case in which the proliferation index is studied. Similar results [37] have been found with the MCF-7 cells treated with E2, OP and NP and other endocrine disruptors at concentrations different from those used now. In that case the growth rate was not studied with enzyme treated solutions. In the second approach, due to the toxicity shown on Saccaromices cervisiae cells by the high initial concentration of APs used in our bioreactor model (data not shown), we decided to investigate with the YES test some dilutions of the untreated and bioremediated solutions. In particular we operated with a solution 1 × 10−6 M and a solution of 0.5 × 10−6 M of both OP and NP. Solutions of 17␤-estradiol at 1 × 10−9 , 1 × 10−10 , 1 × 10−11 M, were assayed as positive control. In addition the YES test was also applied, as control, to the Buffer diluted 1:100 and to distilled water. The latter was chosen to see if the trace of EDCs were present ab initio. The results are reported in Table 5. Results show, as expected, that: i) the estrogenic response is concentration dependent of the compound used; ii) OP and NP at a concentration 1 × 10−6 M show an estrogenic power similar to E2 at 1 × 10−10 M; iii) the apparent estrogenic power of NP is higher than that of OP. Finally, the bioremediated APs solutions lost any estrogenic effect. 4. Conclusions At the aim of removing the OP or NP from water systems polluted by these Endocrine Disruptors, belonging to the class of alkylphenols, a fluidized bed reactor, filled with laccase-based PAN beads, has been employed. Results confirmed the practical usefulness of a bioreactor. In particular the Octylphenol was removed at higher rate than Nonylphenol. In consideration of the estrogenic activity of the two chemical compounds, the loss of estrogenic activity of the polluted solutions has been assessed by studying the proliferation index of MPP89 cells, a human mesothelioma cell line, in presence of OP or NP solutions, untreated or laccase treated. The constant growth rates compared with control experiment, such as normal growth medium or medium enriched by 17-␤-estradiol, demonstrated not only the chemical removal of the pollutants but also the lost of estrogenic activity of the enzyme reaction product. The

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