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Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation
Enzyme and Microbial Technology 25 (1999) 769 –773
Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation W. Edwards, W.D. Leukes, P.D. Rose, S.G. Burton* Goldfields Biotechnology Centre, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa Received 16 December 1998; received in revised form 22 July 1999; accepted 10 August 1999
Abstract Internally skinned polysulphone capillary membranes were coated with a viscous chitosan gel and used as an immobilization matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors then were used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilized polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. This facilitated an increase in the productivity of the immobilized enzyme. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Polyphenol oxidase; Polysulphone capillary membranes; Chitosan; Phenol
1. Introduction The selective recognition and oxidation of phenols by the enzyme polyphenol oxidase to produce reactive o-quinones (Fig. 1) can be successfully exploited as a waste minimization strategy for effluent treatment [1,2,3]. We have shown previously that polyphenol oxidase can be immobilized on capillary membranes facilitating the conversion of phenols to the corresponding o-quinones [4]. However, although polyphenol oxidase is effective in converting phenol and a number of associated derivatives to their corresponding o-quinones, these o-quinones and the low-molecular-weight polymers formed from them remain in the treated effluent. At low concentrations the remaining colored products do not achieve high enough degrees of polymerization to effectively precipitate from solution [2,5,3,6]. Thus, the goal of reducing the toxicity of the phenol-containing effluent is achieved, but the resultant treated stream is highly colored, which is unacceptable for discharge purposes. Therefore, to complete the wastewater treatment process, it is necessary to remove the polyphenol oxidase-generated polymerization products from the bioreactor permeate stream. In addition, a * Corresponding author. Tel.: ⫹1-27-46-6038443; fax: ⫹1-27-466223984. E-mail address: [email protected] (S.G. Burton)
major drawback of polyphenol oxidase is that product inhibition is caused by the o-quinones binding within the active site of the enzyme [7,8]. Recently, increasing interest has been directed at the use of membrane technologies for the removal of particulates, microorganisms, and colloidal matter from effluent streams. In particular, ultrafiltration can replace conventional physicochemical methods of clarification and disinfection, as pores in the ultrafiltration skin-layer fall within the 10 to 50 nm size range [9], and, hence, components contributing to color and turbidity can be removed [10,11]. Removal of the color-generating quinone and polymeric products can also be mediated using chitosan [5]. Chitin (an N-acetylglucosamine polymer) is obtained from abundant natural sources such as crustaceans, where the shells are the major component of seafood processing waste [5]. Chitosan, (134)-2-amino-2-deoxy--D-glucan, is a polycationic polymer obtained commercially by the alkaline deacetylation of chitin [12,13,14]. Due to its solubility in dilute organic acids, chitosan can be processed into different geometrical configurations including membranes, fibers, hollow fibers, capsules, and beads [15]. Thus, it was proposed that coating capillary membranes with chitosan would increase the surface area of the chitosan, thereby increasing the quinone removal effectiveness. Increasing product removal from the microenvironment of
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W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
Science (Stellenbosch University, S.A.). Membranes were manufactured using the wet-phase inversion process according to the fabrication protocol of Jacobs et al. [11]. The capillary membrane dimensions are: internal diameter, 0.7 mm; external diameter, 1.2 mm; and length, 110 mm. Total effective membrane area in the bioreactor was 4.838 cm2. Fig. 1. Reaction of polyphenol oxidase-mediated conversion of phenols to o-quinones
the enzyme would also facilitate an increased half-life for the enzyme, which is characteristically subject to product inhibition. The chitosan is capable of adsorbing o-quinones, but because it does not adsorb phenols, unconverted substrate remains in solution. In this study, a three-step strategy for the selective removal of phenols from a dilute industrial effluent stream was proposed. First, the semipermeable characteristics of an ultrafiltration membrane were used to remove stream contaminants, thus contributing to decreased chemical oxygen demand levels in the reactor permeate. Second, diffusion of phenolic components in the stream across the membrane, on which polyphenol oxidase was immobilized, facilitated enzyme-substrate contact. Third, product removal from the micro-environment of the enzyme was achieved by adsorption on the chitosan coating.
2. Materials and methods 2.1 Chemicals Mushroom polyphenol oxidase (E.C. 1.14.18.1) was obtained from Sigma Chemical Co. (St. Louis, MO, USA) with a specific activity of 780 Units/mg. Activity was determined by using L--3,4-dihydroxyphenylalanine as the substrate according to the method of Burton et al. [16], and protein content was determined according to the method of Bradford [17]. One unit of activity is defined as the amount of enzyme that catalyzes the formation of dopachrome from L--3,4-dihydroxyphenylalanine at a rate of 1 mol/min where the extinction coefficient (僆) of dopachrome is 3600 M⫺1 cm⫺1 under these conditions. Chitosan was obtained from Sigma. A phenolic industrial effluent sample was obtained from a South African coal– gas plant. Detailed chemical analysis was provided with the received samples. After 1 : 10 dilution, the phenolic effluent had a chemical oxygen demand of 1673 mg/L, and the phenol concentration was 0.0197 mM. The industrial effluent was not subject to any pretreatment other than pH adjustment using 0.1 M NaOH.
2.3 Capillary membrane bioreactor construction and operation The capillary membrane bioreactor consisted of a single polysulphone capillary membrane in a shell-and-tube configuration. The single capillary membrane was encased in a borosilicate glass manifold 110 mm long with an outer diameter of 7 mm and inlet and outlet tubes of 3 mm outer diameter. Assembly involved inserting the capillary membrane into the glass manifold and applying an epoxy sealant at either end of the glass manifold. The epoxy was cured for 48 h before use. The capillary membrane bioreactor was operated in a semi-continuous mode similar to that of Prenosil and Hediger [18], and the substrate solution was recirculated, i.e. permeate was not recycled into the feed solution. In terms of operation, the configuration used was therefore similar to the reactor configuration of Reiken et al. [19] However, it was operated by using a combination of convective and diffusional transport, where hydrostatic pressure was applied to facilitate flux across the membrane, as opposed to the dialysis configuration of Reiken et al. [19], in which flux across the membrane was primarily due to diffusion only. Furthermore the “blank” solution on the shell side (outer side) was not circulated as in the dialysis configuration, to allow monitoring of concentration of the reaction products. The capillary membrane bioreactor was operated at a gauge pressure of 50 kPa, with a feed flow rate of 19.8 L/h through the lumen, resulting in a transmembrane flux of 303.8 L/m2/h. 2.4 Preparation of chitosan-coated capillary membranes Chitosan-coated capillary membranes were prepared according to the following procedure: 1% chitosan (w/v) was dissolved in 0.8% acetic acid (v/v) and stirred overnight at ambient temperatures. Undissolved chitosan and debris was removed by centrifugation at 5000 rev./min for 20 min. The viscous chitosan solution was circulated over the shell side of the membrane to coat the entire outer surface, and the solution was neutralized with 8% NaOH, forming the gel coating. The coated membranes then were rinsed with 0.1 M K2HPO4/KH2PO4 buffer until a pH of 6.80 was reached.
2.2 Polysulphone capillary membranes
2.5 Immobilization of polyphenol oxidase on chitosan-coated capillary membranes
Anisotropic internally skinned polysulphone capillary membranes were obtained from the Institute for Polymer
Polyphenol oxidase was immobilized by cross-flow circulation in 0.1 M K2HPO4/KH2PO4 buffer (pH 6.80) on the
W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
shell (outer) side of the membranes, for 1 h at 25°C. Initially, 171 units of polyphenol oxidase was added to the bioreactor, giving a concentration of 24 units/mL. The solution was circulated over the outer side of the membrane at a rate of 1 mL/min. The system was operated at zero pressure, and thus no permeate was collected from the lumen. 2.6 Enzyme loading capacity of chitosan-coated membranes The extent of enzyme immobilization on chitosan-coated and noncoated membranes was compared by sampling during the immobilization procedure. After immobilization, the membranes were washed with K2HPO4/KH2PO4 buffer (pH 6.71) to remove residual protein that was not immobilized by adsorption. 2.7 Determination of substrate conversion 2.7.1 Control experiments Initially, the shell side of the capillary membrane reactor was filled with 0.1 M K2HPO4/KH2PO4 buffer (pH 6.80). The capillary membrane reactor was operated as described above, and the perfusing substrate solution displaced the phosphate buffer solution from the shell side of the reactor. This permeate from the shell side was collected at regular time intervals for analysis by high-performance liquid chromatography, i.e. the composition of the permeate was thus a mixture of the perfusing substrate solution and the buffer being displaced. The effective substrate concentration therefore increased until a constant concentration was reached, once mixing was complete and residual buffer in the shell side had been displaced. 2.7.2 Immobilized-enzyme experiments To measure the enzyme-catalyzed substrate conversion by polyphenol oxidase immobilized on coated and noncoated membranes, the procedure outlined above was followed using capillary membrane reactors in which the enzyme had been immobilized as described above. The difference in permeate substrate concentration between the control experiments and the immobilized-enzyme experiments, measured at equivalent time intervals, was quantitatively indicative of the amount of substrate that had undergone enzymatic conversion. 2.8 Effect of chitosan on color removal In an experiment to compare the effects of chitosan as a viscous gel, as described above, and untreated chitosan, the permeate from a bioreactor where no chitosan layer was present, was stirred overnight with chitosan flakes. The UV spectrum of this solution was compared with that from a bioreactor in which polyphenol oxidase was immobilized with chitosan gel.
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Table 1 Substrate conversion by chitosan-coated, and noncoated, membraneimmobilized polyphenol oxidase
Chitosan-coated membranes Noncoated membranes
Immobilised enzyme
Total substrate converted
Substrate converted per unit enzyme
143 U 55 U
1224.4 mg 20.3 mg
8.6 mg 䡠 U 0.4 mg 䡠 U
2.9 Sample analysis Samples were analyzed by high-performance liquid chromatography (Beckman Instruments, Inc., San Ramon, USA) by using a reverse-phase Nucleosil 5 m C18 (250 ⫻ 4.6 mm inner diameter) column (Machery-Nagel, Du¨ren, Germany). The mobile phase consisted of water/acetonitrile (6:4; v/v) with a flow rate of 1.0 mL/min. Peaks were detected using a Beckman 168 diode array detector at 254 nm and analyzed using Beckman System Gold® software version 6.0. Spectra to determine the effect of chitosan on color removal were obtained using a Shimadzu UV-160A spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
3. Results and discussion 3.1 Phenol removal capacity of polyphenol oxidase on chitosan-coated membranes Significant differences in the capacity to bind polyphenol oxidase and to remove phenol were observed between bioreactors where polyphenol oxidase was immobilized on chitosan-coated, and noncoated, membranes (Table 1). Thus, of the initial inoculum of 171 Units of polyphenol oxidase, 83.7% was immobilized on the coated membranes, compared with 32.5% on the noncoated membranes. Over an 8-h period, a maximum total phenol conversion of 1224.4 mg phenol was achieved using 143 Units polyphenol oxidase on chitosan-coated membranes as compared with 20.3 mg removed using 55 Units of polyphenol oxidase with noncoated capillary membranes (Table 1). While loss in activity was observed for polyphenol oxidase on both coated and noncoated membranes, the activity retention was far greater for polyphenol oxidase immobilized on the coated membranes. The levels of phenol conversion in this case decreased to 53.9 mg removed over a final 2-h period. In comparison, for noncoated membranes, phenol conversion decreased to 4.6 mg in the final 2 h. The greater productivity can be attributed, first, to the increased amount of immobilized enzyme on the coated membranes, and second, to the removal of the resultant o-quinones from the immediate environment of the enzyme due to adsorption by the chitosan, which would decrease the o-quinone-related inactivation of polyphenol oxidase. The quinone removal would be facilitated primarily by permeate flux. However,
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Fig. 2. Spectrophotometric analysis showing membrane-facilitated color removal of the phenolic effluent, measured as absorbance in a 1-cm path length cuvette. Dotted line, Untreated effluent; solid line, uncoated membrane permeate.
in the absence of the chitosan-mediated adsorption, the residence time of the free-circulating quinones on the shell side of the membrane would be significant, resulting in increased contact between the quinones and the immobilized enzyme and consequently, increased inhibition. 3.2 Membrane- and chitosan-mediated color removal The industrial effluent used in this study had considerable color initially, and the ultrafiltration capability of the membrane was found to remove a significant amount of suspended solids, which contributed to this initial brown color. Fig. 2 shows the color removal of the effluent facilitated by the untreated nascent membrane itself. This is also illustrated in Fig. 3, permeate 1. Chemical oxygen demand levels in the effluent also decreased from 1673 ⫾ 70 mg/L to 1393 ⫾ 94 mg/L. Comparisons then were made of color in the permeates from bioreactors in which polyphenol oxidase was immobilized in the presence and absence of the chitosan coating, and with chitosan which had not been
subjected to acetic acid and NaOH treatment. The color generated by the o-quinones was almost completely removed (Fig. 3, permeate 2) by the viscous chitosan coating, as opposed to a significantly lower color removal capacity when the permeate was treated with untreated chitosan flakes that had not been solubilized, and stirred overnight (Fig. 3, permeate 3). No color was removed from enzymetreated pemeate in the absence of chitosan. In this process, accumulation of reaction products would eventually lead to saturation of the adsorbent, and consequently, removal of spent adsorbent by cleaning of the membranes would be required. Because both chitosan and mushroom polyphenol oxidase are readily available and inexpensive, this would be a feasible procedure, and current research is focusing on this aspect. This report demonstrates the potential for further optimization and scale-up of the bioremediation process. Based on the results presented here, a conversion rate of 3.16 g/m2/h would be possible, and thus a bioreactor with membrane area 1 m2 could potentially be used to treat effluent containing 76 g of phenolic solutes per day, requiring approximately 100 g of chitosan to achieve this. Research on the development of novel membrane bioreactors suitable for such applications is ongoing in our laboratories and will be reported in due course.
4. Conclusions This study has shown that one advantage of forming a gel-like layer of chitosan on the capillary membranes is that greater protein loading capacities can be achieved, as compared with noncoated membranes. The effect of chitosan providing an in situ product removal function in combination with a greater enzyme loading capacity, allows for high levels of phenol removal to be achieved. Two functions are therefore served using chitosan-coated capillary membranes
Fig. 3. Spectrophotometric analysis showing the decrease in permeate color as a result of quinone removal facilitated by chitosan. Permeate 1, untreated effluent after ultrafiltration through the capillary membrane; permeate 2, permeate from the bioreactor in the presence of membrane-immobilized polyphenol oxidase; permeate 3, permeate from the bioreactor containing immobilized polyphenol oxidase and chitosan coating.
W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
and immobilized polyphenol oxidase: an initial highly efficient process for removal of phenolic pollutants from water, and effective in situ color removal from the resultant permeate. In addition, the presence of chitosan contributes considerably to decreasing the product inhibition which is characteristic of polyphenol oxidase. Acknowledgments The authors thank the Water Research Commission, South Africa, and the Rhodes University Joint Research Council for funding this research. The authors also thank Dr. E.P. Jacobs (Institute for Polymer Science, Stellenbosch University) for supplying the membranes. References [1] Sun W-Q, Payne, G. F. Tyrosinase-containing chitosan gels: a combined catalyst and sorbent for selective phenol removal. Biotechnol Bioeng 1996;51:79 – 86. [2] Wada S, Ichikawa H, Tatsumi, K. Removal of phenols and wastewater by a combination treatment with tyrosinase and a coagulant. Biotechnol Bioeng 1995;45:304 –9. [3] Payne GF, Sun W-Q, Sohrabi, A. Tyrosinase reaction/chitosan adsorption for selectively removing phenols from aqueous mixture. Biotechol Bioeng 1992;40:1011– 8. [4] Edwards W, Bownes R, Leukes WD, Jacobs EP, Sanderson RD, Rose PD, Burton SG. A capillary membrane bioreactor using immobilised polyphenol oxidase for the removal of phenols from industrial effluents. Enzyme Microb Tech 1999;209 –17. [5] Wada S, Ichikawa H, Tatsumi K. Removal of phenols from wastewater by soluble and immobilised tyrosinase. Biotechnol Bioeng 1993;42:854 – 8. [6] Sun W-Q, Payne GF, Moas MSGL, Chu JH, Wallace KK. Tyrosinase reaction/chitosan adsorption for removing phenols from wastewater. Biotechnol Progr 1992;8:179 – 86.
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[7] Funaki T, Takanohashi Y, Fukazawa H, Kuruma I. Estimation of kinetic parameters in the inactivation of an enzyme by a suicide substrate. Biochim Biophys Acta 1991;1078:43– 6. [8] Robb DA. Tyrosinase. In: Lontie R, editor. Copper proteins and Copper enzymes, volume II. Boca Raton, FL: CRC Press, 1984, p. 208 – 40. [9] Aptel P, Buckley CA. Categories of membrane operation. In: Water treatment membrane processes. Mallevialle J, Odendaal, PE, Wiesner MR, editors. New York: McGraw-Hill, 1996, p. 2.1–2.24. [10] Maartens A, Swart P, Jacobs, EP. Enzymatic cleaning of ultrafiltration membranes fouled in wool-scouring effluent. Water SA 1998; 24:71– 6. [11] Jacobs EP, Botes JP, Bradshaw SM, Saayman HM. Ultrafiltration in potable water production. Water SA 1997;23:1– 6. [12] Siso MIG, Lang E, Carreno˜–Go´mez B, Becerra M, Espinar FO, Me´ndez JB. Enzyme encapsulation on chitosan micro-beads. Process Biochem 1997;32:211–216. [13] Krajewska B, Zaborska W, Leszko M. Chitosan membrane-immobilised urease. Stability against inhibition by boric acid. International Congress On Membranes and Membrane processes (ICOM). Yokohama. Japan 1996. [14] Hall LD, Yalpani M. Formation of branched chain, soluble polysaccharides from chitosan. J Chem Soc Chem Comm 1980;1153– 4. [15] Muzzarelli RAA, Tanfani F, Emanuelli M, Mariotti S. N-(Carboxymethylidene) chitosans and N-(carboxymethyl) chitosans: novel chelating polyampholytes obtained from chitosan glyoxylate. Carbohyd Res 1982;107:199 –214. [16] Burton SG, Duncan JR, Kaye PT, Rose PD. Activity of mushroom polyphenol oxidase in organic medium. Biotechnol Bioeng 1993;42: 938 – 44. [17] Bradford MM. A rapid and sensitive method for the quantification of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976;72:248 –54. [18] Prenosil JE, Hediger T. Performance of membrane fixed biocatalyst reactors. I: membrane reactor systems and modelling. Biotechnol Bioeng 1988;31:913–21. [19] Reiken SR, Knob RJ, Briedis DM. Evaluation of intrinsic immobilised kinetics in hollow fibre reactor systems. Enzyme Microb Tech 1990;12:736 – 42.
Immobilization of polyphenol oxidase on chitosan-coated polysulphone capillary membranes for improved phenolic effluent bioremediation W. Edwards, W.D. Leukes, P.D. Rose, S.G. Burton* Goldfields Biotechnology Centre, Department of Biochemistry and Microbiology, Rhodes University, Grahamstown, 6140, South Africa Received 16 December 1998; received in revised form 22 July 1999; accepted 10 August 1999
Abstract Internally skinned polysulphone capillary membranes were coated with a viscous chitosan gel and used as an immobilization matrix for polyphenol oxidase. Bench-scale, single-capillary membrane bioreactors then were used to determine the influence of the chitosan coating on product removal after substrate conversion by immobilized polyphenol oxidase during the treatment of industrial phenolic effluents. The results indicate that greater efficiency was achieved in the removal of polyphenol oxidase-generated products by the chitosan membrane coating, as compared with chitosan flakes. This facilitated an increase in the productivity of the immobilized enzyme. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Polyphenol oxidase; Polysulphone capillary membranes; Chitosan; Phenol
1. Introduction The selective recognition and oxidation of phenols by the enzyme polyphenol oxidase to produce reactive o-quinones (Fig. 1) can be successfully exploited as a waste minimization strategy for effluent treatment [1,2,3]. We have shown previously that polyphenol oxidase can be immobilized on capillary membranes facilitating the conversion of phenols to the corresponding o-quinones [4]. However, although polyphenol oxidase is effective in converting phenol and a number of associated derivatives to their corresponding o-quinones, these o-quinones and the low-molecular-weight polymers formed from them remain in the treated effluent. At low concentrations the remaining colored products do not achieve high enough degrees of polymerization to effectively precipitate from solution [2,5,3,6]. Thus, the goal of reducing the toxicity of the phenol-containing effluent is achieved, but the resultant treated stream is highly colored, which is unacceptable for discharge purposes. Therefore, to complete the wastewater treatment process, it is necessary to remove the polyphenol oxidase-generated polymerization products from the bioreactor permeate stream. In addition, a * Corresponding author. Tel.: ⫹1-27-46-6038443; fax: ⫹1-27-466223984. E-mail address: [email protected] (S.G. Burton)
major drawback of polyphenol oxidase is that product inhibition is caused by the o-quinones binding within the active site of the enzyme [7,8]. Recently, increasing interest has been directed at the use of membrane technologies for the removal of particulates, microorganisms, and colloidal matter from effluent streams. In particular, ultrafiltration can replace conventional physicochemical methods of clarification and disinfection, as pores in the ultrafiltration skin-layer fall within the 10 to 50 nm size range [9], and, hence, components contributing to color and turbidity can be removed [10,11]. Removal of the color-generating quinone and polymeric products can also be mediated using chitosan [5]. Chitin (an N-acetylglucosamine polymer) is obtained from abundant natural sources such as crustaceans, where the shells are the major component of seafood processing waste [5]. Chitosan, (134)-2-amino-2-deoxy--D-glucan, is a polycationic polymer obtained commercially by the alkaline deacetylation of chitin [12,13,14]. Due to its solubility in dilute organic acids, chitosan can be processed into different geometrical configurations including membranes, fibers, hollow fibers, capsules, and beads [15]. Thus, it was proposed that coating capillary membranes with chitosan would increase the surface area of the chitosan, thereby increasing the quinone removal effectiveness. Increasing product removal from the microenvironment of
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W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
Science (Stellenbosch University, S.A.). Membranes were manufactured using the wet-phase inversion process according to the fabrication protocol of Jacobs et al. [11]. The capillary membrane dimensions are: internal diameter, 0.7 mm; external diameter, 1.2 mm; and length, 110 mm. Total effective membrane area in the bioreactor was 4.838 cm2. Fig. 1. Reaction of polyphenol oxidase-mediated conversion of phenols to o-quinones
the enzyme would also facilitate an increased half-life for the enzyme, which is characteristically subject to product inhibition. The chitosan is capable of adsorbing o-quinones, but because it does not adsorb phenols, unconverted substrate remains in solution. In this study, a three-step strategy for the selective removal of phenols from a dilute industrial effluent stream was proposed. First, the semipermeable characteristics of an ultrafiltration membrane were used to remove stream contaminants, thus contributing to decreased chemical oxygen demand levels in the reactor permeate. Second, diffusion of phenolic components in the stream across the membrane, on which polyphenol oxidase was immobilized, facilitated enzyme-substrate contact. Third, product removal from the micro-environment of the enzyme was achieved by adsorption on the chitosan coating.
2. Materials and methods 2.1 Chemicals Mushroom polyphenol oxidase (E.C. 1.14.18.1) was obtained from Sigma Chemical Co. (St. Louis, MO, USA) with a specific activity of 780 Units/mg. Activity was determined by using L--3,4-dihydroxyphenylalanine as the substrate according to the method of Burton et al. [16], and protein content was determined according to the method of Bradford [17]. One unit of activity is defined as the amount of enzyme that catalyzes the formation of dopachrome from L--3,4-dihydroxyphenylalanine at a rate of 1 mol/min where the extinction coefficient (僆) of dopachrome is 3600 M⫺1 cm⫺1 under these conditions. Chitosan was obtained from Sigma. A phenolic industrial effluent sample was obtained from a South African coal– gas plant. Detailed chemical analysis was provided with the received samples. After 1 : 10 dilution, the phenolic effluent had a chemical oxygen demand of 1673 mg/L, and the phenol concentration was 0.0197 mM. The industrial effluent was not subject to any pretreatment other than pH adjustment using 0.1 M NaOH.
2.3 Capillary membrane bioreactor construction and operation The capillary membrane bioreactor consisted of a single polysulphone capillary membrane in a shell-and-tube configuration. The single capillary membrane was encased in a borosilicate glass manifold 110 mm long with an outer diameter of 7 mm and inlet and outlet tubes of 3 mm outer diameter. Assembly involved inserting the capillary membrane into the glass manifold and applying an epoxy sealant at either end of the glass manifold. The epoxy was cured for 48 h before use. The capillary membrane bioreactor was operated in a semi-continuous mode similar to that of Prenosil and Hediger [18], and the substrate solution was recirculated, i.e. permeate was not recycled into the feed solution. In terms of operation, the configuration used was therefore similar to the reactor configuration of Reiken et al. [19] However, it was operated by using a combination of convective and diffusional transport, where hydrostatic pressure was applied to facilitate flux across the membrane, as opposed to the dialysis configuration of Reiken et al. [19], in which flux across the membrane was primarily due to diffusion only. Furthermore the “blank” solution on the shell side (outer side) was not circulated as in the dialysis configuration, to allow monitoring of concentration of the reaction products. The capillary membrane bioreactor was operated at a gauge pressure of 50 kPa, with a feed flow rate of 19.8 L/h through the lumen, resulting in a transmembrane flux of 303.8 L/m2/h. 2.4 Preparation of chitosan-coated capillary membranes Chitosan-coated capillary membranes were prepared according to the following procedure: 1% chitosan (w/v) was dissolved in 0.8% acetic acid (v/v) and stirred overnight at ambient temperatures. Undissolved chitosan and debris was removed by centrifugation at 5000 rev./min for 20 min. The viscous chitosan solution was circulated over the shell side of the membrane to coat the entire outer surface, and the solution was neutralized with 8% NaOH, forming the gel coating. The coated membranes then were rinsed with 0.1 M K2HPO4/KH2PO4 buffer until a pH of 6.80 was reached.
2.2 Polysulphone capillary membranes
2.5 Immobilization of polyphenol oxidase on chitosan-coated capillary membranes
Anisotropic internally skinned polysulphone capillary membranes were obtained from the Institute for Polymer
Polyphenol oxidase was immobilized by cross-flow circulation in 0.1 M K2HPO4/KH2PO4 buffer (pH 6.80) on the
W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
shell (outer) side of the membranes, for 1 h at 25°C. Initially, 171 units of polyphenol oxidase was added to the bioreactor, giving a concentration of 24 units/mL. The solution was circulated over the outer side of the membrane at a rate of 1 mL/min. The system was operated at zero pressure, and thus no permeate was collected from the lumen. 2.6 Enzyme loading capacity of chitosan-coated membranes The extent of enzyme immobilization on chitosan-coated and noncoated membranes was compared by sampling during the immobilization procedure. After immobilization, the membranes were washed with K2HPO4/KH2PO4 buffer (pH 6.71) to remove residual protein that was not immobilized by adsorption. 2.7 Determination of substrate conversion 2.7.1 Control experiments Initially, the shell side of the capillary membrane reactor was filled with 0.1 M K2HPO4/KH2PO4 buffer (pH 6.80). The capillary membrane reactor was operated as described above, and the perfusing substrate solution displaced the phosphate buffer solution from the shell side of the reactor. This permeate from the shell side was collected at regular time intervals for analysis by high-performance liquid chromatography, i.e. the composition of the permeate was thus a mixture of the perfusing substrate solution and the buffer being displaced. The effective substrate concentration therefore increased until a constant concentration was reached, once mixing was complete and residual buffer in the shell side had been displaced. 2.7.2 Immobilized-enzyme experiments To measure the enzyme-catalyzed substrate conversion by polyphenol oxidase immobilized on coated and noncoated membranes, the procedure outlined above was followed using capillary membrane reactors in which the enzyme had been immobilized as described above. The difference in permeate substrate concentration between the control experiments and the immobilized-enzyme experiments, measured at equivalent time intervals, was quantitatively indicative of the amount of substrate that had undergone enzymatic conversion. 2.8 Effect of chitosan on color removal In an experiment to compare the effects of chitosan as a viscous gel, as described above, and untreated chitosan, the permeate from a bioreactor where no chitosan layer was present, was stirred overnight with chitosan flakes. The UV spectrum of this solution was compared with that from a bioreactor in which polyphenol oxidase was immobilized with chitosan gel.
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Table 1 Substrate conversion by chitosan-coated, and noncoated, membraneimmobilized polyphenol oxidase
Chitosan-coated membranes Noncoated membranes
Immobilised enzyme
Total substrate converted
Substrate converted per unit enzyme
143 U 55 U
1224.4 mg 20.3 mg
8.6 mg 䡠 U 0.4 mg 䡠 U
2.9 Sample analysis Samples were analyzed by high-performance liquid chromatography (Beckman Instruments, Inc., San Ramon, USA) by using a reverse-phase Nucleosil 5 m C18 (250 ⫻ 4.6 mm inner diameter) column (Machery-Nagel, Du¨ren, Germany). The mobile phase consisted of water/acetonitrile (6:4; v/v) with a flow rate of 1.0 mL/min. Peaks were detected using a Beckman 168 diode array detector at 254 nm and analyzed using Beckman System Gold® software version 6.0. Spectra to determine the effect of chitosan on color removal were obtained using a Shimadzu UV-160A spectrophotometer (Shimadzu Corporation, Kyoto, Japan).
3. Results and discussion 3.1 Phenol removal capacity of polyphenol oxidase on chitosan-coated membranes Significant differences in the capacity to bind polyphenol oxidase and to remove phenol were observed between bioreactors where polyphenol oxidase was immobilized on chitosan-coated, and noncoated, membranes (Table 1). Thus, of the initial inoculum of 171 Units of polyphenol oxidase, 83.7% was immobilized on the coated membranes, compared with 32.5% on the noncoated membranes. Over an 8-h period, a maximum total phenol conversion of 1224.4 mg phenol was achieved using 143 Units polyphenol oxidase on chitosan-coated membranes as compared with 20.3 mg removed using 55 Units of polyphenol oxidase with noncoated capillary membranes (Table 1). While loss in activity was observed for polyphenol oxidase on both coated and noncoated membranes, the activity retention was far greater for polyphenol oxidase immobilized on the coated membranes. The levels of phenol conversion in this case decreased to 53.9 mg removed over a final 2-h period. In comparison, for noncoated membranes, phenol conversion decreased to 4.6 mg in the final 2 h. The greater productivity can be attributed, first, to the increased amount of immobilized enzyme on the coated membranes, and second, to the removal of the resultant o-quinones from the immediate environment of the enzyme due to adsorption by the chitosan, which would decrease the o-quinone-related inactivation of polyphenol oxidase. The quinone removal would be facilitated primarily by permeate flux. However,
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W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
Fig. 2. Spectrophotometric analysis showing membrane-facilitated color removal of the phenolic effluent, measured as absorbance in a 1-cm path length cuvette. Dotted line, Untreated effluent; solid line, uncoated membrane permeate.
in the absence of the chitosan-mediated adsorption, the residence time of the free-circulating quinones on the shell side of the membrane would be significant, resulting in increased contact between the quinones and the immobilized enzyme and consequently, increased inhibition. 3.2 Membrane- and chitosan-mediated color removal The industrial effluent used in this study had considerable color initially, and the ultrafiltration capability of the membrane was found to remove a significant amount of suspended solids, which contributed to this initial brown color. Fig. 2 shows the color removal of the effluent facilitated by the untreated nascent membrane itself. This is also illustrated in Fig. 3, permeate 1. Chemical oxygen demand levels in the effluent also decreased from 1673 ⫾ 70 mg/L to 1393 ⫾ 94 mg/L. Comparisons then were made of color in the permeates from bioreactors in which polyphenol oxidase was immobilized in the presence and absence of the chitosan coating, and with chitosan which had not been
subjected to acetic acid and NaOH treatment. The color generated by the o-quinones was almost completely removed (Fig. 3, permeate 2) by the viscous chitosan coating, as opposed to a significantly lower color removal capacity when the permeate was treated with untreated chitosan flakes that had not been solubilized, and stirred overnight (Fig. 3, permeate 3). No color was removed from enzymetreated pemeate in the absence of chitosan. In this process, accumulation of reaction products would eventually lead to saturation of the adsorbent, and consequently, removal of spent adsorbent by cleaning of the membranes would be required. Because both chitosan and mushroom polyphenol oxidase are readily available and inexpensive, this would be a feasible procedure, and current research is focusing on this aspect. This report demonstrates the potential for further optimization and scale-up of the bioremediation process. Based on the results presented here, a conversion rate of 3.16 g/m2/h would be possible, and thus a bioreactor with membrane area 1 m2 could potentially be used to treat effluent containing 76 g of phenolic solutes per day, requiring approximately 100 g of chitosan to achieve this. Research on the development of novel membrane bioreactors suitable for such applications is ongoing in our laboratories and will be reported in due course.
4. Conclusions This study has shown that one advantage of forming a gel-like layer of chitosan on the capillary membranes is that greater protein loading capacities can be achieved, as compared with noncoated membranes. The effect of chitosan providing an in situ product removal function in combination with a greater enzyme loading capacity, allows for high levels of phenol removal to be achieved. Two functions are therefore served using chitosan-coated capillary membranes
Fig. 3. Spectrophotometric analysis showing the decrease in permeate color as a result of quinone removal facilitated by chitosan. Permeate 1, untreated effluent after ultrafiltration through the capillary membrane; permeate 2, permeate from the bioreactor in the presence of membrane-immobilized polyphenol oxidase; permeate 3, permeate from the bioreactor containing immobilized polyphenol oxidase and chitosan coating.
W. Edwards et al. / Enzyme and Microbial Technology 25 (1999) 769 –773
and immobilized polyphenol oxidase: an initial highly efficient process for removal of phenolic pollutants from water, and effective in situ color removal from the resultant permeate. In addition, the presence of chitosan contributes considerably to decreasing the product inhibition which is characteristic of polyphenol oxidase. Acknowledgments The authors thank the Water Research Commission, South Africa, and the Rhodes University Joint Research Council for funding this research. The authors also thank Dr. E.P. Jacobs (Institute for Polymer Science, Stellenbosch University) for supplying the membranes. References [1] Sun W-Q, Payne, G. F. Tyrosinase-containing chitosan gels: a combined catalyst and sorbent for selective phenol removal. Biotechnol Bioeng 1996;51:79 – 86. [2] Wada S, Ichikawa H, Tatsumi, K. Removal of phenols and wastewater by a combination treatment with tyrosinase and a coagulant. Biotechnol Bioeng 1995;45:304 –9. [3] Payne GF, Sun W-Q, Sohrabi, A. Tyrosinase reaction/chitosan adsorption for selectively removing phenols from aqueous mixture. Biotechol Bioeng 1992;40:1011– 8. [4] Edwards W, Bownes R, Leukes WD, Jacobs EP, Sanderson RD, Rose PD, Burton SG. A capillary membrane bioreactor using immobilised polyphenol oxidase for the removal of phenols from industrial effluents. Enzyme Microb Tech 1999;209 –17. [5] Wada S, Ichikawa H, Tatsumi K. Removal of phenols from wastewater by soluble and immobilised tyrosinase. Biotechnol Bioeng 1993;42:854 – 8. [6] Sun W-Q, Payne GF, Moas MSGL, Chu JH, Wallace KK. Tyrosinase reaction/chitosan adsorption for removing phenols from wastewater. Biotechnol Progr 1992;8:179 – 86.
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