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Acid azo dye degradation by free and immobilized horseradish peroxidase (HRP) catalyzed process
Chemosphere 58 (2005) 1097–1105 www.elsevier.com/locate/chemosphere
Acid azo dye degradation by free and immobilized horseradish peroxidase (HRP) catalyzed process S. Venkata Mohan, K. Krishna Prasad, N. Chandrasekhara Rao, P.N. Sarma
*
Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, India Received 11 June 2003; received in revised form 6 September 2004; accepted 15 September 2004
Abstract Acid azo (Acid Black 10 BX) dye removal by plant based peroxidase catalyzed reaction was investigated. Horseradish peroxidase (HRP) was extracted from horseradish roots and its performance was evaluated in both free and immobilized form. HRP showed its ability to degrade the dye in aqueous phase. Studies are further carried out to understand the process parameters such as aqueous phase pH, H2O2 dose, dye and enzyme concentrations during enzyme-mediated dye degradation process. Experimental data revealed that dye (substrate) concentration, aqueous phase pH, enzyme and H2O2 dose play a significant role on the overall enzyme-mediated reaction. Acrylamide gel immobilized HRP showed effective performance compared to free HRP and alginate entrapped HRP. Alginate entrapped HRP showed inferior performance over the free enzyme due to the consequence of non-availability of the enzyme to the dye molecule due to polymeric immobilization. Standard plating studies performed with Pseudomonas putida showed enhanced degradation of HRP catalyzed dye compared to control. 2004 Elsevier Ltd. All rights reserved. Keywords: Horseradish peroxidase; Azo dye; H2O2; pH; Enzyme activity; Immobilization; Alginate; Acrylamide
1. Introduction Dyes are complex aromatic compounds, which are normally used for coloration of various substrates. They are sometimes fused with heavy metals on the structural interface and are considered to have relatively bad consequence on the surrounding environment due to its toxic and inhibitory nature (Correia et al., 1994; Stolz, 2001; Venkata Mohan et al., 2002a,b). Among all the
*
Corresponding author. Fax: +91 40 27193626. E-mail addresses: [email protected], kousik@iict. ap.nic.in (P.N. Sarma).
chemical classes of dyes, azo dyes are considered to be recalcitrant, non-biodegradable and persistent. Treatment of dye based effluents is considered to be one of the challenging tasks in environmental fraternity. Even though physico-chemical methods are effective in the removal of dyes, the overall cost, regeneration problem, secondary pollutant/sludge generation limits their usage (Venkata Mohan and Karthikeyan, 1999). Also, dye based effluents are normally not amenable for conventional biological wastewater treatment due to their recalcitrant and inhibitory nature (Kulla, 1981). However, microbial methods are highly useful and potentially advantageous for the treatment of toxic compounds due to their effectiveness, ecofriendly nature, energy saving and less usage of chemicals (Koller et al., 2000). Researchers have been focusing their attention to study
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.09.070
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enzymatic pretreatment as a potential and viable alternative to conventional methods, due to its highly zselective nature. Further, inhibition by toxic substances is minimum in enzymatic treatment and the process can operate over a broad aromatic concentration range with low retention time (Kasam and Niceu, 1997). Enzymes can act on specific recalcitrant pollutants to remove them by precipitation or transformation to other (innocuous) products and also can change the characteristics of a given waste to render it more amenable for treatment. The catalytic action of enzymes is extremely efficient and selective compared to chemical catalysts due to higher reaction rates, milder reaction conditions and greater stereospecificity. They can catalyze reactions at relatively low temperature and in the entire aqueous phase pH range. Though much attention has been paid in the utilization of biocatalysts in several fields, their involvement has been felt very recently in solving the environmental problems (Kasam and Niceu, 1997; Venkata Mohan et al., 2002c). Extracellular fungal peroxidases are reported to oxidatively catalyze the polymerization of toxic aromatic compounds in aqueous solution and are reported to oxidize various pollutants (Hammel and Tardone, 1988; Dec and Bollag, 1990; Valli and Gold, 1991; Arseguel and Baboulene, 1994; Nicell, 1994; Manimekalai and Swaminathan, 2000). Enzymes from various sources (fungus and plant based) are applied for the treatment of dye based compounds (Novotny et al., 2001). The source of the selected enzyme and its nature along with system conditions are found to have significant influence on the overall performance for pollutant removal. Fungal extracted enzymes are studied quite significantly in the process of dye removal (Glenn and Gold, 1983; Manimekalai and Swaminathan, 2000; Novotny et al., 2001; Maximo and Costa-Ferreira, 2004; Hou et al., 2004). Relatively, plant based peroxidases in the removal of pollutants are less documented (Nicell, 1994; Koller et al., 2000; Bhunia et al., 2001). Several limitations prevent the use of free enzymes as the stability and catalytic ability of free enzymes decrease with the complexity of the effluents (Zille et al., 2003). Some of these limitations are overcome by the use of enzymes in immobilized form which can be used as catalysts with long lifetime (Rogalski et al., 1995; Zille et al., 2003). Immobilization with different polymeric materials is studied for enzyme encapsulation along with their application in treatment of various pollutants (Zille et al., 2003). However, appropriate selection of encapsulation material specific to the enzyme and optimization of process conditions is still under investigation. This communication reports results pertaining to systematic evaluation of hydrogen peroxidase oxidoreductase extracted from horseradish (EC 1.11.1.7) also called as horseradish peroxidase (HRP) in the process of acid azo dye removal. Effect of parameters such as
aqueous phase pH, H2O2 and HRP concentration, contact time, repeated application of immobilized HRP and dye concentration has been investigated to optimize the system conditions. Also, evaluation of immobilized HRP (in alginate and acrylamide polymeric matrix) performance in the process of dye removal was evaluated in order to study its reusability.
2. Materials and methods 2.1. Dye Acid Black 10 BX, an acid application group of dye belonging to azo chemical was studied for HRP catalyzed experiments. Dye was gifted by M/s Atul Chemical Ltd., India. Detailed properties of the dye along with the structure were presented in Table 1. The aqueous solution of dye was prepared prior to the experiments by way of dissolving the requisite amount of dye in double distilled water. 2.2. HRP HRP was extracted from horseradish roots purchased from local vegetable market as per the procedure given by Bhunia et al. (2001). The roots after cleaning with water were crushed in a wet grinder without addition of water and the extract was centrifuged (10 000g, 6 min, 4 C). The resulting supernatant was dialyzed using 12KD membrane against 0.1 M acetate buffer (pH 4.5) at 4 C. The dialyzed enzyme extract was stored (4 C) and used in the dye removal studies.
Table 1 Characteristics of Acid Black 10 BX Name of the dye CI name CI number Chemical name Solubility Hue Dischargeability Chemical class Structure:
Acid Black 10 BX Disazo 27260 7-Amino,1,3-naphthalenedisulphonic acid Soluble in water and slightly soluble in ethanol Violet in soluble state (kmax—617 nm) Poor Di-azo HO
NaO3 S N
N
N
SO3 Na
N
NaO 3 S SO 3 Na
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2.3. Immobilization of HRP Acrylamide gel was prepared by modifying the procedure given by Benny et al. (1998). 3.25 ml of potassium phosphate buffer (0.1 M, pH 7.0) was mixed with 2.7 ml of acrylamide solution (3 g acrylamide and 0.08 g of bisacrylamide in 10 ml potassium phosphate buffer) and 80 ll of ammonium persulfate solution (10% ammonium per sulfate in potassium phosphate buffer) and the resulting mixture was mixed in 20 ml vial. Subsequently, 3 ml of HRP solution (containing 2.94 units) was added followed by 10 ll of TEMED (N,N,N,N-tetra methyle thylenediamine) reagent and the mixture was vortexed. The solution became opaque in a few minutes and complete polymerization was observed between 20 to 30 min. Gel was transferred subsequently to vacuum filter system to remove the solution and subsequently washed with phosphate buffer. Gel was broken by aspiration using a sharp knife into small equal size pieces and stored at 4 C prior to use. For alginate immobilization, 25 ml of HRP (containing 2.94 units and specific activity 0.52) was dissolved in sodium alginate solution (2%) followed by uniform stirring. The resulting mixture of alginate and enzyme was dropped through a fine nozzle to form small droplets into the 0.1 M CaCl2 solution to obtain fine and uniform size beads. Subsequently, the beads were stored at 4 C in double distilled water. 2.4. Dye removal studies Experiments were conducted to assess the HRP catalyzed removal of acid azo dye in aqueous phase by free and immobilized enzyme to determine the equilibrium time required for the dye removal. The experiments were carried out at a constant temperature (25 C) by varying the process parameters such as pH, dye concentration and HRP concentration. Initially kinetics were carried out in a series of vials (at 20 mg l 1 concentration) by keeping aqueous phase pH at 2.0, HRP concentration (2.94 units) and H2O2 dose (0.2 ll l 1) constant. The reaction mixtures in vials were kept for agitation on a horizontal shaker at 100 rpm for the requisite contact time and the solutions were analyzed for residual dye concentration in aqueous phase after centrifugation (5000g, 5 min, 24 C). Each vial was removed at a predetermined time and residual dye concentration in aqueous phase was estimated to know the optimum contact time. Subsequent series of experiments were performed by varying the aqueous phase pH (from 2 to 9), dye concentration (from 5 to 40 mg l 1) and H2O2 dose (from 0.1 to 0.8 ll l 1) to understand the optimum conditions for dye removal by keeping the agitation for the optimum contact time. Repeated application of immobilized HRP was studied by repeated use of immobilized HRP beads for dye removal (dye concentration 20 mg l 1, H2O2 dose
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0.1 ll l 1, pH 2.0, 3 g of immobilized HRP (2.79 units) and experiments of free enzyme addition were repeated and the residual dye color was estimated. The enhanced degradation of the dye in aqueous phase after HRP catalyzed reaction was assessed by adopting standard plating technique (control and enzyme treated dye on 2% agar plates). Pseudomonas putida isolated in our laboratory was used as inoculum for plating. The enzyme treated dye solution and control were used to prepare agar plates and P. putida was streaked. Plates were incubated at 30 C for 5 days prior to monitoring the growth. 2.5. Analytical assay 2.5.1. HRP activity HRP activity was assessed by employing 4-aminoantipyrene method involving calorimetric estimation using phenol and H2O2 as substrates and 4-aminoantipyrene (Am-NH2) as chromogen (Bhunia et al., 2001). The assay was performed at 25 C by adding phosphate buffer (pH 7.4) containing 1.0 · 10 2 M phenol, 2.4.0 · 10 3 M Am-NH2 and 2.0 · 10 4 M H2O2. The rate of H2O2 consumption was estimated by measuring the absorption of the colored product at 510 nm. HRP extracted from horseradish roots was found to contain 2.94 units ml 1 of the enzyme after dialysis. 2.5.2. Dye assay Quantitative estimation of the dye in the aqueous phase was carried out by colorimetry. A solution of 20 mg l 1 concentration of the dye was scanned over a wavelength range of 200–800 nm by using the UV–VIS Spectrophotometer (Bechman, USA) and optimum wavelength was determined (kmax—617 nm, absorbance—1.0212). Standard calibration curve was prepared at maximum wavelength and used for the estimation of the dye concentration in aqueous phase. After HRP treatment, the sample was centrifuged and the supernatant was assayed for the residual dye concentration. The analytical procedures were adopted from the Standard Methods (APHA, 1998). 2.5.3. HPLC analysis High performance liquid chromatography (HPLC) was employed to understand the dye removal during the enzyme catalyzed treatment. HPLC (Shimadzu LC8A) with a reverse phase column (Hypersil BDS, C 18, 250 · 4.6 mm packed with 5 lm particle size) was used for the dye estimation. The separated components were detected at 225 nm. Methanol:water in the ratio of 80:20 was used as mobile phase with a flow rate of 1 ml min 1. The control sample (dye without degradation) and sample after degradation were used for HPLC injection (10 min) after diluting with distilled water. HPLC analysis of the control sample (dye without degradation) and
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sample after degradation was carried out after diluting with the distilled water.
80 70
3.1. Studies with free HRP 3.1.1. Optimum contact time Initially experiments were performed in order to assess the optimum contact time required for the dye removal. To a series of vials containing 5 ml of dye solution (20 mg l 1), 2.94 units of enzyme, 0.2 ll l 1 of H2O2 were added and the reaction mixture (24 C, pH 2) was agitated for a period of 90 min. For every 10 min time interval, one vial was removed and analyzed for the residual dye concentration (Fig. 1). It is evident from the figure that, 45 min of the reaction time is sufficient for the maximum dye removal. After 45 min of contact time, negligible dye removal was noticed up to remaining 90 min of the contact time. Subsequent experiments were performed for 45 min of reaction time. 3.1.2. Optimum pH Enzymes have an optimum pH range at which their activity is maximum and optimum pH of any enzyme is not necessarily identical to its normal intracellular surroundings. pH optimization studies were carried out on the Acid Black 10 BX dye by varying aqueous phase pH of the reaction mixture between a pH from 2 to 9 by keeping all dye concentration (20 mg l 1), enzyme concentration (2.94 units), H2O2 dose (0.2 ll l 1), reaction temperature (24 C) and contact time (45 min) constant. Variation of dye removal at various pH values is depicted in Fig. 2. From this figure, it is observed that about 67% of the dye was found to be removed due to HRP catalyzed reaction at an aqueous phase of pH 2
70
Dye removed (%)
60 50 40 30 20 10 0 0
15
30
45
60
75
90
Dye removed (%)
60
3. Results and discussion
50 40 30 20 10 0 2
4
6
8
10
Aqueous phase pH
Fig. 2. Effect of pH on free HRP catalyzed dye removal.
with the specified experimental conditions. With increase in pH above 2, dye removal was found to drop significantly (pH from 3 to 7) and the same trend continued up to an aqueous phase of pH 9. Aqueous phase of pH 2 resulted in higher HRP activity compared to other pH ranges (3–9). 3.1.3. Optimum concentration of H2O2 Hydrogen peroxide acts as a co-substrate to activate the enzymatic action of peroxidase radical. It contributes in the catalytic cycle of peroxidase, to oxidize the native enzyme to form an enzymatic intermediate, which accepts the aromatic compound to carry out its oxidation to a free radical form. Experiments were carried out to find out the optimum H2O2 dose required to bring out the conversion of dye by varying the H2O2 dose (0.1–0.8 ll l 1) in the reaction mixture by keeping all the other experimental conditions constant (dye concentration—20 mg l 1; temperature—24 C; enzyme concentration—2.94 units; reaction time—45 min). Studies were conducted in series at two aqueous phase pH conditions (2 and 7). The results obtained were presented in a graph relating dye removal with the function of H2O2 dose (Fig. 3). From the data, it is evident that H2O2 dose of 0.6 ll l 1 was sufficient for the maximum dye degradation at the specified experimental conditions. It can also be observed that at both studied pH values, the activity of enzyme in presence of different dosages was also shown in Fig. 3. The enzyme activity was more or less same below 0.6 ll l 1 of H2O2 dose, where maximum activity was observed. Compared to aqueous phase pH of 7, pH 2 yielded more enzyme activity. It can be deduced that 0.6 ll l 1 of H2O2 was optimum for acid azo dye removal.
105
Reaction Time (min)
Fig. 1. Dye removal pattern with free HRP as a function of contact time.
3.1.4. Optimum concentrate of dye Concentration of the substrate present in the aqueous phase has significant influence on any enzyme-mediated
S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105 17
1101
2.96 Dye removal HRP activity 2.94
16 2.92 15
7 2.42 6 5
Fig. 4. Effect of dye concentration on free HRP catalyzed dye removal.
2.4 pH 7 2.38
3 2
2.36 1
Dye removal HRP activity
0
2.34 0.1
0.2
0.4
0.6
0.8
H2O2 dose Fig. 3. Effect of H2O2 dose on free HRP catalyzed dye removal.
reaction. If the amount of enzyme concentration is kept constant and the substrate concentration is gradually increased, the velocity of the reaction will increase until it reaches the maximum. After obtaining the equilibrium state any further addition of the substrate will not change the rate of reaction. Studies were carried out at different concentrations of the dye (5–40 mg l 1), keeping all the other parameters constant (H2O2—0.6 ll l 1; aqueous phase pH—2; reaction time—45 min; temperature—24 C) and the results are shown in Fig. 4. With the increase in dye concentration, the removal was found to be effective up to 30 mg l 1 of dye concentration. Subsequent increase in dye concentration above 30 mg l 1 resulted in relatively low dye removal. This may be presumed to be the cut-off concentration of the dye for the optimum removal at the specified experimental conditions.
an optimum relationship between the concentration of enzyme and substrate for achieving maximum activity. To study the effect of enzyme concentration on the reaction, the reaction must be kept independent of the substrate concentration so that any variation in the amount of product formed is a function of enzyme concentration. To study the optimum dose of HRP, experiments were carried out at various HRP doses ranging from 0.735 to 4.41 units ml 1 at specified experimental conditions (dye—20 mg l 1; pH—2; temperature— 25 C, contact time—45 min, H2O2—0.6 ll l 1) and the results are shown in Fig. 5. The enzyme dose was found to have significant influence on dye removal reaction. The increase in the HRP dose from 0.735 units ml 1 to 2.205 units ml 1 might have resulted in a gradual increase in the dye removal rates (62–84%). However,
18
8
17 7 16 6 15 14
5
13 4 12 3 11
3.1.5. Optimum dose of enzyme Normally removal of the aromatic compound is dependent on the amount of catalyst added since the catalyst has a finite lifetime and also the conversion is found to be dependent on the contact time. There is
Dye removal Dye remaining
10
2 0.735
1.47
2.205
2.94
3.675
4.41
HRP dose (units)
Fig. 5. Effect of free HRP dose on dye removal.
Dye remaining (mg l-1)
4
Dye removal (mg l-1)
Dye removal (%)
2.44
8
HRP activity (units)
2.9
pH 2
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subsequent increase in HRP dose up to 4.41 units ml 1 might have yielded significantly low impact on HRP dye catalyzed reaction (0.5%). It can be presumed that the enzyme dose of 2.205 units ml 1 was found to be the optimum dose for maximum dye removal at specified experimental conditions. 3.1.6. Effect of repeated application of free HRP and H2O2 Repeated application of HRP along with co-activator (H2O2) is having significant effect on the overall enzyme catalyzed reaction. In order to find out the effect of repeated addition of HRP and H2O2 alone and in combination, several studies were performed and the results obtained were depicted in Fig. 6. From this figure, it can be visualized that the addition of enzyme, H2O2 and HRP combined with H2O2 has resulted in effective performance throughout. On the contrary, second addition of H2O2 dose resulted in low dye removal. However, HRP alone comparatively showed effective removal of dye. This can be due to the residual H2O2 present in solution, which might have resulted in the simulation of the HRP activity. The relative low initial dye removal with H2O2 and subsequent increase may be reasoned due to the radical mediated oxidation of the resulting product from enzyme catalyzed reaction. 3.2. Studies with immobilized HRP Application of free enzyme in industrial processes is not economically viable, while immobilization/entrapment of enzyme results in repeated application and is more economical. In the present study, two types of poly80 a
70
c
b
d
Dye removal (mg l-1)
60 50 40 30 20 10 0
0
30
60
90
Time (min)
Fig. 6. Variation of dye removal with repeated application of free HRP and H2O2 (a—no further addition of reactants; b— addition of H2O2; c—addition of enzyme; d—addition of H2O2 + enzyme).
90 Acrylamide gel beads Alginate beads
80 70 Dye removed (%)
1102
60 50 40 30 20 10 0 0
15
30
45
60
75
90
Contact time (min)
Fig. 7. Dye removal pattern with immobilized HRP as a function of contact time.
meric materials viz. alginate and acrylamide for the entrapment of peroxidase have been used in order to study their relative efficiency in dye removal. Experiments were carried out separately with both entrapped HRP at a dye concentration of 20 mg l 1 (H2O2— 0.6 ll l 1; pH—2.0; temperature—25 C, contact time— 45 min). The results are shown in Fig. 7. It can be observed from Fig. 7 that acrylamide gel was more efficient in dye removal when compared to alginate matrix. About 79% of dye removal was observed with acrylamide gel immobilized beads, while only 54% of dye removal was found with alginate matrix. Gel immobilized HRP resulted in effective dye removal when compared to free HRP (67%), while alginate immobilized HRP showed inferior performance compared to the free HRP. Normally, the enzyme immobilization is expected to provide stabilization effect (Rogalski et al., 1995) restricting the protein unfolding process as a result of the introduction of random intra and intermolecular cross-links. Zille et al. (2003) reported less availability of the enzyme for interaction with anionic dyes due to the immobilization in a particular matrix. The objective of the immobilization is the reusability of the matrix in the process. Therefore investigations were carried out to assess repeated usability of entrapped HRP beads for dye removal. The results obtained are shown in Fig. 8. In case of acrylamide entrapped HRP, repeated applications resulted in 10% reduction in the dye removal capacity for second application. Subsequent application resulted in 26%, 39% and 50% in the dye removal efficiency up to the fifth application. In the case of alginate HRP beads, second application resulted in 23% of reduction in dye removal and subsequent application resulted in 3% of consistent reduction in dye removal efficiency.
S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105
1103
90 80 70
Dye removal (%)
60 50 40 30 20 10
Fig. 8. Dye removal pattern at 20 mg l cation of immobilized HRP.
1
with repeated appli-
Effect of aqueous phase pH on the enzyme catalyzed degradation with immobilized HRP was studied (Fig. 9). The results obtained showed that increase in pH resulted in decrease in the dye removal capacity for the both types of the entrapped matrices studied. About 78% and 68% of dye removal was observed at an aqueous phase of pH 2 for acrylamide and alginate entrapped beads, respectively. This observation correlates well with the performance of free HRP reported in this paper. The relatively poor performance of alginate immobilized
0 2
3
4
5
6
7
Aqueous phase pH
Fig. 9. Effect of pH on immobilized HRP catalyzed dye removal.
HRP compared to acrylamide may be reasoned due to the less availability of the peroxidase structure to the dye molecule in alginate matrix compared to acrylamide. The effective performance of acrylamide entrapped beads may be also attributed to the non-ionic nature of the beads which results in minimum modification of the enzyme properties and unaffected nature of the charged substrate as well as product diffusion.
Fig. 10. HPLC Chromatograph (a—control [dye], b—sample [HRP treated dye]).
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3.3. Monitoring of dye degradation by HPLC The degradation of the dye was monitored by HPLC. HPLC profile of the control sample (a) showed a peak at a retention time of 2.13 (Fig. 10). After HRP treatment, the HPLC profile of the dye shifted the retention time of the peak along with the formation of two additional peaks at 2.67 and 3.21 min (b) indicating the possible breakdown of the parent molecule. Comparison of HPLC chromatogram of enzyme treated sample with control, showed 65% of enhanced dye degradation due to the HRP catalyzed treatment. 3.4. Biodegradation studies Enhanced dye degradation due to HRP catalyzed reaction was assessed by standard plating technique using P. putida. The inoculum was grown on plates with nutrient agar (2%) along with dye as a single carbon source (control-untreated and HRP treated) to understand the relatively enhanced degradation. The plates were incubated at 30 C for 5 days. After incubation, growth was continuously monitored up to 10 days. In enzyme treated plates, formation of colonies was observed after the 2nd day of incubation and subsequently profuse growth of colonies were seen. In case of the control plates, growth was seen only after 6 days of incubation. This observation correlates with the fact that enhanced degradation was observed due to HRP catalyzed treatment in the treated samples compared to the control dye plates due to inhibition (colony formation seen after long incubation period).
4. Conclusions The experimental results obtained in the present work revealed the effectiveness of the peroxidase catalyzed enzymatic reaction in the treatment of an acid azo dye in aqueous phase. However, the performance of HRP catalyzed reaction for dye removal was found to be dependent upon the reaction time, dye concentration, enzyme concentration, H2O2 dose and aqueous phase pH. Performance of free HRP verses immobilized HRP (alginate and acrylamide polymeric matrix) was evaluated in the process of dye removal in order to assess the reusability of HRP. Immobilized HRP in acrylamide matrix resulted in effective performance over the free HRP, while alginate entrapped HRP yielded inferior performance over the free one. Repeated application of enzyme was observed to be feasible with immobilized HRP beads. Standard plating studies revealed the enhanced biodegradability of the enzyme treated dye compared to the control. On the whole, the HRP catalyzed treatment seems to be effective for
enhancing biodegradability of recalcitrant azo dye effluents.
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Acid azo dye degradation by free and immobilized horseradish peroxidase (HRP) catalyzed process S. Venkata Mohan, K. Krishna Prasad, N. Chandrasekhara Rao, P.N. Sarma
*
Biochemical and Environmental Engineering Centre, Indian Institute of Chemical Technology, Hyderabad 500 007, India Received 11 June 2003; received in revised form 6 September 2004; accepted 15 September 2004
Abstract Acid azo (Acid Black 10 BX) dye removal by plant based peroxidase catalyzed reaction was investigated. Horseradish peroxidase (HRP) was extracted from horseradish roots and its performance was evaluated in both free and immobilized form. HRP showed its ability to degrade the dye in aqueous phase. Studies are further carried out to understand the process parameters such as aqueous phase pH, H2O2 dose, dye and enzyme concentrations during enzyme-mediated dye degradation process. Experimental data revealed that dye (substrate) concentration, aqueous phase pH, enzyme and H2O2 dose play a significant role on the overall enzyme-mediated reaction. Acrylamide gel immobilized HRP showed effective performance compared to free HRP and alginate entrapped HRP. Alginate entrapped HRP showed inferior performance over the free enzyme due to the consequence of non-availability of the enzyme to the dye molecule due to polymeric immobilization. Standard plating studies performed with Pseudomonas putida showed enhanced degradation of HRP catalyzed dye compared to control. 2004 Elsevier Ltd. All rights reserved. Keywords: Horseradish peroxidase; Azo dye; H2O2; pH; Enzyme activity; Immobilization; Alginate; Acrylamide
1. Introduction Dyes are complex aromatic compounds, which are normally used for coloration of various substrates. They are sometimes fused with heavy metals on the structural interface and are considered to have relatively bad consequence on the surrounding environment due to its toxic and inhibitory nature (Correia et al., 1994; Stolz, 2001; Venkata Mohan et al., 2002a,b). Among all the
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Corresponding author. Fax: +91 40 27193626. E-mail addresses: [email protected], kousik@iict. ap.nic.in (P.N. Sarma).
chemical classes of dyes, azo dyes are considered to be recalcitrant, non-biodegradable and persistent. Treatment of dye based effluents is considered to be one of the challenging tasks in environmental fraternity. Even though physico-chemical methods are effective in the removal of dyes, the overall cost, regeneration problem, secondary pollutant/sludge generation limits their usage (Venkata Mohan and Karthikeyan, 1999). Also, dye based effluents are normally not amenable for conventional biological wastewater treatment due to their recalcitrant and inhibitory nature (Kulla, 1981). However, microbial methods are highly useful and potentially advantageous for the treatment of toxic compounds due to their effectiveness, ecofriendly nature, energy saving and less usage of chemicals (Koller et al., 2000). Researchers have been focusing their attention to study
0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.09.070
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enzymatic pretreatment as a potential and viable alternative to conventional methods, due to its highly zselective nature. Further, inhibition by toxic substances is minimum in enzymatic treatment and the process can operate over a broad aromatic concentration range with low retention time (Kasam and Niceu, 1997). Enzymes can act on specific recalcitrant pollutants to remove them by precipitation or transformation to other (innocuous) products and also can change the characteristics of a given waste to render it more amenable for treatment. The catalytic action of enzymes is extremely efficient and selective compared to chemical catalysts due to higher reaction rates, milder reaction conditions and greater stereospecificity. They can catalyze reactions at relatively low temperature and in the entire aqueous phase pH range. Though much attention has been paid in the utilization of biocatalysts in several fields, their involvement has been felt very recently in solving the environmental problems (Kasam and Niceu, 1997; Venkata Mohan et al., 2002c). Extracellular fungal peroxidases are reported to oxidatively catalyze the polymerization of toxic aromatic compounds in aqueous solution and are reported to oxidize various pollutants (Hammel and Tardone, 1988; Dec and Bollag, 1990; Valli and Gold, 1991; Arseguel and Baboulene, 1994; Nicell, 1994; Manimekalai and Swaminathan, 2000). Enzymes from various sources (fungus and plant based) are applied for the treatment of dye based compounds (Novotny et al., 2001). The source of the selected enzyme and its nature along with system conditions are found to have significant influence on the overall performance for pollutant removal. Fungal extracted enzymes are studied quite significantly in the process of dye removal (Glenn and Gold, 1983; Manimekalai and Swaminathan, 2000; Novotny et al., 2001; Maximo and Costa-Ferreira, 2004; Hou et al., 2004). Relatively, plant based peroxidases in the removal of pollutants are less documented (Nicell, 1994; Koller et al., 2000; Bhunia et al., 2001). Several limitations prevent the use of free enzymes as the stability and catalytic ability of free enzymes decrease with the complexity of the effluents (Zille et al., 2003). Some of these limitations are overcome by the use of enzymes in immobilized form which can be used as catalysts with long lifetime (Rogalski et al., 1995; Zille et al., 2003). Immobilization with different polymeric materials is studied for enzyme encapsulation along with their application in treatment of various pollutants (Zille et al., 2003). However, appropriate selection of encapsulation material specific to the enzyme and optimization of process conditions is still under investigation. This communication reports results pertaining to systematic evaluation of hydrogen peroxidase oxidoreductase extracted from horseradish (EC 1.11.1.7) also called as horseradish peroxidase (HRP) in the process of acid azo dye removal. Effect of parameters such as
aqueous phase pH, H2O2 and HRP concentration, contact time, repeated application of immobilized HRP and dye concentration has been investigated to optimize the system conditions. Also, evaluation of immobilized HRP (in alginate and acrylamide polymeric matrix) performance in the process of dye removal was evaluated in order to study its reusability.
2. Materials and methods 2.1. Dye Acid Black 10 BX, an acid application group of dye belonging to azo chemical was studied for HRP catalyzed experiments. Dye was gifted by M/s Atul Chemical Ltd., India. Detailed properties of the dye along with the structure were presented in Table 1. The aqueous solution of dye was prepared prior to the experiments by way of dissolving the requisite amount of dye in double distilled water. 2.2. HRP HRP was extracted from horseradish roots purchased from local vegetable market as per the procedure given by Bhunia et al. (2001). The roots after cleaning with water were crushed in a wet grinder without addition of water and the extract was centrifuged (10 000g, 6 min, 4 C). The resulting supernatant was dialyzed using 12KD membrane against 0.1 M acetate buffer (pH 4.5) at 4 C. The dialyzed enzyme extract was stored (4 C) and used in the dye removal studies.
Table 1 Characteristics of Acid Black 10 BX Name of the dye CI name CI number Chemical name Solubility Hue Dischargeability Chemical class Structure:
Acid Black 10 BX Disazo 27260 7-Amino,1,3-naphthalenedisulphonic acid Soluble in water and slightly soluble in ethanol Violet in soluble state (kmax—617 nm) Poor Di-azo HO
NaO3 S N
N
N
SO3 Na
N
NaO 3 S SO 3 Na
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2.3. Immobilization of HRP Acrylamide gel was prepared by modifying the procedure given by Benny et al. (1998). 3.25 ml of potassium phosphate buffer (0.1 M, pH 7.0) was mixed with 2.7 ml of acrylamide solution (3 g acrylamide and 0.08 g of bisacrylamide in 10 ml potassium phosphate buffer) and 80 ll of ammonium persulfate solution (10% ammonium per sulfate in potassium phosphate buffer) and the resulting mixture was mixed in 20 ml vial. Subsequently, 3 ml of HRP solution (containing 2.94 units) was added followed by 10 ll of TEMED (N,N,N,N-tetra methyle thylenediamine) reagent and the mixture was vortexed. The solution became opaque in a few minutes and complete polymerization was observed between 20 to 30 min. Gel was transferred subsequently to vacuum filter system to remove the solution and subsequently washed with phosphate buffer. Gel was broken by aspiration using a sharp knife into small equal size pieces and stored at 4 C prior to use. For alginate immobilization, 25 ml of HRP (containing 2.94 units and specific activity 0.52) was dissolved in sodium alginate solution (2%) followed by uniform stirring. The resulting mixture of alginate and enzyme was dropped through a fine nozzle to form small droplets into the 0.1 M CaCl2 solution to obtain fine and uniform size beads. Subsequently, the beads were stored at 4 C in double distilled water. 2.4. Dye removal studies Experiments were conducted to assess the HRP catalyzed removal of acid azo dye in aqueous phase by free and immobilized enzyme to determine the equilibrium time required for the dye removal. The experiments were carried out at a constant temperature (25 C) by varying the process parameters such as pH, dye concentration and HRP concentration. Initially kinetics were carried out in a series of vials (at 20 mg l 1 concentration) by keeping aqueous phase pH at 2.0, HRP concentration (2.94 units) and H2O2 dose (0.2 ll l 1) constant. The reaction mixtures in vials were kept for agitation on a horizontal shaker at 100 rpm for the requisite contact time and the solutions were analyzed for residual dye concentration in aqueous phase after centrifugation (5000g, 5 min, 24 C). Each vial was removed at a predetermined time and residual dye concentration in aqueous phase was estimated to know the optimum contact time. Subsequent series of experiments were performed by varying the aqueous phase pH (from 2 to 9), dye concentration (from 5 to 40 mg l 1) and H2O2 dose (from 0.1 to 0.8 ll l 1) to understand the optimum conditions for dye removal by keeping the agitation for the optimum contact time. Repeated application of immobilized HRP was studied by repeated use of immobilized HRP beads for dye removal (dye concentration 20 mg l 1, H2O2 dose
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0.1 ll l 1, pH 2.0, 3 g of immobilized HRP (2.79 units) and experiments of free enzyme addition were repeated and the residual dye color was estimated. The enhanced degradation of the dye in aqueous phase after HRP catalyzed reaction was assessed by adopting standard plating technique (control and enzyme treated dye on 2% agar plates). Pseudomonas putida isolated in our laboratory was used as inoculum for plating. The enzyme treated dye solution and control were used to prepare agar plates and P. putida was streaked. Plates were incubated at 30 C for 5 days prior to monitoring the growth. 2.5. Analytical assay 2.5.1. HRP activity HRP activity was assessed by employing 4-aminoantipyrene method involving calorimetric estimation using phenol and H2O2 as substrates and 4-aminoantipyrene (Am-NH2) as chromogen (Bhunia et al., 2001). The assay was performed at 25 C by adding phosphate buffer (pH 7.4) containing 1.0 · 10 2 M phenol, 2.4.0 · 10 3 M Am-NH2 and 2.0 · 10 4 M H2O2. The rate of H2O2 consumption was estimated by measuring the absorption of the colored product at 510 nm. HRP extracted from horseradish roots was found to contain 2.94 units ml 1 of the enzyme after dialysis. 2.5.2. Dye assay Quantitative estimation of the dye in the aqueous phase was carried out by colorimetry. A solution of 20 mg l 1 concentration of the dye was scanned over a wavelength range of 200–800 nm by using the UV–VIS Spectrophotometer (Bechman, USA) and optimum wavelength was determined (kmax—617 nm, absorbance—1.0212). Standard calibration curve was prepared at maximum wavelength and used for the estimation of the dye concentration in aqueous phase. After HRP treatment, the sample was centrifuged and the supernatant was assayed for the residual dye concentration. The analytical procedures were adopted from the Standard Methods (APHA, 1998). 2.5.3. HPLC analysis High performance liquid chromatography (HPLC) was employed to understand the dye removal during the enzyme catalyzed treatment. HPLC (Shimadzu LC8A) with a reverse phase column (Hypersil BDS, C 18, 250 · 4.6 mm packed with 5 lm particle size) was used for the dye estimation. The separated components were detected at 225 nm. Methanol:water in the ratio of 80:20 was used as mobile phase with a flow rate of 1 ml min 1. The control sample (dye without degradation) and sample after degradation were used for HPLC injection (10 min) after diluting with distilled water. HPLC analysis of the control sample (dye without degradation) and
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sample after degradation was carried out after diluting with the distilled water.
80 70
3.1. Studies with free HRP 3.1.1. Optimum contact time Initially experiments were performed in order to assess the optimum contact time required for the dye removal. To a series of vials containing 5 ml of dye solution (20 mg l 1), 2.94 units of enzyme, 0.2 ll l 1 of H2O2 were added and the reaction mixture (24 C, pH 2) was agitated for a period of 90 min. For every 10 min time interval, one vial was removed and analyzed for the residual dye concentration (Fig. 1). It is evident from the figure that, 45 min of the reaction time is sufficient for the maximum dye removal. After 45 min of contact time, negligible dye removal was noticed up to remaining 90 min of the contact time. Subsequent experiments were performed for 45 min of reaction time. 3.1.2. Optimum pH Enzymes have an optimum pH range at which their activity is maximum and optimum pH of any enzyme is not necessarily identical to its normal intracellular surroundings. pH optimization studies were carried out on the Acid Black 10 BX dye by varying aqueous phase pH of the reaction mixture between a pH from 2 to 9 by keeping all dye concentration (20 mg l 1), enzyme concentration (2.94 units), H2O2 dose (0.2 ll l 1), reaction temperature (24 C) and contact time (45 min) constant. Variation of dye removal at various pH values is depicted in Fig. 2. From this figure, it is observed that about 67% of the dye was found to be removed due to HRP catalyzed reaction at an aqueous phase of pH 2
70
Dye removed (%)
60 50 40 30 20 10 0 0
15
30
45
60
75
90
Dye removed (%)
60
3. Results and discussion
50 40 30 20 10 0 2
4
6
8
10
Aqueous phase pH
Fig. 2. Effect of pH on free HRP catalyzed dye removal.
with the specified experimental conditions. With increase in pH above 2, dye removal was found to drop significantly (pH from 3 to 7) and the same trend continued up to an aqueous phase of pH 9. Aqueous phase of pH 2 resulted in higher HRP activity compared to other pH ranges (3–9). 3.1.3. Optimum concentration of H2O2 Hydrogen peroxide acts as a co-substrate to activate the enzymatic action of peroxidase radical. It contributes in the catalytic cycle of peroxidase, to oxidize the native enzyme to form an enzymatic intermediate, which accepts the aromatic compound to carry out its oxidation to a free radical form. Experiments were carried out to find out the optimum H2O2 dose required to bring out the conversion of dye by varying the H2O2 dose (0.1–0.8 ll l 1) in the reaction mixture by keeping all the other experimental conditions constant (dye concentration—20 mg l 1; temperature—24 C; enzyme concentration—2.94 units; reaction time—45 min). Studies were conducted in series at two aqueous phase pH conditions (2 and 7). The results obtained were presented in a graph relating dye removal with the function of H2O2 dose (Fig. 3). From the data, it is evident that H2O2 dose of 0.6 ll l 1 was sufficient for the maximum dye degradation at the specified experimental conditions. It can also be observed that at both studied pH values, the activity of enzyme in presence of different dosages was also shown in Fig. 3. The enzyme activity was more or less same below 0.6 ll l 1 of H2O2 dose, where maximum activity was observed. Compared to aqueous phase pH of 7, pH 2 yielded more enzyme activity. It can be deduced that 0.6 ll l 1 of H2O2 was optimum for acid azo dye removal.
105
Reaction Time (min)
Fig. 1. Dye removal pattern with free HRP as a function of contact time.
3.1.4. Optimum concentrate of dye Concentration of the substrate present in the aqueous phase has significant influence on any enzyme-mediated
S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105 17
1101
2.96 Dye removal HRP activity 2.94
16 2.92 15
7 2.42 6 5
Fig. 4. Effect of dye concentration on free HRP catalyzed dye removal.
2.4 pH 7 2.38
3 2
2.36 1
Dye removal HRP activity
0
2.34 0.1
0.2
0.4
0.6
0.8
H2O2 dose Fig. 3. Effect of H2O2 dose on free HRP catalyzed dye removal.
reaction. If the amount of enzyme concentration is kept constant and the substrate concentration is gradually increased, the velocity of the reaction will increase until it reaches the maximum. After obtaining the equilibrium state any further addition of the substrate will not change the rate of reaction. Studies were carried out at different concentrations of the dye (5–40 mg l 1), keeping all the other parameters constant (H2O2—0.6 ll l 1; aqueous phase pH—2; reaction time—45 min; temperature—24 C) and the results are shown in Fig. 4. With the increase in dye concentration, the removal was found to be effective up to 30 mg l 1 of dye concentration. Subsequent increase in dye concentration above 30 mg l 1 resulted in relatively low dye removal. This may be presumed to be the cut-off concentration of the dye for the optimum removal at the specified experimental conditions.
an optimum relationship between the concentration of enzyme and substrate for achieving maximum activity. To study the effect of enzyme concentration on the reaction, the reaction must be kept independent of the substrate concentration so that any variation in the amount of product formed is a function of enzyme concentration. To study the optimum dose of HRP, experiments were carried out at various HRP doses ranging from 0.735 to 4.41 units ml 1 at specified experimental conditions (dye—20 mg l 1; pH—2; temperature— 25 C, contact time—45 min, H2O2—0.6 ll l 1) and the results are shown in Fig. 5. The enzyme dose was found to have significant influence on dye removal reaction. The increase in the HRP dose from 0.735 units ml 1 to 2.205 units ml 1 might have resulted in a gradual increase in the dye removal rates (62–84%). However,
18
8
17 7 16 6 15 14
5
13 4 12 3 11
3.1.5. Optimum dose of enzyme Normally removal of the aromatic compound is dependent on the amount of catalyst added since the catalyst has a finite lifetime and also the conversion is found to be dependent on the contact time. There is
Dye removal Dye remaining
10
2 0.735
1.47
2.205
2.94
3.675
4.41
HRP dose (units)
Fig. 5. Effect of free HRP dose on dye removal.
Dye remaining (mg l-1)
4
Dye removal (mg l-1)
Dye removal (%)
2.44
8
HRP activity (units)
2.9
pH 2
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subsequent increase in HRP dose up to 4.41 units ml 1 might have yielded significantly low impact on HRP dye catalyzed reaction (0.5%). It can be presumed that the enzyme dose of 2.205 units ml 1 was found to be the optimum dose for maximum dye removal at specified experimental conditions. 3.1.6. Effect of repeated application of free HRP and H2O2 Repeated application of HRP along with co-activator (H2O2) is having significant effect on the overall enzyme catalyzed reaction. In order to find out the effect of repeated addition of HRP and H2O2 alone and in combination, several studies were performed and the results obtained were depicted in Fig. 6. From this figure, it can be visualized that the addition of enzyme, H2O2 and HRP combined with H2O2 has resulted in effective performance throughout. On the contrary, second addition of H2O2 dose resulted in low dye removal. However, HRP alone comparatively showed effective removal of dye. This can be due to the residual H2O2 present in solution, which might have resulted in the simulation of the HRP activity. The relative low initial dye removal with H2O2 and subsequent increase may be reasoned due to the radical mediated oxidation of the resulting product from enzyme catalyzed reaction. 3.2. Studies with immobilized HRP Application of free enzyme in industrial processes is not economically viable, while immobilization/entrapment of enzyme results in repeated application and is more economical. In the present study, two types of poly80 a
70
c
b
d
Dye removal (mg l-1)
60 50 40 30 20 10 0
0
30
60
90
Time (min)
Fig. 6. Variation of dye removal with repeated application of free HRP and H2O2 (a—no further addition of reactants; b— addition of H2O2; c—addition of enzyme; d—addition of H2O2 + enzyme).
90 Acrylamide gel beads Alginate beads
80 70 Dye removed (%)
1102
60 50 40 30 20 10 0 0
15
30
45
60
75
90
Contact time (min)
Fig. 7. Dye removal pattern with immobilized HRP as a function of contact time.
meric materials viz. alginate and acrylamide for the entrapment of peroxidase have been used in order to study their relative efficiency in dye removal. Experiments were carried out separately with both entrapped HRP at a dye concentration of 20 mg l 1 (H2O2— 0.6 ll l 1; pH—2.0; temperature—25 C, contact time— 45 min). The results are shown in Fig. 7. It can be observed from Fig. 7 that acrylamide gel was more efficient in dye removal when compared to alginate matrix. About 79% of dye removal was observed with acrylamide gel immobilized beads, while only 54% of dye removal was found with alginate matrix. Gel immobilized HRP resulted in effective dye removal when compared to free HRP (67%), while alginate immobilized HRP showed inferior performance compared to the free HRP. Normally, the enzyme immobilization is expected to provide stabilization effect (Rogalski et al., 1995) restricting the protein unfolding process as a result of the introduction of random intra and intermolecular cross-links. Zille et al. (2003) reported less availability of the enzyme for interaction with anionic dyes due to the immobilization in a particular matrix. The objective of the immobilization is the reusability of the matrix in the process. Therefore investigations were carried out to assess repeated usability of entrapped HRP beads for dye removal. The results obtained are shown in Fig. 8. In case of acrylamide entrapped HRP, repeated applications resulted in 10% reduction in the dye removal capacity for second application. Subsequent application resulted in 26%, 39% and 50% in the dye removal efficiency up to the fifth application. In the case of alginate HRP beads, second application resulted in 23% of reduction in dye removal and subsequent application resulted in 3% of consistent reduction in dye removal efficiency.
S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105
1103
90 80 70
Dye removal (%)
60 50 40 30 20 10
Fig. 8. Dye removal pattern at 20 mg l cation of immobilized HRP.
1
with repeated appli-
Effect of aqueous phase pH on the enzyme catalyzed degradation with immobilized HRP was studied (Fig. 9). The results obtained showed that increase in pH resulted in decrease in the dye removal capacity for the both types of the entrapped matrices studied. About 78% and 68% of dye removal was observed at an aqueous phase of pH 2 for acrylamide and alginate entrapped beads, respectively. This observation correlates well with the performance of free HRP reported in this paper. The relatively poor performance of alginate immobilized
0 2
3
4
5
6
7
Aqueous phase pH
Fig. 9. Effect of pH on immobilized HRP catalyzed dye removal.
HRP compared to acrylamide may be reasoned due to the less availability of the peroxidase structure to the dye molecule in alginate matrix compared to acrylamide. The effective performance of acrylamide entrapped beads may be also attributed to the non-ionic nature of the beads which results in minimum modification of the enzyme properties and unaffected nature of the charged substrate as well as product diffusion.
Fig. 10. HPLC Chromatograph (a—control [dye], b—sample [HRP treated dye]).
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3.3. Monitoring of dye degradation by HPLC The degradation of the dye was monitored by HPLC. HPLC profile of the control sample (a) showed a peak at a retention time of 2.13 (Fig. 10). After HRP treatment, the HPLC profile of the dye shifted the retention time of the peak along with the formation of two additional peaks at 2.67 and 3.21 min (b) indicating the possible breakdown of the parent molecule. Comparison of HPLC chromatogram of enzyme treated sample with control, showed 65% of enhanced dye degradation due to the HRP catalyzed treatment. 3.4. Biodegradation studies Enhanced dye degradation due to HRP catalyzed reaction was assessed by standard plating technique using P. putida. The inoculum was grown on plates with nutrient agar (2%) along with dye as a single carbon source (control-untreated and HRP treated) to understand the relatively enhanced degradation. The plates were incubated at 30 C for 5 days. After incubation, growth was continuously monitored up to 10 days. In enzyme treated plates, formation of colonies was observed after the 2nd day of incubation and subsequently profuse growth of colonies were seen. In case of the control plates, growth was seen only after 6 days of incubation. This observation correlates with the fact that enhanced degradation was observed due to HRP catalyzed treatment in the treated samples compared to the control dye plates due to inhibition (colony formation seen after long incubation period).
4. Conclusions The experimental results obtained in the present work revealed the effectiveness of the peroxidase catalyzed enzymatic reaction in the treatment of an acid azo dye in aqueous phase. However, the performance of HRP catalyzed reaction for dye removal was found to be dependent upon the reaction time, dye concentration, enzyme concentration, H2O2 dose and aqueous phase pH. Performance of free HRP verses immobilized HRP (alginate and acrylamide polymeric matrix) was evaluated in the process of dye removal in order to assess the reusability of HRP. Immobilized HRP in acrylamide matrix resulted in effective performance over the free HRP, while alginate entrapped HRP yielded inferior performance over the free one. Repeated application of enzyme was observed to be feasible with immobilized HRP beads. Standard plating studies revealed the enhanced biodegradability of the enzyme treated dye compared to the control. On the whole, the HRP catalyzed treatment seems to be effective for
enhancing biodegradability of recalcitrant azo dye effluents.
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