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Decolorization of textile dyes and effluents using potato (Solanum tuberosum) phenoloxidase
International Biodeterioration & Biodegradation 72 (2012) 42e45
Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Decolorization of textile dyes and effluents using potato (Solanum tuberosum) phenoloxidase Nikola Lon car a, *, Barbara Janovi c a, Miroslava Vuj ci c b, Zoran Vuj ci ca a b
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Institute of Chemistry, Technology and Metallurgy-Center of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2012 Received in revised form 27 April 2012 Accepted 3 May 2012 Available online 30 May 2012
Potatoes are desirable source for polyphenol oxidase (PPO, EC 1.14.18.1) purification because this enzyme can be purified from the food industry waste such are potato peels from potato chips industry. This paper presents data concerning decolorization of 7 different, so far untested textile dyes and 3 real samples (industry effluents) by a partially purified PPO. Under optimized conditions 93e99.9% removal of dyes was achieved after 1 h using 424e1700 U ml1 of PPO, depending on dye. Optimum pH for decolorization process of all dyes was found to be 3.0. Potato PPO was capable of removing reactive dyes and textile dye effluents without requiring any mediator. Decolorization was accomplished via insoluble polymers formations that were separated by filtration. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polyphenol oxidase Potato Decolorization Reactive dyes Dye effluent
1. Introduction Industrial dyeing of textile consumes large amounts of water and energy, while 5e40% of the dyestuffs used are released in the effluent (Silva et al., 2010). Released colors may seriously jeopardize both photosynthetic activities of surface water ecosystems since they interfere with the absorption of solar radiation and ecosystem integrity due to their toxicity. Since some of those dyes are recalcitrant to direct microbial degradation there is an ongoing search for improvement of existing technologies and introduction of clean and more efficient technologies that will enable degradation of these compounds (Verma et al., 2003). Promising alternative technology is the use of oxidative enzymes as crude or partially purified to reduce overall treatment costs. It is known that polyphenol oxidases (PPO) and peroxidases can quickly and nonselectively oxidize a broad spectrum of structurally diverse organic molecules. PPOs (EC 1.14.18.1) from potatoes are the enzymes with highest potential with respect to its availability and cost (Khan et al., 2006). It has been shown previously that PPO activity was the highest in the tuber exterior, including the skin and
* Corresponding author. Tel.: þ381 113282393. E-mail address: [email protected] (N. Lon car). 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2012.05.001
cortex tissue 1e2 mm beneath the skin (Thygesen et al., 1995). Potatoes are desirable source for PPO purification because this enzyme can be purified from the food industry waste such as potato peels from potato chips industry. The enzymatic treatment efficiency was found to be independent of the enzyme purity and therefore, it was possible to utilize a crude or partially purified preparation that is protected from inactivation due to the significant quantity of protein present instead of a purified one (Cooper and Nicell, 1996). However, use of partially purified enzymes is preferable over crude extracts due to many reasons such as removal of undesired phenolics from plant source during purification processes and avoidance of excess of proteins in crude extracts that may adversely increase biochemical oxygen demand (BOD) in the treated wastewater. The present study was performed to extend our previous work on application of cheap enzyme source such as potato PPO (Loncar and Vujcic, 2011). We now describe the usability of PPO partially purified by ion-exchange chromatography for removal of recalcitrant textile dyes. Seven dyes widely used in textile industries for cotton dyeing have been selected for the study as well as three different dye effluents collected directly from local textile-dying industry. Optimum conditions for efficient removal of dyes by precipitation from real samples and model systems were investigated.
N. Loncar et al. / International Biodeterioration & Biodegradation 72 (2012) 42e45
43
(%) ¼ [(A0 A1)/A0] 100, where A0 was the absorbance of the untreated dye and A1 was the absorbance after treatment.
2. Material and methods 2.1. Materials Potato (Solanum tuberosum) tubers were obtained from the local market. All reagents used in this study were of analytical grade. They were purchased from Merck (Darmstadt, Germany) and SigmaeAldrich (St. Louis, MO, USA). The textile effluents were collected at the outlet of the neutralization tank from a textile dyeing industry, located in Sombor, Serbia. 2.2. Polyphenol oxidase activity assay PPO was purified as described previously (Loncar and Vuj ci c, 2011). Briefly, potatoes were cooled to 3 C over night and then homogenized in commercial juicer. The homogenate was centrifuged and obtained supernatant was desalted against 10 mM Naphosphate buffer pH 7.3 using Sephadex G25 Coarse column. Equilibrated and deaerated ion-exchanger QAE Sephadex A-50 was added to extract and mixed with magnetic stirrer 30 min in oxygen-free atmosphere. Unbound proteins were washed with 3 volumes of starting buffer and enzyme was eluted with 0.75M NaCl in starting buffer. Obtained enzyme preparation was stored at 20 C until use. PPO activity was determined using L-DOPA as a substrate at 25 C by measuring the initial rate of dopachrome formation at 475 nm (Kwon and Kim, 1996). 2.3. Effect of pH on the decolorization of reactive textile dyes Concentrations and characteristic wavelength maximums (lmax) for four azo and three aminochlorotriazine dyes used in this study are given in Table 1. These concentrations were chosen in order to have starting dye solution with absorbance of 1.0 at specific lmax. Each dye was incubated with PPO (424 U ml1) in the appropriate 50 mmol l1 buffers (sodium acetate, pH 3.0 and pH 5.0; sodium phosphate, pH 7.0; glycineesodium hydroxide, pH 9.0) at 25 C for 1 h, after which the solutions were liberated from produced polymers by filtration through Whatman No. 1 or by centrifugation at 600 g and tested for remaining dye content by measuring absorbance at lmax. Starting absorbance at characteristic lmax for each dye (control) was designated as 100%. The extent of decolorization rate (Rd) was defined by following formula: Rd
2.4. Effect of PPO concentration and time of incubation on the decolorization of dyes To test effect of PPO concentration decolorization rates of all dyes were analyzed with increasing concentrations of PPO in range 212e1700 U ml1 in 50 mmol l1 sodium acetate buffer, pH 3.0 at 25 C for 1 h. Effects of incubation time was monitored by incubation of dye solutions with PPO (283 U ml1) in the 50 mmol l1 sodium acetate buffer, pH 3.0 at 25 C for different period of time. Dye decolorization was monitored at specific lmax as described above. 2.5. Vis and FTIR spectrometry analysis of textile effluents treated with PPO The textile effluents were kept at an ambient temperature, original pH being 7.1. pH adjustments to pH 3.0 were done by addition of acetic acid. Acidification had no visible influence on effluents. Decolorization treatments were done by addition of 424 U ml1 of PPO to each effluent. Vis spectrums of effluents were recorded using Cintra 40 Spectrometer in 380e800 nm range before and after treatment with PPO. FTIR spectra of Reactive blue 52 (RB52) and its polymer product were obtained in a range of 400e4000 nm by using an attenuated total reflectance (ATR) technique on FTIR spectrophotometer (Nicolet 6700FT-IR, Thermo Scientific) as described by Aktas et al. (2000, 2003). Polymer product was collected by centrifugation after treatment with PPO and dried until constant weight using Eppendorf Vacuum Concentrator Model 5301. 2.6. Statistics All experimental results reported in the next sections were based on averaging results of repeated experimental runs (triplicates), with the SD ranging from 2 to 6% of the reported average. Statistical significance is confirmed by Student’s t-test. 3. Results and discussion Potato peels from chips industry are free source of enzymes for biotechnology due to potatoes availability during whole year (Vujcic et al., 2010; Lon car et al., 2011) Endogenous polyphenols must be removed during purification procedure since phenoxy
Table 1 Effect of initial solution pH on decolorization of tested dyes with PPO (424 U mL1).
lmax C
(mg L1)
Aminochlorotriazine dyes Procion yellow 420 65 (PY) Procion red 460 50 brown (PRB) Procion dark 600 50 blue (PDB) Azo dyes Reactive blue 52 (RB52) Reactive green 15 (RG15) Reactive yellow 125 (RY125) Congo red (CR) a
% of remaining dyea pH 3
pH 5
pH 7
pH 9
7 0.46 50 1.06 95 1.15 100 26 0.86 67 2.60 97 1.2
100
47 1.60 93 1.65 100
100
5 0.17 71 2.15 91 1.24 92 1.7
615
50
625
100
9 0.31 59 1.69 93 1.0
390
65
11 0.29 54 1.87 97 0.9
100
522
65
0.5 0.04
90 0.8
5 0.03 83 1.1
Statistical significance is confirmed by Student’s t-test.
93 1.2
Fig. 1. Influence of PPO concentration on decolorization of reactive dyes.
44
N. Loncar et al. / International Biodeterioration & Biodegradation 72 (2012) 42e45
Fig. 2. Visible spectrums of treated and untreated effluents. Spectrums of starting solution are shown in solid line and spectra of treated solutions in dashed line. A) pink effluent, B) brownish effluent, C) dark green effluent.
radicals generated in the catalytic cycle attack the active site of the enzyme thereby eliminating its catalytic ability (Kwon and Kim, 1996). Optimization of purification process that deals with above mentioned problems and can be easily scaled-up to industrial level have been recently published (Lon car and Vuj ci c, 2011). 3.1. Effect of pH on the decolorization of reactive textile dyes The effects of pH on the decolorization of textile dyes by potato PPO are summarized in Table 1. The decolorization rate was significantly higher at lower pH having maximum at pH 3.0. There were several earlier reports regarding the maximum decolorization of dyes by various plant peroxidases (Akhtar et al., 2005) and microbial polyphenol oxidases (Unyayar et al., 2005) at acidic pH values. When pH is increased above 5.0 the extent of decolorization was decreased rapidly. This is advantage from industrial application point of view since some dye effluents are slightly acidic (de Souza et al., 2007). In other cases any cheap acid can be used to adjust pH to 3.0. 3.2. Effect of PPO concentration and time of incubation on the decolorization of dyes Optimization of minimal enzyme concentration necessary for decolorization of colored wastewater is needed in order to create economically attractive process (de Souza et al., 2007). Histogram shown in Fig. 1 demonstrates the effect of varying concentrations of PPO on the decolorization of tested dyes. The rate of dye decolorization was continuously enhanced with increasing the amount of PPO. However, for all tested dyes 212 U ml1 of PPO was insufficient for removal, although in case of CR approximately 75% dye was removed at this stage. When 424 U ml1 of PPO were used RB52, RG15, RY125, CR and PY were decolorized maximally and further increase of PPO amount did not give better results (Fig. 1). PRB and PDB showed higher resistance to PPO oxidation and it was necessary to use 848 U ml1 to decolorize them almost completely within 1 h (Fig. 1). Decolorization of textile reactive dyes with potato PPO was examined by varying the time of incubation (data not shown). Apparition of colored precipitate was observable even after 15 min in cases of RB52, RG15 and CR, while in cases of other tested dyes precipitate has gradually formed. However, incubations beyond time period of 1 h did not result in better decolorization of tested dyes. Advantage of precipitate mediated decolorization versus decolorization without precipitation (peroxidases based treatment) is that carbon content is effectively lowered while at the same time in the latter case carbon content remains unchanged.
3.3. Vis and FTIR spectrometry analysis of textile effluents treated with PPO The decolorization of dye effluents was analyzed with Vis spectroscopy (Fig. 2). After treatment, a remarkable diminution in absorbance peaks in whole visible region was observed. This is a result of breakdown of chromophoric groups present in the dyes (Akhtar et al., 2005). Although dye effluents are of unknown composition since dye mixtures used in industry are kept as company’s secret, it is reasonable to assume that some of individually tested dyes (or related structures) are components of these effluents. The treatments of dye effluents resulted in the formation of insoluble precipitate due to quinones-derivative formation, which mediates the aggregation of aromatic pollutants (Khan and Husain, 2007). The treatment of dyes and phenols with PPO resulted in the formation of large insoluble aggregate, which could be easily removed from the reaction mixture (Wada et al., 1995; Akhtar et al., 2005). Analysis of parent dye (RB 52) and its oxidized product showed disappearance of some peaks in parent compound and appearance of some new peaks in the polymeric product when subjected to FTIR analysis (Fig. 3). The spectrum of the product with broader bands is indicative of polymer formation. Several important bands were absent in the spectrum of the product such as the band at 1580 cm1 for N]N stretching vibrations (Telke et al., 2010) and the band at 1188 cm1 of phenolic CeO stretching. The prominent new bands at 1640 cm1 and 1530 cm1 could be ascribed to oquinone C]O and C]N stretching vibrations, respectively. The bands due to sulpho groups, amino groups and aromatic rings were
Fig. 3. FTIR spectrums: upper spectra shows RB52 and lower spectra shows dried precipitate obtained after treatment of RB52 with PPO.
N. Loncar et al. / International Biodeterioration & Biodegradation 72 (2012) 42e45
present in the spectra of both the dye and the product. The new peaks in FTIR spectra of the product as compared to parent dye may be ascribed to the polymer formation. Similar type of results was obtained when oxidative polymerization of a-naphthol and catechol was carried out by laccase (Aktas et al., 2000, 2003). Since similar results were obtained for all tested dyes only one is shown. Acknowledgments This work was supported by the Serbian Ministry of Science and Technological Development (Grant No. 172048). References Akhtar, S., Khan, A.A., Husain, Q., 2005. Partially purified bitter gourd (Momordica charantia) peroxidase catalyzed decolorization of textile and other industrially important dyes. Bioresource Technology 96, 1804e1811. Aktas, N., Kibarer, G., Tanyolac, A., 2000. Effects of reaction conditions on laccase catalyzed a-naphthol polymerization. Journal of Chemical Technology Biotechnology 75, 840e846. Aktas, N., Sahiner, N., Kantoglu, O., Salih, B., Tanyolac, A., 2003. Biosynthesis and characterization of laccase catalyzed poly(catechol). Journal of Polymers and the Environment 11, 123e128. Cooper, V.A., Nicell, J.A., 1996. Removal of phenols from a foundry wastewater using horseradish peroxidase. Water Research 30, 954e964. de Souza, S.M.A.G.U., Forgiarini, E., de Souza, A.A.U., 2007. Toxicity of textile dyes and their degradation by the enzyme horseradish peroxidase (HRP). Journal of Hazardous Materials 147, 1073e1078. Khan, A.A., Akhtar, S., Husain, Q., 2006. Direct immobilization of polyphenol oxidase on Celite 545 from ammonium sulphate fractionated proteins of potato (Solanum tuberosum). Journal of Molecular Catalysis B: Enzymatic 40, 58e63.
45
Khan, A.A., Husain, Q., 2007. Decolorization and removal of textile and non-textile dyes from polluted wastewater and dyeing effluent by using potato (Solanum tuberosum) soluble and immobilized polyphenol oxidase. Bioresource Technology 98, 1012e1019. Kwon, D.Y., Kim, W.Y., 1996. Purification of the glycosylated polyphenol oxidase from potato tuber. Journal of Biochemistry and Molecular Biology 29, 163e168. Lon car, N., Bo zi c, N., AnCelkovi c, I., Milovanovi c, A., Dojnov, B., Vuj ci c, M., Rogli c, G., Vuj ci c, Z., 2011. Removal of aqueous phenol and phenol derivatives by immobilized potato polyphenol oxidase. Journal of Serbian Chemical Society 76, 513e522. Lon car, N., Vuj ci c, Z., 2011. Tentacle carrier for immobilization of potato phenol oxidase and its application for halogenophenols removal from aqueous solutions. Journal of Hazardous Materials 196, 73e78. Silva, M.R., Sa, L.R.V., Russo, C., Scio, E., Ferreira-Leitao, V.S., 2010. The use of HRP in decolorization of reactive dyes and toxicological evaluation of their products. Enzyme Research. doi:10.4061/2010/703824. Telke, A.A., Joshi, S.M., Jadhov, S.U., Tamboli, D.P., Govindwar, S.P., 2010. Decolorization and detoxification of Congo red and textile industry effluent by an isolated bacterium Pseudomonas sp. SU-EBT. Biodegradation 21, 283e296. Thygesen, P.W., Dry, I.B., Robinson, S.P., 1995. Polyphenol oxidase in potato (a multigene family that exhibits differential expression patterns). Plant Physiology 109, 525e531. Unyayar, A., Mazmanci, M.A., Atacag, H., Erkurt, E.A., Coral, G., 2005. A Drimaren blue X3LR dye decolorizing enzyme from Funalia trogii: one step isolation and identification. Enzyme and Microbial Technology 36, 10e16. Verma, P., Baldrian, P., Nerud, F., 2003. Decolorization of structurally different synthetic dyes using cobalt(II)/ascorbic acid/hydrogen peroxide system. Chemosphere 50, 957e979. Vuj ci c, Z., Lon car, N., Dojnov, B., Milovanovi c, A., Vuj ci c, M., Bo zi c, N., 2010. Characterization of leucylaminopeptidase from Solanum tuberosum tuber. Food Chemistry 121, 418e423. Wada, S., Ichikawa, H., Tatsumi, K., 1995. Removal of phenols and aromatic amines from wastewater by a combination treatment with tyrosinase and a coagulant. Biotechnology and Bioengineering 45, 304e309.
Contents lists available at SciVerse ScienceDirect
International Biodeterioration & Biodegradation journal homepage: www.elsevier.com/locate/ibiod
Short communication
Decolorization of textile dyes and effluents using potato (Solanum tuberosum) phenoloxidase Nikola Lon car a, *, Barbara Janovi c a, Miroslava Vuj ci c b, Zoran Vuj ci ca a b
Faculty of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia Institute of Chemistry, Technology and Metallurgy-Center of Chemistry, University of Belgrade, Studentski trg 12-16, Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2012 Received in revised form 27 April 2012 Accepted 3 May 2012 Available online 30 May 2012
Potatoes are desirable source for polyphenol oxidase (PPO, EC 1.14.18.1) purification because this enzyme can be purified from the food industry waste such are potato peels from potato chips industry. This paper presents data concerning decolorization of 7 different, so far untested textile dyes and 3 real samples (industry effluents) by a partially purified PPO. Under optimized conditions 93e99.9% removal of dyes was achieved after 1 h using 424e1700 U ml1 of PPO, depending on dye. Optimum pH for decolorization process of all dyes was found to be 3.0. Potato PPO was capable of removing reactive dyes and textile dye effluents without requiring any mediator. Decolorization was accomplished via insoluble polymers formations that were separated by filtration. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Polyphenol oxidase Potato Decolorization Reactive dyes Dye effluent
1. Introduction Industrial dyeing of textile consumes large amounts of water and energy, while 5e40% of the dyestuffs used are released in the effluent (Silva et al., 2010). Released colors may seriously jeopardize both photosynthetic activities of surface water ecosystems since they interfere with the absorption of solar radiation and ecosystem integrity due to their toxicity. Since some of those dyes are recalcitrant to direct microbial degradation there is an ongoing search for improvement of existing technologies and introduction of clean and more efficient technologies that will enable degradation of these compounds (Verma et al., 2003). Promising alternative technology is the use of oxidative enzymes as crude or partially purified to reduce overall treatment costs. It is known that polyphenol oxidases (PPO) and peroxidases can quickly and nonselectively oxidize a broad spectrum of structurally diverse organic molecules. PPOs (EC 1.14.18.1) from potatoes are the enzymes with highest potential with respect to its availability and cost (Khan et al., 2006). It has been shown previously that PPO activity was the highest in the tuber exterior, including the skin and
* Corresponding author. Tel.: þ381 113282393. E-mail address: [email protected] (N. Lon car). 0964-8305/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibiod.2012.05.001
cortex tissue 1e2 mm beneath the skin (Thygesen et al., 1995). Potatoes are desirable source for PPO purification because this enzyme can be purified from the food industry waste such as potato peels from potato chips industry. The enzymatic treatment efficiency was found to be independent of the enzyme purity and therefore, it was possible to utilize a crude or partially purified preparation that is protected from inactivation due to the significant quantity of protein present instead of a purified one (Cooper and Nicell, 1996). However, use of partially purified enzymes is preferable over crude extracts due to many reasons such as removal of undesired phenolics from plant source during purification processes and avoidance of excess of proteins in crude extracts that may adversely increase biochemical oxygen demand (BOD) in the treated wastewater. The present study was performed to extend our previous work on application of cheap enzyme source such as potato PPO (Loncar and Vujcic, 2011). We now describe the usability of PPO partially purified by ion-exchange chromatography for removal of recalcitrant textile dyes. Seven dyes widely used in textile industries for cotton dyeing have been selected for the study as well as three different dye effluents collected directly from local textile-dying industry. Optimum conditions for efficient removal of dyes by precipitation from real samples and model systems were investigated.
N. Loncar et al. / International Biodeterioration & Biodegradation 72 (2012) 42e45
43
(%) ¼ [(A0 A1)/A0] 100, where A0 was the absorbance of the untreated dye and A1 was the absorbance after treatment.
2. Material and methods 2.1. Materials Potato (Solanum tuberosum) tubers were obtained from the local market. All reagents used in this study were of analytical grade. They were purchased from Merck (Darmstadt, Germany) and SigmaeAldrich (St. Louis, MO, USA). The textile effluents were collected at the outlet of the neutralization tank from a textile dyeing industry, located in Sombor, Serbia. 2.2. Polyphenol oxidase activity assay PPO was purified as described previously (Loncar and Vuj ci c, 2011). Briefly, potatoes were cooled to 3 C over night and then homogenized in commercial juicer. The homogenate was centrifuged and obtained supernatant was desalted against 10 mM Naphosphate buffer pH 7.3 using Sephadex G25 Coarse column. Equilibrated and deaerated ion-exchanger QAE Sephadex A-50 was added to extract and mixed with magnetic stirrer 30 min in oxygen-free atmosphere. Unbound proteins were washed with 3 volumes of starting buffer and enzyme was eluted with 0.75M NaCl in starting buffer. Obtained enzyme preparation was stored at 20 C until use. PPO activity was determined using L-DOPA as a substrate at 25 C by measuring the initial rate of dopachrome formation at 475 nm (Kwon and Kim, 1996). 2.3. Effect of pH on the decolorization of reactive textile dyes Concentrations and characteristic wavelength maximums (lmax) for four azo and three aminochlorotriazine dyes used in this study are given in Table 1. These concentrations were chosen in order to have starting dye solution with absorbance of 1.0 at specific lmax. Each dye was incubated with PPO (424 U ml1) in the appropriate 50 mmol l1 buffers (sodium acetate, pH 3.0 and pH 5.0; sodium phosphate, pH 7.0; glycineesodium hydroxide, pH 9.0) at 25 C for 1 h, after which the solutions were liberated from produced polymers by filtration through Whatman No. 1 or by centrifugation at 600 g and tested for remaining dye content by measuring absorbance at lmax. Starting absorbance at characteristic lmax for each dye (control) was designated as 100%. The extent of decolorization rate (Rd) was defined by following formula: Rd
2.4. Effect of PPO concentration and time of incubation on the decolorization of dyes To test effect of PPO concentration decolorization rates of all dyes were analyzed with increasing concentrations of PPO in range 212e1700 U ml1 in 50 mmol l1 sodium acetate buffer, pH 3.0 at 25 C for 1 h. Effects of incubation time was monitored by incubation of dye solutions with PPO (283 U ml1) in the 50 mmol l1 sodium acetate buffer, pH 3.0 at 25 C for different period of time. Dye decolorization was monitored at specific lmax as described above. 2.5. Vis and FTIR spectrometry analysis of textile effluents treated with PPO The textile effluents were kept at an ambient temperature, original pH being 7.1. pH adjustments to pH 3.0 were done by addition of acetic acid. Acidification had no visible influence on effluents. Decolorization treatments were done by addition of 424 U ml1 of PPO to each effluent. Vis spectrums of effluents were recorded using Cintra 40 Spectrometer in 380e800 nm range before and after treatment with PPO. FTIR spectra of Reactive blue 52 (RB52) and its polymer product were obtained in a range of 400e4000 nm by using an attenuated total reflectance (ATR) technique on FTIR spectrophotometer (Nicolet 6700FT-IR, Thermo Scientific) as described by Aktas et al. (2000, 2003). Polymer product was collected by centrifugation after treatment with PPO and dried until constant weight using Eppendorf Vacuum Concentrator Model 5301. 2.6. Statistics All experimental results reported in the next sections were based on averaging results of repeated experimental runs (triplicates), with the SD ranging from 2 to 6% of the reported average. Statistical significance is confirmed by Student’s t-test. 3. Results and discussion Potato peels from chips industry are free source of enzymes for biotechnology due to potatoes availability during whole year (Vujcic et al., 2010; Lon car et al., 2011) Endogenous polyphenols must be removed during purification procedure since phenoxy
Table 1 Effect of initial solution pH on decolorization of tested dyes with PPO (424 U mL1).
lmax C
(mg L1)
Aminochlorotriazine dyes Procion yellow 420 65 (PY) Procion red 460 50 brown (PRB) Procion dark 600 50 blue (PDB) Azo dyes Reactive blue 52 (RB52) Reactive green 15 (RG15) Reactive yellow 125 (RY125) Congo red (CR) a
% of remaining dyea pH 3
pH 5
pH 7
pH 9
7 0.46 50 1.06 95 1.15 100 26 0.86 67 2.60 97 1.2
100
47 1.60 93 1.65 100
100
5 0.17 71 2.15 91 1.24 92 1.7
615
50
625
100
9 0.31 59 1.69 93 1.0
390
65
11 0.29 54 1.87 97 0.9
100
522
65
0.5 0.04
90 0.8
5 0.03 83 1.1
Statistical significance is confirmed by Student’s t-test.
93 1.2
Fig. 1. Influence of PPO concentration on decolorization of reactive dyes.
44
N. Loncar et al. / International Biodeterioration & Biodegradation 72 (2012) 42e45
Fig. 2. Visible spectrums of treated and untreated effluents. Spectrums of starting solution are shown in solid line and spectra of treated solutions in dashed line. A) pink effluent, B) brownish effluent, C) dark green effluent.
radicals generated in the catalytic cycle attack the active site of the enzyme thereby eliminating its catalytic ability (Kwon and Kim, 1996). Optimization of purification process that deals with above mentioned problems and can be easily scaled-up to industrial level have been recently published (Lon car and Vuj ci c, 2011). 3.1. Effect of pH on the decolorization of reactive textile dyes The effects of pH on the decolorization of textile dyes by potato PPO are summarized in Table 1. The decolorization rate was significantly higher at lower pH having maximum at pH 3.0. There were several earlier reports regarding the maximum decolorization of dyes by various plant peroxidases (Akhtar et al., 2005) and microbial polyphenol oxidases (Unyayar et al., 2005) at acidic pH values. When pH is increased above 5.0 the extent of decolorization was decreased rapidly. This is advantage from industrial application point of view since some dye effluents are slightly acidic (de Souza et al., 2007). In other cases any cheap acid can be used to adjust pH to 3.0. 3.2. Effect of PPO concentration and time of incubation on the decolorization of dyes Optimization of minimal enzyme concentration necessary for decolorization of colored wastewater is needed in order to create economically attractive process (de Souza et al., 2007). Histogram shown in Fig. 1 demonstrates the effect of varying concentrations of PPO on the decolorization of tested dyes. The rate of dye decolorization was continuously enhanced with increasing the amount of PPO. However, for all tested dyes 212 U ml1 of PPO was insufficient for removal, although in case of CR approximately 75% dye was removed at this stage. When 424 U ml1 of PPO were used RB52, RG15, RY125, CR and PY were decolorized maximally and further increase of PPO amount did not give better results (Fig. 1). PRB and PDB showed higher resistance to PPO oxidation and it was necessary to use 848 U ml1 to decolorize them almost completely within 1 h (Fig. 1). Decolorization of textile reactive dyes with potato PPO was examined by varying the time of incubation (data not shown). Apparition of colored precipitate was observable even after 15 min in cases of RB52, RG15 and CR, while in cases of other tested dyes precipitate has gradually formed. However, incubations beyond time period of 1 h did not result in better decolorization of tested dyes. Advantage of precipitate mediated decolorization versus decolorization without precipitation (peroxidases based treatment) is that carbon content is effectively lowered while at the same time in the latter case carbon content remains unchanged.
3.3. Vis and FTIR spectrometry analysis of textile effluents treated with PPO The decolorization of dye effluents was analyzed with Vis spectroscopy (Fig. 2). After treatment, a remarkable diminution in absorbance peaks in whole visible region was observed. This is a result of breakdown of chromophoric groups present in the dyes (Akhtar et al., 2005). Although dye effluents are of unknown composition since dye mixtures used in industry are kept as company’s secret, it is reasonable to assume that some of individually tested dyes (or related structures) are components of these effluents. The treatments of dye effluents resulted in the formation of insoluble precipitate due to quinones-derivative formation, which mediates the aggregation of aromatic pollutants (Khan and Husain, 2007). The treatment of dyes and phenols with PPO resulted in the formation of large insoluble aggregate, which could be easily removed from the reaction mixture (Wada et al., 1995; Akhtar et al., 2005). Analysis of parent dye (RB 52) and its oxidized product showed disappearance of some peaks in parent compound and appearance of some new peaks in the polymeric product when subjected to FTIR analysis (Fig. 3). The spectrum of the product with broader bands is indicative of polymer formation. Several important bands were absent in the spectrum of the product such as the band at 1580 cm1 for N]N stretching vibrations (Telke et al., 2010) and the band at 1188 cm1 of phenolic CeO stretching. The prominent new bands at 1640 cm1 and 1530 cm1 could be ascribed to oquinone C]O and C]N stretching vibrations, respectively. The bands due to sulpho groups, amino groups and aromatic rings were
Fig. 3. FTIR spectrums: upper spectra shows RB52 and lower spectra shows dried precipitate obtained after treatment of RB52 with PPO.
N. Loncar et al. / International Biodeterioration & Biodegradation 72 (2012) 42e45
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