Probing horseradish peroxidase catalyzed degradation of azo dye from tannery wastewater
© Preethi et al.; licensee Springer. 2013
Received: 17 May 2013
Accepted: 19 July 2013
Published: 24 July 2013
Biocatalysis based effluent treatment has outclassed the presently favored physico-chemical treatments due to nil sludge production and monetary savings. Azo dyes are commonly employed in the leather industry and pose a great threat to the environment. Here, we show the degradation of C. I. Acid blue 113 using horseradish peroxidase (HRP) assisted with H2O2 as a co-substrate. It was observed that 0.08 U HRP can degrade 3 mL of 30 mg/L dye up to 80% within 45 min with the assistance of 14 μL of H2O2 at pH 6.6 and 30°C. The feasibility of using the immobilized HRP for dye degradation was also examined and the results show up to 76% dye degradation under similar conditions to that of free HRP with the exception of longer contact time of 240 min. Recycling studies reveal that the immobilized HRP can be recycled up to 3 times for dye degradation. Kinetics drawn for the free HRP catalyzed reaction marked a lower K m and higher V max values, which denotes a proper and faster affinity of the enzyme towards the dye, when compared to the immobilized HRP. The applicability of HRP for treating the actual tannery dye-house wastewater was also demonstrated.
Synthetic dyes are extensively used in a variety of industries including leather and textiles. Conversion of skin into leather generates huge quantity of wastewater comprising a mixture of biogenic matter of skins and a large variety of organic and inorganic chemicals. Wastewater from tanneries usually contains high concentrations of dyes, neutral salts, aliphatic and aromatic polymeric substances as well as poly-phenols (Murugananthan et al. 2004). Acid azo dyes are much frequently used and are potentially toxic if left untreated and there is a high risk to the natural flora (Mohan et al. 2005). The chemical constituents of the dye are mainly phenolic compounds. Since these dye molecules are often toxic and hard to degrade in the conventional wastewater treatment systems, urgency for proper treatment of the colored effluent is required.
Although a variety of physico-chemical treatments such as adsorption, precipitation, chemical degradation, electrochemical, photochemical, etc. is available to decolorize the dye-house wastewater, they have inherent disadvantages such as demand for an external reagent, large sludge generation, and are expensive and tedious (Gozmen et al. 2009; Sauer et al. 2006; Rodriguez et al. 2010; Lachheb et al. 2002). Hence, alternative treatment processes based on biotechnological principles have gained popularity in recent years (Palmieri et al. 2005; Ollikka et al. 1993). Enzymatic treatment systems are simpler and easy to operate in comparison to the microbial treatment systems (Mohan et al. 2005; Kandelbauer et al. 2004). The catalytic action of enzymes is efficient, selective, have higher reaction rates and require mild reaction conditions compared to chemical catalysts. Oxidative enzymes such as lignin peroxidase, horseradish peroxidase (HRP), manganese peroxidase and phenoloxidase (laccase) are extensively employed to remove color from effluent by oxidative degradation of colored compounds and to degrade toxic polyphenols, polyaromatic hydrocarbons, polychlorinated biphenyls, etc. (Mohan et al. 2005; Ollikka et al. 1993; Kandelbauer et al., 2004; Regalado et al. 2004). Reduction of peroxides at the expense of electron donating substrates makes peroxidases useful in oxidative breakdown of synthetic azo dyes (Regalado et al. 2004).
HRP (EC: 126.96.36.199) is known to degrade a wide spectrum of aromatic compounds such as phenols, anilines as well as dyes in the presence of H2O2 (Mohan et al. 2005; Wagner and Nicell 2001; Ulson de Souza et al. 2007; Onder et al. 2011; Arslan 2011; Gholami-Borujeni et al. 2011). The enzyme has relatively high thermal stability and wide distribution (Regalado et al. 2004). H2O2 helps to oxidize the enzyme into a catalytically active form that is capable of reacting with the phenolic contaminant. Peroxidases act by generating free-radical compounds followed by spontaneous polymerization, the polymers can then be removed from the aqueous phase (Wagner and Nicell 2001). The use of immobilized HRP for treating effluent is becoming popular since it can offer long lifetime, stability and recyclability (Mohan et al. 2005; Arslan 2011; Alemzadeh and Nejati 2009). However, appropriate selection of encapsulation material specific to the enzyme and optimization of process conditions is still a challenge (Mohan et al. 2005). Here, we show the ability of HRP both in its free and immobilized form to decolorize an industrially important azo dye, C.I. Acid blue 113. Various parameters such as pH, temperature, contact time, H2O2 and HRP concentration, and dye concentration have been investigated to optimize the treatment conditions. Further, the immobilized HRP performance was evaluated in the process of dye removal along with its recyclability. The enzyme kinetics and ability to treat real tannery effluent were also examined.
Optimization of process parameters for the removal of color using free HRP
Where A 0 is the absorbance of the dye solution before enzymatic treatment at 566 nm A T is the absorbance of the dye solution after enzymatic treatment at 566 nm.
Immobilization of HRP
0.08 U of HRP was added to 1 mL of 0.02 g/mL sodium alginate solution and mixed well. The mixture was dropped into 50 mL of 0.1 M calcium chloride solution through a pipette to form beads while the bottom of the flask was continuously shaken. The beads were left undisturbed in calcium chloride solution to attain stability at 30°C for 40 min. The beads were then filtered and used for further studies.
Optimization of process parameters for the removal of color using immobilized HRP
Optimization of various process parameters such as contact time, temperature, H2O2 and HRP concentration was carried out. Batch reactions were conducted at room temperature maintained at 30°C using borosilicate vials of 15 mL capacity. Each reaction mixture consisted of 3 mL of 30 mg/L dye, 14 μL of H2O2 and 0.08 U of immobilized HRP for all the test series except during H2O2 and HRP optimization. The vials containing the reaction mixture were not subjected to stirring or any kind of agitation. The reaction mixture displayed a pH of 6.6. Dye decolorization was measured spectrophotometrically using a UV-Visible spectrophotometer (Model UV-160A; Shimadzu) based on the absorbance at 566 nm (λmax). Similar experiments were performed by varying the contact time (0, 15, 30, 60, 90, 130, 180, 210, 240, 270 min), immobilized HRP concentration (0.02, 0.04, 0.06, 0.08, 0.10, 0.12 U), H2O2 concentration (4, 8, 12, 14, 16, 20 μL) and temperature (4, 30, 50°C). Experiments were performed in triplicate and the mean values of the results are presented along with error bars.
Recycling studies for the removal of color using immobilized HRP
Each reaction mixture consisted of 3 mL of 30 mg/L dye, 14 μL of H2O2 and 0.08 U of immobilized HRP were kept for different time intervals from 60 to 240 min at 30°C and pH 6.6 for the first cycle experiments. The extent of decolorization after each point of time was analyzed spectrophotometrically as above. Experiments were performed in triplicate and the mean values of the results are presented along with error bars. After the experiments, the beads were collected, washed and reused for the second and third cycle of experiments.
The kinetic experiments for the free HRP were performed by varying the concentration of dye (20, 30, 40 and 50 mg/L) and the contact time (15, 30, 45, 60, 75 min) using constant free enzyme and H2O2 concentration under the optimum conditions at pH 6.6 and 30°C (see Additional file 1). Similarly, the kinetic experiments for the immobilized HRP were performed by varying the concentration of dye (20, 30, 40 and 50 mg/L) and the contact time (25, 85, 125, 200 min) using constant immobilized enzyme and H2O2 concentration under the optimum conditions at pH 6.6 and 30°C (see Additional file 1). The maximum rate of decolorization reaction (V max) and Michaelis-Menten constant (K m) of free as well as immobilized HRP were determined by linear regression and the Lineweaver – Burk plots.
Results and discussion
Optimization of process parameters using free HRP
Optimization of process parameters using immobilized HRP
Recyclability of immobilized HRP
Kinetics of free and immobilized HRP
Decolorization of tannery effluent using free HRP
The performance of both the free and immobilized HRP depends on the reaction time, pH, temperature, HRP and H2O2 concentration. Ambient conditions such as room temperature (30°C) and near-neutral pH (6.6) were suitable for the action of HRP (0.08 U) in both the free and immobilized forms on the optimized dye concentration (30 mg/L) with the assistance of 14 μl H2O2. Free HRP has a faster reaction time (45 min) than immobilized HRP (4 h). The latter can be recycled for at least 3 times thereby demonstrating its potential for application at industrial level. The kinetic parameters for this study are also in line with the optimization experimental results. Free HRP was used for treating actual tannery effluent and the results obtained were satisfactory. This study demonstrates a feasible method for treating tannery effluent. This method could be used to achieve a sustainable and greener environment.
PT and MA wish to thank the CSIR, New Delhi, for providing funding under the Young Scientist Award project scheme.
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