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Multifunctional biomagnetic capsules for easy removal of phenol and bisphenol A

Cristina R. Ispas, Matthew T. Ravalli, Ashley Steere, Silvana Andreescu*
Department of Chemistry and Biomolecular Science, Clarkson University, PO Box 5810, Potsdam, NY 13699-5810, United States

article info
Article history: Received 26 June 2009 Received in revised form 19 November 2009 Accepted 26 November 2009 Available online xxx Keywords: Bisphenol A Phenol Removal Microencapsulation Tyrosinase Chitosan Biocapsules

abstract
This paper reports fabrication, optimization and characterization of multifunctional biocapsules with immobilized enzyme using a layer-by-layer conguration and their application for removal of phenol and bisphenol A (BPA). The method is based on the combined use of enzymatic oxidation of the BPA and subsequent binding of the reaction product onto a chitosan core biopolymer. This platform has multiple functions including: (1) enzymatic degradation of BPA, (2) adsorption of the degraded compound within the core material, (3) colorimetric quantication and (4) magnetic capabilities. We examined various congurations of core/shell structures of alginate and chitosan and determined the stability and the optimum conditions in which these structures provide the most effective removal capacity. The amount of BPA that can be removed per capsule is 5.6 ppm while phenol can be removed up to 10 ppm per capsule within 15 h. 2009 Elsevier Ltd. All rights reserved.

1.

Introduction

Phenol and phenolic derivatives are major industrial (petroleum, chemical and plastic) and agricultural byproducts that can be found in surface waters as well as in food and clinical samples. Among these, bisphenol A (BPA), which is used to fabricate polycarbonate plastics, has recently received considerable attention due to its endocrine disrupting activity and possible toxic environmental and health impacts (Ballesteros-Gomez et al., 2009; Kang et al., 2006; Le et al., 2008). BPA levels in low mg/L range could be found in clinical (blood, urine, saliva), food and water samples (Dekant and Voelkel, 2008; Volkel et al., 2008). The removal of phenols and phenolic derivatives with estrogenic activity is an important environmental problem.

Several methods currently exist to remove these chemicals from environmental matrices including activated carbon adsorption, electrochemical, ozonization, biological and chemical procedures (Busca et al., 2008; Dabrowski et al., 2005; Deborde et al., 2008; Lin et al., 2009; Yu et al., 2009; Zhang et al., 2008). Enzymatic degradation of phenols into less toxic forms has been suggested as a treatment method (Wu and Zhou, 2001). Several enzymes catalyze the oxidation of phenols and have been used for this purpose: horseradish peroxidase (HRP), laccase, tyrosinase and chloroperoxidase (Buchanan and Nicell, 1997; Ikehata and Nicell, 2000a; Kim and Nicell, 2006; Yu et al., 1994). Studies on the enzymatic oxidation of BPA have been less reported. With HRP, phenols are oxidized in the presence of hydrogen peroxide (Buchanan and Nicell, 1997; Ghan et al., 2004) to produce unstable phenoxy radicals that spontaneously react with each other or with another

* Corresponding author. E-mail address: eandrees@clarkson.edu (S. Andreescu). 0043-1354/$ see front matter 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2009.11.049

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phenol monomer to give rise to an insoluble high molecular weight polymer. The polymer can then be removed from the solution by ltration and sedimentation. However, the method has several limitations such as the residual toxicity of soluble small molecular weight reaction products that remain in the aqueous phase (Wagner and Nicell, 2002), the enzyme inactivation and the addition of H2O2 as the oxidant, which makes the system more complicated. As an alternative, tyrosinase catalyzes the oxidation of phenols to quinones in the presence of molecular oxygen (Solomon et al., 1996) and provides a simpler and more cost effective strategy (Edwards et al., 1999). The o-quinones are toxic soluble products that do not precipitate spontaneously in solution (Ikehata and Nicell, 2000b), and thus cannot be removed by ltration and sedimentation. However, they can chemically bind to chitosan through its amino groups (Kumar et al., 1999; Payne et al., 1996) and coagulate to produce a gel-like polymeric structure with lower toxicities than those of solutions treated with peroxidase enzymes (Kumar et al., 1999; Sun et al., 1992; Yamada et al., 2005). The majority of reported works have used the enzyme in solution and only few systems with immobilized enzyme have been explored for this purpose. Seetharam and Savillehave studied the degradation of phenol by tyrosinase immobilized on siliceous supports (Seetharam and Saville, 2003). Ensuncho et al. have studied the removal of aqueous phenol using tyrosinase immobilized with chitosan in a bench and pilot scale three-phase uidized reactor and have found that the activity of tyrosinase was maintained within the chitosan matrix (Ensuncho et al., 2005). There are no reports on the use of this concept for removal of BPA. Herein, we describe development, optimization and characterization of a biomagnetic capsule platform with immobilized enzyme as an environmentally benign method incorporating both sensing and removal capabilities for BPA. To fabricate the biocapsules we used natural biopolymers chitosan and alginate and a layer-by-layer assembly approach (Taqieddin and Amiji, 2004). The method is based on the combined use of enzymatic oxidation of BPA by tyrosinase and subsequent covalent binding of the reaction product onto the corresponding core biopolymer. This binding induces a distinct color change in the presence of BPA that could be explored as a quantitative way to probe the reaction in the interior of the capsule and detect the presence of BPA. The platform described in this present study has multiple functions including: (1) enzymatic degradation of BPA, (2) adsorption of the degraded compound within the core material for removal, (3) colorimetric quantication and (4) magnetic capabilities so that the entire assembly can be easily manipulated. We demonstrate here the feasibility of this concept for removal with phenol and BPA.

purchased from SigmaAldrich Chemical Co., St. Louis, MO. The activity of dry solid tyrosinase was 3900 Units/mg and stock solution of 100 U/mL, stored at 20 C was prepared by dissolving solid enzyme in 0.1 M phosphate buffer (pH 6.5). Glutaraldehyde 25% (w/v) solution was purchased from Fischer Scientic, Morris Plains, N.J. A 1.25% (w/v) stock chitosan solution was prepared by dissolving chitosan in 0.1 M acetic acid and stirring for 4 h. Stock alginate solution of 2% (w/v) was prepared in distilled water (Millipore). Chitosan and alginate solutions were kept at 8 C for up to 4 weeks. A 2% (w/v) glutaraldehyde, 1.5% NaTPP (w/v) and 0.34 M CaCl2 stock solutions were also prepared using distilled water. Stock solutions of 100 mM BPA and phenol were prepared in methanol. Subsequent dilutions were made with distilled water. Amino functionalized Fe3O4 magnetic particles with average diameter of 10 nm were fabricated according to a published procedure (Rossi et al., 2004).

2.2.

Fabrication of biomagnetic capsules

Two types of capsules were fabricated using chitosan and alginate and were labeled A and B as follows: chitosan core (A) and chitosan core/alginate shell (B). For each of the two types, two cross-linking agents were used: NaTPP and glutaraldehyde (glut). To fabricate the chitosan core, 15 mL of tyrosinase (100 U/mL) were added to 400 mL of chitosan stock solution and mixed thoroughly. The chitosan/tyrosinase solution was then dropped by a syringe into a bath of 1.5% NaTPP solution and allowed to crosslink for 1 h; these beads were labeled A-NATPP. To fabricate A-glut, chitosan cores were immersed for 10 min in a 2% glutaraldehyde solution for additional cross-linking. To fabricate the core/shell chitosanalginate capsules, A-NaTPP and A-glut capsules were rst immersed into a 0.34 M CaCl2 solution to adsorb a supercial layer of calcium chloride onto the chitosan and allow rapid cross-linking of the alginate. They were then immersed into a bath of 2% alginate solution to form an outer alginate shell and immediately returned to the calcium chloride for full cross-linking. Capsules A-NaTPP with an alginate shell were labeled B-NaTPP, whereas A-Glut with alginate shell were labeled B-Glut. All capsules were stored in distilled water until use. Three different storage conditions were tested to determine the optimum conditions: room temperature, 8 C and 20 C. The magnetic biocapsules were fabricated in the same manner, but with the alginate shell made by adding aminofunctionalized Fe3O4 nanoparticles to the alginate solution in a 1:2 (w/v) ratio.

2.3.

Methods

2.3.1. UV/VIS study of the biocatalytic conversion of BPA in the presence of chitosan

2.
2.1.

Materials and methods

Materials

Chitosan, from crab shells (practical grade), alginic acid (Na salt), sodium triphosphate pentabasic or NaTPP (practical grade), phenol (99% purity), BPA (99% purity), tyrosinase from mushroom, and calcium chloride (96% purity) were

The tyrosinase catalytic conversion of BPA to quinone and the chemical binding of the generated quinone to chitosan were rst followed up by using UVVIS spectroscopy. For that, 100 mL of an aqueous solution mixture of 400 mL chitosan (1.25 w/v) and 15 mL tyrosinase stock solution was added to 250 mL BPA solutions of concentrations ranging from 0.01 to 0.8 mM. The chitosan/tyrosinase/BPA solution was mixed using a vortex and then incubated for a xed period of time to allow the

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enzymatic and the subsequent chemical binding reactions to proceed. Then, UVVIS spectra for the various BPA concentrations were recorded.

2.3.5.

Application to environmental water samples

2.3.2.

Study of enzyme activity and leaching

UV/VIS measurements were carried out using a Shimadzu UVVIS spectrophotometer (model Shimadzu UV-2401PC) equipped with a 1-cm path length cell. The activity of the tyrosinase was measured using a colorimetric assay according to established procedures (Worthington, 1993). Enzyme leaching was evaluated by measuring the amount of enzyme in the supernatant solution after incubation for 1, 2 and 3 days of two capsules in 1.0 mL distilled water. The absorbance of the supernatant at 280 nm was measured repeatedly once a day in order to determine if any enzyme was present.

Functionality and stability of the capsules were tested in real environmental water samples. The water was collected from the local Water Treatment Plant, Potsdam, NY, prior treatment and purication. The Plant draws its water from Raquette River, Potsdam, NY. The samples were ltered before use. No other treatment was applied. For this study, 2 capsules were incubated in 1 mL water without added phenol and in samples spiked with 0.3 mM phenol.

3.

Results and discussion

3.1. UVVIS spectral change of BPA in the presence of chitosan with and without tyrosinase
Formation of enzymatically degraded BPA bound to chitosan was monitored by UVVIS measurements after incubation of BPA in the presence of tyrosinase and chitosan. Fig. 1A presents the changes in the UVVIS absorption spectra of the tyrosinase catalyzed reaction of BPA in the presence of chitosan. BPA is characterized by two strong absorption bands with maximums at w224 and 275 nm. In the presence of tyrosinase (without chitosan), the absorbance of the two BPA characteristic peaks increased at the same wavelength and a new peak appeard at 390 nm, due to the formation of a quinoid compound (Andreescu and Sadik, 2004). The color of this solution was light yellow. In contrast, in the presence of both chitosan and tyrosinase, a new well-dened absorption peak with a maximum at around 610 nm and a wide shoulder at w350 nm were observed, accompanied by the

2.3.3. Evaluation of capsule reproducibility, long term storage, pH and ionic strength
In order to test the reproducibility of the capsules, 5 separate vials containing the same number of capsules per vial and the same concentration of phenol (0.1 mM) were prepared. After 20 h, the supernatant of each vial was analyzed to determine the amount of residual phenol. Long term storage stability, effect of pH and ionic strength were determined only for the most efcient type of capsules (B-glut). To test the stability, three groups of vials containing 2 capsules per vial were stored in three different conditions: room temperature, or approx. 2225 C, in the refrigerator at w28 C, and in the freezer at approx. 20 C. Capsules were then removed periodically and their removal capacity was tested every 3 days. For this purpose, the capsules were incubated with 1.0 mL of known concentration of phenol for 16 h and after exposure, the residual phenol was determined. The effect of pH and ionic strength were tested using a similar procedure by incubating 2 capsules (B-glut) in 1 mL of phenol solutions (0.3 mM). Acetate and phosphate (0.1 M) were used to prepare buffer solutions with pH ranging from 4.5 to 7.5. The effect of ionic strength was tested with NaCl solutions of concentrations ranging from 1 to 1000 mM in acetate buffer solutions at pH 6.5.

A
Absorbance

1.2 1 0.8 0.6 0.4 0.2 0.3 mM 0.2 mM 0.1mM 0.05 mM 0.01 mM 0.02 mM
300 400 500 600 700 800

2.3.4.

Study of phenol and BPA removal

0
200

Batch experiments to study the removal of phenol and BPA were performed in glass vials by incubating three capsules with 1 mL of known concentrations of phenol or BPA. For comparison purposes, capsules with and without enzyme were tested and the supernatant was analyzed after 2, 15, 24 and 39 h respectively. The concentration of residual phenol and BPA in aqueous solution was determined using a calibration curve with a linear range of 0.56 mM for phenol and 0.11.4 mM for BPA. Calibration and quantitative analysis was performed by spectrophotometry using a Shimadzu UV VIS spectrophotometer (UV-2401PC) or by HPLC with a HewlettPackard Series 1050 HPLC with UV detection with a reverse-phase Luna 5m C18 (2) (250 4.60 mm, 5 micron) analytical column (Phenomenex, Torrance, CA). Structural changes of the chitosan core before and after exposure to phenol were estimated by infrared spectroscopy (FTIR, BRUKER Equinox 55 spectrophotometer) of dried core samples.

Wavelength (nm)

Fig. 1 (A) UVVIS changes of chitosan and tyrosinase in the presence of 0.01, 0.02, 0.05, 0.1, 0.2, 0.3 mM BPA after 24 h incubation. (B) Color change of a cross-linked chitosan/ tyrosinase lm with varying concentrations of phenol (up-red) and BPA (down-green): from left to right: 0, 0.05, 0.1, 0.3, 0.5 and 0.8 mM BPA. (For interpretation of the references to colour in this gure legend, the reader is referred to the web version of this article.)

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formation of a green-bluish colored compound, within several hours of incubation (Fig. 1A and B). These spectral changes were not observed in the absence of enzyme, suggesting that BPA alone does not bind to chitosan. The new peaks at 350 and 610 nm increased gradually with increasing the concentration of BPA. This change is characteristically different than that of the BPA generated quinone without chitosan and indicates a conversion of quinone into a strongly colored compound. This compound is formed through covalent binding between the quinone carbonyl groups and the nucleophilic amino groups of the chitosan via a Schiff based reaction. Similar spectral changes in the presence of chitosan and tyrosinase were reported for phenol (Payne et al., 1996; Sun et al., 1992), catechin (Wu et al., 2002) and p-cresol (Kumar et al., 2000), with the appearance of a reddish-brown compound. The same mechanism applies for p-alkylphenols like nonylphenol and octylphenol (Yamada et al., 2006). We determined the reaction kinetic and compared the enzymatic conversion followed by the chemical binding of BPA to chitosan with that of phenol and observed that BPA required longer reaction time compared to phenol. For phenol, the color appeared almost immediately after the contact with the chitosan/tyrosinase mixture. However, the kinetic of BPA reaction was slow; the color change was visible after w30 min incubation and developed over time. The maximum intensity was reached after 8 h for BPA versus 4 h for phenol. This difference may be explained by the bulkier structure of the BPA that could induce conformational constraints and restrict the accessibility to the active site, affecting the reaction kinetics. The consequence of this slower kinetic is the slower binding of BPA, and thus reduced removal efciency as compared to phenol.

3.2. Fabrication and optimization of the biocatalytic capsules 3.2.1. Biocapsule concept

The enzymatic degradation of BPA by tyrosinase, followed by the subsequent covalent binding of the enzymatically generated quinone to chitosan was utilized to fabricate microcapsules and apply this concept to the removal of BPA. The quinoneimine cross-linked chitosan is a gel-like structure with residual toxicity and it is difcult to remove. In this study, we further exploited this principle by specically tailoring the binding of the enzymatically generated quinone to a chitosan core, which forms the internal cavity of a microcapsule. The resulting quinone-bound chitosan is then trapped inside the capsule and retained by the shell, thus preventing further release of the generated quinone in the environment. As opposed to other works, which utilized this principle for phenol removal and in which chitosan lms (Payne et al., 1996; Sun et al., 1992) or membranes (Edwards et al., 1999) were used, here we describe a layer-by-layer assembly to create a core-shell structure with magnetic properties for easy handling and removal. To fabricate the microcapsules, chitosan, a positively charged polyelectrolyte was used as core material, while alginate, a negatively charged polyelectrolyte, was used to form the protective shell. Alginate is a well known biocompatible and biodegradable polymer, traditionally used for encapsulating biological materials. Alginate and chitosan

can be assembled in a sequential layer-by-layer conguration via electrostatic interactions or in core-shell microcapsules using different manufacturing procedures (Gaserod et al., 1998, 1999). Enzymes can be encapsulated in the interior of the microcapsule with preservation of catalytic activity and stability over long periods of time (Lee et al., 2003; Santos et al., 2001). These structures have found various applications in biotechnology, biomedicine and drug delivery (Itoh et al., 2006) but have been less exploited for environmental purposes. Chitosan-alginate beads have previously been studied for the removal of heavy metal contaminants from wastewaters (Huang et al., 1996). The biocompatibility and stability of the chitosan/alginate microcapsule provide enhanced storage stability and functionality for the immobilized enzyme. The enzyme was encapsulated into the core of the microcapsule to ensure biocatalytic properties while amino-functionalized magnetic nanoparticles were uniformly dispersed in the alginate shell material. To avoid leaching of the particles, the Fe3O4 nanoparticles were functionalized with amino functional groups (Rossi et al., 2004) to bind with the carboxyl groups in the alginate. Thus, the chitosan core containing the enzyme acts as both biocatalytic and adsorption site, due to the presence of amino groups on the glucosamine unit, while the shell prevents enzyme leaching, stabilizes the capsule and provides magnetic capabilities for easy removal and handling. In addition, the o-quinones formed in the reaction are chemically bound to chitosan through its amino groups to yield Schiff bases (Fig. 2) or Michael adducts (Kumar et al., 2000), thereby reducing the inhibitory effects on the enzyme. This binding was further conrmed by FTIR spectroscopy (supplementary information section) to obtain structural analysis of the core containing the enzyme before and after exposure to phenol, conrming changes in the functional amino groups of the chitosan after the binding of phenol. The core-shell assembly could be regarded as a magnetic carrier with multifunctional capabilities that can be magnetically controlled, separated and directed to specic sites for environmental removal. The functioning principle is schematically presented in Fig. 2. Phenolic compounds diffuse through the alginate permeable shell to the chitosan core, where they are degraded by tyrosinase, producing a quinoid compound. The quinone subsequently binds to chitosan to produce a high molecular weight polymer that is contained within the capsule and protected by the alginate shell.

3.2.2.

Optimization and characterization of the biocapsules

To optimize the system, four different types of capsules were studied for their mechanical stability, enzyme stability, reproducibility, long-time functioning and phenol degrading capabilities. To determine the role of the shell chitosan core and core-shell (chitosan-alginate) congurations were tested. The capsules varied physically by their biopolymer composition and cross-linking agents. NaTPP and glutaraldehyde were tested as cross-linking agents for chitosan. NaTPP allows crosslinking via its triphosphate units and the resulting NATPPchitosan layers have a spongy-like structure with increased permeability (Taqieddin and Amiji, 2004). Glutaraldehyde was reported as an efcient cross-linking agent for chitosan, generating stronger polymeric units (Schiffman and Schauer, 2007). Capsules were rst optimized and tested with phenol as

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Fig. 2 Schematic diagram of the functioning principle of the biomagnetic capsule for the removal of BPA. Microcapsules are made of chitosan core and alginate shell. Tyrosinase is encapsulated in the chitosan core. Fe3O4 magnetic nanoparticles are incorporated in the alginate shell. The size of a capsule is around 2 mm in diameter.

a model phenolic compound to demonstrate the functioning principle and evaluate their stability and removal capability. A summary of the optimization results with the four capsule congurations are presented in Table 1. First to be investigated was the enzyme leaching. In order to be functional and effective in the degradation of phenol, tyrosinase must be active and contained within the microcapsule. Therefore it was necessary to explore the possibility of enzyme leaching. For this, capsules stored at room temperature in distilled water were tested for the presence of the enzyme in the supernatant once a day for four days. The results of the UVVIS absorbance measurements at 280 nm demonstrated that with the exception of the A-NaTPP no signicant enzyme leaching occurred for any of the capsules (results not shown). This is consistent with the observation that enzymes encapsulated within polyelectrolyte layers have good stability with no enzyme leaching (Ghan et al., 2004). Capsules A without shell appeared less mechanically stable than core-shell type B capsules. Capsule stability was greatly improved when glutaraldehyde was used as a cross-linking agent. However, chitosan core cross-linked with glutaraldehyde (A-glut) without alginate shell were fractioning and

tended to disintegrate in solution. In the same conditions, chitosan cross-linked with NaTPP showed very low removal rate, probably due to enzyme leaching.

3.2.3. Study of reproducibility and long- term storage stability

Experiments were conducted to determine the reproducibility of the capsules. The three most efcient types of capsules were used in these studies: A-NaTPP, A-Glut, and B-Glut. The average percentage of phenol remaining in vials containing one capsule and 1.0 mL of 0.1 mM phenol, and the corresponding standard deviation for ve replicate analysis was as follows: A-NaTPP 54.97 (20.76)%, A-Glut. 50.36 (1.46)%, and B-Glut. 49.71 (1.37)%. The results show that all three types of capsules removed nearly equivalent percentages of phenol over the given period of time. However, it is evident from the standard deviations that the A-Glut and B-Glut capsules are much more reproducible than the A-NaTPP capsules. To determine the long term functionality, A-Glut capsules were stored in distilled water at varying temperatures and then assessed for their phenol removing properties; a constant concentration of phenol of 0.3 mM and a 16 h incubation time

Table 1 Summary results of the four different capsule congurations and percent phenol removal. Batch experiments were carried out with two capsules exposed to 0.3 mM phenol (1 mL) for a 24 h time period at room temperature. Capsule Conguration
A-NaTPP A-Glut. B-NaTPP B-Glut.

Capsule composition/ cross-linking agent

Chitosan core/NaTPP Chitosan core/glutaraldehyde Chitosan core alginate shell/NaTPP Chitosan core alginate shell/glutaraldehyde

% of Phenol Removed
3.01 86.82 71.19 92.09

Experimental observations
Stable capsules Possible enzyme leaching Instability and fracturing Capsules tend to disintegrate Stable capsules No enzyme leaching Capsules with enhanced mechanical stability No enzyme leaching

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was selected for these studies (Fig. 3). The three sets of capsules performed almost similarly in the three storage conditions. This indicates that (1) the enzyme encapsulated in the polyelectrolyte layer is stable and functional for multiple days even at room temperature and (2) no special storage conditions is required. With the exception of those stored at 20 C, that were occasionally disintegrated due to freezing, capsules were mechanically stable. The capsules with chitosan core/alginate shell cross-linked with glutaraldehyde (BGlut) showed superior stability and no enzyme leaching. These were functional for multiple days even at room temperature indicating that the enzyme encapsulated in the polyelectrolyte layer is stable. Capsules can be produced reproducibly with an average value and coefcient of variation for phenol removal of 49.71 (1.37)% for ve replicate analysis (Fig. 3).

A
% Phenol Remaining

100 80 60 40 20 0

Enzyme Enzyme+Magnetic particles

0.2

0.4

0.6

0.8

Phenol concentration (mM)

B
% Phenol Remaning

Control-no enzyme

100 80 60 40 20 0

Enzyme

3.3.

Removal studies

Removal capacity and removal rate were rst established for phenol. Each type of capsule was tested in ve different concentrations of phenol after 2, 15, 24 and 39 h of exposure (Fig. 4). After two hours, all types of capsules appeared to be removing phenol at similar rates. However, after 15 h, the removal properties of B-NaTPP, B-Glut and A-Glut were superior to that of the A-NaTPP. This suggests that glutaraldehyde plays an important role in the stabilization of the capsules. The system with chitosan core/alginate shell and cross-linked with glutaraldehyde (B-Glut) was the most effective and mechanically stable, providing the higher removal rate, with a concentration of w0.1 mM phenol per capsule in less than 1 h contact. These results indicate that the alginate shell plays an important role in the stability of the capsule. Fig. 4A shows phenol removal capability, quantied by HPLC, obtained with three capsules in a concentration range of 0.050.8 mM after 15 h exposure with and without magnetic particles. The presence of magnetic particles is not

10

20

30

40

50

Incubation time (hours)

Fig. 4 (A) phenol removal capability, quantied by HPLC, obtained with 3 capsules (1 mL solution) with and without magnetic particles in a concentration range of 0.050.8 mM after 15 h exposure (average results from three independent experiments). (B) percentage of phenol remaining after 0, 2, 15, 24 and 39 h for capsules exposed to 0.3 mM and stored at room temperature.

Fig. 3 The percentage of phenol remaining after 3,6, or 12 days of storage at various conditions followed by 16 h of phenol exposure. All capsules were A-Glut. Capsules were stored in distilled water at the various temperatures: 22 258C (room temperature), 288C, and wL208C for 3, 6, or 12 days at which point the capsules were removed from storage and placed in vials containing 1.0 mL of 0.3 mM phenol at room temperature for 16 h.

affecting the removal capacity. Up to 95% phenol was fully removed after 39 h incubation (Fig. 4B). Control experiments in the absence of enzyme (Fig. 4B), show less than 10% removal (vs 90% with enzyme), indicating that the enzymatic conversion is essential to initiate the proposed mechanism. Further tests for BPA removal were performed only with the most efcient conguration, B-Glut. Fig. 5A and B shows the effect of pH and ionic strength on the removal capacity of the biocapsules with phenol. The capsules effectively removed phenol over the pH range from 4.5 to 8, with a slightly higher removal rate at pH 5. The functionality of the system over this broad range of pH was similar to that reported by Ikehata and Nicell (Ikehata and Nicell, 2000b) for homogenous solutions of phenol catalyzed by tyrosinase in the presence of chitosan. At a pH lower the 4 the chitosan is soluble and the enzyme activity is lower; thus the capsules are not stable and the ability of the enzyme to catalyze the transformation of phenol is low. The removal efciency was enhanced by increasing NaCl concentrations up to 100 mM and then decreased for 1000 mM (Fig. 5B). This salinity prole could be explained by an activation effect of the immobilized enzyme by the

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A 100
% Phenol Remaining 80 60 40
100

20
80 73.04 66.55 60 48.62 40 26.66 20 % BPA Remaining

84.95

4.5

5.5

6 pH

6.5

7.5

100 80 60 40 20 0

% Phenol Remaining

0 0.1 0.5 0.8 1 BPA concentration (mM) 1.5

Fig. 6 BPA removal after 15 h of exposure with biomagnetic capsules. Inset: vials used in batch experiment with 3 capsules per vial in 0, 0.1, 0.5, 0.8, 1 and 1.5 mM BPA after 5 h exposure.

50 NaCl (mM)

100

1000
as efcient, the biomagnetic capsules are still successful in removing BPA in the lower mM range. Fig. 7 shows the fabricated microcapsules in the absence (A) and presence (B) of phenol, indicating a visible color change of the capsule core as a result of the enzymatically generated quinone. Inclusion of magnetic nanoparticles into the polymeric structure provides magnetic capabilities and possibilities for simple handling and separation as seen in Fig. 7D. It is obvious from these images that the quinone does not bind to alginate, which was used as shell material. No color change was observed when alginate alone was incubated with phenol or BPA in the presence of tyrosinase and this conrms that binding occurs through the amino groups of the chitosan.

Fig. 5 Effect of pH (A) and ionic strength, NaCl (B) on the removal capacity of 0.3 mM phenol (1 mL solution).

presence of sodium ions, inducing an increase in the oxidation rate, or by possible changes in the porosity of the alginate shell at high salt concentrations. Fig. 6 shows BPA removal, quantied by HPLC, obtained with three B-Glut magnetic capsules in a concentration range of 0.11.5 mM after 15 h exposure. Up to 74% of the 0.1 mM BPA was converted after 15 h incubation. As expected, a distinct color change of the chitosan core occurred when the enzymatically produced quinone was bound to the chitosan core, allowing for simple visualization of the reaction. No color change was detected in the absence of enzyme for neither phenol nor BPA. The color starts to develop gradually over time after w30 min exposure and is proportional to BPA concentration (Fig. 6 inset). By comparing the removal capacity of BPA with that of the phenol, we noticed that BPA is more slowly and less efciently removed. For example, in the same experimental conditions after 15 h incubation, a concentration of 0.5 mM BPA was removed in a proportion of 54%, as compared to 86% for the same concentration of phenol. The main cause for the less efcient removal can be attributed to steric hindrance and diffusion constraints of the BPA through the alginate shell. In addition, the resulting quinone-bound-polymer is of a larger size and could prevent further penetration of BPA to the enzyme and chitosan sites. Increasing the porosity of the alginate and chitosan layer for easier diffusion is possible; however, that could induce enzyme leaching and decrease capsule stability. Though not

4. Application of the biocapsules to environmental water samples

The stability of the capsules in real environmental water was rst investigated by incubating capsules in environmental raw water collected from the Potsdam Water Treatment Plant, which draws its water from Raquette River, Potsdam, NY. While the water developed visible microbial growth over time, the capsules were stable and robust for at least two weeks. The alginate shell acts as a semi-permeable membrane restricting the access of larger contaminants to the biologically active core of the capsule thus providing enhanced stability and protective action. The polymeric core/shell structure of the capsules was robust and did not show structural changes over time. The capsules were further used for the removal of phenol from the environmental water. Since

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Fig. 7 Core-shell chitosan-alginate microcapsules containing tyrosinase in the absence (A) and presence (B) of phenol. (C): biomagnetic capsules (D): magnetic properties under an external magnetic eld. (E): side view of a capsule.

the samples had no detectable phenol concentrations, for the removal tests, these were spiked with 0.3 mM phenol. As with distilled water, the color of the chitosan core changed within the rst minutes of incubation with the spiked water, conrming the biocatalytic transformation and chemical binding of phenol inside the core. Removal rates of 45% were obtained. In the environmental eld, these capsules can be used to design batch adsorption systems for the removal of phenolics. The capsules could be added to phenol-containing water tanks at room temperature and incubated for a period of several hrs to days (e.g. 10 h 3 days). Because the capsules are functional in a wide pH range, adjustments of pH are not necessary. The capsules can then be retrieved through the use of a permanent magnet, eliminating the need for additional ltration or separation steps.

structures are fully functional, stable at room temperature and robust; they can be used and stored for at least 12 days without special storage conditions and work well in real environmental water samples. In addition, chitosan and alginate are both biodegradable and nontoxic, which makes them an excellent choice for use in environmental remediation. In the future this concept could be extended to other environmental phenolics.

Acknowledgements
This work was supported by grant NSF-DMR-0804506. SA and AS acknowledge NSF-REU grants EEC-0452789 and EEC0755345. MTR acknowledges funding from the NSF GK-12 program DGE-0338216. We thank Potsdam Water Treatment Plant for providing the environmental water samples.

5.

Conclusions

In summary, by the use of natural and environmentally safe materials, core-shell biomagnetic capsules with immobilized tyrosinase were fabricated in a layer-by-layer conguration and studied for the removal of BPA. The enzyme was efciently immobilized within the biopolymer layer, which conferred enhanced stability with no leaching. Capsules with a chitosan core/alginate shell cross-linked with glutaraldehyde showed superior stability, faster removal rate and greater reproducibility. The removal of phenol was efcient within several hours and the capsules were able to operate in source and river waters. The removal capacity for BPA was slightly decreased due to the bulkier molecular size of BPA compared to phenol. The amount of BPA that can be removed per capsule is 5.6 ppm while phenol can be removed up to 10 ppm per capsule. The use of iron oxide magnetic particles had no effect on removal, and greatly improved ease of handling. The biomagnetic capsules can be applied to treatment of phenol contaminated waters and of other aqueous solutions containing amounts of phenolics in the ppm range. Binding of the enzymatically degraded phenolics to the chitosan core produced a distinct color change that could be used as a simple tool for visualization and conrmation of the reaction in the interior of the capsule. In summary, this study demonstrated the potential of core/shell microcapsules in environmental remediation of phenol and BPA. These

Supplementary material
Supplementary data associated with this article can be found in the online version, at doi:10.1016/j.watres.2009.11.049.

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