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 horseradishperoxidase (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. Horserad-

ish peroxidase (HRP) was extracted from horseradish roots and its performance was evaluated in both free and immo-

bilized 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 de-

gradation 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 con-

sequence 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

0045-6535/$ - see front matter � 2004 Elsevier Ltd. All rights reserv

doi:10.1016/j.chemosphere.2004.09.070

* 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. Treat-

ment 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 re-

moval 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 conven-

tional biological wastewater treatment due to their recal-

citrant 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 sav-

ing and less usage of chemicals (Koller et al., 2000).

Researchers have been focusing their attention to study

ed.

Table 1

Characteristics of Acid Black 10 BX

Name of the dye Acid Black 10 BX

CI name Disazo

CI number 27260

Chemical name 7-Amino,1,3-naphthalenedisulphonic acid

Solubility Soluble in water and slightly

soluble in ethanol

Hue Violet in soluble state (kmax—617nm)

Dischargeability Poor

Chemical class Di-azo

Structure:

N N

NaO3S

NaO 3S

N N

SO3NaHO

SO3Na

1098 S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105

enzymatic pretreatment as a potential and viable alter-

native 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 (innoc-

uous) products and also can change the characteristics

of a given waste to render it more amenable for treat-

ment. The catalytic action of enzymes is extremely effi-

cient 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 oxi-

datively catalyze the polymerization of toxic aromatic

compounds in aqueous solution and are reported to oxi-

dize 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. Fun-

gal 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 re-

moval 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 de-

crease 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 encapsu-

lation material specific to the enzyme and optimization

of process conditions is still under investigation.

This communication reports results pertaining to sys-

tematic evaluation of hydrogen peroxidase oxidoreduc-

tase 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, con-

tact 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) per-

formance 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 cata-

lyzed 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 solu-

tion 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 pur-

chased from local vegetable market as per the proce-

dure 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 centri-

fuged (10000g, 6min, 4 �C). The resulting supernatant

was dialyzed using 12KD membrane against 0.1M

acetate buffer (pH4.5) at 4 �C. The dialyzed enzyme

extract was stored (4 �C) and used in the dye removal

studies.

S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105 1099

2.3. Immobilization of HRP

Acrylamide gel was prepared by modifying the proce-

dure given by Benny et al. (1998). 3.25ml of potassium

phosphate buffer (0.1M, pH7.0) was mixed with 2.7ml

of acrylamide solution (3g acrylamide and 0.08g of bis-

acrylamide in 10ml potassium phosphate buffer) and

80ll of ammonium persulfate solution (10% ammonium

per sulfate in potassium phosphate buffer) and the

resulting mixture was mixed in 20ml vial. Subsequently,

3ml of HRP solution (containing 2.94units) was added

followed by 10ll of TEMED (N,N,N,N-tetra methyle

thylenediamine) reagent and the mixture was vortexed.

The solution became opaque in a few minutes and com-

plete polymerization was observed between 20 to 30min.

Gel was transferred subsequently to vacuum filter sys-

tem 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, 25ml of HRP (contain-

ing 2.94units and specific activity 0.52) was dissolved in

sodium alginate solution (2%) followed by uniform stir-

ring. The resulting mixture of alginate and enzyme was

dropped through a fine nozzle to form small droplets

into the 0.1M CaCl2 solution to obtain fine and uniform

size beads. Subsequently, the beads were stored at 4 �Cin double distilled water.

2.4. Dye removal studies

Experiments were conducted to assess the HRP cata-

lyzed 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 20mgl�1 concentration) by

keeping aqueous phase pH at 2.0, HRP concentration

(2.94units) and H2O2 dose (0.2ll l�1) constant. The

reaction mixtures in vials were kept for agitation on a

horizontal shaker at 100rpm for the requisite contact

time and the solutions were analyzed for residual dye

concentration in aqueous phase after centrifugation

(5000g, 5min, 24 �C). Each vial was removed at a prede-

termined 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 concen-

tration (from 5 to 40mgl�1) and H2O2 dose (from 0.1 to

0.8ll l�1) to understand the optimum conditions for dye

removal by keeping the agitation for the optimum con-

tact time. Repeated application of immobilized HRP

was studied by repeated use of immobilized HRP beads

for dye removal (dye concentration 20mgl�1, H2O2 dose

0.1ll l�1, pH2.0, 3g of immobilized HRP (2.79units)

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 en-

zyme treated dye on 2% agar plates). Pseudomonas put-

ida 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 5days prior

to monitoring the growth.

2.5. Analytical assay

2.5.1. HRP activity

HRP activity was assessed by employing 4-amino-

antipyrene method involving calorimetric estimation

using phenol and H2O2 as substrates and 4-aminoanti-

pyrene (Am-NH2) as chromogen (Bhunia et al., 2001).

The assay was performed at 25 �C by adding phosphate

buffer (pH7.4) containing 1.0 · 10�2M phenol, 2.4.0 ·10�3M Am-NH2 and 2.0 · 10�4M H2O2. The rate of

H2O2 consumption was estimated by measuring the

absorption of the colored product at 510nm. HRP ex-

tracted from horseradish roots was found to contain

2.94unitsml�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

20mgl�1 concentration of the dye was scanned over a

wavelength range of 200–800nm by using the UV–VIS

Spectrophotometer (Bechman, USA) and optimum

wavelength was determined (kmax—617nm, absor-

bance—1.0212). Standard calibration curve was pre-

pared 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 concen-

tration. 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 LC-

8A) with a reverse phase column (Hypersil BDS, C 18,

250 · 4.6mm packed with 5lm particle size) was used

for the dye estimation. The separated components were

detected at 225nm. Methanol:water in the ratio of 80:20

was used as mobile phase with a flow rate of 1mlmin�1.

The control sample (dye without degradation) and sam-

ple after degradation were used for HPLC injection

(10min) after diluting with distilled water. HPLC analy-

sis of the control sample (dye without degradation) and

80

1100 S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105

sample after degradation was carried out after diluting

with the distilled water.

0

10

20

30

40

50

60

70

2 4 6 8 10

Aqueous phase pH

Dye

rem

oved

(%

)

Fig. 2. Effect of pH on free HRP catalyzed dye removal.

3. Results and discussion

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 5ml of dye solu-

tion (20mgl�1), 2.94units of enzyme, 0.2ll l�1 of H2O2

were added and the reaction mixture (24�C, pH2) was

agitated for a period of 90min. For every 10min time

interval, one vial was removed and analyzed for the

residual dye concentration (Fig. 1). It is evident from

the figure that, 45min of the reaction time is sufficient

for the maximum dye removal. After 45min of contact

time, negligible dye removal was noticed up to remain-

ing 90min of the contact time. Subsequent experiments

were performed for 45min 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 sur-

roundings. 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 (20mgl�1), enzyme con-

centration (2.94units), H2O2 dose (0.2ll l�1), reaction

temperature (24 �C) and contact time (45min) constant.

Variation of dye removal at various pH values is de-

picted 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 pH2

0

10

20

30

40

50

60

70

0 15 30 45 60 75 90 105

Reaction Time (min)

Dye

rem

oved

(%)

Fig. 1. Dye removal pattern with free HRP as a function of

contact time.

with the specified experimental conditions. With in-

crease in pH above 2, dye removal was found to drop

significantly (pH from 3 to 7) and the same trend contin-

ued up to an aqueous phase of pH9. Aqueous phase of

pH2 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 ac-

cepts 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

(0.1–0.8ll l�1) in the reaction mixture by keeping all

the other experimental conditions constant (dye concen-

tration—20mgl�1; temperature—24 �C; enzyme concen-

tration—2.94units; reaction time—45min). 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 0.6ll 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

0.6ll l�1 of H2O2 dose, where maximum activity was

observed. Compared to aqueous phase pH of 7, pH2

yielded more enzyme activity. It can be deduced that

0.6ll l�1 of H2O2 was optimum for acid azo dye removal.

3.1.4. Optimum concentrate of dye

Concentration of the substrate present in the aqueous

phase has significant influence on any enzyme-mediated

Fig. 4. Effect of dye concentration on free HRP catalyzed dye

removal.

15

16

17

2.44

2.9

2.92

2.94

2.96Dye removalHRP activity

0

1

2

3

4

5

6

7

8

0.1 0.2 0.4 0.6 0.8

Dye

rem

oval

(%

)

2.34

2.36

2.38

2.4

2.42

HR

P a

ctiv

ity

(uni

ts)

Dye removalHRP activity

pH 2

pH 7

H2O2 dose

Fig. 3. Effect of H2O2 dose on free HRP catalyzed dye removal.

10

11

12

13

14

15

16

17

18

0.735 1.47 2.205 2.94 3.675 4.41

HRP dose (units)

2

3

4

5

6

7

8

Dye removal

Dye

rem

oval

(m

g l-1

)

Dye remaining

Dye

rem

aini

ng (

mg

l-1)

Fig. 5. Effect of free HRP dose on dye removal.

S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105 1101

reaction. If the amount of enzyme concentration is kept

constant and the substrate concentration is gradually in-

creased, 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 (5–40mgl�1), keeping

all the other parameters constant (H2O2—0.6ll l�1;

aqueous phase pH—2; reaction time—45min; tempera-

ture—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 30mgl�1 of dye concentra-

tion. Subsequent increase in dye concentration above

30mgl�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 experimen-

tal conditions.

3.1.5. Optimum dose of enzyme

Normally removal of the aromatic compound is

dependent on the amount of catalyst added since the cat-

alyst has a finite lifetime and also the conversion is

found to be dependent on the contact time. There is

an optimum relationship between the concentration of

enzyme and substrate for achieving maximum activity.

To study the effect of enzyme concentration on the reac-

tion, the reaction must be kept independent of the sub-

strate concentration so that any variation in the

amount of product formed is a function of enzyme con-

centration. To study the optimum dose of HRP, exper-

iments were carried out at various HRP doses ranging

from 0.735 to 4.41unitsml�1 at specified experimental

conditions (dye—20mgl�1; pH—2; temperature—

25 �C, contact time—45min, H2O2—0.6ll 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 0.735unitsml�1 to

2.205unitsml�1 might have resulted in a gradual in-

crease in the dye removal rates (62–84%). However,

0

10

20

30

40

50

60

70

80

90

0 15 30 45 60 75 90

Contact time (min)

Dye

rem

oved

(%

)

Acrylamide gelbeadsAlginate beads

Fig. 7. Dye removal pattern with immobilized HRP as a

function of contact time.

1102 S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105

subsequent increase in HRP dose up to 4.41unitsml�1

might have yielded significantly low impact on HRP

dye catalyzed reaction (0.5%). It can be presumed that

the enzyme dose of 2.205unitsml�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-activa-

tor (H2O2) is having significant effect on the overall en-

zyme catalyzed reaction. In order to find out the effect of

repeated addition of HRP and H2O2 alone and in com-

bination, 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 per-

formance 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 prod-

uct from enzyme catalyzed reaction.

3.2. Studies with immobilized HRP

Application of free enzyme in industrial processes is

not economically viable, while immobilization/entrap-

ment of enzyme results in repeated application and is

more economical. In the present study, two types of poly-

0

10

20

30

40

50

60

70

80

0 30 60 90

Time (min)

a b c d

Dye

rem

oval

(m

g l-1

)

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).

meric materials viz. alginate and acrylamide for the

entrapment of peroxidase have been used in order to

study their relative efficiency in dye removal. Experi-

ments were carried out separately with both entrapped

HRP at a dye concentration of 20mgl�1 (H2O2—

0.6ll l�1; pH—2.0; temperature—25 �C, contact time—

45min). The results are shown in Fig. 7. It can be ob-

served 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 re-

sulted in effective dye removal when compared to free

HRP (67%), while alginate immobilized HRP showed

inferior performance compared to the free HRP. Nor-

mally, 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 immobiliza-

tion is the reusability of the matrix in the process. There-

fore 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 acryl-

amide 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.

Fig. 8. Dye removal pattern at 20mgl�1 with repeated appli-

cation of immobilized HRP.

0

10

20

30

40

50

60

70

80

90

2 3 4 5 6 7

Aqueous phase pH

Dye

rem

oval

(%)

Fig. 9. Effect of pH on immobilized HRP catalyzed dye

removal.

S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105 1103

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 pH2 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

Fig. 10. HPLC Chromatograph (a—control

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.

[dye], b—sample [HRP treated dye]).

1104 S.V. Mohan et al. / Chemosphere 58 (2005) 1097–1105

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 3.21min (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 under-

stand the relatively enhanced degradation. The plates

were incubated at 30 �C for 5days. After incubation,

growth was continuously monitored up to 10days. In

enzyme treated plates, formation of colonies was ob-

served after the 2nd day of incubation and subsequently

profuse growth of colonies were seen. In case of the con-

trol plates, growth was seen only after 6days of incuba-

tion. This observation correlates with the fact that

enhanced degradation was observed due to HRP cata-

lyzed 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 cata-

lyzed 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 concentra-

tion, 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 as-

sess the reusability of HRP. Immobilized HRP in acryl-

amide matrix resulted in effective performance over the

free HRP, while alginate entrapped HRP yielded infe-

rior performance over the free one. Repeated applica-

tion of enzyme was observed to be feasible with

immobilized HRP beads. Standard plating studies re-

vealed 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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