Decolorization of Synthetic Wastewater Containing Azo Dyes in a

15 Jul 2010 - Biodecolorization of synthetic wastewater containing azo dyes, Direct Red-80 (DR-80) and Mordant Blue-9. (MB-9), both individually and ...
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Ind. Eng. Chem. Res. 2010, 49, 7484–7487

Decolorization of Synthetic Wastewater Containing Azo Dyes in a Batch-Operated Rotating Biological Contactor Reactor with the Immobilized Fungus Phanerochaete chrysosporium Kannan Pakshirajan* and Sukhwinder Singh Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati -781039, India

Biodecolorization of synthetic wastewater containing azo dyes, Direct Red-80 (DR-80) and Mordant Blue-9 (MB-9), both individually and together, using immobilized Phanerochaete chrysosporium in a batch-operated rotating biological contactor (RBC) reactor was investigated. Following initial startup of the RBC reactor, which took almost 1 month, results on dye decolorization and enzyme activities of lignin peroxidase (LiP) and manganese peroxidase (MnP) by the fungus were obtained. From experiments involving the individual dyes, decolorization efficiencies were found to be in the range of 94-100%, and from experiments in which the dyes were added together, the decolorization efficiencies of the dye mixture were between 77% and 97% at the end of 24 h. Results of LiP and MnP activities by the fungus revealed a strong role played by the enzymes in the dye decolorization process. As compared to the previous results obtained in batch shake flasks, the results in the present study revealed excellent performance of the bioreactor in decolorizing the wastewater containing the azo dyes. Introduction Environmental problems associated with production and utilization of dyes have attracted considerable attention in the recent past. Among all of the textile dyestuffs produced and used in the industries, azo dyes account for about 70%.1 Also, because this particular group of dyes possesses complicated structure and varies in concentration in the effluents, wastewaters containing such dyes are considered highly recalcitrant.2,3 The other most common dye groups employed in industries are anthraquinon and phthalocyanine dyes. All of these dyes react with the nucleophilic groups on the fabric, forming covalent bonds giving a good fixation.4 However, 10-50% of these dyes show up in the effluent as they react strongly with hydroxyl ions in aqueous solution.4 While reactive dyes have shown to pass ordinary aerobic sewage treatment unaffected, effluents containing the other dyes, which can be otherwise treated either by aerobic or by anaerobic treatment methods, are known to be toxic due to the intermediates, such as aromatic amines formed during the process.5,6 Among the various aerobic methods available for treating the dyes, white-rot fungus has shown promising results in batch shake flasks.7-9 This is mainly due to its lignolytic enzymes that are substrate nonspecific and that are capable of degrading a wide variety of recalcitrant compounds, including complex mixtures of pollutants.10 Because the enzymes are extracellular, diffusion limitation of substrates into the cell that is generally encountered in bacteria is not observed.7 However, proper realization of this fungus in real wastewater treatment systems depends on the right choice of reactor. A reactor that has gained considerable attention is the rotating biological contactor (RBC) reactor in which degrading organisms are grown by attachment onto the rotating discs of the reactor.11 Although this type of reactor system is not new, it offers good performance for large-scale application, and its potential in treating colored wastewaters particularly using the white-rot fungus has not been fully evaluated.11 In the literature, * To whom correspondence should be addressed. Tel.: +91-3612582210. Fax: +91-361-2690762. E-mail: [email protected].

decolorization of azo dyes (DR-80 and MB-9) by Phanerochaete chrysosporium in batch shake flasks with efficiencies greater than 90% has been reported.8 However, decolorization of a mixture of dyes in RBC reactor system using Phanerochaete chrysosporium has not been investigated so far in the literature. Therefore, in the present study, a laboratory scale RBC reactor with the immobilized fungus Phanerochaete chrysosporium was investigated under batch mode of operation for studying decolorization of synthetic wastewater containing the two commercial azo dyes Direct Red-80 (DR-80) and Mordant Blue-9 (MB-9) present both individually and as mixtures. Results of dye decolorization and enzyme activities obtained in the study were also compared to those obtained previously in batch shake flasks using Phanerochaete chrysosporium. Materials and Methods Chemicals and Reagents. The azo dyes DR-80 and MB-9, ABTS-2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), and veratryl alcohol 96% (3,4-dimethoxybenzyl alcohol) were purchased from Sigma Chemicals (St. Louis, MO). All other chemicals and solvents, which were all of Guaranteed Reagent (GR) grade, were purchased from either High Media Pvt. Ltd., Mumbai (India), Sisco Research Laboratory (India), or Merck India Ltd. Microorganism and Culture Media. Phanerochaete chrysosporium MTCC 787 used in the study was procured from the Institute of Microbial Technology (IMTECH), Chandigarh, India, and was maintained by growing on potato dextrose agar slants at 25 °C for 2-5 d and stored in a refrigerator at 4 °C. Media used for dye decolorization were optimized previously by Singh et al. (2010), and it consisted of glucose, 13.46 g L-1; veratryl alcohol, 9.30 mM; KH2PO4, 24.52 g L-1; CaCl2, 2.18 g L-1; MgSO4, 9.89 g L-1; NH4Cl2, 4.68 g L-1; tween 20, 0.050%; and trace elements, MgSO4, 3 g L-1; MnSO4, 0.5 g L-1; NaCl, 1 g L-1; FeSO4 · 7H2O, 0.1 g L-1; CoCl2, 0.1 g L-1; ZnSO4 · 7H2O, 0.1 g L-1; CuSO4, 0.1 g L-1; AlK(SO4)2 · 12H2O, 10 mg L-1; H3BO3, 10 mg L-1; Na2MoO4 · 2H2O, 10 mg L-1; nitrilotriacetate, 1.5 g L-1; and

10.1021/ie1007079  2010 American Chemical Society Published on Web 07/15/2010

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Table 1. Design Specifications of the RBC Reactor specifications

value

number of stages number of discs in each stage diameter of each disc spacing between each disc total working volume % disc submergence

02 10 16 cm 1 cm 3L 40%

other ingredients, 2,2-dimethylsuccinate, 0.1 M (pH 4.2); thiamine, 100 mg L-1.9,12 Experimental Setup. A lab scale RBC reactor was constructed from a methacrylic cylinder of 4 mm thickness, 60 cm length, and 18 cm diameter. The cylinder was cut into two halves with the top half acting as the lid, and the bottom half was closed at the ends to form a chamber. The RBC reactor had 20 circular discs with equally spaced three concentric rings and four ribs made from a 1 cm thick perspex sheet. The discs were covered with polystyrene mesh material, closed at the ends, and mounted on a 3.5 cm diameter aluminum rod supported with bearings at each end of the reactor through a hole at the center of the discs. The discs were made rotatable at the desired speed with the help of a variable speed motor (1-12 rpm) mounted outside the reactor. Table 1 lists the design specifications of the RBC, and the experimental setup is shown in Figure 1. The fungal biomass was immobilized by growing onto the RBC discs covered with polyurethane foam (PUF), which acted as the biosupport material in the reactor. For the immobilization, media, as mentioned before but without any dye, were added to the reactor and inoculated with the fungal spores. The reactor was then recharged with a fresh medium every 4 days for over a month, and, after this period, a thick layer of the fungal biomass was observed on the discs, and dye decolorization experiments were initiated subsequently. However, no attempts were made to quantify the amount of biomass immobilized in the reactor. Batch Dye Decolorization Experiments. Single Dye Decolorization. Batch decolorization of synthetic wastewater containing the two individual azo dyes was performed with the RBC reactor one after the other using different initial dye concentrations (10, 50, 100, 150, and 200 mg L-1) in the culture media. Molecular structures of the two commercial dyes belonging to the same azo group but different classes, Mordant and Direct dyes, are shown in Figure 2a and b, respectively. For operating the reactor under batch mode, while the rotation speed of the discs was maintained at 6 rpm, the disc submergence in the dye wastewater was 40% throughout the experiments. Samples were collected from sampling ports of the reactor at regular time intervals of every 2 h for analyzing the activities of lignin peroxidase (LiP) and manganese peroxidase (MnP), and residual dye concentration in the culture media. At the end of every batch experiment in the reactor, any dye adsorbed onto the fungal biomass was desorbed by applying

Figure 1. Schematic of the experimental setup showing the RBC reactor.

Figure 2. (a) Mordant Blue-9. (b) Direct Red-80.

Figure 3. Decolorization of DR-80 and MB-9 in the single dye decolorization experiments.

10% (v/v) methanol solution, and the amount of dye adsorbed was determined by spectroscopy measurements, which was, however, found to be insignificant. For analysis of dye and enzyme activity, samples collected were centrifuged at 10 000g for 10 min at 4 °C to remove the fungal biomass. After the cells were separated, one portion of the supernatant containing the enzymes was assayed for LiP activity at 310 nm using a UV-visible spectrophotometer, which was based on the oxidation of veratryl alcohol to veratralaldehyde. The other portion was assayed for MnP activity by monitoring the oxidation of 1 mM MnSO4 in 50 mM sodium malonate buffer (pH 4.5) in the presence of 0.1 mM H2O2.13,14 One unit of the enzyme was defined as the amount of enzyme (LiP/MnP) that oxidized l µmol of substrate per minute, and its activities were reported in U L-1. The residual concentration of DR-80 or MB-9 in the media was analyzed using the supernatant as obtained above by measuring its absorbance at 528 nm (λmax of DR-80) and 516 nm (λmax of MB-9) in a UV-visible spectrophotometer (Carry 100, Varian, USA). Mixed Dye Decolorization. To evaluate the performance of the batch-operated RBC reactor in decolorizing synthetic wastewater containing a mixture of the two dyes, a two-level factorial design of experiments involving the dyes at various concentrations was employed. The results obtained were also

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of determination (R2 ≈ 98.95) between the model predicted and added initial dye concentrations in the mixtures.8 Enzyme activities of LiP and MnP in all of the experimental combinations were also analyzed, as described before, for finding their role in dye decolorization by the fungus. Earlier results obtained from batch shake flask experiments were also compared to those obtained from the reactor experiments.

Table 2. (a) DR-80 Decolorization Efficiency, and Maximum LiP and MnP Activities Obtained in the Single Dye Decolorization Experiments for Various Initial Concentrations of the Dye in the Synthetic Wastewater; (b) MB-9 Decolorization Efficiency, and Maximum LiP and MnP Activities Obtained in the Single Dye Decolorization Experiments for Various Initial Concentrations of the Dye in the Synthetic Wastewater initial dye concentration (mg L-1) 10 50 100 150 200 initial dye concentration (mg L-1) 10 50 100 150 200

enzyme activitiesa (U L-1) LiP

decolorization efficiency (%)

MnP

1230 ( 0.36 1127 ( 0.37 1012 ( 0.36 989 ( 0.75 1007 ( 1.36 721 ( 0.39 984 ( 1.93 449 ( 0.73 442 ( 0.92 221 ( 0.1.38 enzyme activitiesa (U L-1)

Results and Discussion

100 100 98 96 94

LiP

MnP

decolorization efficiency (%)

1192 ( 0.835 972 ( 0.363 735 ( 0.982 524 ( 1.377 237 ( 1.376

892 ( 0.643 640 ( 0.386 492 ( 0.393 372 ( 0.393 196 ( 1.363

100 100 100 97 94

Single Dye Decolorization. Dye decolorization profiles obtained in the batch-operated RBC reactor are presented in Figure 3, which shows that the rate and extent of decolorization of the individual dyes depended on the nature and concentration of the dyes in the synthetic wastewater. It is also quite evident from this figure that, in general, the culture took much less time to decolorize wastewater containing DR-80 than that containing MB-9 under similar conditions; however, the decolorization efficiency of both of the dyes was more than 94% within a time period of only 24 h of operating the reactor. It has already been reported that, unlike the direct class of dyes, the mordant class of dyes form insoluble color complexes with its substrates due to the metal chelating (mordant) group present within their structures, thereby rendering these class of dyes more resistant toward decolorization than the direct dyes.8 Moreover, the results indicated that the dyes were completely decolorized (100% decolorization efficiency) in their respective media when presented at low initial concentrations. These results on the dye decolorization in the RBC reactor were also superior to those obtained previously in simple batch shake flasks.8 Further, LiP and MnP activities were analyzed to establish the role of these enzymes in the dye decolorization process. Table 2a and b summarizes the results of activities of LiP and MnP and dye decolorization efficiencies in the study. The results indicate that, in the case of DR-80, the LiP and MnP activities were usually high at all concentrations and were maximum at 1230 and 1127 U L-1, respectively; in the case of MB-9, the maximum LiP and MnP activities were slightly lower as compared to the values obtained using DR80. Thus, it could be said that the maximum activities of LiP and MnP by the fungus were found to vary depending upon the dye and its initial concentration in the synthetic wastewater (Table 2a and b). Nevertheless, the activities of both enzymes using the RBC reactor were almost ∼8-10 times more than the activities previously obtained in the batch shake flasks study involving the same dyes. That the fungus exhibited maximum

a The observed values of activities were the mean values of duplicates with standard deviation (mean ( SD).

compared to those obtained in simple batch shake flasks. As before, samples were collected during the experiments and analyzed for enzyme activities and residual concentrations of the dyes. The individual dye concentrations from the respective mixtures were quantified by a simple classical least squares (CLS) method.15,16 Briefly, this method involved the application of multiple linear regression (MLR) to the classical expression of the Beer-Lambert law of spectroscopy. Thus, the model equations used for estimating dye concentrations in a mixture were: Abs528 )

rY pX + q+X s+Y

(1)

Abs516 )

cY aX + b+X d+Y

(2)

where Abs528 was the absorbance maxima of DR-80 in a mixture and Abs516 was that of MB-9. While X represented the concentration of DR-80, Y represents the concentration of MB9; other parameters such as a, b, c, etc., were the constants in the model. For solving the model equations using the solver function of Microsoft Excel, values of the constants were chosen by trial and error method that gave the best value of coefficient

Table 3. Comparison of Dye Decolorization Efficiency and Maximum Enzyme Activities Obtained in the RBC Reactor with Those Obtained in Batch Shake Flasks for Different Initial Concentrations of DR-80 and MB-9 in the Mixed Dye Experiments cecolorization efficiency of the dye mixture (%) -1

-1

a

enzyme activitiesb (U L-1) in shake flaska

in RBC reactor

exp. run no.

MB-9 conc. (mg L )

DR-80 conc. (mg L )

in shake flask

in RBC reactor

LiP

MnP

LiP

MnP

1 2 3 4 5 6 7 8 9 10 11 12

200 200 10 200 200 200 10 10 10 200 10 10

10 200 200 10 200 200 200 10 10 10 200 10

16 12 26 16 12 12 26 29 29 16 26 29

89.5 77.0 89.0 86.5 77.5 77.5 93.5 97.0 97.5 88.5 84.0 98.0

67.47 ( 1.36 42.91 ( 1.33 97.36 ( 1.39 69.45 ( 0.92 44.97 ( 0.63 43.93 ( 0.87 98.37 ( 0.75 135.76 ( 1.36 130.73 ( 1.38 64.48 ( 0.37 93.77 ( 0.36 135.53 ( 0.32

43.30 ( 0.38 21.28 ( 0.85 78.63 ( 0.75 44.72 ( 0.98 24.62 ( 0.42 22.17 ( 0.37 77.36 ( 0.32 127.73 ( 0.74 129.73 ( 0.55 45.75 ( 0.75 73.74 ( 0.76 128.73 ( 0.85

983 ( 0.36 437 ( 0.37 563 ( 0.75 569 ( 0.53 429 ( 0.21 421 ( 0.64 638 ( 0.23 882 ( 0.32 892 ( 0.31 592 ( 0.45 419 ( 0.21 932 ( 0.43

728 ( 0.53 382 ( 0.42 499 ( 0.72 482 ( 0.43 398 ( 0.36 419 ( 0.32 595 ( 0.47 752 ( 0.74 754 ( 0.43 588 ( 0.94 398 ( 0.83 872 ( 0.82

a

Age of the fungal culture was 5 d, and volume of the shake flask was 100 mL. b Observed values of enzyme activity ) mean value (SD.

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LiP and MnP activities depending on the dye and its initial concentration in the wastewater together with the finding that maximum decolorization of the dyes occurred when the activities of the enzymes peaked in the media suggest a strong role played by these enzymes in the dye decolorization process. The performance of the reactor in decolorizing the wastewater containing the individual dyes was also in agreement with those of Swaminathan et al. (2004) and Pakshirajan et al. (2009).17,18 Mixed Dye Decolorization. To monitor decolorization of the dye mixture in the synthetic wastewater, the following model eqs 3 and 4 were obtained as described before: Abs516 )

73 632.44 × Y 9.38 × X + 482.24 + X 2 727 402.23 + Y

(3)

Abs528 )

52 711.34 × Y 62.28 × X + 1512.35 + X 323 672.29 + Y

(4)

Table 3 presents the concentration combinations of the two dyes, as per the 22 full factorial design of experiments, followed in the study. Decolorization efficiencies of the dye mixtures in the various experimental runs are also shown in the table, which indicate that the values ranged from 77% to 98%. It is also clear that decolorization values were very high as compared to those obtained previously in batch shake flasks (Table 3). To support the variations in decolorization efficiency of the dye mixture in the study, activities of LiP and MnP in the corresponding experimental runs were analyzed. It could be seen that decolorization of the dye mixture highly depended on the activities of both of the enzymes shown by the fungus. Further, it is quite evident from the table that, while the activities of LiP and MnP were generally lower than the values obtained in the individual dye containing system, decolorization efficiencies of the dyes varied depending upon their initial concentration combinations. For instance, at high initial concentrations combinations of the dyes (150 mg L-1 and above), both enzyme activities and dye decolorization efficiencies were slightly less as compared to the values at lower concentration combinations (100 mg L-1 and below). Bonnarme et al. (1991) also reported a similar trend in enzyme activities and dye decolorization by P. chrysosporium for treating dye mixture in a batch-operated reactor system.19 These results of dye decolorization and activities of the enzymes clearly manifest the efficiency of the immobilized fungus in treating a mixture of dyes in the wastewater. Further, when comparing the results obtained in previous batch shake flasks, which took more than 6 days for complete decolorization of a mixture of the dyes, the results in the present bioreactor study reveal its superior performance (the time taken was only 1 day). Overall, the study clearly demonstrates the potential of the batch-operated RBC reactor system in decolorizing colored industrial wastewaters. However, keeping in mind that for practical reasons it would be essential to establish the system under continuous mode of operation, further work in this direction seems necessary. Conclusion A batch-operated RBC reactor containing immobilized P. chrysosporium was efficient in decolorizing synthetic wastewater containing two model azo dyes, DR-80 and MB-9, both individually and together. Decolorization efficiencies of the dyes, however, varied depending upon the dye initial concentrations, with lower concentration favoring easier and complete decolorization as compared to higher concentrations. Further, the activities of LiP and MnP by the fungus positively influenced

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dye decolorization in the study, which suggests the role of the extracellular peroxidase enzyme system of the fungus in the dye decolorization process. These results of dye decolorization and enzyme activities in the batch-operated bioreactor were also found to be superior to those obtained previously in batch shake flasks. Acknowledgment This study was funded by the Council of Scientific and Industrial Research (CSIR), India, under Scheme No. 38(1171)/ 07/EMR-II. Literature Cited (1) Carliell, C. M.; Barclay, S. J.; Naidoo, N.; Buckley, C. A.; Mulholland, D. A.; Senior, M. Microbial decolorization of a reactive azo dye under anaerobic conditions. Water SA 1995, 21, 61. (2) Pandey, A.; Singh, P.; Iyengar, L. Review bacterial decolorization and degradation of azo dyes. Int. Biodeterior. Biodegrad. 2007, 59, 73. (3) Supaka, N.; Juntongjin, K.; Damronglerd, S.; Delia, M. L.; Strehaiano, P. Microbial decolorization of reactive azo dyes in a sequential anaerobic-aerobic system. Chem. Eng. J. 2004, 99, 169. (4) Carliell, C. M.; Barclay, S. J.; Buckley, C. A. Treatment of exhausted reactive dyebath effluent using anaerobic digestion: laboratory and fullscale trials. Water SA 1996, 22, 225. (5) Panswad, T.; Luangdilok, W. Decolorization of reactive dyes with different molecular structures under different environmental conditions. Water Res. 2000, 4, 4177. (6) Brown, M. A.; De Vito, S. C. Predicting azo dye toxicity. Crit. ReV. EnViron. Sci. Technol. 1993, 23, 249. (7) Adosinda, M.; Martinsa, M.; Limaa, N.; Silvestreb, A. J. D.; Queiroz, M. J. Comparative studies of fungal degradation of single or mixed bioaccessible reactive azo dyes. Chemosphere 2003, 52, 967. (8) Singh, S.; Pakshirajan, K. Enzyme activity and decolourization of single and mixed azo dyes by the white rot fungus Phanerochaete chrysosporium. Int. Biodeterior. Biodegrad. 2010, 64, 146. (9) Singh, S.; Pakshirajan, K.; Daverey, A. Screening and optimization of media constituents for decolourization of Mordant Blue-9 dye by Phanerochaete chrysosporium. Clean Technol. EnViron. Policy 2010, 12, 313. (10) Spadaro, J. T.; Gold, M. H.; Renganathan, V. Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chrysosporium. Appl. EnViron. Microbiol. 1992, 58, 2397. (11) Antonie, R. L. Fixed Biological Surfaces - Wastewater Treatment: The Rotating Biological Contactor; CRC Press: Boca Raton, FL, 1976. (12) Tien, M.; Kirk, T. K. Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 1988, 161, 238. (13) Linko, S.; Haapala, R. A critical study of lignin peroxidase activity assay by veratryl alcohol oxidation. Biotechnol. Tech. 1993, 7, 75. (14) Wariishi, H.; Valli, K.; Gold, M. H. Manganese (II) oxidation by manganese peroxidase from basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 1992, 26, 23688. (15) Haaland, D. M.; Easterllng, R. G. Improved sensitivity of infrared spectroscopy by the application of least squares methods. Appl. Spectrosc. 1980, 34, 539. (16) Haaland, D. M.; Easteriing, R. G. Application of new least-squares methods for the quantitative infrared analysis of multi component samples. Appl. Spectrosc. 1982, 36, 665. (17) Swaminathan, T.; Rene, E. R.; Jagannathan, K.; Pakshirajan, K. Treatment of effluents containing hazardous pollutants using rotating biological contactor. Second Int. Symp. Southeast Asian Water EnVironment Hanoi, Vietnam 2004, 16. (18) Pakshirajan, K.; Rene, E. R.; Swaminathan, T. Decolourization of azo dye containing synthetic wastewater in a rotating biological contactor reactor: A factorial design study. Int. J. EnViron. Pollut. 2009, 37, 266. (19) Bonnarme, P.; Delattrea, M.; Corrieua, G.; Asthera, M. Peroxidase secretion by pellets or immobilized cells of Phanerochaete Chrysosporium BKM-F-1767 and INA-12 in relation to organelle content. Enzyme Microb. Technol. 1991, 9, 727.

ReceiVed for reView March 23, 2010 ReVised manuscript receiVed June 22, 2010 Accepted July 5, 2010 IE1007079