Decolorization and removal of textile and non-textile dyes from polluted wastewater and dyeing euent by using potato

(Solanum tuberosum) soluble and immobilized polyphenol oxidase

Amjad Ali Khan, Qayyum Husain
*
Department of Biochemistry, Faculty of Life Science, Aligarh Muslim University, Aligarh 202 002, UP, India Received 21 February 2006; received in revised form 14 April 2006; accepted 17 April 2006 Available online 9 June 2006

Abstract Celite bound potato polyphenol oxidase preparation was employed for the treatment of wastewater/dye euent contaminated with reactive textile and non-textile dyes, Reactive Blue 4 and Reactive Orange 86. The maximum decolorization was found at pH 3.0 and 4.0 in case of Reactive Blue 4 and Reactive Orange 86, respectively. Immobilized potato polyphenol oxidase was signicantly more eective in decolorizing the individual dye and complex mixtures of dyes as compared to soluble enzyme. The absorption spectra of the treated and untreated dye mixture and dyeing euent exhibited a marked dierence in the absorption value at various wavelengths. The polluted water contaminated with an individual dye or mixtures of dyes treated with soluble and immobilized potato polyphenol oxidase resulted in the remarkable loss in total organic carbon. 2006 Elsevier Ltd. All rights reserved.
Keywords: Polyphenol oxidase; Dyes; Celite 545; Immobilization; Decolorization

1. Introduction Textile and several other industries generate a number of visual pollutants, which they discharge to the surrounding environment without any further treatment. These pollutants not only add color to water but they also cause extensive toxicity to the aquatic and other forms of life. About 1015% of the total dyes from various textile and other industries get discharged in wastewater causing extensive pollution (Robinson et al., 2001; Keharia and Madamvar, 2003). Therefore, the treatment of industrial euents containing aromatic compounds becomes necessary prior to their nal discharge to the environment. Conventional methods for the eective removal of phenols,
Abbreviations: PPO; polyphenol oxidase; S-PPO; soluble polyphenol oxidase; I-PPO; immobilized polyphenol oxidase; TOC; total organic carbon. * Corresponding author. Tel.: +91 571 2700741; fax: +91 571 2721776. E-mail address: qayyumhusain@redimail.com (Q. Husain). 0960-8524/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.04.008

aromatic amines and dyes are outdated due to their certain inherent limitations (Robinson et al., 2001; Abadulla et al., 2000; Sumathi and Manju, 2000). The idea of using oxidoreductive enzymes for wastewater treatment was developed in early 1980s. An advantage of enzymatic approach is that these enzymes can react with a broad range of aromatic compounds under dilute conditions. The rate of decolorization and their removal is quite fast and are less sensitive to operational upsets as compared to the microbial ora (Husain and Jan, 2000; Duran and Esposito, 2000; Torres et al., 2003). Several peroxidases and laccases have been used for the treatment of wastewater containing dyes. The procedure of dye decolorization by peroxidases adds additional cost to the processes due to use of expensive hydrogen peroxide as a co-substrate (Akhtar et al., 2005a,b). The rare availability and high cost of purication of laccases limits its use for the decolorization studies. Plant polyphenol oxidases are the simple and cheaper alternative for the decolorization of aromatic pollutants due to its

utilization of free molecular oxygen as an oxidant (Wada et al., 1995; Duran and Esposito, 2000). The major reason that enzymatic treatments have not yet been applied on an industrial scale is due to the huge volume of polluted wastewater demanding remediation. Free enzymes suer from certain drawbacks such as thermal instability, reusability, susceptibility to attack by proteases, activity inhibition etc. (Husain and Jan, 2000). To overcome all these limitations use of immobilized enzyme has been suggested due to its stability and reusability etc. (Khan et al., 2005; Akhtar et al., 2005c; Kulshrestha and Husain, 2006). Polyphenol oxidases like other enzymes have been successfully immobilized on various natural and synthetic carriers (Duran and Esposito, 2000; Husain and Jan, 2000; Duran et al., 2002; Khan et al., 2006). Covalent immobilization of enzymes often resulted in the loss of activity (Husain et al., 1992). Several investigators have reported the leaching of enzymes from the entrapped gels, which limits its applications in the continuous use (Palmieri et al., 1994; Musthapa et al., 2004; Matto and Husain, in press). There is always a search for cheaper support and enzyme for preparing an immobilized enzyme preparation for such applications. Here, an eort has been made to use an inexpensive Celite bound potato polyphenol oxidase (PPO) preparation for the decolorization of a number of reactive textile dyes present in polluted water. Potato PPO has been successfully immobilized on a cheaper support like Celite 545 (diatomaceous earth). Celite bound potato PPO preparation was then exploited for the treatment of polluted wastewater containing textile and non-textile dyes, mixture of reactive textile dyes and dyeing euent. Dye removal was monitored by using spectrophotometry and total organic carbon (TOC) analyzer.

according to the procedure described by Khan et al. (2006). 2.3. Eect of pH on the decolorization of textile reactive dyes Dye solutions were prepared in the concentration range of 50120 mg/l in distilled water. The solutions of two dyes (Reactive Blue 4 and Reactive Orange 86) were independently incubated with soluble and immobilized potato PPO (1.5 EU/ml) in dierent buers having pH from 3.0 to 10.0 at 37 C for 1 h (50 mM buer solutions). The reaction mixtures were centrifuged at 3000g for 5 min on a Remi Centrifuge. Dye decolorization by PPO was monitored at predetermined specic wavelength (kmax) using Cintra 10e spectrophotometer. The percent decolorization was calculated by taking untreated dye solution in each buer as control (100%). 2.4. Decolorization of textile reactive dyes Eight textile reactive dyes used in textile industry (Reactive Blue 4, Reactive Blue 160, Reactive Blue 171, Reactive Orange 4, Reactive Orange 86, Reactive Red 11, Reactive Red 120 and Reactive yellow 84) were selected and their solutions were prepared in the concentration range of 50 120 mg/l in distilled water. Each dye was incubated with soluble and immobilized potato PPO (1.5 EU/ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. The disappearance of color by PPO was monitored at predetermined kmax of the respective dye solution. The percent decolorization was calculated by taking the maximum absorbance of each untreated dye solution as control (100%).The optical density was measured using Cintra 10e spectrophotometer. 2.5. Decolorization of mixtures of textile reactive dyes

2. Methods 2.1. Materials Bromophenol Blue, Catechol, Coomassie Brilliant Blue R250 and PAGE Blue 83 were obtained from Sigma Chemicals Co. Ltd. (St. Louis, MO) USA. Carmine, Naphthalene Black 12 B, 1,2-Naphthaquinone 4-sulphonic acid and Tropaeolin were the products of BDH Ltd., Poole, England. Reactive dyes were a gift from Atul India Ltd., Gujarat, India. Chelpark Red, Chelpark Black and Chelpark Blue were purchased from Chelpark Chem. Pvt Ltd., New Delhi, India. Celite 545 (2045 l mesh) was purchased from Serva Labs. Heidelberg, Germany. Potato was obtained from the local market. Dyeing euent was collected from a local dyer. All other chemicals and reagents employed were of analytical grade and were used without any further purication. 2.2. Immobilization of potato PPO Potato PPO was partially puried by ammonium sulphate fractionation and was immobilized on Celite 545 Dierent mixtures of four reactive dyes (Reactive Blue 160, Reactive Blue 171, Reactive Yellow 84 and Reactive Red 11), (Reactive Red 120, Reactive Orange 86, Reactive Orange 4 and Reactive Blue 4), (Reactive Blue 160, Reactive Orange 86, Reactive Yellow 84 and Reactive Orange 4), (Reactive Blue 4, Reactive Blue 160, Reactive Red 120 and Reactive Red 11) were prepared by taking each dye with equal absorbance in equal volume. Each mixture was incubated with soluble and immobilized potato PPO (1.5 EU/ ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. The reaction mixture was centrifuged before measuring the absorbance of the respective dye. Dye decolorization of the mixture was measured at their specic wavelength. The percent decolorization was calculated by taking each untreated dye mixture as control (100%). The optical density was measured using Cintra 10e spectrophotometer. 2.6. Decolorization of dyeing euent Euent from the local dyer was collected and the insoluble material was removed by centrifugation. Prior to the

enzymatic treatment, the euent was suitably diluted with 50 mM sodium citrate buer, pH 3.0 to evaluate the kmax. Using Cintra 10e spectrophotometer the wavelength maximum, kmax of the suitably diluted dye euent was recorded. Two hundred milliliter of diluted euent was treated independently with 150 EU of soluble and immobilized PPO for 4 h at room temperature (30 C) under stirring conditions. After treatment, the aliquots of euent were centrifuged and clear supernatant was analyzed for remaining color. 2.7. Decolorization of non-textile dyes Non-textile dyes; Bromophenol Blue, Carmine, Chelpark Blue, Chelpark Black, Chelpark Red, Coomassie Brilliant Blue R 250, 1,2-Naphthaquinone-4-sulphoncic acid, Naphthalene Black 12 B, PAGE Blue 83 and Tropaeolin were selected for this study. Dye solutions were prepared in the concentration range of 50100 mg/l in distilled water. Each dye was treated with soluble and immobilized potato PPO (1.5 EU/ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. The treated samples were rst centrifuged before measuring the absorbance at a particular wavelength of a dye. Untreated dyes were considered as control for the calculation of percent decolorization. 2.8. Monitoring of absorption spectra of a mixture of dyes and dyeing euent Before taking absorption spectra of mixture of dyes and dyeing euent, these were treated as described in Sections 2.6 and 2.7, respectively. Absorption spectra for the mixtures of dyes (Reactive Blue 4, Reactive Blue 160, Reactive Red 120 and Reactive Red 11) and dyeing euent before and after treatment with soluble and immobilized potato PPO were recorded on Cintra 10e spectrophotometer. 2.9. Determination of total organic carbon (TOC) of treated dyes All the textile reactive dyes either individually or in mixtures were treated with soluble or immobilized potato PPO (1.5 EU/ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. After 1 h incubation, their insoluble products were removed by centrifugation. Clear solutions were used for the measurement of TOC content. TOC contents of each control and treated dye or dye mixtures were measured by using a TOC analyzer (Analytic Jena, Multi N/ C 2000). 2.10. Assay of potato PPO activity, protein concentration and calculation of percent dye decolorization The method of Vieira and Fatibello-Filho (1998) was used with some modications to assay the PPO activity. The reaction was initiated by adding a dened quantity

of enzyme to a reaction mixture containing 1.0 ml of 20 mM catechol in a total volume of 3.0 ml with 50 mM sodium citrate buer, pH 4.0. The reaction mixture was incubated at 37 C for 1.53.0 min. Reddish colored complex formed due to oxidation of catechol was measured at 420 nm using Cintra 10e spectrophotometer. There was a linear rise in the formation of colored complex for 3.0 min. One unit of PPO activity was dened as the amount of enzyme protein that catalyzed the formation of 1.0 lmole of colored complex after the oxidation of catechol per minute at 420 nm. In case of immobilized enzyme the procedure for assaying activity was same except that the reaction mixture was continuously stirred during the progress of reaction. After the reaction was over, the mixture was centrifuged at 3000 g for 5 min before measuring absorbance. The protein concentration was determined by the method of Lowry et al. (1951). Bovine serum albumin was used as a standard. The decolorization was calculated for each dye or mixture of dyes. The parameter percent decolorization was dened as
Percent decolorization Absorbance of the untreated dye Absorbance after treatment Absorbance of the untreated dye 100

Each value represents the mean of three independent experiments performed in duplicate with average standard deviation <5%. 3. Results 3.1. Decolorization of textile dyes The eect of pH on the rate of dye decolorization by using soluble and immobilized PPO is shown in Table 1. Reactive Blue 4 was decolorized maximally at pH 4.0

Table 1 Eect of pH on the decolorization of textile reactive dyes by soluble and immobilized potato PPO pH % Dye decolorization Reactive Blue 4 S-PPO 3 4 5 6 7 8 9 10 80 1.7 83 2.0 53 1.2 48 1.3 11 0.6 5 0.4 0 0 I-PPO 87 1.1 93 2.3 60 1.6 47 1.2 16 0.6 5 0.4 0 0 Reactive Orange 86 S-PPO 78 1.5 62 1.6 50 1.3 41 1.7 20 1.4 10 0.3 0 0 I-PPO 96 0.9 78 1.7 67 1.0 48 1.3 30 0.8 15 0.6 11 0.4 7 0.2

Both dyes were treated independently with soluble and immobilized potato PPO (1.5 EU/ml) in buers of pH 310 at 37 C for 1 h. Each value represents the mean for three-independent experiments performed in duplicate, with average standard deviation <5%.

Table 2 Decolorization of textile reactive dyes by soluble and immobilized potato PPO Name of reactive dyes kmax (nm) % Dye decolorization TOC content (ppm) Control TOC S-PPO Reactive Reactive Reactive Reactive Reactive Reactive Reactive Reactive Blue 4 Blue 160 Blue 171 Red 11 Red 120 Orange 4 Orange 86 Yellow 84 600 616 607 545 511 489 413 414 80 3.0 96 1.4 97 1.0 85 1.7 91 1.4 78 1.5 78 1.8 91 1.7 I-PPO 87 2.2 98 1.2 100 0.0 91 1.2 97 1.1 86 2.2 96 1.6 95 1.4 167 5.1 182 4.6 150 6.0 171 6.6 156 6.3 192 4.2 144 2.8 174 3.2 Remaining TOC after treatment withS-PPO 89 1.4 105 2.6 71 1.8 98 1.9 74 1.7 99 1.3 120 2.7 104 2.5 I-PPO 59 0.9 79 1.4 52 1.6 62 1.9 52 1.6 72 2.2 80 2.1 86 2.3

Each dye was treated independently with soluble and immobilized potato PPO (1.5 EU/ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. Centrifugation of each treated sample was done before monitoring TOC content. Each value represents the mean for three-independent experiments performed in duplicate, with average standard deviation <5%.

Table 3 Decolorization of mixtures of textile reactive dyes and dyeing euent by soluble and immobilized potato PPO Name of mixture of dyes kmax (nm) % Decolorization TOC content (ppm) Control TOC S-PPO Reactive Blue160 + Reactive Blue 171 + Reactive Yellow 84 + Reactive Red 11 Reactive Red 120 + Reactive Orange 86 + Reactive Orange 4 + Reactive Blue 4 Reactive Blue 160 + Reactive Orange 86 + Reactive Yellow 84 + Reactive Orange 4 Reactive Blue 4 + Reactive Blue 160 + Reactive Red 120 + Reactive Red 11 Dyeing euent 551 510 483 544 635 40 1.3 52 1.0 24 0.3 50 0.7 82 3.1 I-PPO 51 1.9 56 1.5 34 1.8 63 1.5 95 3.3 174 5.1 184 6.2 177 6.1 162 5.8 Remaining TOC after treatment withS-PPO 100 3.1 105 3.3 96 2.3 106 3.5 I-PPO 82 2.2 74 1.3 86 3.8 78 4.1

Each dyes mixture was independently treated with soluble and immobilized potato PPO (1.5 EU/ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. Centrifugation of each treated sample was done before monitoring TOC level. Each value represents the mean for three-independent experiments performed in duplicate, with average standard deviation <5%.

while the Reactive Orange 86 exhibited its maximum decolorization at pH 3.0. However, the immobilized PPO preparation was more useful in removing higher percentage of color, both at lower and higher pH values of pH-optima as compared to its soluble counterpart. Table 2 describes the decolorization of eight textile reactive dyes by soluble and Celite bound potato PPO. Soluble enzyme was capable of removing 7897% of the color of the dyes while the immobilized enzyme was more eective in decolorizing higher percent of color (86100%) under similar experimental conditions. The decolorization of dyes from the complex mixture was quite a slow process and each mixture was decolorized to a lesser extent as compared to single dye. However, immobilized enzyme preparation decolorized more color from the dye mixtures as compared to the soluble enzyme (Table 3). 3.2. Decolorization of dye industry euent Treatment of a dyeing euent by soluble and immobilized potato PPO resulted in a signicant loss of color.

The disappearance of color was followed by the formation of an insoluble precipitate. The observations suggested that immobilized potato PPO could remove more color from the dyeing euent as compared to soluble enzyme (Table 3). Soluble potato PPO decolorized 82% of the dyeing euent while the immobilized PPO decolorized more than 95% of the dyeing euent under identical experimental conditions. 3.3. TOC content of potato PPO treated dyes TOC content of soluble and immobilized potato PPO treated individual dye and mixtures of dyes are shown in Tables 2 and 3. As compared to control, the level of TOC was decreased in both soluble and immobilized potato PPO treated polluted water. However, the immobilized PPO treated dye solutions exhibited more loss of TOC from the polluted water. Soluble potato PPO removed 4153% of TOC in case of single reactive dye treatment. However, Reactive Orange 86 was found to be recalcitrant to PPO action and only 17% of TOC was removed. Immobilized

potato PPO removed TOC to a large extent and it was found to be between 45% and 67%. TOC removal from mixture of dyes was observed to a lesser extent than the single dye treatment. When mixture of four treated textile reactive dyes ware analyzed for the TOC content, soluble potato PPO removed 3546%. However, immobilized enzyme removed 5260% TOC under similar incubation conditions (Table 3). 3.4. Decolorization of non-textile dyes Table 4 illustrates the eect of soluble and immobilized potato PPO on the decolorization of ten industrially important non-textile dyes. Removal of color from all tested dyes by immobilized potato PPO was signicantly higher as compared to the soluble enzyme. All the dyes were decolorized by more than 60% by immobilized potato PPO. However, two dyes; 1,2-Naphthaquinone-4-sulphonic acid and Tropaeolin were decolorized slowly as compared to other dyes by the same amount of potato PPO. 3.5. UVvisible absorbance spectra of the treated complex mixtures of textile reactive dyes and dyeing euent The UVvisible absorbance spectra of complex mixture of dyes before and after treatment with soluble and immobilized PPO are shown in Fig. 1. The diminution in the peaks of the dyes in both visible and UV regions took place due to the removal of dyes in the form of an insoluble precipitate after treatment with PPOs. However, the peak was diminished by 50% by the treatment with soluble PPO and immobilized PPO reduced the peak to 63% in visible region as compared to the control. However, in UV region there

Table 4 Decolorization of industrially important non-textile dyes by soluble and immobilized potato PPO Name of dye kmax (nm) 590 518 581 484 493 556 400 617 557 446 % Dye decolorization S-PPO Bromophenol Blue Carmine Chelpark Blue Chelpark Black Chelpark Red Coomassie Brilliant Blue R 250 1,2-Naphthaquinone4-sulphonic acid Naphthalene Black 12B PAGE Blue Tropaeolin 59 1.4 60 1.9 87 2.1 63 2.1 84 2.1 87 1.7 25 0.9 63 1.6 80 2.1 30 2.2 I-PPO 63 1.1 77 2.0 93 1.5 80 2.1 94 1.3 98 1.0 37 1.7 80 2.1 87 2.0 45 1.1

All dyes were treated independently with soluble and immobilized potato PPO (1.5 EU/ml) in 50 mM sodium citrate buer, pH 3.0 at 37 C for 1 h. Each value represents the mean for three-independent experiments performed in duplicate, with average standard deviation <5%.

was a remarkable diminution in the peaks both by the soluble and the immobilized potato PPO. Fig. 2 demonstrates the spectra of dye euent before and after treatment with soluble and immobilized potato PPO. There was a remarkable decrease in absorbance peaks of dyeing euent in both visible and UV regions after treatment with soluble and immobilized potato PPO. 4. Discussion The use of free enzymes has several drawbacks such as the stability, reusability and catalytic eciency in decolorizing industrial waste due to complexity of the euents

Fig. 1. Spectra of a mixture of reactive dyes (Reactive Blue 4 + Reactive Blue 160 + Reactive Red 120 + Reactive Red 11) before and after treatment with soluble and immobilized potato PPO. The spectra of this mixture were taken before treatment (control) and after its treatment with soluble and immobilized PPO (treated). For the procedure of dye treatment refer to the assay procedure of dye decolorization. Spectra for treated and untreated dyes mixture are labeled in the gure.

Fig. 2. Absorption spectra of a dyeing euent obtained from a local dyer. The spectra of this dyeing euent were drawn before treatment (control) and after its treatment with soluble and immobilized PPO. For the procedure of dye treatment refer to the assay of dye decolorization. Spectra for treated and untreated dye are labeled in the gure.

(Zille et al., 2003). Some of these limitations can be overcome by the use of immobilized enzymes, which could be used as catalysts with longer lifetime in continuous reactors (Zille et al., 2003; Kandelbauer et al., 2004). Mohan et al. (2005) have reported that polyacrylamide gel immobilized HRP was more eective in decolorizing Acid Black 10BX compared to alginate entrapped and free HRP. Celite adsorbed PPO preparation had very high stability against several forms of denaturants. In view of its high stability and cost of preparation, it could be exploited for the treatment of wastewater contaminated with dyes. In order to make the preparation more successful for the decolorization of textile reactive dyes, various conditions were optimized to obtain maximum activity of immobilized potato PPO. The decolorization of Reactive Blue 4 with soluble and immobilized PPO was maximum at pH 4.0, while Reactive Orange 86 was decolorized maximally in the buer of pH 3.0. Immobilized PPO preparation was more successful in removing higher percentage of color both at lower and higher pH values than its soluble counterpart (Table 1). Some workers have already shown earlier that the degradation of industrially important dyes by HRP (Bhunia et al., 1997; Mohan et al., 2005), bitter gourd peroxidase (Akhtar et al., 2005a,b), manganese peroxidase (Christian et al., 2003) and laccase (Palmieri et al., 2005; Camarero et al., 2005) was also maximum in the buer of lower pH values. Present results suggested that the immobilized preparation of potato PPO was more eective in removing the higher percent of color from dye solutions as compared to the soluble enzyme (Tables 24). Potato PPO decolorized markedly more color of dyes even in the absence of free redox mediators, which have been used by a number of workers for other oxidoreductases (Soares et al., 2001; Akhtar et al., 2005a; Rodriguez-Couto et al., 2005).

Complex mixtures of dyes were also treated with immobilized potato PPO. Immobilized enzyme decolorized signicantly more color from the dye mixtures as compared to the soluble enzyme (Table 3). These observations suggested that the industrial euents containing mixture of dyes could be successfully treated with immobilized potato PPO preparation. The decolorization of dyes in the dye mixture was relatively a slow process as compared to the decolorization of individual dye. In an earlier study, it has been demonstrated that the removal of phenols from their mixtures was also quite slow as compared to individual phenols (Kahru et al., 2000). This was probably due to the competition between various phenols or aromatic compounds for the active site of the enzyme or certain substituted phenols become recalcitrant or slowly transformed by the action of enzyme. After successful treatment of model wastewater containing textile and non-textile dyes or mixture of dyes by immobilized PPOs, an eort was made to investigate the eect of potato PPO on dyeing euent. Immobilized potato PPO treated dyeing euent showed a signicant loss of color. The disappearance of color was followed by insoluble precipitate formation. The observations suggested that immobilized potato PPO could remove more color from the dyeing euents as compared to soluble enzyme (Table 3). The signicance of using immobilized enzyme was that such type of enzyme preparation could be reused with signicantly high stability against several tested denaturing agents present in wastewaters. The diminution in absorbance peaks in visible as well as in UV regions were observed when the dye mixtures and dyeing euent were treated with soluble and immobilized potato PPO. This decrease in the peaks of the complex mixture of dyes in visible and UV regions was more pronounced in case of immobilized PPO treated samples (Figs. 1 and 2).

Earlier, workers have described an eective removal and decolorization of industrially important dyes by using immobilized peroxidases (Shaqu et al., 2002; Akhtar et al., 2005b). The applications of peroxidases in wastewater treatment added additional cost due to the use of expensive co-substrate hydrogen peroxide. The use of stable and reusable PPOs could avoid the use of H2O2 because these enzymes catalyze reactions in the presence of molecular O2 (Husain and Jan, 2000). The use of laccases and peroxidases in the decolorization of various reactive and non-reactive dyes required redox mediators for eective treatment of wastewaters (Soares et al., 2001; Akhtar et al., 2005b; Rodriguez-Couto et al., 2005). However, these redox mediators are expensive compounds and their presence could add pollution to the environment. To avoid the use of such compounds, plant PPOs were considered to be better alternatives than laccases and peroxidases. The level of TOC was signicantly decreased in both soluble and immobilized potato PPO treated polluted water. However, the immobilized PPO treated dye solutions exhibited more loss of TOC from the wastewater (Tables 2 and 3). These observations suggested that major toxic compounds could be removed out of the potato PPO treated samples. Immobilized HRP has been shown to remove 88% of TOC from model wastewater containing mixture of chlorophenols (Tatsumi et al., 1996). Recently we have reported that a signicant amount of TOC from polluted water containing dyes/dye-mixtures and dyeing euent was removed when treated with soluble and immobilized bitter gourd peroxidase (Akhtar et al., 2005b). Several earlier workers have described the removal of dye from wastewater in the form of TOC by treating them with certain chemical methods such as ozonation (Koch et al., 2002). Finally, as a whole this work has clearly demonstrated that the wastewater containing dierent types of textile and non-textile dyes and their mixtures could be easily decolorized by the partially puried PPOs obtained from cheaper plant sources like potato and brinjal. Immobilized enzymes were more useful due to their higher stability and reusability and could be exploited at large scale treatment of industrial euents contaminated with organic pollutants such as aromatic dyes. 5. Conclusions It was concluded that Celite bound potato (Solanum tuberosum) PPO were eective for the decolorization/ removal of mixtures of industrially important dyes. Thus, PPOs could be eective tools for the treatment of industrial euents containing several kinds of organic pollutants even if they are present in the complex compositions. Acknowledgements Financial assistance from the Council of Scientic and Industrial Research, New Delhi, India is gratefully acknowledged.

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